uses of a composition prepared from dried gold kiwi fruit

专利摘要:
The present disclosure encompasses compositions prepared from kiwi fruit. In particular, the invention encompasses compositions prepared from the golden kiwi varieties of Actinidia chinensis. Methods for preparing these compositions are also encompassed. Furthermore, methods of using these compositions are further encompassed, in particular, to treat or prevent disorders of the gastrointestinal system, including but not limited to: inflammation, constipation, bowel irregularity, microbiota imbalances, irritable bowel syndrome and inflammatory bowel disease.
公开号:BR112017011078B1
申请号:R112017011078-4
申请日:2015-11-27
公开日:2021-05-11
发明作者:Juliet ANSELL；Paul BLATCHFORD
申请人:Anagenix Ip Limited；
IPC主号:

专利说明:

RELATED PATENT APPLICATIONS
[001] This application claims the benefit of New Zealand patent application number 702454 filed November 28, 2014 and New Zealand patent application number 706405 filed March 27, 2015, with the contents of incorporated herein by reference. FIELD OF THE INVENTION
[002] The present invention relates to compositions prepared from golden kiwi fruit, Actinidia chinemis. Also listed are methods of making such compositions and methods of using such compositions, including methods of treating or preventing disorders of the gastrointestinal system. BACKGROUND OF THE INVENTION
[003] The gastrointestinal tract contains about 1014 microbial cells, consisting of more than 1000 species or phylotypes, most of which reside in the colon (Rajilic-Stojanovic and Vosm 2014; Qin et al. 2010; Egert et al. 2006). The large intestine is a metabolically active fermentation site, characterized by diverse and intricate microbial relationships that are an integral part of human health (Backhed et al. 2005). Experimental data have shown the extent to which the colonic microbiota and its human host exist in a delicate state of equilibrium. The microbiota has been shown to be associated with a wide range of health benefits, including improved maturation and immune function, modified behavior, satiety regulation, pathogen inhibition, increased mineral absorption, and maintenance of energy balance (Geurts et al. 2014 ; Parnell and Reimer 2012; Bravo et al. 2011; Buffie and Pamer 2013).
[004] This interdependence between host-microbiota was illustrated in a study using mice and zebrafish, where the native microbiota of each were transplanted to germ-free representatives of other species. It has been found that the composition can be reversed to resemble that of the native host microbiota (Rawls et al. 2006). Imbalances in the composition of the microbial community caused by diet, genetics, age, stress or xenobiotics can induce a state of dysbiosis that can promote a more susceptible microbiota to the disease. Consumption of certain types of dietary components has a major influence on colonic microorganism communities, with changes typically seen within 24 hours of consumption (Wu et al. 2011; Parkar et al. 2012).
[005] In the human gastrointestinal tract, Faecalibacterium prausnitzii is one of the most populous species, typically being observed in more than 5% of the total proportion of the colonic microbiota of healthy adults (Miquel et al. 2013). Members of the Firmicutes phylum, F. prausnitzii, are commensal inhabitants of the human large intestine, with anti-inflammatory properties demonstrated in vivo (Sokol et al. 2009; Furet et al. 2010). Low levels of F. prausnitzii have been repeatedly associated with a variety of bowel disorders including irritable bowel syndrome (IBS), atopy, diabetes and inflammatory bowel disease (IBD) such as Crohn's disease (CD) and ulcerative colitis ( UC) (Sokol et al. 2009; Furet et al. 2010; Rajilic-Stojanovic et al. 2011; Candela et al. 2012; Willing et al. 2010). These consistent observations show that a depleted concentration of F. prausnitzii is an undesirable end point and therefore any treatment that can selectively stimulate its proliferation is likely to be valuable.
[006] The mechanisms by which F. prausnitzii facilitate its health-promoting effects have been attributed to butyrate production and anti-inflammatory effects. A mouse study by Sokol and colleagues found that the supernatant of F. prausnitzii or F. prausnitzii reduced the severity of chemical-induced colitis, promoted the synthesis of anti-inflammatory cytokines, and attenuated the production of pro-inflammatory cytokines, suggesting that anti-inflammatory effects are mediated by secreted metabolites (Sokol et al. 2008). In addition, F. prausnitzii generates large amounts of butyrate, as well as some lactate and formate, as a result of carbohydrate fermentation (Duncan et al. 2002; Duncan et al. 2004).
[007] Constipation is a common condition that can significantly affect the quality of life of an individual, the deficiency being compared to that of severe chronic conditions such as diabetes and osteoarthritis. It is estimated that up to 20% of the world population suffers from this condition, with women and individuals over 65 years of age being the most affected (Attaluri et al. 2011; Udani & Bloom 2013). Complications that can arise from constipation include anal fissures, rectal prolapse, and fecal impaction. The effort to pass stool can lead to hemorrhoids. In later stages of constipation, the abdomen can become distended, hard, and diffusely tender. Severe cases may present with symptoms of intestinal obstruction, eg vomiting and painful abdomen, as well as encopresis, where soft stools from the small intestine exceed the mass of impacted fecal matter in the colon.
[008] It is well known that disturbance of the balance of microbiota in the digestive tract can alter intestinal motility, resulting in constipation (Husebye et al. 2001; Rhee et al. 2001). In addition, patients with chronic constipation have an imbalance in the microbiota, characterized by a relative decrease in beneficial bacteria and an increase in potentially pathogenic bacteria and fungi (Khalif et al. 2005). These patients show a significant increase in E. coli, S. aureus, and enterobacteria counts (Khalif et al. 2005).
[009] In addition, longer disease progression from chronic constipation leads to higher scores for undesirable microorganisms, and lower scores for desirable ones (Khalif et al. 2005). In a study of children diagnosed with chronic constipation, intestinal dysbiosis was found in the stool, with a significant increase in Clostridium, Bacteroides and E. coli counts (Zoppi et al. 1998). One particular study found a significant decrease in Bifidobacterium and Lactobacillus strain counts in the stool of constipated individuals; such a decrease is up to ten times smaller for the Bifidobacterium genus (Chassard et al. 2012).
[010] Intervention options for constipation remain difficult and challenging, and many individuals are dissatisfied with current therapies and medications. These include lifestyle and dietary modifications such as increased consumption of fruits and vegetables, fiber supplementation (methylcellulose, Konjac glucomannan, psyllium), increased fluid intake and exercise, as well as pharmacological intervention with stool softeners, stimulant laxatives and osmotic laxatives (Attaluri et al. 2011; Leung et al. 2011; Liu 2011).
[011] Routine use of laxatives is contraindicated, as patients may come to depend on their use. Enemas can be used to provide a form of mechanical stimulation. On the other hand, enemas are typically only useful for stool in the rectum, not the intestinal tract. A growing range of herbs and other natural products (including aloe and rhubarb (Udani & Bloom 2013)) advertised to aid laxation became available on the market. However, there is only anecdotal evidence about its effectiveness and success in providing relief from constipation.
[012] In contrast, fiber is known to be important in digestion - soluble fiber attracts water which helps slow digestion, and insoluble fiber adds bulk to the stool to aid laxation. Enzymes also aid digestion by breaking down large dietary compounds into smaller, controllable pieces. For example, amylase breaks down starch into simple sugars and proteases breaks down proteins into amino acids. However, fiber and enzyme supplementation alone is largely ineffective for slow transit constipation (prolonged delay in the transit of stool through the colon) and defecatory disorders. Furthermore, such supplementation can have unwanted side effects such as excessive gas production and uncomfortable bloating.
[013] Green kiwi fruit (eg Actinidia delicious var. Hayward) have been highlighted as an effective product in the area of digestion with several studies now demonstrating their effectiveness (Stonehouse et al. 2012). The current consensus is that the laxation effect of green kiwi fruit appears to be primarily due to its dietary fiber and enzyme content (Chang et al. 2010; Rush et al. 2002, Stonehouse et al. 2012, Drummond & Gearry 2013).
[014] In contrast, golden kiwi fruit (eg, Actinidia chinensis var. Hortl6A) are not traditionally associated with laxation (Ferguson 2003; Rush 2002). Gold kiwi has been found to have little/no actinidin content and less dietary fiber than green kiwi. The Hortl6A variety of kiwi fruit has been investigated for its effect on immunity (Hunter et al 2012, Skinner 2012), with the assertion that the reduced or minor laxative effects of the golden kiwi make it an ideal choice for naturally enhancing immunity, particularly in children where increased laxation may not be desirable (Adaim 2010).
[015] Given the occurrence of gastrointestinal disorders in the population, there is a need for new compositions, particularly compositions derived from natural sources, to restore and maintain digestive health. SUMMARY OF THE INVENTION
[016] In one aspect, the invention comprises a method of preparing a kiwi fruit composition, comprising: obtaining golden kiwi fruit that is a variety of GoId3 or a genetic derivative thereof; remove the kiwi skins and form a puree to a sieve size of less than 1 mm; and dry the puree and produce a powder.
[017] In several aspects: The method also comprises the removal of the kiwi seeds before the formation of puree. The method further comprises purifying the kiwi to obtain puree with a Brix value of 16 to 21°. The method further comprises purifying the kiwi fruit to obtain puree having a viscosity of 11.0 to 13.0, or 10.0 to 14.0, measured at 12.5° Brix, 20°C. The method further comprises freeze-drying the puree and milling to produce the powder. The method further comprises enriching the composition for polyphenols. The method further comprises adding polyphenols to the composition. The method further comprises drying the puree for 24 to 56 hours. The method further comprises lyophilizing the puree for 40 to 56 hours.
[018] In another aspect, the invention comprises a composition prepared from dry gold kiwi, wherein the dry gold kiwi is a Gold3 kiwi or a genetic derivative thereof. Powder can be produced by a method of any of the above aspects.
[019] In several respects: The composition is formulated for enteral administration. The composition is formulated for oral administration. The composition is formulated as a tablet or capsule. The capsule is a gel capsule. The tablet or capsule is formulated to comprise 400 to 800 mg of the powder. Alternatively, the capsule is formulated to comprise 100 to 1000 mg of the powder. The composition is formulated as a liquid. The liquid is formulated to comprise 400 to 800 mg of the powder per dosage unit. The composition is formulated as a jelly or a sachet. The composition is formulated in combination with an additional digestive aid. The composition is formulated in combination with one or more compositions from a prebiotic, probiotic or symbiotic composition. The composition is formulated in combination with fiber and/or a digestive enzyme. The composition is supplemented with polyphenols.
[020] In another aspect, the invention comprises a method for treating or preventing constipation, or for maintaining or improving bowel regularity, comprising: administering to an individual a composition of any of the foregoing aspects, thereby treating or preventing constipation, or maintaining or improving bowel regularity, in the individual.
[021] In several respects: The composition is administered enterally. The composition is administered orally or rectally. The composition is administered as a tablet, capsule or liquid. The composition is administered as a jelly or a sachet. The capsule is a gel capsule. The composition is administered in a dosage of 2000 to 4000 mg of powder per day, or a liquid equivalent. Alternatively, the dosage is 250 to 2500 mg of powder per day, or a liquid equivalent thereof. The composition is administered in combination with an additional digestive aid. The composition is administered in combination with one or more compositions from a prebiotic, probiotic or symbiotic composition. The composition is administered in combination with fiber and/or a digestive enzyme. The composition is supplemented with polyphenols.
[022] In another aspect, the invention comprises a method for treating or preventing microbial imbalance in the digestive tract, comprising: administering to an individual a composition of any of the foregoing aspects, thereby treating or preventing microbial imbalance in the digestive tract. individual.
[023] In several respects: The composition is administered enterally. The composition is administered orally or rectally. The composition is administered as a tablet, capsule or liquid. The composition is administered as a jelly or a sachet. The capsule is a gel capsule. The composition is administered in a dosage of 2000 to 4000 mg of powder per day, or a liquid equivalent. Alternatively, the dosage is 250 to 2500 mg of powder per day, or a liquid equivalent thereof. The composition is administered in combination with an additional digestive aid. The composition is administered in combination with one or more compositions from a prebiotic, probiotic or symbiotic composition. The composition is administered in combination with fiber and/or a digestive enzyme. The composition is supplemented with polyphenols.
[024] In yet another aspect, the invention comprises a method for maintaining or increasing beneficial bacteria in the digestive tract, comprising: administering to an individual a composition of any of the foregoing aspects, thereby maintaining or increasing beneficial bacteria in the tract digestive in the individual.
[025] In several respects: The composition is administered enterally. The composition is administered orally or rectally. The composition is administered as a tablet, capsule or liquid. The composition is administered as a jelly or a sachet. The capsule is a gel capsule. The composition is administered in a dosage of 2000 to 4000 mg of powder per day, or a liquid equivalent. Alternatively, the dosage is 250 to 2500 mg of powder per day, or a liquid equivalent thereof. The composition is administered in combination with an additional digestive aid. The composition is administered in combination with one or more compositions from a prebiotic, probiotic or symbiotic composition. The composition is administered in combination with fiber and/or a digestive enzyme. The composition is supplemented with polyphenols.
[026] The beneficial bacteria are selected from: the group of Bacteroides-Prevotella-Porphyromonas, Bifidobacterium spp., Lactobacillus spp., and the group of Lachnospiraceae.
[027] The beneficial bacteria are selected from: Faecalibacterium prausnitzii, Clostridium coccoides, Bacteriodes fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides cellulosilyticus, Roseburia intestinalis, Roseburia inulinovorans, Ruminococcus brococcus, and Ruminococcus.
[028] In yet another aspect, the invention comprises a method for maintaining or increasing Faecalibacterium prausnitzii in the digestive tract, comprising: administering to an individual a composition of any of the foregoing aspects, thereby maintaining or increasing Faecalibacterium prausnitzii in the digestive tract in the individual.
[029] In several respects: The composition is administered as one or more of a tablet, capsule, liquid, jelly or sachet. The composition is administered as one or more of a tablet, capsule, liquid, jelly or sachet. The capsule is a gel capsule. The composition is administered in a dosage of 2000 to 4000 mg of powder per day, or a liquid equivalent. The composition is administered in a dosage of 250 to 2500 mg of powder per day, or a liquid equivalent. The composition is administered in combination with an additional digestive aid. The composition is administered in combination with one or more compositions from a prebiotic, probiotic or symbiotic composition. The composition is administered in combination with fiber and/or a digestive enzyme. The composition is supplemented with polyphenols. The individual has one or more symptoms of inflammation. The individual has one or more symptoms of: Crohn's disease, ulcerative colitis, irritable bowel syndrome, inflammatory bowel disease, gastrointestinal cancer, allergy, atopy, or diabetes.
[030] In another aspect, the invention comprises a method of treating or preventing irritable bowel syndrome or inflammatory bowel disease, comprising: administering to a subject a composition of any of the foregoing aspects, thereby treating or preventing irritable bowel syndrome or inflammatory bowel disease in the individual.
[031] In several respects: The composition is administered as one or more of a tablet, capsule, liquid, jelly or sachet. The composition is administered as one or more of a tablet, capsule, liquid, jelly or sachet. The capsule is a gel capsule. The composition is administered in a dosage of 2000 to 4000 mg of powder per day, or a liquid equivalent. The composition is administered in a dosage of 250 to 2500 mg of powder per day, or a liquid equivalent. The composition is administered in combination with an additional digestive aid. The composition is administered in combination with one or more compositions from a prebiotic, probiotic or symbiotic composition. The composition is administered in combination with fiber and/or a digestive enzyme. The composition is supplemented with polyphenols.
[032] In yet another aspect, the invention comprises the use of the composition of any of the foregoing aspects for the preparation of a medicament to: treat or prevent constipation, or maintain or improve bowel regularity, in an individual; treating or preventing a microbiota imbalance in an individual; maintain or increase beneficial bacteria in an individual's digestive tract; maintain or increase Faecalibacterium prausnitzii in an individual's digestive tract; or
[033] to treat or prevent irritable bowel syndrome or inflammatory bowel disease in an individual.
[034] The above brief summary broadly describes the characteristics and technical advantages of certain embodiments of the present invention. Other technical advantages will be described in the detailed description of the invention and examples below.
[035] New features believed to be features of the invention will be better understood from the detailed description of the invention when considered in relation to any attached figures and examples. However, the figures and examples provided herein are intended to help illustrate the invention or to aid in developing an understanding of the invention, and are not intended to limit the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS
[036] Figure 1; The polyphenol content of green kiwi (Hayward) and gold (Gold3) powder pre- and post-digestion.
[037] Figure 2; Change in the growth of bacterial strains when exposed to water and DMSO extracts from digested powdered kiwifruit (Gold3)
[038] Figure 3; Effect of kiwi gold (Gold3) powder on the production of short-chain fatty acids in vitro using a mixed fermentation model.
[039] Figure 4; Treatment testing scheme, washout periods and sampling points.
[040] Figure 5A: Improvement in the Digestive Health Index in participants with functional constipation after treatment with GOLD (Gold3).
[041] Figure 5B: Net difference in the abundance of Faecalibacterium prausnitzii in participants with functional constipation after treatment with GOLD (Gold3)
[042] Figure 5C: Quantitative PGR data (transformed LOG) showing the net difference in Faecalibacterium prausnitzii levels in participants with functional constipation after treatment with GOLD (Gold3).
[043] Figure 6; LightCycler® 480 qPCR data showing bacterial groups as mean 16S rRNA gene copy number/gram of fecal sample (upper row LOG transformed, lower row natural data) calculated and standard error of the mean (SEM) as error bars before and after each treatment period in the healthy group.
[044] Figure 7; LightCycler® 480 qPCR data showing bacterial groups as mean 16S rRNA gene copy number/gram of fecal sample (upper line LOG transformed, lower line natural data) calculated and standard error of the mean (SEM) before and after each period of treatment in the group with functional constipation. Detailed description of the invention
[045] The following description presents numerous exemplary configurations, parameters and the like. It should be recognized, however, that such description is not intended to be a limitation on the scope of the present invention, but is rather provided as a description of exemplary embodiments.
[046] All references, including patents and patent applications, cited in this specification are hereby incorporated by reference. Any reference is not admitted to constitute the prior art. Nor does discussion of any reference constitute an admission that such reference forms part of the common general knowledge in the art, in New Zealand or in any other country. Definitions
[047] In each case herein, in the descriptions, embodiments and examples of the present invention, the terms "comprising", "including", etc., should be read expansively, without limitation. Thus, unless the context clearly requires otherwise, throughout the description and claims, the words "comprises", "comprising", and the like, should be interpreted in an inclusive sense as opposed to an exclusive sense, ie , in the sense of "including, but not limited to".
[048] "Bowel regularity" means having regular bowel movements, for example, on a daily or weekly basis. Although regularity differs from person to person, an expected number of bowel movements can range from at least four times a week to at least once a day. An “improvement” in regularity means an increase in at least one bowel movement per week.
[049] The term "bowel irregularity" means that the regularity of bowel movements has been compromised. This could mean, for example, an increased time period between bowel movements, or a decrease in the expected number of bowel movements per day or per week. For example, a reduction in at least one bowel movement per week may indicate an intestinal irregularity.
[050] As used herein "constipation" means having one or more symptoms of being constipated, for example one or more of: straining during a bowel movement; hard or lumpy stools; sensation of anorectal blockage or blockage; feeling of incomplete evacuation; manual maneuvers to facilitate defecation; or three or fewer bowel movements in a week.
[051] A "dietary aid" is a composition that aids in digestion or other aspects of the digestive system, eg, soluble and insoluble fiber, digestive enzymes, probiotics, prebiotics and symbiotics. Included as digestive aids are compositions comprising one or more of psyllium, methylcellulose, glucomannan, magnesium, linseed, papule, ginger, aloe (e.g., aloe vera juice) and rhubarb.
[052] A "digestive enzyme" is an enzyme that aids in the function of the gastrointestinal system. Included are actinidine, amylase, protease, lipase, lactase, maltase, sucrase and cellulase.
[053] "Digestive tract" and "gastrointestinal tract" refer to the digestive system (ie, gastrointestinal system) of a human being and of another animal. This includes the esophagus, stomach, small intestine including duodenum, jejunum and ileum, large intestine including cecum, ascending colon, transverse, descending, sigmoid and rectum. "Intestine" is a synonym for the intestinal tract.
[054] A "disorder" of the digestive tract includes an illness or other condition that affects the digestive system (ie, gastrointestinal system), which may be an acute or chronic state, such as inflammation, constipation, bowel irregularity, imbalance of microbiota (eg, reduced levels of beneficial organisms, altered ratios of beneficial organisms, and/or increased levels of harmful organisms). Particular disorders include inflammatory bowel disease and irritable bowel syndrome. Other disorders are described in detail here.
[055] "Gold3" refers to a particular variety of gold kiwis also known as ‘Zesy002’, and marketed as ZESPRI® SUNGOLD kiwis. A New Zealand plant variety rights application was made on June 25, 2009 for Gold3 under application number K1W042. The botanical name for the Gold3 kiwi fruit is Actinidia chinensis Planch.
[056] A "genetic derivative" of a golden kiwi variety (eg, Gold3 variety) refers to offspring cultivars, mutants (sports) or other cultivars that are obtained from the original gold kiwi stock. This includes offspring obtained from a genetic cross with the original golden kiwi, eg F1 progeny or F2 progeny. The term "genetic derivative" can refer to the derived plant itself, or its fruit.
[057] "Inflammation" refers to a condition characterized by one or more of: vasodilation, heat, redness, pain, swelling, edema, lesions, fissures, ulcerations, leukocyte extravasation, and loss of function. Both acute and chronic forms of inflammation are included, the latter of which includes inflammatory disorders, eg autoimmune diseases. Inflammatory bowel disease is particularly included. Other inflammatory disorders are described elsewhere in this document.
[058] As noted here, the terms "lyophilization" and "freeze drying" are used synonymously. It will be understood that the terms "freeze drying"/"lyophilization" do not exclude the use of higher temperatures (i.e., higher than freezing temperatures). For example, higher temperatures can be used to remove residual moisture during the secondary drying stage for freeze drying/freeze drying procedures.
[059] "Microbiota", as used herein, refers to populations of microorganisms that live in the digestive tracts of humans and other animals. The synonymous terms are "microflora" and "microbiome".
[060] A "microbial imbalance" (also called "dysbiosis") is a state whereby the number of beneficial organisms in the digestive system is reduced, one or more ratios of beneficial organisms are altered, and/or the number of organisms harmful effects in the digestive system is increased. Beneficial organisms can include, for example, lactic acid producing bacteria and butyrate producing bacteria. Particular beneficial organisms include, but are not limited to, strains of Bifidobacterium, strains of Bacteroidaceae such as Bacteriodes fragilis, and also faecal bacteria such as beneficial bacteria of the phylogenetic group Clostridium leptum, including Faecalibacterium prausnitzii. Also included are Clostridium coccoides, Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides cellulosilyticus, Roseburia intestinalis, Roseburia inulinovorans, Ruminococcus bromii, and Ruminococcus flavefaciens. Also included are beneficial organisms from the groups of Bacteroides-Prevotella-Porphyromonas, Lachnospiraceae, and lactobacilli. The deleterious organisms can include, as non-limiting examples, strains of Staphylococcus and Salmonella, as well as members of the groups Enterobacteriaceae, Pasteurellacaea, Veillonellaceae, and Fusobacteriaceae.
[061] A "prebiotic" is a composition that increases the number and/or activity of beneficial organisms in the digestive system. Typically, a prebiotic comprises fermentable components and produces positive changes (eg, improved levels, activities, or ratios) in populations of beneficial bacteria in the gastrointestinal microbiota. This includes, for example, lactic acid producing bacteria and/or butyrate producing bacteria. Specific examples of beneficial organisms include Bifidobacterium strains and Faecalibacterium strains, including Faecalibacterium prausnitzii strains, and also Bacteroidaceae strains such as Bacteriodes fragilis. Also included are Clostridium coccoides, Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides cellulosilyticus, Roseburia intestinalis, Roseburia inulinovorans, Ruminococcus bromii, and Ruminococcus flavefaciens. Also included are the groups of Bacteroides-Prevotella-Porphyromonas, Lachnospiraceae, and lactobacilli, as well as other organisms described herein.
[062] A "probiotic" is a composition comprising one or more beneficial organisms that colonize the digestive system, including the colon, for example, lactic acid producing bacteria and/or butyrate producing bacteria. Specific beneficial organisms include Bifidobacterium strains and Faecalibacterium strains, including Faecalibacterium prausnitzii strains, and also Bacteroidaceae strains such as Bacteriodes fragilis, as well as other organisms described herein.
[063] A "symbiotic" is a composition that combines one or more prebiotic agents and one or more probiotic organisms.
[064] As used herein, a "subject" can be a human or non-human animal, particularly a mammal, including cattle, sheep, goats, swine, horses and other livestock, including also dogs, cats and others domesticated animals.
[065] "Treatment", as used herein, is intended to mean the reduction, amelioration or resolution of a disorder, for example, a gastrointestinal disorder, such as a disease or other condition of the gastrointestinal system. A treatment will result in the reduction, improvement or elimination of one or more symptoms of the disorder.
[066] "Prevention", as used herein, is intended to mean stopping or delaying the onset of a disorder, for example, a gastrointestinal disorder, such as a disease or other condition of the gastrointestinal system. A preventive measure will result in the stoppage or delay of one or more symptoms of the disorder, or a decrease in symptoms, if they arise. Kiwi gold and associated bioactivity
[067] Gold3 is a new variety of golden kiwi fruit developed by Zespri®, which was found to have a tolerance to Pseudomonas syringae pv actinidiae (Psa; a bacterial disease of the kiwi vine) and is replacing the Hortl 6A variety as the new variety of commercial gold of choice in New Zealand. The Gold3 kiwi variety is similar in nutritional composition to its predecessor, having less dietary fiber and actinidin content than the green kiwi (Table 1). Table 1: Nutritional content of all kiwi fruit

[068] Since gold kiwi varieties contain lower levels of fiber and actinidin, the components considered to be primarily responsible for the laxation effects of green kiwi fruit, one would expect that a gold kiwi-derived product would have a lesser laxative effect than than a derivative of the green Hayward kiwi.
[069] Surprisingly, the inventors found that Gold3 kiwi-derived powder (GOLD) improved laxation to the same degree as Hayward Green kiwi-derived powder (ACTAZIN™) in healthy subjects (see Examples 5 and 6, hereafter) . These results suggest that other bioactive kiwi components present in Gold3 powder may have a greater impact on the observed laxation effect than current convention dictates. The compositions are therefore useful for improving or maintaining bowel regularity and/or treating or preventing constipation.
[070] Although not intending to be bound by any theory, it is postulated that polyphenols may be important active ingredients in golden kiwi varieties, in particular in the Gold3 kiwi variety. It is postulated that the observed efficacy for laxation may be attributable to the combination of fiber, enzymes, prebiotic carbohydrates, and polyphenols in the fruit.
[071] Polyphenols are compounds that occur naturally in plants, characterized by the presence of one or more phenol units. It is believed that at least some of the health benefits of polyphenols arise by favoring the growth of beneficial bacteria and by bacteria converting the polyphenols into other bioactive compounds that are absorbed into the bloodstream and exert effects both in the intestine and in other parts of the body. body (Seeram 2014; Cardona et al. 2013).
[072] It is estimated that 90 to 95% of total polyphenol consumption accumulates in the colon where phenolic compounds undergo metabolism by intestinal microbes to generate low molecular weight metabolites. These metabolites are absorbable and may be attributable to the observed health effects of polyphenols (Cardona et al. 2013).
[073] Based on the results presented here, the inventors believe that polyphenols from the golden kiwi varieties, eg Gold3 or its derivatives, may have the ability to act as prebiotics. Researchers in the field have described prebiotics as "selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastrointestinal microflora, thereby conferring benefit(s) on the health of the host" (Gibson et al. 2010).
[074] In particular, gold kiwi has higher polyphenol contents than green kiwi (see eg Drummond 2013; see also Table 5 in this document). Furthermore, the inventors' in vitro studies have shown that digested kiwi gold Gold3 powder (simulated upper gastrointestinal digestion) has an altered polyphenol profile compared to pre-digestion (see Example 2); however, the overall phenolic content is maintained. Although powdered green kiwi also has an altered post-digestion polyphenol profile, the overall content is reduced by digestion (see Example 2).
[075] From there, the inventors conclude that the polyphenols present in powdered Gold3 kiwi are surviving digestion in the stomach better than those in powdered green kiwi. This means that gold kiwi polyphenols such as the Gold3 kiwi variety are available for biotransformation into different phenolic compounds.
[076] Without sticking to theory, Gold3 kiwi fruit is believed to have a unique profile rich in chlorogenic acid, E-caffeoyl-3-glycoside, phlorizine, procyanidin B2, and quercetin. These phenolic compounds are believed to remain 'undigested' and therefore transit to the colon, where they are metabolized by the intestinal microbiota into metabolites (phenolic derivatives). These derivatives can be absorbed and start to interact in other metabolic pathways and bring health benefits, including facilitated laxation.
[077] The phenylpropanoid pathway is known to produce most of the phenolic compounds present in plants. Phenylpropanoids control plant repair, growth and defense systems (immune systems), and their interaction with beneficial microbes and beneficial predators (trophic systems). Thus, it is possible that the phenolic compounds in the Gold3 variety may be responsible for both the plant's resistance to the pest and the digestive benefits of the Gold3-derived powder as disclosed herein.
[078] The inventors have also found that Gold3 gold kiwi powder contains components resistant to digestion, which are known to have prebiotic properties (see Example 4). These components aid in the modulation of the gut microbiota, and stimulate the production of metabolites, such as short-chain fatty acids (SCFAs; see Example 4), which are attributed with several health benefits.
[079] Furthermore, the inventors have found that the powdered GoId3 gold kiwi fruit influences the growth pattern of the intestinal microflora, stimulating the growth of beneficial bacteria over harmful bacteria (see Example 3). Notably, the inventors also found a significant increase in the relative abundance of Faecalibacterhim prausnitzii in faecal samples from patients with constipation treated with gold kiwi Gold3 powder (Examples 7 and 8). This increase in beneficial bacteria, including F. prausnitzii, was confirmed by quantitative PCR analysis (Examples 9 and 10).
[080] This is a significant finding since depleted concentrations of F. prausnitzii are associated with gastrointestinal disorders, and in particular, with inflammatory states of the gastrointestinal tract. It is noted that reduced levels of F. prausnitzii have been specifically associated with irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), as well as other clinical conditions. See, for example, Sokol et al. 2008; Sartor 2011. See also, below.
[081] Thus, it is evident that Gold3 Gold kiwis and their genetic derivatives can be used in compositions to: treat or prevent constipation, maintain or improve bowel regularity, treat or prevent microbiota imbalance, maintain or increase beneficial bacteria in the digestive tract, maintain or increase F. prausnitzii in the digestive tract, and/or treat or prevent irritable bowel syndrome or inflammatory bowel disease. Other uses for composition are described in detail in this document. Methods for producing kiwi compositions
[082] The present invention relates, in general, to a composition prepared from gold kiwi fruit. In a particular aspect, the composition is prepared from Actinidia chinensis. Preferably, the Gold3 (also known as G3) variety of gold kiwi is used. In other aspects, one or more genetic derivatives of the golden kiwi variety may be used. For example, it may be desirable to use the F1 or F2 progeny of a genetic cross that includes the main stock of the golden kiwi variety. Alternatively, any mutant cultivars or other cultivars obtained from the original can be used.
[083] The composition may be prepared in powder form, for example a lyophilized powder, or in any other suitable dosage form. In certain respects, it may be desirable to formulate the powder into tablets (including tablets if rapidly dissolving) or capsules (including sustained-release capsules). Tablets can be scored tablets, chewable tablets, effervescent tablets, orodispersible tablets, or compressed to form a suspension. Capsules can be gelatin capsules, including gelatin capsules produced by one-piece gel encapsulation and two-piece gel encapsulation. Non-gelatin capsules are also included, as well as pills. The powder can be supplied in free-flowing form or as a solid cake. The composition can be supplied as a powder to form a suspension, a powder to form a solution, oral bulk granules, or bulk oral powder. Alternatively, the composition may be formulated as a tonic, elixir, linctus, concentrate, syrup, solution, suspension, emulsion, slurry, puree, paste, or as drops. In other aspects, the composition can be formulated as a gel or jelly. The composition may be provided in sachet form, for example a powder sachet, or a gel or jelly sachet. Also included are formulations that comprise thin strips, or that comprise solids in a capsule for mixing with food or beverage. Other formulas are also possible, as described here below.
[084] The compositions of the invention can be prepared from a golden kiwi puree obtained from one or more commercial sources. Preferably, the golden kiwi puree had both the seeds and skin removed. It is also preferred that the puree has been prepared with a sieve size of about 1 mm or less. It is further preferred that the puree has a viscosity (measured at 12.5° Brix, 20°C) of about 12.0; or can range from 10.0 to 14.0; or from 11.0 to 13.0; or from 11.75 to 12.25; or one from 11.8 to 12.2; or from 11.9 to 12.1; or it can be from about 11.0, from about 11.25, from about 11.7, from about 11.8, from about 11.9, from about 12.1, from about 12, 2, of about 12.3, or of about 12.5.
[085] The pH of the puree can range from 3.2 to 3.8; or 3.0 to 4.0; or 3.1 to 3.9; or it can be from about 3.1, from about 3.2, from about 3.3, from about 3.4, from about 3.5, from about 3.6, from about 3, 7, of about 3.8, of about 3.9, or of about 4.0. In some circumstances, it may be desirable to adjust the pH of the puree or final composition to approximate physiological levels. In particular, obtaining a pH range of 6.0 to 8.0 may be useful; or from 6.5 to 7.5; or from 6.8 to 7.2; or a pH of about 6.5, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7, 3, about 7.4, or about 7.5.
[086] In certain aspects, the compositions of the invention can be prepared by the "soft pulping" technology referred to in New Zealand Patent No. 235972 (which is hereby incorporated by reference), which can be adapted to produce a golden kiwi juice pulpy.
[087] In early preparatory stages, golden kiwifruit may be subjected to a pre-treatment process which may include the well-known steps of ripening, inspection, sorting and/or selection of kiwifruit. With respect to ripening, it is preferable to use ripened or ripe golden kiwi fruit when producing the compositions according to the invention; however, rotting or decaying material is preferably avoided.
[088] Ripening can be assessed using methods widely known and used in the art. Ripening can be measured before harvesting or processing the gold kiwi fruit. In particular, ripening can be measured using the Brix system. The golden kiwi has a sugar level ranging from 16 to 21°Brix; or 14° to 23°Brix; or 15° to 22°Brix; or about 14°, about 15°, about 16°, about 17°, about 18°, about 19°, about 20°, about 21°, about 22°, or about 23 °Brix, may be indicative of ripeness.
[089] Gold kiwi that exceeds this Brix level may be overripe or fermenting and may not produce an ideal composition. Kiwi fruit with a sub-optimal Brix level can be artificially ripened before use. The time left in storage may be enough to reach ripening. For example, golden kiwi fruit harvested at about 5°Brix can rise above 10°Brix in 4 to 6 weeks in cold storage at 0°C. This fruit will ripen to reach 12°Brix or more after removal from cold storage. Other changes in chemistry also occur during ripening so that the kiwi fruit is within the optimal range of ripeness to provide an optimized product.
[090] As part of processing, golden kiwis can be sterilized. Fruit can be passed through an assembly having one or more rotating brushes to remove any adhering foreign matter. Conventional washing techniques can then be used. For example, you can use a series of spray nozzles to wash kiwi fruit. Wash additives that aid in cleaning or reducing kiwifruit bacteria counts can be used in accordance with local regulations and requirements. For example, the fruit can be washed with a chlorine wash and/or an ozone-impregnated water wash followed by a fresh water wash.
[091] The sterilized gold kiwi can then be transported into a dispenser. This can be tapered to form a funnel to direct the kiwi fruit one by one into the cutting assembly. The cutting assembly may include a cutting device such as a water laser or the like, which has the advantage of preventing damage to the seed so that the fruit seed does not contaminate the pulp. Other suitable cutting devices include rotating circular blades, reciprocating blades, fluid jet cutting devices, oscillating blades, etc.
[092] The cutting device can cut the gold kiwi fruit substantially in half, for example, to its length. Alternatively, the cutting device can be replaced with a soft crushing device capable of breaking the kiwifruit skin. Preferably, this is done without causing significant cellular damage to the kiwi fruit. For example, the gold kiwi fruit can be driven between the rolls to get the kiwi's skin to break. In particular, kiwi fruit can be popped by passing the fruit through spaced rollers slanted towards each other. This method can be used to squeeze the fruit so that the skin is separated. The busted kiwi fruit remains substantially intact but easily separable into large fragments. Other methods of disruption can be used.
[093] After cutting, the golden kiwi segments can be passed through a pressing assembly designed to separate the skin from the pulp. The press assembly can be adapted to carry out a pulping or crushing process. Such a process can be relatively light and gentle ("soft pulping") compared to conventional fruit pulping techniques. With soft pulping, there is no significant disintegration or lysis of fruit cells or components. Preferably, only a small proportion (generally less than 5 to 10%) of seeds are fragmented by this process. Excluded from soft pulping processes are chemical and/or enzymatic lysis methods, thermal techniques, techniques aimed at decomposing cells, and mechanical techniques involving excessive spraying of fruit material.
[094] In one embodiment, the press assembly softly pulps the gold kiwi by pressing the kiwi segments between a converging double belt press. Press belts can be multiple loops rotated around a series of pulleys. The distance separating the press belts can decrease in the direction of travel of the kiwi. In this way, an increase in force can be exerted on the kiwi as it travels along the length of the pressing assembly. This can produce pulping of the kiwi fruit without significant damage to the seeds. This, in turn, prevents the seeds from contaminating the pulp.
[095] The pulp generated from the pressing set can be directed to a screening process in order to separate the seeds from the pulp. In particular, the pulp can be separated from the seed using a soft mechanical sorting technique. For example, a pulp finisher can be used. This includes a rotating flexible impeller that is rotated within a cone-shaped sieve with openings of a predetermined size. In particular aspects, the size of the openings is selected to allow the kiwi pulp and juice to pass through the sieve, keeping a substantial portion, if not all, of the seeds within the internal cavity defined by the sieve.
[096] In certain respects, it may be preferable to use a paste rather than a puree of the golden kiwi. A kiwi paste can be made as a concentrate. For example, the fruit can be heated for several hours, strained, and reduced to a thick, concentrated form. The fruit can be heated after removing the skins, or after the pulping or pureing process. The fruit can be heated gradually, and then kept warm to a moderate temperature with mixing. After thickening, the paste can be spread onto a flat sheet, or transferred to a package, for example, a bag, tube, bottle, bottle or other container. The paste can be transferred aseptically in such a way that it is suitable for human consumption. Preferably, the kiwi paste is produced from mature golden kiwi fruit. Preferably, the paste is prepared from pulped fruit. The paste may be a smooth preparation, and may comprise a concentrate of about 40° Brix; or from 30° to 50° Brix; or from 35° to 45° Brix; or about 35°, about 36°, about 37°, about 38°, about 39°, about 41°, about 42°, about 43°, about 44°, or about 45 ° Brix.
[097] The pulp (eg in the form of paste or puree) can then be processed by a freezing step. This can be followed or used in conjunction with a drying step. In an alternative embodiment, the pulp is dried and processed to form a powder without an intermediate freezing step. For example, methods involving drum drying can be used. In the drum drying process, a puree or paste can be dried at relatively low temperatures on high capacity rotating drums, which produce drum dried product sheets. In certain respects, an additive can be used to speed up or otherwise assist in the drying process. For example, pea starch or other drying aids can be used. The dried product can then be ground into a finished flake or powder form. Advantageously, drum drying techniques can be used to produce a dry composition which retains its main components, eg phenolic compounds, and which can be easily reconstituted using liquid. For example, drum dried products can be made to be soluble in cold water. As other alternatives, belt drying or convection drying can be used. Such drying methods are widely known and used in the art.
[098] If freezing is used, it is preferable to freeze the pulp as soon as possible after it is produced to maintain freshness. However, freezing can be carried out within 24 or 48 hours as needed. Freezing methodologies are well known and do not need to be described in greater detail here. Quick freezing is particularly preferred for use in accordance with the invention. The pulp can be frozen in standard size stakes, which are used to collect fresh pulp after processing. The pulp can be stored frozen (eg at -18°C) until compounding is required.
[099] Frozen pulp can be freeze-dried, that is, lyophilized. Freeze drying techniques are widely known and commonly used. The freeze-drying cycle can be about 48 hours; or can range from 40 to 56 hours; or from 12 to 36 hours; or 36 to 60 hours; or about 40 hours, about 42 hours, about 44 hours, about 46 hours, about 48 hours, about 50 hours, about 52 hours, or about 54 hours. A longer freeze-drying cycle, for example at least 48 hours ("mild freeze-drying"), can be used to retain maximum activity. In particular aspects, the process can be carried out in such a way that the formation of water is avoided, and the moisture content is minimized during processing.
[100] It may be desirable to use a particular freeze-drying process to obtain the dry product. For example, a freeze drying program can be used as part of an automated drying system. The freeze-drying process can include several drying steps, for example, with gradual increases and decreases in temperature. Preferably, a primary drying setting is used for sublimation, followed by one or more secondary drying settings which are used to remove residual moisture. In particular aspects, the highest temperature of the freeze-drying process does not exceed 70°C. In other aspects, the temperature of the freeze-drying process ranges from -10°C to 70°C. In another aspect, lyophilization for up to 48 hours is used.
[101] The resulting dry product can then be ground to form a powder which can then be used as appropriate. Milling methods are well known and widely used in the art. Standard mesh sizes can be used to produce the powder, for example US 20, US 23, US 30, US 35, US 40, US 45, or US 50 mesh sizes can be used. powder can range from 1.0 to 0.3 mm; or 0.84 to 0.4 mm; or 0.71 to 0.5 mm; or it can be about 1.0mm, about 0.84mm, about 0.71mm, about 0.59mm, about 0.5mm, about 0.47mm , about 0.465 mm, about 0.437 mm, about 0.4 mm, about 0.355 mm, or about 0.3 mm.
[102] To ensure minimal degradation of the kiwi ingredients, the preparation process can be carried out at a temperature below 40°C. In various embodiments, the process is carried out at a temperature ranging from -4°C to 40°C; or from -1°C to 10°C; or from 1°C to 6°C; or from approximately 0°C, approximately 1°C, approximately 2°C, approximately 3°C, approximately 4°C, approximately 5°C, or approximately 6°C. These temperatures can be maintained throughout the entire brewing process, including the storage of whole fruits, before they are broken, and during the pulping/mashed process. For optimal results, these temperatures are maintained at least from the point at which the fruit was broken. The use of such temperatures prevents oxidation of the fruit and the use of reducing agents. Under certain circumstances, it may be possible to obtain organic certification.
[103] The processing method is preferably carried out so as to avoid or at least minimize any damage or effects on the active material in the kiwi gold. To ensure optimal production methods, the resulting compositions can be monitored for activity, for example, for polyphenol content. The composition can be expected to contain at least the following polyphenolic compounds: chlorogenic acid, B-caffeoyl-3-glycoside, neochlorogenic acid, phlorizin, procyanidin B2, and quercetin rhamnoside. Also present may be: catechin, epicatechin, E-caffeoyl-4-glycoside, and quercetin rutinoside. The levels of these polyphenols are indicated here below. Assays for polyphenols are well known in the art and are also described below. In particular, it is possible to measure gallic acid equivalents (GAE) to determine the total content of polyphenols. For example, the Folin-Ciocalteu method (employing the Folin-Ciocalteu reagent, also called the Folin phenol reagent or Foiin-Denis reagent) can be used for in vitro colorimetric assays of phenolic compounds (Singleton et al. 1999). ).
[104] Alternatively or additionally, compositions can be tested to stimulate the growth of beneficial organisms, eg Lactobacillus and/or Bifidobacterium, or to increase levels of short-chain fatty acids, eg acetate, butyrate, and/or propionate. These growth levels and organic acid levels are indicated here below. Corresponding assays are widely known and also described in detail in this document. Preferably, the activities observed in stimulating bacterial growth and organic acid levels, and the polyphenol content observed are still present after digestion of the composition, for example, as tested by in vitro digestion. In particular aspects, in vitro digestion assays can be used as described by the inventors in this document. Other in vitro assays are known and used in the art (see, for example, Kaur et al. 2010).
[105] In some circumstances, it may be possible to use a genetic derivative of gold kiwi stock (eg, Gold3 stock) to obtain the compositions of the invention. A composition obtained from such a derivative is expected to share one or more of the characteristics of the compositions obtained from the gold kiwi stock. Exemplary characteristics include: polyphenol levels and polyphenol profiles, actinidin levels, fiber levels, vitamin levels, stimulating the growth of beneficial organisms, and improving organic acid levels, as mentioned above and disclosed in detail in this document.
[106] In relation to the fruit itself, it is expected that kiwi fruit obtained from a genetic derivative may share a similar composition constituted as the original golden kiwi fruit. For example, the vitamin C content of the fruit can be from 100 to 150 mg; or from 90 to 200 mg; or from 80 to 220 mg; or from about 80 mg, from about 90 mg, from about 100 mg, from about 115 mg, from about 130 mg, from about 140 mg, from about 150 mg, from about 160 mg, from about 170 mg, or about 180 mg, per 100 g of fruit. The actinidin content of the fruit can be from 60 to 110 FU; or from 70 to 100 FU; or 80 to 90 FU; or about 60 FU, about 70 FU, about 80 FU, about 90 FU, about 100 FU, about 110 FU, or about 120 FU, per 100 g of fruit. The total polyphenol content of the fruit can be from 250 to 450 mg of GAE; or from 200 to 800 mg of GAE; or from 300 to 600 mg of GAE; or from 270 to 430 mg of GAE; or from 280 to 420 mg of GAE; or from 290 to 410 mg of GAE; or from 300 to 400 mg of GAE; or from 310 to 390 mg of GAE; or from 320 to 380 mg of GAE, per 100 g of fruit. Compositions comprising gold kiwi fruit
[107] The inventors have found that Gold3 Gold Kiwi Powder includes beneficial ingredients that are useful for maintaining the health of the digestive system, as well as the treatment and prevention of digestive problems and/or gastrointestinal disorders. The inventors have shown that Gold3 Gold Kiwi Powder is particularly effective in improving bowel regularity. The GoId3 powder was also effective in stimulating the growth of beneficial bacteria over harmful bacteria, and generating increases in F. prausnitzii specifically.
[108] Thus, the golden kiwi compositions of the present invention can be used to support or improve overall gut health and/or to treat or prevent various diseases or other disorders of the digestive tract, including inflammation, constipation, microbiota imbalance , irritable bowel syndrome, and inflammatory bowel disease. In addition, the compositions can be used to maintain or improve bowel regularity, and to maintain or augment beneficial bacteria in the digestive tract, including F. prausnitzii.
[109] Gold kiwi powder can be encapsulated, compressed, or added to or incorporated into other products. Particularly encompassed are delayed-release formulas, extended-release formulas as well as formulas for rapid disintegration. Gelatin capsules are specifically included, as are sachets and chewable tablets. Combination formulas are further included which include the powder of the invention mixed with other beneficial agents, for example, one or more probiotics, prebiotics, symbiotics, or other digestive aids. In alternative embodiments, the powder may be reconstituted as a liquid, for example, a concentrate, syrup, suspension, or tonic for oral administration, or as an enema or enema composition for rectal administration. Rectal suppositories are also included.
[110] In preferred aspects, the resulting gold kiwi powder is encapsulated, and each capsule contains about 500 mg or about 600 mg of the dry powder; or ranging from 50 to 650 mg; from 150 to 850 mg; or from 200 to 800 mg; from 300 to 700 mg; or from 550 to 750 mg; or approximately 50mg, approximately 100mg, approximately 150mg, approximately 200mg, approximately 250mg, approximately 300mg, approximately 350mg, approximately 400mg, approximately 450mg, approximately 500mg, approximately 550mg, approximately 575mg, approximately 590 mg, approximately 610 mg, approximately 625 mg, approximately 650 mg, approximately 675 mg, approximately 700 mg, approximately 750 mg, approximately 800 mg, or approximately 850 mg, of the dry powder.
[111] In certain circumstances, it may be desirable to isolate or enrich polyphenols from the gold kiwi fruit. In particular, it may be advantageous to use gold kiwi fruit to obtain polyphenol-enriched compositions, phenolic concentrates, or compositions comprising isolated phenolic compounds. For example, the compositions of the invention can be enriched in polyphenols in such a way that their concentration is increased relative to that of the other components of the golden kiwi, for example fibers, sugars and/or proteins. In particular aspects, the compositions of the invention can include those polyphenols that have been isolated (e.g., purified) from the other components of the golden kiwi fruit.
[112] Polyphenol enrichment and extraction methods are widely known in the art (see, for example, Sun-Waterhouse et al. 2009; Eidenberger et al. 2014). Preferably, the resulting composition has at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 10 times the amount of polyphenols compared to the composition prepared without enrichment or isolation steps of polyphenol. Polyphenol-enriched compositions, phenolic concentrates, and compositions comprising isolated phenolic compounds can be dried as a powder, and used in accordance with the present invention. In particular aspects, such powder is encapsulated, and each capsule contains approximately 100 mg, approximately 150 mg, approximately 200 mg, approximately 250 mg, approximately 300 mg, or approximately 350 mg, or approximately 400 mg, of powder; or ranging from 100mg to 200mg; or from 100 to 300 mg; or from 200 to 400 mg of powder.
[113] The dosage form can contain excipients, for example, one or more non-sticks, binders, coatings, disintegrants, flavors, dyes, sweeteners, lubricants, glidants, flow agents, anti-caking agents, sorbents or preservatives. Useful excipients include, but are not limited to: stearin, magnesium stearate, and stearic acid; saccharides and their derivatives, eg disaccharides: sucrose, lactose; polysaccharides and their derivatives, for example starches, cellulose or modified cellulose such as macrocrystalline cellulose and cellulose ethers such as hydroxypropyl cellulose; sugar alcohols such as isomalt, xylitol, sorbitol and maltitol; proteins such as gelatin; synthetic polymers such as polyvinylpyrrolidone, polyethylene glycol; fatty acids, waxes, shellac, plastics and vegetable fibers, eg corn protein zein; hydroxypropyl methylcellulose; cross-linked polymers, for example, cross-linked polyvinylpyrrolidone (crospovidone), and cross-linked carboxymethyl cellulose sodium (croscarmellose sodium); sodium starch glycolate; silicon dioxide, fumed silica, talc and magnesium carbonate.
[114] The golden kiwi compositions of the present invention are expected to include various components, for example, carbohydrates, dietary fiber, polyphenols, and actinidine. In many ways, it can have 10 to 20 mg of carbohydrates; 0.5 to 5.5 mg of dietary fiber; from 0.1 to 0.5 mg of polyphenol GAE; and from 100 to 500 AU of actinidine, per 25 mg of powder. In a specific aspect, it can have approximately 18 mg of carbohydrates, approximately 3.0 mg of dietary fiber, approximately 0.28 mg of polyphenol GAB, and approximately 230 AU of actinidine, per 25 mg of powder.
[115] The golden kiwi compositions of the present invention may include certain polyphenols as active components. Polyphenols in the composition may still be present after digestion of the composition, for example, in vitro digestion, as described in detail herein. For example, after digestion, the chlorogenic acid content of the composition can be at least 40 μg/g, at least 45 μg/g, at least 50 μg/g, or at least 55 μg/g; or from 40 to 60 µg/g; or from 45 to 55 μg/g, based on dry weight. The E-caffeoyl-3-glycoside content of the composition may be at least 45 μg/g, at least 50 μg/g, at least 55 μg/g, at least 60 μg/g, at least 65, at least 70 μg /g, or at least 75 µg/g; or from 40 to 80 µg/g; or from 50 to 70 μg/g, based on dry weight. The epicatechin content of the composition may be at least 6 μg/g, at least 7 μg/g, at least 8 μg/g, at least 9 μg/g, at least 10 μg/g, at least 11 μg/g, at least 12 μg/g, at least 13 μg/g, or at least 14 μg/g; or from 8 to 12 µg/g; or from 9 to 11 μg/g, based on dry weight. The neochlorogenic acid content of the composition may be at least 15 μg/g, at least 18 μg/g, at least 20 μg/g, at least 22 μg/g, at least 25, at least 30 μg/g, or at least minus 35 µg/g; or from 10 to 30 µg/g; or from 15 to 25 μg/g, based on dry weight.
[116] According to other aspects, after digestion, the phlorizin content of golden kiwi compositions may be at least 45 μg/g, at least 50 μg/g, at least 55 μg/g, at least 60 μg/ g, at least 65, at least 70 µg/g, or at least 75 µg/g; or from 40 to 80 µg/g; or from 50 to 70 μg/g, based on dry weight. The procyanidin B2 content may be at least 15 μg/g, at least 20 μg/g, at least 25 μg/g, at least 30, at least 35 μg/g, at least 40 μg/g, or at least 45 µg/g; or from 20 to 40 µg/g; or from 25 to 35 μg/g, based on dry weight. The rhamnoside quercetin content of the composition may be at least 15 μg/g, at least 18 μg/g, at least 20 μg/g, at least 22 μg/g, at least 25, at least 30 μg/g, or at least minus 35 µg/g; or from 10 to 30 µg/g; or from 15 to 25 μg/g, based on dry weight.
[117] In still other aspects, after digestion, the catechin content of golden kiwi compositions may be at least 0.5 μg/g, at least 1 μg/g, at least 2 μg/g, or at least 3 µg/g; or from 1 to 4 µg/g; or from 2 to 3 μg/g, based on dry weight. The E-caffeoyl-4-glycoside content of the composition may be at least 3 μg/g, at least 4 μg/g, at least 5 μg/g, at least 6 μg/g, at least 7 μg/g, at least minus 8 μg/g, or at least 9 μg/g; or from 4 to 8 µg/g; or from 5 to 7 μg/g, based on dry weight. The quercetin rutinoside content of the composition may be at least 1 μg/g, at least 2 μg/g, at least 3 μg/g, at least 4 μg/g, or at least 5 μg/g; or from 1 to 5 µg/g; or from 2 to 4 μg/g, based on dry weight.
[118] In the absence of digestion, the golden kiwi compositions of the present invention may include a total polyphenol content of 1000 to 1200 mg of GAE; or from 900 to 1300 mg of GAE; or from 800 to 1400 mg of GAE, per 100 g. The E-caffeoyl-3-glycoside content can be from 60 to 120 μg/g; or from 80 to 100 µg/g; or from 90 to 110 µg/g; or from about 80 μg/g, from about 90 μg/g, from about 100 μg/g, from about 110 μg/g, from about 115 μg/g, or from about 120 μg/g, based on dry weight. The epicatechin content can be from 20 to 60 µg/g; or from 30 to 50 µg/g; or from about 20 μg/g, from about 30 μg/g, from about 40 μg/g, from about 45 μg/g, from about 50 μg/g, or from about 60 μg/g, based on dry weight. The neochlorogenic acid content can be from 20 to 60 μg/g; or from 30 to 50 µg/g; or from about 20 μg/g, from about 30 μg/g, from about 39 μg/g, from about 40 μg/g, from about 41 μg/g, from about 50 μg/g, or about 60 μg/g, based on dry weight. The content of procyanidin B2 can be from 40 to 120 µg/g; or from 50 to 100 µg/g; or from 65 to 90 µg/g; or from 60 to 80 µg/g; or about 40 μg/g, or about 50 μg/g, or about 60 μg/g, or about 70 μg/g, or about 75 μg/g, or about 80 µg/g, or about 90 µg/g, or about 100 µg/g, based on dry weight.
[119] The golden kiwi compositions of the present invention may also include fiber as an active component. In particular aspects, the total fiber content can be from 12.4 to 12.7%, based on dry weight; or from 12.0 to 13.0%; or from 10 to 15%; or about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%, based on dry weight. In particular aspects, the soluble fiber content can be from 3.4 to 3.5% based on dry weight; or from 3.0 to 4.0%; or from 3.2 to 3.8%; or from about 3.0%, from about 3.1%, from about 3.2%, from about 3.3%, from about 3.4%, from about 3.5%, from about 3.6%, about 3.7%, about 3.8%, about 3.9%, or about 4.0%, based on dry weight. In additional aspects, the insoluble fiber content can be from 8.9 to 9.3%, based on dry weight; or from 8.0 to 10.0%; or from 8.5 to 9.5%; or about 8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0%, based on dry weight.
[120] The golden kiwi compositions of the invention may also include various sugars, including neutral sugars and uronic acids. Pectic polysaccharides are specifically included in the compositions of the invention. With respect to neutral sugars, the compositions of the invention can include one or more of the following: rhamnose, arabinose, galactose and glucose.
[121] The golden kiwi compositions of the present invention may further include the actinidin enzyme. In particular aspects, actinidine levels can be from 8,000 to 11,000 AU; or from 6,000 to 16,000 AU; from or from 7,000 to 11,000 AU; or from 8,000 to 10,000 AU; or about 8,000 AU, about 9,000 AU, about 10,000 AU, about 11,000 AU, or about 15,000 AU, per gram. Methods to measure the level of actinidine are widely known and used in the art. See, for example, Drummond 2013 and Kaur et al. 2010. For example, it is possible to measure the digestion of N-α-CBZ-lys-p-nitrophenol (Z-lys-pNp; Sigma Aldrich Pty Ltd), for example, at 25°C (ie 77°F) to determine actinidin levels/activity (Boland & Hardman 1972). Measurements can be expressed as AU/g or AU/mg for the composition. Alternatively, it is possible to measure actinidin levels using fluorescent assays to assess cysteine protease activity (Nieuwenhuizen et al. 2012; Maddumage 2013). In particular, the fluorescent substrates Z-FRAMC(benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin), H-D-Ala-Leu-Lys-AMC, or Bz-Arg-AMC (Feinchemikalien AG) can be used. These measurements can be expressed as FU/g or FU/mg for the composition.
[122] The golden kiwi compositions of the present invention are expected to be active in stimulating the growth of beneficial enteric organisms (eg, beneficial bacteria) such as members of the Lactobacillus or Bifidobacterium groups, as well as certain members of the Clostridiales group, including Faecalibacterium prausnitzii, as shown here. Other beneficial organisms may also show increased growth, for example, strains of Erysipelotrichales and Bacteroidales. Beneficial organisms are also included which include Clostridium coccoides, Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides cellulosilyticus, Roseburia intestinalis, Roseburia inulinovorans, Ruminococcus bromii, and Ruminococcus flavefaciens. Also included are beneficial organisms within the groups Bacteroides-Prevotella-Porphyromonas, and Lachnospiraceae, as well as other organisms described herein.
[123] In various aspects of the invention, the golden kiwi composition (eg, 25 mg powder, which can be diluted to 0.3 to 0.5 mg/ml powder) can enhance the growth of one or more strains. of Lactobacillus and/or Bifidobacterium (eg starting with 1 billion colony forming units (cfu) in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least at least 70%, at least 80%, at least 90%, at least 100%, or at least 10%. In a particular aspect, 25 mg of powder added to 1 billion cfu of probiotic organism(s) can be used to obtain a growth rate greater than 50%, relative to the control, which has been normalized to 0%. Such increases can still be observed after digestion of the composition, for example, in vitro digestion, as described herein in detail. Preferably, there are no concomitant increases in enteric harmful organisms (eg harmful bacteria) such as Salmonella and/or Staph strains. ylococcus. In some cases, it may be possible to obtain reductions in one or more harmful organisms.
[124] The golden kiwifruit compositions of the invention may also be active in stimulating the growth of faecal bacteria, including Clostridiales bacteria, such as those of the phylogenetic group Clostridium leptum, and in particular strains of Faecalibacterium, including Faecalibacterium prausnitzii. Increases in the relative abundance of Faecalibacterium prausnilzii can be at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least minus 100%, at least 150%, or at least 200%; or 20% to 200%; or 50% to 150%; or 70% to 100%, in individuals, for example, individuals with constipation, after treatment with the powdered gold kiwi.
[125] The golden kiwi compositions of the invention show activity in increasing levels of short-chain fatty acids. In particular aspects of the invention, the composition (for example, 10 mg of powder) can increase levels of one or more of acetate, butyrate, and propionate by at least 10%, at least 15%, at least 18%, at least 20%, at least 22%, at least 25%, at least 28%, or at least 30%. Such increases can still be observed after digestion of the composition, for example, in vitro digestion, as described in detail herein. Methods of using kiwi compositions
[126] As noted above, the golden kiwi compositions of the present invention can be used to support or improve overall gut health and/or to treat or prevent various digestive tract disorders, including inflammation, constipation, microbiota imbalance, syndrome irritable bowel disease, and inflammatory bowel disease. In addition, the compositions can be used to maintain or improve bowel regularity, and to maintain or augment beneficial bacteria in the digestive tract, including F. prausnitzii.
[127] Constipation may be caused by a bowel function disorder or a structural problem, and may have one or more symptoms including: reduced bowel movements, assisted and incomplete bowel movements, straining to pass, hard or lumpy forms of stool, abdominal distension , flatulence, abdominal pain, and laxative dependence. Common causes of constipation are: inadequate water intake; inadequate dietary fiber; a break from regular or routine diet; inappropriate activity or exercise; consumption of large quantities of dairy products; stress; avoidance of bowel movement (eg, due to pain); abuse of laxatives or stool softeners; hypothyroidism; neurological conditions such as Parkinson's disease or multiple sclerosis; antacid medications containing calcium or aluminum; medications (especially pain medications such as narcotics, antidepressants, or iron supplements); depression; eating disorders; irritable bowel syndrome; pregnancy; colon cancer; and lack of nerve and muscle function in the intestine.
[128] The aetiology of microbiota imbalances (eg, decreases in beneficial organisms, altered ratios of beneficial organisms, and/or increases in harmful organisms) is complex and has not yet been fully elucidated. Imbalances can be caused by certain medications such as antibiotics, digestive tract disorders, or dietary deficiencies. An imbalance in the gut microbiota may present as one or more symptoms of: diarrhea, in particular, antibiotic-associated diarrhea, watery stools, constipation, and/or bloating.
[129] Microbiota imbalances are correlated with various disorders of the gastrointestinal system as well as other disorders. In particular, an imbalance in the microbiota may be associated with inflammatory bowel disease, irritable bowel syndrome, celiac disease, Crohn's disease, ulcerative colitis, necrotizing enterocolitis, intestinal inflammation, enteric infections, carcinogenesis including gastric carcinomas, colorectal carcinomas, cholelithiasis, endotoxemia, liver disease such as cirrhosis, and hepatic encephalopathy.
[130] Microbial imbalances in the digestive tract are associated with several different health conditions, including:



[131] Inflammation of the digestive tract can be associated with a variety of conditions including atrophic glossitis, angular cheilitis, orofacial granulomatosis, esophagitis, gastritis, including atrophic gastritis, pyloric stenosis, colitis, ileitis, Crohn's disease, celiac disease, inflammatory disease. bowel, irritable bowel syndrome, lesions, fissures, and various ulcers, including ulcers of the mouth, esophagus, stomach and intestines, and specifically including ulcerative colitis. Inflammation can also be associated with harmful organisms such as bacterial, protozoan and/or viral organisms, including the groups/organisms of Enterobacteriaceae, Pasteurellaceae, Veillonellaceae, Fusobacteriaceae, Proteobacteria, Campylobacter, Shigella, Yersinia, Listeria, Salmonella, Escherichia coli , Staphylococcus aureus, Clostridium difficile, Helicobacter pylori, Mycobacterium avium, Enterococcus faecalis, Fusobacterium varium, Giardia, Entamoeba histolytica, rotavirus, norovirus, adenovirus, astrovirus, and measles virus. Under certain circumstances, inflammation can result from injury, medication, or surgery.
[132] The golden kiwifruit compositions of the invention can be used in the treatment or prevention of gastrointestinal inflammation, constipation, bowel irregularity, microbiota imbalance, irritable bowel syndrome, inflammatory bowel disease, or other conditions described herein. As exemplary dosages, the compositions can be administered from 250 to 2500 mg; 500 to 5000 mg; 1000 to 4000 mg; or from 1500 to 4500 mg; or from 2000 to 3000 mg; or to about 250mg, about 500mg, about 600mg, about 800mg, about 1000mg, about 1200mg, about 1500mg, about 1700mg, about 1800mg, about 2000 mg, about 2200 mg, about 2300 mg, about 2400 mg, about 2500 mg, about 2600 mg, about 2700 mg, about 2800, about 2900, about 3000 mg, about 3200, about of 3500, about 3600mg, about 4000mg, about 4200mg, about 4800mg, about 5000mg, about 5400mg, about 6000mg, or about 6600mg, of the powdered gold kiwi by day, or equivalent dosage, if a liquid form is used. Administration can be performed once a day, twice a day, or three times a day. Administration can be done with food or before a meal. The pharmaceutical form and proper dosage will be readily determined by a person skilled in the art.
[133] Various routes of administration can be used for the golden kiwi compositions of the invention, including enteral administration, oral administration, and rectal administration. Oral administration may be by tablet, capsule, sachet, drops, elixir, linctus, solution, emulsion, suspension, sipping amounts, puree, paste, syrup, gel, jelly, tonic, or other known means. Enteral administration can be via duodenal tubing or via gastric tubing, including nasogastric tubing. Rectal administration can be by enema, suppository, or other suitable means. Different means of administration are known in the art and can be used by a person skilled in the art. The compositions of the invention are not limited to a particular form of administration.
[134] In particular aspects, the compositions of the invention can be administered in combination with one or more probiotic organisms. For example, the golden kiwi composition can be formulated as a dosage form combined with one or more probiotics. Alternatively, the golden kiwi composition can be administered as a separate dosage form, together with one or more probiotics. Exemplary probiotic organisms include, but are not limited to: Bacillus coagulans, for example, strains GBI-30 and 6086; Bifidobacterium longum, for example, subsp. children 35624; Lactobacillus acidophilus, for example, NCFM and CL1285 strains; Lactobacillus paracasei, for example St11 and NCC2461 strains; Lactobacillus johnsonii, for example La1 and NCC533 strains; Lactobacillus LC1; Lactobacillus plantarum, for example, 299v and HEAL 9 strains; Lactobacillus reuteri, for example ATCC 55730, SD2112, Protectis (DSM 17938, daughter strain of ATCC 55730), Prodentis (DSM 17938/ATCC 55730 and ATCC PTA 5289 in combination), and RC-14®;Saccharomyces boulardii; Lactobacillus rhamnosus, for example, a GR-1® strain; Lactobacillus casei, for example an LBC80R strain; Lactobacillus bulgaricus; Streptococcus thermophilus; and Lactobacillus bifidus. Other organisms that may be useful as probiotics include Faecalibacterium strains, including Faecalibacterium prausnitzii, and also Bacteroidaceae strains such as Bacteriodes fragilis.
[135] In other aspects, the golden kiwi compositions of the invention may be administered in combination with one or more agents, e.g., prebiotics, as a combined dosage form or in separate dosage forms. As previously identified, prebiotic agents consist of fermentable compounds or non-digestible fibers that pass through the upper part of the digestive tract and stimulate the growth and/or activity of beneficial organisms that colonize the large intestine, acting as a substrate for them.
[136] These agents can be short-chain, long-chain, or full-spectrum prebiotics. Short-chain prebiotics include 2 to 8 bonds per saccharide molecule. Long-chain prebiotics include 9 to 60 bonds per saccharide molecule. Full spectrum prebiotics include a full range of molecular bond lengths from 2 to more than 60 bonds per saccharide molecule. Exemplary prebiotic agents include, but are not limited to; oligofructose, inulin, inulin enriched with oligofructose, fructooligosaccharides, xylooligosaccharides, polydextrose, galactooligosaccharides, transgalactooligosaccharides, mannan oligosaccharides, lactulose, tagatose and starch.
[137] Based on the results presented here, it is believed that the polyphenols present in the disclosed compositions may be acting as prebiotic agents. Thus, it may be useful to add one or more phenolic compounds to the compositions of the invention to complement their prebiotic activity. Exemplary compounds include, but are not limited to: phenolic derivatives such as phenolic acid, and flavonoids such as lignins, proanthocyanidins, anthocyanins, anthocyanidins, isoflavones, catechins, tannins, quercetin, naringenin and hesperidin. Particularly encompassed are phenolic compounds extracted from one or more of: tea, cocoa, wine, soybeans, feijoa, citrus fruits, apples, grapes, berries, and kiwis, particularly golden kiwis, including Hort16 and Gold3. Gold3 kiwi fruit specific phenolics include, but are not limited to: catechin, chlorogenic acid, E-caffeoyl-3-glycoside, E-caffeoyl-4-glycoside, epieatechin, neochlorogenic acid, phlorizin, procyanidin B1 and B2, quercetin rhamnoside , and keratin rutinoside.
[138] According to other aspects, the compositions of the invention may be administered in combination with one or more symbiotic agents (prebiotics and probiotics combined). For example, powdered gold kiwi fruit can be formulated as a dosage form combined with one or more symbiotics. Alternatively, the golden kiwi can be administered as a separate dosage form along with one or more symbiotics. As examples, Bifid bacteria or Lactobacilli can be combined with the fructooligosaccharides or inulins or galactooligosaccharides. Specific symbiotic combinations include, but are not limited to: Bifid bacteria and fructooligosaccharides; Lactobacillus rhamnosus, for example, a GG strain, and inulins.
[139] As additional aspects, the compositions of the invention can be administered in combination with fiber and/or digestive enzymes. For example, powdered kiwi fruit can be formulated as a pharmaceutical form combined with one or more compositions comprising fiber and/or digestive enzymes. Alternatively, the powdered kiwi fruit can be formulated as administered as a separate dosage form, with one or more compositions comprising fiber and/or digestive enzymes. Exemplary fiber compositions include soluble and/or insoluble fiber compositions, for example, compositions including one or more of wheat dextrin, calcium polycarbophil, psyllium, inulin, methylcellulose, glucomannan, flax, flaxseed, wheat grass, senegal acacia , and rhubarb. Exemplary digestive enzymes include, but are not limited to: actinidine, protease, lipase, amylase, cellulose, pancreatin, pepsin, bromelain, papain, trypsin, and chymotrypsin. Combinations of digestive enzymes can also be used, for example combinations of protease, lipase and amylase, including those with or without added actinidine.
[140] According to other aspects, the golden kiwi compositions of the present invention can be administered in combination with one or more anti-inflammatory agents and/or antimicrobial agents. Of particular interest is the use of the composition of the invention as a prebiotic supplement during and/or after antibiotic treatment. For example, powdered golden kiwi fruit can be formulated as a dosage form combined with one or more anti-inflammatory/antimicrobial agents. Alternatively, the golden kiwi can be administered as a separate dosage form, together with one or more anti-inflammatory/antimicrobial agents. Exemplary anti-inflammatory/antimicrobial agents include, but are not limited to, aminosalicylates, e.g., mesalazine (e.g., Pentasa®) and sulfasalazine (e.g., Salazopyrin®), corticosteroids, e.g., budesonide (e.g., Entocort ®) and hydrocortisone acetate (eg Colifoam®), and also include azathioprine (eg Azasan®, Imuran®), mercaptopurine (eg Purinethol®, Purixan™), cyclosporine (eg Gengraf®, Neoral ®, Sandimmune®), infliximab (eg Remicade®), adalimumab (eg Humira®), golimumab (eg Simponi®), methotrexate (eg Rheumatrex™, Methoblastin™), natalizumab (eg. Tysabri™), vedolizumab (eg Entyvio™), ustekinumab (eg Stelara®), and antibiotics including metronidazole (eg Flagyl®) and ciprofloxacin (eg Cipro®, Ciplox™). EXAMPLES
[141] The examples described herein are provided for the purpose of illustrating specific embodiments of the invention and are not intended to limit the invention in any way. Example 1 Preparation of Kiwi Powder
[142] Frozen gold kiwi puree (Single strength frozen gold kiwi puree (seedless)) was obtained from Kiwifruit Processing Company Ltd, Tauranga, New Zealand. The puree was produced from 100% New Zealand gold kiwi (Actinidia chinensis G3) grown to Zespri® export standards, and graded by hand. The puree was produced by manufacturing processes to remove the skin and seeds to leave a smooth, enriched puree.
[143] The product specification of the frozen gold kiwi puree is as follows:
[144] The color is kiwi gold, with some variation in color being normal. The taste is typical of ripe golden kiwi fruit. The texture is smooth and seedless. The puree has zero amounts of rotten or fermented fruit or foreign bodies, and contains no detectable E coli. The Brix value is from 16 to 21°. The viscosity (at 12.5°Brix, 20°C) is about 12.0, but it can vary when the fruit is kept for a season. The pH is 3.2 to 3.8. The sieve size is < 1 mm. The product is kept frozen at -18°C until use.
[145] In addition, frozen green kiwi puree (Single strength frozen green kiwi puree (seedless)) was obtained from Kiwifruit Processing Company Ltd, Tauranga, New Zealand. The puree was made from 100% New Zealand green kiwi (Actinidia delicious, Hayward variety) grown to Zespri® export standards, and graded by hand. The puree was produced by manufacturing processes to remove the skin and seeds to leave a smooth, enriched puree.
[146] The product specification of the frozen green kiwi puree is as follows:
[147] The color is kiwi green, some variation in color is normal. The taste is typical of ripe green kiwi fruit. The texture is smooth and seedless. The puree has zero amounts of rotten or fermented fruit or foreign bodies, and contains no detectable E. coli. The Brix value is from 13 to 18°. The viscosity (at 12.5°Brix, 20°C) is about 12.0, but it can vary when the fruit is kept for a season. The pH is 3.2 to 3.8. The sieve size is < 1 mm. The product is kept frozen at -18°C until use. The process for obtaining powdered kiwi fruit was as follows: 1) Frozen Gold3 puree was purchased from Kiwifruit Processing Company Ltd, Tauranga, New Zealand. 2) The frozen puree was lyophilized (freeze-dried). The lyophilization process was typically carried out for up to about 48 hours, at temperatures not exceeding 70°C. The resulting dry material was then milled through a US 20 mesh to produce a free flowing powder. 3) The same method was used to obtain green kiwi powder. Example 2 Measurement of Polyphenols for Kiwi Powders
[148] The polyphenol profiles of green kiwi and gold kiwi powders were tested. Freeze dried (freeze-dried) kiwi powders of green (Hayward) and gold (Gold3) were prepared according to Example 1.
[149] Powders were digested using an in vitro upper gastrointestinal model as described in Monro et al. 2010. Briefly, 20 mL of water and 5 mL of 20% saline were added to 5 g of samples at pH 2.5 prior to vortexing. Then, 1 ml of 1% pepsin in 0.05 M hydrochloric acid was added, followed by 30 minutes of incubation at 37°C with slow constant mixing (220 rpm). The samples were adjusted to pH 6.5, followed by the addition of 5 mL of 2.5% bile extract obtained from Sigma Aldrich®, and 1 mL of 5% pancreatin in 3% sodium chloride (NaCl).
[150] The samples were then shaken and incubated at 37°C with slow constant mixing (220 rpm) for 2 hours. The digested samples were then transferred to dialysis bags (500 Da MWCO, obtained from Thermofisher Scientific) and dialyzed in 10 mM NaCl at 4°C overnight, followed by a change in dialysis fluid and a further 2 hours at 2°C, which represents absorption in the small intestine. Samples were stored at -80°C and then lyophilized.
[151] Pre- and post-digested samples of freeze-dried green kiwi and gold kiwi powder (100 mg) were extracted with 5 mL of ethanol/formic acid/water (80:20:1). The extraction mixture was sonicated for 30 minutes, then stored overnight. Samples were centrifuged at full speed to remove particles and diluted 2x before analysis by liquid chromatography - mass spectrometry (LC-MS). Details are provided in Table 2, below.Table 2: LC-MS analysis for pre- and post-digested kiwi powders


[152] Compound concentrations were calculated using calibration curves from authentic standards.
[153] As shown in Figure 1, digested Gold3 powder (simulated upper gastrointestinal digestion) has an altered polyphenol profile compared to pre-digestion (Figure 1); however, the overall phenolic content is retained. In particular, pre-digestion of 269 μg/g of polyphenol content versus post-digestion of 264 μg/g of phenol content was observed for Gold3 powder (Figure 1; combined total for tested phenolic compounds). Green kiwi powder also has an altered post-digestion polyphenol profile, but the overall content is reduced by digestion (Figure 1). For green kiwifruit powder, the total polyphenol content was 258 μg/g pre-digestion versus 153 μg/g post-digestion (Figure 1; combined total for phenolic compounds tested). Example 3 High Microbial Yield Assays for Kiwi Powders
[154] Gold3 kiwi powder was obtained from a paste formula (prepared by Cedenco Foods, New Zealand), which was drum dried with pea starch (14.28:1 wet weight). A sample (5 g) of the powder was digested in vitro using an upper gastrointestinal model as described in Example 2.
[155] The digested material was then solubilized in water or dimethyl sulfoxide (DMSO) at a uniform concentration of 100 mg/mL. Samples were then diluted 100-fold with sterile deionized water to reduce DMSO to a controllable concentration to prevent bacterial lysis. Water and DMSO 'extracts' were added to high microbial yield assay wells at a final concentration of 1.0 mg/mL.
[156] Organisms used in the study included probiotics from Escherichia coli Nissle 1917, Lactobacillus rhamnosus HN001 (DR20), Bifidobacterium lactis HN019 (DR10), and pathogenic Salmonella enterica serovar Typhimurium ATCC 1772 and Staphylococcus aureus ATCC 25932.
[157] A ninety-six-well microplate growth bioassay measuring optical density (OD) as described in Rosendale et al. 2008, was used for this work. The change in growth (Δgrowth) was calculated and used to represent the magnitude of the effect. This was calculated by converting the OD to a percentage of the control OD, then subtracting 100, effectively normalizing the growth control to a zero baseline value.
[158] One strain of bacteria per microplate was used and each extract was analyzed in quadruplicate per microplate at a range of concentrations (0, 0.0124, 0.037, 0.111, 0.333, 1 mg/mL). Microplates were inoculated with an equal volume (50 µL) of bacterial inoculum and the OD measured immediately at a wavelength of 595 nm with a plate reader (FLUOstar Optima®) to determine the growth blank/zero value. Microplates were incubated at 37°C for 24 hours, then the OD was determined to measure the growth of cultures.
[159] The results showed that Gold3 kiwi powder supported the growth of Lactobacilli and bifid Bacteria strains without having a significant effect on the number of pathogenic bacteria, relative to the control (Figure 2). Example 4 Mixed fermentation model for testing kiwi powders
[160] Gold3 kiwi powder was obtained from a paste formula (prepared by Cedenco Foods, New Zealand), which was drum dried with pea starch (14.28:1 wet weight). A sample (5g) of powder was digested according to Example 2. The digested sample was solubilized in water at a final concentration of 10mg/ml.
[161] Freshly obtained fecal samples were collected from three healthy human volunteers. The digested gold kiwi powder was incubated in a test tube inoculated with mixed microbiota from a single fecal donor, which was repeated for each of the three donors. Hungate tubes containing pre-reduced fermentation media (9 mL) under carbon dioxide were supplemented with 1 mL of sample or control and inoculated with 1 mL of a 10% (w/v) solution of fecal suspension. Tubes were incubated at 37°C with gentle orbital shaking (150 rpm). Subsamples were periodically removed (0, 5, 10, 24, 48 hours) with a syringe and a 22 gauge needle through the rubber septum to avoid exposing the sample to the atmosphere.
[162] Samples taken from the fermentation tubes were collected in 1.5 mL Eppendorf® tubes and centrifuged immediately at 13,000 x g for 5 minutes. Particle and cell-free supernatants were collected in fresh Eppendorf® tubes and frozen at -80°C until necessary.
[163] The supernatant was thawed and diluted 1:4 in 0.01 M phosphate-buffered saline with 2-ethylbutyrate (5 mM final concentration) as an internal standard. The sample was then centrifuged at 3000 x g for 5 minutes at 4°C. The supernatant (0.25 ml) was acidified with concentrated hydrochloric acid (0.125 ml), diethyl ether (0.5 ml) was added then vortexed and centrifuged at 10,000 xg for 5 minutes (4 °C). The upper diethyl ether phase was collected, dried over magnesium sulfate, and derivatized with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide with 1% tert-butyldimethylchlorosilane by heating at 80°C for 20 minutes.
[164] The concentrations of microbial organic acid metabolites in the fermentation samples were quantified by gas chromatography (GC) as indicated in Table 3 below. Table 3: GC analysis of organic acid metabolites

[165] Standard mixtures of organic acid of known concentration were analyzed along with the samples and used to create standard curves with relative peak areas normalized to the response to 2-ethylbutyrate. Results were expressed as μmol of organic acid/mL of fermentation.
[166] As shown in Figure 3, the presence of Gold3 kiwi powder promoted an increase in organic acid production, particularly after the first 12 hours of exposure. Given the observed increase, this indicates that the non-digestible fermentable components (carbohydrates and polyphenols) in the gold kiwi powder were decomposed and used as a substrate for energy generation and growth. This is consistent with acting as a prebiotic. Example 5 Clinical trial to test GoId3 gold kiwi powder to improve regularity
[167] Clinical protocol overview
[168] The study was designed as a placebo-controlled, double-blind, randomized crossover trial with participants consuming four different interventions for 4 weeks each, with 2 weeks of washout periods between each intervention. A schematic view of the study model is shown in Figure 4.
[169] Clinical trial participants
[170] Two groups of participants were recruited through newspaper and community radio advertisements in local district health departments, Canterbury University (Christchurch, New Zealand), Lincoln University (Lincoln, New Zealand), newsletters, posters in general practitioners' offices and through our existing database of past participants.
[171] Twenty-nine total participants were recruited from fifty-one total volunteers. Participants were divided into two cohorts: cohort 1 ("healthy"): participants without clinical symptoms of constipation; and cohort 2 ("functionally constipated"): participants classified as having C3 functional constipation based on Rome III criteria.
[172] Rome III criteria: 1) Must include two or more of the following procedures: a) exertion during at least 25% of defecations; b) hard or lumpy stools in at least 25% of defecations; c) sensation of anorectal obstruction or blockage during at least 25% of defecations; d) feeling of incomplete evacuation during at least 25% of defecations; e) manual maneuvers to facilitate at least 25% of defecations; f) three or less bowel movements per week; 2) Soft stools are rarely present, without the use of laxatives; and 3) Insufficient criteria for irritable bowel syndrome.
[173] Inclusion criteria were: age 18 to 60; BMI limits between 19 and 30 K/m2; fasting blood glucose less than 5.6 mmol/L; subjects were asked to be willing to maintain their usual food and beverage intake (other than substituting study foods for similar products) and physical activity pattern throughout the study period; subjects were asked to exclude high-fiber supplements such as Metamucil®, Benefibre® and Phloe™, as well as refrain from eating fresh kiwi fruit during the study period; Participants were asked to avoid traveling abroad during the study period because of the impact this can have on diet.
[174] Exclusion criteria were: the presence of gastrointestinal alarm symptoms (including blood in the stool, frequent diarrhea, constant abdominal pain); dieters or people on a vegan diet, raw food diets or very high fiber diets; gastroparesis or lactose intolerance; weight loss surgery (gastric band or gastric bypass); pregnant women; clinically significant renal, hepatic, endocrine, cardiac, pulmonary, pancreatic, neurological, hematological, or biliary disorders; and known allergy or sensitivity to kiwi fruit.
[175] The "healthy" group consisted of participants who were without clinical symptoms of constipation. The "functionally constipated" (CF) group was selected based on Rome III criteria (Drossman 2006) as having C3 functional constipation. Twenty participants (2 men and 18 women) were recruited into the healthy group. The mean age was 38 years (range 23 to 56 years) and the mean body mass index was 23 kg/m2 (range 19 to 29 kg/m2). Nine participants (1 male and 8 female) were recruited into the CF group. The mean age was 44 years (range 38 to 54 years) and the mean body mass index was 25 kg/m2 (range 21 to 29 kg/m2). See also Table 6 below.
[176] Nineteen of the twenty participants in the healthy group completed the study. A female participant withdrew due to personal reasons. All nine CF group participants completed the study. The order in which participants were allocated to their intervention was randomized by a biostatistician using a Williams Latin Square model and computer-generated random numbers. Upon completion of the analyses, the study was unblinded to reveal the order of intervention. Ethical approval was obtained from the New Zealand Human Disability and Ethics Committee “New Zealand Human Disability Committee” under expedited review (Request number 12/STH/72/AM01), and has been registered with the Australia New Zealand Clinical Trials Registry (Australia and New Zealand Clinical Trials Register). ACTRN registration number: 12612001270808.
[177] Clinical Test Methods
[178] Interventions were administered in 4 x 600 mg capsules provided by Anagenix Limited (Wellington, New Zealand) prepared to appear identical to preserve blind intervention (Table 4).


[179] All participants consumed four different intervention combinations: Placebo (green isomalt) (2400 mg/day), ACTAZIN™ L (600 mg/day), ACTAZIN™ H (2400 mg/day) and GOLD (2400 mg/day) mg/day) for 28 days at each intervention, with a 14-day washout period between each treatment phase.
[180] ACTAZIN™ L (low dose, green kiwi) and ACTAZIN™ H (high dose, green kiwi) were formulated from 'Hayward' green kiwi, cold processed Actinidia delicious and GOLD was formulated from the pulp of Gold3 'Zesy002' gold kiwi, Actinidia chinensis, cold processed. See Example 1.
[181] At the beginning and end of each 4-week intervention period, participants were asked to provide a faecal sample. The 2-week washout period was chosen to allow sufficient time for bowel habits to return to baseline for the measured parameters (microbial ecology, microbial metabolites).
[182] At the beginning and end of each intervention period, participants were also asked to provide a faecal sample and complete the Birmingham Irritable Bowel Syndrome (IBS) symptom questionnaire (Johnston et al. 2010) as well as a IBS-specific quality of life questionnaire (Patrick et al. 1998) relating to well-being.
[183] For each day from baseline to the end of the study, participants recorded several parameters in the daily reports provided. These parameters included: 1) number of bowel movements; 2) incomplete and assisted bowel movements; 3) effort to pass feces; 4) stool shape as determined by Bristol stool scale; 5) bloating; 6) flatulence; 7) abdominal pain; 8) use of laxatives.
[184] Data were analyzed using analysis of variance (ANOVA) in GenStat (v.16, 2013, VSNi Ltd., Hemel Hempstead, UK). Data from the two cohorts were analyzed separately. Results from each observation period were analyzed as a complete block model, with participant and participant x phase as blocks, and phase (1, 2, 3, or 4) and intervention (including washout) as factors. Mean washout data were used as a baseline for statistical analysis purposes. Residues were inspected to ensure that ANOVA expectations were met; when necessary, the data were log-transformed to stabilize the variance.
[185] Post hoc subgroup analysis was performed in the healthy cohort using the Cochran Q test to compare participants who responded to each intervention (ie, with an increase of at least one bowel movement per week over the previous washout period ). Participants were then classified as responsive (showed a response to at least one of the non-placebo interventions) or unresponsive; data from the responder subgroup (14 of 19 participants (74%)) were analyzed using ANOVA.
[186] The primary outcome of the study was a significant increase in bowel movement frequency. Example 6 Treatment results in improving regularity
[187] Participant demographics are shown in Table 6. Adherence within the healthy and functionally constipated cohorts was 98% ± 9 and 99% ± 8, respectively.Table 6: Study participant demographics

[188] The results of this study are summarized in Tables 7 and 8.
[189] In the healthy group (n = 19) significant differences were observed between interventions and washout for the number of daily bowel movements (p = 0.002), Bristol stool score (p = 0.036), strain (p = 0.044 ) and flatulence (p = 0.007). The number of daily bowel movements for ACTAZIN-H and GOLD interventions was significantly higher (p = 0.014 and p = 0.009, respectively) compared to washout, with an approximate increase of 0.8 bowel movements per week with each intervention. Table 7.
[190] In the responsive subgroup (n = 14), consumption of ACTAZIN-L, ACTAZIN-H and GOLD resulted in a significant increase in daily bowel movements (p = 0.005, p <0.001 and p = 0.001, respectively) when compared to washout. Table 7.
[191] Consumption of the 28-day interventions was generally well tolerated, with no serious adverse events reported, and no effect (p < 0.05) on well-being parameters was recorded on the daily questionnaires except for self-reported flatulence, which was higher in the ACTAZIN-H group of the healthy cohort (p = 0.007). Table 7.
[192] In subjects with functional constipation (n = 9), dietary interventions did not significantly increase the frequency of bowel movements compared with washout. Table 8. This was attributed to the small size of the study sample, which has limitations in its statistical power to detect significant differences in the frequency of bowel movements. However, the functionally constipated group showed overall improvement in digestive parameters, and a reduction in other constipation symptoms.
[193] In the functionally constipated group, overall improvement was seen in the Digestive Health Index. Figure 5A. Eight of nine functionally constipated participants showed improvement in gastrointestinal health parameters, as calculated from the sum of the mean washout change for: effort, incomplete evacuation, manual maneuvers, bloating, gas, laxative use, and pain. Figure 5A. The score for each parameter was obtained from the reports with the participants' daily notes.
[194] An increase of more than one bowel movement per week in a symptomatic population is considered a clinically significant magnitude of effect (Food and Drug Administration, 2012) and would potentially improve symptoms in people suffering from mild or occasional constipation. ACTAZIN™ and GOLD, which are derived from green (Hayward) and gold (Gold3) kiwi fruit, have demonstrated this degree of effectiveness in a healthy population. In particular, an increase of about 1.5 bowel movements per week each was observed in the responsive subgroup of healthy cohorts compared to the washout.
[195] In summary, this study showed that ACTAZIN™ and GOLD improved regularity and laxation without affecting stool shape for the healthy cohort. For the functionally constipated group, GOLD improved overall digestive parameters and alleviated major constipation symptoms, including exertion, incomplete evacuation, manual maneuvers, bloating, gas, laxative use, and pain. Table 7: Analysis of results of variance for reports with daily notes, Birmingham IBS symptoms and IBS quality of life questionnaires in the healthy cohort




Example 7 Clinical trial to evaluate the effects of Gold3 kiwi powder on colon microbial populations Overview
[196] The human intervention study described in Example 5 was used to determine the effect of kiwi-derived supplements on microbial colonic composition and metabolism. As described, two kiwi-derived supplements, ACTAZIN™ green kiwi pulp (Actinidia delicious 'Hayward') and GOLD kiwi pulp (Actinidia chinensis 'Zesy002' Gold3) (Anagenix Ltd, Wellington, New Zealand), were used as dietary interventions in the rehearsal. These capsules were cold processed food supplements formulated to maintain the integrity of the innate kiwi compounds. The preparation was carried out in accordance with Example 1 above.
[197] Results for ACTAZIN™ (2400 mg and 600 mg) and GOLD (2400 mg) kiwifruit supplements were evaluated. Participants in the clinical trial were recruited into a healthy group of 19 participants and a functionally constipated group of 9 participants, each of whom underwent all treatments and a placebo for 4 weeks in a randomized crossover with 2-week washout periods. The modification of the composition of the colonic microbiota was determined by sequencing the 16S rRNA gene and the final metabolic products were measured using gas chromatography.
[198] In the functionally constipated group, the relative abundance of Faecalibacterium prausnitzii was observed to increase significantly (P = 0.024) from 3.4% to 7.0% after supplementation with GOLD. Lower proportions of F. praiisnitzii are often associated with gastrointestinal disorders; especially those with an inflammatory pathology. The finding that supplementation with GOLD increased the abundance of F. praiisnitzii offers a strategy to correct dysbiosis of the colonic microbiota, as F. prausnitzii is an important producer of butyrate and has also been shown to exert anti-inflammatory effects. DNA extraction, PCR and 16S rRNA gene sequencing
[199] All faecal samples were shipped on dry ice to Plant & Food Research, Palmerston North, where they were received and stored at -20°C. Two hundred and fifty milligrams of each sample were weighed into a sterile microtube and DNA was extracted from each sample using the MO-BIO PowerSoil® DNA Isolation Kit (MO-BIO Laboratories, Carlsbad, CA, USA #12888).
[200] PCR was performed to amplify the variable regions V3-V4 of the 16S rRNA gene (positions 341-805 in the rRNA gene of Escherichia coli), using the forward primer Bakt_341FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG (SEQ ID NO: 1) and reverse primer5CCATGATCTGGGTGGAT_GTTGGT_GTAGGCAT_GTAGGTG ID NO: 2) Herlemann et al. 2000; W=A/T, H=A/C/T, V=G/C/A, N=G/A/T/C). The primer sequence shown in bold was used for binding to the amplicon region of interest and the rest of the primer was used for binding to Illumina® index adapters.
[201] Each PCR was performed in a total volume of 50 μL of consisting of 25 μL of HotStarTaq® master mix (QIAGEN, Melbourne, Australia), 1 μL of template or negative control DNA (sterile H2O) and 12 μL of each one of the forward and reverse initiators at a final concentration of 0.1 µM. PCR conditions included an initial denaturation at 95°C for 15 minutes, followed by thirty cycles of 30 seconds of denaturation at 95°C, 30 seconds of annealing at 55°C, 30 seconds of extension at 72°C, and ending with a 5 minute extension step at 72°C.
[202] PCR products were column purified using the QIAquick PCR purification kit (Qiagen, Melbourne, Australia), quantified using the Qubit® 2.0 fluorimeter (Life Technologies™), and shipped to New Zealand Genomics Ltd (NZGL), Massey Genome Service (MGS), New Zealand (NZ). In NZGL, the second step of PCR was performed, the amplicons were checked in the QC library, diluted and pooled. The libraries were then loaded onto the Illumina® MiSeq instrument over three paired end (PE) runs of 2 x 250 bp. Bioinformatics
[203] Illumina® MiSeq sequencing data was analyzed using Quantitative Insights into Microbial Ecology (QIIME) software version 1.8.0; Caporaso et al. 2010). To assemble the paired-end readings into a single continuous sequence, PANDASeq with parameters of at least 40 bp overlapping, a minimum length of 350 bp and a maximum length of 500 bp was used (Masella et al. 2012). Putative chimeras were filtered from the sequences and the reads were grouped into operational taxonomic units (OTUS) based on a 97% identity threshold value using USEARCH and UCLUST (Edgar 2010). A subsample of the total readings was taken to allow faster processing of samples and to normalize to approximately 15,000 readings per sample, which is sufficient for phylogenetic and taxonomic attribution (Caporaso et al. 2011; Schloss et al. 2012).
[204] Sequence alignment was performed using PyNAST (Caporaso et al. 2010) with reference to the Greengenes core reference database (version13_8) (DeSantis et al. 2006). Taxonomic assignment was done using the Bayesian Naive RDP classifier (Wang et al., 2007). The healthy and CF groups were analyzed separately and the effect of each of the four treatments on the composition of the microbial community was determined by comparing the average abundance of each genus of bacteria after each treatment (greater than 1% abundance in at least one of the eight samples) with the mean value before treatment. Quantification of organic acid by GC
[205] A 500 to 1000 mg portion of each fecal sample was weighed into a clean tube and diluted 1:10 in phosphate-buffered saline (PBS). Internal standard (ethyl butyrate) was included to generate a final concentration of 5 mM. Organic acids were quantified by GC using a modified method as previously described (Richardson et al., 1989).
[206] The analysis was performed on a Shimadzu gas chromatography system (GC-17A, Kyoto, Japan) equipped with a flame ionization detector and equipped with an HP-1 column (Agilent Technologies, Santa Clara, CA, USA). The instrument was controlled and chromatograms were acquired using GC Solution Chromatography Data System software (Shimadzu, Version 2.3). Standard curves were prepared following the analysis of standard solutions of formic, acetic, propionic, isobutyric, butyric, lactic, succinic, isovaleric, valeric, heptanoic, and hexanoic acids. Organic acid concentrations were expressed as μmol/g feces. Statistical analysis
[207] Statistical calculations were conducted in R Studio using the statistics package (R Studio 2012). Wilcoxon signed-rank test was performed to assess significant differences between the taxonomy at the gender level and significant differences between organic acid concentrations before and after each treatment. A P value of less than 0.05 was considered significant after correction for multiple comparisons using the False Discovery Rate (FDR) method as a function of the p adjustment. (Benjamini and Hochberg 1995). Example 8 Treatment results on colonic microbial populations 16S rRNA gene sequencing
[208] High-throughput sequencing was performed for variable regions of the 16S rRNA gene, amplified from bacterial DNA derived from fecal specimens. This resulted in 26.3 million reads. After quality filtration, chimera removal and subsampling, a total of 3.72 million reads were obtained with an average of 14879 (14139 minimum-14999 maximum) sequences per sample.
[209] Across all samples, 218 species-level phylotypes were observed at a sequence identity threshold of 97%. In the healthy group, Clostridiales increased significantly after GOLD supplementation from 5.0% to 7.6% (P = 0.042) (Table 9). In the CF group, Faecalibacteriwn prausnitzii significantly increased after GOLD treatment from 3.4% to 7.0% (P = 0.024), a two-fold increase, while Dorea spp. increased from 0.9% to 1.4% (P = 0.008) after treatment with ACTAZIN™ H (Table 10). For the FC group, Rwninococcus spp. decreased from 9.9% to 5.6% (P = 0.024) after the placebo treatment period (Table 10).
[210] Of the nine participants who consumed GOLD in the CF group, eight experienced an increase in F. prausnitzii, with only a slightly smaller decrease being observed (Figure 5B). When comparing the healthy and CF groups, many of the genera observed were significantly different between the groups, the most notable being the species Bacteroides spp. and Ruminococcaceae which were significantly higher in the healthy group and Akkennansia spp. which was significantly elevated in the CF group. Organic acid production
[211] Lactate, formate, and isovalerate were not detected in faecal samples in this study. Hexanoate significantly decreased at a concentration of 0.6 μmol/g to 0.2 μmol/g (P = 0.030) after treatment with GOLD in the healthy group (Table 11). Succinate significantly decreased at a concentration of 2.3 µmol/g to 1.7 µmol/g (P = 0.040) after treatment with placebo in the CF group (Table 12). No other significant changes in organic acid concentrations were observed in any of the treatments. Quantitative differences between before and after each treatment were generally modest, with the exception of acetate which increased or decreased by up to 13 μmol/g after some treatments.






Discussion
[212] It was determined that several bacterial groups were significantly altered in abundance after consumption of kiwi-derived supplements. Clostridiales increased by 2.6% after supplementation with GOLD (Gold3) in the healthy group, although the implications of this increase are still under investigation. The relative abundance of F. prausnitzii was significantly elevated (3.6%) in the FC group after a 4-week period of supplementation with the gold kiwi-based GOLD.
[213] In particular, F. prausnitzii is a known butyrate producer. Butyrate is the preferred energy source for colonic epithelial cells and plays a role in relieving inflammation as well as attenuating carcinogenesis, pathogenic colonization, and oxidative stress (Hamer et al. 2008; Macfarlane and Macfarlane 2011). In a recent study, administration of a butyrate-producing bacterium, Clostridium tyrobutyricum, was shown to protect mice from sodium dextran sulfate-induced colitis (Hudcovic et al. 2012). Therefore, increasing the amount of F. prausnitzii in the colon can help to alleviate the symptoms of gastrointestinal disorders through increased production of butyrate.
[214] As the human gastrointestinal tract consists of about 1014 microbial cells (Egert et al. 2006) and the abundance of F. prausnitzii is about 5% (Miquel et al. 2013), this equates to a concentration of approximately five trillion F. prausnitzii cells, which can exert a significant effect on microbial activities and metabolism. The present study showed a total proportion of 5.6% abundance of F. prausnitzii from all samples in the FC group (the mean taken across all samples in the FC group, including all treatments and washout), while 6.1 % was observed in the healthy group. Samples from participants in the CF group before administration of GOLD supplementation had a relatively low abundance of F. prausnitzii of 3.4%, which increased twice to 7.0% after GOLD treatment. A decrease from 7.2% to 5.3% was observed in the healthy group which may be due to already elevated levels at baseline. This has been observed in other studies, where the reference concentration of a bacterial group has a substantial influence on the magnitude of the effect observed in response to a dietary intervention (Kolida et al. 2007; Tuohy et al. 2001).
[215] There were small differences between organic acids after treatments that can be attributed to the use of faecal samples as a substitute for in situ determination of organic acid composition and concentration. Organic acid concentrations decrease distally in the large intestine, which is due to secondary fermentation, absorption into the bloodstream and/or the use of organic acids (particularly butyrate) by colonocytes (Bach et al. 2000). Therefore, measuring organic acids in faecal samples can greatly underestimate the concentration of organic acids in situ (Millet et al. 2010).
[216] Butyrate was slightly higher in faecal samples from participants in the CF group, which is in line with the increased abundance of F. prausnitzii. Lactate, formate, and isovalerate were not detected in fecal samples from both study groups. Lactate is not normally detected in high concentrations in stool samples from healthy individuals as it is an intermediary in many metabolic networks and is consumed by members of the microbiota as part of normal metabolism (Duncan et al. 2004; Belenguer et al. 2011). Formate also acts as an intermediate and can be converted to methane, carbon dioxide and water, as well as being only produced in the early stages of fermentation, usually in the proximal colon (Pryde et al. 2002; Huda-Faujan et al. 2010) .
[217] F. prausniizii is well characterized in terms of its metabolic capabilities and fermentation profile, despite being an oxygen-sensitive bacterium that is difficult to cultivate in the laboratory (Duncan et al. 2002). Despite its numerical dominance in the gut and butyrate-producing nature, F. prausnitzii has not yet been used to improve digestive health. Given that an impoverished abundance of F. prausnitzii is observed in various gastrointestinal disorders, it has been proposed that F. prausnitzii could be formulated as a probiotic and administered to people suffering from IBS or IBD (Sokol et al. 2008; Sartor 2011). However, this work is still ongoing. The oxygen sensitivity of F. prausnitzii makes its formulation difficult as a component of probiotic compositions.
[218] The gold kiwi-derived supplement, GOLD, provides a means of selectively stimulating the proliferation of F. prausnitzii, commensal, and is therefore considered particularly useful for inflammation-related gastrointestinal disorders. In conclusion, GOLD improved F. prausnitzii impoverished dysbiosis in participants with functional constipation. As such, Gold3 Gold Kiwi Powder can be used to restore the microbiota to a healthy state with anti-inflammatory benefits and higher concentrations of butyrate in situ. Example 9: Microbiome qPCR analysis for human clinical trial summary
[219] This study investigated the effect of ACTAZIN™ (2400 mg) and GOLD (2400 mg) kiwifruit supplements on faecal microbial concentrations in a human intervention study as well as in vitro fermentation experiments. The objective was to determine the effect of kiwi fruit-based interventions on the microbial composition of the samples. The modification of the composition of the colonic microbiota was determined by real-time quantitative polymerase chain reaction (qPCR) which complements the existing 16S rRNA gene sequencing data obtained previously. See Examples 7 and 8, above.
[220] Real-time qPCR is a common method used to investigate the microbial ecology of the gastrointestinal tract. It has been used in several studies and can give a quantitative indication of microbial numbers. Quantification was performed using the Roche LightCycler® 480 instrument. In addition to total bacteria, individual bacterial groups quantified were the Clostridium coccoides group, the Lachnospiraceae group, Bacteroides-Prevotella-Porphyromonas, Bifidobacteria, Lactobacilli and Faecalibacterium prausnitzii. The bacterial groups above were also quantified for the in vitro fermentation work with the exception of Lactobacilli, which were previously tested.
[221] In the functionally constipated group, the concentration of F. prausnitzii increased after supplementation with GOLD, falling short of being statistically significant. Lower proportions of F. praiisnitzii are often associated with gastrointestinal disorders; especially those with an inflammatory pathology. The finding that GOLD supplementation increased F. prausnitzii concentrations is in line with the significant increase in abundance found in the sequencing data and provides support for the prebiotic effects of GOLD consumption. Background
[222] Green kiwi fruit has been shown to improve aspects of gastrointestinal health, including altering the microbial ecology of the colonic environment (Blatchford et al. 2015a; Blatchford et al. 2015b; Parkar et al. 2012). These benefits are believed to derive from inherent levels of digestion resistant carbohydrates (DRC), polyphenols, and vitamin C (Chan et al. 2007; Ferguson & Ferguson 2003). The benefits of the gold kiwi have not been clearly established. This study details experiments performed using samples from the existing human clinical trial (described in Example 5). The placebo-controlled, randomized, double-blind, crossover trial examined the role of two kiwi-derived ingredients, ACTAZIN™ (green kiwi) and GOLD (gold kiwi Gold3), on digestive health.
[223] The primary end points measured in the original trial were stool frequency, stool shape, and quality of life scores. See Examples 5 and 6. Secondary end points included the measurement of short-chain fatty acids (SCFA), and also the measurement of changes in the relative abundances of faecal microbial populations using 16S rRNA gene sequencing. Sequencing analysis of the 16S rRNA gene generated a global picture of the microbial ecology of the samples (as percentages of relative abundance), but did not yield quantitative results. See Examples 7 and 8, above.
[224] Therefore, the following DNA samples obtained from the original trial were used for the current trial: 2400 mg of pre- and post- GOLD intervention samples (n = 52), 2400 mg of intervention samples (n = 52) of ACTAZIN™ and placebo samples (n = 52). These samples were used to determine the concentration of selected bacterial groups using qPCR, which produces a quantitative measure of the concentration of bacteria in a sample. Materials and methods
[225] As detailed in Example 5, the study design was a randomized, double-blind, placebo-controlled, crossover trial with participants consuming four different treatments in four weeks, with two weeks of washout between each treatment. Interventions were administered in 4 x 600 mg capsules formulated to look the same to keep treatment blinded, as shown in Table 13, below. See also Example 5. As described, the powdered ingredients were prepared from New Zealand green kiwi and gold kiwi, respectively. See Examples 1 and 5.

[226] The selected bacteria were used as the representative genera for each of the seven bacterial groups evaluated (Table 14). All isolates were cultured anaerobically at 37°C overnight using Hungate tubes sealed with butyl rubber stoppers, unless otherwise indicated. All media were obtained from Oxoid (Adelaide, Australia) unless otherwise noted.
[227] Lactobacillus reuteri (DPC 16) was grown in Man-Rogosa-Sharpe broth (MRS); Bifidobacterium bifidum (DSM 20082) were grown in MRS broth supplemented with 0.05% (w/v) L-cysteine hydrochloride (Sigma-Aldrich); Roseburia intestinalis (DSM 14610) was cultivated in ruminal bacteria medium (DSMZ Medium 330); Lachnospira multipara (ATCC 19207) was grown in RM02 medium supplemented with filtered ruminal fluid (Leahy et al. 2010); Bacteroides fragilis (ATCC 25285) was grown in anaerobic Wilkins-Chalgren broth supplemented with 0.05% (w/v) L-cysteine hydrochloride (Sigma-Aldrich) for 2 days; Faecalibacterium prausnitzii (DSM 17677) was grown in brain-heart infusion broth (BHI) supplemented with 0.5% (w/v) yeast extract, 0.0005% (w/v) haemin (Sigma-Aldrich) ), 0.0005% (w/v) vitamin K (Sigma-Aldrich) and 0.2% L-cysteine hydrochloride (Sigma-Aldrich); Escherichia coli (Nissle) was cultivated in tryptic soybean broth (TSB) at 37°C aerobically.


[228] The specificity of each standard strain of primers was assessed using in silico analysis in the SnapGene® software (www.snapgene.com). Cell density was determined using a Neubauer hemocytometer and cultures were diluted or concentrated as required to 1.0 x 108 or 1.0 x 109 cells/ml. DNA was then extracted using the MO-BIO PowerSoil® DNA Isolation Kit (MO-BIO Laboratories, Carlsbad, CA, US #12888). Standard curves were constructed using dilution series of representative bacterial strains from each group. Samples and standards were run in triplicate by absolute quantification on the Roche LightCycler® 480 real-time PCR instrument. Roche Green I SYBR master mix detection chemistry (04707516001) was used to detect double-stranded DNA amplification . The total reaction volume was 20 µL, consisting of 10 µL of SyBr green I master mix, 4 µL of forward primer (5X concentrate at 2.5 µM), 4 µL of reverse primer (5X concentrate at 2.5 µM ) and 2 μL of DNA template or sterile water (no template control).
[229] Each qPCR test included an activation cycle (95°C) for 5 minutes, test cycles of 32-40 (including denaturation step at 95°C (30s), annealing step as in Table 14 and extension step at 72°C for 1 minute), and a melting curve cycle (60 to 95°C at 0.1°C per second, with continuous fluorescence acquisition) followed by a cooling cycle at 40°C . The cycle called the Tm melting curve made it possible to differentiate between the target product and the non-specific double-stranded product, such as primer dimers. Primers were diluted in PCR grade water to a concentration of 2.5 µM. This was then diluted 1:5 to obtain a concentrated 0.5 μM solution in the PCR reaction.
[230] Statistical calculations were conducted in R using the statistics package (R Development Core Team 2008). Wilcoxon signed-rank test was performed to assess significant differences before and after each treatment. For in vitro fermentations, significant differences were determined by comparing time 0 with other time points. A P value less than 0.05 was considered significant after correction for multiple comparisons using the False Discovery Rate (FDR) method as a function of the p adjustment. in Studio R (Benjamini and Hochberg 1995). Example 10 Results of microbiome qPCR analysis in human clinical trial
[231] The qPCR data results portray a relatively stable microbial profile over time for each bacterial group evaluated. Moderate increases or decreases were observed after each treatment. Table 15 presents LightCycler® 480 qPCR data showing bacterial groups as the mean 16S rRNA gene copy number/gram of fecal sample (transformed LOG) calculated and the standard error of the mean (SEM) before and after each treatment period in the healthy group. Table 16 presents LightCycler® 480 qPCR data showing bacterial groups as the average number of 16S rRNA gene copies/gram of fecal sample (transformed LOG) calculated and the standard error of the mean (SEM) before and after each treatment period in the functionally constipated group.
[232] As shown in Table 15 and Figure 6, in the healthy group after treatment with GOLD, moderate increases in total bacteria, C. coccoides, Lachnospiraceae, Lactobacilli, Bifidobacteria and Bacteroides-Prevotella-Porphyromonas groups were observed, in which the concentration of F. prausnitzii remained constant. With ACTAZIN™ supplementation, slight decreases were seen in the total bacteria, Bacteroides-Prevotella-Porphyromonas, Lactobacilli, Bifidobacteria, and F. prausnitzii groups, where the C. coccoides group remained constant and the Lachnospiraceae group increased slightly. Placebo treatment showed high concentrations of total bacteria and Bacteroides-Prevotella-Porphyromonas and small decreases in concentrations of groups of C. coccoides, Lachnospiraceae, Lactobacilli, Bifidobacteria, and F. prausnitzii.
[233] As shown in Table 16 and Figure 7, in the functionally constipated group after treatment with GOLD, moderate increases in total bacteria, Clostridium coccoides, Lachnospiraceae, Bacteroides-Prevotella-Porphyromonas, and Lactobacilli groups, and a smaller decrease were observed in Bifidobacterium spp. The concentration of F. prausnitzii increased mostly from log 7.03 to 7.45 which almost reached significance (P = 0.0503) and is in agreement with a similar elevation reported in the 16S rRNA gene sequencing data. The increase in real numbers is 231.9 million copy numbers of the 16S rRNA gene, which is a 179% increase after GOLD treatment. After treatment with ACTAZIN™, decreases were observed in all groups of bacteria. Eight of nine of the functionally constipated participants showed an increase in F. prausnitzii levels after treatment with GOLD. See Figure 5C. Placebo treatment showed high concentrations in all groups, except for Lachnospiraceae which remained constant.



[234] Figures 6 and 7 show bacterial concentration data graphically in both log-transformed and natural forms for the healthy and functionally constipated groups, respectively. Viewing the data in this form highlights how stable the composition of the faecal microbiota is over time.
[235] Bacteroides-Prevotella-Porphyromonas group bacteria are noted to constitute a considerable collection of primary degraders in the human colonic community. They play an important role in accessing unabsorbed carbohydrate using specialized elimination mechanisms. Among these are cellulose, starch, inulin, and xylan degraders such as Bacteroides thetaiotaomicron, Bacteroides ovatus, and Bacteroides cellulosilyticus (Flint et al. 2008; Walker). et al. 2008). Roseburia intestinalis, Roseburia inulinovorans, Ruminococcus bromii, and Ruminococcus flavefaciens are also from the group of major primary degradants that are members of the C. coccoides group (Kurakawa et al. 2015).
[236] The C. coccoides group constitutes the largest subgroup of bacteria in the colon, typically constituting 25-60% of total bacterial abundance (Hold et al. 2002) and is composed of a large proportion of butyrate producers (Louis & Flint 2009). Two main microorganisms with proven probiotic states are Bifida Bacteria and Lactobacilli. Bifid bacteria have often been associated with healthy microbiota and are acetate producers, which can have beneficial effects on the health of the host. Lactobacilli are a group of commensal lactic acid bacteria that have been exploited for centuries in the production of dairy products and have been studied in hundreds of trials for their probiotic effectiveness (Reid 1999).
[237] The increase in F. prausnitzii concentration was too small to be significant (P = 0.0503) after the 4-week supplementation period with the kiwi-gold Gold3-based GOLD. F. prausnitzii is one of the most populous species in the human gastrointestinal tract, being typically observed in more than 5% of the total proportion of the colonic microbiota of healthy adults (Miquel et al. 2013). Members of Firmicutes phylum, F. prausnitzii are commensal inhabitants of the human large intestine, which have demonstrated anti-inflammatory properties in vivo (Furet et al. 2010; Sokol et al. 2009). F. prausnitzii generate butyrate as a result of carbohydrate fermentation, as well as lactate and formate (Duncan et al. 2002; Duncan et al. 2004). Butyrate is the preferred energy source for colonic epithelial cells and plays a role in relieving inflammation as well as attenuating carcinogenesis, pathogenic colonization, and oxidative stress (Hamer et al. 2008; Macfarlane & Macfarlane 2011). GOLD selectively stimulates the proliferation of F. prausnitziicomensals and therefore may be considered useful for inflammation-related gastrointestinal disorders.
[238] In summary, the qPCR data confirm the relative microbial abundance data and show that the Gold3 kiwi-derived supplement increased the concentration of F. prausnitzii in functionally constipated participants. This demonstrates a beneficial result as F. prausnitzii is known to grow in the large intestine and generate butyrate, which has protective/healthy health functions in the intestine.
[239] These results are considered to be significant, given that F. prausnitzii is highly sensitive to oxygen (Rigottier-Gois 2013), and therefore a challenge for formulation and storage as a probiotic agent. Therefore, the gold kiwi compositions of the invention provide an efficient and effective means to increase F. prausnitzii contents that would otherwise be difficult to modulate.
[240] Those skilled in the art can use the descriptions and teachings in this document to produce other modalities and variations without undue experimentation. All such modalities and variations are considered to form part of the present invention.
[241] Thus, a person skilled in the art will understand immediately from the disclosure that subsequent modifications, substitutions and/or variations perform substantially the same function or achieve substantially the same result as the modalities described herein can be used in accordance with such related embodiments of the present invention. Thus, the invention aims to cover, within its scope, modifications, substitutions, and variations in processes, manufacturing, compositions of matter, compounds, means, methods and/or steps described herein. References
[242] Abdollahi-Roodsaz S, Joosten LA, Koenders MI, Devesa I, Roelofs MF, Radstake TR, Heuvelmans-Jacobs M, Akira S, Nicklin MJ, Ribeiro-Dias F, van den Berg WB 2008. Stimulation of TLR2 and TLR4 differentially skews the balance of T cells in a mouse model of arthritis. Journal of Clinical Investigation 118(1): 205-16.
[243] Abrahamsson TR, Jakobsson HE, Andersson AF, Bjorksten B, Engstrand L, Jenmalm MC 2012. Low diversity of the gut microbiota in infants with atopic eczema. J Allergy Clin Immunol 129(2): 434-40, 440 e1-2.
[244] Adaim A. 2010. Investigating the effect of gold kiwifruit consumption on the incidence and symptoms of upper respiratory tract infections in preschool children: the thesis presented in partial fulfillment of the requirements for the degree of Masters of Sciences in Human Nutrition and Human Health, Massey University, Auckland, New Zealand.
[245] Attaluri A, Donahoe R, Valestin J, Brown K, Rao SSC 2011. psyllium for constipation. Alimentary Pharmacology & Therapeutics 33(7): 822-828.
[246] Bach Knudsen KE, Jorgensen H, Canibe N. Quantification of absorption of nutrients derived from carbohydrate assimilation: model experiment with catheterized pigs fed on wheat-or oat-based rolls. The British Journal of Nutrition. 2000; 84: 449-58.
[247] Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science. 2005; 307: 1915-20.
[248] Benjamini Y, Hochberg Y. Controlling the false discovery rate - a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society Series B-Methodological. 1995; 57: 289-300.
[249] Belenguer A, Holtrop G, Duncan SH, Anderson SE, Calder AG, Flint HJ, et al. Rates of production and utilization of lactate by microbial communities from the human colon. FEMS Microbiol. Ecology 2011; 77: 107-19.
[250] Bentley-Hewitt KL, Blatchford PA, Parkar SG, Ansell J, Pernthaner A. Digested and fermented green kiwifruit increases human beta-defensin 1 and 2 production in vitro. Plant Foods for Human Nutrition. 2012; 67: 208-14.
[251] Blatchford P, Bentley-Hewitt KL, Stoklosinski H, McGhie T, Gearry R, Gibson G, Ansell J 2015a. In vitro characterization of the fermentation profile and prebiotic capacity of gold-fleshed kiwifruit. Benef Microbes pages 1-12. Published online at DOI: http://dx.doi.org/10.3920/BM20150006.
[252] Blatchford P, Stoklosinski H, Walton G, Swann J, Gibson G, Gearry R, Ansell J 2015b. Kiwifruit fermentation drives positive gut microbial and metabolic changes irrespective of initial microbiota composition. Bioactive Carbohydrates and Dietary Fiber 6(1): 37-45.
[253] Boland, M.J., Hardman, M.J. 1972. Kinetic studies on the thiol protease from Actinidia chinensis. FEBS Letters, 27(2): 282-284.
[254] Bravo JA, Forsythe P, Chew MV, Escaravage E, Savignac HM, Dinan TG, et al. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proceedings of the National Academy of Sciences of the United States of America. 2011; 108: 16050-5.
[255] Buffie CG, Pamer EG. Microbiota-mediated colonization resistance against intestinal pathogens. Nature Reviews Immunology. 2013; 13: 790-801.
[256] Candela M, Rampelli S, Turroni S, Severgnini M, Consolandi C, De Bellis G, et al. Unbalance of intestinal microbiota in atopic children. BMC Microbiology. 2012; 12:95.
[257] Caporaso JG, Bittinger K, Bushman FD, DeSantis TZ, Andersen GL, Knight R. PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics. 2010; 26: 266-7.
[258] Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nature Methods. 2010; 7: 335-6.
[259] Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, et al. Global patterns of 16S rRNA diversity at depth of millions of sequences per sample. Proceedings of the National Academy of Sciences of the United States of America. 2011; 108 Suppl 1: 4516-22.
[260] Cardona F, Andres-Lacueva C, Tulipani S, Tinahones FJ, Queipo-Ortuno MI. 2013. Benefits of polyphenols on gut microbiota and implications in human health. Journal of Nutritional Biochemistry, 24: 1415-1422.
[261] Chan AO, Leung G, Tong T, Wong NY. Increasing dietary fiber intake in terms of kiwifruit improves constipation in Chinese patients. World Journal of Gastroenterology: WJG. 2007; 13: 4771-5.
[262] Chang C, Lin T, Lu Y, Liu T, Liu J. 2010. Kiwifruit improves bowel function in patients with irritable bowel syndrome with constipation. Asia Pacific Journal of Clinical Nutrition 19(4): 451-457.
[263] Chassard, C, Dapoigny, M, Scott, KP, Crouzet, L, Del'homme, C, Marquet, P, Martin, JC, Pickering, G, Ardid, D, Eschalier, A, Dubray, C, Flint , HJ and Bernalier-Donadille, A. 2012. Functional dysbiosis within the gut microbiota of patients with constipated-irritable bowel syndrome. Alimentary Pharmacology & Therapeutics 35: 828-838.
[264] Chen W, Liu F, Ling Z, Tong X, Xiang C 2012. Human intestinal lumen and mucosa-associated microbiota in patients with colorectal cancer. PloS One 7(6): e39743.
[265] DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, et al. Greengenes, the chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol. 2006; 72: 5069-72.
[266] by Sousa Moraes LF, Grzeskowiak LM, by Sales Teixeira TF, Gouveia Peluzio Mdo C 2014. Intestinal microbiota and probiotics in celiac disease. Clinical Microbiology Reviews 27(3): 482-9.
[267] by Vos WM, by Vos EA 2012. Role of the intestinal microbiome in health and disease: from correlation to causation. Nutrition Revision 70 Supplement 1: S45-56.
[268] by Weerth C, Fuentes S, Puylaert P, by Vos WM 2013. Intestinal microbiota of infants with colic: development and specific signatures. Pediatrics 131(2): e550-8.
[269] Desbonnet L, Garrett L, Clarke G, Bienenstock J, Dinan TG 2008. The probiotic Bifidobacteria infantile: An assessment of potential antidepressant properties in the rat. Journal of Psychiatric Research 43(2): 164-74.
[270] Drossman DA. The functional gastrointestinal disorders and the Rome III process. Gastroenterology. 2006; 130: 1377-90.
[271] Drummond L. 2013. The composition and nutritional value of kiwifruit. Advances in Food and Nutrition Research 68: 33-57.
[272] Drummond L, Gearry RB. 2013. Kiwifruit modulation of gastrointestinal motility. Advances in Food and Nutrition Research 68: 219-232.
[273] Duncan SH, Hold GL, Harmsen HJ, Stewart CS, Flint HJ. Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen. nov., comb. Nov. International Journal of Systematic and Evolutionary Microbiology. 2002; 52:21416.
[274] Duncan SH, Louis P, Flint HJ. Lactate-using bacteria, isolated from human feces, that produce butyrate as a major fermentation product. Appl. Environ. Microbiol. 2004; 70: 5810-7.
[275] Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010; 26: 2460-1.
[276] Egert M, by Graaf AA, Smidt H, by Vos WM, Venema K. Beyond diversity: functional microbiomics of the human colon. Trends in Microbiology. 2006; 14: 86-91.
[277] Eidenberger T, Selg M, Fuerst S, Krennhuber K. 2014. In-vitro inhibition of human lipase PS by polyphenols from kiwi fruit. Journal of Food Research. 3(4): 71-77.
[278] Ferguson AR, Ferguson LR. Are kiwifruit really good for you In: Huang HW, editor. Proceedings of the Fifth International Symposium on Kiwifruit. 2003. 131-8.
[279] Flint HJ, Bayer EA, Rincon MT, Lamed R, White BA 2008. Polysaccharide analysis by gut bacteria: potential for new insights from genomic. Nat Rev Microbiol 6(2): 121-31.
[280] Fu CJ, Carter JN, Li Y, Porter JH, Kerley MS 2006. Comparison of agar plate and real-time PCR on enumeration of Lactobacillus, Clostridium perfringens and total anaerobic bacteria in dog faeces. Letters in Applied Microbiology 42(5): 490-494.
[281] Fujimoto T, Imaeda H, Takahashi K, Kasumi E, Bamba S, Fujiyama Y, Andoh A 2013. Decreased abundance of Faecalibacterium prausnitzii in the gut microbiota of Crohn's disease. J Gastroenterol Hepatol 28(4): 613-9.
[282] Furet JP, Kong LC, Tap J, Poitou C, Basdevant A, Bouillot JL, et al. Differential adaptation of human gut microbiota to bariatric surgery-induced weight loss: links with metabolic and low-grade inflammation markers. Diabetes. 2010; 59: 3049-57.
[283] Gibson GR, Scott KP, Rastall RA, Tuohy KM, Hotchkiss A, Dubert-Ferrandon A, Gareau M, Murphy EF, Sulnier D, Loh G, MacFarlane S, Delzenne N, Ringel Y, Kozianowski G, Dickmann R, Lenoir-Wijnkoop I, Walker C, Buddington R. 2010. Dietary prebiotics: current status and new definition. Food Science and Technology Bulletin: Functional Foods, 7: 1-19.
[284] Geurts L, Neyrinck AM, Delzenne NM, Knauf C, Cani PD. Gut microbiota controls adipose tissue expansion, gut barrier and glucose metabolism: novel insights into molecular targets and interventions using prebiotics. Beneficial Microbes. 2014; 5: 3-17.
[285] Gostner A, Blaut M, Schaffer V, Kozianowski G, Theis S, Klingeberg M, et al. Effect of isomalt consumption on faecal microflora and colonic metabolism in healthy volunteers. Br. J. Nutr. 2006; 95: 40-50.
[286] Guidance for industry: irritable bowel syndrome - clinical evaluation of drugs for treatment. U.S. Department of Health and Human Services. Food and Drug Administration. Center for Drug Evaluation and Research. http://www.fda.gov/downloads/Drugs/Guidances/UCM205269.pdf
[287] Hamer HM, Jonkers D, Venema K, Vanhoutvin S, Troost FJ, Brummer RJ. Revised article: The role of butyrate on colonic function. Food Pharmacol. The R. 2008; 27: 104-19.
[288] Hazenberg MP, van de Merwe JP, Pena AS, Pennock-Schroder AM, van Lieshout LM. Antibodies to Coprococcus comes in sera of patients with Crohn's disease. Isolation and purification of the agglutinating antigen tested with an ELISA technique. Journal of Clinical & Laboratory Immunology. 1987; 23: 143-8.
[289] Herlemann DPR, Labrenz M, Juergens K, Bertilsson S, Waniek JJ, Andersson AF. Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea. ISME Journal. 2011; 5: 1571-9.
[290] Hold GL, Pryde SE, Russell VJ, Furrie E, Flint HJ 2002. Assessing microbial diversity in human colonic samples by 16S rDNA sequence analysis. FEMS Microbiol Ecol 39(1): 33-9.
[291] Huda-Faujan N, Abdulamir AS, Fatimah AB, Anas OM, Shuhaimi M, Yazid AM, et al. The impact of the level of the intestinal short chain fatty acids in inflammatory bowel disease patients versus healthy subjects. The Open Biochemistry Journal. 2010; 4:53-8.
[292] Hudcovic T, Kolinska J, Klepetar J, Stepankova R, Rezanka T, Srutkova D, et al. Protective effect of Clostridium tyrobutyricum in acute dextran sodium sulphate-induced colitis: differential regulation of tumor necrosis factor-alpha and interleukin-18 in severe BALB/c and combined immunodeficiency mice. Clinical and Experimental Immunology. 2012; 167: 356-65.
[293] Husebye E, Hellstrom PM, Sundler F, Chen J, and Midtvedt T. 2001. Influence of microbial species on small intestinal myoelectric activity and transit in germ-free rats. American Journal of Physiology, Gastrointestinal and Liver Physiology 280: G368-G380.
[294] Hunter DC, Skinner MA, Wolber FM, Booth CL, Loh JMS, Wohlers M, Stevenson LM, Kruger MC. 2012. Consumption of gold kiwifruit reduces severity and duration of selected upper respiratory tract infection symptoms and increases plasma vitamin C concentration in healthy older adults. British Journal of Nutrition 108: 1235-1245.
[295] Jeffery IB, O'Toole PW, Ohman L, Claesson MJ, Deane J, Quigley EM, Simren M 2012. Gut 61(7): 997-1006.
[296] Kalliomaki M, Satokari R, Lahteenoja H, Vahamiko S, Gronlund J, Routi T, Salminen S 2012. Expression of microbiota, Toll-like receptors, and their regulators in the small intestinal mucosa in celiac disease. Journal of Pediatric Gastroenterology and Nutrition 54(6): 727-32.
[297] L Kaur, SM Rutherfurd, PJ Moughan, L Drummond, and MJ Boland. 2010. Actinidin enhances protein digestion in the small intestine as advertised using an in vitro digestion model, J. Agric. Food Chem. 58 (8): 5074-5080.
[298] Khalif IL, Quigley EMM, Konovitch EA and Maximova ID. 2005. Alterations in the colonic flora and intestinal permeability and evidence of immune activation in chronic constipation. Digestive and Liver Disease 37: 838-849.
[299] Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, Britt EB, Fu X, Wu Y, Li L, Smith JD, DiDonato JA, Chen J, Li H, Wu GD, Lewis JD , Warrier M, Brown JM, Krauss RM, Tang WH, Bushman FD, Lusis AJ, Hazen SL 2013. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nature Medicine 19(5): 576-85.
[300] Kok RG, DeWaal A, Schut F, Welling GW, Weenk G, Hellingwerf KJ 1996. Specific detection and analysis of a probiotic Bifidobacterium strain in infant feces. Applied and Environmental Microbiology 62(10): 3668-3672.
[301] Kolida S, Gibson GR. 2007. Prebiotic capacity of inulin-type fructans. The Journal of Nutrition, 137(11): 2503S-2506S.
[302] Kolida S, Meyer D, Gibson GR. A double-blind placebo-controlled study to establish the bifidogenic dose of inulin in healthy humans. Eur. J. Clin. Nutrition 2007; 61: 1189-95.
[303] Koren O, Spor A, Felin J, Fak F, Stombaugh J, Tremaroli V, Behre CJ, Knight R, Fagerberg B, Ley RE, Backhed F 2011. Human oral, gut, and plaque microbiota in patients with atherosclerosis. Proceedings of the National Academy of Sciences of the United States of America 108 Supplement 1: 4592-8.
[304] Kurakawa T, Ogata K, Matsuda K, Tsuji H, Kubota H, Takada T, Kado Y, Asahara T, Takahashi T, Nomoto K 2015. - quantitative PCR. PLoS One 10(5): e0126226.
[305] Leahy SC, Kelly WJ, Altermann E, Ronimus RS, Yeoman CJ, Pacheco DM, Li D, Kong Z, McTavish S, Sang C, Lambie SC, Janssen PH, Dey D, Attwood GT 2010. The genome of sequence the rumen methanogen Methanobrevibacter ruminantium reveals new possibilities for controlling ruminant methane emissions. PLoS One 5(1).
[306] Lee YK, Menezes JS, Umesaki Y, Mazmanian SK 2011. Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis. Proceedings of the National Academy of Sciences of the United States of America 108 Supplement 1: 4615-22.
[307] Lepage P, Hasler R, Spehlmann ME, Rehman A, Zvirbliene A, Begun A, Ott S, Kupcinskas L, Dore J, Raedler A, Schreiber S 2011. ulcerative colitis. Gastroenterology 141(1): 227-36.
[308] Leung L, Riutta T, Kotecha J, Rosser W. 2011. Chronic constipation: An evidence-based review. The Journal of the American Board of Family Medicine 24(4): 436-451.
[309] Ley RE, Turnbaugh PJ, Klein S, Gordon JI 2006. Microbial ecology - Human gut microbes associated with obesity. Nature 444(7122): 1022-1023.
[310] Liu LWC. 2011. Chronic constipation: current treatment options. Canadian Journal of Gastroenterology 25(Suppl B): 22B-28B.
[311] Louis P, Flint HJ 2009. Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol Lett 294(1): 1-8.
[312] Maccaferri S, Candela M, Turroni S, Centanni M, Severgnini M, Consolandi C, Cavina P, Brigidi P 2012. IBS-associated phylogenetic unbalances of the intestinal microbiota are not reverted by probiotic supplementation. Gut Microbes 3(5): 406-13.
[313] Macfarlane GT, Macfarlane S. Fermentation in the human large intestine: its physiologic consequences and the potential contribution of prebiotics. J. Clin. Gastroenterol. 2011; 45 Supply: S120-7.
[314] R Maddumage, NJ Nieuwenhuizen, SM Bulley, JM Cooney, SA Green, RG Atkinson. 2013. Diversity and relative levels of actinidin, kiwellin, and thaumatin-like allergens in 15 varieties of kiwifruit (Actinidia). J.Agric. Food Chem. 61: 728-739.
[315] Masella AP, Bartram AK, Truszkowski JM, Brown DG, Neufeld JD. PANDAseq: PAired-eND Assembler for Illumina sequences. BMC Bioinformatics. 2012; 13:31.
[316] Matsuki T, Watanabe K, Fujimoto J, Miyamoto Y, Takada T, Matsumoto K, Oyaizu H, Tanaka R 2002. Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces. Appl Environ Microbiol 68(11): 5445-51.
[317] Metcalf AM, Phillips SF, Zinsmeister AR, MacCarty RL, Beart RW, Wolff BG. Simplified assessment of segmental colonic transit. Gastroenterology. 1987; 92: 40-7.
[318] Millet S, Van Oeckel MJ, Aluwe M, Delezie E, De Brabander DL. Prediction of in vivo short-chain fatty acid production in hindgut fermenting mammals: problems and pitfalls. Crit. Rev. Food Sci. Nutr. 2010; 50:605-19.
[319] Miquel S, Martin R, Rossi O, Bermudez-Humaran LG, Chatel JM, Sokol H, et al. Faecalibacterium prausnitzii and human intestinal health. Current Opinion in Microbiology. 2013; 16: 255-61.
[320] Monro JA, Mishra S, Venn B. 2010. Baselines representing blood glucose clearance improve in vitro prediction of the glycaemic impact of customarily consumed food quantities. British Journal of Nutrition, 103(2): 295-305.
[321] Murata Y, Ohmori H, Ichikawa M, Harada A, Agari M, Fukuba H, Yamasaki M, Yasuzawa M, Takemoto M, Ikeda M, Matsumoto N, Tamura T, Sumimoto R, Namba K 2013. Fecal microbiota and fecal characteristics of patients with Parkinson's disease. Journal of Intestinal Microbiology 27(4): 211-215.
[322] Nadkarni MA, Martin FE, Jacques NA, Hunter N 2002. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology-Sgm 148: 257-266.
[323] Neufeld KM, Kang N, Bienenstock J, Foster JA 2011. Reduced anxiety-like behavior and central neurochemical change in germ-free mice. Neurogastroenterology and Motility 23(3): 255-64, e119.
[324] NJ Nieuwenhuizen, R Maddumage, GK Tsang, LG Fraser, JM Cooney, HN De Silva, S Green, KA Richardson, RG Atkinson. 2012. Mapping, complementation, and targets of the cysteine protease actinidin in kiwifruit. Plant Physiology 158(1): 376-388.
[325] Parkar SG, Rosendale D, Paturi G, Herath TD, Stoklosinski H, Phipps JE, et al. In vitro utilization of gold and green kiwifruit oligosaccharides by human gut microbial populations. Plant Foods for Human Nutrition. 2012; 67: 200-7.
[326] Parnell JA, Reimer RA. Prebiotic fibers dose-dependently increase satiety hormones and alter Bacteroidetes and Firmicutes in lean and obese JCR:LA- cp rats. The British Journal of Nutrition. 2012; 107: 601-13.
[327] Paturi G, Butts CA, Bentley-Hewitt KL, Ansell J 2014. Influence of green and gold kiwifruit on indices of large bowel function in healthy rats. J Food Sci 79(8): H1611-20.
[328] Pryde SE, Duncan SH, Hold GL, Stewart CS, Flint HJ. The microbiology of butyrate formation in the human colon. FEMS Microbiology Letters. 2002; 217: 1339.
[329] Qin JJ, Li RQ, Raes J, Arumugam M, Burgdorf KS, Manichanh C, et al. A human gut microbial gene catalog established by metagenomic sequencing. Nature. 2010; 464: 59-U70.
[330] R Development Core Team 2008. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. .
[331] RStudio. RStudio: Integrated development environment for R (Version 0.97.551) Computer software. Boston, MA. http://www.rstudio.org/. 2012.
[332] Rajilic-Stojanovic M, Biagi E, Heilig HG, Kajander K, Kekkonen RA, Tims S, et al. Global and deep molecular analysis of microbiota signatures in fecal samples from patients with irritable bowel syndrome. Gastroenterology. 2011; 141: 1792801.
[333] Rajilic-Stojanovic M, by Vos WM. The first 1000 cultured species of the human gastrointestinal microbiota. FEMS Microbiology Reviews. 2014; 38: 9961047.
[334] Ramirez-Farias C, Slezak K, Fuller Z, Duncan A, Holtrop G, Louis P 2009. Effect of inulin on the human gut microbiota: stimulation of Bifidobacterium adolescentis and Faecalibacterium prausnitzii. British Journal of Nutrition 101(4): 541-550.
[335] Rawls JF, Mahowald MA, Ley RE, Gordon JI. Reciprocal gut microbiota transplants from zebrafish and mice to germ-free recipients reveal host habitat selection. Cell. 2006; 127: 423-33.
[336] Reid G 1999. The scientific basis for probiotic strains of Lactobacillus. Appl Environ Microbiol 65(9): 3763-6.
[337] Rhee SH, Pothoulakis C, and Mayer EA. 2009. Principles and clinical implications of the brain-gut-enteric microbiota axis. Nature Reviews Gastroenterology & Hepatology 6: 306-314.
[338] Richardson AJ, Calder AG, Stewart CS, Smith A. 1989. Simultaneous determination of volatile and non-volatile acidic fermentation products of anaerobes by capillary gas-chromatography. Letter Appl. Microbiol. 9:5-8.
[339] Rigottier-Gois L. 2013. Dysbiosis in inflammatory bowel diseases: the oxygen hypothesis. The ISME Journal 7: 1256-1261.
[340] Rinttila T, Kassinen A, Malinen E, Krogius L, Palva A 2004. Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR. Journal of Applied Microbiology 97(6): 1166-1177.
[341] Rosendale DI, Maddox IS, Miles MC, Rodier M, Skinner M, Sutherland J. 2008. High-throughput microbial bioassays to screen potential New Zealand functional food ingredients intended to manage the growth of probiotic and pathogenic gut bacteria. International Journal of Food Science and Technology, 43(12): 2257-2267.
[342] Rumah KR, Linden J, Fischetti VA, Vartanian T 2013. Isolation of Clostridium perfringens type B in an individual at first clinical presentation of multiple sclerosis provides clues for environmental triggers of the disease. PloS One 8(10): e76359.
[343] Rush EC, Patel M, Plank LD, Ferguson LR. 2002. Kiwifruit promotes laxation in the elderly. Asia Pacific Journal of Clinical Nutrition 11(2): 164-168.
[344] Sanchez B, Champomier-Verges MC, Collado Mdel C, Anglade P, Baraige F, Sanz Y, et al. Low-pH adaptation and the acid tolerance response of Bifidobacterium longum biotype longum. Appl. Environ. Microbiol. 2007; 73: 64509.
[345] Sartor RB. Efficacy of probiotics for the management of inflammatory bowel disease. Gastroenterology & Hepatology. 2011; 7: 606-8.
[346] Saulnier DM, Spinler JK, Gibson GR, Versalovic J. Mechanisms of probiosis and prebiosis: considerations for enhanced functional foods. Curr. Opinion Biotechnol. 2009; 20: 135-41.
[347] Savino F, Pelle E, Palumeri E, Oggero R, Miniero R 2007. Lactobacillus reuteri (American Type Culture Collection Strain 55730) versus simethicone in the treatment of infantile colic: a prospective randomized study. Pediatrics 119(1): e124-30.
[348] Scheperjans F, Aho V, Pereira PA, Koskinen K, Paulin L, Pekkonen E, Haapaniemi E, Kaakkola S, Eerola-Rautio J, Pohja M, Kinnunen E, Murros K, Auvinen P 2014. Gut microbiota are related to Parkinson's disease and clinical phenotype. Movement Disorders.
[349] Schloss PD, Schubert AM, Zackular JP, Iverson KD, Young VB, Petrosino JF. Stabilization of the murine gut microbiome following weaning. Gut Microbes. 2012; 3:383-93.
[350] Seeram N. Associate Professor of Bioactive Botanical Research Laboratory, University of Rhode Island. Digestive Health Forum, NutraIngredients- USA, 25 September 2014.
[351] Singleton, Vernon L, Orthofer, Rudolf, Lamuela-Raventós, Rosa M. 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent 299: 152.
[352] Skinner MA. 2012. Wellness foods based on the health benefits of fruit: gold kiwifruit for immune and reducing symptoms of colds and influenza. Journal of Food and Drug Analysis 20(1): 261-264.
[353] Sokol H, Pigneur B, Watterlot L, Lakhdari O, Bermudez-Humaran LG, Gratadoux JJ, et al. Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc. Natl. Academic Sci. USA 2008; 105: 16731-6.
[354] Sokol H, Seksik P, Furet JP, Firmesse O, Nion-Larmurier I, Beaugerie L, et al. Low counts of Faecalibacterium prausnitzii in colitis microbiota. Inflammatory Bowel Diseases. 2009; 15: 1183-9.
[355] Stonehouse W, Gammon CS, Beck KL, Conlon CA, Von Hurst PR, Kruger R. 2012. Kiwifruit: our daily prescription for health. Canadian Journal of Physiology and Pharmacology 91(6): 442-447.
[356] D Sun-Waterhouse, I Wen, R Wibisono, LD Melton, S Wadhwa. 2009. Evaluation of the extraction efficiency for polyphenol extracts from by-products of green kiwifruit juicing. International Journal of Food Science & Technology. 44(12): 2644-2652.
[357] Taras D, Simmering R, Collins MD, Lawson PA, Blaut M. Reclassification of Eubacterium formicigenerans Holdeman and Moore 1974 as Dorea formicigenerans gen. nov., comb. nov., and description of Dorea longicatena sp. nov., isolated from human faces. International Journal of Systematic and Evolutionary Microbiology. 2002; 52: 423-8.
[358] Timm D, Willis H, Thomas W, Sanders L, Boileau T, Slavin J. The use of a wireless motility device (SmartPill(R)) for the measurement of gastrointestinal transit time after a dietary fiber intervention. The British Journal of Nutrition. 2011; 105: 1337-42.
[359] Tuohy KM, Kolida S, Lustenberger AM, Gibson GR. The prebiotic effects of biscuits containing partially hydrolysed guar gum and fructo-oligosaccharides--a human volunteer study. The British Journal of Nutrition. 2001; 86: 341-8.
[360] Turnbaugh PJ, Baeckhed F, Fulton L, Gordon JI 2008. Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome. Cell Host & Microbe 3(4): 213-223.
[361] Udani J, Bloom D. 2013. Effects of kivia powder on Gut health in patients with occasional constipation: a randomized, double-blind, placebo-controlled study. Nutrition Journal 12(1): 78.
[362] Vaahtovuo J, Munukka E, Korkeamaki M, Luukkainen R, Toivanen P 2008. Fecal microbiota in early rheumatoid arthritis. Journal of Rheumatology 35(8): 1500-5.
[363] Vogt JA, Pencharz PB, Wolever TMS. L-Rhamnose increases serum propionate in humans. American Journal of Clinical Nutrition. 2004; 80: 89-94.
[364] Walker AW, Duncan SH, McWilliam Leitch EC, Child MW, Flint HJ. pH and peptide supply can radically alter bacterial populations and short-chain fatty acid ratios within microbial communities from the human colon. Appl. Environ. Microbiol. 2005; 71: 3692-700.
[365] Walker AW, Duncan SH, Harmsen HJ, Holtrop G, Welling GW, Flint HJ 2008. The species composition of the human intestinal microbiota differs between particle-associated and liquid phase communities. Environ Microbiol 10(12): 3275-83.
[366] Walker AW, Ince J, Duncan SH, Webster LM, Holtrop G, Ze X, et al. Dominant and diet-responsive groups of bacteria within the human colonic microbiota. ISME Journal. 2011; 5: 220-30.
[367] Wang L, Christophersen CT, Sorich MJ, Gerber JP, Angley MT, Conlon MA 2011. Low relative abundances of the mucolytic bacterium Akkermansia muciniphila and Bifidobacterium spp. in feces of children with autism. Applied and Environmental Microbiology 77(18): 6718-21.
[368] Wang T, Cai G, Qiu Y, Fei N, Zhang M, Pang X, Jia W, Cai S, Zhao L 2012. Structural segregation of gut microbiota between colorectal cancer patients and healthy volunteers. ISME Journal 6(2): 320-9.
[369] Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 2007; 73: 5261-7.
[370] Williams BL, Hornig M, Parekh T, Lipkin WI 2012. Application of novel PCR-based methods for detection, quantification, and phylogenetic characterization of Sutterella species in intestinal biopsy samples from children with autism and gastrointestinal disturbances. mBio 3(1).
[371] Willing BP, Dicksved J, Halfvarson J, Andersson AF, Lucio M, Zheng Z, et al. A pyrosequencing study in twins shows that gastrointestinal microbial profiles vary with inflammatory bowel disease phenotypes. Gastroenterology. 2010; 139: 1844-54 e1.
[372] Wong JMW, de Souza R, Kendall CWC, Emam A, Jenkins DJA. Colonic health: Fermentation and short chain fatty acids. Journal of Clinical Gastroenterology. 2006; 40: 235-43.
[373] Wu GD, Chen J, Hoffmann C, Bittinger K, Chen YY, Keilbaugh SA, et al. Linking long-term dietary patterns with gut microbial enterotypes. Science. 2011; 334: 105-8.
[374] Wu X, Ma C, Han L, Nawaz M, Gao F, Zhang X, Yu P, Zhao C, Li L, Zhou A, Wang J, Moore JE, Millar BC, Xu J 2010. Molecular characterisation of the faecal microbiota in patients with type II diabetes. Current Microbiology 61(1): 69-78.
[375] Ze X, Duncan SH, Louis P, Flint HJ. Ruminococcus bromii is a keystone species for the degradation of resistant starch in the human colon. ISME Journal. 2012; 6: 1535-43.
[376] Zoppi G, Cinquetti M, Luciano A, Benini A, Miner A, and Bertazzoni ME. 1998. The intestinal ecosystem in chronic functional constipation. Pediatric Minutes 87: 836841.

权利要求:
Claims (14)
[0001]
1. Use of a composition prepared from dried gold kiwi, characterized in that it is for the manufacture of a drug to increase the levels of Faecalibacterium prausnitzii in the digestive tract of an individual, where the dried gold kiwi is a kiwi Actinidia chinensis Zesy002 or a genetic derivative thereof.
[0002]
2. Use according to claim 1, characterized in that the drug is formulated for enteral, oral or rectal administration.
[0003]
3. Use according to claim 1 or 2, characterized in that the drug is formulated as one or more of a tablet, capsule, liquid, jelly or sachet.
[0004]
4. Use according to any one of claims 1 to 3, characterized in that the drug is formulated for administration at a dosage of: (i) 2000 to 4000 mg of powder per day or a liquid equivalent thereof; (ii) 250 to 2500 mg of powder per day or a liquid equivalent thereof; (iii) approximately 2400 mg of powder per day or a liquid equivalent thereof; or (iv) approximately 600 mg of powder per day or a liquid equivalent thereof.
[0005]
5. Use according to any one of claims 1 to 4, characterized in that the drug is formulated for co-administration with one or more of: (i) an additional digestive aid; (ii) one or more probiotic, prebiotic or symbiotic compositions; or (iii) fiber and/or a digestive enzyme.
[0006]
6. Use according to any one of claims 1 to 5, characterized in that the drug is additionally supplemented with polyphenols.
[0007]
7. Use according to any one of claims 1 to 6, characterized in that the individual has one or more symptoms of constipation, bowel irregularity, Crohn's disease, ulcerative colitis, irritable bowel syndrome, inflammatory bowel disease or cancer gastrointestinal.
[0008]
8. Use of a composition prepared from dried gold kiwi, characterized by the fact that it is for the manufacture of a drug to improve the decrease in the levels of Faecalibacterium prausnitzii in the digestive tract of an individual, in which the dried gold kiwi is a kiwi Actinidia chinensis Zesy002 or a genetic derivative thereof.
[0009]
9. Use according to claim 8, characterized in that the drug is formulated for enteral, oral or rectal administration.
[0010]
10. Use according to claim 8 or 9, characterized in that the drug is formulated as one or more of a tablet, capsule, liquid, jelly or sachet.
[0011]
11. Use according to any one of claims 8 to 10, characterized in that the drug is formulated for administration at a dosage of: (i) 2000 to 4000 mg of powder per day or a liquid equivalent thereof; (ii) 250 to 2500 mg of powder per day or a liquid equivalent thereof; (iii) approximately 2400 mg of powder per day or a liquid equivalent thereof; or (iv) approximately 600 mg of powder per day or a liquid equivalent thereof.
[0012]
12. Use according to any one of claims 8 to 11, characterized in that the drug is formulated for co-administration with one or more of: (i) an additional digestive aid; (ii) probiotic, prebiotic or symbiotic compositions; or (iii) fiber and/or a digestive enzyme.
[0013]
13. Use according to any one of claims 8 to 12, characterized in that the drug is additionally supplemented with polyphenols.
[0014]
14. Use according to claim 3 or 10, characterized in that the drug is formulated as a gel capsule.

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同族专利:

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公开号 | 公开日

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CA2967263A1|2016-06-02|

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EP3223838A4|2018-08-01|

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EP3223838A1|2017-10-04|

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WO2016085356A1|2016-06-02|

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JP2017537156A|2017-12-14|

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KR20170088970A|2017-08-02|

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BR112017011078A2|2017-12-26|

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US20170326190A1|2017-11-16|

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CN106999523A|2017-08-01|

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US20200197460A1|2020-06-25|

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US11090348B2|2021-08-17|

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US10512663B2|2019-12-24|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

JP2750767B2|1990-02-21|1998-05-13|雪印乳業株式会社|New sugar alcohol|

---

CN1107308A|1994-02-25|1995-08-30|中国癌症研究基金会|Natural Actinidia chinensis nutrient oral liquid|

---

JPH10313822A|1997-05-20|1998-12-02|Pola Chem Ind Inc|Intestinal bacterial flora improver and composition containing the same|

---

AT503521A1|2006-05-05|2007-11-15|Omnica Gmbh|USE OF AN EXTRACT OF KIWI FRUIT|

---

BR112017011078B1|2014-11-28|2021-05-11|Anagenix Ip Limited|uses of a composition prepared from dried gold kiwi fruit|BR112017011078B1|2014-11-28|2021-05-11|Anagenix Ip Limited|uses of a composition prepared from dried gold kiwi fruit|

---

KR101833348B1|2016-12-26|2018-03-02|주식회사 엠디헬스케어|Method for diagnosis of breast cancer using analysis of bacteria metagenome|

---

KR101833503B1|2016-12-26|2018-03-05|주식회사 엠디헬스케어|Method for diagnosis of lung cancer in chronic obstructive pulmonary disease patients using analysis of bacteria metagenome|

---

KR101940426B1|2016-12-28|2019-01-18|주식회사 엠디헬스케어|Method for diagnosis of colon tumor using analysis of bacteria metagenome|

---

KR101942197B1|2016-12-28|2019-01-24|주식회사 엠디헬스케어|Method for diagnosis of prostate disease using analysis of bacteria metagenome|

---

US10881675B2|2017-10-11|2021-01-05|Jennifer L. Gu|Gut health compositions|

---

KR102130485B1|2017-10-13|2020-07-06|주식회사 엠디헬스케어|Method for diagnosis of Alzheimer's dementia using analysis of bacteria metagenome|

---

KR102007786B1|2017-10-18|2019-08-07|주식회사 엠디헬스케어|Method for diagnosis of head and neck cancer using analysis of bacteria metagenome|

---

CA3088630A1|2017-12-15|2019-06-20|Solarea Bio, Inc.|Microbial compositions and methods for treating type 2 diabetes, obesity, and metabolic syndrome|

---

KR101940950B1|2018-01-23|2019-01-21|주식회사 엠디헬스케어|Method for diagnosis of bile duct cancer using analysis of bacteria metagenome|

---

KR101944662B1|2018-02-14|2019-02-01|주식회사 엠디헬스케어|Method for diagnosis of stroke using analysis of bacteria metagenome|

---

JP2022517237A|2019-01-18|2022-03-07|シー・ピー・ケルコ・ユー・エス・インコーポレイテツド|Prebiotic compositions and their use|

---

CN110279814A|2019-06-10|2019-09-27|湖北虹玮农业有限公司|A kind of functional food for person in middle and old age's chronic constipation|

法律状态:
2019-10-08| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-10-06| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-11-03| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-02-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-05-11| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 27/11/2015, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

---

申请号 | 申请日 | 专利标题

---

NZ70245414|2014-11-28|

---

NZ702454|2014-11-28|

---

NZ706405|2015-03-27|

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NZ70640515|2015-03-27|

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PCT/NZ2015/050200|WO2016085356A1|2014-11-28|2015-11-27|Gold kiwifruit compositions and methods of preparation and use therefor|

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