• 专利附录
  • 专利说明
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    • 专利摘要:
    The invention is in the field of prebiotics and relates in particular to a method for preparing a stabilized and inactivated cell culture of Saccharomyces Cerevisiae. Stabilized and inactivated cell cultures of Saccharomyces Cerevisiae, which can be achieved with this method, and their applications are also described.
    公开号:CH715559A2
    申请号:CH01443/19
    申请日:2019-11-13
    公开日:2020-05-15
    发明作者:Veneroni Flavio
    申请人:Grisotech Sa；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION
    The invention is located in the field of prebiotics and relates in particular to a method for preparing a stabilized and inactivated cell culture of Saccharomyces Cerevisiae. Furthermore, stabilized and inactivated cell cultures of Saccharomyces Cerevisiae, which can be achieved with this method, and their applications are described.
    STATE OF THE ART
    The human microbiome is the set of symbiotic microorganisms that coexist with the human organism without harming it, a good example of mutualism between different kinds of organisms, each of which gives an advantage.
    The human microbiome develops in the course of the first days of life and survives, except in the case of disease, surprisingly long. Every individual has their own microbiome.
    In humans there are between 500 and 10,000,000 different types of microorganisms, the most numerous of which are bacteria and, to a lesser extent, fungi and viruses.
    The majority of the bacteria are anaerobic, more or less obligate or facultative (many survive in the absence of oxygen and some tolerate its presence).
    The most numerous intestinal bacteria in humans are lactobacilli, bifidobacteria and Bacillus, including Bacillus subtilis.
    [0007] An important function of the human microbiome is the decomposition of substances that our system cannot break down, such as cartilage and cellulose molecules. Another important function is the synthesis of essential substances, for example vitamin K, which plays an essential role in blood clotting.
    The microbiome performs functions that we are unable to perform in any other way. These functions include the ability to assimilate otherwise indigestible components of our diet, such as vegetable polysaccharides.
    [0009] The growth and activity of the intestinal microbiome are promoted by prebiotics.
    As prebiotic, not to be confused with probiotic, any substance present in food is defined that is not absorbed by the organism, but used by the intestinal microbiome.
    Examples of prebiotics are water-soluble fibers that do not gel, including non-starchy polysaccharides or beta-glucans, fructans, fructooligosaccharides, inulin, lactitol, lactosucrose, lactulose, pyrodextrins and soy oligosaccharides.
    As a result of some diseases, it is necessary to restore the altered intestinal flora (dysbiosis) by promoting the growth of the microbiome.
    Nutritional yeast is characterized by its high supply of proteins, vitamins, antioxidants and mineral salts.
    Nutritional yeast is very often a strain of Saccharomyces cerevisiae which, in contrast to brewer's yeast or baker's yeast (Baker's Yeast), is inactivated.
    The particular difficulties encountered with prebiotics such as nutritional yeast as a result of the inactivation procedures commonly used relate to the ability to retain all of their natural characteristics until they reach the microbiome. The method relating to the subject matter of the present invention, however, preserves in the subsequent product the characteristic resistance to general chemical-physical stress, including, for example, that which is generated by gastric juices or by the action of antibiotic substances.
    While nutritional yeast, as its name suggests, is used to supply people and / or animals with nutritional factors (food supplements), the subject of the present patent product is the symbiotic microbial cells (microbiome), so that they supply a whole range of factors for eubiosis and thus for the welfare of the host (prebiotic use).
    SUMMARY OF THE INVENTION
    The invention therefore relates to a method for preparing a stabilized and inactivated cell culture of Saccharomyces Cerevisiae, the method comprising the following phases: a. Addition to a fresh cell culture of Saccharomyces Cerevisiae: sodium chloride in an amount ranging from 0.2% to 3% w / w; - Process activators from the group consisting of mono- and disaccharides, magnesium sulfate, lithium, calcium or magnesium chloride, calcium or magnesium nitrate, salts of organic calcium or sodium acids of acetic, lactic, gluconic or pantothenic acid; - Stabilizers of the suspension selected from the group consisting of micrograin cellulose, carboxymethyl cellulose, gum arabic or other natural substances suitable for forming colloids, hydrogenated silica or silicates; b. Shake the from phase a. obtained suspension for a period of 0.5-5 hours at a temperature between 45 ° C and 70 ° C; c. Adding a stabilizer to the shaking phase b. from the group consisting of: - benzoic, sorbic, propionic, formic, acetic, tartaric acid, ascorbic or lactic acid or lactic ferments, - phenols, hydroquinones or derivatives, benzyl alcohol or derivatives, - alkyl esters of para-hydroxybenzoic acid and - Ammonium formate; d. Cooling the in phase c. obtained suspension to 20 ° C-25 ° C to obtain a suspension containing a stabilized and inactivated cell culture of Saccharomyces Cerevisiae.
    According to a second aspect, the invention relates to a stabilized and inactivated cell culture of Saccharomyces Cerevisiae, which can be achieved by the method according to the invention.
    According to a further aspect, the invention relates to the use of a stabilized and inactivated cell culture of Saccharomyces cerevisiae, which can be achieved by the method according to the invention, as a prebiotic.
    DESCRIPTION OF THE FIGURES
    The invention will now be described in detail with reference to the accompanying figures, in which: FIG. 1 shows a graph which shows the characteristic growth curve of Lactobacillus acidophilus, which is used as a reference strain of the product according to the invention and is described in Example 3. FIG. 2 shows the graph with the growth curves of the reference Lactobacillus acidophilus in the presence or absence of different types of yeast under the test conditions described in Example 3.
    DETAILED DESCRIPTION OF THE INVENTION
    The present invention relates to a method for preparing an inactivated cell culture of Saccharomyces Cerevisiae which contains the cell bodies and all products that are formed during the cell reproduction process.
    If in the present invention the definition:"Inactivated and stabilized yeast" or "inactivated and stabilized cell culture of Saccharomyces Cerevisiae" is used, this means a yeast strain, preferably a strain of Saccharomyces Cerevisiae, whose cells are inactivated and stabilized and can therefore no longer reproduce. These cells, which contain the cell bodies and all the products that are formed during the cell reproduction process, are stabilized in order to maintain the integrity and therefore the original cell material unchanged for a long period of time, but they are dead cells.
    [0023] The invention therefore relates to a method for preparing a stabilized and inactivated cell culture of Saccharomyces Cerevisiae, the method comprising the following phases: a. Addition to a fresh cell culture of Saccharomyces Cerevisiae: sodium chloride in an amount ranging from 0.2% to 3% w / w; - Process activators from the group consisting of mono- and disaccharides, magnesium sulfate, lithium, calcium or magnesium chloride, calcium or magnesium nitrate, salts of organic calcium or sodium acids of acetic, lactic, gluconic or pantothenic acid; - Stabilizers of the suspension selected from the group consisting of micrograin cellulose, carboxymethyl cellulose, gum arabic or other natural substances suitable for forming colloids, hydrogenated silica or silicates; b. Shake the from phase a. obtained suspension for a period of 0.5-5 hours at a temperature between 45 ° C and 70 ° C; c. Adding a stabilizer to the shaking phase b. from the group consisting of: - benzoic, sorbic, propionic, formic, acetic, tartaric acid, ascorbic or lactic acid or lactic ferments, - phenols, hydroquinones or derivatives, benzyl alcohol or derivatives, - alkyl esters of para-hydroxybenzoic acid, - Ammonium formate; d. Cooling of the phase c. obtained suspension to 20 ° C-25 ° C.
    In a preferred form, the cooling takes place by induction.
    The product obtained is a thick and homogeneous liquid (or liquid cream) containing a very high concentration of Saccharomyces Cerevisiae cells, which are stabilized by a treatment with special active ingredients that are generated by heat during an operative phase that the The propagation phase follows, and are inactivated by a final cooling phase brought about by induction to a temperature of 20 ° C to 25 ° C.
    The effect of inactivating and stabilizing the cells is to inhibit their biological activity and at the same time to keep all the typical properties of the yeast unchanged. In particular, the resistance to acids (for example gastric juices) and antibiotics.
    These features of the product make it particularly effective as a human, plant and animal food supplement as a regulator and booster of the microbiome. The product according to the invention surprisingly makes it possible to achieve improved growth of the microbial symbionts, such as, for example, lactobacilli, bifidobacteria, Bacillus subtilis and enterobacteria.
    Its action is both prophylactic and curative under conditions in which the organism of the microbiome is involved.
    Surprisingly, the product according to the invention does not experience the same difficulties as occur with prebiotics such as nutritional yeast, since the method described here allows all of its natural characteristics to be retained until the microbiome is reached and the product has the characteristic resistance to general chemical-physical stress to be preserved, such as that caused by gastric juices or by the action of antibiotic substances, as described above.
    In a preferred form, the cell reproduction process according to the invention is carried out by adding the following ingredients to the fresh yeast (also in the form of a compressed mass), which consists of selected strains of Saccharomyces Cerevisiae:Sodium chloride in an amount of 0.2-3% by weight of the total:Process activators, that is: mono- and disaccharides (dextrose, lactose, etc.) in the amount of 0.5-5% by weight of the total; Inositol or magnesium sulfate in the amount of 0.04-0.25%; Lithium, calcium or magnesium chloride in the amount of 0.2-1.5%; Calcium or magnesium nitrate, calcium salts of organic acids such as lactate, acetate, gluconate or pantothenate, sodium acetate, sodium pyruvate or glutathione in an amount of 0.01-2%Suspension stabilizers such as micrograin cellulose, carboxymethyl cellulose, gum arabic or other natural substances capable of forming colloids, hydrogenated silica or silicates:
    The suspension is kept at a temperature between 45 ° and 70 ° C for a period of 0.5-5 hours with shaking.
    The cell culture mentioned above is stabilized according to the present invention by adding one or more of the stabilizing additives mentioned below and again keeping the suspension at a temperature of 45 ° -70 ° C. for a period of between 5 and 10 minutes with shaking becomes.
    The stabilizers include:Benzoic, sorbic, propionic, formic, acetic, tartaric, ascorbic or lactic acid or lactic ferments,Ammonium formate,Phenols, hydroquinones and derivatives and benzyl alcohol and derivatives andAlkyl esters of p-hydroxybenzoic acid.
    The amount of stabilizer to be added varies over a wide range depending on the substance or mixture of substances used.
    For the process, the suspension is cooled to a temperature of 20-25 ° C., for example by contact with the cold surface of the reactor.
    The product, which is obtained in the form of a thick, homogeneous brown liquid, can in any case be used as such or absorbed in hydrogenated silica.
    Alternatively, it can be dried in a fluidized bed in the presence of amorphous silica or spray dried.
    In a preferred embodiment, the salts of organic calcium or sodium acid are selected from the group consisting of calcium lactate, calcium acetate, calcium gluconate, calcium pantothenate, sodium acetate and sodium pyruvate in the process according to the invention.
    In a further preferred embodiment, the inventive method has an additional phase after phase d. In which the phase d. suspension obtained is absorbed in hydrogenated silica.
    In yet another preferred embodiment, the inventive method has an additional phase after phase d. In which the phase d. suspension obtained is dried by a fluidized bed in the presence of amorphous silica.
    According to a further aspect, the present invention relates to a stabilized and inactivated cell culture of Saccharomyces Cerevisiae, which can be achieved by the method according to the invention.
    It has surprisingly been found that the product according to the invention has a booster effect on the flora of the microbiome, which mainly consists of lactobacilli, bifidobacteria and Bacillus subtilis. The improved activity of the microbiome enables an improvement in the metabolic processes of the organism, a better resistance to stress and diseases and a lower need for drugs.
    Exemplary embodiments of the present invention are given below.
    According to a further aspect, the invention relates to the use of a stabilized and inactivated cell culture of Saccharomyces cerevisiae, which can be achieved by the method according to the invention, as a prebiotic.
    EXAMPLES
    example 1
    2000 kg of fresh yeast as starting material are placed in a steel heating and cooling reactor with a capacity of 5000 liters, and the suspension is heated to 60 ° C with shaking; then the following additives are added: 40 kg of pure sodium chloride, 30 kg of dextrose and k kg of magnesium sulfate.
    The mixture is kept shaking for 60 minutes in order to obtain a homogeneous suspension, after which 2 kg of methyl or ethyl ester of p-hydroxybenzoic acid are added.
    Shaking is continued for a further 10 minutes, followed by a cooling phase to 22 ° C to give a final product in the form of a thick, homogeneous liquid that can be used as such or absorbed in hydrogenated silica in an amount of 25 kg is added. Cooling down to 22 ° C.
    Example 2
    1000 kg of fresh yeast as starting material are placed in an enamelled reactor with a capacity of 5000 liters, and the suspension is heated to 45 ° C with shaking; then the following additives are added: 25 kg of sodium chloride, 20 kg of calcium gluconate, 2 kg of magnesium sulfate, 10 kg of lactose (official pharmacopoeia), 15 kg of micrograin cellulose and 2 kg of carboxymethyl cellulose.
    The mixture is kept shaking for 4 hours in order to obtain a homogeneous suspension.
    30 kg of sorbic acid and 20 kg of ascorbic acid (official pharmacopoeia) are added to the mixture.
    Shaking is continued for a further 8 minutes, after which the mixture is cooled to 20 ° C to obtain an inactivated final product which can be used as such or dried in the presence of silica.
    Example 3
    Evaluation of the bioactivity of inactivated yeast cells with the routine dosing method used as quality control of the product.
    The analytical method, biological in nature, is aimed at determining the bioactivity index of the dead / inactivated yeast, which is considered to be the ability to stimulate the growth of a strain of Lactobacillus acidophilus. The experimental principle for determining the bioactivity is based on the measurement of the growth of Lactobacillus acidophilus by turbidimetry at different times from the inoculation, with or without the presence of the yeast according to the invention as a prebiotic and in comparison to other commercially available yeasts.
    The difference between the growth with or without our product (yeast according to the invention), as well as the other yeast (s), allows the bioactivity index to be extrapolated.
    Preparation of the reagents
    A) base medium
    The following substances were dissolved in 500 ml of water, then the volume was brought to 1,000 ml: 1. Glucose 6.25 g 2. Sodium acetate 6.25 g 3. Ascorbic acid 6.25 g 4. Ammonium sulfate 3.15 g 5. Potassium phosphate 3.15 g 6. Urea 1.25 g 7. Tween 80 1.00 ml
    B) vitamin solution
    The following vitamins were dissolved in water and then poured into the base medium: 1. Panthenol 30 mg / 100 ml 2.15 ml 2. Riboflavin phosphate 42 mg / 100 ml 2.15 ml 3. Nicotinic acid 30 mg / 50 ml 2 , 15 ml 4. Pyroxidine 30 mg / 50 ml 2.15 ml 5. Thiamine 30 mg / 50 ml 2.15 ml 6. Folic acid 2 mg / 200 ml 0.50 ml 7. p-aminobenzoic acid 2 mg / 20 ml 0, 50 ml 8. Biotin 2 mg / 20 ml 0.50 ml
    C) Solution with mineral traces
    The following salts were dissolved in 250 ml of water. 1. Magnesium sulfate 4 g 2. Manganese sulfate 140 mg 3. Iron sulfate 200 mg 4. Calcium chloride 20 mg
    3.15 ml of this solution are removed and added to the base medium. After adding the vitamin solution and the solution with mineral traces to the base medium, it is made up to 1,000 ml with water. The base medium has a pH between 4.8 and 5.2, which is corrected to 7.05 after use by adding a 10% sodium hydroxide solution.
    PREPARATION AND STORAGE OF THE LACTOBACILLUS STEM.
    The L.acidophilus strain is stored in the refrigerator at 4 ° C in a dark test tube containing MRS agar.
    Before it is used in the experiment, the Lactobacillus strain stored in the refrigerator must be reactivated as follows:
    A strip with Lactobacillus culture is removed and dispersed in 20 ml previously sterilized MRS broth, then shake the Lactobacillus suspension and incubate at 37 ° C for 17-18 hours before use. It is important not to exceed 24 hours of incubation in order to obtain the highest level of the Lactobacillus population.
    CONTROL OF THE ACTIVITY OF THE L. ACIDOPHILUS STRAIN
    The activity of the L. Acidophilus strain prepared as above is assessed by turbidimetric determination carried out hourly over 12 hours and then after 24 hours.
    From the graph in FIG. 1 we can see that the maximum of Lactobacillus growth is reached between the 10th and 24th hour of incubation.
    PREPARATION OF SAMPLES TO BE TESTED FOR BIOACTIVITY
    Around 1.00 g of each sample (yeast according to the invention: patent, dry yeast and live yeast) are resuspended in 100 ml of distilled water. After thorough homogenization, the samples are sterilized in the autoclave for 30 minutes at 0.5 atm and then left to rest for 20 h at 4 ° C. before they are used.
    The supernatant solution of the sample is used in the experiment.
    TEST METHOD
    For each sample, three tests are carried out according to the following method: 1. 1 ml of lactobacillus suspension is diluted in 9 ml of sterile physiological solution and the optical density is read at 600 nm, where usually a value of 0.850 +/- 0.050 is found; if the determined value is different, it can be adjusted using the Lactobacillus suspension or the physiological solution depending on the correction. 2. The culture medium is divided into 9 ml portions in 12 test tubes (3 test tubes per test sample) for the test.Each sample is prepared in triplicate, 1 ml of each sample is added to the test tubes: Sample A (stabilized and inactivated cell culture of Saccharomyces Cerevisiae according to the present invention: Grisotech), Sample B (dry yeast - Diamond V XP culture), Sample C (live yeast) ; while adding 1 ml of physiological solution to form Sample D (negative control).0.5 ml of the Lactobacillus solution is added to the first two tubes of each group; 0.5 ml of the physiological solution (blank for each group) is added to the third tube of each group. 3. After the wavelength of the spectrophotometer has been set to 600 nm, the optical density of each sample is read in comparison with the blank sample of each group. 4. Each test tube is then incubated at 37 ° for a period of 24 hours, the optical density being checked every hour for 12 consecutive hours and then after 24 hours. 5. After this period, all optical density readings are collected and the bioactivity index and microbial growth curve are extrapolated.
    RESULTS
    Table 1 shows the average readings of the duplicate samples at 600 nm of each of the three Lactobacillus growth curves, both with the culture medium and in the presence of the yeast according to the invention (Grisotech) or the dry yeast and live yeast.
    Summary of the readings on the spectrophotometer at 600 nm for the three experiments on lactobacillus growth, incubated in a culture medium without enrichment (sample D) and with enrichment of: yeast according to the invention (sample A), dry yeast (sample B) and live yeast ( Sample C).
    The bioactivity index is expressed by the following equation:CB yeast according to the invention = (SLT11- SLT0) / (SL11- SL0) = (0.4891 - 0.0660) / (0.1380 - 0.0634)= 6.6CB dead dry yeast = (SLT11- SLT0) / (SL11- SL0) = (0.1602-0.0649) / (0.1380-0.0634) = 1.2CBLive yeast = (SLT11- SLT0) / (SL11- SL0) = (0.1583 - 0.0645) / (0.1380 - 0.0634) = 1.2in whichCB = bioactivity coefficient or indexSL0 = absorbance of the lactobacillus suspension (control) a t = oSL11 = absorbance of the lactobacillus suspension (control) a t = 11 hSLT0 = absorbance of the lactobacillus suspension + test sample (treated) a t = oSLT11 = absorbance of the lactobacillus suspension + test sample (treated) a t = 11 h
    Using the results shown in FIG. 2, we can observe that in the medium which has been enriched with the yeast according to the invention with the name Grisotech, there is a significant increase in Lactobacillus growth compared to the base medium with a CB equal to 6.6 is. The media enriched with dry yeast (culture Diamond V XP =, on the other hand, showed extremely low growth, practically similar to or equal to the base medium (CB = 1.2).
    The advantages achieved by the method according to the invention clearly emerge from the detailed description and the examples given above. In particular, this method has surprisingly and advantageously proven to be suitable for the generation of stabilized and inactivated cell cultures of Saccharomyces Cerevisiae.
    权利要求:
    Claims (6)
    [1]
    1. A method for preparing a stabilized and inactivated Saccharomyces Cerevisiae cell culture, the method comprising the following phases:a. Add to a fresh Saccharomyces Cerevisiae cell culture from:Sodium chloride in an amount ranging from 0.2% to 3% w / w;Process activators from the group consisting of mono- and disaccharides,Magnesium sulfate,Lithium, calcium or magnesium chlorideCalcium or magnesium nitrate,Salts of organic calcium or sodium acids of acetic, lactic, gluconic or pantothenic acid;- Stabilizers of the suspension from the group consisting of micro-granular cellulose, carboxymethyl cellulose, gum arabic or other natural substances which are suitable for forming colloids, hydrogenated silica or silicates;b. Shake out of phase a. suspension obtained for a period of 0.5-5 hours at a temperature between 45 ° C and 70 ° C;c. Adding a stabilizer to the shaking phase b. from the group consisting of:- Benzoic, sorbic, propionic, formic, acetic, tartaric, ascorbic or lactic acid or milk ferments- phenols, hydroquinones or derivatives, benzyl alcohol or derivatives- Alkyl esters of para-hydroxybenzoic acid- ammonium formate;d. Cooling of the phase c. suspension obtained at 20 ° C-25 ° C.
    [2]
    2. The method of claim 1, wherein the salts of organic calcium or sodium acid are selected from the group consisting of calcium lactate, calcium acetate, calcium gluconate, calcium pantothenate, sodium acetate and sodium pyruvate.
    [3]
    3. The method according to any one of claims 1 or 2, which the additional phase of absorption by phase d. suspension obtained in hydrogenated silica.
    [4]
    4. The method according to any one of claims 1 or 2, which the additional phase of drying of the phase d. suspension obtained through a fluidized bed in the presence of amorphous silica.
    [5]
    5. A stabilized and inactivated Saccharomyces Cerevisiae cell culture which can be obtained by the method according to any one of claims 1 to 4.
    [6]
    6. Use of the stabilized and inactivated Saccharomyces Cerevisiae cell culture obtainable by the method according to any one of claims 1 to 4 as a prebiotic.
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    同族专利:
    公开号 | 公开日
    IT201800010330A1|2020-05-14|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    IT1155422B|1982-12-10|1987-01-28|Dox Al Italia Spa|PROCEDURE FOR THE PREPARATION OF AN INTEGRAL CULTURE OF SACCHAROMYCES CEREVISIAE CELLS|
    IT1312090B1|1999-04-22|2002-04-04|Ecoser S R L|INACTIVATED MICROORGANISMS INCLUDING SOLUBLE AND / SOLUBILIZABLE SUBSTANCES FOR PHARMACOLOGICAL ACTIVITY AND / OR NUTRITIONAL SUBSTANCES|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    IT102018000010330A|IT201800010330A1|2018-11-14|2018-11-14|SACCHAROMYCES CEREVISIAE AS A MICROBIOTA BOOSTER|
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