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    • 专利摘要:
    New strains of lactobacillus reuteri species for use in pan-mothers masses. The present invention relates to the use of strains cect 9148 and/or cect 9224 of lactobacillus reuteri for the production of vitamin b12, of media enriched in vitamin b12, for the manufacture of functional foods, nutritional and/or pharmaceutical supplements, as well as a method for the production of vitamin b12 in acid or sourdough sourdough by said strains. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2604355A1
    申请号:ES201631584
    申请日:2016-12-13
    公开日:2017-03-06
    发明作者:Marina DIANA PEREZ；Laura GONZALEZ LERIDA；Joan QUILEZ GRAU
    申请人:EUROPASTRY S A；EUROPASTRY SA；
    IPC主号:
    专利说明:

    New strains of the LactobaciIJus reuteri species for the production of panary mother masses. The present invention relates to the CECT 9148 and CECT 9224 strains of Lactobacillus reuteri and their use for the production of Vitamin B'2 or cobalamin, of
    10 means enriched in Vitamin B'2 or cobalamin, for the manufacture of functional foods, nutritional and / or pharmaceutical supplements, as well as a method for the production of Vitamin B'2 or cobalamin in sourdough sourdoughs of bread or sourdough by means of said strains .
    15 Vitamin 812 or cobalamin is a water-soluble vitamin essential for the functioning of the organism in both humans and animals. It is necessary for the metabolic function of two enzymes: methionine synthase and methylmalonylCoAmutase, involved in the metabolism of amino acids, fatty acids and carbohydrates, and in the synthesis and regulation of DNA.
    20 Except for bacteria and archaea, no other organism has the enzymes necessary for the synthesis of this vitamin. For this reason, it is necessary to include in the diet foods that serve as a source of cobalamin to meet the Recommended Daily Allowance (RDA) of 2.5¡Jg / day. Most foods of animal origin
    25 contain Vitamin 8 12, such as meat, fish, eggs, milk and their derivatives; while foods of plant origin do not, or contain very small amounts. Vitamin 8 12 deficiency, due to low intake, can cause various clinical manifestations. This type of deficiency is, therefore, more common in vegan, vegetarian and older people. However, anemia
    30 pernicious, which is the most severe deficiency, does not have its origin in dietary problems, but is the result of a problem in the absorption of vitamins in the small intestine.
    Structurally it is one of the most complex vitamins, formed by a corrin ring with a central cobalt atom attached to 4 pyrrolic rings, a lower a-ligand and a higher p-ligand. It includes three naturally occurring forms (adenosylcobalamin, methylcobalamin and hydroxycobalamin) and a chemically transformed form (cyanocobalamin), which differ in the superior ligand.
    5 In aqueous solution, Vitamin B12 is sensitive to light, oxidizing and reducing agents and at elevated temperatures. Its stability can also be affected by the presence of other vitamins. The most stable and inexpensive form is obtained by the reaction of natural vitramics produced by bacteria (mainly hydroxycobalamin) with cyanide, forming cyanocobalamin, which is what is used
    10 In fortified foods and in pharmaceutical preparations. The industrial production of Vitamin B12 is carried out through biosynthetic fermentation processes, using highly producing strains of said vitamin, such as Propionibacterium freudenreichii and Pseudomonas denilrificans. Recently it has been discovered that Laclobacillus reuleri is also capable of producing the active form of Vitamin B12
    15 in fermentation processes. This microorganism is part of the lactic acid bacteria (BAL), a heterogeneous group that is naturally present in a wide range of ecological niches, such as food and the gastrointestinal tract of animals, including man. LABs, especially the genus LactobaciJ / us, are widely used as probiotics, helping to restore the
    20 intestinal microbiota after a gastric process and preventing infectious diseases and food allergies, among other benefits. In addition to acting as probiotics, some strains of lactic acid bacteria can produce, release or even increase certain beneficial compounds in food. These compounds, called nutritional, can be macronutrients, micronutrients
    25 (such as vitamins) or non-nutritive molecules, and with them they develop what we know as functional foods, which are foods that are consumed as part of a normal diet and that contain biologically active components beneficial to health.
    30 Lactic acid bacteria play an important role in the fermentation of sourdoughs that are used to make bread. This fact, together with the ability of certain strains to produce Vitamin B'2, offers the possibility of obtaining a naturally enriched bread with this vitamin.
    Background of the invention
    The taste for traditional bread made with sourdough is currently being recovered. The fermentation of this sourdough improves the texture and taste of the bread, and also improves its nutritional characteristics. If, in addition, the fermentation of this mass is carried out by a microorganism capable of producing cobalamin in a manner
    Naturally, a functional food that brings health benefits is achieved, without this implying a higher cost for the final consumer.
    Studies such as that of Edelmann et al. (2016) in which the stability of Vitamin 812 was analyzed, both added and produced in situ by 10 Propionibacterium freudenreichii, in the production of bread. The quantification of the vitamin was carried out both by ultra high precision chromatography (UHPLC) and by microbiological assay (MBA). The cyanocobalamin concentration obtained with this latter method was approximately 0.5 g / g dry weight. Propionibacterium freudenreichii, despite having GRAS status, is missing
    15 in the mother masses in a natural way, so it is likely that it does not contribute the same organoleptic characteristics to bread as an 8AL.
    Other studies are known to fortify a bread naturally with a cobalamin-producing microorganism, but none that is done using 20 Lactobacillus reuteri. Other authors have used this microorganism to increase the concentration of Vitamin 8'2 in different food products. Malina et al. (2012) used the lactobacillus reuteri CRL 1098 lactic acid bacterium to biofortify a soy milk and evaluate its efficiency in the prevention of symptoms produced by Vitamin 812 nutritional deficiency in pregnant female murine 25 females. What's up. (2015) used another strain of Lactobacillus reuteri to increase the content of Vitamin 8 12 in a soy yogurt, achieving a concentration of 18 IJg / 100mL. Both studies use biofortification on a product derived from soybeans. This is due to the popularity achieved among vegetarians and the elderly for its taste, its easy digestibility and its association with health. However, the bread is
    30 an accessible food in practically the entire planet, very economical and that does not require energy expenditure for its conservation (e.g. refrigeration), making it the ideal candidate to combat Vitamin 8'2 deficiency.
    Patents of Lactobacillus reuteri strains and their 35 different applications are also known, such as W02015025072, CN105219683, US2015250836, G82535177 And others, but none of them refers to the use of strains
    of LaclobacilJus reuteri to elaborate mother masses enriched in Vitamin 8 12.
    5 Explanation of the invention
    The present invention relates to new strains of the species LactobaciIJus reuteri registered in the Spanish Type Culture Collection (CECT) with the numbers CECT 9148 and CECT 9224 isolated for use in mass production
    10 panary mothers and in the manufacture of bread as a functional food through the inclusion of sourdough or sourdough produced through these strains as well as for the manufacture of any food product considered functional food or nutritional and / or pharmaceutical supplement through the source Vitamin 812 concentrate for ingestion or oral administration.
    The invention also relates to the process for the use of these isolated strains in the elaboration of mother masses enriched in Vitamin 812 which has the
    inoculum preparation, propagation in a culture medium to obtain cell biomass, resuspension and / or inoculation of the biomass obtained in substrates
    20 or solutions and fermentation thereof.
    Based on knowledge of the biochemical effects of Vitamin B12 and of
    the multiple studies that have corroborated it, in the data of this description thus it is demonstrated, the high capacity of cobalamin production by the strains
    25 CECT 49148 and CECT 9224 of Lactobacillus reuteri, which have been isolated from fermented food products.
    Lactic acid bacteria (SAL) are essential for the production of fermented foods. Its presence in the raw material is well known, even without being added (102 ~ 103 grams), quickly dominating fermentation. Its ability to lower the pH of the food matrix producing acids through the fermentation of carbohydrates allows to obtain a final product with remarkable organoleptic characteristics. Modern manufacturing practices involve the use of commercial starters allowing large-scale production without risk. However, its use leads to obtaining products with a microbiological profile
    Very standardized and less variable compared to those that have been naturally fermented.
    The advantages of this invention include the possibility of using5 natural fermentation substrates at low cost allowing the use of thesemicroorganisms and their biosynthesis of Vitamin B12 • a type of fermentation that
    It allows a high physiological value enriched naturally in Vitamin B12 with the advantage of being able to be preserved for long periods of time and the possibility of creating a potentially functional food suitable for human consumption.
    10 Materials and methods
    Media and reagents
    15 All reagents used were analytical grade. Sodium acetate, acetic acid, sodium cyanide and Aspergillus oryzae a-amylase (or taka-diastase) were obtained from Sigma-Aldrich (Steinheim, Germany). MRS agar was purchased at
    Biokar Diagnostics (Beauvais, France) and the test medium of Vitamin B'2 and theTween 80 in Sigma-Aldrich (Steinheim, Germany). The API suspension medium (from 2
    20 and 5 mL), the API 50CHL medium, the API 50CH strips and the McFarland standard were provided by Biomérieux (Marcy-I'Étoile, France). The D - (+) - monohydrated maltose was obtained from Sigma-Aldrich (Steinheim, Germany). The KHCOJ of PanReacApplichem (Castellar del Vallés, Barcelona). The VitaFast kit used for the determination and quantification of Vitamin B12 (Cyanocobalamin) and the standard of
    25 recovery of Vitamin 8'2 were obtained from R-Biopharm (Darmstadt, Germany).
    Microorganisms
    As experimental control, two strains of Laclobacillus reuleri were used. One of
    30 they were Laclobacillus reuler; JCM 1112, obtained from the Japan Col / ectian of Microorganisms (JCM) and the other was obtained from a pharmaceutical probiotic preparation called Reuteri Drops (BioGaia, Casen Fleet SLU Laboratories, Zaragoza) containing Lactobacillus reuteri Protectis® DSM 17938. These strains served guide to compare the results with those of our isolated strains.
    Isolation of strains from fermented products
    For this study, 47 food products were analyzed (44 of them fermented). Listed in Table 1. 10 grams of sample were weighed in a bag with 5 filters (AES Chemunex, Terrassa), a 1/10 dilution was made with peptone water and triturated in a stomacher homogenizer (UIL Instruments, Barcelona, Spain ). From the filtered sample, 8 serial dilutions were made in peptone water and the last 4 dilutions in Petri dishes were seeded by immersion with a previously melted Vitamin B12 agar test medium. Once the agar solidified, the plates were incubated in an anaerobiosis jar at 37 ° C for 48 hours. In agar plates that showed growth, colony forming units (cfu) counting was performed using a colony counter and several of them were randomly selected to perform a pre-extraction culture. Each of these colonies was stung and reseeded in a falcon tube with skirt, provided with broth of test medium of
    15 Vitamin B12 previously prepared and autoclaved. The tubes were incubated for 48h at 37 ° C. After that incubation period, the pellet of each tube was homogenized and Vitamin B12 was extracted.
    Extraction and quantification of Vitamin 8 12
    20 To extract cyanocobalamin, the extraction protocol provided in the instructions of the VitaFast Vitamin B12 kit (R-Biopharm, Germany) was followed. First, 1 g of the homogenized pellet was weighed into another falcon tube and 20 mL of acetate buffer (pH 4.5), prepared previously, was added. Then
    25 was added to each tube 250 ~ L of NaCN (1%, freshly prepared), 300 mg of takadiastase (Aspergillus oryzae a-amylase) and stirred well in a vortex. The entire procedure was performed trying to keep the tubes in the dark, covering them with aluminum foil. After the addition of all reagents, the tubes were incubated for 1 h in the dark at 37 ° C, shaking every ten minutes. After. be
    30 the tubes were made up to 40 mL with distilled water and the extracts were heated for 30 minutes in a 95 ° C water bath. During the process, the tubes were shaken at least 5 times and kept in the dark, covering the bathroom. After that time, the tubes were cooled rapidly below 30 ° C, centrifuged and stored at 4 ° C in the dark until the moment of carrying out the
    35 kit
    For the quantification of cyanocobalamin, the VitaFastVitamin 812 kit (from R-8iopharm) was used, consisting of a microtiter plate consisting of 12 strips with 8 wells each (coated with Lactobacillus delbrueckii subsp. Lactis), a medium
    5 of Vitamin 8 12 and a vial with a vitamin standard that is used to perform a standard curve. The medium and standards were prepared fresh each time the kit was made, following the instructions provided therein. The extractions were filtered (using 0.20 IJm 0 syringe filters) in a 2 mL eppendorf and, when necessary, diluted with sterile distilled water. 150 IJL of the medium was pipetted
    10 growth in each well, followed by another 150 IJL of the standards or 150 IJL of the samples in the assigned wells, both in duplicate, following a template. After removing the bubbles formed in the wells with the help of sterile pipette tips, the strips were covered with an adhesive sheet and incubated at 37 ° C in darkness for 48h. After incubation, the adhesive sheet was pressed to avoid
    After the contents of the wells were poured, the microplate was placed face down on a table and carefully shaken to homogenize the contents of the wells. Then, the adhesive sheet was removed diagonally, holding the strips carefully to avoid removing them from the microtiter plate, bubbles that had appeared during agitation with sterile pipette tips were destroyed and
    20 turbidity measurement was performed at 630 nm using a ChroMate microplate absorbance reader (Awareness Technology, Palm City, Florida). The generated data was analyzed using the RIDA® 80FT Win software from R-8iopharm.
    To verify that the cyanocobalamin extraction procedure is
    25 performed correctly, a recovery test was carried out, in which 200IJL of a solution of Vitamin 8 12 of known concentration was added to a falcon tube with 1 gr of sample and the same extraction and analysis protocol was followed as with the rest of samples. Finally, the result obtained was compared with that of the sample without the recovery solution and the recovery percentage was calculated.
    ID
    The strains that produced a concentration of Vitamin 812 of interest for this study in VitaFast were identified by the API 50CH gallery (8iomérieux, 35 France), following the instructions indicated by the manufacturer. For this, a part of the pellet obtained in the isolation process was seeded on the surface in MRS agar, and incubated for 48 hours at 30 ° C in anaerobiosis. With a Kolle handle, a dense suspension was made in a 2mL medium suspension API ampoule. Then, with a pasteur pipette, a certain number of drops (n) were transferred to a SmL ampoule of 5 API medium suspension, reaching a turbidity equal to McFarland pattern 2. To achieve the final inoculum, twice as many drops (2n) were transferred to an AP / 50CHL medium ampoule and, after homogenization, it was inoculated in API SOCH galleries, placed on its support with distilled water to prevent evaporation of the medium. . Once the 50 galleries were inoculated, they were covered with paraffin oil to generate a
    10 anaerobic environment and incubated at 30 ° C for 24-48h. Using the API web reading program (www.apiweb.biomerieux.com), isolated strains were identified. Finally, those that were of interest were transferred to tubes with cryoballs to preserve them better and be able to use them in subsequent procedures.
    15 Preparation of mother and bread doughs
    Once the 8 '2 Vitamin-producing strains were obtained and identified, fermentation tests of liquid mother masses were made at two different scales: laboratory scale in an Erlenmeyer flask and pilot plant scale in a bioreactor of
    20 SL (8iostat A plus, Sartorius, Germany). In both cases it was necessary to prepare an inoculum of the strain isolated above. For this, two falcon tubes with 8'2 Vitamin test broth were prepared and 2 cryoballs were deposited in each. After incubating them for 48h at 37 ° C, the tubes were centrifuged, the medium was decanted and the pellet was washed carefully with sterile distilled water, preventing it
    25 detached from the bottom of the tube. It was then resuspended in 20 mL of distilled water to each tube, vortexed for a few minutes and turbidity was measured by a spectrophotometer to calculate the microorganism concentration from which the mother mass would start.
    30 In the case of laboratory tests, for each strain tested, a white mother mass (without inoculum) and one with cyanocobalamin producing strain were prepared in duplicate. Whites were prepared by mixing tap water and who / egrain wholemeal flour in proportion 80% and 20%, respectively. The masses with inoculum maintained the same proportion but included the 20 mL with the
    35 resuspended strain. Both the blanks and the samples were incubated at 30 ° C for 48 hours, with manual agitation from time to time. During that period, the pH and titratable total acidity (TIA) of 10 g of the homogenized mother masses with 90 mL of distilled water were measured in an automatic meter (Titroline pH MATIC 23, Crison Instruments, Spain). TIA, expressed as the amount (mL) of 0.1 NaOH
    5 M needed to reach a pH of 8.4, and the pH were analyzed at 3 different times (lo = Oh, t1 = 24h and t2 = 48h). Finally, the extraction and quantification of Vitamin 8 12 from the fermented mother masses was carried out, following the same procedure explained above, including recovery.
    In the case of the pilot plant scale tests, the inoculum preparation was exactly the same (including duplicate) as in the laboratory scale, except the final step. After adding 20 mL of water and vortexing, the strains were transferred to two Erlenmeyers, flushed at 200 mL and then turbidity was determined in the spectrophotometer. Fermentation of the mother mass in the bioreactor was performed
    15 two different modes. In one of them the same proportion of ingredients used in Erlenmeyer flasks was respected (80% tap water and 20% wholegrain wholemeal flour) while in the other it was optimized, adding maltose to the flour and maintaining the pH at 5 during the 48h of process. The proportion of ingredients that was followed in this case was the following: wholegrain whole wheat flour (17%), D - (+)
    20 maltose monohydrate (3%) and tap water (80%), where the diluted strain was included. During the procedure the fermentation temperature (30 ° C), pH and stirring (rpm) were monitored. After 48 hours, Vitamin 8 12 was extracted and TIA was determined in the same way as with the laboratory-scale mother masses, all in duplicate. In addition, a plate count was also made
    25 the final concentration of microorganisms in the mother mass, sowing in the medium of Vitamin 8 12 agar. The obtained mother doughs were frozen for later use in bread making.
    To finalize the project, bread was made from the optimized mother masses
    30 in the bioreactor. All bread tests were performed in duplicate. In the pan of the bakery (Moulinex, Alen90n, France), the following ingredients were weighed in this order: 160 grams of sourdough, 10 grams of salt, 150 grams of water, 500 grams of bread flour and 5 grams of yeast fresh A quick bread program (98 minutes) was selected. Once cooled and unmold, we proceeded to extract and
    35 quantify Vitamin 812. To extract, a piece of bread was crushed with the help of a knife mill (M20-IKA Labortechnik, Brisgovia, Germany) and weighed 1 gr in a falcon tube. Then it was followed with the same procedure of extraction and quantification previously detailed.
    5 Results
    Screening of Vitamin 8 producing strains 12
    Table 1 shows that, of the 47 samples analyzed, 34 gave a result
    10 positive in terms of plate growth, while the remaining 13 do not. The number of plate colony forming units obtained from the different products was very varied, with counts between 106ufclgr (Banon de Chévre goat cheese Fermier Xavier, France) up to 109ufclgr (VolI-Damn beer-based mother dough, produced by Europastry).
    The Vitamin 8 12 test medium used to perform the screening is a medium that lacks this vitamin and is intended for the growth of acid-lactic bacteria capable of producing coba lamina or for those that grow independently of their presence. With this in mind, we start from the
    20 following hypothesis: strains capable of growing in that medium could be producers of cobalamin and be useful for this study or, on the contrary, they could be strains that did not require this vitamin for growth, producing results known as "false positives" . To confirm this hypothesis and rule out the possible false positives obtained, the culture of 5 colonies chosen at
    25 random samples of each sample (170 colonies in total) in Vitamin 8 12 broth and the vitamin was extracted (with a recovery percentage of 98.77%) and then quantified using the VitaFast Vitamin B12 kit. Of the 170 colonies analyzed, 19 were found to be real positive, producing a sufficient amount of cyanocobalamin to be quantified with the kit. The others
    30 produced a value lower than the detection limit (0.21 ~ g! 100gr). Despite the positive results, only 8 colonies (belonging to two samples analyzed) gave values close to those obtained in the analysis of the two control strains (Lactobacillus reuteri JCM 1112 and Lactobacillus reuteri Protectis® DSM 17938). Cyanocobalamin values were 4.00 ± 0.11 and 4.22 ± 0.05 ~ g! 1 OOg, Y
    35 corresponded to two strains isolated from two mother doughs obtained from a craft bakery in Tarragona, which correspond to strains CECT 9148 and CECT 9224.
    Table 1. Foods analyzed: t: screeening results.
    Sample Milk originGrowth in the middle of ('nsa ~' O from Vita min to BuC FU / mlVita mine Bu (Jlg / IOO g)
    Which ones
    Cabbage AND
    C-2 G+1.80109<0.03
    C-3 C+4.56 '108<0.03
    C-4 G+4.00 '107<0.03
    C-S AND+9.65 '107<0.03
    C-6 C+9.48 'lOs<0.03
    C-) M+Uncountable(' )<0.03
    C-8 AND+5.62 '106<0.03
    C-9 G+1.96108<0.03
    C_IO C+6.41 '107<0.03
    C-II G+1.00106<0.03
    C-12 C+4.98 '107<0.03
    C-13 B
    C-14 C
    C-15 C
    C-16 G
    C-1 7 C
    C-18 G+3.45. 107<0.03
    C-19 C
    C-20 AND+1.50108<0.03
    Lacto products
    D-21 AND+Uncountable (.)<0.03
    D-22 G
    D-23 C+2.18. the'<0.03
    D-24 C
    D-25 C+5.72. 10 '<0.03
    D-26 C+Incontablc ('f<0.03
    D-27 D-28 C C+ +2.21. 107 4.05 '107<0.03 <0.03
    Masses mother
    Sun +1.38. 10 '<0.03
    S-2 +3.84. 10 '<0.03
    S-3 +5.4710 '<0.03
    S-4 +4.90 'lO'<0.03
    S-5 +3.0 1. the<0.03
    S-6 +4.3210 '<0.03
    S-) +1.80 10 '0.030 ± 0.008
    S-8 +1.00 '10'0.030 ± 0.002
    S-9 +1.5010 '0.030 ± 0.004
    ST-I +2.04. 10 '4.10 ± 0.OO6
    ST-2 +3.00 10 '4.22 ± 0.OO9
    S-lO +2.03. 10 '<0.03
    S-II +1.7010 '<0.03
    S-12 +7.10. 10 '0.031 ± O.I
    Others
    0-29: Ilarina Int egral wheat
    0-30: Beer
    0-31: Ccrvew.
    0-32: Ccrvew.
    0-33: Salami +9.47107<0.03
    E, sheep; B, buffalo; G, eabra; C, see; M, mixed.The cobalamin concentration is presented as mean ± standard deviation.
    (o) Very small colonies.
    Laboratory mass production at laboratory scale
    In the laboratory-scale tests performed in Erlenmeyer flasks, the results observed in Table 2 were obtained. The pH and TTA of all the mother masses were evaluated every 24 hours. During the first 24 hours of incubation, a drastic decrease in pH was observed both in the white mother masses and in those inoculated with Lactobacillus reuteri. In the case of the mother masses without inoculum, the pH dropped from 6.29 ± 0.07 to 3.69 ± 0.07, while in the mother masses inoculated with the experimental strains it decreased from 6.16 ± 0.04 to 3.24 ± 0.06 in Lactobacillus reuter; CECT 9148 and from 6.20 ± 0.08 to 3.21 ± 0.06 in CECT 9224. The total titratable acidity also varied considerably during the first 24 hours but, unlike pH, increased. This increase was more noticeable in the mother masses inoculated with the CECT 9148 and CECT 9224 strains, doubling the acidity of the white mother mass. In the next 24 hours, a difference in the evolution of some types of mother masses was observed. While the targets and those prepared with control strain followed the same trend (pH and TTA continued to decrease and increase, respectively), in the mother masses prepared with strains CECT 9148 and CECT 9224 a peak was observed at 48h in the There is a slight increase in pH and a decrease in TTA.
    As for the Vitamin 8 12 produced, the white mother masses produced a small amount (average of 0.11 ± 0.03 Jg / 100gr). This very small concentration can be caused by the presence of wild bacteria that produce coba lamina in the same flour or by interference from it in the kit, being irrelevant in the whole of this study. The mother masses incubated with the colonies of Lactobacillus reuteri CECT 9148 and CECT 9224 produced an amount of cobalamin much higher than those of the white mother masses and even higher than that of the control strains Laetobaeillus reuter; JCM 1112 (0.68 ± 0.03 ~ g / 1 OOgr) and Laetobaeillus reuter; Proteetis · DSM 17938 (1.06 ± 0.03 ~ g / 1 OOgr). The concentrations of these mother masses were 2.36 ± 0.32 Jg / 100gr in the strain Laetobaeillus reuter; CECT 9148 and 2.28 ± 0.23 ~ g / 1 OOgr in Laetobaeillus reuter; CECT 9224, with a recovery percentage of 97.33%.

    Table 2. Results laboratory tests (Erlenmeyer flask).
    , ~,
    ., TTA ml 0.1 M NaOH!
    Vitamin 812 (lfII / l00g ') ü "ÜO
    ME,. UFCJml '"" "'" ""
    L. reureri 1.48 W 6.22.1 0.01 3.25.1 0.04 3, lhO.03 0.51 ~ 0.1t 10.09 ~ 0.32 11.45 ~ 0.22 1.00.1 0.03
    DSM ' , ..,, o '
    L, ", uren 6.21.1 0.04 3.48.1 0.21 3.16.1 0.00 0.52 .1 0.05 8.02.1 1.92 10.93 .1 0.44 0.68.1 0, 03
    JCM '
    L, l8IJIeti 2.75 W6.16.10.04 3.24.1 0.06 3.39.1 0.02 0.60 .1 0.00 12.22 .1 O, SS 11.61.1 0.41 2.07.1 0.25
    ""
    L, ", uten 3.10 'lo" 6.20.1 O. or 3.21.1 0.06 3.40.1 0.02 0.65.10.11 12.80 .1 2.04 11.96 .1 0.46 2, 22.10.18
    ""
    ""
    ,. ~ 6.29.1 0.07 3.69.1 0.07 3.5hO, 14 0.49 ~ 0.03 5.1013.36 6.96 1 3.72 0.09 1 0.02
    The values correspond to the average t standard deviation.'Control strains.
    5 Preparation of mother masses at pilot plant scale
    Table 3 shows the results of the tests carried out in the mother masses prepared at pilot plant scale. In the mother mass without inoculum ('BIOBLANC')
    obtained a final count of 3.20'1 O 'ufclmL This result evidences the presence of
    10 microorganisms in the flour itself and that grow throughout the fermentation. The count of the non-optimized mother masses remained practically the same throughout the fermentation. However, the final count of the optimized mother masses increased with respect to the initial count. In the case of the OPTIFARM and OPTIJAP mother masses (inoculated with Lae / obaeil / us reuteri Protectis® DSM 17938 and
    15 Lactobacillus reuteri JCM 1112, respectively) the final count increased 1 logarithm, while the mother masses OPTITARR 1 and OPTITARR 2 (inoculated with strains of CECT 9148 and CECT 9224, respectively) increased their final plate count from 108 to 1010 • The total titratable acidity in the non-optimized mother masses was much higher than that obtained in those made at constant pH and with
    20 maltose, In determining the concentration of cyanocobalamin, a
    result of 2.55 ± 0.07 ~ g / 100gr in OPTITARR 1 and 2.67 ± 0.15 ~ gf100gr in OPTITARR 2, with 5.10 ± 0.07 ~ gf100gr and 4.67 ± 0.88 ~ gf100gr those obtained in OPTIFARM and OPTIJAP, respectively, The recovery percentage of Vitamin B12 was 107.57%,
    Table 3. Test results at pilot plant scale (Bioreactor).
    Mother mass CFU / mL 11 = 0)CFU / mL 11 = 48h)initial pHfinal pHFinal TTA (mLNaOH O, 1MIVitamin B12 concentration! L;! Af100arl
    Do not
    imized opt
    BIOBLANC 3.2010 '6.203.649.89 ± 0.310.03 ± 0.00
    BIOFARM 1.25 10 '2.38. 10 '6.153.4112.62 ± 2.121.40 ± 0.11
    BIOJAp 2.4210 '8.90. 10 '6.303.4011.49 ± 0.051.19 ± 0.02
    BIOTARR 1+ 3.2510 '6.2710 '6.203.3813.02 ± 0.180.73 ± 0.02
    BIOTARRi 2.9010 '2.49. 1086.153.3412.22 ± 0.100.92 ± 0.05
    Optimized
    OPTIFARM " 2.9010 '4.8510 '6.144.954.51 ± 0.265.10 ± 0.07
    OPT1JAp 2.7510 '2.0410 '6.344.954.30 ± 0.084.67 ± 0.07
    OPTITARR 1+ 2.6710 '6.0010106.254.954.79 ± 0.252.55 ± 0.07
    OPTITARRi 3.12. 10 '1.70. 10106.264.954.86 ± 0.042.67 ± 0.15
    • Mother masses inoculated with the control strains Lactobacillus reuteri ProtectiS® DSM 17938 and Lactobacillus reuteri JCM 1112.
    5 • Mother masses inoculated with the strain Lactobacillus reuleri CECT 9148.~ Mother masses inoculated with the strain Lactobacillus reuleri CECT 9224.
    Bread making with optimized sourdough
    10 From each optimized sourdough in the bioreactor a bread was made in duplicate. After the extraction of Vitamin B12 (with a recovery of 89.37%), the following results were obtained: 0.12 ± 0.00 1J9 / 100gr in BIOBLANC; 0.63 ± 0.07 ~ g / 100gr for those made with the control strains (OPTIFA RM and OPTIJAP): 0.33 ± 0.05 ~ g / 100gr in OPTITARR 1 AND 0.48 ± 0.07 ~ g / 100gr in OPTITARR 2.
    Evolution of Vitamin 8 12 with the scaling process
    Figure 1 shows how the cyanocobalamin quantification of the LactobaciJJus reuteri strains that have been studied at all levels varies (isolated colony 20, laboratory scale, pilot scale and bread). The concentration of Vitamin B12 decreases as the complexity of the sample studied increases. The increase in concentration that can be seen in the step from laboratory scale to scale
    Pilot plant is due to the fact that, in the latter case, the fermentation was optimized by adding maltose and maintaining the pH at 5 during the process.
    In the last step of the escalation, knowing the percentage of mother mass that is
    5 used to make the bread (32%), it was possible to calculate the concentration of Vitamin 8 '2 that would be expected to be obtained and, when compared with the real one, the percentage that was maintained and the percentage that was lost was calculated (Table 4). The bread that best conserved Vitamin 8 '2 was the one prepared with the strain of CECT 9224, preserving 56.32% of the present in the sourdough. On the contrary, the worst result obtained was that of processed bread
    10 OPTIFARM, in which only 38.60% of Vitamin 812 was preserved • On average, a loss of 55.71% was quantified.
    Table 4. Concentration of theoretical and real Vitamin B11 in bread.
    ~ g / 100 gr 1J9 / 1 OOgr Vitamin IJg / 1 OOgr Vitamin% Vitamin
    % MM
    Vitamin B12 bread in 8 12 expected in 8 12 obtained in
    B "
    in bread
    bioreaclor bread preserved bread OPTIFARM-5.10 ± 0.07 32% 1.63 ± 0.02 0.63 ± 0.07 38.60 OPTIJAP-4.671 0.88 32% 1.50 ± 0.28 0.63 ± 0.07 41, 94 OPTITARR 13 2.55 ± 0.07 32% 0.82 ± 0.02 0.33 ± 0.05 40.29 OPTI TARR 2b 2.67 t 0.15 32% 0.85 ± 0.05 0.48 ± 0.07 56.32
    • Bread made with the mother masses of the control strains (Lactobacillus reuteri Proteclis® DSM 17938 and
    15 Lactobacillus reuteri JCM 1112). ~ Bread made with the sourdough of the Lactobacillus reuteri strain CECT 91 48. 1> Bread made with the sourdough of the Lactobacillus reuteri strain CECT 9224.
    20 Screening of Vitamin-producing strains 8 12
    Cobalamin is only synthesized by some groups of bacteria and archaea. Among them we find microorganisms such as Propionibacterium freudenreichii, Propionibacterium shermanii, Pseudomonas denitrificans, Salmonella
    25 typhimurium, Bacillus megaterium, Rhizobium cobalaminogenum, Rhodopseudomonas protamicus and Streptomyces olivaceus. Of all those mentioned, Pseudomonas denitrificans is the most industrially exploited, since its productivity can reach 300 mg / L. However, this microorganism has not obtained GRAS status (from the Generally Recognized As Safe) of the Agency
    30 US Food and Drug Administration (FDA), so its use in the food industry is not approved. In 2003 Taranto et al. described for the first time the ability of a lactic acid bacterium to synthesize cobalamin, specifically Laclobacillus reuleri CRL 1098. SALs, unlike Pseudomonas denitrificans, do have FDA GRAS status, so they can be used in the food industry to provide nutritional characteristics and
    5 beneficial organoleptics to processed food products. This group of bacteria plays an important role in the production of a large number of fermented products, such as cheeses, yogurts, sausages, sourdoughs and even alcoholic beverages. For the reasons stated, in the present study the screening of lactic acid strains in these products has been carried out.
    It is not the first time that an analysis of this nature is carried out. The strain of Laclobacillus reuleri (Laclobacillus reuleri CRL 1098 studied by Taranta el al. (2003) was isolated from a mother mass. Miescher Schwenninger al. (2004) isolated 1,424 strains from various food products (raw milk, cheese, yogurt,
    15 black olives, mother masses and salami, among others), to later test their antifungal activity on the predominant fungal contaminants in yogurt and fruit. Recently, Diana on 81. (2014) performed the screening of 58 artisan cheeses with the purpose of selecting strains of lactobacilli producing y-aminobutyric acid (GASA), a molecule with hypotensive effect (11), for its
    20 subsequent use in breads with mothers mastered by these isolates. This last study is the most similar to the one presented in this paper. Of the 580 colonies analyzed (10 colonies per cheese) by Diana al. (2014), 65 gave a positive result in the production of GASA (11, 2%). In this, of the 170 colonies analyzed in total, 19 produced a positive result regarding the synthesis of
    25 Vitamin S'2 (11.2%). However, it is necessary to take into account that only 65 colonies were isolated from the cheese samples, and of those 65, only 2 (3.1%) produced a quantifiable amount of cobalamin. To perform the screening, cheeses from multiple geographical origins were purchased (Table 1), preferably artisanal. The choice of these cheeses is due to the type of strains
    30 found in them. While standard starter cultures are often used in cheeses made in more industrialized processes, those that follow an artisanal process contain the wild strains found in the milk with which they are made. Despite the selection of the cheeses analyzed (handmade), only Ardi Gazta Ekia 0.0 cheeses. Roncal (Roncal Valley,
    35 Navarra) and Banon de Chévre Fermier Xavier D.O.P. (Provence -Alpes -Cote d'Azur, France) provided positive results. Both differ in practically everything: the first is made with sheep's milk, while in the second the milk is goat's, and they are not geographically close either. The only characteristics that they share are the state of the milk with which they are made (raw milk) and that both have Protected Designation of Origin (D.O. or D.O.P.), which guarantees an artisanal production only with the ingredients of a certain area. The others, despite their artisanal appearance, it is possible that they carry some type of starter culture, that the wild strains they contain are not producing cobalamin or that their concentration is so negligible that it cannot be detected with the microbiological method of VitaFast.
    Of the other products analyzed, only cobalamin producing strains of 6 mother masses were obtained (Table 1). In 2005, Corsettiet al. isolated and identified a new species of lactobacillus, called Lactobacillus rossiae DSM 15814, from an Italian mother mass in the course of a study in which it was intended to analyze the production of antimicrobial substances by the LABs present. The mother masses are considered complex ecosystems in which lactic acid bacteria are the prevalent microorganisms. Of all these bacteria, the genus Lactobacillus is responsible for the acidification of the mass, for that reason it is a good base product from which to isolate different strains of this microorganism. Later, from Angeliset al. (2014) performed a complete genomic analysis (including genomic annotation, comparative analysis and reconstruction of metabolic pathways) of Lactobacillus rossiae DSM 15814 and discovered the genetic cluster allegedly involved in the biosynthesis of cobalamin. In addition, to demonstrate this biosynthetic ability, he performed a microbiological assay (MBA) using the Lactobacillus rossiae cell extract for the growth of Lactobacillus leichmannii subsp. lactis, an auxotrophic microorganism for Vitamin B'2. Unlike the present study, De Angelis et al. It did not quantify the concentration of vitamin produced, but was satisfied with the growth of the auxotrophic microorganism. Apart from genomic identification, no further analysis has been performed to demonstrate the presence of the genes involved in cobalamin biosynthesis in strains CECT 9148 and CECT 9224, but could be part of a future project.
    Preparation and optimization of mother masses
    In the present study, it was decided to carry out a laboratory scale test (using autoclaved Erlenmeyer flasks) to see if the selected strains were capable of synthesizing cobalamin in a more complex fermentation medium than the one used in screening, such as the mixture of water and flour that is used in the
    5 mother mass production. These types of laboratory-scale tests are very common. For example, Madhu et al. (20 10) carried out a small-scale fermentation to verify that the Lactobacillus planlarum strain isolated from kanjika (a fermented product used as a medicinal food in India) produced Vitamin
    812. In that study, to the fermentation medium (which contained macro and micronutrients)
    10 Glycerol was added as it is shown to induce the synthesis of cobalamin. In the laboratory-scale fermentation carried out in this study, no source of external carbon was added to the flour itself, since it was only intended to corroborate the production of Vitamin 8 12 from the study strains. In the results obtained in the study of Madhu et al. they used another analytical method
    15 and expressed the concentration in ng of Vitamin B12 / g of dry biomass. Chamlagain al. (2015) performed a test similar to that performed in this study. This group used two matrices of two cereals (rye and barley) as a means of fermentation, but, unlike the one used here, they were previously processed (cooked for 2 minutes in boiling water and autoclaved). In addition, the microorganism
    20 he used was Propionibaclerium freudenreichií subsp. Shermanií instead of Laclobacillus reuleri. The result of cyanocobalamin obtained was 28.80 ng / gr (28.8 ~ gI100gr) in the mass of cenlen and 25.00 nglgr (25 ~ gI100gr) in that of barley, values higher than those obtained in the present study (Table 2).
    25 Once the production of Vitamin 812 in a small-scale mother dough was corroborated, several master doughs were developed at pilot plant scale. Seeing that the cyanocobalamin results obtained without adding any source of exogenous carbon were so low, it was decided to optimize the fermentation, as other authors had done in previous studies. Mohammed al. (2014) described the capacity
    30 of Lactobacillus reuteri to produce an active form of Vitamin B12 optimizing fermentation conditions. In that study, o-aminolevulinic acid (critical precursor at the beginning of the biosynthetic pathway) and 5,6-dimethylbenzimidazole (the lower aligand responsible for the production of the active form of cobalamin) were added. In this study it was decided not to use these chemical compounds for the optimization of
    35 fermentation with the intention of not generating additional costs and preserving the
    naturalness of the product Madhu al. (2010) optimized fermentation using glycerol as a carbon source. Also in 2010, Hugenschmidt al. He used serum permeate as a carbon source instead of glucose in the fermentation he performed with Lactobacillus reuteri and 99 strains of propionic bacteria (PAB) to determine if 5 were able to synthesize cobalamin. In a project carried out by Xiaet al. (2015) In order to develop an effective and economical fermentation medium for the industrial production of Vitamin B12 (with P. denitrificans), malt syrup and maceration maize syrup (both by-products of the corn industry) were used as fermentation medium, the first being a source of carbon and the second 10 a source of nitrogen. After a 180h process Xia el al. it obtained a concentration of 198.27 ± 4.60 mg / L of Vitamin 812. Although the microorganism and the purpose of this study differed from the present one, it was very interesting the possibility of using an effective and economical carbon source at the time of optimize fermentation in the bioreactor, since that would not affect the price of the final product 15 made. For that reason it was decided to add maltose to the fermentation mixture. Xia el al. (2015) used maltose syrup as the only carbon source, hence its high concentration in the medium (80.3%). In this study, however, only 3% was added to check if there was an improvement in the amount of synthesized cobalamin. After a 48h fermentation (Table 3), it is
    20 obtained a lower result than the one obtained in Xiaet al. (2015). This difference may be due to several factors: the use of another microorganism other than Laclobacillus reuteri, the high proportion of maltose used or the duration of the process (180h).
    25 In the present study, the fermentation pH was also optimized. So far, in all fermentations carried out there was a drastic decrease in pH, from values close to neutrality to an important acidity (Table 3). Although the decrease in pH is normal in the fermentation of mother masses, it can cause degradation of cobalamin, since it is sensitive to changes in pH. Xia el
    30 al. (2015) showed that the use of maltose as a source of carbon instead of glucose produced a lower decrease in pH, maintaining a stable range close to 6.5. Edelmann al. (2016) studied the stability of Vitamin B12, both added and produced in situ by Propionibaclerium freudenreichii in bread making. The malt extract used by Edelmann al. in dough making
    35 mother also resulted in a lower drop in pH, keeping close
    a 5. Due to these results, it was decided to maintain the pH at 5 during the 48h of fermentation, automatically and constantly supplying a solution of 0.1 M KHC03. This base was used and not NaOH to avoid an increase in sodium in the mother mass and that this salt will not affect the study strains.
    Bread making fortified with Vitamin 8 '2
    The main source of Vitamin 812 is found in animal foods, such as meat, fish, milk and eggs. An omnivorous diet (commonly known as "varied diet") ensures the intake of a sufficient amount of Vitamin 812 to meet daily needs (RDA = 2.5 ~ g / day). Vitamin 8 12 deficiency can cause different pathological manifestations, affecting the hematopoietic system, the neurological system and even the cardiovascular system. A more severe type of deficiency is pernicious anemia, a pathology caused by the lack of production of a glycoprotein that facilitates the absorption of the vitamin in the small intestine. It has been shown that oral administration of the strain Lactobacillus reuteri CRL1098 helps prevent symptoms caused by this hypovitaminosis. There are several population groups with a high risk of suffering from pathologies derived from a Vitamin deficiency 8 12 • Among these we find 20 vegan and vegetarian people, who do not consume any food of animal origin (except lacto-ova-vegetarians, who consume milk , dairy products and eggs). Another group that is very prone to suffer from this deficiency is that formed by the elderly (it has been estimated that between 10 and 30% of the population over 50 years of age has an absorption capacity of cabala mina
    25 reduced) .Finally, people living in countries with low consumption of animal products are also exposed to this deficiency.
    Due to insufficient intake by the aforementioned risk groups, some institutions recommend the use of vitamin supplements and / or foods enriched with cyanocobalamin, which represents an additional expense for these people. According to Pawlak al. (2013), in the United States, many cereals and soy milk are fortified with this vitamin. The fortification of these foods is given by the addition of cyanocobalamin, which is synthesized on an industrial scale in fermenters to subsequently be purified and added to the desired food. The use of Vitamin 812 producing microorganisms during the process of
    Fermentation of some foods is a more natural fortification alternative that is currently being studied.
    Description of the drawings
    To complement the description that is being made, and in order to help a better understanding of the features of the invention, the present specification is attached, as an integral part thereof, Figure 1 showing a graph with the Vitamin concentration 812 at different levels of scaling of Lactobacillus reuteri DSM, Lactobacillus reuteri JCM, Lactobacillus reuteri CECT 9148 and Laetobaeillus reuteri CECT 9224.
    Preferred Embodiment of the Invention
    As a conclusion to all the tests of the study that have been detailed, a preferred embodiment of the invention would be the one related to the use of the two isolates of the species Laetobaeillus reuteri CECT 9148 and Laetobacillus reuteri CECT 9224 together or separated interchangeably for use in masses panary mothers.
    Likewise, bread would be manufactured through the inclusion of sourdough or sourdough produced by means of these strains in the manufacture of any food product considered functional food or nutritional and / or pharmaceutical supplement through the concentrated source of Vitamin 812 for its ingestion or oral administration by using these strains of Lactobacillus reuteri CECT 9148 and CECT 9224.
    The process for the preparation of the sourdoughs enriched in Vitamin 812 and the subsequent use of these sourdoughs for the manufacture of bread or food enriched in Vitamin 812 comprises the following sections:
    a) Preparation of inoculum of the strains of Lactobacillus reuteri CECT 9148 and / or LactobacilJus reuteri CECT 9224 at 37 ° C for 48 hours.
    b) Propagation in a culture medium of the Lactobacillus reuteri strains
    CECT 9148 and / or Lactobacillus reuter; CECT 9224 at 37'C for 48h for
    Obtaining the biomass or cell pellet.
    and) ResuspensionOr inoculationfromthebiomassobtainedinsubstratesor
    5 flour solutions (mother masses).
    d) Fermentation of inoculated flour substrates or solutions.
    权利要求:
    Claims (4)
    [1]
     Claims
    1.-New strains of the species Lactobacillus reuteri for use in masses 5 panary mothers characterized by the fact that strains CECT 9148 and CECT 9224 are of
    Lactobacillus reuteri.
    [2]
    2.-Use of the strains of claim 1 for the manufacture of bread as a functional food through the inclusion of sourdough or sourdough produced by means of these strains.
    [3]
    3.-Use of the strains of claim 1 for the manufacture of any food product considered functional food or nutritional and / or pharmaceutical supplement by means of the concentrated source of Vitamin 8'2 for ingestion or administration
    15 oral.
    [4]
    4. Use of the strains according to claim 1 for the preparation of mother masses enriched in Vitamin 8 '2 by a process comprising the following sections:
    20 a) Preparation of inoculum of Lactobacillus reuteri CECT 9148 and / or Lactobacillus reuteri CECT 9224 strains at 37 ° C for 48 hours.
    b) Propagation in a culture medium of the strains of Lactobacillus reuteri CECT 9148 and / or Lactobacillus reuteri CECT 9224 at 37 ° C for 48 hours to obtain the biomass or cell pellet.
    C) Resuspension or inoculation of the biomass obtained in substrates or flour solutions. d) Fermentation of inoculated flour substrates or solutions.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    CO5700171A1|2004-09-01|2006-11-30|Consejo Nac Invest Cient Tec Conicet|A PURE CROP OF LACTOBACILLUS REUTERI WITH A CAPACITY TO PRODUCE COBALAMINS, USE OF THE SAME AND FOOD PRODUCTS THAT USE IT|
    WO2011154820A2|2010-06-11|2011-12-15|Probiotical S.P.A.|Vitamin b12 producing probiotic bacterial strains|
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