CN117264852A - Method for cultivating probiotics composite flora by simulating human intestinal microecology co-evolution - Google Patents
Method for cultivating probiotics composite flora by simulating human intestinal microecology co-evolution Download PDFInfo
- Publication number
- CN117264852A CN117264852A CN202311497068.0A CN202311497068A CN117264852A CN 117264852 A CN117264852 A CN 117264852A CN 202311497068 A CN202311497068 A CN 202311497068A CN 117264852 A CN117264852 A CN 117264852A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- lactobacillus
- culturing
- human intestinal
- hbm11
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000000968 intestinal effect Effects 0.000 title claims abstract description 82
- 239000002131 composite material Substances 0.000 title claims abstract description 45
- 239000006041 probiotic Substances 0.000 title claims abstract description 41
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000012258 culturing Methods 0.000 claims abstract description 70
- 244000199866 Lactobacillus casei Species 0.000 claims abstract description 43
- 235000013958 Lactobacillus casei Nutrition 0.000 claims abstract description 43
- 241000191996 Pediococcus pentosaceus Species 0.000 claims abstract description 43
- 229940017800 lactobacillus casei Drugs 0.000 claims abstract description 43
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 42
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 42
- 241000186604 Lactobacillus reuteri Species 0.000 claims abstract description 42
- 241000218588 Lactobacillus rhamnosus Species 0.000 claims abstract description 42
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 42
- 229940001882 lactobacillus reuteri Drugs 0.000 claims abstract description 42
- 230000000529 probiotic effect Effects 0.000 claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 19
- 230000004151 fermentation Effects 0.000 claims abstract description 19
- 239000001963 growth medium Substances 0.000 claims description 150
- 239000007788 liquid Substances 0.000 claims description 71
- 230000001580 bacterial effect Effects 0.000 claims description 63
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 44
- 235000020246 buffalo milk Nutrition 0.000 claims description 38
- 235000006040 Prunus persica var persica Nutrition 0.000 claims description 31
- 239000002609 medium Substances 0.000 claims description 30
- 244000144730 Amygdalus persica Species 0.000 claims description 29
- 150000004676 glycans Chemical class 0.000 claims description 29
- 229920001282 polysaccharide Polymers 0.000 claims description 29
- 239000005017 polysaccharide Substances 0.000 claims description 29
- 230000001954 sterilising effect Effects 0.000 claims description 27
- 238000011081 inoculation Methods 0.000 claims description 24
- 230000002496 gastric effect Effects 0.000 claims description 21
- 239000003833 bile salt Substances 0.000 claims description 20
- 239000002054 inoculum Substances 0.000 claims description 19
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 18
- 230000001079 digestive effect Effects 0.000 claims description 18
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 18
- 239000012530 fluid Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 16
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 229930006000 Sucrose Natural products 0.000 claims description 16
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 16
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 claims description 16
- 229940040526 anhydrous sodium acetate Drugs 0.000 claims description 16
- 229940041514 candida albicans extract Drugs 0.000 claims description 16
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 claims description 16
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 16
- 239000003792 electrolyte Substances 0.000 claims description 16
- 239000008103 glucose Substances 0.000 claims description 16
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 16
- 229920000053 polysorbate 80 Polymers 0.000 claims description 16
- 239000005720 sucrose Substances 0.000 claims description 16
- 239000012138 yeast extract Substances 0.000 claims description 16
- 150000001875 compounds Chemical class 0.000 claims description 15
- 244000068988 Glycine max Species 0.000 claims description 13
- 235000010469 Glycine max Nutrition 0.000 claims description 13
- 239000001888 Peptone Substances 0.000 claims description 13
- 108010080698 Peptones Proteins 0.000 claims description 13
- 230000029087 digestion Effects 0.000 claims description 13
- 235000019319 peptone Nutrition 0.000 claims description 13
- 230000003213 activating effect Effects 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 9
- 210000000813 small intestine Anatomy 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 239000011248 coating agent Substances 0.000 claims description 7
- 238000000576 coating method Methods 0.000 claims description 7
- 238000012136 culture method Methods 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 238000005070 sampling Methods 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 6
- 102000057297 Pepsin A Human genes 0.000 claims description 4
- 108090000284 Pepsin A Proteins 0.000 claims description 4
- 102000004142 Trypsin Human genes 0.000 claims description 4
- 108090000631 Trypsin Proteins 0.000 claims description 4
- 229940111202 pepsin Drugs 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000012588 trypsin Substances 0.000 claims description 4
- 229960001305 cysteine hydrochloride Drugs 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims 3
- 239000007853 buffer solution Substances 0.000 claims 1
- 235000011187 glycerol Nutrition 0.000 claims 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 13
- 210000001035 gastrointestinal tract Anatomy 0.000 abstract description 12
- 210000004211 gastric acid Anatomy 0.000 abstract 2
- 238000010276 construction Methods 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 25
- 230000004083 survival effect Effects 0.000 description 21
- 238000009630 liquid culture Methods 0.000 description 18
- 239000007787 solid Substances 0.000 description 9
- 238000004321 preservation Methods 0.000 description 8
- 238000004659 sterilization and disinfection Methods 0.000 description 8
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 244000005700 microbiome Species 0.000 description 5
- 230000002411 adverse Effects 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 238000004088 simulation Methods 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 3
- 235000012000 cholesterol Nutrition 0.000 description 3
- 210000004051 gastric juice Anatomy 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 230000002906 microbiologic effect Effects 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 240000005809 Prunus persica Species 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000036772 blood pressure Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- YJLUBHOZZTYQIP-UHFFFAOYSA-N 2-[5-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NN=C(O1)CC(=O)N1CC2=C(CC1)NN=N2 YJLUBHOZZTYQIP-UHFFFAOYSA-N 0.000 description 1
- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical compound C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 description 1
- 206010000050 Abdominal adhesions Diseases 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 206010057669 Colon injury Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 206010019909 Hernia Diseases 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 208000012873 acute gastroenteritis Diseases 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000112 colonic effect Effects 0.000 description 1
- 201000002758 colorectal adenoma Diseases 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 208000002551 irritable bowel syndrome Diseases 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/245—Lactobacillus casei
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a method for culturing a probiotic composite flora by simulating human intestinal microecology co-evolution, which is a method capable of simultaneously culturing lactobacillus reuteri, lactobacillus plantarum, pediococcus pentosaceus, lactobacillus rhamnosus and lactobacillus casei. The existing probiotics are generally single strain fermentation, are not resistant to gastrointestinal tract bad factors such as gastric acid and the like, and are difficult to effectively keep activity to reach intestinal tract colonisation, so that the construction of a composite culture fermentation system which mimics the gastrointestinal tract environment of a human body is very necessary. The method can enable the five selected strains to co-produce and evolve and culture in the human micro-ecological environment, not only can enable the five strains to effectively resist bad factors such as gastric acid in the gastrointestinal tract environment and the like, keep higher activity to reach intestinal tract colonisation, exert the probiotics of lactobacillus reuteri, lactobacillus plantarum, pediococcus pentosaceus, lactobacillus rhamnosus and lactobacillus casei, but also can generate the co-synergistic effect to proliferate the probiotic composite flora, and has great industrial value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for culturing a probiotic composite flora by simulating human intestinal microecology co-evolution, in particular to a method capable of simultaneously culturing lactobacillus reuteri, lactobacillus plantarum, pediococcus pentosaceus, lactobacillus rhamnosus and lactobacillus casei.
Background
The general research shows that probiotics participate in the microecology regulation of intestinal flora of human body, improve the immunity of the organism, and in addition, the probiotics can also treat acute gastroenteritis and irritable bowel syndrome; regulating cholesterol content; involved in gene expression regulation and lipid metabolism; reducing inflammatory response in mice, etc. The lactobacillus reuteri has remarkable curative effect on infant hernia; lactobacillus casei has the functions of efficiently reducing blood pressure and cholesterol, promoting cell division, preventing cancer, inhibiting tumor growth and the like; the lactobacillus casei can relieve colon injury of mice caused by DSS, inhibit APCmin/+ colorectal adenoma of mice, and relieve injury of Salmonella to intestinal mucosa; lactobacillus rhamnosus promotes apoptosis of human colorectal cancer cells, improves oxidative damage, reduces blood pressure and the like; the lactobacillus plantarum has strong intestinal adhesion capability, and can rapidly reduce blood sugar and relieve ulcerative colitis; pediococcus pentosaceus can inhibit food-borne pathogenic bacteria, regulate intestinal tract immunity, reduce cholesterol, resist tumor, etc.
Currently, probiotics have been widely used in the food, pharmaceutical, livestock industries, and the like. However, the precondition that the probiotics can survive and proliferate after entering the gastrointestinal tract is that the gastrointestinal tract is in a low-acid environment and a large amount of digestive enzymes exist, so that the probiotics are stressed by the bad environment of the gastrointestinal tract after being ingested, the number of the probiotics is greatly reduced, and the probiotics cannot effectively play a role. Therefore, through in vitro simulation of gastrointestinal tract experiments, probiotics have certain tolerance to gastrointestinal fluid, and through adjusting culture components, the probiotics can have higher viable count under the conditions of enzyme tolerance and low acid environment. Probiotics health food declaration and evaluation (claiming opinion manuscript) GB 7101-2015 (food safety Standard beverage) prescribes that the number of lactobacillus in the product marked with viable bacteria should be more than or equal to 1×10 6 CFU/mL (g). At present, the culture method of single bacteria is gradually perfected, and the aim of high-activity bacteria number of single probiotics can be realized by high-density culture, fed-batch feeding and other methods. Complex flora with 1+1>2, for example, there is a synergistic effect between bacillus subtilis and lactic acid bacteria, bacillus subtilis acts as an aerobic bacteria, consuming oxygen to provide an anaerobic environment for lactic acid bacteria, promoting proliferation of lactic acid bacteria. However, for the culture of complex bacterial groups, there are few reports in the literature.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provides a method for cultivating probiotic composite flora by simulating human intestinal microecology co-evolution.
The aim of the invention is achieved by the following technical scheme:
a method for culturing probiotic composite flora by human intestinal microecological co-evolution comprises the steps of respectively continuously carrying out passage for 2-3 times on lactobacillus reuteri HBM11-69 (with the preservation number of GDMCC NO: 63608), pediococcus pentosaceus MPL5 (with the preservation number of GDMCC NO: 63606), lactobacillus plantarum La-10 (with the preservation number of GDMCC NO: 63605), lactobacillus rhamnosus HBM11-35 (with the preservation number of GDMCC NO: 63607) and lactobacillus casei C7-2 (with the preservation number of GDMCC NO: 63609), taking bacterial liquid of the last passage according to the proportion, inoculating the bacterial liquid into a co-evolution composite culture medium for fermentation culture for the last passage for carrying out human intestinal microecological system culture, wherein the 2-3 times are 16-24 hours.
(1) Inoculating to A culture medium, fermenting at 35-37 deg.C for 0-24 hr, adding human intestinal digestion liquid, culturing at 35-37 deg.C for 0-8 hr or
(2) Inoculating to B culture medium, fermenting at 35-37 deg.C for 0-24 hr, adding human intestinal tract-imitating digestive juice, culturing at 35-37 deg.C for 0-8 hr or
(3) Inoculating the mixture to a C culture medium, fermenting and culturing for 0-24 hours at 35-37 ℃, and then adding human intestinal digestion simulating liquid for culturing for 0-8 hours at 35-37 ℃;
wherein, the A culture medium comprises the following components: 15-25g of sucrose, 17-30g of maltose, 16-30g of glucose, 30-50g of yeast extract powder, 25-40g of soybean peptone, 1-3g of dipotassium hydrogen phosphate, 2-5g of diammonium hydrogen citrate, 5-10g of anhydrous sodium acetate, 0.2-1g of L-cysteine hydrochloride, 0.1-1g of bile salt and MnSO (total of 1L-cysteine hydrochloride) 4 0.05-0.1g、MgSO 4 •7H 2 0.5-1g of O, 0.5-2 mL of Tween-80 and 5-15Mmol/L of glycerol; sterilizing at 121 deg.c and pH of 6.0-7.0 for 15-30 min;
the culture medium B is a culture medium A containing 5% -20% (v/v) buffalo milk; sterilizing at 121 deg.c and pH of 6.0-7.0 for 15-30 min; wherein, the buffalo milk is sterilized independently, and the temperature is 60 ℃ for 30min;
the culture medium C is a culture medium A containing 0.5% -5% (m/v) peach gum polysaccharide; sterilizing at 121 deg.c and pH of 6.0-7.0 for 15-30 min.
Preferably, the probiotic composite flora imitates human intestinal microecology co-evolution composite culture method is to continuously passage lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 for 3 times respectively, wherein the 3 rd passage culture time is 22 hours. And (3) proportionally taking the bacterial liquid of the last passage, and inoculating the bacterial liquid into a co-evolution composite culture medium for fermentation culture.
Further preferably, the probiotic composite flora imitates human intestinal microecology co-evolution composite culture method is to continuously passe lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 for 3 times respectively, wherein the time of passaging for the 3 rd time is 22 hours. According to the total inoculation amount of 4%, lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 strains are inoculated into the same co-evolution compound culture medium according to the proportion of 2:2:1:3:3.
Preferably, the bacterial liquid is inoculated into the co-evolution composite culture medium in proportion for fermentation culture, and the culture time is 0-14 hours.
Further preferably, the bacterial liquid is inoculated into the co-evolution compound culture medium in proportion for fermentation culture, and the culture time is 0-4 hours.
Most preferably, the bacterial liquid is proportionally inoculated into the co-evolution compound culture medium for fermentation culture, and the culture time is 1 hour.
Preferably, the A medium comprises the following components: based on 1L, 18-21g of sucrose, 20-23g of maltose, 18-21g of glucose, 40-45g of yeast extract powder, 30-35g of soybean peptone, 2-3g of dipotassium hydrogen phosphate, 2-4g of diammonium hydrogen citrate, 7-9g of anhydrous sodium acetate, 0.2-0.6g of L-cysteine hydrochloride, 0.4-0.7g of bile salt and MnSO 4 0.08-0.1g、MgSO 4 •7H 2 0.6-0.9g of O, 7-10 Mmol/L of Tween-80 and 0.7-0.9g of glycerol.
Most preferably, the formula of the culture medium A is as follows: 19.32g of sucrose, 21.84g of maltose, 20.07g of glucose, 42.53g of yeast extract powder, 32g of soytone, 2g of dipotassium hydrogen phosphate, 3g of diammonium hydrogen citrate, 8g of anhydrous sodium acetate, 0.46g of L-cysteine hydrochloride, 0.5g of bile salt and MnSO (total of 1L) 4 0.096g、MgSO 4 •7H 2 O0.71 g, tween80 1mL, 8Mmol/L glycerol, adjusting pH to 6.5, sterilizing at 121deg.C for 15min.
Preferably, the culture medium B is a culture medium A containing 5% -20% (v/v) buffalo milk.
Further preferably, the B medium is A medium containing 10% (v/v) buffalo milk.
Most preferably, the composition of the B medium is as follows: 19.32g of sucrose, 21.84g of maltose, 20.07g of glucose, 42.53g of yeast extract powder, 32g of soybean peptone, 2g of dipotassium hydrogen phosphate, 3g of diammonium hydrogen citrate, 8g of anhydrous sodium acetate, 0.46g of L-cysteine hydrochloride, 0.5g of bile salt and MnSO (total of 1.1L) 4 0.096g、MgSO 4 •7H 2 O0.71 g, tween-80 1mL, glycerol 8Mmol/L, buffalo milk 100ml.
Preferably, the culture medium C is a culture medium A containing 0.5% -5% (m/v) peach gum polysaccharide.
Further preferably, the medium C is medium A containing 1% (m/v) peach gum polysaccharide.
Most preferably, the composition of the medium C is as follows: 19.32g of sucrose, 21.84g of maltose, 20.07g of glucose, 42.53g of yeast extract powder, 32g of soytone, 2g of dipotassium hydrogen phosphate, 3g of diammonium hydrogen citrate, 8g of anhydrous sodium acetate, 0.46g of L-cysteine hydrochloride, 0.5g of bile salt and MnSO (total of 1L) 4 0.096g、MgSO 4 •7H 2 O0.71 g, tween-80 1mL, glycerol 8Mmol/L, and peach gum polysaccharide 10g.
Preferably, the human intestinal tract-imitating digestive juice comprises the following components in parts by weight:
(1) Simulated gastric fluid, gastric electrolyte, 2.80-3.50g NaCl;0.70-1.40g KCl;0.05-0.2g CaCl 2 ；0.50-1.00g NaHCO 3 Deionized water is used for fixing the volume to 1000mL; adding 30.00-40.00mg pepsin into 150mL gastric electrolyte, adding 1.5mL CH3COONa buffer (1.0 mol/L, pH 5.0), magnetically stirring at room temperature for 10min, and adjusting pH to 4.5 (0.5 mol/L HCI);
(2) Simulated small intestine liquid, small intestine electrolyte, 3.0-8.0g NaCl;0.50-1.00g KCl;0.10-0.50g CaCl 2 Deionized water is fixed to 1000mL; 25mL of small intestine electrolyte, 50mL of 4% bile salt solution and 25mL7% pancreatic acid supernatant was mixed and 13mg trypsin was added;
(3) Simulated gastrointestinal fluid simulated gastric fluid and simulated intestinal fluid were mixed in a 1:1 (v/v) ratio.
Further preferably, the human intestinal tract-imitating digestive juice comprises the following components:
(1) Simulated gastric fluid, gastric electrolyte, 3.10g NaCl;1.10g KCl;0.15g CaCl 2 ；0.60g NaHCO 3 Deionized water is used for fixing the volume to 1000mL; to 150mL of the gastric electrolyte was added 35.40mg of pepsin, 1.5mL of CH3COONa buffer (1.0 mol/L, pH 5.0) was added, and the mixture was magnetically stirred at room temperature for 10 minutes to adjust the pH to 4.5 (0.5 mol/L HCI);
(2) Simulated small intestine electrolyte, 5.40g NaCl;0.65g KCl;0.25g CaCl 2 Deionized water is fixed to 1000mL; 25mL of small intestine electrolyte, 50mL of 4% bile salt solution and 25mL of 7% pancreatic acid supernatant were mixed, and 13mg of trypsin was added;
(3) Simulated gastrointestinal fluid simulated gastric fluid and simulated intestinal fluid were mixed in a 1:1 (v/v) ratio.
Preferably, the human intestinal canal imitation micro-ecological system is: simulated gastrointestinal fluid (10-30%) is co-evolution composite culture medium (70-80%).
Further preferably, the human intestinal canal imitation micro-ecological system is: simulated gastrointestinal fluid (10% -20%): co-evolution composite culture medium (80% -90%).
Most preferably, the human intestinal canal imitation micro-ecological system is: simulated gastrointestinal fluid (15%) co-evolving complex medium (85%).
Preferably, the probiotic composite flora imitates human intestinal microecology co-evolution composite culture method comprises the following steps:
step 1: activating strains: marking off (first generation) Lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and Lactobacillus casei C7-2 respectively, picking out single bacterial colony grown out, inoculating into liquid MRS culture medium, culturing at 37deg.C for 16-18h (second generation), inoculating bacterial liquid into fresh liquid MRS culture medium according to 3% (third generation), and culturing at 37deg.C for 22h;
step 2: total inoculum size was 4% and lactobacillus reuteri HBM11-69: pediococcus pentosaceus MPL5: lactobacillus plantarum La-10: lactobacillus rhamnosus HBM11-35: inoculating Lactobacillus casei C7-2 into co-evolution composite culture medium A at an inoculation ratio of 2:2:1:3:3, culturing at 37deg.C for 1 hr, adding 15% volume of human intestinal digestive juice of the culture medium A, culturing at 37deg.C for 0, 2, 4, 6, 8 hr, sampling, and coating for counting; or alternatively
Step 2: total inoculum size was 4% and lactobacillus reuteri HBM11-69: pediococcus pentosaceus MPL5: lactobacillus plantarum La-10: lactobacillus rhamnosus HBM11-35: inoculating Lactobacillus casei C7-2 into an A culture medium co-evolution composite culture medium (B culture medium) containing 5% (v/v) buffalo milk, 10% (v/v) buffalo milk, 15% (v/v) buffalo milk or 20% (v/v) buffalo milk according to the inoculation ratio of 2:2:1:3:3, culturing at 37 ℃ for 1h, adding 15% volume of human intestinal digestion liquid simulated by the B culture medium, culturing at 37 ℃ for 0, 2, 4, 6 and 8h, sampling, coating and counting; or alternatively
Step 2: total inoculum size was 4% and lactobacillus reuteri HBM11-69: pediococcus pentosaceus MPL5: lactobacillus plantarum La-10: lactobacillus rhamnosus HBM11-35: lactobacillus casei C7-2 is inoculated with co-evolution compound culture medium (C culture medium) containing 0.5% (m/v) peach gum polysaccharide, 1% (m/v) peach gum polysaccharide, 3% (m/v) peach gum polysaccharide or 5% (m/v) peach gum polysaccharide A culture medium according to the inoculation proportion of 2:2:1:3:3, after being cultured for 1h at 37 ℃, 15% volume of human intestinal digestion liquid of the C culture medium is added, and the mixture is cultured for 0, 2, 4, 6 and 8h at 37 ℃ for sampling, and the coating count is carried out.
Further preferably, the strain activation operation is as follows: the Lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and Lactobacillus casei C7-2 are respectively streaked (first generation), the single bacterial colony which grows out is selected to be connected with a liquid MRS culture medium, the liquid MRS culture medium is cultured for 16-18 hours at 37 ℃, then the bacterial liquid is connected with a fresh liquid MRS culture medium (third generation) according to 3 percent, and the liquid MRS culture medium is cultured for 22 hours at 37 ℃.
The probiotic composite flora imitates human intestinal microecology co-evolution composite culture, and is prepared by the method.
The application of the probiotic composite flora in the field of probiotic products.
Compared with the prior art, the invention has the following advantages and effects: (1) Provides a co-evolution culture medium, so that a plurality of probiotics can co-produce and the culture cost is reduced. 2) Through in vitro simulation experiments, the results well verify that the compound flora can survive in a large amount in the gastrointestinal tract, and indicate that the compound flora can effectively resist adverse factors in the gastrointestinal tract environment and reach colonic colonisation so as to play a probiotic effect. (3) By improving the culture medium, the viable count is greatly improved, and after the culture medium is subjected to adverse environmental stress, the viable count is still higher.
Drawings
FIG. 1 shows the effect of buffalo milk on the number of viable bacteria in a complex population.
FIG. 2 is a graph showing the effect of buffalo milk on complex bacterial flora survival.
FIG. 3 shows the effect of peach gum polysaccharide on the viable count of complex flora.
Figure 4 is the effect of peach gum polysaccharide on complex flora survival.
The graph represents a significant difference compared to the control group (CK).
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The Lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35, lactobacillus casei C7-2 strains used in the examples described below were obtained from the Proc. Of the university of agricultural food, proc. Of China, group Lin Junfang, assigned by the present company and stored in the microorganism strain collection, guangdong province. Other reagents used in the examples were purchased commercially, wherein buffalo milk was fresh milk produced by buffalo, and accepted according to GB19301-2010 (national food safety standard raw milk). Peach gum polysaccharide was purchased from Shandong Youcao chemical technology Co.
The lactobacillus plantarum La-10 is classified and named Lactiplantibacillus plantarum, is preserved in the Guangdong province microorganism strain preservation center, and has the preservation number of GDMCC No. 63605, the preservation time of 2023 and 6 months and 30 days, and the preservation unit address of Guangzhou national institute of sciences of Guangdong province, namely, no. 100 of Mitsui, no. 59 building 5 of the university, is adopted; pediococcus pentosaceus MPL5, classified and named Pediococcus pentosaceus, is deposited in the Guangdong province microorganism strain deposit center, with deposit number GDMCC No. 63606, deposit time 2023, 6 months and 30 days, and deposit unit address of Guangzhou national institute of sciences microbiological study, guangdong province, no. 100 university, no. 59 building 5; lactobacillus rhamnosus HBM11-35, classified and named Lacticaseibacillus rhamnosus, is deposited at the cantonese institute of microbiological bacterial deposit under accession number GDMCC No. 63607, deposit time 2023, 6 months and 30 days, and deposit unit address is 5 th floor of 100 th institute of middle road, first, and 59 th floor of the cantonese institute of microbiological study; lactobacillus reuteri HBM11-69, classified and named Limosilactobacillus reuteri, is deposited in the Guangdong province microorganism strain collection center, with the deposit number of GDMCC No. 63608, the deposit time of 2023, 6 months and 30 days, and the deposit unit address of Guangzhou national institute of sciences, no. 59 building 5, of Xianzhonglu 100 university, guangdong province; lactobacillus casei C7-2, classified and named Lacticaseibacillus casei, is deposited in the Guangdong province microorganism strain deposit center with the deposit number GDMCC No. 63609, the deposit time of 2023, 6 and 30 days, and the deposit unit address of Guangzhou national institute of sciences of Guangdong province, no. 100, no. 59, no. 5 building.
The human digestive juice-like liquid used in the following examples specifically comprises the following formula:
(1) Simulate gastric juice (gastric electrolyte: 3.10g NaCl;1.10g KCl;0.15g CaCl) 2 ；0.60g NaHCO 3 The method comprises the steps of carrying out a first treatment on the surface of the Volume was set to 1000mL with deionized water). To 150mL of gastric electrolyte was added 35.40mg of pepsin and 1.5mL of CH 3 COONa buffer (1.0 mol/L, pH 5.0). Magnetic stirring at room temperature for 10min, adjusting pH to 4.5 (0.5 mol/L HCI)
(2) Simulated intestinal fluid (intestinal electrolyte), 5.40g NaCl;0.65g KCl;0.25gCaCl 2 The method comprises the steps of carrying out a first treatment on the surface of the Deionized water to 1000 mL). 25mL of small intestine electrolyte, 50mL of 4% bile salt solution and 25mL of 7% pancreatic acid supernatant were mixed, and 13mg of trypsin was added.
(3) Simulated gastric juice, simulated gastric juice and simulated small intestinal juice are mixed according to the volume ratio of 1:1.
Example 1
(1) Activating strains:
and (3) on a MRS solid culture medium flat plate, taking and separating preserved Lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5 lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and Lactobacillus casei C7-2 bacterial liquid, culturing at 37 ℃ for 48 hours, inoculating the single colony on the flat plate into the MRS liquid culture medium, culturing at 37 ℃ for 18 hours, inoculating the single colony into the fresh MRS liquid culture medium again according to the inoculum size of 3%, and culturing for 22 hours to obtain activated bacterial liquid.
(2) The sterilization conditions of the buffalo milk: 30min at 60 ℃.
(3) Preparing a culture medium:
medium a: sucrose 19.32g, maltose 21.84g, glucose 20.07g, yeast extract 42.53g, soybean peptone 32g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 3g, anhydrous sodium acetate 8g, L-cysteine hydrochloride 0.46g, bile salt 0.5g and MnSO 4 0.096g、MgSO 4 •7H 2 O0.71 g, tween-80 1mL, glycerol 8Mmol/L, adding water to constant volume to 1L, adjusting pH to 6.5, and sterilizing at 121deg.C for 15min.
B culture medium: 42.5mL of sterilized A culture medium is taken, 2.125mL of buffalo milk (calculated by 5% of the volume of the culture medium) is added, and the total co-evolution compound culture medium volume is 44.625mL (accounting for 85% of the human intestinal microecological system).
(4) Fermentation culture:
taking activated lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 bacterial liquid, inoculating the bacterial liquid into a B culture medium (the total inoculum size is 4%) according to the inoculation proportion of 2:2:1:3:3, and culturing for 1h at 37 ℃;
(5) Human intestinal canal simulated micro-ecological environment culture:
after 1h of culture, 7.875mL of human intestinal tract-imitating digestive juice (15% of human intestinal tract-imitating microecological system) is added into the culture medium, evenly mixed, sampled immediately (0 h sample) and cultured for 2, 4, 6 and 8 h. Samples were taken and diluted and coated, and the number of viable bacteria and the survival rate were calculated, and the results are shown in tables 1 and 2.
Example 2
(1) Activating strains:
and (3) on a MRS solid culture medium flat plate, taking and separating preserved Lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5 lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and Lactobacillus casei C7-2 bacterial liquid, culturing at 37 ℃ for 48 hours, inoculating the single colony on the flat plate into the MRS liquid culture medium, culturing at 37 ℃ for 18 hours, inoculating the single colony into the fresh MRS liquid culture medium again according to the inoculum size of 3%, and culturing for 22 hours to obtain activated bacterial liquid.
(2) The sterilization conditions of the buffalo milk: 30min at 60 ℃.
(3) Preparing a culture medium:
medium a: sucrose 19.32g, maltose 21.84g, glucose 20.07g, yeast extract 42.53g, soybean peptone 32g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 3g, anhydrous sodium acetate 8g, L-cysteine hydrochloride 0.46g, bile salt 0.5g and MnSO 4 0.096g、MgSO 4 •7H 2 O0.71 g, tween-80 1mL, glycerol 8Mmol/L, adding water to constant volume to 1L, adjusting pH to 6.5, and sterilizing at 121deg.C for 15min.
B culture medium: 42.5mL of sterilized A culture medium is taken, 4.25mL of buffalo milk (calculated by 10% of the volume of the culture medium) is added, and the total co-evolution compound culture medium volume is 46.75mL (accounting for 85% of the human intestinal microecological system).
(4) Fermentation culture:
taking activated lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 bacterial liquid, inoculating the bacterial liquid into a B culture medium (the total inoculum size is 4%) according to the inoculation proportion of 2:2:1:3:3, and culturing for 1h at 37 ℃;
(5) Human intestinal canal simulated micro-ecological environment culture:
after 1h of culture, 8.25mL of human intestinal tract-imitating digestive juice (15% of human intestinal tract-imitating microecological system) is added into the culture medium, evenly mixed, sampled immediately (0 h sample) and cultured for 2, 4, 6 and 8 h.
Samples were taken and diluted and coated, and the number of viable bacteria and the survival rate were calculated, and the results are shown in tables 1 and 2.
Example 3
(1) Activating strains:
and (3) on a MRS solid culture medium flat plate, taking and separating preserved Lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5 lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and Lactobacillus casei C7-2 bacterial liquid, culturing at 37 ℃ for 48 hours, inoculating the single colony on the flat plate into the MRS liquid culture medium, culturing at 37 ℃ for 18 hours, inoculating the single colony into the fresh MRS liquid culture medium again according to the inoculum size of 3%, and culturing for 22 hours to obtain activated bacterial liquid.
(2) The sterilization conditions of the buffalo milk: 30min at 60 ℃.
(3) Preparing a culture medium:
medium a: sucrose 19.32g, maltose 21.84g, glucose 20.07g, yeast extract 42.53g, soybean peptone 32g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 3g, anhydrous sodium acetate 8g, L-cysteine hydrochloride 0.46g, bile salt 0.5g and MnSO 4 0.096g、MgSO 4 •7H 2 O0.71 g, tween-80 1mL, glycerol 8Mmol/L, adding water to constant volume to 1L, adjusting pH to 6.5, and sterilizing at 121deg.C for 15min.
B culture medium: 42.5mL of sterilized A culture medium is taken, 6.375mL of buffalo milk (calculated by 15% of the volume of the culture medium) is added, and the total co-evolution compound culture medium volume is 48.875mL (accounting for 85% of the human intestinal microecological system).
(4) Fermentation culture:
taking activated lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 bacterial liquid, inoculating the bacterial liquid into a B culture medium (the total inoculum size is 4%) according to the inoculation proportion of 2:2:1:3:3, and culturing for 1h at 37 ℃;
(5) Human intestinal canal simulated micro-ecological environment culture:
after 1h of culture, 8.625mL of human intestinal tract-imitating digestive juice (15% of human intestinal tract-imitating microecological system) is added into the culture medium, evenly mixed, sampled immediately (0 h sample) and cultured for 2, 4, 6 and 8 h.
Samples were taken and diluted and coated, and the number of viable bacteria and the survival rate were calculated, and the results are shown in tables 1 and 2.
Example 4
(1) Activating strains:
and (3) on a MRS solid culture medium flat plate, taking and separating preserved Lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5 lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and Lactobacillus casei C7-2 bacterial liquid, culturing at 37 ℃ for 48 hours, inoculating the single colony on the flat plate into the MRS liquid culture medium, culturing at 37 ℃ for 18 hours, inoculating the single colony into the fresh MRS liquid culture medium again according to the inoculum size of 3%, and culturing for 22 hours to obtain activated bacterial liquid.
(2) The sterilization conditions of the buffalo milk: 30min at 60 ℃.
(3) Preparing a culture medium:
medium a: sucrose 19.32g, maltose 21.84g, glucose 20.07g, yeast extract 42.53g, soybean peptone 32g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 3g, anhydrous sodium acetate 8g, L-cysteine hydrochloride 0.46g, bile salt 0.5g and MnSO 4 0.096g、MgSO 4 •7H 2 O0.71 g, tween-80 1mL, glycerol 8Mmol/L, adding water to constant volume to 1L, adjusting pH to 6.5, and sterilizing at 121deg.C for 15min.
B culture medium: taking 42.5mL of sterilized A culture medium, adding 8.5mL of buffalo milk (calculated by 20% of the volume of the culture medium), and the total co-evolution compound culture medium volume is 51mL (accounting for 85% of the human intestinal microecological system).
(4) Fermentation culture:
taking activated lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 bacterial liquid, inoculating the bacterial liquid into a B culture medium (the total inoculum size is 4%) according to the inoculation proportion of 2:2:1:3:3, and culturing for 1h at 37 ℃;
(5) Human intestinal canal simulated micro-ecological environment culture:
after 1h of culture, 9mL of human intestinal tract-imitating digestive juice (15% of human intestinal tract-imitating microecological system) is added into the culture medium, evenly mixed, sampled immediately (0 h sample) and cultured for 2, 4, 6 and 8 h.
Samples were taken and diluted and coated, and the number of viable bacteria and the survival rate were calculated, and the results are shown in tables 1 and 2.
The results of the above examples are shown in FIGS. 1 and 2, respectivelyThe influence of buffalo milk on the viable count of the complex bacterial flora and the survival rate of the complex bacterial flora after 2, 4, 6 and 8 hours of culture are shown. As shown in FIG. 1, when 10% buffalo milk was added to the medium, the viable count could reach 10 after 1 hour of cultivation 8 However, with the addition of the simulated digestive system, the viable count tends to decrease and then increase, and after 8 hours of culture, the viable count still remains 7.8X10 7 As shown in fig. 2, when 10% buffalo milk is added, the survival rate of the complex bacterial group is relatively low, which is about 50%; compared with a non-added digestion simulation system, after 1h of culture, 10% buffalo milk obviously enhances the growth and reproduction of flora. The buffalo milk has a certain growth promoting effect on the compound flora and has a certain effect of resisting adverse environment.
Example 5
(1) Activating strains:
and (3) on a MRS solid culture medium flat plate, taking and separating preserved Lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5 lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and Lactobacillus casei C7-2 bacterial liquid, culturing at 37 ℃ for 48 hours, inoculating the single colony on the flat plate into the MRS liquid culture medium, culturing at 37 ℃ for 18 hours, inoculating the single colony into the fresh MRS liquid culture medium again according to the inoculum size of 3%, and culturing for 22 hours to obtain activated bacterial liquid.
(2) Preparing a culture medium:
medium a: sucrose 19.32g, maltose 21.84g, glucose 20.07g, yeast extract 42.53g, soybean peptone 32g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 3g, anhydrous sodium acetate 8g, L-cysteine hydrochloride 0.46g, bile salt 0.5g and MnSO 4 0.096g、MgSO 4 •7H 2 O0.71 g, tween-80 1mL, glycerol 8Mmol/L, adding water to constant volume to 1L, adjusting pH to 6.5, and sterilizing at 121deg.C for 15min.
C culture medium: 42.5mL of A medium with pH adjusted to 6.5 is taken, 0.213g (calculated as 0.5% (m/v) of the medium) of peach gum polysaccharide is added, and sterilization is carried out at 121 ℃ for 15min.
(3) Fermentation culture:
taking activated lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 bacterial liquid, inoculating the bacterial liquid into a C culture medium according to an inoculation proportion of 2:2:1:3:3 (the total inoculation amount is 4%), and culturing for 1h at 37 ℃;
(4) Human intestinal canal simulated micro-ecological environment culture:
after 1h of culture, 7.5mL of human intestinal tract-imitating digestive juice (15% of human intestinal tract-imitating microecological system) is added into the culture medium, evenly mixed, sampled immediately (0 h sample) and cultured for 2, 4, 6 and 8 h.
Samples were taken and diluted and coated, and the number of viable bacteria and the survival rate were calculated, and the results are shown in tables 1 and 2.
Example 6
(1) Activating strains:
and (3) on a MRS solid culture medium flat plate, taking and separating preserved Lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5 lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and Lactobacillus casei C7-2 bacterial liquid, culturing at 37 ℃ for 48 hours, inoculating the single colony on the flat plate into the MRS liquid culture medium, culturing at 37 ℃ for 18 hours, inoculating the single colony into the fresh MRS liquid culture medium again according to the inoculum size of 3%, and culturing for 22 hours to obtain activated bacterial liquid.
(2) Preparing a culture medium:
medium a: sucrose 19.32g, maltose 21.84g, glucose 20.07g, yeast extract 42.53g, soybean peptone 32g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 3g, anhydrous sodium acetate 8g, L-cysteine hydrochloride 0.46g, bile salt 0.5g and MnSO 4 0.096g、MgSO 4 •7H 2 O0.71 g, tween-80 1mL, glycerol 8Mmol/L, adding water to constant volume to 1L, adjusting pH to 6.5, and sterilizing at 121deg.C for 15min.
C culture medium: 42.5mL of A medium with pH adjusted to 6.5 is taken, 0.425g (calculated as 1% (m/v) of the medium) of peach gum polysaccharide is added, and sterilization is carried out for 15min at 121 ℃.
(3) Fermentation culture:
taking activated lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 bacterial liquid, inoculating the bacterial liquid into a C culture medium according to an inoculation proportion of 2:2:1:3:3 (the total inoculation amount is 4%), and culturing for 1h at 37 ℃;
(4) Human intestinal canal simulated micro-ecological environment culture:
after 1h of culture, 8.625mL of human intestinal tract-imitating digestive juice (15% of human intestinal tract-imitating microecological system) is added into the culture medium, evenly mixed, sampled immediately (0 h sample) and cultured for 2, 4, 6 and 8 h.
Samples were taken and diluted and coated, and the number of viable bacteria and the survival rate were calculated, and the results are shown in tables 1 and 2.
Example 7
(1) Activating strains:
and (3) on a MRS solid culture medium flat plate, taking and separating preserved Lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5 lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and Lactobacillus casei C7-2 bacterial liquid, culturing at 37 ℃ for 48 hours, inoculating the single colony on the flat plate into the MRS liquid culture medium, culturing at 37 ℃ for 18 hours, inoculating the single colony into the fresh MRS liquid culture medium again according to the inoculum size of 3%, and culturing for 22 hours to obtain activated bacterial liquid.
(2) Preparing a culture medium:
medium a: sucrose 19.32g, maltose 21.84g, glucose 20.07g, yeast extract 42.53g, soybean peptone 32g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 3g, anhydrous sodium acetate 8g, L-cysteine hydrochloride 0.46g, bile salt 0.5g and MnSO 4 0.096g、MgSO 4 •7H 2 O0.71 g, tween-80 1mL, glycerol 8Mmol/L, adding water to constant volume to 1L, adjusting pH to 6.5, and sterilizing at 121deg.C for 15min.
C culture medium: 42.5mL of A medium with pH adjusted to 6.5 is taken, 0.85g (calculated as 2% (m/v) of the medium) of peach gum polysaccharide is added, and sterilization is carried out at 121 ℃ for 15min.
(3) Fermentation culture:
taking activated lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 bacterial liquid, inoculating the bacterial liquid into a C culture medium according to an inoculation proportion of 2:2:1:3:3 (the total inoculation amount is 4%), and culturing for 1h at 37 ℃;
(4) Human intestinal canal simulated micro-ecological environment culture:
after 1h of culture, 8.625mL of human intestinal tract-imitating digestive juice (15% of human intestinal tract-imitating microecological system) is added into the culture medium, evenly mixed, sampled immediately (0 h sample) and cultured for 2, 4, 6 and 8 h.
Samples were taken and diluted and coated, and the number of viable bacteria and the survival rate were calculated, and the results are shown in tables 1 and 2.
Example 8
(1) Activating strains:
and (3) on a MRS solid culture medium flat plate, taking and separating preserved Lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5 lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and Lactobacillus casei C7-2 bacterial liquid, culturing at 37 ℃ for 48 hours, inoculating the single colony on the flat plate into the MRS liquid culture medium, culturing at 37 ℃ for 18 hours, inoculating the single colony into the fresh MRS liquid culture medium again according to the inoculum size of 3%, and culturing for 22 hours to obtain activated bacterial liquid.
(2) Preparing a culture medium:
medium a: sucrose 19.32g, maltose 21.84g, glucose 20.07g, yeast extract 42.53g, soybean peptone 32g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 3g, anhydrous sodium acetate 8g, L-cysteine hydrochloride 0.46g, bile salt 0.5g and MnSO 4 0.096g、MgSO 4 •7H 2 O0.71 g, tween-80 1mL, glycerol 8Mmol/L, adding water to constant volume to 1L, adjusting pH to 6.5, and sterilizing at 121deg.C for 15min.
C culture medium: 42.5mL of A medium with pH of 6.5 was added with 1.275g (calculated as 3% (m/v) of medium) of peach gum polysaccharide, and sterilized at 121℃for 15min.
(3) Fermentation culture:
taking activated lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 bacterial liquid, inoculating the bacterial liquid into a C culture medium according to an inoculation proportion of 2:2:1:3:3 (the total inoculation amount is 4%), and culturing for 1h at 37 ℃;
(4) Human intestinal canal simulated micro-ecological environment culture:
after 1h of culture, 7.5mL of human intestinal tract-imitating digestive juice (15% of human intestinal tract-imitating microecological system) is added into the culture medium, evenly mixed, sampled immediately (0 h sample) and cultured for 2, 4, 6 and 8 h.
Samples were taken and diluted and coated, and the number of viable bacteria and the survival rate were calculated, and the results are shown in tables 1 and 2.
Example 9
(1) Activating strains:
and (3) on a MRS solid culture medium flat plate, taking and separating preserved Lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5 lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and Lactobacillus casei C7-2 bacterial liquid, culturing at 37 ℃ for 48 hours, inoculating the single colony on the flat plate into the MRS liquid culture medium, culturing at 37 ℃ for 18 hours, inoculating the single colony into the fresh MRS liquid culture medium again according to the inoculum size of 3%, and culturing for 22 hours to obtain activated bacterial liquid.
(2) Preparing a culture medium:
medium a: sucrose 19.32g, maltose 21.84g, glucose 20.07g, yeast extract 42.53g, soybean peptone 32g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 3g, anhydrous sodium acetate 8g, L-cysteine hydrochloride 0.46g, bile salt 0.5g and MnSO 4 0.096g、MgSO 4 •7H 2 O0.71 g, tween-80 1mL, glycerol 8Mmol/L, adding water to constant volume to 1L, adjusting pH to 6.5, and sterilizing at 121deg.C for 15min.
C culture medium: 42.5mL of A medium with pH adjusted to 6.5 is taken, 2.125g (calculated as 5% (m/v) of the medium) of peach gum polysaccharide is added, and sterilization is carried out at 121 ℃ for 15min.
(3) Fermentation culture:
taking activated lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 bacterial liquid, inoculating the bacterial liquid into a C culture medium according to an inoculation proportion of 2:2:1:3:3 (the total inoculation amount is 4%), and culturing for 1h at 37 ℃;
(4) Human intestinal canal simulated micro-ecological environment culture:
after 1h of culture, 7.5mL of human intestinal tract-imitating digestive juice (15% of human intestinal tract-imitating microecological system) is added into the culture medium, evenly mixed, sampled immediately (0 h sample) and cultured for 2, 4, 6 and 8 h.
Samples were taken and diluted and coated, and the number of viable bacteria and the survival rate were calculated, and the results are shown in tables 1 and 2.
Examples 5 to 6 show the results of FIGS. 3 and 4, respectively, showing the effect of adding peach gum to the culture system on the number of viable bacteria of the complex bacterial groupSurvival rate of complex bacterial groups after 2, 4, 6 and 8 hours of cultivation. As shown in FIG. 1, when 1% peach gum polysaccharide is added into the culture medium, a digestion simulation system is added, the number of viable bacteria is gradually increased after 2 hours of culture, and the number of viable bacteria reaches 6.6X10 after 8 hours of culture 7 The method comprises the steps of carrying out a first treatment on the surface of the As shown in fig. 2, the survival rate of the complex bacterial group is gradually increased, and the survival rate is more than 100% after 4 hours; the peach gum can promote the growth of fungus groups and has better effect on resisting adverse environments.
The total inoculation amount in the example is calculated according to the total co-evolution compound culture medium volume, and then the bacterial liquid is inoculated into each culture medium according to the inoculation proportion.
The control group is 42.5mL of A culture medium, lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 are inoculated into the A culture medium according to the inoculation proportion of 2:2:1:3:3, after being cultured for 1h at 37 ℃, 7.5mL of human intestinal digestion liquid is added, evenly mixed, and cultured for 0, 2, 4, 6 and 8h at 37 ℃ for sampling, dilution coating is carried out, and the number of living bacteria and the survival rate are calculated.
Comparative example 1
The same as in examples 1-8 except that no buffalo milk or peach gum polysaccharide was added to the A medium, and after 1 hour of incubation, the human-simulated digestive juice was added, and after 0, 2, 4, 6, 8 hours of incubation at 37℃were sampled, diluted and spread, and the viable count and viability were calculated.
The total viable count measurement results of the complex bacterial groups of examples 1 to 8 and comparative examples 1 to 3 are shown in tables 1 and 2 below:
TABLE 1 results of total viable count of composite strains per hour
| Additive component | Number of viable bacteria for 0h | Number of viable bacteria for 2 hours | Number of viable bacteria for 4 hours | Number of viable bacteria for 6 hours | Number of viable bacteria for 8 hours | |
| Example 1 | 5% buffalo milk | 5.24×10 7 | 5.14×10 7 | 4.29×10 7 | 4.18×10 7 | 4.05×10 7 |
| Example 2 | 10% buffalo milk | 12.0×10 7 | 8.41×10 7 | 7.8×10 7 | 8.32×10 7 | 7.87×10 7 |
| Example 3 | 15% buffalo milk | 6.08×10 7 | 5.23×10 7 | 4.16×10 7 | 5.98×10 7 | 4.36×10 7 |
| Example 4 | 20% buffalo milk | 8.05×10 7 | 7.98×10 7 | 8.06×10 7 | 10.6×10 7 | 8.37×10 7 |
| Example 5 | Peach gum polysaccharide 0.5% | 5.52×10 7 | 5.68×10 7 | 4.87×10 7 | 4.75×10 7 | 5.6×10 7 |
| Example 6 | 1% peach gum polysaccharide | 5.7×10 7 | 4.93×10 7 | 5.2×10 7 | 5.63×10 7 | 6.6×10 7 |
| Example 7 | 2% peach gum polysaccharide | 6.77×10 7 | 6.0×10 7 | 5.57×10 7 | 5.12×10 7 | 6.97×10 7 |
| Example 8 | 3% peach gum polysaccharide | 5.87×10 7 | 5.05×10 7 | 6.14×10 7 | 6.49×10 7 | 5.43×10 7 |
| Example 9 | 5% peach gum polysaccharide | 6.33×10 7 | 4.45×10 7 | 4.88×10 7 | 5.1×10 7 | 5.98×10 7 |
| Comparative example 1 (blank) | 5.14×10 7 | 3.93×10 7 | 4.05×10 7 | 3.59×10 7 | 3.47×10 7 |
TABLE 2 survival of Complex microbial populations after digestion with simulated gastrointestinal fluids
| Survival rate of 0-2 h | Survival rate of 0-4 h | Survival rate of 0-6 h | Survival rate of 0-8 h | |
| Example 1 | 98.09% | 81.87% | 79.77% | 77.29% |
| Example 2 | 70.08% | 65% | 69.33% | 65.58% |
| Example 3 | 86.02% | 68.42% | 98.36% | 71.71% |
| Example 4 | 99.13% | 100.12% | 131.68% | 103.98% |
| Example 5 | 103.02% | 88.22% | 86.10% | 101.51% |
| Example 6 | 86.49% | 91.23% | 98.77% | 115.79% |
| Example 7 | 88.67% | 82.27% | 76.35% | 102.96% |
| Example 8 | 86.02% | 104.72% | 110.63% | 92.61% |
| Example 9 | 70.26% | 77.11% | 80.53% | 94.37% |
| Comparative example 1 (blank) | 76.46% | 78.79% | 69.84% | 67.51% |
The embodiments described above are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be made in the equivalent manner, and are included in the scope of the present invention.
Claims (10)
1. A method for culturing a probiotic composite flora by simulating human intestinal microecology co-evolution is characterized by comprising the following steps: lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 strain are respectively continuously passaged for 2-3 times, the 2-3 times of passaging time is 16-24 hours, bacterial liquid of the last passaging is proportionally taken and added into the following co-evolution compound culture medium for fermentation culture, and human intestinal microecological system-simulating culture is carried out, wherein the co-evolution compound culture medium is a culture medium A, a culture medium B or a culture medium C:
(1) Inoculating the mixture into a culture medium A, fermenting and culturing for 0-24 h at 35-37 ℃, and then adding human intestinal digestion liquid, and culturing for 0-8 h at 35-37 ℃, or alternatively;
(2) Inoculating the mixture into a B culture medium, fermenting and culturing for 0-24 h at 35-37 ℃, and then adding human intestinal digestion liquid, and culturing for 0-8 h at 35-37 ℃, or alternatively;
(3) Inoculating the mixture into a C culture medium, fermenting and culturing for 0 to 24 hours at the temperature of between 35 and 37 ℃, and then adding human intestinal digestion liquid, and culturing for 0 to 8 hours at the temperature of between 35 and 37 ℃;
wherein, the A culture medium comprises the following components: 15-25g of sucrose, 17-30g of maltose, 16-30g of glucose, 30-50g of yeast extract powder, 25-40g of soybean peptone, 1-3g of dipotassium hydrogen phosphate, 2-5g of diammonium hydrogen citrate, 5-10g of anhydrous sodium acetate, 0.2-1g of L-cysteine hydrochloride, 0.1-1g of bile salt and MnSO (total of 1L-cysteine hydrochloride) 4 0.05-0.1g、MgSO 4 •7H 2 O0.5-1 g, tween-80 0.5-2 mL and glycerol5-15Mmol/L; sterilizing at 121 deg.c and pH of 6.0-7.0 for 15-30 min;
the culture medium B is a culture medium A containing 5-20% v/v buffalo milk; sterilizing at 121 deg.c and pH of 6.0-7.0 for 15-30 min; wherein, the buffalo milk is sterilized independently, and the temperature is 60 ℃ for 30min;
the culture medium C is a culture medium A containing 0.5-5% m/v peach gum polysaccharide; sterilizing at 121 ℃ for 15-30 min at pH 6.0-7.0.
2. The method for cultivating a probiotic composite flora by human intestinal microecological co-evolution according to claim 1, wherein the method comprises the following steps: the probiotic composite flora imitates human intestinal microecology co-evolution composite culture method comprises the steps of continuously passaging lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 strain for 3 times respectively, wherein the passaging time for the 3 rd time is 22 hours, and proportionally taking bacterial liquid of the last passaging to enter a culture medium for fermentation culture.
3. The method for culturing the probiotic composite flora by simulating human intestinal microecology co-evolution according to claim 1 or 2, wherein the method comprises the following steps of: the probiotic composite flora imitates human intestinal microecological co-evolution composite culture method comprises the steps of respectively passaging lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 for 3 times, wherein the 3 rd time passaging time is 22 hours, and the culture time is according to lactobacillus reuteri HBM11-69: pediococcus pentosaceus MPL5: lactobacillus plantarum La-10: lactobacillus rhamnosus HBM11-35: lactobacillus casei C7-2=2:2:1:3:3 was inoculated into the co-evolving complex medium.
4. The method for culturing the probiotic composite flora by simulating human intestinal microecology co-evolution according to claim 1 or 2, wherein the method comprises the following steps of:
and inoculating the bacterial liquid into a co-evolution composite culture medium for fermentation culture, wherein the culture time is 1 hour.
5. The method for culturing the probiotic composite flora by simulating human intestinal microecology co-evolution according to claim 1 or 2, wherein the method comprises the following steps of:
the A culture medium comprises the following components: 19.32g of sucrose, 21.84g of maltose, 20.07g of glucose, 42.53g of yeast extract powder, 32g of soytone, 2g of dipotassium hydrogen phosphate, 3g of diammonium hydrogen citrate, 8g of anhydrous sodium acetate, 0.46g of L-cysteine hydrochloride, 0.5g of bile salt, 0.096g of MnSO4, 0.71g of MgSO4.7H2O, 80 mL of tween-80 and 8Mmol/L of glycerin; sterilizing at 121 ℃ for 15min at pH 6.0-7.0;
the culture medium B is a culture medium A containing 5-20% v/v buffalo milk; sterilizing at 121 deg.c and pH of 6.0-7.0 for 15min; wherein, the buffalo milk is sterilized independently, and the temperature is 60 ℃ for 30min;
the culture medium C is a culture medium A of 0.5-5% m/v peach gum polysaccharide; sterilizing at 121 deg.c and pH of 6.0-7.0 for 15min.
6. The method for culturing the probiotic composite flora by simulating human intestinal microecology co-evolution according to claim 1 or 2, wherein the method comprises the following steps of:
the human intestinal tract-imitating digestive juice comprises the following components in parts by weight:
(1) Simulated gastric fluid, gastric electrolyte, 2.80-3.50g NaCl;0.70-1.40g KCl;0.05-0.2g CaCl 2 ；0.50～1.00g NaHCO 3 Deionized water is used for fixing the volume to 1000mL; adding 30.00-40.00mg pepsin into 150mL gastric electrolyte, adding 1.5mL CH with concentration of 1.0mol/L and pH of 5.0 3 COONa buffer solution, magnetically stirring at room temperature for 10min, adding HCI with the concentration of 0.5mol/L, and adjusting the pH to 4.5;
(2) Simulated small intestine liquid, namely small intestine electrolyte, 3.0-8.0g NaCl;0.50-1.00g KCl;0.10 to 0.50g CaCl 2 Deionized water is fixed to 1000mL; 25mL of small intestine electrolyte, 50mL of 4% bile salt solution and 25mL of 7% pancreatic acid supernatant were mixed, and 13mg of trypsin was added;
(3) Simulated gastric fluid and simulated intestinal fluid are mixed in a ratio of 1:1 v/v.
7. The method for culturing the probiotic composite flora by simulating human intestinal microecology co-evolution according to claim 1 or 2, wherein the method comprises the following steps of:
the human intestinal canal imitation micro-ecological system is v/v 15% human intestinal canal imitation digestive juice and 85% co-evolution composite culture medium.
8. The method for culturing the probiotic composite flora by simulating human intestinal microecology co-evolution according to claim 1 or 2, wherein the method comprises the following steps of:
the probiotic composite flora imitates human intestinal microecology co-evolution composite culture method comprises the following steps:
step 1: activating strains: marking off Lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and Lactobacillus casei C7-2 to form the first generation, selecting single bacterial colony, inoculating into liquid MRS culture medium, culturing at 37deg.C for 16-18h to form the second generation, inoculating bacterial liquid into fresh liquid MRS culture medium according to 3%, and culturing at 37deg.C for 22h to form the third generation;
step 2: total inoculum size was 4% and lactobacillus reuteri HBM11-69: pediococcus pentosaceus MPL5: lactobacillus plantarum La-10: lactobacillus rhamnosus HBM11-35: inoculating Lactobacillus casei C7-2 into co-evolution composite culture medium A at an inoculation ratio of 2:2:1:3:3, culturing at 37deg.C for 1 hr, adding 15% volume of human intestinal digestive juice of the culture medium A, culturing at 37deg.C for 0, 2, 4, 6, 8 hr, sampling, and coating for counting; or alternatively
Step 2: total inoculum size was 4% and lactobacillus reuteri HBM11-69: pediococcus pentosaceus MPL5: lactobacillus plantarum La-10: lactobacillus rhamnosus HBM11-35: inoculating Lactobacillus casei C7-2 into co-evolution composite culture medium B containing 5% v/v buffalo milk, 10% v/v buffalo milk, 15% v/v buffalo milk or 20% v/v buffalo milk according to the inoculation ratio of 2:2:1:3:3, culturing at 37deg.C for 1 hr, adding 15% volume of human intestinal digestion simulating liquid of B culture medium, culturing at 37deg.C for 0, 2, 4, 6, 8 hr, sampling, coating, and counting; or alternatively
Step 2: total inoculum size was 4% and lactobacillus reuteri HBM11-69: pediococcus pentosaceus MPL5: lactobacillus plantarum La-10: lactobacillus rhamnosus HBM11-35: lactobacillus casei C7-2 is inoculated with a co-evolution compound culture medium C culture medium containing 0.5% m/v peach gum polysaccharide, 1% m/v peach gum polysaccharide, 3% m/v peach gum polysaccharide or 5% m/v peach gum polysaccharide according to the inoculation proportion of 2:2:1:3:3, after being cultured for 1h at 37 ℃, 15% volume of human intestinal digestion liquid is added into the C culture medium, and the mixture is cultured for 0, 2, 4, 6 and 8h at 37 ℃ for sampling, coating and counting.
9. A probiotic composite flora article, obtainable by the process of any one of claims 1-8.
10. Use of a probiotic composite flora preparation according to claim 9 for the preparation of a probiotic preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311497068.0A CN117264852B (en) | 2023-11-10 | 2023-11-10 | Method for cultivating probiotics composite flora by simulating human intestinal microecology co-evolution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311497068.0A CN117264852B (en) | 2023-11-10 | 2023-11-10 | Method for cultivating probiotics composite flora by simulating human intestinal microecology co-evolution |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117264852A true CN117264852A (en) | 2023-12-22 |
| CN117264852B CN117264852B (en) | 2024-01-23 |
Family
ID=89204789
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311497068.0A Active CN117264852B (en) | 2023-11-10 | 2023-11-10 | Method for cultivating probiotics composite flora by simulating human intestinal microecology co-evolution |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117264852B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20220354907A1 (en) * | 2019-06-19 | 2022-11-10 | Solarea Bio, Inc. | Microbial compositions and methods for producing combined probiotic assemblages |
| CN116606784A (en) * | 2023-07-18 | 2023-08-18 | 华南农业大学 | Application of novel Lactobacillus reuteri anti-freezing protective agent in vacuum freeze drying process |
-
2023
- 2023-11-10 CN CN202311497068.0A patent/CN117264852B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20220354907A1 (en) * | 2019-06-19 | 2022-11-10 | Solarea Bio, Inc. | Microbial compositions and methods for producing combined probiotic assemblages |
| CN116606784A (en) * | 2023-07-18 | 2023-08-18 | 华南农业大学 | Application of novel Lactobacillus reuteri anti-freezing protective agent in vacuum freeze drying process |
Non-Patent Citations (3)
| Title |
|---|
| ILIYA D. KWOJI 等: "Multi-Strain Probiotics: Synergy among Isolates Enhances Biological Activities", 《BIOLOGY》, vol. 10, no. 4 * |
| 崔美兰 等: "益生菌复合培养及发酵条件研究", 《中国食品科学技术学会第十一届年会论文摘要集》, pages 281 - 282 * |
| 王彦玲 等: "3种益生菌的混合发酵探索", 《饲料研究》, no. 2, pages 48 - 50 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117264852B (en) | 2024-01-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102660461A (en) | Microbial preparation for shortening tobacco fermentation period and application of microbial preparation | |
| CN107541477B (en) | Method for culturing photosynthetic bacteria by using lactobacillus fermentation liquor | |
| CN113604402A (en) | Specific lactic acid bacteria culture medium and culture method and application thereof | |
| CN116676225B (en) | Lactobacillus rhamnosus LR-28 strain with nerve soothing and sleep aiding effects, fermentation product, hypnotic fungus group mixture and application | |
| KR101134324B1 (en) | Media of powder for mass culture of microorganism | |
| CN112608861A (en) | Composite preparation containing clostridium butyricum and pediococcus acidilactici as well as preparation method and application of composite preparation | |
| WO2022083007A1 (en) | Fermentation method for producing high-content bacillus coagulans spore probiotics by means of using cocoa powder and use thereof | |
| CN102417892A (en) | Feeding lactobacillus johnsonii high-density fermentation medium and application thereof | |
| CN117264852B (en) | Method for cultivating probiotics composite flora by simulating human intestinal microecology co-evolution | |
| CN110607253B (en) | Streptococcus thermophilus and proliferation culture method and application thereof | |
| CN102071165A (en) | Method for improving biomass of lactic acid bacteria at low pH by adding glutamic acid | |
| CN106148212A (en) | A kind of feeding enterococcus faecalis high density fermentation culture medium and fermentation process thereof | |
| CN107299071A (en) | A kind of enteric probiotics preparation | |
| CN114958655B (en) | Clostridium Ding Suanxing produced from pit mud produced by white spirit brewing and application thereof | |
| CN102399728B (en) | Lactobacillus and fermentation method thereof | |
| CN113293101B (en) | Inactivation method and application of lactic acid bacteria | |
| CN114854647A (en) | Lactobacillus fermentum and culture and application thereof | |
| CN109536422B (en) | Aerobic high-density culture method of lactic acid bacteria | |
| KR100801143B1 (en) | Method for culturing of mixture of bacillus polyfermenticus and saccharomyces cerevisiae | |
| CN110846241B (en) | Bifidobacterium animalis capable of decomposing and utilizing human milk oligosaccharide, culture method thereof and food or medicine | |
| CN108690820B (en) | High ampicillin resistance lactobacillus plantarum and breeding method and application thereof | |
| CN113397170A (en) | Marine prebiotic composition for regulating human intestinal flora and application thereof | |
| CN111647521A (en) | Lactobacillus GM _1 and breeding method thereof | |
| US20230310523A1 (en) | Use of lactic acid bacteria or fermentation product thereof for maintaining or improving gastrointestinal condition | |
| CN107723251A (en) | A kind of saccharomyces boulardii high-density cultivation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |