CN117264852B - 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
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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, probiotics are able to survive and survive the human gastrointestinal tract on the premise that they exert their probiotic effectsThe proliferation, because the gastrointestinal tract is in a low-acidity environment and a large amount of digestive enzymes exist, the probiotic bacteria are stressed by the adverse environment of the gastrointestinal tract after being ingested, so that the number of the viable bacteria of the probiotic bacteria is greatly reduced, and the probiotic effect cannot be effectively exerted. 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 0.71g of O, 1mL of Tween-80, 8Mmol/L of glycerol, adjusting pH to 6.5, and 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 and 0.46g of L-cysteine hydrochloride according to 1.1Lg. Bile salt 0.5g, mnSO 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 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.
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 temperature10min, adjusting to pH 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, which show the effect of adding buffalo milk to the culture system on the number of viable bacteria of the complex bacterial groups, and the survival rate of the complex bacterial groups after 2, 4, 6 and 8 hours of culture, respectively. 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%; general purpose medicineCompared 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.
The results of examples 5 to 6 are shown in FIGS. 3 and 4, and show the effect of adding peach gum to the culture system on the number of viable bacteria of the complex bacterial groups, and the survival rate of the complex bacterial groups after 2, 4, 6 and 8 hours of culture, respectively. 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 group and resistThe protective effect on the bad environment is better.
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 gumPolysaccharide | 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 (8)
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 subculture time is 16-24 hours, bacterial liquid of the last passaging is proportionally taken to be inoculated into a co-evolution composite culture medium for fermentation culture, and human intestinal microecological system-simulating culture is carried out, wherein the co-evolution composite culture medium is a B culture medium or a C culture medium:
(1) 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;
(2) 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 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, wherein the pH is 6.0-7.0;
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 deposit number of the lactobacillus reuteri HBM11-69 is GDMCC NO:63608, the Pediococcus pentosaceus MPL5 has a deposit number of GDMCC NO:63606 the Lactobacillus plantarum La-10 has a deposit number of GDMCC NO:63605 the deposit number of lactobacillus rhamnosus HBM11-35 is GDMCC NO:63607, the Lactobacillus casei C7-2 has accession number GDMCC NO:63609.
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: lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 are respectively continuously passaged for 3 times, the time of the 3 rd passaging is 22 hours, and the bacterial liquid of the last passaging is proportionally taken to be added into 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 total inoculation amount is 4%, after lactobacillus reuteri HBM11-69, pediococcus pentosaceus MPL5, lactobacillus plantarum La-10, lactobacillus rhamnosus HBM11-35 and lactobacillus casei C7-2 strain are respectively passaged for 3 times, the 3 rd time of subculture is 22 hours, and the lactobacillus reuteri HBM11-69: pediococcus pentosaceus MPL5: lactobacillus plantarum La-10: lactobacillus rhamnosus HBM11-35: lactobacillus casei C7-2 was inoculated into the co-evolving complex medium at an inoculation ratio of 2:2:1:3:3.
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 preparation method of the human intestinal tract-imitating digestive juice comprises the following steps:
(1) Simulated gastric fluid, gastric electrolyte, 2.80-3.50g NaCl;0.70-1.40g KCl;0.05-0.2g CaCl2; 0.50-1.00g NaHCO3, and deionized water is used for fixing the volume to 1000mL; adding 30.00-40.00mg of pepsin into 150mL of gastric electrolyte, adding 1.5mL of CH3COONa buffer solution with the concentration of 1.0mol/L and the pH of 5.0, magnetically stirring for 10min at room temperature, and adding HCI with the concentration of 0.5mol/L to adjust the pH to 4.5;
(2) Simulated small intestine liquid, namely small intestine electrolyte, 3.0-8.0g NaCl;0.50-1.00g KCl;0.10-0.50g CaCl2, deionized water 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 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 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.
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