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CN-121975651-A - Composite lactobacillus, metaplasia and preparation method and application thereof

CN121975651ACN 121975651 ACN121975651 ACN 121975651ACN-121975651-A

Abstract

The invention discloses a composite lactobacillus, a metazoan and a preparation method and application thereof. The synergistic effect of various strains is exerted by the compound compatibility of probiotics in the yak yoghourt separated from Qinghai-Tibet plateau, the negative influence caused by using a medicine means for treatment is avoided, no side effect is caused, and the problems of single beneficial bacteria group, low regulatory capacity and the like in the prior art are solved. The addition of the selective auxiliary materials not only improves the survival rate of the composite probiotics after freeze-drying treatment, but also enables the probiotics to resist the bad environment of the gastrointestinal tract more effectively, solves the problems that the probiotics are easily damaged by gastric acid, the survival rate is reduced in the storage process, a large amount of the probiotics are inactivated before entering the intestinal tract and the like in the prior art, improves the field planting rate of the probiotics, and has longer and more stable effect. The synergistic compatibility of the compound probiotics and the functional prebiotics derived from the characteristic plant resources of Qinghai-Tibet plateau not only improves the regulating capability of intestinal flora balance, but also increases the beneficial effects of effectively improving constipation, obesity and other symptoms.

Inventors

  • TANG XIAOHONG
  • DANG JUN
  • TANG HONGYU
  • WANG QILAN
  • SUN CE

Assignees

  • 青海瑞湖生物资源开发有限公司
  • 中国科学院西北高原生物研究所

Dates

Publication Date
20260505
Application Date
20251210

Claims (10)

  1. 1. A compound lactic acid bacterium, characterized in that the compound lactic acid bacterium comprises two or more of Lactiplantibacillus plantarum、Lacticaseibacillus rhamnosus、Lactobacillus delbrueckii、Streptococcus thermophiles、Bifidobacterium longum、Bifidobacterium animalissubsp. lactis、 Bifidobacterium breve、Lacticaseibacillus paracasei、Lactobacillus acidophilus、Pediococcus acidilactici、Bifidobacterium adolescentis、Bifidobacterium bifidum; the serial numbers are NWIPB-1, NWIPB-2, NWIPB-3, NWIPB-4, NWIPB-5, NWIPB-6, NWIPB-7, NWIPB-8, NWIPB-9, NWIPB-10, NWIPB-11 and NWIPB-12 respectively, are separated from self-made yak yoghourt and are preserved in the common microorganism center of China Committee for culture Collection of microorganisms, NWIPB-1 with preservation number of CGMCC No. 1.65177 and preservation date of 2025, 11 and 5 days, wherein the nucleotide sequence of the 16SrRNA and the pheS gene sequence of NWIPB-1 are shown as SEQ ID NO. 2; NWIPB-2 with preservation number of CGMCC No. 1.65185 and preservation date of 2025, 11 months and 5 days, wherein the nucleotide sequence of the 16SrRNA and the dnaK gene sequence of NWIPB-2 are shown as SEQ ID NO. 2; NWIPB-3 with preservation number of CGMCC No. 1.65178 and preservation date of 2025, 11 months and 5 days, wherein the nucleotide sequence of the 16SrRNA and the pheS gene sequence of NWIPB-3 are shown as SEQ ID NO. 2; NWIPB-4 with preservation number of CGMCC No. 1.65186 and preservation date of 2025, 11 and 5 days, wherein the nucleotide sequence of the 16SrRNA and recN gene sequence of NWIPB-4 are shown as SEQ ID NO. 2; NWIPB-5 with preservation number of CGMCC No. 1.65179 and preservation date of 2025, 11 months and 5 days, wherein the nucleotide sequence of the 16SrRNA and the tuf gene sequence of NWIPB-5 are shown as SEQ ID NO. 2; NWIPB-6 with preservation number of CGMCC No. 1.65194 and preservation date of 2025, 11 and 21 days, wherein the nucleotide sequence of the 16SrRNA and the tuf gene sequence of NWIPB-6 are shown as SEQ ID NO. 2; NWIPB-7 with preservation number of CGMCC No. 1.65193 and preservation date of 2025, 11 and 21 days, wherein the nucleotide sequence of the 16SrRNA and the tuf gene sequence of NWIPB-7 are shown as SEQ ID NO. 2; NWIPB-8 with preservation number of CGMCC No. 1.65181 and preservation date of 2025, 11 and 5 days, wherein the nucleotide sequence of the 16SrRNA and the dnaK gene sequence of NWIPB-8 are shown as SEQ ID NO. 2; NWIPB-9 with preservation number of CGMCC No. 1.65182 and preservation date of 2025, 11 and 5 days, wherein the nucleotide sequence of the 16SrRNA and the pheS gene sequence of NWIPB-9 are shown as SEQ ID NO. 2; NWIPB-10 with preservation number of CGMCC No. 1.65183 and preservation date of 2025, 11 months and 5 days, wherein the nucleotide sequence of the 16SrRNA and the pheS gene sequence of NWIPB-10 are shown as SEQ ID NO. 2; NWIPB-11 with preservation number of CGMCC No. 1.65184 and preservation date of 2025, 11 month and 5 days, wherein the nucleotide sequence of the 16SrRNA and the tuf gene sequence of NWIPB-11 are shown as SEQ ID NO. 2; NWIPB-12 with preservation number of CGMCC No. 1.65187 and preservation date of 2025, 11 months and 5 days, and the nucleotide sequence of the 16SrRNA and tuf gene sequence of NWIPB-12 are shown as SEQ ID NO. 2.
  2. 2. The method for preparing the composite lactic acid bacteria according to claim 1, which is characterized by comprising the following steps: step 1, bacterial strain separation and purification Step 1.1, collecting a Tibetan homemade yak yoghourt sample for later use; Step 1.2, taking a small amount of yoghurt sample, carrying out gradient dilution by using sterile normal saline, selecting 7 dilutions, sucking 0.3mL of fungus suspension, coating the fungus suspension on an MRS agar culture medium, an improved MRS culture medium or an MC culture medium flat plate, and placing the fungus suspension in a 37 ℃ constant temperature incubator for culturing for 24 hours under facultative or strict anaerobic conditions; step 1.3, selecting single bacterial colonies with the diameter of 1-2 mm, smooth and convex surface, complete edge, milky white, glossy and soft texture on an MRS agar culture medium, an improved MRS culture medium or an MC culture medium plate; Step 1.4, inoculating the picked single colony to an MRS agar culture medium, an improved MRS culture medium or an MC culture medium by a streaking method, placing the single colony into a 37 ℃ constant temperature incubator, culturing for 24 hours under facultative or strict anaerobic conditions, collecting fungus coating on a flat plate, placing the fungus coating into 10% skimmed milk powder solution, fully and uniformly mixing, sub-packaging the fungus coating into pre-sterilized glass freeze-drying pipes, and filling 0.2-0.3 mL of each freeze-drying pipe with a sterile cotton plug; Step 1.5, placing the glass freeze-dried tube after sub-packaging in a vacuum freeze dryer for pre-cooling for 24 hours at the temperature of minus 80 ℃, transferring the pre-cooled freeze-dried tube into the vacuum freeze dryer, drying for 24 hours at the low temperature under the condition that the temperature is minus 52 ℃ and the vacuum degree is 0.1 mbar, connecting the fully dried freeze-dried tube to a vacuum device, sealing the freeze-dried tube by using flame firing after the pressure in the tube is reduced to below 10Pa, and then placing the freeze-dried tube in a refrigerator at the temperature of minus 20 ℃ for later use; Step 2, detecting physiological and biochemical indexes of strain Step 2.1, testing the catalase and oxidase activities of the strain, wherein the catalase and the oxidase are negative; Step 2.2, API 50CH kit test isolates with different carbohydrate production capacities of glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, adonitol, beta-methyl-D-xyloside, D-galactose, D-glucose, D-fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, mannitol, sorbitol, alpha-methyl-D-mannoside, alpha-methyl-D-glucoside, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, D-melibiose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabinitol, gluconate, 2-ketonic acid salts; step 3, strain identification, namely activating each pure culture strain for 12-24 hours at the temperature of 32-37 ℃ and carrying out continuous three times of subculture on an MRS slant culture medium to carry out strain identification; Step 3.1, cell morphology identification, namely picking a single colony smear, carrying out gram staining, and observing whether the staining effect of cells is consistent with the cell morphology or not through an optical microscope, wherein the cell morphology is rod-shaped, and the gram staining is positive; step 3.2, extracting genome DNA of the strain, namely picking 1-3 colonies by using a sterile inoculating loop, adding 25 μl of 0.5% Chelex solution, fully oscillating and uniformly mixing, carrying out boiling water bath for 10-15 min, and centrifuging at 12000rpm for 1min, wherein the supernatant is the extracted genome DNA solution, and preserving at-20 ℃ for later use; Step 3.3, PCR amplification and sequencing: the 16s rRNA gene amplification primers were as follows: 8F:5'-AgA GTT TgA TCC Tgg CTC Ag-3'; 1492R:5'-ggT TAC CTT gTT ACg ACT T-3'; the PCR reaction conditions were 95℃pre-denaturation for 5min,95℃denaturation for 1min,56℃annealing for 1min, 72℃extension for 1.5min,30 cycles, 72℃extension for 10min, the obtained amplified product length was 1.5kb, and 4℃storage; sequencing the amplified product, submitting the sequencing result of the 16S rRNA gene amplified fragment to NCBI for sequence comparison, and preliminarily determining the phylogenetic status of the strain; Step 3.4, sequencing, namely amplifying pheS, dnaK, recN and tuf specific conserved functional genes by PCR according to the comparison result of the 16S rRNA gene sequence, and sequencing the amplified products; the pheS specific conserved functional gene amplification primer: pheS-f primer 5'-CAY CCN GCH CGY GAY ATG C-3' PheS-r primer 5'-CCC WAR VCC RAA RGC AAA RCC-3' The PCR reaction conditions are 95 ℃ pre-denaturation for 5min,95 ℃ denaturation for 1min,48 ℃ annealing for 1min,72 ℃ extension for 1.5min and 30 cycles, 72 ℃ extension for 10min, and the obtained amplification product has a length of 500bp and is stored at 4 ℃; dnaK specific conserved functional gene amplification primers: 180F primer 5'-GTG AAG ACR CCR CCC ATG G-3'; 940R primer 5'-Arg CAN CCA Agg ATg CCg g-3'; The PCR reaction conditions comprise pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 1 min, annealing at 52 ℃ for 1 min, extension at 72 ℃ for 1.5min for 30 cycles, extension at 72 ℃ for 10min, and preservation at 4 ℃ with the obtained amplification product length of 800 bp; recN specific conserved functional gene amplification primers: recNf primer 5'-GGA ART CAN TYA TYA TTG ATG C-3'; recNr primer 5'-WAA CWC CNG TAT CAN CYT CAT C-3'; the PCR reaction conditions were 95℃pre-denaturation for 5min, 95℃denaturation for 1min,50℃annealing for 1min, 72℃extension for 1.5min,30 cycles, 72℃extension for 10 min, the obtained amplification product length was 1.3 kb, and 4℃storage; tuf specific conserved functional gene amplification primers: bif-1 primer 5'-gAg TAC gAC TTC AAC CAg-3'; Bif-2 primer 5'-CAg gCg Agg ATC TTg gT-3'; The PCR reaction conditions are 95 ℃ pre-denaturation for 5min,95 ℃ denaturation for 1min,52 ℃ annealing for 1min, 72 ℃ extension for 1.5min and 30 cycles, 72 ℃ extension for 10min, and the obtained amplification product has a length of 800bp and is stored at 4 ℃; Step 3.5, classifying status, integrating cell morphology, physiological and biochemical characteristics, 16S rRNA gene and specific conserved functional gene sequence of the strain, and determining that each isolated strain is Lactobacillus plantarum、Lactobacillus rhamnosus、Lactobacillus delbrueckii、Streptococcus thermophilus、Bifidobacterium longum、Bifidobacterium lactis、Bifidobacterium breve、Lactobacillus paracasei、Lactobacillus acidophilus、Pediococcus acidilactici、Bifidobacterium adolescentis、Bifidobacterium bifidum.
  3. 3. The method for producing a complex lactic acid bacterium according to claim 1, wherein NWIPB-1, NWIPB-2, NWIPB-3, NWIPB-8, NWIPB-9, NWIPB-10 are isolated and purified using MRS agar medium, NWIPB-4 is isolated and purified using MC medium, and NWIPB-5, NWIPB-6, NWIPB-7, NWIPB-11, NWIPB-12 are isolated and purified using modified MRS medium.
  4. 4. The preparation method of the composite lactobacillus according to claim 1, wherein the MRS agar culture medium is prepared by adding 10.0g of peptone, 5.0g of beef powder, 4.0g of yeast powder, 20.0g of glucose, 1.0mL of tween 80, 2.0g of K 2 HPO 4 •7H 2 O, 5.0g of sodium acetate 3H 2 O, 2.0g of tri-ammonium citrate, 0.05g of MgSO 4 •7H 2 O 0.2g、MnSO 4 •4H 2 O and 15.0g of agar powder into 1000mL of distilled water, heating and dissolving, adjusting the pH to 6.2+/-0.2, and autoclaving at 121 ℃ for 20min after split charging; The MC culture medium is prepared by adding 5.0g of soybean peptone, 3.0g of beef powder, 3.0g of yeast powder, 20.0g of glucose, 20.0g of lactose, 10.0g of calcium carbonate, 15.0g of agar and 5.0mL of 1% neutral red solution into 1000mL of distilled water, heating to dissolve, regulating pH to 6.0+ -0.2, adding the neutral red solution, sub-packaging, and sterilizing at 121deg.C under high pressure for 20min; the improved MRS culture medium is prepared by (1) weighing 50 mg Mopirocin lithium salt, adding into 50mL distilled water, filtering with 0.22 μm microporous membrane, and sterilizing to obtain Mopirocin lithium salt stock solution; (2) Then weighing 250 mg cysteine hydrochloride, adding the cysteine hydrochloride into 50mL distilled water, filtering and sterilizing the solution by using a 0.22 mu m microporous filter membrane to obtain a cysteine hydrochloride stock solution for later use; (3) Adding the components of the MRS culture medium into 950 mL distilled water, heating for dissolving, adjusting pH to 6.0+ -0.2, sub-packaging, and autoclaving at 121deg.C for 20min for use; The agar was melted by heating in a water bath and cooled to 48℃and the above-mentioned mupirocin lithium salt stock solution and cysteine hydrochloride stock solution were added to the melted MRS agar medium by a syringe with a microporous membrane of 0.22. Mu.m, so that the concentration of mupirocin lithium salt in the medium was 50. Mu.g/mL and the concentration of cysteine hydrochloride was 500. Mu.g/mL.
  5. 5. The method for producing a lactic acid bacterium composition according to claim 1, wherein NWIPB-1, NWIPB-2, NWIPB-3, NWIPB-4, NWIPB-8, NWIPB-9, NWIPB-10 are cultured under facultative anaerobic conditions, and NWIPB-5, NWIPB-6, NWIPB-7, NWIPB-11, NWIPB are cultured under strictly anaerobic conditions.
  6. 6. The use of a complex lactic acid bacteria according to claim 1, characterized in that the complex lactic acid bacteria are used for the preparation of complex lactic acid bacteria metazoans having a regulating function of the intestinal flora and generally stool-moistening probiotic properties.
  7. 7. The method for preparing the composite lactic acid bacteria metazoan according to claim 6, wherein the specific preparation method is as follows: step 1, activating the strain, namely activating the identified strain in an MRS liquid culture medium for 2 times, and culturing for 12-24 hours at the temperature of 32-37 ℃; step 2, preparing seed liquid, namely respectively inoculating activated strains into 25-50 mL MRS liquid culture medium, and culturing for 12-24 hours at 32-37 ℃ to obtain the seed liquid; Step 3, preparing fermentation liquor, namely inoculating each seed liquor into an MRS liquid culture medium added with 2% of whey protein or casein according to the mass ratio of 5-8%, and fermenting at 32-37 ℃ for 24-48 hours to obtain fermentation bacteria liquor; Step 4, preparing bacterial powder, namely inactivating each fermentation bacterial liquid in a hot water bath at 90-92 ℃ for 30-60 min, carrying out heat inactivation treatment, adding 1-60% of auxiliary materials by mass ratio, and carrying out freeze drying for 24-36 h at the temperature of-50 to-55 ℃ and the vacuum degree of 0.1-0.3 mbar to obtain bacterial powder; And 4, mixing the bacterial powder, namely mixing the bacterial powder according to the mass ratio of 3-5%, and adding the prebiotics with the mass ratio of 10-15% to obtain the metazoan.
  8. 8. The method for preparing the composite lactobacillus metazoan according to claim 7, wherein the MRS liquid culture medium is prepared by mixing 10.0g of peptone, 5.0g of beef powder, 4.0g of yeast powder, 20.0g of glucose, 1.0mL of Tween 80, 2.0g of K 2 HPO 4 •7H 2 O, 5.0g of sodium acetate 3H 2 O, 2.0g of tri-ammonium citrate, 0.05 g of MgSO 4 •7H 2 O 0.2g、MnSO 4 •4H 2 O and 1000mL of distilled water, adjusting pH to 6.5+/-0.2, and autoclaving at 121 ℃ for 15min after sub-packaging.
  9. 9. The preparation method of the composite lactic acid bacteria metagen according to claim 7, wherein the auxiliary material is one or more of sucrose, lactose, trehalose, fructo-oligosaccharide, xylo-oligosaccharide, galacto-oligosaccharide, maltodextrin, alpha-cyclodextrin, inulin and skim milk powder, and the metagen is any one of medlar fruit powder, sea buckthorn fruit powder and black medlar fruit powder.
  10. 10. The use of a complex lactic acid bacteria metagen according to claim 6, characterized in that the metagen is used in a dietary fiber food for regulating intestinal flora.

Description

Composite lactobacillus, metaplasia and preparation method and application thereof Technical Field The invention relates to the technical field of microorganisms, in particular to a composite lactobacillus, a metazoan, a preparation method and application thereof. Background With the acceleration of modern life pace and the change of human dietary structure, intestinal health problems are increasingly prominent. More and more studies have demonstrated that probiotics are closely related to human health and that they enhance the immunity of the body by modulating intestinal flora, improving the nutritional utilization of the host, promoting maturation of the host's immune system, and stimulating an immune response. The probiotics are taken as a promising means for improving the intestinal health, and greatly relieve the intestinal health problem which puzzles people to live. In recent years, probiotics are widely used as an important means for regulating intestinal microecology and improving immunity. For example, lactobacillus plantarum ZJ316 has been proved to inhibit colonitis and regulate intestinal micro-ecology of mice, bifidobacterium lactis HN019 of animals can increase defecation frequency of patients with functional constipation, pediococcus acidilactici can reduce serum cholesterol and inhibit proliferation of staphylococcus aureus and escherichia coli, bifidobacterium bifidum B1 regulates gastrointestinal transmitter level of hosts by increasing acetic acid level and improves antioxidant capacity of hosts, defecation frequency and fecal property are improved, constipation is relieved, bifidobacterium bifidum CCFM16 indirectly participates in regulation of related physiological activities such as energy metabolism in peripheral blood and brain by regulating substances such as butyric acid, indole-3 formaldehyde, xanthine and the like in intestinal tracts, and recovers abnormal brain volume and brain density of an autism organism, thereby relieving autism, and probiotics and intestinal microorganisms are becoming potential targets for treating various diseases such as obesity, diabetes, liver diseases, cancers and even neurodegenerative diseases. Although probiotics are able to supplement certain species lacking in the body to some extent, they are able to improve the intestinal flora environment in a short period of time, there are still a number of limitations. After the probiotics enter the intestinal tract, the problems of low survival rate, insufficient intestinal tract colonization capability, unstable action and the like of the probiotics exist due to the complex microenvironment of the intestinal tract, and the healthy ecology of the intestinal tract is difficult to maintain for a long time, so that the ideal health improvement effect is achieved. While the beneficial effects of the metazoan on the host are similar to those of the probiotics, the stability and safety of the metazoan are far superior to those of the probiotics, and the metazoan not only contains inactivated probiotic thalli, but also metabolic products generated by the probiotics in the growth metabolic process and beneficial effects exerted by the products on the host. The yak yoghourt takes the milk of the semi-wild grazing yaks in Qinghai-Tibet plateau as the raw material, has pure milk quality and high nutrition density, and has better contents of protein, amino acid and microelements (such as calcium, iron and zinc) than common milk. The yak yoghourt is prepared by adopting a traditional natural fermentation mode and slowly fermenting by using wild lactic acid bacteria in a plateau environment. The lactobacillus in the yak yoghourt has good probiotics characteristics and fermentation characteristics after long-term natural selection, can keep the original flavor and active probiotics of the yak yoghourt, and plays an important role in regulating the health and stability of intestinal flora of a human body. Therefore, the invention provides a compound lactobacillus, a metagen, a preparation method and application thereof, and aims to solve the problems. Disclosure of Invention Based on the technical problems, the invention aims to provide a preparation method and application of a composite lactobacillus metazoan. Solves the problems of low survival rate, insufficient colonization capability and easy inactivation in the gastrointestinal tract environment of probiotics in the prior art. The invention provides a compound lactobacillus which comprises two or more of Lactiplantibacillus plantarum、Lacticaseibacillus rhamnosus、Lactobacillus delbrueckii、Streptococcus thermophiles、Bifidobacterium longum、Bifidobacterium animalissubsp. lactis、Bifidobacterium breve、Lacticaseibacillus paracasei、Lactobacillus acidophilus、Pediococcus acidilactici、Bifidobacterium adolescentis）、Bifidobacterium bifidum, the serial numbers of which are NWIPB-1, NWIPB-2, NWIPB-3, NWIPB-4, NWIPB-5, NWIPB-6, NWIPB-7, NWIPB-8, NWIPB-9, NWIPB-10, NWIPB-11 and N