CN111154694A - Microbial fermentation inoculant and preparation method and application thereof - Google Patents
Microbial fermentation inoculant and preparation method and application thereof Download PDFInfo
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Classifications
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- 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
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- A—HUMAN NECESSITIES
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- 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
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
- A23L19/20—Products from fruits or vegetables; Preparation or treatment thereof by pickling, e.g. sauerkraut or pickles
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- 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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
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- 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
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
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- 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
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- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
Abstract
The invention relates to the technical field of microbial agents, and particularly relates to a microbial fermentation agent, and a preparation method and application thereof. According to the microbial fermentation inoculant disclosed by the invention, three inocula can grow in a culture medium with the pH of 3.0 and the concentration of bile salt of 0.3%, and show certain tolerance to acid and bile salt; has antioxidant and free radical scavenging effects; meanwhile, the microbial inoculum has the capacity of degrading cholesterol, and the degradation rates of the three microbial inocula are equivalent when the concentration of the cholesterol is 0.1 mg/mL; in addition, the microbial inoculum is used for assisting fruits, vegetables and Chinese herbal medicines in fermentation to prepare the ferment, so that the antioxidant activity of the ferment and the quantity of probiotics can be effectively improved, and the method has important significance for improving the quality of the ferment and improving the fermentation process of the ferment.
Description
Technical Field
The invention relates to the technical field of microbial agents, and particularly relates to a microbial fermentation agent as well as a preparation method and application thereof.
Background
Probiotics (Probiotics) originated in latin, meaning "beneficial to life". The ElieMetchnikoff research at the beginning of the 20 th century found that the secret of longevity of Bulgaria people is related to the daily drinking of the yoghourt, and points out that the yoghourt contains certain substances which can inhibit harmful bacteria in the intestinal tract, thereby proposing the hypothesis of probiotic concept and the effect of prolonging life. Since then, with the intensive research on probiotics, the definition thereof is revised many times. In 1974, Parker defined probiotics as: feed preparations for promoting healthy growth of animals by improving the balance of their intestinal flora are defined, but this definition is only a simple understanding of their action and does not exclude other substances, such as antibiotics, having an equivalent effect. In 1989, Fuller further defined probiotics as: probiotics are a class of live bacterial preparations that benefit the health of the host by improving the balance of the host's intestinal flora. In 1992, Havenaar et al further defined probiotics as: a live bacterial preparation consisting of a single or a plurality of microorganisms capable of producing beneficial effects on human and animals by improving the balance of the indigenous flora of animals or humans. Until 2001, the food and agriculture organization/world health organization (FAO/WHO) of the united nations has regulated the summary of probiotics: probiotics are a class of live microorganisms that have a health benefit to the host when ingested in sufficient quantities of viable bacteria. With the continuous development of biotechnology, probiotics are widely concerned by experts in various fields, the concept of the probiotics is gradually improved, and the existing consensus is formed, namely the probiotics are active viable bacteria, and can be planted in a host after being taken into the organism by oral administration or other administration modes, so that the micro-ecological balance of the host is improved and the probiotics effect is played.
The screened probiotics need to meet the following standards of ① having the function of benefiting the host, ② having the function of resisting the low pH value of gastric juice and having a certain tolerance to acid, ③ having a certain tolerance to bile salt, ④ having an inhibiting effect on pathogenic bacteria, ⑤ having the function of being planted on the surface of the gastrointestinal epithelium, ⑥ being convenient to process and store and the like.
The research on the ferment in China is at the beginning stage, and the research on the ferment is mainly carried out in a natural fermentation mode, namely, the ferment is carried out only through microorganisms carried on the surfaces of fruits and vegetables without adding any exogenous microorganisms for auxiliary fermentation. However, the fermentation method is easily affected by raw materials, temperature and the like, and may cause pathogenic bacteria pollution, so that the product quality is difficult to control, and the development and utilization of the enzyme product are affected.
Therefore, the invention provides a microbial fermentation inoculant with functions of regulating intestinal flora, resisting oxidation, degrading cholesterol and the like, and a preparation method and application thereof.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a microbial fermentation inoculant as well as a preparation method and application thereof, wherein the three inoculants can grow in a culture medium with the pH of 3.0 and the concentration of bile salt of 0.3 percent and show certain tolerance to acid and bile salt; has antioxidant and free radical scavenging effects; the three microbial inoculums have the capacity of degrading cholesterol, and the degradation rates of the three microbial inoculums are equivalent when the concentration of the cholesterol is 0.1 mg/mL; in addition, the microbial inoculum is used for assisting fruits, vegetables and Chinese herbal medicines in fermentation to prepare the ferment, so that the antioxidant activity of the ferment and the quantity of probiotics can be effectively improved, and the method has important significance for improving the quality of the ferment and improving the fermentation process of the ferment.
The purpose of the invention is realized by the following technical scheme:
a microbial fermentation agent comprises Lactobacillus casei (Lactobacillus casei), Lactobacillus harbourbonensis (Lactobacillus harbinensis) and Enterococcus faecium (Enterococcus faecium).
Further, the total viable count of each viable bacterium in the microbial fermentation agent is 2.4 multiplied by 109CFU/mL or more. Preferably, the total viable count of each viable bacterium in the microbial fermentation agent is 2.4 multiplied by 109～2.7×109CFU/mL。
Furthermore, the lactobacillus casei is lactobacillus casei QH16, the accession number of 16SrDNA of lactobacillus casei QH16 in Genbank is MN809276,
the lactobacillus haber is lactobacillus haber QH21, the landing number of 16SrDNA of lactobacillus haber QH21 on Genbank is MN809279,
the enterococcus faecium is enterococcus faecium QH19, and the accession number of 16SrDNA of enterococcus faecium QH19 on Genbank is MN 809277.
Further, the microbial fermentation inoculum also includes Saccharomyces cerevisiae. Preferably, the saccharomyces cerevisiae comprises saccharomyces cerevisiae QH1, saccharomyces cerevisiae QH2 and saccharomyces cerevisiae QH8, the accession number of 18SrDNA of the saccharomyces cerevisiae QH1 on Genbank is MN759772, the accession number of 18SrDNA of the saccharomyces cerevisiae QH2 on Genbank is MN759439, and the accession number of 18SrDNA of the saccharomyces cerevisiae QH8 on Genbank is 918288.
Further, the total viable count of the microbial fermentation inoculum is 2.8 multiplied by 109CFU/mL or more. Preferably, the total viable count of each viable bacterium in the microbial fermentation agent is 2.8 multiplied by 109～3.0×109CFU/mL。
Further, the microbial fermentation inoculum also comprises Bacillus subtilis. Preferably, the bacillus subtilis comprises bacillus subtilis QH10 and bacillus subtilis QH12, the accession number of the 16SrDNA of the bacillus subtilis QH10 on Genbank is MN809144, and the accession number of the 16SrDNA of the bacillus subtilis QH12 on Genbank is MN 809275.
Further, the total viable count of the microbial fermentation inoculum is 3.8 multiplied by 109CFU/mL or more. Preferably, the total viable count of each viable bacterium in the microbial fermentation agent is 3.8 multiplied by 109～4.2×109CFU/mL。
Furthermore, the lactobacillus casei is lactobacillus casei QH16, the accession number of 16SrDNA of lactobacillus casei QH16 in Genbank is MN809276,
the lactobacillus haber is lactobacillus haber QH21, the landing number of 16SrDNA of lactobacillus haber QH21 on Genbank is MN809279,
the enterococcus faecium is enterococcus faecium QH19, the landing number of 16SrDNA of enterococcus faecium QH19 on Genbank is MN809277,
the saccharomyces cerevisiae comprises saccharomyces cerevisiae QH1, saccharomyces cerevisiae QH2 and saccharomyces cerevisiae QH8, the accession number of 18SrDNA of the saccharomyces cerevisiae QH1 on the Genbank is MN759772, the accession number of 18SrDNA of the saccharomyces cerevisiae QH2 on the Genbank is MN759439, the accession number of 18SrDNA of the saccharomyces cerevisiae QH8 on the Genbank is 918288,
the bacillus subtilis comprises bacillus subtilis QH10 and bacillus subtilis QH12, the accession number of 16SrDNA of the bacillus subtilis QH10 on Genbank is MN809144, and the accession number of 16SrDNA of the bacillus subtilis QH12 on Genbank is MN 809275.
Furthermore, in the microbial inoculum, the viable bacteria ratio of lactobacillus casei QH16, lactobacillus haber QH21, enterococcus faecium QH19, saccharomyces cerevisiae QH1, saccharomyces cerevisiae QH2, saccharomyces cerevisiae QH8, bacillus subtilis QH10 and bacillus subtilis QH12 is 1.0-2.5: 1.0-2.5: 2.0-3.0: 2.0-2.5: 3.0-3.5: 3.0-3.5: 1.5-2.0: 2.0 to 2.5.
The application method of the microbial fermentation inoculant is used for fruit and vegetable fermentation.
A preparation method of a microbial fermentation inoculant takes at least one of mulberry enzyme, kiwi fruit enzyme, grape enzyme, Hami melon enzyme, mulberry compound enzyme stock solution, yoghourt and probiotic as raw materials, and is prepared by the following steps:
step S1, placing the raw material enzymes in an MRS liquid culture medium for constant-temperature culture to obtain an enrichment culture solution;
step S2, separating and purifying the enrichment culture solution, and separating the pure strains from the culture solution by adopting a gradient dilution method;
step S3, acid-resistant screening and cholate-resistant screening are respectively carried out on each pure strain obtained by separation,
the acid-resistant screening is specifically that the pure strains of the two activated generations are respectively inoculated into an MRS liquid culture medium with the pH value of 3.5, an MRS liquid culture medium with the pH value of 6.5, an YPD liquid culture medium with the pH value of 3.5 and an YPD liquid culture medium with the pH value of 6.5 according to the inoculum concentration of 1 percent, after the pure strains are cultured in a constant-temperature shaking table at the temperature of 35 ℃ for 24 hours, the light absorption value of the pure strains under 600nm is measured, the ratio of the two light absorption values in the same type of culture medium is taken as the survival rate of the strains under the pH value of 3.5, the strains with the survival rate of more than 50 percent based on the MRS liquid culture medium and the strains with the survival rate of more than 80 percent based on the YPD;
the bile salt resistant screening is specifically that the pure strains after two generations of activation are respectively inoculated into an MRS liquid culture medium with bile salt concentration of 0.3%, an MRS liquid culture medium without bile salt, an YPD liquid culture medium with bile salt concentration of 0.3% and an YPD liquid culture medium without bile salt according to the inoculum concentration of 1%, after the pure strains are cultured in a constant temperature shaking table at the temperature of 35 ℃ for 24 hours, the light absorption value of the pure strains under 600nm is measured, the ratio of the two light absorption values in the same type of culture medium is taken as the survival rate of the strains under the bile salt concentration of 0.3%, the strains with the survival rate of more than 20% based on the MRS liquid culture medium are screened, and the survival rate of more than 45% based on the YPD liquid culture medium is screened;
and S4, compounding strains which simultaneously meet acid resistance screening and cholate resistance screening to obtain the microbial fermentation inoculant.
Further, in step S4, before the strain is compounded, acid-resistant and bile-salt-resistant acclimatization of the strain is further performed, wherein the acid-resistant and bile-salt-resistant acclimatization specifically includes performing acid-resistant and bile-salt-resistant acclimatization on the strain obtained by screening, and the method of gradually reducing the pH value of the acid culture medium or gradually increasing the concentration of bile salt in the bile salt culture medium is adopted to acclimatize the strain to improve the survival rate of the strain.
Further, in the step S4, the compounding specifically includes inoculating the screened and domesticated three strains of lactobacillus casei QH16, lactobacillus haber QH21, and enterococcus faecium QH19 to an MRS medium according to 20% -25%, and 50% -60% of the total bacterial volume, respectively, and culturing in a constant temperature shaker at 35 ℃ for 24 hours to obtain a microbial inoculum I, which has the advantages of cost saving, strong bile salt resistance, and good cholesterol reduction capability when the cholesterol concentration is low; on the basis, the saccharomyces cerevisiae QH1, the saccharomyces cerevisiae QH2 and the saccharomyces cerevisiae QH8 are continuously added and are respectively inoculated into an MRS culture medium according to 20-25%, 20-30%, 5-10% and 15-30% of the total bacterial volume, and are cultured in a constant temperature shaking table at the temperature of 35 ℃ for 24 hours, so that the microbial inoculum II is obtained, and the advantage of the microbial inoculum II is that the antioxidant activity is stronger; on the basis, continuously adding bacillus subtilis QH10 and bacillus subtilis QH12, respectively inoculating the bacillus subtilis QH10 and the bacillus subtilis QH12 into an MRS culture medium according to 5-12.5%, 10-15%, 10-12.5%, 15-17.5%, 7.5-10% and 10-12.5% of the total bacterial volume, and culturing in a constant-temperature shaking table at the temperature of 35 ℃ for 24 hours to obtain a microbial inoculum III which has the advantages of strong acid resistance and obvious cholesterol reduction capability when the cholesterol concentration is high.
Further, in step S1, specifically, under aseptic operation, each enzyme 5 in unit volume is transferred to a sterilized MRS liquid medium in 100 in unit volume, and cultured in a shaker at a rotation speed of 150r/min and a temperature of 35 ℃ for 48 hours, so as to obtain a primary culture solution; taking 1 unit volume of the primary culture solution to be cultured in 100 unit volume of sterilized MRS liquid culture medium in a constant temperature shaking table with the rotating speed of 150r/min and the temperature of 35 ℃ for 48h to obtain an enriched culture solution.
Further, in the step S2, 1 unit volume of the enriched culture broth is added to 9 unit volumes of sterilized normal saline, and diluted to 10 dilution-1Diluting the bacterial liquid; then 1 unit volume 10 is taken-1Adding the diluted bacterial liquid into 9 unit volume of sterilized normal saline, and diluting to 10 dilution-2Diluting the bacterial liquid, repeating the above operation to dilute the enrichment culture liquid to 10 respectively-3-10-8Diluting the bacterial liquid; respectively take 0.2 unit volume 10-3-10-8Adding the diluted bacterial liquid into a sterilized MRS plate culture medium and a sterilized YPD plate culture medium, uniformly coating, and then, inversely culturing in an incubator at 35 ℃ for 72 hours; selecting the strain which grows well in a culture medium and has obvious single colony, respectively carrying out streak purification on an MRS plate culture medium and an YPD plate culture medium, inverting the strain and culturing the strain in an incubator at 35 ℃ for 72h, and repeating the operation for multiple times until the single colony which is completely consistent appears on the plate, thereby obtaining the pure strain.
Further, the MRS liquid culture medium comprises the following raw materials: 10.0 parts by weight of peptone, 4.0 parts by weight of yeast powder, 5.0 parts by weight of beef powder, 20.0 parts by weight of glucose, 1.0 part by volume of tween 80, and 5.0 parts by weight of CH3COONa·3H2O, 2.0 parts by weight of K2HPO4·7H2O, 2.0 parts by weight of triammonium citrate, 0.2 parts by weight of MgSO4·7H2O, 0.05 weight part of MnSO4·4H2O and 1000 parts by volume of distilled water.
Further, the YPD liquid medium comprises the following raw materials: 20 parts by weight of peptone, 10 parts by weight of yeast powder, 20 parts by weight of glucose, and 1000 parts by volume of distilled water.
Further, when the strain is placed in a liquid culture medium and cultured in a constant temperature shaking table, the rotating speed is controlled to be 150 r/min.
The invention has the beneficial effects that: according to the microbial fermentation inoculant disclosed by the invention, three inocula can grow in a culture medium with the pH of 3.0 and the concentration of bile salt of 0.3%, and show certain tolerance to acid and bile salt; has antioxidant and free radical scavenging effects; meanwhile, the microbial inoculum has the capacity of degrading cholesterol, and the degradation rates of the three microbial inocula are equivalent when the concentration of the cholesterol is 0.1 mg/mL; in addition, the microbial inoculum is used for assisting fruits, vegetables and Chinese herbal medicines in fermentation to prepare the ferment, so that the antioxidant activity of the ferment and the quantity of probiotics can be effectively improved, and the method has important significance for improving the quality of the ferment and improving the fermentation process of the ferment.
Drawings
FIG. 1 is a diagram showing the results of acid-resistance detection of a microbial inoculum I, a microbial inoculum II and a microbial inoculum III in an experimental example of the present invention;
FIG. 2 is a diagram showing the results of detection of cholate resistance of microbial inoculum I, microbial inoculum II and microbial inoculum III in the test example of the present invention;
FIG. 3 is a diagram showing the results of the reduction power measurements of the microbial inoculum I, the microbial inoculum II and the microbial inoculum III in the test example of the present invention;
FIG. 4 is a graph showing the results of scavenging rates of superoxide radicals by the bacterial agent I, the bacterial agent II and the bacterial agent III in the experimental examples of the present invention;
FIG. 5 is a graph showing the results of hydroxyl radical scavenging rates of bacterium I, bacterium II and bacterium III in the experimental examples of the present invention;
FIG. 6 is a graph showing the results of cholesterol lowering ability tests of the microbial inoculum I, the microbial inoculum II and the microbial inoculum III in the test example of the present invention (the cholesterol concentration is 0.1 mg/mL);
FIG. 7 is a graph showing the results of cholesterol lowering ability tests of bacterium I, bacterium II and bacterium III in the test examples of the present invention (cholesterol concentrations of 3mg/mL, 4mg/mL and 5mg/mL, respectively).
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to specific embodiments and drawings, but the scope of the present invention is not limited to the following.
Examples
A preparation method of a microbial fermentation inoculant takes at least one of mulberry enzyme, kiwi fruit enzyme, grape enzyme, Hami melon enzyme, mulberry compound enzyme stock solution, yoghourt and probiotic as raw materials, and comprises the following steps:
step S1, under aseptic operation, respectively transferring 5mL of various enzymes into 100mL of sterilized MRS liquid culture medium, and culturing in a shaking table at the rotating speed of 150r/min and the temperature of 35 ℃ for 48h to obtain a primary culture solution; taking 1mL of primary culture solution, placing in 100mL of sterilized MRS liquid culture medium, and culturing in a constant temperature shaking table at the rotation speed of 150r/min and the temperature of 35 ℃ for 48h to obtain an enrichment culture solution;
step S2, adding 1mL of enrichment culture bacterial liquid into 9mL of sterilized normal saline, and diluting to 10 dilution-1Diluting the bacterial liquid; then take 1mL 10-1Adding the diluted bacterial liquid into 9mL of sterilized normal saline, and diluting to 10 dilution-2Diluting the bacterial liquid, repeating the above operation to dilute the enrichment culture liquid to 10 respectively-3-10-8Diluting the bacterial liquid; respectively transferring 200 μ L10-3-10-8Adding the diluted bacterial liquid into a sterilized MRS plate culture medium and a sterilized YPD plate culture medium, uniformly coating, and then, inversely culturing in an incubator at 35 ℃ for 72 hours; selecting and taking a strain which grows well in a culture medium and has obvious single bacterial colonies, respectively carrying out lineation purification on an MRS plate culture medium and an YPD plate culture medium, inverting the strain and culturing the strain for 72 hours in an incubator at 35 ℃, repeating the operation for multiple times until the single bacterial colonies which are completely consistent appear on the plate, screening to obtain 8 strains (respectively numbered QH-1-QH-8) based on the YPD culture medium, screening to obtain 13 strains (respectively numbered QH-9-QH-21) based on the MRS culture medium, and totaling 21 pure strains;
step S3, acid-resistant screening and cholate-resistant screening are respectively carried out on each pure strain obtained by separation,
the acid-resistant screening is specifically that the pure strains of the two activated generations are respectively inoculated in an MRS liquid culture medium with the pH value of 3.5, an MRS liquid culture medium with the pH value of 6.5, an YPD liquid culture medium with the pH value of 3.5 and an YPD liquid culture medium with the pH value of 6.5 according to the inoculation amount of 1 percent (strains obtained based on the YPD culture medium screening are subjected to acid-resistant screening only in the YPD culture medium, strains obtained based on the MRS culture medium screening are subjected to acid-resistant screening only in the MRS culture medium), the light absorption value of the strains under 600nm is measured after the strains are cultured in a constant temperature shaking table at the rotating speed of 150r/min and the temperature of 35 ℃ and the light absorption value ratio of the two light absorption values in the same type culture medium is taken as the survival rate of the strains under the pH value of 3.5, and the results are,
TABLE 1 tolerance of strains to acids
Selecting strains with the survival rate of more than 50 percent based on MRS liquid culture medium, and respectively selecting QH-1, QH-2 and QH-8; respectively screening out QH-10, QH-12, QH-16, QH-19 and QH-21 based on the strains with the survival rate of YPD liquid culture medium being more than 80%;
the bile salt resistance screening is specifically that the pure strains after two generations are respectively inoculated in an MRS liquid culture medium with bile salt concentration of 0.3%, a bile salt-free MRS liquid culture medium, an YPD liquid culture medium with bile salt concentration of 0.3% and a bile salt-free YPD liquid culture medium according to the inoculation amount of 1% (the strains obtained based on the YPD culture medium screening are subjected to acid resistance screening only in the YPD culture medium, the strains obtained based on the MRS culture medium screening are subjected to acid resistance screening only in the MRS culture medium), after the strains are cultured in a constant temperature shaking table at the rotating speed of 150r/min and the temperature of 35 ℃ for 24 hours, the light absorption value of the strains under 600nm is measured, the ratio of two light absorption values in the same type culture medium is taken as the survival rate of the strains under the bile salt concentration of 0.3%, and the results are shown in Table 2,
TABLE 2 tolerance of strains to bile salts
Selecting strains with the survival rate of more than 16 percent based on MRS liquid culture medium, and respectively selecting QH-10, QH-12, QH-16, QH-19 and QH-21; respectively screening out QH-1, QH-2 and QH-8 based on strains with the survival rate of YPD liquid culture medium being more than 45%;
s4, performing acid and bile salt resistance domestication on the strain, wherein the acid and bile salt resistance domestication specifically comprises the steps of performing acid and bile salt resistance domestication on the screened strain, and domesticating the strain to improve the survival rate of the strain by adopting a method of gradually reducing the pH value of an acid culture medium or gradually increasing the concentration of bile salt in a bile salt culture medium; after domestication, compounding strains which simultaneously meet acid resistance screening and cholate resistance screening, specifically, inoculating three strains of lactobacillus casei QH16, lactobacillus haber QH21 and enterococcus faecium QH19 obtained by screening and domestication into an MRS culture medium according to 20-25%, 20-25% and 50-60% of the total bacterial volume respectively, and culturing for 24 hours in a constant-temperature shaking table at 35 ℃ to obtain a microbial inoculum I, wherein the microbial inoculum I has the advantages of cost saving, strong cholate resistance and good cholesterol reduction capability when the cholesterol concentration is low; on the basis, the saccharomyces cerevisiae QH1, the saccharomyces cerevisiae QH2 and the saccharomyces cerevisiae QH8 are continuously added and are respectively inoculated into an MRS culture medium according to 20-25%, 20-30%, 5-10% and 15-30% of the total bacterial volume, and are cultured in a constant temperature shaking table at the temperature of 35 ℃ for 24 hours to obtain a microbial inoculum II, and the microbial inoculum II has the advantage of strong antioxidant activity; on the basis, continuously adding bacillus subtilis QH10 and bacillus subtilis QH12, respectively inoculating the bacillus subtilis QH10 and the bacillus subtilis QH12 into an MRS culture medium according to 5-12.5%, 10-15%, 10-12.5%, 15-17.5%, 7.5-10% and 10-12.5% of the total bacterial volume, and culturing in a constant-temperature shaking table at the temperature of 35 ℃ for 24 hours to obtain a microbial inoculum III which has the advantages of strong acid resistance and obvious cholesterol reduction capability when the cholesterol concentration is high.
Specifically, the preparation of the MRS liquid culture medium comprises the following steps: mixing 10.0g peptone, 4.0g yeast powder, 5.0g beef powder, 20.0g glucose, 1.0mL Tween 80, 5.0g CH3COONa·3H2O, 2.0g of K2HPO4·7H2O, 2.0g of triammonium citrate, 0.2g of MgSO4·7H2O, 0.05g MnSO4·4H2Dissolving O and 1000mL of distilled water by heating, adjusting pH to 6.5 with HCl, subpackaging, and sterilizing at 121 ℃ for 20min for later use; if a solid medium is prepared, 20g of agar powder is added.
Specifically, preparation of the YPD liquid medium: heating and dissolving 20g of peptone, 10g of yeast powder, 20g of glucose and 1000mL of distilled water, and sterilizing at 121 ℃ for 20min for later use; if a solid medium is prepared, 20g of agar powder is added.
Respectively carrying out strain identification on the strains QH-1, QH-2, QH-8, QH-10, QH-12, QH-16, QH-19 and QH-21 obtained by screening, and identifying that the QH-1, the QH-2 and the QH-8 are Saccharomyces cerevisiae (Saccharomyces cerevisiae), the QH-10 and the QH-12 are Bacillus subtilis (Bacillus subtilis), the QH-16 is Lactobacillus casei (Lactobacillus casei), the QH-19 is Enterococcus faecium (Enterococcus faecium) and the QH-21 is Lactobacillus haber (Lactobacillus binensis).
Test examples
Acid resistance
The microbial inoculum I, the microbial inoculum II and the microbial inoculum III obtained by compounding in the experimental example are measured for the growth conditions at different pH values (based on MRS liquid culture medium for detection), and the acid tolerance of the compound microbial inoculum is evaluated, and the result is shown in figure 1; the pH value of normal gastric juice of a human body is between 0.8 and 1.5, when the pH value is more than 1.0 and less than or equal to 2.0, the influence of an acid environment on the microbial inoculum III is relatively small, and when the pH value is 2.0, the survival rate is more than 70 percent. The bacteria agent III has strong acid resistance and can meet the requirement of normal gastric juice of human body. After a human body eats, gastric juice is diluted after meal, the pH can be increased to about 3.5, when the pH is more than or equal to 3.0 and less than or equal to 5.0, the three bactericides are only slightly influenced by an acid environment, and the survival rates of the three bactericides are more than 65 percent, which shows that the three bactericides can better survive when the pH is more than 3.0, and can survive in vivo to play a probiotic effect when the human body eats. In conclusion, the three microbial inoculum types have better acid resistance, and under the condition of higher acid environment, the microbial inoculum III has stronger acid resistance than the microbial inoculum I and the microbial inoculum II relatively, and can be taken by human bodies when the human bodies do not eat, and the other two microbial inoculum types are preferably taken after meals.
Resistance to bile salts
The microbial inoculum I, the microbial inoculum II and the microbial inoculum III which are obtained by compounding in the experimental example are measured for the growth conditions under different pig bile salt concentrations (based on MRS liquid culture medium for detection), and the tolerance capability of the microbial inoculum to bile salts is evaluated, and the result is shown in figure 2; as can be seen from FIG. 2, the three bacterial agents are slightly reduced by increasing the concentration of the bile salt in the bacterial liquid concentration in the pig bile salt culture with different concentrations; when the concentration of the bile salt is 0.1-0.5%, the bile salt does not have obvious inhibiting effect on three bactericides, the survival rate of the three bactericides reaches more than 70%, and the three bactericides have stronger bile salt resistance, and the bile salt resistance of the bactericide I is slightly higher than that of the bactericide II and the bactericide III; it is known that the bile juice content in the small intestine of a normal human body is between 0.03% and 0.3%, and when the concentration of bile salts is 0.3%, the survival rates of the three bactericides are 90.62%, 83.62% and 83.69% respectively, which indicates that the three bactericides can better survive when the concentration of bile salts is 0.3%, and when the concentration of bile salts is lower than 0.3%, the survival rates of the three bactericides are more than 94%, more than 85% and more than 84% respectively, which indicates that the influence on the bactericides I is small, and the influence on the bactericides II and 3 is small, and when the concentration of bile salts is higher than 0.3%, the survival rates of the three bactericides are more than 84%, more than 80% and more than 73% respectively, which indicates that the influence on the three bactericides is certain, but the influence is not large.
Antioxidant function
The reducing power is a common index for in vitro oxidation resistance detection, the reducing power of the strain is determined by adopting a potassium ferricyanide reducing method, and the reducing power of the strain is represented by the size of a light absorption value; the results of the reducing power of the somatic cells, fermentation supernatant and cell-free extract of the three microbial agents, namely microbial agent I, microbial agent II and microbial agent III, are shown in FIG. 3; as can be seen from FIG. 3, the three bacterial agents all show high reducing power, the difference of the reducing power of different parts is large, and the reducing power of the cell-free extracts of the three bacterial agents is obviously superior to that of the bacterial cells and the fermentation supernatant. And the reducing power of the microbial inoculum III is slightly higher than that of the microbial inoculants I and 2.
The superoxide radical can induce lipid peroxidation in vivo, accelerate human body aging, easily induce skin diseases, cardiovascular diseases, cancer and the like, and seriously harm human health; the results of eliminating superoxide radical by somatic cells, fermentation supernatant and cell-free extract of the three bacterial agents are shown in FIG. 4; as can be seen from FIG. 4, the clearance rates of different parts of the three bactericides for superoxide radicals are different, the clearance rates of the bactericides I and the bactericides II for superoxide radicals are mainly located in fermentation supernatant and bacterial cells, the bactericides III are mainly located in the fermentation supernatant and cell-free extracts, wherein the clearance rates of the fermentation supernatant of the three bactericides for superoxide radicals are respectively 48.73%, 50.73% and 45.1%, the clearance rates of the bacterial cells for superoxide radicals are respectively 60.38%, 62.38% and 25.57%, and the clearance rates of the cell-free extracts for superoxide radicals are respectively 35.23%, 37.23% and 36.31%.
The hydroxyl free radical can kill red blood cells, destroy cell membranes and polysaccharide compounds, and damage cells of a human body, thereby reducing the immune function of the human body and accelerating aging; the results of the hydroxyl radical scavenging ability of the two bacterial agents, the bacterial cells, the fermentation supernatant and the cell-free extract are shown in figure 5; as can be seen from FIG. 5, the hydroxyl radical scavenging rate of the bacterial cells and the fermentation supernatant of the three bacterial agents is obviously superior to that of the cell-free extract, which indicates that the substances with the hydroxyl radical scavenging ability of the three bacterial agents mainly exist in the bacterial cells and the fermentation supernatant, and a small amount of the substances exists in the cell-free extract. Wherein the hydroxyl radical clearance rates of three microbial inoculum fermentation supernatants are respectively 66.56%, 74.56% and 73.79%, the hydroxyl radical clearance rates of thallus cells are respectively 88.47%, 90.47% and 87.62%, and the hydroxyl radical clearance rates of cell-free extracts are respectively 20.84%, 30.84% and 25.62%.
Capacity of degrading cholesterol
The three microbial inoculums are respectively adopted to carry out degradation tests on cholesterol with different concentrations, the test results are shown in fig. 6-7, and it can be seen from fig. 6 that under the condition of low cholesterol concentration (0.1mg/mL), the cholesterol reduction capabilities of the three microbial inoculums are not greatly different, and the cholesterol degradation rates are respectively 68.18%, 70.18% and 72.41%, which shows that the three microbial inoculums have better degradation capabilities on the cholesterol; as known by national standards, the normal amount of total cholesterol in human body is below 2.2mg/mL, thus, the cholesterol-lowering ability of the three inocula at high cholesterol concentrations (3mg/mL, 4mg/mL and 5mg/mL) was determined, as can be seen from FIG. 7, the three bacterial agents have a reduced degradation rate of cholesterol at a lower concentration under the condition of high concentration of cholesterol, but still have a certain degradation capability, the degradation rates of the three microbial inoculum are respectively more than 22%, 28% and 34%, which shows that the three microbial inoculum still has certain degradation capability to cholesterol under the condition of high-concentration cholesterol, in conclusion, under the condition of low concentration cholesterol, the three bactericides have equivalent cholesterol-reducing effect and can be used, however, when the concentration of cholesterol is higher, the degradation rate of the microbial inoculum III is obviously better than that of the microbial inoculum I and the microbial inoculum II, so that the microbial inoculum III is preferably used.
In conclusion, the microbial inoculum I has the advantages that the cost can be saved, the cholate resistance is strongest, and the cholesterol degradation rate is equivalent to that of the microbial inoculum II and the microbial inoculum III under the condition of low concentration cholesterol; the microbial inoculum II has the advantages that the antioxidant activity is strongest, and the cholesterol reducing capability of the microbial inoculum II is obviously better than that of the microbial inoculum I under the condition of high concentration cholesterol; the microbial inoculum III has the advantages of strongest acid resistance, higher survival rate under the condition of pH2.0, and obvious advantage of cholesterol reduction capability under the condition of higher cholesterol concentration, and is obviously superior to the microbial inoculum I and the microbial inoculum II.
Application of microbial agent
The microbial inoculum I obtained in the experimental example is used for preparing the mulberry enzyme, brown sugar is used as fermentation glycogen, when the fermentation is finished, the total phenol content of the prepared mulberry enzyme is 14.935mg/mL, the Vc content reaches 90.348mg/mL, the reducing power is 0.283, the superoxide radical clearance rate is 12.2%, the hydroxyl radical clearance rate is 34.1%, and the total phenol content, the Vc content, the reducing power and the hydroxyl radical clearance rate are respectively increased by 23%, 8%, 43%, 11% and 53% compared with the natural fermentation.
The microbial inoculum II obtained in the experimental example is used for preparing grape ferment, brown sugar is used as fermentation glycogen, when fermentation is finished, the total phenol content in the prepared grape ferment is 2.155mg/mL, the Vc content reaches 17.642mg/mL, the reducing power is 0.635, the superoxide radical clearance rate is 25.283%, the hydroxyl radical clearance rate is 25.685%, and the total phenol content is respectively increased by 31%, 8%, 1%, 518% and 33% compared with natural fermentation.
The microbial inoculum I obtained in the experimental example is used for preparing apple ferment, brown sugar is used as fermentation glycogen, when the fermentation is finished, the total phenol content in the prepared apple ferment is 1.747mg/mL, the Vc content reaches 25.230mg/mL, the reducing power is 0.335, the clearance rate of superoxide radical is 20.6%, the clearance rate of hydroxyl radical is 26.3%, and the clearance rates are respectively increased by 4.86%, 11.28%, 15.12%, 3.6% and 6.7% compared with the natural fermentation.
The microbial inoculum II obtained in the experimental example is used for preparing apple ferment, brown sugar is used as fermentation glycogen, when the fermentation is finished, the total phenol content in the prepared apple ferment is 1.714mg/mL, the Vc content reaches 25.750mg/mL, the reducing power is 0.332, the clearance rate of superoxide radicals is 19.3%, the clearance rate of hydroxyl radicals is 25.3%, and the clearance rates are respectively increased by 2.88%, 13.58%, 14.09%, 2.3% and 5.7% compared with the natural fermentation.
The microbial inoculum III obtained in the experimental example is used for preparing apple ferment, brown sugar is used as fermentation glycogen, when the fermentation is finished, the total phenol content in the prepared apple ferment is 2.588mg/mL, the Vc content reaches 41.177mg/mL, the reducing power is 0.322, the clearance rate of superoxide radical is 20.6%, the clearance rate of hydroxyl radical is 21.9%, and the clearance rates are respectively increased by 16.97%, 30.46%, 13.39%, 3.6% and 2.3% compared with the natural fermentation.
The foregoing is illustrative of the preferred embodiments of this invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and that various other combinations, modifications, and environments may be resorted to, falling within the scope of the concept as disclosed herein, either as described above or as apparent to those skilled in the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A microbial fermentation inoculum, which is characterized by comprising Lactobacillus casei (Lactobacillus casei), Lactobacillus harbinensis (Lactobacillus harbinensis) and Enterococcus faecium (Enterococcus faecium).
2. The microbial fermentation inoculum according to claim 1, wherein the total viable count of each viable organism in the microbial fermentation inoculum is 2.4 x 109CFU/mL or more.
3. The microbial fermentation inoculant according to claim 1, further comprising Saccharomyces cerevisiae.
4. The microbial fermentation inoculum of claim 3, wherein the total viable count of the microbial fermentation inoculum is 2.8 x 109CFU/mL or more.
5. The microbial fermentation inoculant according to claim 3, further comprising Bacillus subtilis.
6. The microbial fermentation inoculum of claim 5, wherein the total viable count of the microbial fermentation inoculum is 3.8 x 109CFU/mL or more.
7. The microbial fermentation inoculum according to claim 5,
the lactobacillus casei is lactobacillus casei QH16, the accession number of 16SrDNA of the lactobacillus casei QH16 in Genbank is MN809276,
the lactobacillus haber is lactobacillus haber QH21, the landing number of 16SrDNA of lactobacillus haber QH21 on Genbank is MN809279,
the enterococcus faecium is enterococcus faecium QH19, the landing number of 16SrDNA of enterococcus faecium QH19 on Genbank is MN809277,
the saccharomyces cerevisiae comprises saccharomyces cerevisiae QH1, saccharomyces cerevisiae QH2 and saccharomyces cerevisiae QH8, the accession number of 18SrDNA of the saccharomyces cerevisiae QH1 on the Genbank is MN759772, the accession number of 18SrDNA of the saccharomyces cerevisiae QH2 on the Genbank is MN759439, the accession number of 18SrDNA of the saccharomyces cerevisiae QH8 on the Genbank is 918288,
the bacillus subtilis comprises bacillus subtilis QH10 and bacillus subtilis QH12, the accession number of 16SrDNA of the bacillus subtilis QH10 on Genbank is MN809144, and the accession number of 16SrDNA of the bacillus subtilis QH12 on Genbank is MN 809275.
8. The microbial fermentation inoculum of claim 7, wherein the viable bacteria ratio of lactobacillus casei QH16, lactobacillus haber QH21, enterococcus faecium QH19, saccharomyces cerevisiae QH1, saccharomyces cerevisiae QH2, saccharomyces cerevisiae QH8, bacillus subtilis QH10 and bacillus subtilis QH12 in the inoculum is 1.0-2.5: 1.0-2.5: 2.0-3.0: 2.0-2.5: 3.0-3.5: 3.0-3.5: 1.5-2.0: 2.0 to 2.5.
9. The application method of the microbial fermentation inoculant according to any one of claims 1 to 8, wherein the microbial fermentation inoculant is used for fruit and vegetable fermentation.
10. The preparation method of the microbial fermentation inoculant is characterized in that at least one of grape ferment, Hami melon ferment, mulberry ferment, kiwi ferment, yoghourt and probiotic is used as a raw material, and the microbial fermentation inoculant is prepared by the following steps:
step S1, placing the raw material enzymes in an MRS liquid culture medium for constant-temperature culture to obtain an enrichment culture solution;
step S2, separating and purifying the enrichment culture solution, and separating the pure strains from the culture solution by adopting a gradient dilution method;
step S3, acid-resistant screening and cholate-resistant screening are respectively carried out on each pure strain obtained by separation,
the acid-resistant screening is specifically that the pure strains of the two activated generations are respectively inoculated into an MRS liquid culture medium with the pH value of 3.5, an MRS liquid culture medium with the pH value of 6.5, an YPD liquid culture medium with the pH value of 3.5 and an YPD liquid culture medium with the pH value of 6.5 according to the inoculum concentration of 1 percent, after the pure strains are cultured in a constant-temperature shaking table at the temperature of 35 ℃ for 24 hours, the light absorption value of the pure strains under 600nm is measured, the ratio of the two light absorption values in the same type of culture medium is taken as the survival rate of the strains under the pH value of 3.5, the strains with the survival rate of more than 50 percent based on the MRS liquid culture medium and the strains with the survival rate of more than 80 percent based on the YPD;
the bile salt resistant screening is specifically that the pure strains after two generations of activation are respectively inoculated into an MRS liquid culture medium with bile salt concentration of 0.3%, an MRS liquid culture medium without bile salt, an YPD liquid culture medium with bile salt concentration of 0.3% and an YPD liquid culture medium without bile salt according to the inoculum concentration of 1%, after the pure strains are cultured in a constant temperature shaking table at the temperature of 35 ℃ for 24 hours, the light absorption value of the pure strains under 600nm is measured, the ratio of the two light absorption values in the same type of culture medium is taken as the survival rate of the strains under the bile salt concentration of 0.3%, the strains with the survival rate of more than 20% based on the MRS liquid culture medium are screened, and the survival rate of more than 45% based on the YPD liquid culture medium is screened;
and S4, compounding strains which simultaneously meet acid resistance screening and cholate resistance screening to obtain the microbial fermentation inoculant.
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