CN116121166A - Preparation method of lactobacillus reuteri whole grain fermentation system with high viable count - Google Patents
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Abstract
The invention discloses a preparation method of a lactobacillus reuteri whole grain fermentation system with high viable count, which takes natural grains as a culture medium, and comprises the steps of sterilizing at high temperature, cooling, adding lactobacillus reuteri for anaerobic fermentation to obtain the lactobacillus reuteri with the viable count of 10-15Log 10 CFU/mL Lactobacillus reuteri whole grain fermentation system; the natural grains can obviously promote the growth of lactobacillus reuteri and realize the high-density culture of lactobacillus reuteri; the lactobacillus reuteri whole grain fermentation system tightly combines lactobacillus reuteri and whole grains to obtain lactobacillus reuteri whole grain fermentation liquor with high viable count, and the fermentation liquor has the advantages of whole grains and probiotics, and can be used for producing fermented milk, fermented milk beverage and functional food.
Description
Technical Field
The invention belongs to the field of probiotics and functional foods, and particularly relates to a preparation method of a lactobacillus reuteri whole grain fermentation system with high viable count.
Background
Probiotics are active microorganisms that produce health benefits by modulating the microbial flora in the host after ingestion in sufficient amounts, mainly from normal physiologically active bacteria in the intestinal and parenteral tracts of fermented foods, healthy humans and animals. Beneficial effects of probiotics on health include improving intestinal flora, enhancing the immune system, lowering serum cholesterol, preventing cancer, treating irritable bowel syndrome, antihypertensive, etc. Today, downstream products of the probiotic industry have spanned many fields of food, healthcare, daily chemicals, and the like. It is not to be neglected that all probiotic preparations should follow the three basic criteria "sufficient number", "live bacterial status", "health function" together. Therefore, the improvement of the viable count of the probiotics in the fermentation process becomes a core technology of attack of vast scientific researchers and probiotics production enterprises. The number of viable bacteria in the current probiotic preparation and related products is mainly in the trillion level or the trillion level of bacteria powder per gram of raw material, and how to break through the trillion level still has technical problems which need to be solved urgently.
As one of the stable microorganism members in human body, the lactobacillus reuteri has good biocompatibility and safety, and has various probiotic functions of resisting bacteria, enhancing immunity, improving oral health, reducing blood fat, improving irritable bowel syndrome and the like, so that the related products using the lactobacillus reuteri as the fermentation strain have immeasurable market value. It is worth noting that the viable count of lactobacillus reuteri in fermented products is not only critical for its probiotic function, but also an important factor in determining the market competitiveness of the product. Patent publication No. CN114304620A discloses that raw materials of medicinal mulberries, blueberries and blackcurrants are cleaned, pulped respectively and mixed according to a proportion to obtain mixed fruit pulp, and the mixed fruit pulp is refrigerated for later use; activating and culturing the stored lactobacillus plantarum, lactobacillus delbrueckii, lactobacillus reuteri, lactobacillus rhamnosus, lactobacillus paracasei and lactobacillus helveticus under the culture condition of 37 ℃ and the viable count of 10 percent 8 CFU/mL or more; adding pectinase, cellulase and hemicellulase into the mixed pulp according to the mass ratio, uniformly stirring, and performing enzymolysis for 3-4 hours to obtain enzymolysis mixed pulp; adding sucrose into the obtained enzymolysis mixed pulp, uniformly mixing, adjusting the TSS value to be 19.0, transferring into a fermentation tank, heating and sterilizing, wherein the sterilizing temperature is 95 ℃, the sterilizing time is 30min, naturally cooling to 34 ℃ after sterilizing, inoculating compound bacteria under aseptic condition for anaerobic fermentation, and the fermenting temperature is 32-36 ℃ and the fermenting time is 20-25h; sterilizing at 95deg.C for 25min, cooling to 30deg.C, centrifuging, filtering to obtain supernatant, filling into nitrogen-filled brown bottle, sterilizing at 100deg.C for 18min, and collecting the supernatantThen cooling, keeping away from light, and preserving at low temperature to prepare and obtain Xinjiang medicine mulberry compound ferment; and the patent with publication number of CN106754482A discloses a preparation method of a probiotic starter for producing functional sugar, comprising the following steps: lactobacillus griseus, lactobacillus reuteri or lactobacillus plantarum are taken as fermentation strains, the fermentation strains are inoculated into a proliferation culture medium and fed in stages to add a carbon source, anaerobic fermentation culture is carried out for 8-16 hours at 35-40 ℃, then lactobacillus cells are obtained by centrifugation under aseptic conditions, the lactobacillus cells and a freeze-drying protective agent are mixed at-55-45 ℃ for prefreezing for 1-3 hours, and then freeze-drying is carried out to obtain the solid-state bacterial powder of the ferment, wherein the viable count in the solid-state bacterial powder of the ferment is 2.1x10 10 cfu/g or more. However, the existing technology adopting lactobacillus reuteri can not achieve trillion viable count rapidly, or concentrate on the optimization of artificial culture medium or the synergistic promotion effect of various complex bacteria. At present, the high-density culture of lactobacillus reuteri by taking natural grains as a culture matrix has not been reported. Therefore, the high-density culture of the lactobacillus reuteri by taking the natural grains as a culture medium has great significance for developing lactobacillus reuteri products with high viable count and whole grain foods.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method of a lactobacillus reuteri whole grain fermentation system with high viable count.
The method is realized by the following technical scheme:
a method for preparing whole grain fermentation system of Lactobacillus reuteri with high viable count comprises sterilizing natural grain as culture medium, cooling, inoculating Lactobacillus reuteri seed solution, and performing proliferation fermentation for 12-24 hr to obtain a viable count of 10-15Log 10 CFU/mL lactobacillus reuteri whole grain fermentation system.
The natural grains include, but are not limited to, one of coix seed, quinoa, millet, red rice, black rice and brown rice or a plurality of mixtures according to any ratio.
The culture medium includes, but is not limited to, powder-conditioned, emulsion-type, and enzymatic-type.
The preparation method of the powder-conditioned medium comprises the following steps: drying natural grains, pulverizing, sieving with 60-120 mesh sieve, weighing appropriate amount of powder in 250mL conical flask, adding distilled water according to feed-liquid ratio (1:8-10), mixing, sterilizing at 121deg.C for 20min, and cooling to room temperature to obtain the culture medium.
The preparation method of the emulsion type culture medium comprises the following steps: weighing natural grains in a 250mL conical flask, adding distilled water, repeatedly cleaning for several times, adding distilled water according to a feed-liquid ratio (1:8-10), soaking, transferring the grains and distilled water into a pulping machine for grinding together after the soaking is completed, filtering with 60-120 mesh filter cloth after grinding, sterilizing the filtrate at 90-95 ℃ for 10-15min, and cooling to room temperature to obtain the emulsion type culture medium.
The preparation method of the enzymolysis type culture medium comprises the following steps: gelatinizing the ground grain suspension at 90 ℃ for 20-30min, cooling to 60-70 ℃, adding 0.05% -1% of alpha-amylase, carrying out enzymolysis for 10-30min at 65-70 ℃, adding 0.05% -1% of saccharifying enzyme after enzymolysis, carrying out enzymolysis for 10-30min at 60-70 ℃, filtering the suspension with 60-120 mesh filter cloth after enzymolysis, collecting filtrate, subpackaging the filtrate in a 250mL conical flask, sterilizing for 20min at 121 ℃, and cooling to room temperature to obtain the enzymolysis type culture medium.
The preparation method of the lactobacillus reuteri seed solution comprises the following steps: fully dissolving lactobacillus reuteri bacteria powder by using sterile water, absorbing 1mL of bacteria suspension, coating the bacteria suspension on an MRS agar culture medium, carrying out anaerobic culture for 24 hours at 37 ℃, selecting single bacterial colony from the MRS agar culture medium which is cultured for the first time, transferring the single bacterial colony into the MRS broth culture medium, carrying out anaerobic culture for 24 hours at 37 ℃, activating for 3 times continuously, carrying out anaerobic culture for 12-16 hours at 37 ℃ on activated lactobacillus reuteri in the MRS broth culture medium, centrifuging for 5 minutes at 3000-6000r/min, discarding the supernatant, washing for a plurality of times by using sterile saline, and adding the saline with the same volume as that of the culture to carry out resuspension after washing to obtain lactobacillus reuteri fermentation seed liquid.
The MRS agar medium is as follows: 10.0g of tryptone, 5.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 5.0g of sodium acetate, 2.0g of triamine citrate, 1mL of Tween 80, 2.0g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate and 15.0g of agar, adding 1L of distilled water, fully dissolving and uniformly mixing, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20min.
The MRS broth culture medium is as follows: 10.0g of tryptone, 5.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 5.0g of sodium acetate, 2.0g of triamine citrate, 1mL of Tween 80, 2.0g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate and 0.05g of manganese sulfate are added with 1L of distilled water to be fully dissolved and uniformly mixed, the pH is adjusted to 7.0, and the mixture is sterilized for 20min at 121 ℃.
The inoculation amount of the lactobacillus reuteri seed solution is 5-10% of the culture medium, and the inoculation viable count is 10 6 -10 7 CFU/mL。
The lactobacillus reuteri whole grain fermentation system is applied to preparation of fermented milk, fermented beverage and live bacteria preparation.
The beneficial effects are that:
the invention provides a method for obtaining a lactobacillus reuteri whole grain fermentation system with high viable count, which combines lactobacillus reuteri with whole grains, takes natural whole grains as a culture medium, takes lactobacillus reuteri as a fermentation strain, and remarkably improves the viable count of lactobacillus reuteri after anaerobic fermentation, wherein the viable count reaches trillion levels, thereby laying a foundation for the development of related high viable probiotic foods. Meanwhile, the lactobacillus reuteri fermentation improves the defects of nutrition resistance, indigestion, easy initiation of irritable bowel syndrome and the like of whole grains, and the functional activity is obviously enhanced.
The natural cereal adopted by the invention can promote the growth of lactobacillus reuteri, realizes the high-density culture of lactobacillus reuteri, has the advantages of both whole cereal and probiotics, can be used for producing fermented milk, fermented milk beverage and functional food, and takes the natural cereal as a culture medium prepared by raw materials, thereby greatly reducing the cost of the culture medium, and the prepared powder-type, emulsion-type and enzymolysis-type culture medium has the advantages of simple and easy preparation method, rich and comprehensive nutrition, low requirements on fermentation conditions, no special control on conditions such as temperature and humidity, and the like.
The method of the invention is simple, the cost is low, the production period is short, the whole grains are easier to combine with other products, and the novel food with richer product types and more tastes is formed.
Drawings
FIG. 1 is a graph showing the trend of viable count over time of Lactobacillus reuteri in a powder-conditioned medium prepared from different grains;
FIG. 2 shows the viable count of Lactobacillus reuteri in a powder conditioned medium made from different grains at the end of fermentation;
FIG. 3 is a graph showing the trend of viable count over time of Lactobacillus reuteri in emulsion-type culture media made from different grains;
FIG. 4 shows the number of viable bacteria of Lactobacillus reuteri in emulsion-type culture medium made of different grains at the end of fermentation;
FIG. 5 is a graph showing the trend of viable count over time of Lactobacillus reuteri in an enzymatic culture medium made from different grains;
FIG. 6 shows the number of viable bacteria of Lactobacillus reuteri in an enzymatic culture medium made from different grains at the end of fermentation.
Detailed Description
The following detailed description of the invention is provided in further detail, but the invention is not limited to these embodiments, any modifications or substitutions in the basic spirit of the present examples, which still fall within the scope of the invention as claimed.
Example 1: preparing a lactobacillus reuteri powder-regulated whole grain fermentation system with high viable count;
1. preparation of powder-conditioned medium
Respectively drying Coicis semen, quinoa, semen Setariae, red rice, fructus Zizaniae Caduciflorae and brown rice, pulverizing, sieving with 60 mesh sieve, weighing appropriate amount of powder, adding distilled water according to feed-liquid ratio (1:8) into 250mL conical flask, mixing, sterilizing at 121deg.C for 20min, and cooling to room temperature to obtain powder-conditioned culture medium;
2. activation of the strains and seed fermentation
(1) Strain activation: before the experiment, fully dissolving lactobacillus reuteri bacteria powder by using sterile water, sucking 1mL of bacteria suspension, coating the bacteria suspension on an MRS agar culture medium, and carrying out anaerobic culture for 24 hours at 37 ℃; selecting single colony from the MRS agar culture medium for primary culture, transferring into MRS broth culture medium, anaerobic culturing at 37deg.C for 24 hr, and continuously activating for 3 times to ensure strain activity;
(2) Preparing fermentation seed liquid: anaerobic culturing activated lactobacillus reuteri in MRS broth culture medium at 37deg.C for 8 hr, centrifuging at 3000r/min for 5min, discarding supernatant, washing with sterile physiological saline for several times, and adding physiological saline equal in volume to culture for resuspension;
(3) Inoculating and fermenting: inoculating the fermentation seed liquid into different treated cereal matrixes at an inoculum size of 5%, and performing anaerobic culture at 37 ℃ for 12-24 hours;
3. determination of viable count
Accurately sucking 1mL of fermentation liquor, and carrying out 10-time gradient dilution by using sterile physiological saline. After dilution is completed, three diluted gradient bacterial solutions are selected, 1mL of diluted bacterial solutions are respectively absorbed and uniformly coated in an MRS agar culture medium, a coated flat plate is placed in an anaerobic workstation, and is cultured for 48 hours at 37 ℃ to perform colony counting, and the counting result is expressed as CFU/mL; the results of this example are shown in FIG. 1.
Example 2: preparing a lactobacillus reuteri emulsion type whole grain fermentation system with high viable count;
1. preparation of emulsion-type culture substrate
Weighing a proper amount of coix seeds, quinoa, millet, red rice, black rice and brown rice in a 250mL conical flask, adding distilled water, repeatedly cleaning for a plurality of times, adding distilled water according to a feed liquid ratio (1:10), soaking, and after the soaking is finished, transferring grains and distilled water into a pulping machine together for grinding; grinding, filtering with 20 mesh filter cloth, sterilizing the filtrate at 92 deg.C for 15min, and cooling to room temperature to obtain emulsion culture matrix;
2. activation of the strains and seed fermentation
(1) Strain activation: before the experiment, fully dissolving lactobacillus reuteri bacteria powder by using sterile water, sucking 1mL of bacteria suspension, coating the bacteria suspension on an MRS agar culture medium, and carrying out anaerobic culture for 24 hours at 37 ℃; selecting single colony from the MRS agar culture medium for primary culture, transferring into MRS broth culture medium, anaerobic culturing at 37deg.C for 24 hr, and continuously activating for 3 times to ensure strain activity;
(2) Preparing fermentation seed liquid: anaerobic culturing activated lactobacillus reuteri in MRS broth culture medium at 37deg.C for 16 hr, centrifuging at 6000r/min for 5min, discarding supernatant, washing with sterile physiological saline for several times, and adding physiological saline equal in volume to culture for resuspension;
(3) Inoculating and fermenting: inoculating the fermentation seed liquid into different treated cereal matrixes at an inoculum size of 5%, and performing anaerobic culture at 37 ℃ for 12-24 hours;
3. determination of viable count
Accurately sucking 1mL of fermentation liquor, and carrying out 10-time gradient dilution by using sterile physiological saline. After dilution is completed, three diluted gradient bacterial solutions are selected, 1mL of diluted bacterial solutions are respectively absorbed and uniformly coated in an MRS agar culture medium, a coated flat plate is placed in an anaerobic workstation, and is cultured for 48 hours at 37 ℃ to perform colony counting, and the counting result is expressed as CFU/mL; the results of this example are shown in FIG. 2.
Example 3: preparing a high viable count lactobacillus reuteri enzymolysis type whole grain fermentation system;
1. preparation of enzymolysis type culture medium
Gelatinizing the ground grain suspension at 90deg.C for 25min, cooling to 65deg.C, adding 0.08% alpha-amylase, performing enzymolysis at 65deg.C for 20min, adding 0.05-1% saccharifying enzyme, performing enzymolysis at 65deg.C for 20min, filtering the suspension with 100 mesh filter cloth, collecting filtrate, packaging the filtrate in 250mL conical flask, sterilizing at 121deg.C for 20min, and cooling to room temperature to obtain enzymolysis culture medium;
2. activation of the strains and seed fermentation
(1) Strain activation: before the experiment, fully dissolving lactobacillus reuteri bacteria powder by using sterile water, sucking 1mL of bacteria suspension, coating the bacteria suspension on an MRS agar culture medium, and carrying out anaerobic culture for 24 hours at 37 ℃; selecting single colony from the MRS agar culture medium for primary culture, transferring into MRS broth culture medium, anaerobic culturing at 37deg.C for 24 hr, and continuously activating for 3 times to ensure strain activity;
(2) Preparing fermentation seed liquid: anaerobic culturing activated lactobacillus reuteri in MRS broth culture medium at 37deg.C for 12 hr, centrifuging at 4500r/min for 5min, discarding supernatant, washing with sterile physiological saline for several times, and adding physiological saline equal to that of culture for resuspension;
(3) Inoculating and fermenting: inoculating the fermentation seed liquid into different treated cereal matrixes at an inoculum size of 5%, and performing anaerobic culture at 37 ℃ for 12-24 hours;
3. determination of viable count
Accurately sucking 1mL of fermentation liquor, and carrying out 10-time gradient dilution by using sterile physiological saline. After dilution is completed, three diluted gradient bacterial solutions are selected, 1mL of diluted bacterial solutions are respectively absorbed and uniformly coated in an MRS agar culture medium, a coated flat plate is placed in an anaerobic workstation, and is cultured for 48 hours at 37 ℃ to perform colony counting, and the counting result is expressed as CFU/mL; the results of this example are shown in FIG. 3.
As can be seen from FIGS. 1 and 2, in the powder-type whole grain fermentation system, the maximum viable count of Lactobacillus reuteri in the Coicis semen matrix can reach 14.12Log 10 CFU/mL(1.33×10 14 CFU/mL). The maximum viable count of lactobacillus reuteri in quinoa matrix is 12.79Log 10 CFU/mL(6.20×10 12 CFU/mL). The maximum viable count of lactobacillus reuteri in the millet matrix reaches 12.05Log 10 CFU/mL(1.12×10 12 CFU/mL). The maximum viable count of the lactobacillus reuteri in the black rice matrix reaches 10.81Log 10 CFU/mL(6.50×10 10 CFU/mL). The maximum viable count of Lactobacillus reuteri in brown rice and red rice matrix is 10.79Log 10 CFU/mL(6.33×10 10 CFU/mL) and 10.93Log 10 CFU/mL(8.5×10 10 CFU/mL)。
As can be seen from FIGS. 3 and 4, in the emulsion type whole grain fermentation system, the maximum viable count of Lactobacillus reuteri in the coix seed matrix reaches 14.48Log 10 CFU/mL(3.10×10 14 CFU/mL). The maximum viable count of lactobacillus reuteri in quinoa matrix is 13.09Log 10 CFU/mL(1.25×10 13 CFU/mL). In millet matrix, the maximum viable count of lactobacillus reuteri is 12.18Log 10 CFU/mL(1.53×10 12 CFU/mL). The maximum viable count of lactobacillus reuteri in the millet matrix is 11.57Log 10 CFU/mL(3.73×10 11 CFU/mL). In a fermentation system taking black rice and brown rice as matrixes, the maximum viable count of lactobacillus reuteri is 11.18Log respectively 10 CFU/mL(1.55×10 11 CFU/mL) and 11.07Log 10 CFU/mL(1.15×10 11 CFU/mL). The maximum viable count of lactobacillus reuteri in the red rice matrix is 10.87Log 10 CFU/mL(7.10×10 10 CFU/mL)。
As can be seen from FIGS. 5 and 6, in the enzymatic whole grain fermentation system, the maximum viable count of Lactobacillus reuteri in the coix seed matrix reaches 14.31Log 10 CFU/mL(2.07×10 14 CFU/mL). The maximum viable count of lactobacillus reuteri in quinoa and millet matrixes is 13.01Log respectively 10 CFU/mL(1.03×10 13 CFU/mL) and 12.13Log 10 CFU/mL(1.6×10 12 CFU/mL). In the red rice and black rice matrixes, the maximum viable count of the lactobacillus reuteri is 11.12Log respectively 10 CFU/mL(1.33×10 11 CFU/mL) and 10.92Log 10 CFU/mL(8.23×10 10 CFU/mL). In the brown rice matrix, the maximum viable count of the lactobacillus reuteri is 10.51Log 10 CFU/mL(3.27×10 10 CFU/mL)。
Taken together, the 6 grains selected all supported the growth of lactobacillus reuteri, but there were significant differences in growth among the 6 grain substrates. From the result of viable count, coix seed is an ideal substrate for high-density culture of lactobacillus reuteri. The viable count of the lactobacillus reuteri reaches 10 under the condition of no other exogenous nutrient substances 14 CFU/mL. Under the same fermentation condition, the viable count in the quinoa matrix reaches 10 13 CFU/mL, while the number of viable bacteria in the matrix of red rice, black rice, brown rice and millet is 10 10 CFU/mL or more, compared with the fermentation (the viable count at the time of inoculation is about 10) 7 CFU/mL) are greatly improved.
Claims (10)
1. A process for preparing the whole-grain fermentation system of lactobacillus reuteri with high living bacteria number includes such steps as sterilizing natural grains, cooling, inoculating the seed liquid of lactobacillus reuteri, and fermenting for 12-24 hr to obtain the product with living bacteria number of 10-15Log 10 CFU/mL lactobacillus reuteri whole grain fermentation system.
2. The method of claim 1, wherein the natural cereal includes, but is not limited to, one or more of coix seed, quinoa, millet, red rice, black rice and brown rice, or a mixture thereof in any ratio.
3. The method of claim 1, wherein the culture medium comprises, but is not limited to, a modified-flour type, an emulsion type, and an enzymatic type.
4. The method for preparing the lactobacillus reuteri whole grain fermentation system with high viable count as claimed in claim 3, wherein the method for preparing the powder-conditioned medium comprises the following steps: drying natural grains, pulverizing, sieving with 60-120 mesh sieve, weighing appropriate amount of powder in 250mL conical flask, adding distilled water according to feed-liquid ratio (1:8-10), mixing, sterilizing at 121deg.C for 20min, and cooling to room temperature to obtain the culture medium.
5. The method for preparing the lactobacillus reuteri whole grain fermentation system with high viable count as claimed in claim 3, wherein the method for preparing the emulsion type culture medium comprises the following steps: weighing natural grains in a 250mL conical flask, adding distilled water, repeatedly cleaning for several times, adding distilled water according to a feed-liquid ratio (1:8-10), soaking, transferring the grains and distilled water into a pulping machine for grinding together after the soaking is completed, filtering with 60-120 mesh filter cloth after grinding, sterilizing the filtrate at 90-95 ℃ for 10-15min, and cooling to room temperature to obtain the emulsion type culture medium.
6. The method for preparing the lactobacillus reuteri whole grain fermentation system with high viable count as claimed in claim 3, wherein the method for preparing the enzymolysis type culture medium comprises the following steps: gelatinizing the ground grain suspension at 90 ℃ for 20-30min, cooling to 60-70 ℃, adding 0.05% -1% of alpha-amylase, carrying out enzymolysis for 10-30min at 65-70 ℃, adding 0.05% -1% of saccharifying enzyme after enzymolysis, carrying out enzymolysis for 10-30min at 60-70 ℃, filtering the suspension with 60-120 mesh filter cloth after enzymolysis, collecting filtrate, subpackaging the filtrate in a 250mL conical flask, sterilizing for 20min at 121 ℃, and cooling to room temperature to obtain the enzymolysis type culture medium.
7. The method for preparing the lactobacillus reuteri whole grain fermentation system with high viable count according to claim 1, wherein the method for preparing the lactobacillus reuteri seed solution is as follows: fully dissolving lactobacillus reuteri bacteria powder by using sterile water, absorbing 1mL of bacteria suspension, coating the bacteria suspension on an MRS agar culture medium, carrying out anaerobic culture for 24 hours at 37 ℃, selecting single bacterial colony from the MRS agar culture medium which is cultured for the first time, transferring the single bacterial colony into the MRS broth culture medium, carrying out anaerobic culture for 24 hours at 37 ℃, activating for 3 times continuously, carrying out anaerobic culture for 12-16 hours at 37 ℃ on activated lactobacillus reuteri in the MRS broth culture medium, centrifuging for 5 minutes at 3000-6000r/min, discarding the supernatant, washing for a plurality of times by using sterile saline, and adding the saline with the same volume as that of the culture to carry out resuspension after washing to obtain lactobacillus reuteri fermentation seed liquid.
8. The method for preparing a lactobacillus reuteri whole grain fermentation system with high viable count as claimed in claim 7, wherein the MRS agar medium is: 10.0g of tryptone, 5.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 5.0g of sodium acetate, 2.0g of triamine citrate, 1mL of Tween 80, 2.0g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate and 15.0g of agar, adding 1L of distilled water, fully dissolving and uniformly mixing, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20min.
9. The method for preparing a lactobacillus reuteri whole grain fermentation system with high viable count as claimed in claim 7, wherein the MRS broth culture medium is: 10.0g of tryptone, 5.0g of beef extract, 4.0g of yeast powder, 20.0g of glucose, 5.0g of sodium acetate, 2.0g of triamine citrate, 1mL of Tween 80, 2.0g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate and 0.05g of manganese sulfate are added with 1L of distilled water to be fully dissolved and uniformly mixed, the pH is adjusted to 7.0, and the mixture is sterilized for 20min at 121 ℃.
10. A highly viable bacterium according to claim 1The preparation method of the whole grain fermentation system of lactobacillus reuteri is characterized in that the inoculation amount of the lactobacillus reuteri seed liquid is 5-10% of the culture medium, and the inoculation viable count is 10 6 -10 7 CFU/mL。
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| CN116333910A (en) * | 2022-09-23 | 2023-06-27 | 华中农业大学 | Culture medium and method for promoting lactobacillus reuteri growth |
| CN116333910B (en) * | 2022-09-23 | 2024-09-20 | 华中农业大学 | Culture medium and method for promoting lactobacillus reuteri growth |
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