CN110106119B - Lactobacillus rhamnosus M9 separated from breast milk and application thereof - Google Patents
Lactobacillus rhamnosus M9 separated from breast milk and application thereof Download PDFInfo
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- CN110106119B CN110106119B CN201910453691.3A CN201910453691A CN110106119B CN 110106119 B CN110106119 B CN 110106119B CN 201910453691 A CN201910453691 A CN 201910453691A CN 110106119 B CN110106119 B CN 110106119B
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- 238000005406 washing Methods 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- 235000008924 yoghurt drink Nutrition 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
Images
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
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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
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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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
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/10—Cereal-derived products
- A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/12—Antidiarrhoeals
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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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- 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
- C12N1/205—Bacterial isolates
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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
- 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
- A23Y—INDEXING SCHEME RELATING TO LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23Y2220/00—Lactobacillus
- A23Y2220/73—Rhamnosus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
Abstract
The application provides a Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) separated from breast milk, the microorganism preservation number of the Lactobacillus rhamnosus M9 is CGMCC No.16002, and the Lactobacillus rhamnosus M9 has excellent gastrointestinal fluid tolerance and good bile salt tolerance and has basic conditions as probiotics; the probiotic characteristics of enhancing immunity, improving the diversity of intestinal flora and increasing the stability of the intestinal flora are achieved; the Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) can be used as probiotic bacteria to be added into various common foods, health products and the like.
Description
Technical Field
The application relates to Lactobacillus rhamnosus (Lactobacillus rhamnosus), in particular to Lactobacillus rhamnosus M9 separated from breast milk and application thereof.
Background
The gastrointestinal tract of a healthy person is populated by a wide variety of microorganisms, which are referred to as intestinal flora. The intestinal flora is combined according to a certain proportion, the bacteria are restricted and dependent with each other, an ecological balance is formed on the quality and quantity, once the internal and external environments of the organism change, especially when the broad-spectrum antibiotics are applied for a long time, the sensitive intestinal flora is inhibited, and the bacteria which are not inhibited multiply by the organism, so that the flora imbalance is caused, the normal physiological combination of the sensitive intestinal flora is destroyed, the pathological combination is generated, and the clinical symptoms are called as the intestinal flora imbalance.
The intestinal flora imbalance disease takes severe diarrhea or chronic diarrhea as a main clinical manifestation, and some tourists may have intestinal flora imbalance due to changes of climate and environment, which is commonly called as water-soil inadequacy. In recent years, due to the popularization and use of refrigerators, some families store a large amount of meat and vegetables, the food is deteriorated after long-term storage, intestinal flora imbalance is caused after eating, vomiting and diarrhea are caused, and some people have no concentration and even have the mindedness.
Once the balance of the intestinal flora is destroyed, the original balance state is difficult to restore, so that the intestinal flora imbalance is difficult to adjust and cure.
Disclosure of Invention
One of the purposes of the application is to provide a strain of rhamnose Lactobacillus M9(Lactobacillus rhamnosus M9) screened from 540 strains of Lactobacillus and bifidobacteria separated from 100 breast milk samples of healthy women in regions such as Mongolia, Xinjiang, Hubei, Beijing and the like in China, the microorganism preservation number is CGMCC No.16002, and the classification and the nomenclature are as follows: lactobacillus rhamnosus; the preservation unit is China general microbiological culture Collection center; the preservation time is 2018, 6 months and 22 days; and (4) storage address: western road No.1 institute 3, institute of microbiology, china academy of sciences, north chen, chaoyang, china.
In the application, the complete genome of Lactobacillus rhamnosus M9 is determined by using a PacBio SMRT RSII third generation sequencing platform, the genome length is 3002913bp, the GC content is 46.77% (wherein, the GC content refers to the ratio of guanine and cytosine in 4 bases of DNA), and the chromosome genome contains 2965 coding genes and 76 RNA genes.
In the present application, the Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) can be isolated according to a method comprising: diluting 100 collected breast milk samples of healthy women in multiple proportions to obtain 10-1～10-2Diluting the gradient, sucking 200 μ L of each gradient breast milk sample, uniformly coating on MRS solid culture medium plate, and cooling to 37 deg.CAnaerobic culturing for 48-72 h. Selecting monoclonals with different shapes, sizes and colors, inoculating the monoclonals into a liquid culture medium, and culturing the monoclonals in a constant-temperature incubator at 37 ℃ for 24 hours. After the strain grows well, gram staining and microscopic examination are carried out. Isolates were preserved and strain genomic DNA was extracted for subsequent assay analysis.
Wherein the MRS culture medium comprises the following components: 10g of soybean peptone, 5g of beef extract, 4g of yeast powder, 20g of glucose, 801 Tween 801 ml, 2g of sodium dihydrogen phosphate, 5g of anhydrous sodium acetate, 2g of triammonium citrate, 0.02g of manganese sulfate, 0.1g of magnesium sulfate and 1L of distilled water, adjusting the pH to about 6.2, 15g of agar, sterilizing at 121 ℃ for 15 min.
The molecular biological identification method of the Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) comprises the following steps:
inoculating a frozen and preserved test strain into a TPY enrichment liquid culture medium, culturing at a constant temperature of 30 ℃ for 24h, subculturing for 2-3 generations by TPY, placing 2mL of thallus culture at the last logarithmic growth stage into a sterile EP tube for centrifugation, collecting thallus after centrifugation for 3min (4 ℃) at 8000 Xg, removing supernatant, and extracting the genomic DNA of the strain by adopting a CTAB freeze-thawing method special for lactobacillus.
Wherein, the TPY enrichment culture solution comprises the following components: 10g of lactose, 5g of beef extract, 5g of yeast powder, 10g of casein peptone, 5g of soybean peptone, 2.5g of dipotassium hydrogen phosphate, 2.5g of potassium dihydrogen phosphate, 0.1g of magnesium sulfate, 800.25 g of tween, 0.5g of L-cysteine hydrochloride, 15g of agar and 1L of distilled water, sterilizing at 121 ℃ for 15 min.
The PacBio SMRT RSII third generation sequencing platform is adopted to determine the whole genome of Lactobacillus rhamnous M9, the genome length is 3,002,913bp, the GC content is 46.77%, and the chromosome genome contains 2965 coding genes and 76 RNA genes.
The Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) provided by the invention has the following morphological characteristics: the bacterial body is rod-shaped under microscope (5000 times electron microscope), and the cells are in single, paired or chain shape, as shown in figure 1, without flagella or cilia, and will not move.
The Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) of the invention forms obvious colonies on an MRS culture medium, and the colonies are large, have the diameter of 0.5-1mm, have neat edges, and are cream-white opaque colonies, as shown in figure 2.
It is another object of the present application to provide the use of said lactobacillus rhamnosus M9 for the preparation of a modulator of the intestinal flora. The fermentation metabolite of Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) separated by the application has the function of regulating intestinal flora, thereby relieving diarrhea.
Further, the lactobacillus rhamnosus M9 can also be used as a starter for fermented dairy products and fermented cereal products. In particular, the lactobacillus rhamnosus M9 can be used as a dairy product starter, for example, as a starter for yoghurt, drinking yoghurt, fermented milk drinks or fermented soy milk. The lactobacillus rhamnosus M9 can be used as a leaven for fermenting cereal products, for example, as a leaven for fermenting oat, brown rice or coix seed.
The term "probiotic bacteria" as defined by the world health organization means "live microbial preparations ingested in quantities that have a beneficial effect on the health of the host".
The lactobacillus rhamnosus M9 provided by the application can have good tolerance to gastrointestinal digestive juice and bile salts, so the lactobacillus rhamnosus M9 provided by the application belongs to probiotics.
The application also provides a probiotic microbial inoculum prepared from the lactobacillus rhamnosus M9.
The application also provides a composite probiotic, which comprises the lactobacillus rhamnosus M9, wherein the lactobacillus rhamnosus M9 can be a main component and can also be an auxiliary component.
In the present application, the complex probiotics can be in all medically acceptable dosage forms such as powder, tablet, solution or emulsion.
In the present application, the complex probiotic may be used as a modulator for regulating the balance of the intestinal flora. When the compound probiotics is used as a regulator for regulating the balance of intestinal flora, the compound probiotics can be used independently and can also be used together with other substances for regulating the intestinal flora.
Optionally, in the composite probiotics, a metabolite of the lactobacillus rhamnosus M9 can also be included.
The application also provides a Lactobacillus rhamnosus microbial inoculum which is obtained by fermenting the Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9), and specifically comprises Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) and a fermentation metabolite thereof. The microbial inoculum can also be used as a medicine for relieving diarrhea.
Further, the application also provides a composite microbial inoculum comprising the lactobacillus rhamnosus microbial inoculum, and in the composite microbial inoculum, the lactobacillus rhamnosus microbial inoculum can be a main component and can also be an auxiliary component. The compound microbial inoculum can also be used as a medicine for relieving diarrhea.
The applicant finds that the Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) provided by the application has excellent gastrointestinal fluid tolerance and good bile salt tolerance, and has basic conditions as probiotics; the probiotic characteristics of enhancing immunity, improving the diversity of intestinal flora and increasing the stability of the intestinal flora are achieved; the Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) can be used as probiotic bacteria to be added into various common foods, health products and the like.
Drawings
FIG. 1 shows the morphological diagram under electron microscope of Lactobacillus rhamnosus M9;
FIG. 2 shows a colony morphology of Lactobacillus rhamnosus M9;
FIG. 3 shows a graph of the survival rate of Lactobacillus rhamnosus M9 in simulated gastrointestinal fluids;
figure 4 shows the results for the stability of fermentation with lactobacillus rhamnosus M9.
Detailed Description
The features and advantages of the present invention will become more apparent and appreciated from the following detailed description of the invention.
The present invention is described in detail below.
Examples
Example 1 probiotic Properties of the Strain
Acid resistance, cholate resistance and adhesiveness are the primary standards for screening probiotics, and the activity level of the probiotics in intestinal tracts directly relates to the health promotion effect of the probiotics after the probiotics are taken into human bodies, so the acid resistance, cholate resistance and adhesiveness of the probiotics are important indexes of the probiotics as functional dietary supplement factors.
(1) Preparation of bacterial test solution
The cells at the end of the culture cycle of the continuous subculture were collected by centrifugation (2500 Xg, 7min), and washed with PBS (pH 7.2, 0.8% NaCl, 0.02% KH)2PO4、0.115％Na2HPO4W/V, the same as below with PBS) for 2 times, suspending the thalli in 10mL of PBS, then diluting the thalli to a proper multiple in a gradient manner by using sterilized PBS, counting the total number of colonies by adopting an MRS agar medium plate pouring method, and simultaneously determining the OD value of the bacterial suspension at 600nm of the corresponding dilution.
According to the linear relationship between the OD value of each dilution and the corresponding approximate colony count (cfu/ml), the OD value of each thallus at 600nm can be adjusted to make the final concentration of the thallus of the test bacteria liquid be 1-2 × 109cfu/ml。
(2) Preparation of artificial gastric juice and intestinal juice
The preparation method of the simulated gastrointestinal fluid comprises the following steps: sterilizing PBS, adjusting pH to 2.5 with 1mol/L HCl, adding pepsin 3.0mg/ml, filtering with 0.22 μm microporous membrane for sterilization, and making into simulated artificial gastric juice; sterilizing PBS, adjusting pH to 8.0 with 0.1mol/L NaOH, adding 0.1% trypsin and 1.8% ox bile salt, and filtering with 0.22 μm microporous membrane for sterilization to obtain artificial simulated intestinal fluid.
(3) Determination of tolerance of test strain to artificial gastric juice and intestinal juice
(3.1) gastric juice resistance: adding 1.0ml of each test bacterial liquid into 9.0ml of the artificial gastric juice prepared in the step (2), digesting for 3h at 37 ℃, simultaneously counting and determining the number of viable bacteria in a 0h sampling plate pouring method and a 3h sampling plate pouring method respectively, performing 3 parallels on each sample, performing activated culture on the strains obtained by separation and purification for two generations, performing centrifugal bacterial washing twice, and collecting thalli.
(3.2) intestinal juice tolerance: adding 1.0ml of artificial bacteria-containing gastric juice digested by gastric juice for 3h into 9.0ml of artificial intestinal juice prepared in step (2), continuing to culture in water bath at 37 ℃, respectively adding into the intestinal juice for 4h and 8h, namely accumulating with the gastric juice digested for 3h, as shown in figure 4, respectively sampling the bacterial liquid again for 7h and 11h, counting and determining the number of viable bacteria by using an MRS agar medium pouring method, and making 3 samples in parallel.
Strain survival the strain survival can be calculated using the following formula I:
wherein the content of the first and second substances,
n1 represents the number of viable bacteria after strain treatment;
n0 indicates the initial viable count of the strain.
The test results are shown in FIG. 3, and it can be seen from FIG. 3 that: the survival rate of the Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) after being digested in artificial gastric juice with the pH value of 2.5 for 3 hours is 83.72%, and then the digestion in artificial digestive juice with the pH value of 8.0 is continued for 11 hours, and the survival rate is as high as 78.33%.
Therefore, the Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) has good gastrointestinal digestive juice tolerance, can enter human intestinal tracts in a living state and exerts health efficacy, and the characteristics are the basis of probiotics.
The applicant believes that: the Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) provided by the invention can resist adverse effects of the digestive tract environment and well adhere and colonize in the intestinal tract, the living bacteria have the effects of enhancing immunity resistance, improving diversity of intestinal flora and increasing stability of the intestinal flora, and can densely stay on the wall of the digestive tract in the intestine when being used for people and animals, so that the harmful bacteria can not stay, and lactic acid is generated to reduce the pH value in the intestine and inhibit the propagation of the harmful bacteria. In addition, the live Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) can generate bacteriocin and peroxide, so that the propagation of pathogenic bacteria can be inhibited, and the activity of intestinal villi which is responsible for absorbing nutrient components is helped.
(4) Bile salt tolerance
During the passage of the strain, the bile salt tolerance of the strain was determined every 200 passages of culture.
The test method comprises the following steps: the test strain at the end of the culture cycle was inoculated at 1% inoculum size into MRS medium (0.3% Oxgall + 0.05% cysteine + 0.2% sodium thioglycolate) containing bovine bile salts, as control with MRS without added bovine bile salts. The test strain was incubated in a water bath at 37 ℃ and a sample was taken every hour and the absorbance was measured at 620nm until the absorbance increased by 0.3 unit.
The bile salt tolerance of the test strains is evaluated by taking the length of a lag phase as an evaluation standard, wherein the difference of the light absorption values of the test strains and the blank strains increased by 0.3 unit is the delay time (LT, lag time), and each strain is subjected to three parallel tests.
After the strain obtained by separation and purification is activated and cultured for three generations, the strain is respectively inoculated into MRS culture medium containing 0.3% of bovine bile salt (0.2 g/100mL of sodium thioglycolate and 0.3g/100mL of bovine bile salt are added into the culture medium) and MRS culture medium containing no bovine bile salt at the inoculation amount of 1%, and the MRS culture medium is used as a blank control and is placed at 37 ℃ for anaerobic culture. During the culture period, samples were taken at intervals of one hour to determine OD values at 620nm, and triplicate measurements were taken in each group, and the culture was stopped until OD values increased by 0.3 unit or more. And the time required for the strain to grow in MRS medium with and without bile salts to increase the OD by 0.3 units was calculated, the difference being called the Lag Time (LT).
When the Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) is cultured in a medium without bile salts, the time required for increasing the OD620nm value by 0.3 unit is 2.51h, the time required for increasing the OD620nm value in the medium with bile salts is 2.17h, and the delay time is 0.34 h.
In the art, it is generally accepted that resistant strains (lag time ≦ 0.25h), tolerant strains (0.25h < lag time ≦ 0.67h), weakly tolerant strains (0.67h < lag time ≦ 1.00h), and sensitive strains (lag strain ≧ 1.00 h). Therefore, Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) had a lag time of 0.34h and was a tolerant strain.
In the case of living cells, bile salts can destroy the cell membrane of living cells, and the most significant factor affecting probiotic viability in the small intestine is bile salts, so that tolerance to bile salts is one of the important indicators for evaluating probiotics. It can be seen from this example that Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) isolated from breast milk has good probiotic properties.
(6) Fermentation experiment of strain sugar
The sugar fermentation test is a commonly used biochemical reaction for identifying microorganisms, and is particularly important in the identification of intestinal bacteria. Most bacteria can use sugars as a carbon source and an energy source, but they have a large difference in their ability to decompose sugar substances. The operation is performed according to the specification flow of the API 50CH standardized authentication system.
The Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) provided by the present application can utilize lactose and ferment various monosaccharides, such as glucose, arabinose, maltose, etc.
The specific experimental results are shown in table 1:
TABLE 1 results of the Lactobacillus rhamnosus M9 sugar fermentation experiments
Wherein "+" indicates a substrate that can be utilized and "-" indicates a substrate that cannot be utilized. As can be seen from Table 2, Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) can utilize D/L-arabinose, D-glucose, D-galactose, xylitol, etc., but cannot utilize mannitol, L-xylose, starch, glycogen, etc.
In conclusion, the Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) can well utilize lactose, has good gastrointestinal fluid and bile salt tolerance, can enter human intestinal tracts in a living state to play a health role, and is a probiotic strain which can be widely applied to common foods, health care products and the like.
Example 2 application of Lactobacillus rhamnosus M9 to fermented soymilk
The soybean milk is rich in protein, dietary fiber, minerals, vitamins, and a large amount of linoleic acid and linolenic acid which are necessary for human body. The fermented soybean milk is an important component of a soybean product and is a plant fermented food prepared by inoculating lactobacillus on the basis of soybean milk and fermenting. By the fermentation effect of lactobacillus, the fermented soybean milk can improve lipid metabolism without causing obesity; the fermented soybean protein is easier to digest and absorb, and can improve the immunity of the organism and effectively reduce the content of total cholesterol in serum; the decomposed soybean oligosaccharide can promote proliferation of probiotics and keep intestinal health.
However, the fermented soybean milk has the problems of poor strain adaptability, poor tissue state, poor fermentation flavor, beany flavor and the like at present. The Lactobacillus rhamnous M9 added into the fermented soybean milk can well improve the fermentation state and flavor of the soybean milk and effectively remove beany flavor.
The experimental method comprises the following steps: preheating water to 65 deg.C, adding soybean powder, mixing, hydrating at 55 deg.C for 30min, adding 8% white sugar and stabilizer, homogenizing, sterilizing at 90 deg.C for 10min, cooling to 41-43 deg.C, inoculating Streptococcus thermophilus ST-6(5 × 10), and fermenting6CFU/g) and the probiotic Lactobacillus rhamnosus M9 (1X 10)4CFU/g), end of fermentation to pH 4.50 and below, about 6 hours of fermentation, after-ripening, the acidity and viable count change of probiotic were measured every 7 days with sample retention at 10 ℃ and the results are shown in table 2:
TABLE 2 changes in acidity and viable count of M9 during storage of fermented soymilk at 10 ℃
The measurement results in table 2 show that the post-acidification phenomenon of the fermented soybean milk is not aggravated and hardly influenced by adding the probiotic Lactobacillus rhamnosus M9 into the fermented soybean milk; the number of viable bacteria of M9 after fermentation is 109More than CFU/g and kept relatively during storageHigh viable count, demonstrating good probiotic properties of Lactobacillus rhamnosus M9 isolated from breast milk.
Example 3 application of Lactobacillus rhamnosus M9 to fermentation of Coix lacryma-jobi seed beverage
Currently, the probiotics used for fermenting cereal beverages are mainly lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus, lactobacillus bifidus and the like.
In this example, a control group was prepared by fermenting coix seed with a combination of Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) and streptococcus thermophilus S10 to prepare a coix seed beverage, and fermenting coix seed with streptococcus thermophilus S10 alone to prepare a coix seed beverage.
The specific experimental method comprises the following steps: cleaning and soaking the coix seeds overnight, adding water according to the proportion of 1:10, decocting at 90 ℃ for 2 hours, adding high-temperature resistant amylase for hydrolysis, cooling to 65 ℃ after hydrolysis, adding 6 wt% of white granulated sugar, homogenizing under the condition of 30Mpa after uniform mixing, sterilizing at 95 ℃ for 10 minutes, and inoculating bacteria for fermentation when cooling to 37 ℃.
The experimental group was inoculated with Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) (1X 10)6CFU/g) and S10 (2X 10S)6CFU/g) for fermentation; control group was inoculated with S10 (2X 10)6CFU/g), the fermentation was terminated to pH 4.0, the fermentation was carried out for about 20 to 24 hours, sensory evaluation and stability comparison were carried out after the end of the fermentation, and the results are shown in table 3 and fig. 4:
TABLE 3 sensory evaluation of fermented Job's tears seed beverage
The results of table 3 and fig. 4 show that the experimental group added with Lactobacillus rhamnosus M9(Lactobacillus rhamnosus M9) can improve the taste of the fermented coix seed beverage, has sweet and sour taste, has rich and soft taste, and enhances the stability of the fermented coix seeds to a certain extent.
The applicant finds that the biggest problem of cereal beverage is stability, and researches show that probiotics ferment degradable amylopectin to improve the water retention of starch and form strong gel property; in addition, the consumption of sugar by microorganisms, the improvement of protein water retention and emulsifiability by various secretase systems, and the increase of viscosity by metabolites can promote the stability of the grain beverage. The coix seed is a traditional medical and edible cereal resource in China, contains multiple essential amino acids and mineral substances, has high yield in China, is processed into a convenient and easy-to-eat cereal beverage, not only accords with the trend of modern beverages, but also is beneficial to developing deep-processed coix seed foods.
The present application has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to limit the application. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the presently disclosed embodiments and implementations thereof without departing from the spirit and scope of the present disclosure, and these fall within the scope of the present disclosure. The protection scope of this application is subject to the appended claims.
Claims (1)
1. Application of lactobacillus rhamnosus M9 in preparing intestinal flora regulator, wherein lactobacillus rhamnosus M9 (B)Lactobacillus rhamnosus) M9 is separated from breast milk, the microorganism preservation number is CGMCC No.16002, the genome length of the lactobacillus rhamnosus M9 is 3002913bp, the GC content is 46.77%, and the chromosome genome contains 2965 coding genes and 76 RNA genes.
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