CN112029676B - Probiotic composition beneficial to improving immunity and application thereof - Google Patents
Probiotic composition beneficial to improving immunity and application thereof Download PDFInfo
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- CN112029676B CN112029676B CN202010272611.7A CN202010272611A CN112029676B CN 112029676 B CN112029676 B CN 112029676B CN 202010272611 A CN202010272611 A CN 202010272611A CN 112029676 B CN112029676 B CN 112029676B
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- bacillus coagulans
- lactobacillus rhamnosus
- survival rate
- probiotic composition
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- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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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 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
- A23V2400/175—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 invention provides a probiotic composition beneficial to improving immunity, which comprises lactobacillus rhamnosus NJ551 and bacillus coagulans BC01, wherein the lactobacillus rhamnosus NJ551 is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2016157, Bacillus coagulans strain BC01 is preserved in China center for type culture Collection with CCTCC NO of M2017813. The probiotic composition can realize or surpass the effects of restoring or improving the permeability of the intestinal mucosa, the killing rate of NK cells and the contents of serum gamma-interferon (IFN-gamma) and TNF-alpha, which can be realized only by single bacteria at a larger intake amount, under the condition of a smaller intake amount. The probiotic composition of the invention helps to improve immunity.
Description
Technical Field
The invention belongs to microorganisms, and particularly relates to a probiotic composition.
Background
Immunity is the ability of the body to fight disease and maintain self-health. And what performs this function in the human body is the immune system. The immune system comprises immune organs, immune tissues and immune molecules, and the interaction of the immune organs, the immune tissues and the immune molecules forms a huge body defense network. Although the immune system is not fully understood in humans, the current knowledge has demonstrated that 70% of the immune cells of the human body accumulate in the gut, and that the development of the immune system is closely related to the gut flora.
On the intestinal mucosa of human body, thousands of intestinal flora with the quantity of 1000 hundred million inhabit. The intestinal flora and the mucosal immune system of the human body reach a dynamic balance, on one hand, the mucosal system of the human body regulates and maintains the quantity and the variety of the intestinal flora, and on the other hand, the intestinal flora also trains the immune system of the human body to help the immune system of the human body to mature.
The impact of the normal gut flora on the immune system can be reflected in at least 3 aspects: (1) stimulating intestinal epithelial cells to secrete mucus, and enhancing the thickness of a mucous membrane; (2) participate in a mechanical locking mechanism among epithelial cells of the intestinal tract, maintain the permeability of the intestinal mucosa and avoid pathogen from invading human body through the intestinal mucosa; (3) exchange with the immune cells of the intestinal mucosa immune system to improve the activity of the immune cells. If the immune system is compromised, it may in turn affect the permeability of the intestinal epithelium, the thickness of the mucosa and ultimately the composition of the intestinal flora [1 ]. The interaction of the intestinal mucosal immune system with the intestinal flora is the first line of defense in maintaining the body against infection and self-health.
[1]Josie Libertucci and Vincent B.Yong.The role of the microbiota in infectious disease.Nature microbiology,2019,vol4:35-45
Therefore, it is a realistic approach to achieve enhanced immunity (including enhanced mechanical barrier of the gut mucosal system) by probiotic intervention in gut flora composition. The effectiveness of this intervention depends on the particular probiotic strain [2], and different strains of the same species may have completely different effects on immunity. The screening of the bacterial strain capable of improving the intestinal mucosa barrier effect and the immune response and the establishment of the evaluation method of the influence of the bacterial strain on the immunity have important significance for improving the immunity by using probiotic intervention.
[2]Cai,M.,Jolley etc.Animal studies on enhancement of immune function by dietary probiotic supplementation of Lactobacillus acidophilus NCFM and Bifidobaterium latics Bi-07.Chinese Journal of Microbiology,2008,20，(1):17-19
There are several patents which have been filed or are being filed for the use of probiotics in connection with immunization, such as patent application No. 01816819.1, etc. However, these patents mainly use allergy models to evaluate the effect of probiotic intervention, and less often immune suppression models to evaluate probiotic intervention, and hardly evaluate the effect of probiotics on the permeability of the intestinal mucosa. Moreover, these patents do not evaluate the composition versus the effect of the individual bacteria alone when forming the strain composition.
Disclosure of Invention
In order to solve the existing problems, the invention provides a probiotic composition beneficial to improving immunity, which comprises lactobacillus rhamnosus NJ551 and bacillus coagulans BC01, wherein the lactobacillus rhamnosus NJ551 is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2016157, Bacillus coagulans strain BC01 is preserved in China center for type culture Collection with CCTCC NO of M2017813.
The lactobacillus rhamnosus NJ551 has the following characteristics:
the survival rate of 1 in the artificial gastric juice is more than 50 percent after being treated for 6 hours, and the survival rate of 1 in the artificial small intestine juice is more than 26 percent after being treated for 1 hour.
2 can effectively recover or improve the permeability of the intestinal mucosa of the mouse, the killing rate of NK cells, the contents of serum gamma-interferon (IFN-gamma) and tumor necrosis factor (TNF-alpha in the invention) in an immunosuppression mouse model.
3 the survival rate of the freeze-dried powder prepared by the strain is more than 50% after being damaged at 37 ℃ for 10 days, and the survival rate of the freeze-dried powder is more than 90% after being stored at-18 ℃ for 12 months.
The bacillus coagulans BC01 of the invention has the following characteristics:
the survival rate of 1 in the artificial gastric juice after being treated for 6 hours is more than 75 percent, the survival rate of 4 in the artificial small intestine juice is more than 82 percent, and the survival rate of 3 hours after being continuously treated by the artificial gastric juice and the artificial small intestine juice is more than 63 percent.
2 the survival rate of the freeze-dried powder (or spray-dried powder) prepared by the strain is more than 98 percent after being damaged for 10 days at 37 ℃, and the survival rate of the freeze-dried powder (or spray-dried powder) stored at normal temperature for 18 months is more than 98 percent.
3 in its life cycle, there are two life forms, vegetative and spore.
4 in vitro tests prove that the medicine has bacteriostatic effect on escherichia coli and staphylococcus aureus.
5 can effectively recover or improve the permeability of the intestinal mucosa of the mouse, the killing rate of NK cells and the contents of serum gamma-interferon (IFN-gamma) and TNF-alpha in an immunosuppression mouse model.
The invention also provides a probiotic composition consisting of the lactobacillus rhamnosus NJ551 and the bacillus coagulans BC01, and the probiotic composition can realize or surpass the recovery or improvement effect on the intestinal mucosa permeability, NK cell killing rate and serum gamma-interferon (IFN-gamma) and TNF-alpha content of a mouse, which can be realized only by a single bacterium at a larger intake amount, under the condition of a smaller intake amount.
According to one aspect of the technology, the lactobacillus rhamnosus NJ551 in the probiotic composition beneficial to improving the immunity has the following characteristics:
the survival rate of the artificial gastric juice after being treated in the artificial gastric juice for 6 hours is more than 50 percent, and the survival rate of the artificial intestinal juice after being treated in the artificial intestinal juice for 1 hour is more than 26 percent;
can effectively recover or improve the permeability of the intestinal mucosa of the mouse, the killing rate of NK cells and the contents of serum gamma-interferon (IFN-gamma) and TNF-alpha in an immunosuppressed mouse model;
the survival rate of the freeze-dried powder prepared from the strain is more than 50% after being damaged at 37 ℃ for 10 days, and the survival rate of the freeze-dried powder is more than 90% after being stored at-18 ℃ for 12 months;
bacillus coagulans BC01 has the following properties:
the survival rate of the artificial intestinal juice after being treated in the artificial gastric juice for 6 hours is more than 75 percent, the survival rate of the artificial intestinal juice after being treated in the artificial intestinal juice for 4 hours is more than 82 percent, and the survival rate of the artificial intestinal juice after being continuously treated in the artificial gastric juice for 3 hours and the artificial intestinal juice for 3 hours is more than 63 percent:
the survival rate of the freeze-dried powder or spray-dried powder prepared by the strain after being damaged at 37 ℃ for 10 days is more than 98 percent, and the survival rate of the freeze-dried powder or spray-dried powder after being stored at normal temperature for 18 months is more than 98 percent;
two life forms of a vegetative state and a spore state exist in the life cycle of the plant;
in vitro tests prove that the compound has bacteriostatic effect on escherichia coli and staphylococcus aureus;
can effectively recover or improve the permeability of the intestinal mucosa, the killing rate of NK cells and the contents of serum gamma-interferon (IFN-gamma) and TNF-alpha in an immunosuppressed mouse model.
According to one aspect of the technology, the invention provides a probiotic composition beneficial to improving immunity, wherein the lactobacillus rhamnosus NJ551 and the bacillus coagulans BC01 in the probiotic composition can be stored separately or after being mixed.
According to an aspect of the technology, the invention provides a probiotic composition beneficial to improving immunity, wherein the probiotic composition beneficial to improving immunity is a mixture containing lactobacillus rhamnosus NJ551 and bacillus coagulans BC 01. The composition is effective in improving immunity of ingesting people. The concentration (dosage) of the viable bacteria in the composition can be calculated according to the weight of a person who ingests the composition, and a preferable scheme is as follows: intake concentration (dosage) of lactobacillus rhamnosus NJ551 single bacteria: 2.0*105CFU/gram (body weight)/day; intake concentration (dose) of bacillus coagulans BC01 single bacterium: 6.0*104CFU/gram (body weight)/day. The actual intake time can be adjusted within a reasonable range according to the self-sensitivity.
According to one aspect of the technology, the invention provides a probiotic combination method for improving immunity, which is characterized in that lactobacillus rhamnosus NJ551 and bacillus coagulans BC01 are combined for use, lactobacillus rhamnosus NJ551 is preserved in China center for type culture collection, and the preservation number is CCTCC NO: m2016157 and Bacillus coagulans BC01, which is preserved in China Center for Type Culture Collection (CCTCC) with preservation number of M2017813.
According to one aspect of the technology, the invention provides a preparation method of lactobacillus rhamnosus NJ551, which comprises the steps of inoculating 1% of the inoculum size of the inner Mongolia cheese into 10% skim milk culture medium, culturing at 37 ℃, curdling the inner Mongolia cheese, then diluting the inner Mongolia cheese by 10 times of gradient, and sequentially diluting the inner Mongolia cheese to 10 degrees-8And (4) gradient. Selecting 4 appropriate dilutions, mixing 1mL of the dilutions with MRS solid culture medium by a pouring method, and pouring the dilutions onto a flat plate.Culturing in an anaerobic tank at 37 deg.C for 48h, observing colony morphology, selecting colonies of different morphologies, streaking and separating on MRS solid culture medium, repeating streaking and separating for 3 generations to obtain purified strain, observing with gram staining method and microscope, selecting gram staining positive strain, and observing with microscope to obtain spherical, rod-shaped and multi-shaped strains; carrying out anaerobic proliferation and propagation on the 8 strains of bacteria by using an MRS culture medium, then collecting bacterial sludge to carry out 16s rDNA test, and selecting lactobacillus rhamnosus NJ551, wherein the lactobacillus rhamnosus NJ551 is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2016157. The method can also be used as a screening method.
According to one aspect of the technology, the invention provides lactobacillus rhamnosus NJ551, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2016157.
According to one aspect of the technology, the invention provides an application of the lactobacillus rhamnosus NJ551, and the lactobacillus rhamnosus NJ551 is applied to preparation of a probiotic composition beneficial to improving immunity.
According to one aspect of the technology of the present invention, a preparation method of the probiotic composition beneficial for improving immunity is provided, the probiotic composition beneficial for improving immunity comprises the lactobacillus rhamnosus NJ551 and bacillus coagulans BC01, the lactobacillus rhamnosus NJ551 is preserved in the chinese typical culture collection with the preservation number of CCTCC NO: m2016157, the Bacillus coagulans BC01 strain is preserved in China center for type culture Collection with preservation number of CCTCC NO: M2017813, and the preparation method of the probiotic composition for improving immunity comprises the following steps:
and diluting the freeze-dried powder of the lactobacillus rhamnosus NJ551 and the live bacteria powder of the bacillus coagulans BC01 into a mixed bacteria liquid containing the bacillus coagulans BC01 and the lactobacillus rhamnosus NJ 551. The two kinds of bacteria can be drunk by mixing with dry powder, or added into liquid beverage, or fermented.
According to an aspect of the present technology, there is provided the use of the above probiotic composition for enhancing immunity,the probiotic composition beneficial to improving the immunity is applied to preparation of medicines, foods or health-care products for improving the immunity. The lactobacillus rhamnosus NJ551 strain related in the application is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2016157, the Bacillus coagulans strain BC01 is preserved in China Center for Type Culture Collection (CCTCC) with preservation number of M2017813, and the content of the Bacillus coagulans strain NJ 5512.0 x 10 is sufficient5CFU/gram (body weight)/day; bacillus coagulans BC01 single strain 6.0 x 104CFU/g (body weight)/day.
The preferred intake of the two bacteria of the present invention is lactobacillus rhamnosus NJ 5512.0 x 10 per day5CFU/gram (body weight)/day; bacillus coagulans BC01 single strain 6.0 x 104CFU/gram (body weight)/day.
The invention provides a probiotic composition consisting of lactobacillus rhamnosus and bacillus coagulans, which can effectively strengthen intestinal mucosa barrier and improve NK cell killing rate and serum IFN-gamma and TNF-alpha content. Compared with single bacteria, the composition can achieve the effect only by using a small amount of the composition or surpass the effect which can be achieved by using a large amount of the single bacteria, and shows good synergistic effect.
The invention also provides a screening and preparation method and application of the probiotic composition.
Drawings
FIG. 1 is a microscope photograph of Lactobacillus rhamnosus NJ551 according to an embodiment of the present invention;
FIG. 2 shows the tolerance of Lactobacillus rhamnosus NJ551 in artificial gastric acid and artificial intestinal juice according to one embodiment of the present invention;
FIG. 3 shows the viable cell preservation capacity of Lactobacillus rhamnosus NJ551 during storage period according to an embodiment of the present invention;
FIG. 4 is a vegetative form of Bacillus coagulans BC01 in accordance with one embodiment of the present invention;
FIG. 5 shows the spore form of Bacillus coagulans BC01 according to one embodiment of the present invention;
FIG. 6 shows the survival rate of Bacillus tuberculosis BC01 in artificial gastric juice and intestinal juice according to one embodiment of the present invention;
FIG. 7 shows the shelf-life stability of Bacillus coagulans BC01 in one embodiment of the present invention.
FIG. 8 shows the inhibitory effect of Bacillus coagulans BC01 on Staphylococcus aureus in one embodiment of the present invention;
FIG. 9 shows the retention time of lactulose and mannitol standards on HPLC in one embodiment of the present invention.
Detailed Description
The following examples are intended to further illustrate some, but not all, preferred embodiments of the present invention. Other embodiments of the invention based on the present invention, which can be made by a person skilled in the art without inventive step, belong to the scope of protection of the present invention. The invention will be further described with reference to the accompanying drawings.
Example 1 isolation and identification of Lactobacillus rhamnosus NJ551 and preparation of lyophilized powder
1. Test materials:
is prepared from 3 parts of natural fermented cheese from inner Mongolia and fermented pickle jar fermentation liquid from Sichuan peasant family
2. The separation method comprises the following steps:
2.1 inoculating 3 parts of inner Mongolia cheese into 10% skim milk culture medium according to 1% inoculum size, culturing at 37 deg.C, coagulating, diluting by 10 times gradient, and sequentially diluting to 10 times-8And (4) gradient. Selecting 4 appropriate dilutions, mixing 1mL of the dilutions with MRS solid culture medium by a pouring method, and pouring the dilutions onto a flat plate. Culturing in anaerobic tank at 37 deg.C for 48h, observing colony morphology, selecting colony of different morphology, streaking and separating on MRS solid culture medium, repeating streaking and separating for 3 generations, and observing the obtained purified strain with gram staining method and microscope, selecting gram staining positive strain, and observing with microscope to obtain spherical, rod-shaped and polymorphic strains.
2.2 selecting 8 strains together according to the method of 2.1, carrying out anaerobic multiplication and propagation culture on the 8 strains by using an MRS culture medium, and then collecting bacterial sludge for 16s rDNA test. 3 strains of lactobacillus plantarum, 2 strains of lactobacillus rhamnosus, 1 strain of bifidobacterium, 1 strain of enterococcus durans and 1 strain of pediococcus acidilactici are selected through tests. The selected Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium and Pediococcus acidilactici were left for further testing according to the provisions of the Ministry of health, List of probiotic strains available for food. Enterococcus durans was deposited as a strain, but not used.
2.3 further research on the strains selected in the step 2.2 shows that the fermentation liquor of the lactobacillus rhamnosus cannot form firm bacterial sludge sediment by centrifugation, and the bacterial sludge is loose, which indicates that the extracellular polysaccharide is generated more. According to the found documents, a plurality of documents show that the strains with more exopolysaccharides can have stronger function of regulating immunity, so that negative dyeing is carried out on three kinds of bacteria (lactobacillus plantarum, lactobacillus rhamnosus and bifidobacterium) by adopting negative dyeing, the capsular dyeing of the lactobacillus rhamnosus is found to be thickest, and meanwhile, the lactobacillus rhamnosus is selected for the next test because the lactobacillus rhamnosus fermented yoghourt is refrigerated and stored for 15 days without deterioration. And the lactobacillus rhamnosus is named as lactobacillus rhamnosus NJ 551. The lactobacillus rhamnosus NJ551 strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2016157
3. Preparation of freeze-dried powder of lactobacillus rhamnosus NJ551
3.1 amplifying and proliferating lactobacillus rhamnosus NJ551 in MRS culture medium by stages at 37 ℃ according to the inoculation amount of 2.5%. And finally, centrifuging the fermentation liquor, collecting bacterial sludge, mixing the bacterial sludge with a freeze-drying protective agent, and carrying out vacuum freeze-drying to obtain freeze-dried viable bacteria powder of the lactobacillus rhamnosus NJ 551. Counting the freeze-dried viable bacteria powder for dilution when needed.
3.2 the formula of the MRS culture medium is as follows: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of diammonium phosphate, 20 g of glucose, 801.0 mL of tween-sodium acetate, 5 g of sodium acetate, 2 g of dipotassium phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, 1000mL of distilled water and pH of 6.2-6.5; MRS solid medium: adding 18 g of agar on the basis of MRS culture medium; the above culture media were all sterilized at high temperature before use.
Example 2 physiological and Biochemical Properties of Lactobacillus rhamnosus NJ551
1. Gram-positive bacteria are short rods (0.5-1.2 mu m X3.0.0-10.0 mu m), have different properties under different nutritional conditions, and are mainly in a chain shape and a cross arrangement under the microscope condition (figure 1).
As shown in fig. 1: microscope pictures of lactobacillus rhamnosus NJ 551.
2. Tolerance in artificial gastric acid and artificial intestinal juice
The lactobacillus rhamnosus NJ551 has good characteristics of resisting artificial gastric acid and artificial small intestine juice, the survival rate in 2h is more than 75% and the survival rate in 6h is more than 50% in the artificial gastric juice with the pH value of 3.0 at 37 ℃; the survival rate of the artificial small intestine liquid at 37 ℃ and pH7.0 is more than 26% for 1h and more than 13% for 2h (see figure 2). Embodies the strong potential of reaching colon alive.
3. Viable bacteria protector for lactobacillus rhamnosus NJ551 freeze-dried powder in storage period
The results of accelerated destruction tests and-18 ℃ refrigerator freezing storage tests show that the viable bacteria of the lactobacillus rhamnosus NJ551 can be stably stored at the temperature of-18 ℃, and the survival rate of the viable bacteria is more than 90 percent when the lactobacillus rhamnosus NJ551 is stored for 12 months at the temperature of-18 ℃ (figure 3). Facilitating further research on the method.
As shown in fig. 3: storage-period viable bacteria preservation capacity of lactobacillus rhamnosus NJ551
Example 3 Bacillus coagulans BC01
Preparation of live bacterial preparation of Bacillus coagulans BC01
1. Collecting a sample possibly containing bacillus coagulans, wherein the sample is derived from soil, fermented food, plants, human intestinal tracts and the like, then placing the sample in a sterilized sterile bottle, and then obtaining a pure strain by adopting the following two methods:
1.1 obtaining pure strains method 1: adding bacteria-containing sample and 100mL sterile normal saline into sterile bottle to prepare bacteria-containing stock solution, and then diluting the stock solution by gradient 10110 times of210 times of310 times of410 times of510 times of610 times of710 times of810 times of9And (4) doubling. Coating the stock solution and the diluent with each dilution multiple on the enrichment solid culture of the bacillus coagulansOn the medium, culturing for 48 hours at 40 ℃ in a thermostat, selecting a single colony which is fully separated and well grown, inoculating the single colony on the bacillus coagulans enriched liquid culture medium again, and culturing for 24 hours at 40 ℃ in a shaking table at the rotating speed of 220 rpm. Centrifuging the liquid culture medium at 10000rpm, collecting bacterial sludge, performing 16S rDNA analysis, identifying Bacillus coagulans after NCBI comparison, and preserving pure strains for further test analysis such as physiological and biochemical analysis, antibiotic resistance, gastric acid resistance and intestinal juice resistance.
1.2 obtaining pure strains method 2: adding a bacteria-containing sample (Sichuan thick broad-bean sauce) and 100mL of sterile normal saline into a sterile bottle to prepare a bacteria-containing stock solution, directly inoculating the bacteria-containing stock solution into a bacillus coagulans enriched liquid culture medium, and culturing for 48 hours on a shaking table at the rotating speed of 220rpm and the temperature of 40 ℃. Then treating the liquid culture solution for 20 minutes at 80 ℃, coating the liquid culture solution in a bacillus coagulans enriched solid culture medium, selecting and fully separating, inoculating well-growing colonies into a bacillus coagulans enriched solid culture inclined plane, and culturing for 48 hours at 40 ℃ in a constant temperature box. The obtained bacterial sludge is subjected to 16S rDNA analysis, bacillus coagulans is identified after NCBI comparison, and pure strains are preserved for further test analysis such as physiological and biochemical analysis, antibiotic resistance, gastric acid resistance and intestinal juice resistance.
2. The bacillus coagulans enriched liquid culture medium comprises the following components in percentage by weight (g/L): 10 parts of corn starch, 10 parts of beef extract, 5 parts of yeast powder OXIDE and 5 parts of sodium chloride. Adjusting pH to 6.0-6.2 with 0.1mol/L sodium hydroxide or 0.1mol/L hydrochloric acid (solid culture medium added with 2% agar)
3. Obtaining pure strain of Bacillus coagulans, performing amplification culture in a fermentation tank, and regulating OD of Bacillus coagulans in the fermentation solution600And (3) centrifuging at 10000rpm after the spore rate is more than 45 and the spore rate is more than 80%, collecting bacterial sludge, properly diluting the collected bacterial sludge with normal saline, then obtaining the viable bacteria powder of the bacillus coagulans by using methods such as spray drying, fluidized bed drying, vacuum drying and the like, detecting the spore rate and viable bacteria content of the viable bacteria powder, and adding proper amount of auxiliary materials such as maltodextrin, isomaltooligosaccharide and the like to prepare the viable bacteria powder of the bacillus coagulans into preparations with different viable bacteria concentrations.
4. The amplification medium (g/L) of the bacillus coagulans is as follows: glucose 5, corn starch 10, beef extract 10, yeast powder OXIDE 5, magnesium sulfate 7H2O0.4, manganese sulfate 1H2O0.1, Festus 7H2O0.1 and sodium chloride 5.
5. The amplification culture conditions of the bacillus coagulans are as follows: the fermentation temperature is 40 ℃, the rotation speed is 300rpm, DO is more than 80 percent, and the pH value is 5.6-6.0.
The bacillus coagulans BC01 is preserved in China center for type culture collection with the preservation number of CCTCC NO: M2017813. The strain is derived from Sichuan thick broad-bean sauce.
The bacillus coagulans BC01 bacteria are slender rods, gram-positive and spore-end growth, the spore state is just like a small badminton racket, and some spores can fall off.
Plate form: milky white colony with regular edges and the size of about 2-4 mm.
The bacillus coagulans BC01 of the invention has the following characteristics:
1. the physiological and biochemical identification results of bacillus coagulans BC01 are as follows (table 1):
table 1: and (3) physiological and biochemical identification results of the bacillus coagulans. "+": positive, "-": negative of
The strain can be made into viable bacteria lyophilized powder (or spray-dried powder).
The 16SrDNA identification Sanger sequencing of the Bacillus coagulans strain BC01 shows that the strain BC01 is the Bacillus coagulans from the sequencing alignment result.
Example 4 physiobiochemical Properties of Bacillus coagulans BC01
1. Gram-positive bacteria, the growth cycle has two life forms: vegetative forms and spores. The nutrients are distributed in a dispersed manner with single rods, the dyeing is clear, and the two ends are round (figure 4). The spore is terminal spore, and is observed under microscope to form a small badminton racket (figure 5). As shown in fig. 4: vegetative form of bacillus coagulans BC 01; as shown in fig. 5: spore form of bacillus coagulans BC 01.
2. Tolerance of bacillus coagulans BC01 in artificial gastric juice and artificial small intestine juice
The bacillus coagulans BC01 has excellent tolerance in artificial gastric juice and artificial small intestine juice. Treating in artificial gastric juice at 37 deg.C and pH of 3.0 for 2h and 6h to obtain survival rate of more than 90% and 73%; treating at 37 deg.C in pH7.0 for 1 hr and 4 hr to obtain survival rate of more than 92% and 82%; the survival rate of the artificial gastric juice and the artificial small intestine juice is more than 63 percent after passing through the artificial gastric juice for 3 hours and the artificial small intestine juice for 3 hours continuously (figure 6). The tolerance to artificial gastric juice and small intestinal juice is far higher than that of all other lactic acid bacteria. As shown in fig. 6: survival rate of bacillus coagulans BC01 in artificial gastric fluid and small intestine fluid.
3. Viable bacteria stability of bacillus coagulans BC01 in storage period
The bacillus coagulans BC01 is stable in storage period, the survival rate of the bacillus coagulans BC01 is more than 98% when the bacillus coagulans BC01 is damaged for 10 days at 37 ℃ and stored for 12 months at normal temperature (figure 7), and further research on the bacillus coagulans BC01 is facilitated.
Method for testing lactobacillus rhamnosus NJ551 and bacillus coagulans BC01 by using animal model
As shown in fig. 7: shelf-life stability of Bacillus coagulans BC01
4. The bacillus coagulans BC01 in vitro test shows good inhibition effect on staphylococcus aureus (figure 8), the diameter of a staphylococcus aureus inhibition zone is + + (1.28cm), but the bacillus coagulans BC01 does not show inhibition capability on escherichia coli.
FIG. 8: inhibition of staphylococcus aureus by bacillus coagulans BC01
Example 5
The animal model is utilized to test the influence of lactobacillus rhamnosus NJ551, bacillus coagulans BC01 and the combined bacteria consisting of the two single bacteria on the immunity, and the specific technical route is as follows:
1. experimental animals: the weight of the male sex pheromone is about 18-22 g of SPF mice.
2. Testing indexes are as follows: NK cell killing rate of mouse spleen, IFN-gamma and TNF-alpha concentrations of mouse serum and intestinal mucosa permeability rate of mouse
3. The test steps are as follows:
3.1 total 28 SPF-class Kunming mice, divided into 7 experimental groups. The 7 test groups were: blank group, model group, lactobacillus rhamnosus NJ551 single bacterial group (hereinafter referred to as NJ551 group), bacillus coagulans BC01 single bacterial group (hereinafter referred to as BC01 group), lactobacillus rhamnosus NJ551 and bacillus coagulans BC01 combined group (hereinafter referred to as combined group) high, medium and low concentration test group 3;
3.2, molding: except for the blank group, each of the other groups was intraperitoneally injected with cyclophosphamide (50mg/kg/d), an immunosuppressant, on a body mass basis, daily for 3 consecutive days. The blank group was injected with the same amount of normal saline every day for 3 consecutive days;
3.3 probiotic intervention: after the molding is finished, 2mL of normal saline is perfused into the blank group and the model group every day, 1mL of single bacterial liquid is perfused into the NJ551 group every day, 1mL of single bacterial liquid is perfused into the BC01 group every day, 2mL of mixed bacterial liquid is perfused into the combined high-dose group every day, 1mL of mixed bacterial liquid is perfused into the combined medium-dose group every day, 0.5mL of mixed bacterial liquid is perfused into the combined low-dose group every day, and all the test groups are perfused into the stomach continuously for 15 days;
3.4 on day 16, all mice in the test groups were orally fed with 2mL of a mixed solution of lactulose and mannitol (containing 100mg of lactulose and 50mg of mannitol), and urine was collected 24 hours after canning, and 4mg of thimerosal was added to each 20mL of urine and stored at-20 ℃ for testing.
3.5 day 18, jugular venous blood was taken from all mice for peripheral blood gamma-IFN and TNF-alpha content determination. All mice were then sacrificed and spleens were removed for determination of NK cell activity.
4. The preparation method of the lactobacillus rhamnosus NJ551 bacterial liquid, the bacillus coagulans BC01 bacterial liquid and the NJ551 and BC01 combined bacterial liquid comprises the following steps:
4.1 preparation of Lactobacillus rhamnosus NJ551 Single bacterial liquid: firstly, preparing freeze-dried powder of lactobacillus rhamnosus NJ551, measuring the viable count of the freeze-dried powder, weighing a proper weight of the freeze-dried powder according to the measured viable count, and diluting the freeze-dried powder with normal saline to obtain lactobacillus rhamnosus NJ551 with the concentration of 4 x 106CFU/mL of bacterial liquid (called NJ551 single bacterial liquid);
4.2 preparation of a bacillus coagulans BC01 single bacterial liquid: firstly preparing live bacillus coagulans BC01 powder, measuring the number of live bacteria of the live bacteria powder, weighing a proper weight according to the measured number of the live bacteria, and diluting the live bacteria with physiological saline to obtain the bacillus coagulans BC01 with the concentration of 1.2 x 106CFU/mL bacterial liquid (called BC01 single bacterial liquid);
4.3 preparation of mixed bacterial liquid of lactobacillus rhamnosus NJ551 and bacillus coagulans BC 01: weighing a certain weight of the lactobacillus rhamnosus NJ551 freeze-dried powder and the bacillus coagulans BC01 live powder, and diluting the weighed powder with normal saline to obtain the bacillus coagulans BC01 with the concentration of 6 x 105CFU/mL and Lactobacillus rhamnosus NJ551 concentration of 2 x 106CFU/mL of a bacterial suspension (referred to as a mixed bacterial suspension).
5. A method for measuring killing rate of NK cells in mouse spleen comprises the following steps:
measurement of NK cell Activity by LDH method:
5.1 preparation of LDH matrix solution
Lithium lactate 5X 10-2mol/L
Tetrazole nitrochloride (INT) 6.6X 10-4mol/L
Phenazine dimethyl sulfate (PMS) 2.8X 10-4mol/L
Oxidized coenzyme I (NAD) 1.3X 10-3mol/L
The above reagent was dissolved in 0.2mol/L Tris-HCl buffer (pH8.2)
5.2 passages of target cells (YAC-1 cells, mouse lymphoma cells)
The target cells were subcultured 24h before the experiment. The cells were washed 2 times with RPMI1640 complete medium before application, and centrifuged at 1000rpm for 5 minutes after each wash. Finally, the cells were resuspended after washing with 10% RPMI1640 complete medium, the cell concentration was adjusted to 1X 105one/mL.
5.3 preparation of spleen cell suspension (Effector cells)
The mice were dissected and spleens removed. Cleaning with PBS; grinding a syringe needle core in a glass dish by a stainless steel cell sieve of 200 meshes to prepare cell suspension; 5ml PBS was mixed well and cell suspension was collected. Centrifuging at 1000r/min for 10min, and discarding the supernatant. Standing 5ml of erythrocyte lysate at room temperature for 5 min;centrifuging at 1000r/min for 10min, and discarding the supernatant; 5ml PBS heavy suspension centrifugal washing, abandon the supernatant. Resuspending in 1mL of complete RPMI1640 culture medium containing 10% calf serum, diluting with 1% glacial acetic acid, counting (viable cell number should be above 95%), staining with talarol to count viable cell number (should be above 95%), and adjusting cell concentration to 2 × 10 with complete RPMI1640 culture medium7one/mL.
5.4NK cell killing Rate assay
Reaction test group: taking 100 mu L of target cells and effector cells respectively (the effective-target ratio is 50: 1), and adding the target cells and the effector cells into a 96-well culture plate;
natural release group: taking 100 mu L of target cells and culture solution respectively, and adding the target cells and the culture solution into a 96-well culture plate;
maximum release group: taking 100 mu L of target cells and 1% NP40 or 2.5% Triton respectively, and adding the target cells and the 1% NP40 or 2.5% Triton into a 96-well culture plate;
all the above-mentioned materials are equipped with three composite holes, at 37 deg.C and 5% CO2Culturing for 4h in an incubator, centrifuging the 96-well culture plate at 1500r/min for 5min, sucking 100 μ L of supernatant per well, placing in a flat-bottomed 96-well culture plate, adding 100 μ L of LDH matrix solution, reacting for 3min, adding 1mol/L HCl30 μ L per well, and measuring Optical Density (OD) at 490nm of a microplate reader.
The NK cell killing rate was calculated as follows:
NK cell killing rate (reaction absorbance-natural release absorbance)/(maximum release absorbance-natural release absorbance) × 100%
6. Peripheral blood IFN-gamma and TNF-alpha content determination: by using ELISA kit (Shanghai enzyme-linked bioscience)
Company Limited), according to the method of use on the kit.
7. Determination of intestinal mucosal permeability:
the two-sugar probe method utilizes the different absorption characteristics of lactulose and mannitol at different positions of intestinal tract, mannitol is generally absorbed at the top of mucous epithelium, and lactulose is generally absorbed at the loose crypt part of epithelial cell tight junction. When normal, the permeability of mannitol is higher than that of lactulose, intestinal villi fall off and crypt link is relaxed when intestinal mucosa permeability changes, the absorption rate of lactulose is higher than that of mannitol, and the change of absorption of lactulose and mannitol can be reflected in urine of a mouse, so that the change of the intestinal mucosa permeability can be reflected by the change of the ratio (L/M) of lactulose to mannitol in urine.
The specific determination method comprises the following steps:
on the last day after the end of the intervention, all mice of the test groups were fed orally with lactulose and mannose solutions, 2mL per mouse (100 mg lactulose, 50mg mannose). Urine was collected from each mouse over 24 hours after feeding, and 4mg of thimerosal was added to each 20mL of urine and stored at-20 ℃ for testing. The content of lactulose and mannose in mouse urine is measured by high performance liquid chromatography, and the ratio (L/M) of lactulose to mannitol is used for representing the permeability of intestinal mucosa. In the detection, 0.4mL of urine is taken, centrifuged at 12000rpm at 4 ℃ for 30min, and the supernatant is taken. The chromatographic conditions are as follows: the column type is an amino chromatographic column (5 μm, 4.6 mm. times.250 mm); mobile phase acetonitrile and water in a ratio of 72:28 (v/v); the flow rate is 1 mL/min; the column temperature is 30 ℃; the detector is a differential refraction detector; the internal heating temperature was 37 ℃ and the amount of sample was 10. mu.L. The concentrations of mannitol and lactulose in the sample are calculated according to a standard curve, and the discharge rate ratio (L/M) of lactulose (lactulose, L) and mannitol (mannitol, M) in rats is calculated according to the gastric lavage dosage of the mannitol and the lactulose.
FIG. 9: retention time of lactulose and mannitol standard substance on high performance liquid chromatography
8. Test results
8.1 variation in the killing Rate of NK cells in each test group
The results show that: NK cell killing rates were significantly reduced in the model group relative to the blank group after molding with cyclophosphamide, indicating that molding was successful. In the probiotic dry prognosis, the killing rate of NK cells of all intervention groups is remarkably increased relative to a blank group and a model group, and even if lactobacillus rhamnosus NJ551 single bacteria and bacillus coagulans BC01 single bacteria are used, the killing rate of the NK cells is respectively increased by 42 percent and 39.5 percent relative to the model group, which shows that the single bacteria can also play a role in improving the immunity when being used alone. However, the combination group of NJ551 and BC01 showed a more significant improvement in the NK cell killing rate, and the higher the amount of probiotic used in the combination group, the more significant improvement in the NK cell killing rate, and the higher the NK cell killing rate of the low dose group compared to the model group, was 49.3% (table 2). The NK cell killing rate was also significantly increased in the middle and high dose groups of the combined group compared to the single bacterial groups of NJ551 and BC 01. Although the low dose group of the combination group did not significantly improve NK cell killing rate compared to the NJ551 single bacterial group, the average NK cell killing rate was also improved by about 4.8%. The combination of the results shows that NJ551 and BC01 have the functions of improving the killing rate of NK cells and recovering the inhibited immunity independently, but the combination of the two can make the improving effect more obvious, and the effect of single bacterium high dose can be achieved at lower dose.
Table 2: the NK cell killing rate of each test group is measured (the significant difference p is less than 0.05 compared with the model group; the significant difference p is less than 0.05 compared with the blank group; the significant difference p is less than 0.05 compared with the single bacterium group)
8.2 Effect of Each test group on intestinal mucosal permeability
The result shows that the intestinal mucosa permeability of the model mouse is obviously increased relative to that of the blank group, and the damaged intestinal mucosa permeability can be repaired by using probiotic intervention. After the intervention period, the intestinal mucosa permeability of the probiotic intervention group except the NJ551 group is remarkably reduced compared with that of the model group, and the intestinal mucosa permeability of the probiotic intervention group is basically restored to the level of the blank group. Although the intestinal mucosal permeability of the probiotic intervention group was not significantly different from that of the blank group as a whole, the intestinal mucosal permeability of the combination group was also reduced to some extent compared with that of the blank group in the combination group at the high and low dose groups, and the reduction was not achieved to a significantly different level, but could also indicate that the intestinal mucosal barrier could be enhanced by the combination group at the high and medium doses to some extent (table 3).
Table 3: the influence of each test group on the intestinal mucosa permeability (significant difference p < 0.05 compared with the model group; significant difference p < 0.05 compared with the blank group; significant difference p < 0.05 compared with the single bacteria group)
8.3 Effect of Each test group on the content of IFN-gamma and TNF-alpha in peripheral blood
The results show that the serum IFN-gamma and TNF-alpha contents of the model group are obviously reduced compared with those of the blank group. The probiotic intervention group can recover the contents of the two cytokines, and generally speaking, compared with TNF-alpha, the probiotics can stimulate the organism to generate IFN-gamma. The IFN-gamma content of the NJ551 single bacterium group is respectively improved by 6 percent and 72 percent compared with the respective significance (p is less than 0.05) of the blank group and the model group; the level of TNF-a was elevated above the blank but was significant relative to the model group but not relative to the blank. The IFN-gamma content of the BC01 single bacterium group is increased by 2.3% and 66.1% compared with that of a blank group and a model group respectively, but the improvement has no significance for the blank group and has significance for the model group; compared with the model group, the content of TNF-alpha can be increased, and the increase is significant. The high-dose group and the medium-dose group of the combination group can obviously improve the content of IFN-gamma and TNF-alpha compared with the model group or the blank group; compared with single bacterium groups (NJ551 groups and BC01 groups), the content of IFN-gamma can be obviously improved, and the content of TNF-alpha can be obviously improved compared with that of BC01 single bacterium groups. The low-dose group of the combination group can also obviously improve the content of IFN-gamma and TNF-alpha in serum compared with the model group, but the improvement does not represent obvious advantage compared with a single bacterium group. In general, the probiotic intervention group can recover or improve the concentration of serum cytokines inhibited by cyclophosphamide, but the combination group can achieve or even exceed the higher intake of a single bacterium group by utilizing the lower intake of probiotics, thereby showing obvious advantages. The probiotic single bacterium group can also effectively restore the content of IFN-gamma or TNF-alpha even to exceed the level of the blank group, and the effect of effectively improving the immunity is reflected (Table 4).
Table 4: the influence of IFN-gamma in peripheral blood and TNF-alpha in serum in each test group (p is less than 0.05) compared with the model group, p is less than 0.05 compared with the blank group, p is less than 0.05 compared with the single bacterium group)
9. As the animal experiments prove that the combination of the lactobacillus rhamnosus NJ551, the bacillus coagulans BC01 and the two bacteria has obvious help on recovering the destroyed immunity and even improving the immunity, and particularly the combined bacteria have obvious advantages compared with single bacteria. Since both of these bacteria are in the list of strains approved by the ministry of health in china that can be used in food, these two single bacteria and their combinations can be applied to food, health products or drugs. The application mode can be as follows:
1. the freeze-dried powder of the two bacteria is used alone or in combination with other auxiliary materials such as prebiotics, maltodextrin and the like to be mixed into products in the forms of solid powder, tablets, granules, capsules and the like.
2. The two kinds of bacteria or their combination are added into fermented food, such as yogurt, sauerkraut, fermented fruit and vegetable juice, and ferment. The adding mode can be that freeze-dried powder of the two bacteria or the combination of the two bacteria is added after the liquid product is fermented, or the freeze-dried powder is added at the beginning of fermentation and is multiplied in the fermentation process.
3. The two bacteria or combination are added into semisolid food such as jelly, cheese, etc. or other semisolid products.
The foregoing examples are intended to further illustrate some preferred embodiments of the invention, not all embodiments. Other embodiments of the invention based on the present invention, which can be made by a person skilled in the art without inventive step, belong to the scope of protection of the present invention.
Claims (6)
1. The probiotic composition beneficial to improving the immunity is characterized by comprising lactobacillus rhamnosus NJ551 and bacillus coagulans BC01, wherein the lactobacillus rhamnosus NJ551 is preserved in China center for type culture collection with the preservation number of CCTCCNO: m2016157, wherein the Bacillus coagulans strain BC01 is preserved in China Center for Type Culture Collection (CCTCCNO) with a preservation number of M2017813.
2. The probiotic composition for boosting immunity according to claim 1, characterized in that the lactobacillus rhamnosus NJ551 has the following characteristics:
the survival rate of the artificial gastric juice after being treated in the artificial gastric juice for 6 hours is more than 50 percent, and the survival rate of the artificial intestinal juice after being treated in the artificial intestinal juice for 1 hour is more than 26 percent;
can effectively recover or improve the permeability of the intestinal mucosa of the mouse, the killing rate of NK cells and the contents of serum gamma-interferon and TNF-alpha in an immunosuppressed mouse model;
the survival rate of the freeze-dried powder prepared from the strain is more than 50% after being damaged at 37 ℃ for 10 days, and the survival rate of the freeze-dried powder is more than 90% after being stored at-18 ℃ for 12 months;
the bacillus coagulans BC01 has the following characteristics:
the survival rate of the artificial intestinal juice after being treated in the artificial gastric juice for 6 hours is more than 75 percent, the survival rate of the artificial intestinal juice after being treated in the artificial intestinal juice for 4 hours is more than 82 percent, and the survival rate of the artificial intestinal juice after being continuously treated in the artificial gastric juice for 3 hours and the artificial intestinal juice for 3 hours is more than 63 percent;
the survival rate of the freeze-dried powder or spray-dried powder prepared by the strain after being damaged at 37 ℃ for 10 days is more than 98 percent, and the survival rate of the freeze-dried powder or spray-dried powder after being stored at normal temperature for 18 months is more than 98 percent;
two life forms of a vegetative state and a spore state exist in the life cycle of the plant;
in vitro tests prove that the compound has bacteriostatic effect on escherichia coli and staphylococcus aureus;
can effectively recover or improve the permeability of the intestinal mucosa of the mouse, the killing rate of NK cells and the contents of serum gamma-interferon and TNF-alpha in an immunosuppressed mouse model.
3. The probiotic composition for improving immunity according to claim 1, characterized in that the lactobacillus rhamnosus NJ551 and the bacillus coagulans BC01 in the probiotic composition are stored separately or after being mixed.
4. The probiotic composition for boosting immunity according to claim 3, characterized in that it is a mixture containing said Lactobacillus rhamnosus NJ551 and said Bacillus coagulans BC01, said Lactobacillus rhamnosus NJ551 being used in an amount of 2.0 x 105CFU/gram (body weight)/day; the dosage of the bacillus coagulans BC01 is 6.0 x 104CFU/gram (body weight)/day.
5. The preparation method of the probiotic composition for improving the immunity is characterized in that the composition comprises the lactobacillus rhamnosus NJ551 and bacillus coagulans BC01, and the lactobacillus rhamnosus NJ551 strain is preserved in China center for type culture Collection with the preservation number: CCTCC NO: m2016157, the Bacillus coagulans strain BC01 is deposited in China center for type culture Collection under accession number: m2017813, and the preparation method of the probiotic composition for improving immunity comprises the following steps:
and diluting the freeze-dried powder of the lactobacillus rhamnosus NJ551 and the live bacteria powder of the bacillus coagulans BC01 into mixed bacteria liquid.
6. The application of the probiotic composition for improving the immunity is characterized in that the composition of lactobacillus rhamnosus NJ551 and bacillus coagulans BC01 is applied to the preparation of drugs, foods or health products for improving the immunity, the lactobacillus rhamnosus NJ551 strain is preserved in the China center for type culture Collection, and the preservation number is as follows: CCTCC NO: m2016157, the Bacillus coagulans strain BC01 is deposited in China center for type culture Collection under accession number: CCTCC NO: M2017813.
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