The present invention belongs to the technical field of bioengineering, and relates to a strain, a screening method and use thereof, in particular to Bifidobacterium animalis subsp. lactis i797, method for separation and purification thereof and use thereof.
Bifidobacterium was first discovered when Henry Tissier separated it from the feces of breast-fed babies in 1899. Bifidobacterium belongs to Gram-positive bacilli, is very sensitive to oxygen, has poor tolerance to low PH and is easily inactivated in a low PH environment; the optimum PH for Bifidobacterium is 6.5-7.0, and the optimum growth temperature for Bifidobacterium is 37-42° C. Bifidobacterium widely exists in habitats such as digestive tracts, vaginas and oral cavities of human and animal, and is one of the important members of the intestinal flora of human and animal. It has been found that there are 32 subtypes of Bifidobacterium, and there are up to 70 kinds of biological agents containing Bifidobacterium.
Bifidobacterium is a kind of probiotic bacteria that neither produces endotoxins or exotoxins, nor produces pathogenic substances or harmful gases, which is of great significance for maintaining the health of the body. Bifidobacterium can effectively maintain the normal balance of the intestinal flora, and has unique physiological functions against diarrhea, constipation, infection and tumor etc. Bifidobacterium plays an important role in body health, for example, maintaining intestinal microecological balance, inhibiting invasion and colonization of pathogenic bacteria, regulating body immunity and reducing cholesterol content, etc. Therefore, it has a wide application prospect. In addition, the nutrient substances such as vitamins and amino acids produced by Bifidobacterium during the growing process can improve the nutritional value of milk. Therefore, Bifidobacterium is widely applied in milk production.
Bifidobacterium can colonize in the intestinal tract of a newborn baby within a few hours after the birth. The quantity of Bifidobacterium distributed in the gastrointestinal tract decreases as the age increases, and is highest in breast-fed babies. In infants, Bifidobacterium accounts for about 60% of the total intestinal bacteria; in old people over sixty, Bifidobacterium accounts for only 7.9% of the total intestinal bacteria, while putrefactive bacteria such as Clostridium perfringens and Escherichia coli increase greatly; in elderly people, the intestinal tracts are full of putrefactive bacteria, while Bifidobacterium almost disappears. The researches demonstrate that the minimum viable bacteria concentration should be higher than 107 cfu/mL to maintain the function of probiotic bacteria.
Therefore, maintaining the quantity of Bifidobacterium in the intestinal tracts is very important to maintain human health. Research on the mechanism of Bifidobacterium for regulating the intestinal flora is helpful for the industrial application of Bifidobacterium. For example, up to now, some Bifidobacterium strains have been put into industrial use in China to treat the diseases caused by intestinal flora disorder, such as diarrhea and functional constipation, etc., and have achieved remarkable results. Besides, some medical studies demonstrate that long-term use of artificially synthesized probiotic bacteria may result in the phenomenon of intestinal tract gradually losing its ability to reproduce probiotic bacteria by itself and dependency of intestinal tract on artificial probiotic bacteria arising, leading to “probiotic dependence”.
In recent years, the researches on the mechanism of Bifidobacterium for regulating the intestinal flora mainly focused on colonization, specific binding and generated metabolites of Bifidobacterium. However, in-depth researches on how to give full play to the function of Bifidobacterium for regulating the intestinal flora should be carried out, and such researches involve selection and cultivation of strains.
An object of the present invention is to provide a Bifidobacterium animalis subsp. lactis i797, which is separated and screened from the feces of breast-fed infants or babies, and can regulate the balance of the intestinal flora and promote body health.
Another object of the present invention is to provide a method for separating and purifying the above Bifidobacterium animalis subsp. lactis i797.
Another object of the present invention is to provide use of the above Bifidobacterium animalis subsp. lactis i797.
To attain the objects described above, the present invention employs the following technical solution:
A Bifidobacterium animalis subsp. lactis i797, preserved in the China General Microbiological Culture Collection Center (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on Aug. 20, 2019, with a preservation number of CGMCC No. 18403.
As a limitation, the Bifidobacterium animalis subsp. lactis i797 is screened out from the intestinal flora of infants or babies.
As a second limitation, its 16SrRNA sequence is as follows:
As a third limitation, its tuf gene sequence is as follows:
The present invention further provides a method for separating and purifying above Bifidobacterium animalis subsp. lactis i797, comprising the following steps which are carried out in sequence:
obtaining intestinal feces of infants or babies, then adding the intestinal feces into normal saline and mixing thoroughly to obtain a sample A;
adding the sample A into a modified MRS liquid culture medium, and culturing in an anaerobic environment at 35-40° C. for 62-82 hours to obtain a culture solution B;
wherein the modified MRS liquid culture medium is an MRS liquid culture medium added with 0.5 wt % cysteine;
the volume ratio of the sample A to the modified MRS liquid culture medium is 1:10-100;
diluting the culture solution B with 0.9% sterile normal saline by ten-time gradient multiplication, i.e., diluting to 10−1, 10−2, 10−3, 10−4 and 10−5 times sequentially, thus obtaining bacterial suspensions C1-C5 correspondingly;
melting the modified MRS solid culture medium and pouring it into first to fifth culture dishes, thus obtaining culture media D1-D5 after cooling and complete solidification; drawing 0.1 mL bacterial suspensions C1-C5 respectively, and spreading on the culture media D1-D5 respectively in one-to-one correspondence, then turning the plates upside down and culturing in an anaerobic environment at 35-40° C. for culture for 62-82 hours, and observing the growth of the colonies;
the modified MRS solid culture medium is a solid culture medium obtained by adding 15 wt % agar per 1,000 mL modified MRS liquid culture medium;
after typical colonies appear on the plates, picking corresponding single colony E;
picking a selected single colony E and streak-inoculating the single colony E culture on the modified MRS solid culture medium, and culturing in anaerobic environment at 35-40° C. for 62-82 hours to obtain a single colony F;
streak-inoculating the single colony F further on the modified MRS solid culture medium, and culturing in an anaerobic environment at 35-40° C. for 62-82 hours to obtain a single colony G;
further streak-inoculating the single colony G on the modified MRS solid culture medium, and culturing in an anaerobic environment at 35-40° C. for 62-82 hours to obtain a pure culture H, which is the strain of Bifidobacterium animalis subsp. lactis i797.
As a limitation, the strain of Bifidobacterium animalis subsp. lactis i797 is preserved as follows: mixing the pure culture H with 50 wt % sterile glycerol at a ratio of 1:1, placing the mixture in a strain preservation tube, mixing homogeneously and then preserving at −80-70° C.; at the same time, inoculating on the test-tube slant of the modified MRS solid culture medium for temporary storage.
As a second limitation, the components of the modified MRS liquid culture medium include: casein peptone, beef extract, yeast extract, glucose, sodium acetate, diamine citrate, Tween-80, K2HPO4, MgSO4.7H2O, MnSO4.7H2O, cysteine and distilled water;
wherein a dosage ratio of casein peptone:beef extract:yeast extract:glucose:sodium acetate:diamine citrate:Tween-80:K2HPO4:MgSO4.7H2O:MnSO4.7H2O:cysteine:distilled water is 10 g:10 g:5 g:20 g:5 g:2 g:1 g:2 g:0.2 g:0.05 g:0.5 g:1,000 mL.
The present invention further provides use of the Bifidobacterium animalis subsp. lactis i797 in preparation of drinks, foods or medicines.
As a limitation, the drinks are beverages or fermented milk drinks;
the foods are cereals, cereal derivatives, fermented meat products, probiotics or milk foods;
the medicines are in dosage form of capsule, tablet, pill or powder.
As a further limitation, the probiotics are compound probiotics.
With the technical solution described above, compared with the prior art, the present invention achieves the following technical progresses:
The Bifidobacterium animalis subsp. lactis i797 provided by the present invention is suitable for regulating intestinal tract, balancing the intestinal flora, and improving the characteristics of feces.
Hereunder some preferred examples of the present invention will be detailed. It should be understood that the preferred examples described here are only used to describe and explain the present invention, but not intended to limit the present invention.
In this example, a Bifidobacterium animalis subsp. lactis i797, which is separated and screened from the feces of breast-fed infants or babies, and preserved in the China General Microbiological Culture Collection Center (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on Aug. 20, 2019, with a preservation number of CGMCC No. 18403, is provided.
The 16SrRNA sequence of above Bifidobacterium animalis subsp. lactis i797 is as follows:
Its tuf gene sequence is as follows:
In this example, a method for separating and purifying the Bifidobacterium animalis subsp. lactis i797 described in the example 1 is provided, including the following steps which are carried out in sequence:
1 g intestinal feces of infants or babies is obtained and then added into 9 mL normal saline for thorough mixing to obtain a sample A;
2 mL (V1) sample A is added into 100 mL (V2) modified MRS liquid culture medium, and cultured in an anaerobic environment at 37° C. (T1) for 72 hours (t1) to obtain a culture solution B;
1 mL culture solution B1 is diluted with 0.9% sterile normal saline by ten-time gradient multiplication, i.e., diluting to 10−1, 10−2, 10−3, 10−4 and 10−5 times sequentially, thus obtaining bacterial suspensions C11-C15 correspondingly;
A modified MRS solid culture medium is melted and poured into the first to fifth culture dishes, thus culture media D1-D5 are obtained after cooling and complete solidification; 0.1 mL bacterial suspensions C1-C5 are drawn and spread on the culture media D1-D5 respectively in one-to-one correspondence, then the plates are turned upside down and cultured in an anaerobic environment at 37° C. (T2) for 72 hours (t2), and the growth of the colonies is observed;
After typical colonies appear on the plates, corresponding single colony E is selected according to the colony characteristics of standard Bifidobacterium with reference to images in relevant literature;
A selected single colony E is picked and its culture is streak-inoculated on the modified MRS solid culture medium, and cultured in anaerobic environment at 37° C. (T3) for 72 hours (t3) to obtain a single colony F;
The single colony F is further streak-inoculated on the modified MRS solid culture medium, and cultured in an anaerobic environment at 37° C. (T4) for 72 hours (t4) to obtain a single colony G;
The single colony G is further streak-inoculated on the modified MRS solid culture medium, and cultured in an anaerobic environment at 37° C. (T5) for 72 hours (t5) to obtain a pure culture H, which is the strain of Bifidobacterium animalis subsp. lactis i797;
The pure culture H is mixed with 50 wt % sterile glycerol at a ratio of 1:1, the mixture is placed in a strain preservation tube, mixed homogeneously and then preserved at −70° C. (T6); at the same time, it is inoculated on the test-tube slant of the modified MRS solid culture medium for temporary storage.
In this example, the raw materials of the modified MRS liquid culture medium include: casein peptone, beef extract, yeast extract, glucose, sodium acetate, diamine citrate, Tween-80, K2HPO4, MgSO4.7H2O, MnSO4.7H2O, cysteine and distilled water; wherein a dosage ratio of casein peptone:beef extract:yeast extract:glucose:sodium acetate:diamine citrate:Tween-80:K2HPO4:MgSO4-7H2O:MnSO4.7H2O:cysteine:distilled water is 10 g:10 g:5 g:20 g:5 g:2 g:1 g:2 g:0.2 g:0.05 g:0.5 g:1,000 mL; the modified MRS solid culture medium is obtained by adding 15 wt % agar per 1,000 mL modified MRS liquid culture medium.
The examples 3-6 provide a separation and purification method of the example 1 respectively, which is essentially the same as that of the example 2, except that the technical parameters of the separation and purification process are different. The specific parameters are shown in Table 1:
This example illustrates the basic bacteriological characteristics of the Bifidobacterium animalis subsp. lactis i797 in the example 1, which are shown in Table 2:
This example illustrates the sugar fermentation characteristics of the strain of Bifidobacterium animalis subsp. lactis i797 in the example 1. The experimental method for sugar fermentation characteristics is as follows: a single colony of the strain of Bifidobacterium animalis subsp. lactis i797 obtained with the separation and purification method in the example 3 is inoculated into a sterilized modified MRS liquid culture medium, and cultured at 37° C. for 24 hours, then the bacterial suspension is inoculated into a sugar fermentation tube, and cultured under anaerobic condition at 37° C. for 48 hours, and the color change is observed. The identification result of the sugar fermentation characteristics is shown in Table 3:
The modified MRS liquid culture medium used in this example has the same composition as the modified MRS liquid culture medium in the example 2.
The strain of Bifidobacterium animalis subsp. lactis i797 obtained with the separation and purification method in the example 5 is subjected to molecular biological identification, and through DNA extraction, PCR amplification, 16SrRNA sequencing, and NCBI Online Blast, it is finally determined as a Bifidobacterium animalis subsp. lactis.
Its 16SrRNA sequencing result is as follows:
Its tuf gene sequencing result is as follows:
After the strain of Bifidobacterium animalis subsp. lactis i797 to be tested is activated for 3 generations, 1 mL strain is injected in 9 mL filtered and sterilized artificial gastric fluid with a pH of 3.0, the mixture is shaken homogeneously and cultured in an anaerobic environment at 37° C.; samples are taken at the beginning of culturing and after 2 hours culturing respectively, and the viable counts are determined respectively. Then, 1 mL culture solution which has been digested in the artificial gastric fluid with 3.0 pH for 2 hours is inoculated into 9 mL filtered and sterilized artificial intestinal fluid with a pH of 8.0, and is further cultured at 37° C.; the viable count is measured at 0 hour, 4 hours and 6 hours respectively.
Bifidobacterium BB-12 is used as a standard strain for a control experiment, and the experimental parameters are the same as those of the experiment for the strain of Bifidobacterium animalis subsp. lactis i797.
Survival rate (%)=(cfu N1/cfu N0)×100%
where, N1—the viable count after treatment with the artificial digestive fluid for 6 hours; N0—the viable count after treatment with the artificial digestive fluid for 0 hour.
b. Intestinal fluid: 0.9 g Bile Salts (Difco) per 100 mL, the pH is adjusted to 8.0, and the fluid is filtered and sterilized for later use.
The experiment demonstrates that BB-12 has strong tolerance to gastric acid and poor tolerance to intestinal fluid, while the Bifidobacterium animalis subsp. lactis i797 has poor tolerance to gastric acid and strong tolerance to intestinal fluid. By comprehensive comparison, the Bifidobacterium animalis subsp. lactis i797 achieves a better survival rate in the simulated digestive fluid, which is 7.4% and superior to that of BB-12.
A drink containing the Bifidobacterium animalis subsp. lactis i797 is given to the testers for drinking, and statistics on the drinking result of 10 testers is carried out.
The statistical result is shown in Table 5:
Bifidobacterium Animalis Subsp. Lactis i797
In the statistical process:
It can be seen from Table 5: among all indexes, four indexes (frequency of defecation, difficulty in defecation, hardness of feces, and shape of feces) have changed significantly (statistically significant, P<0.05), which indicates that Bifidobacterium animalis subsp. lactis i797 has a regulating effect on the intestinal tracts.
Fresh milk of 97-98 parts and white sugar of 2-3 parts are mixed and blended evenly, homogenized at 65° C. and 15 MPa, sterilized at 95° C. for 300 seconds, and then cooled down to 37° C. to obtain sterilized milk; the Bifidobacterium animalis subsp. lactis i797 is inoculated into the sterilized milk with an inoculation amount of 3 wt % of the sterilized milk, and then fermented at 36° C., and the pH change is detected.
Standard strain of Bifidobacterium BB-12 is used as a control strain for control experiment, and the experimental parameters are the same as those of the above experiment for the strain of Bifidobacterium animalis subsp. lactis i797.
The result of acid production is shown in Table 6.
It can be seen from Table 6: the Bifidobacterium animalis subsp. lactis i797 can control the post-acidification well under a storage condition suitable for the growth of lactic acid bacteria at a higher temperature of 37° C.
This example provides use of the Bifidobacterium animalis subsp. lactis i797 in Example 1, which can be used to prepare drinks, foods or medicines. For example, the strain can be used to prepare cereals and their derivatives, fermented meat products, probiotics and formula milk powder, which have an intestinal regulation function; it can also be used to prepare beverages and fermented yogurts that have an intestinal regulation function; in addition, it can be used to prepare medicines with an intestinal regulation function, in a dosage form of capsule, powder, pill, oral liquid or spray, etc.
The probiotics in the example 13 may be probiotics that only contain the Bifidobacterium animalis subsp. lactis i797; or the probiotics may be compound probiotics prepared by mixing Bifidobacterium animalis subsp. lactis i797, Lactobacillus paracasei N1115, Lactobacillus plantarum N3117 and Streptococcus thermophilus JMCC0003, and the dosage of the compound probiotics may be compound probiotic microcapsule powder.
Number | Date | Country | Kind |
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201911263293.1 | Dec 2019 | CN | national |
202010259210.8 | Apr 2020 | CN | national |
Filing Document | Filing Date | Country | Kind |
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PCT/CN2020/101661 | 7/13/2020 | WO |