Composition for improving intestinal bacterial phase and prolonging colonization rate of beneficial bacteria in intestinal tract

Abstract

The invention provides a composition for improving the intestinal bacterial phase and prolonging the colonization rate of beneficial bacteria in the intestinal tract, the composition comprises Bifidobacterium lactis GKK2, and the composition can be used for improving the intestinal bacterial phase, promoting the colonization rate of the beneficial bacteria in the intestinal tract and prolonging the activity of the beneficial bacteria in the intestinal tract.

Description

Technical Field

[0001] This invention relates to a composition for improving the intestinal flora. Specifically, it relates to a composition comprising Bifidobacterium lactis GKK2. Background Technology

[0002] Gut microbiota and physiological functions

[0003] The gut microbiota refers to a group of microorganisms residing in the human intestine. They regulate neurotransmission, endocrine function, digestion, metabolism, and the immune system, influencing human physiological state and playing a close role in various systemic diseases, thus determining human health. The gut microbiota can be divided into beneficial bacteria (or symbiotic bacteria), harmful bacteria (pathogenic bacteria), and neutral bacteria (or opportunistic bacteria, conditionally pathogenic bacteria). Beneficial bacteria account for approximately 10-20% of the human gut microbiota, harmful bacteria also account for approximately 10-20%, and neutral bacteria account for 60-70%.

[0004] Neutral bacteria generally have a neutral effect on the human body, neither good nor bad. They influence human physiological functions in a way that depends on the composition and proportion of the gut microbiota. Studies have found that when the proportion of beneficial bacteria in the human gut microbiota increases, opportunistic bacteria tend to gravitate towards the dominant bacteria, working together to produce a positive effect on bodily functions. Conversely, when the proportion of harmful bacteria in the human gut microbiota increases, opportunistic pathogenic bacteria tend to gravitate towards the harmful bacteria, working together to produce negative physiological effects, such as the development of disease.

[0005] Beneficial bacteria, harmful bacteria, and harmless bacteria in the gut

[0006] The intestinal bacteria that have been classified as beneficial bacteria include: (1) Lactobacillus sp., including Lactobacillus acidophilus (A bacteria), Lactobacillus casei (C bacteria, Kjeldahl bacteria), Lactobacillus rhamnosus (LGG), Lactobacillus shirota, Lactobacillus paracasei, Lactobacillus gasseri, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus lordii, Lactobacillus bulgaricus (LB), Streptococcus thermophilus (ST), Lactococcus lactis, etc. (2) Bifidobacterium sp., including Bifidobacterium bifidum (B bacteria, Bifidobacterium bifidum), Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium brevis, Bifidobacterium lactis (B bacteria), Bifidobacterium infantis, Bifidobacterium adultis, etc. (3) Others include Bacillus subtilis, Clostridium butyricum (butyric acid bacteria, CB bacteria), etc.

[0007] The following intestinal bacteria have been classified as harmful: Clostridium difficile, Clostridium perfringens, Staphylococcus aureus, Enteropathogenic Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella sp., Shigella sp., Campylobacter jejuni, Vibrio cholerae, and Bacillus cereus.

[0008] The human gut is composed of neutral bacteria, commonly including Enterococcus faecalis, Enterococcus faecium, and non-pathogenic Escherichia coli. Other common gut bacteria whose effects on human physiology are not yet clearly defined or for which there is sufficient evidence to support a positive or negative influence include Bacteroides sp., Parabacteroides sp., Faecalibacterium sp., Akkermansia sp., Ruminococcus sp., and Anaerorotruncus sp.

[0009] Supplementing gut beneficial bacteria and its limitations

[0010] In order to maintain or improve human health, in addition to medical treatments administered by doctors, personal exercise habits, dietary choices, and adjustments to work and rest, supplements are also a common method available on the market. Currently, the mainstream approach to promoting or maintaining human physiological functions by altering the gut microbiota is primarily through direct supplementation with probiotics and their combinations.

[0011] However, gut bacteria are classified into resident (permanent residents) and transient (foreign) bacteria based on the length of time they reside and grow in the gut. Transient bacteria stay for a few days and are then excreted from the body. Most of the probiotic supplements mentioned above on the market are transient gut bacteria. Therefore, although supplementation can increase the number of beneficial gut bacteria, they are excreted from the body within a few days once supplementation is stopped, and cannot maintain the number of beneficial gut bacteria.

[0012] Taking the experimental data published by Taverniti et al. (2019) as an example, the experimental design involved 20 adults without specific diseases as subjects. They ingested a combination containing Bifidobacterium animalis subsp. lactis strain Bl-04 daily for 14 consecutive days. Fecal samples were collected from subjects between day 0 and day 28 of the experiment to analyze the amount of Bifidobacterium animalis subsp. lactis strain Bl-04 in the intestines. The results showed a significant increase in Bifidobacterium lactis in the intestines before and after supplementation with the Bl-04 combination (days 0 and 7 of the experiment). However, after stopping supplementation with the Bl-04 combination (day 14 of the experiment), the amount of Bifidobacterium lactis in the intestines decreased from approximately 10 within four days (day 18 of the experiment). 9 CFU / g decreased to approximately 10 8 CFU / g decreased significantly by 10-fold; one week after stopping Bl-04 supplementation (i.e., day 21 of the trial), the subjects' intestinal Bifidobacteria decreased to approximately 10. 5 The CFU / g level was significantly reduced by 10,000 times compared to the supplementation period (day 14 of the trial).

[0013] For detailed experimental information, please refer to the following publications:

[0014] Taverniti, V., Koirala, R., Dalla Via, A., Gargali, G., Leonardis, E., Arioli, S., & Guglielmetti, S. (2019); Effect of Cell Concentration on the Persistence in the Human Intestine of Four Probiotic Strains Administered through a Multispecies Formulation; Nutrients, 11(2), 285. This indicates that supplementation with probiotic products helps increase the number of beneficial bacteria in the gut, but once supplementation is stopped, the number of beneficial bacteria in the gut can decrease within a week, affecting the positive physiological activity of beneficial bacteria in the human body.

[0015] In response to the limitations of existing probiotic supplements on the market, there is a need for a probiotic that can not only increase the total number of live beneficial bacteria in the human gut, but also increase the colonization rate of beneficial bacteria and prolong their positive activity in the human gut. Summary of the Invention

[0016] To achieve the above objectives, the present invention provides a composition for improving the intestinal flora, comprising Bifidobacterium lactis GKK2, with accession number CGMCC No. 15205, deposited at the China General Microbiological Culture Collection Center.

[0017] Preferably, improving the intestinal flora means increasing the total number of viable Bifidobacterium sp. or Lactobacillus sp.

[0018] To achieve another objective of the present invention, a composition for prolonging the colonization rate of beneficial intestinal bacteria is also provided, comprising Bifidobacterium lactis GKK2, with accession number CGMCC No.15205, deposited at the China General Microbiological Culture Collection Center.

[0019] Preferably, the beneficial bacteria are Bifidobacterium sp. or Lactobacillus sp.

[0020] Preferably, the composition comprises an additive selected from the group consisting of: excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, or combinations thereof.

[0021] Preferably, the composition is a pharmaceutical, feed, beverage, nutritional supplement, dairy product, food, or health food.

[0022] Preferably, the composition is in the form of powder, tablet, granulation, suppository, microcapsule, ampoule, liquid spray or stopper.

[0023] Preferably, the intestine is the human intestine.

[0024] Preferably, the composition contains 10 to 30 wt% lyophilized Bifidobacterium lactis GKK2 powder.

[0025] Preferably, the freeze-dried powder is prepared by the following method:

[0026] The GKK2 strain was inoculated onto MRS agar and cultured in a solid state until colonies formed.

[0027] The colonies were inoculated into MRS liquid medium and incubated at 25 to 40°C for 16 to 48 hours.

[0028] The bacterial culture obtained from the liquid culture was transferred to a fermentation medium for fermentation culture for 16 hours. The fermentation medium contained 1 to 10 wt% sugars, 0.1 to 5 wt% yeast extract, 0.1 to 5 wt% peptone, 0.01 to 2 wt% trace elements, 0.01 to 0.1 wt% cysteine, and 0.05 to 1 wt% Tween-80.

[0029] Centrifuge the fermentation broth at 1000 to 15000 rpm to obtain bacterial sludge;

[0030] The bacterial sludge was freeze-dried using a gradient freeze-drying method to obtain freeze-dried powder.

[0031] Preferably, the gradient freeze-drying process is performed by storing at 20 to 0°C for 1 to 4 hours, then at 0 to -20°C for 4 to 8 hours, and finally freezing at -196 to -30°C for more than 8 hours.

[0032] To achieve another objective of the present invention, a use is provided for Bifidobacterium lactis GKK2 in manufacturing pharmaceutical compositions that improve intestinal flora or prolong the colonization rate of beneficial intestinal bacteria, wherein the accession number of GKK2 is CGMCC No.15205 and is deposited at the China General Microbiological Culture Collection Center.

[0033] Preferably, the improvement of intestinal flora refers to increasing the total number of viable Bifidobacterium sp. or Lactobacillus sp.

[0034] Preferably, the beneficial bacteria are Bifidobacterium sp. or Lactobacillus sp.

[0035] Preferably, the intestine is the human intestine. Attached Figure Description

[0036] Figure 1 To illustrate the number of viable Bifidobacterium bacteria after GKK2 supplementation was stopped.

[0037] Preservation of biological materials for patent procedures:

[0038] Bifidobacterium lactis strain GKK2;

[0039] Date of deposit: January 12, 2018;

[0040] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);

[0041] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China;

[0042] Accession number: CGMCC No. 15205;

[0043] Classification and nomenclature: Bifidobacterium lactis. Detailed Implementation

[0044] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0045] strain source

[0046] The Bifidobacterium lactis strain used in this experiment was GKK2. In a preferred embodiment, this strain was isolated from infant feces. The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 15205 (deposit date: January 12, 2018).

[0047] bacterial culture

[0048] Bifidobacterium lactis was inoculated onto a solid culture medium to activate the bacterial strain. In a preferred embodiment, the solid culture medium was MRS agar. After colony formation, a single colony was picked and inoculated into liquid culture medium for liquid culture. In a preferred embodiment, the strain was cultured at a temperature of 25 to 40°C. In a preferred embodiment, the strain was cultured under anaerobic conditions. In a preferred embodiment, the liquid culture time was 16 to 48 hours. In a preferred embodiment, the liquid culture medium was MRS liquid medium. After liquid culture, fermentation was carried out for 16 hours. In a preferred embodiment, the fermentation medium formulation is shown in Table 1 below.

[0049] Table 1. Fermentation medium formulation

[0050] Element Ratio (by weight percentage) carbohydrate 1～10％ yeast extract 0.1～5％ peptone 0.1～5％ Trace elements 0.01～2％ Cysteine 0.01～0.1％ Tween-80 0.05～1％

[0051] Preparation of freeze-dried powder

[0052] After Bifidobacterium lactis completes its growth through fermentation culture, the fermentation broth containing bacterial cells and culture medium is collected and centrifuged to obtain a bacterial slurry. In a preferred embodiment, centrifugation is performed at a rate of 1000 to 15000 rpm. The resulting bacterial slurry is freeze-dried and stored at low temperature. In a preferred embodiment, the pre-freezing temperature for freeze-drying is set in a gradient: storage at 20 to 0°C for 1 to 4 hours, followed by storage at 0 to -20°C for 4 to 8 hours, and finally freezing at -196 to -30°C for more than 8 hours. In a preferred embodiment, the freeze-drying temperature and time are approximately -40°C for about 2 hours, followed by -20°C for about 2 hours, then 0°C for about 2 hours, and finally 20°C for more than 10 hours. In a preferred embodiment, the cryopreservation temperature is -30 to 4°C. The preserved freeze-dried powder is used as a supplementary ingredient in the following human clinical trials.

[0053] The composition of the present invention comprising Bifidobacterium lactis GKK2 further comprises excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, or combinations thereof. Preferably, the excipients may be lactose, maltodextrin, and / or silicon dioxide; the preservatives may be potassium sorbate, sodium benzoate, and / or methylparaben; the diluents may be lactose, sucrose, and / or maltodextrin; the fillers may be microcrystalline cellulose and / or calcium phosphate; the absorption enhancers may be citric acid, vitamin C, and / or pectin; and the sweeteners may be sucrose, stevia, and / or xylitol.

[0054] This composition is provided as a pharmaceutical, feed, beverage, nutritional supplement, dairy product, food, or health food, and may be in the form of powder, tablets, granules, suppositories, microcapsules, ampoules, liquid sprays, or plugs. The following experiments will test the supplement in granule form.

[0055] Preparation of granule supplements of Bifidobacterium lactis GKK2

[0056] Take 20 wt% of lyophilized Bifidobacterium lactis GKK2 powder, and mix it with 15 wt% corn starch, 12 wt% lactose, 10 wt% fructose powder, 8 wt% galactooligosaccharide, 8 wt% chicory fiber, 7 wt% yogurt powder, 6 wt% apple juice powder, 5 wt% isomaltooligosaccharide syrup, 3 wt% flavoring, 2 wt% ethanol, 2 wt% DL-malic acid, 1 wt% bromelain, and 1 wt% silicon dioxide. The aforementioned wt% refers to the proportion of each component to the total weight of the composition. The proportion of lyophilized powder can be 10 to 30 wt%, preferably 20 wt%, and the remaining components can be adjusted appropriately according to needs.

[0057] During production, all powdered raw materials are first sieved through a 60-mesh sieve to ensure the moisture content meets specifications, and liquid raw materials are prepared. In the mixing stage, corn starch, lactose, and silica are mixed for 5 minutes, then Bifidobacterium lactis GKK2 lyophilized powder is added and mixed for 3 minutes. Next, other powdered raw materials are added sequentially and mixed for 5 minutes each. Finally, liquid raw materials are added and stirred for 10 minutes. Granulation is performed using a vibrating granulator with a mesh size of 20 to 40 mesh at room temperature (20 to 25°C) for 15 to 20 minutes. Then, a fluidized bed dryer is used to dry the granules at 40 to 45°C for 20 to 30 minutes, ensuring a final moisture content of ≤5%. Qualified granules are collected by sieving through a 20 to 40-mesh standard sieve. The finished product undergoes quality testing, including moisture content determination, viable cell count determination, granule uniformity testing, and flowability testing. Each granule supplement (approximately 500 mg) contains approximately 1 x 10⁻⁶ granules. 11 CFU of Bifidobacterium lactis GKK2. Specific Implementation

[0059] Human Experimentation Case Acceptance Criteria and Grouping

[0060] The clinical trial was open to healthy adults aged 20 to 45 years without intestinal diseases, who had not taken any gastrointestinal medications before or during the trial, and who were willing to follow the dietary recommendations. A total of 34 participants were enrolled during the trial, with one person excluded due to their inability to participate in the entire trial.

[0061] A total of 33 participants were actually included in the study. The participants were divided into a placebo group (n=16) and an experimental group (ingesting strain GKK2) (n=17). Basic data of the participants are shown in Table 2 below. There was no significant difference in weight between the two groups before and after the experiment.

[0062] Table 2. Basic Data of Subjects

[0063] Placebo group (16 people) Experimental group (17 people) Age (Y) 27.5±5.1 27.2±6.7 Gender (Male / Female) 7 / 9 3 / 14 Pre-test weight (kg) 66.3±16.9 67.4±14.6 Post-test weight (kg) 66.3±16.8 67.2±14.4

[0064] Clinical trial design

[0065] The experiment lasted three weeks, divided into a blank period (day 0), the initial treatment period (days 1-7), the middle treatment period (days 8-14), and the discontinuation period (days 15-21). Participants in the experimental group (17 individuals) took two 2g packets daily from day 1 to day 14 of a granular supplement containing Bifidobacterium lactis GKK2, each packet containing 20 wt% (400 mg) of Bifidobacterium lactis GKK2. Participants in the placebo group (16 individuals) took a granular supplement without Bifidobacterium lactis during the same period. All participants (placebo and experimental groups) discontinued the granular supplement from day 15 to day 21 of the experimental period.

[0066] All subjects (placebo group and experimental group) had stool samples collected on day 0 (blank period), day 7 (initial supplementation), day 14 (mid-supplementation), and day 21 (discontinuation period) of the experimental period to analyze the viable count of specific target gut microbiota.

[0067] Analysis of viable bacteria count in fecal intestinal flora

[0068] The fecal samples collected in the aforementioned experiment were diluted using a tenfold serial dilution method. Samples were then added to appropriate culture media and incubated anaerobically at 37°C for 48 hours. The number of colonies generated in the culture medium was then calculated. The anaerobic diluent consisted of 50 mL distilled water, 0.2 g gelatin, 50 mL salt solution, and 0.4 mL resazurin solution (25 mg / 100 mL H₂O). The solution was sterilized by boiling for 5 to 10 minutes, cooled, and then 0.05 g cysteine-HCl was added.

[0069] Viable count analysis of specific target microbial communities in fecal samples was performed, analyzing the viable counts of beneficial bacteria, including Lactobacillus sp. and Bifidobacterium sp., and harmful (or non-beneficial) bacteria, including Clostridium perfringens and Coliform bacteria. In this example, the culture medium used for the Lactobacillus sp. viable count analysis was MRS medium (Merck Millipore, catalog number: 105413). In this embodiment, the culture medium used for the viable count analysis of Bifidobacterium sp. was Bifidobacterium iodoacetate medium-25 (Acumedia Neogen, catalog number: 7714A), supplemented with nalidixic acid, polymyxin B sulfate, kanamycin sulfate, iodoacetic acid, and 2,3,5-triphenyltetrazolium chloride. In this embodiment, the culture medium used for the viable count analysis of Clostridium perfringens was Tryptose sulfite D cycloserine medium (Merck Millipore, catalog number: 111972), supplemented with D-cycloserine and egg yolk emulsion. In this embodiment, the culture medium used for the analysis of viable coliform bacteria was Coliform medium (Merck Millipore, catalog number: 110426).

[0070] Data statistics and judgment

[0071] The viable bacterial count in fecal samples was expressed as log CFU (colony forming unit). All data obtained in this case study are expressed as mean ± standard deviation (mean ± SD). Data were analyzed using the Duncan's test in One-Way ANOVA software (SPSS 12.0) to determine differences between the placebo and experimental groups. A p-value < 0.05 was considered statistically significant.

[0072] Improved gut microbiota function was defined as a significant increase in Bifidobacterium sp. and a decrease or no significant change in Clostridium perfringens.

[0073] Example 1: Changes in the viable count of total lactobacilli

[0074] The changes in total lactic acid bacteria flora in the feces of each group of subjects during the trial blank period to the supplementary mid-term (day 0 to day 14 of the trial) are shown in Table 3 below.

[0075] Table 3. Changes in total lactic acid bacteria flora in the feces of the subjects

[0076]

[0077]

[0078] Note 1. The number of viable bacteria is expressed as log CFU (Colony Forming Unit).

[0079] Note 2. Different letters indicate within-group differences (p<0.05).

[0080] In the placebo group, there was no significant change in the total number of lactobacilli in stool samples before and after taking the granular supplement without Bifidobacterium lactis. In the experimental group, there was a significant change in the total number of lactobacilli in stool samples before and after taking the granular supplement containing Bifidobacterium lactis GKK2. If the total number of lactobacilli measured at the blank period (day 0 of the experiment) is taken as 1, the total number of lactobacilli in the experimental group before supplementation with Bifidobacterium lactis GKK2 (day 7 of the experiment) increased by more than 80 times. These results indicate that Bifidobacterium lactis GKK2 supplementation helps increase the number of beneficial live bacteria in the human gut, especially Lactobacillus.

[0081] Example 2: Changes in the viable count of Bifidobacteria

[0082] The changes in Bifidobacterium flora in the feces of each group of subjects during the trial blank period to the supplementary mid-term (day 0 to day 14 of the trial) are shown in Table 4 below.

[0083] Table 4. Changes in Bifidobacterium flora in the feces of the subjects

[0084]

[0085] Note 1. The number of viable bacteria is expressed as log CFU (Colony Forming Unit).

[0086] Note 2. Different letters indicate within-group differences (p<0.05).

[0087] In the placebo group, there was no significant change in the number of Bifidobacterium bacteria in stool samples before and after taking the granular supplement without Bifidobacterium lactis. In the experimental group, there was a significant change in the number of Bifidobacterium lactis in stool samples before and after taking the granular supplement containing Bifidobacterium lactis GKK2. If the number of Bifidobacterium bacteria measured during the blank period (day 0 of the experiment) is taken as 1, the number of Bifidobacterium bacteria in the experimental group increased approximately 6-fold before supplementation with Bifidobacterium lactis GKK2 (day 7 of the experiment). These results indicate that Bifidobacterium lactis GKK2 supplementation helps increase the number of live beneficial bacteria in the human gut, especially Bifidobacterium spp.

[0088] Example 3: Changes in the viable count of Clostridium perfringens

[0089] The changes in Clostridium perfringens flora in the feces of gas-producing subjects in each group during the trial blank period to the supplementary mid-term (days 0 to 14 of the trial) are shown in Table 5 below.

[0090] Table 5. Changes in Clostridium perfringens bacterial community in subjects' feces

[0091]

[0092]

[0093] Note 1. The number of viable bacteria is expressed as log CFU (Colony Forming Unit).

[0094] Note 2. The same letter indicates no significant difference (p>0.05).

[0095] In the experimental group, the number of Clostridium perfringens in fecal samples did not change significantly before and after supplementation with granular formulation containing Bifidobacterium lactis GKK2. This meets the definition of improved intestinal bacterial community function: a significant increase in Bifidobacterium sp. (Table 4) and a decrease or no significant change in Clostridium perfringens (Table 5). Therefore, it can be concluded that Bifidobacterium lactis GKK2 supplementation helps improve the human intestinal flora.

[0096] Example 4: Changes in the viable count of Escherichia coli

[0097] The changes in the coliform flora in the feces of each group of subjects during the trial blank period to the supplementary mid-term (day 0 to day 14 of the trial) are shown in Table 6 below.

[0098] Table 6. Changes in the coliform flora of subjects' feces

[0099]

[0100] Note 1. The number of viable bacteria is expressed as log CFU (Colony Forming Unit).

[0101] Note 2. The same letter indicates no significant difference (p>0.05).

[0102] In the experimental group, the number of coliform bacteria in fecal samples did not change significantly before and after taking the granular supplement containing Bifidobacterium lactis GKK2. This means that, apart from promoting the number of beneficial live bacteria in the human gut (Tables 3 and 4), it did not affect the number of non-beneficial (harmful) bacteria in the human gut, especially the number of coliform bacteria.

[0103] Example 5: Live count of Bifidobacterium spp. after GKK2 supplementation was stopped

[0104] Figure 1 The change in viable Bifidobacterium count after discontinuation of Bifidobacterium lactis GKK2 supplementation (day 21 of the experiment). Before supplementation (day 0 of the experiment), the experimental group had approximately 10 viable Bifidobacterium counts in the intestines. 8 CFU; After starting supplementation with Bifidobacterium lactis GKK2, the number of viable Bifidobacterium spp. in the intestine increased significantly. On days 7 and 14 of the experiment, the number of viable Bifidobacterium spp. in the intestine increased to 10 after one week and two weeks of supplementation with GKK2, respectively. 9 CFU; after stopping GKK2 supplementation for one week (day 21 of the experiment), the number of viable Bifidobacterium spp. in the intestine was still 10. 9 CFU indicates that strain GKK2 not only increases the number of viable Bifidobacterium spp., a beneficial bacterium in the gut, but also promotes the colonization rate of Bifidobacterium spp. in the gut, thus delaying and enhancing the positive functional promotion of beneficial bacteria in the human gut.

[0105] If we take the number of Bifidobacteria measured during the blank period (day 0 of the experiment) as 1, the number of Bifidobacteria in the experimental group increased approximately 6-fold during the initial period of Bifidobacterium lactis GKK2 supplementation (day 7 of the experiment); and the number of Bifidobacterium lactis remained more than 6-fold after the cessation of Bifidobacterium lactis GKK2 supplementation (day 21 of the experiment). This indicates that Bifidobacterium lactis GKK2 supplementation helps to increase the colonization rate of beneficial bacteria in the human gut, enabling the maintenance of a certain number of beneficial bacteria in the gut even after supplementation is stopped, thus prolonging the beneficial activity of beneficial bacteria in the human gut.

[0106] Example 6: Preparation of the Composition

[0107] If the Bifidobacterium lactis GKK2 of the present invention is applied to food use, the following composition 1 is provided as an illustrative example.

[0108] Composition 1: Take 20 wt% of lyophilized Bifidobacterium lactis GKK2 powder, mix thoroughly with benzyl alcohol (8 wt%) as a preservative and glycerol (7 wt%) as a diluent, and dissolve in pure water (65 wt%). Store at 4°C for later use. The aforementioned wt% refers to the proportion of each component to the total weight of the composition.

[0109] If the Bifidobacterium lactis GKK2 of the present invention is used in a liquid dosage form for pharmaceutical purposes, the following sample of composition 2 is provided as an illustrative example.

[0110] Composition 2: 20 wt% of lyophilized Bifidobacterium lactis GKK2 powder is thoroughly mixed with benzyl alcohol (8 wt%) as a preservative, glycerol (7 wt%) as a diluent, and sucrose (10 wt%) as a diluent, and dissolved in pure water (55 wt%). The mixture is then stored at 4°C for later use. The aforementioned wt% refers to the proportion of each component to the total weight of the composition. In summary, the above embodiments demonstrate that the composition containing Bifidobacterium lactis GKK2 of this invention helps improve the human intestinal flora, promotes the colonization rate of beneficial intestinal bacteria, and prolongs the activity of beneficial intestinal bacteria in the human intestine.

[0111] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the invention. Those skilled in the art, upon referring to the above teachings, can make appropriate modifications to the above embodiments while still achieving the effects claimed in this case. Therefore, the scope of protection of the present invention should be determined by the scope of protection.

Claims

1. A composition for improving intestinal flora, comprising Bifidobacterium lactis GKK2, accession number CGMCC No. 15205, deposited at the China General Microbiological Culture Collection Center.

2. The composition of claim 1, wherein improving the intestinal flora refers to increasing the total number of viable Bifidobacterium sp. or Lactobacillus sp.

3. A composition for prolonging the colonization rate of beneficial intestinal bacteria, comprising Bifidobacterium lactis GKK2, with accession number CGMCC No. 15205, deposited at the China General Microbiological Culture Collection Center.

4. The composition of claim 3, wherein the beneficial bacteria is Bifidobacterium sp. or Lactobacillus sp.

5. The composition of claim 1 or 3, comprising an additive selected from the group consisting of: excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, or combinations thereof.

6. The composition according to claim 1 or 3, wherein it is a pharmaceutical, feed, beverage, nutritional supplement, dairy product, food or health food.

7. The composition of claim 1 or 3, wherein the composition is in the form of powder, tablet, granulation, suppository, microcapsule, ampoule, liquid spray or stopper.

8. The composition of claim 1 or 3, wherein the intestine is the human intestine.

9. The composition of claim 1 or 3, comprising 10 to 30 wt% lyophilized Bifidobacterium lactis GKK2.

10. The composition of claim 9, wherein the lyophilized powder is prepared by the following method: (a) Inoculate the GKK2 strain into MRS agar and culture it in a solid state until colonies are formed; (b) Inoculate the colonies into MRS liquid medium and incubate at 25 to 40°C for 16 to 48 hours; (c) The bacterial culture obtained from the liquid culture was transferred to a fermentation medium for fermentation culture for 16 hours. The fermentation medium contained 1 to 10 wt% sugars, 0.1 to 5 wt% yeast extract, 0.1 to 5 wt% peptone, 0.01 to 2 wt% trace elements, 0.01 to 0.1 wt% cysteine ​​and 0.05 to 1 wt% Tween-80. (d) Centrifuge the fermentation broth at 1000 to 15000 rpm to obtain bacterial sludge; (e) The bacterial sludge is freeze-dried using a gradient freeze-drying method to obtain freeze-dried powder.

11. The composition of claim 10, wherein the gradient freeze-drying sequence is storage at 20 to 0°C for 1 to 4 hours, followed by storage at 0 to -20°C for 4 to 8 hours, and finally freezing at -196 to -30°C for more than 8 hours.

12. The use of Bifidobacterium lactis GKK2 in the manufacture of pharmaceutical compositions for improving intestinal flora or prolonging the colonization rate of beneficial intestinal bacteria, wherein the accession number of GKK2 is CGMCC No.15205 and is deposited at the China General Microbiological Culture Collection Center.

13. The use as described in claim 12, wherein the improvement of intestinal flora refers to increasing the total number of viable Bifidobacterium sp. or Lactobacillus sp.

14. The use as claimed in claim 12, wherein the beneficial bacteria is Bifidobacterium sp. or Lactobacillus sp.

15. The use as described in claim 12, wherein the intestine is the human intestine.

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