A composition and product to promote growth of akk bacteria

Abstract

The application discloses a composition and product for promoting growth of AKK bacteria. The composition comprises the following components: phosphatidylserine, bovine colostrum and lacto-N-tetraose; the concentration ratio of the phosphatidylserine, bovine colostrum and lacto-N-tetraose is 1:(0.08-1.67):(8-122), and the concentration of the PS is 0.015 g / L-0.03 g / L; when the concentration of the PS is 0.015 g / L, the concentration of the lacto-N-tetraose is not 0.728 g / L-1.092 g / L. The composition comprises bovine colostrum, PS and LNT, and the synergistic proliferation effect of the three specific components on AKK bacteria is better than that of a composition with single component. From the aspects of nutrition, convenience and wide health benefits, the composition can increase the potential probiotic AKK bacteria in the intestinal tract, improve intestinal health, and has important application value.

Description

Technical Field

[0001] This invention specifically relates to a composition and product for promoting the growth of AKK bacteria. Background Technology

[0002] Bovine colostrum is the milk secreted by cows in the first few days after calving. It is rich in immunoglobulins (IgG, IgA, IgM), growth factors (IGF-1, TGF-β, EGF), lactoferrin, lysozyme, cytokines, and various bioactive peptides. Bovine colostrum not only has multiple physiological functions, such as enhancing immunity, promoting growth and development, and improving gut health, but it has also been shown to regulate the gut microbiota through its bioactive components.

[0003] Phosphatidylserine (PS) is an important membrane phospholipid, mainly found in the inner layer of the cell membrane, and possesses various physiological activities, including regulating cell signaling, maintaining cell membrane fluidity, and promoting nerve cell function. Recent studies have found that PS can improve intestinal barrier function and promote metabolic health by regulating gut microbiota structure. However, there are currently no clear research reports on the specific effects of PS on *Amycosis ulcerans* (AKK).

[0004] Lacto-N-tetraose (LNT) is an important neutral tetrasaccharide structure among human milk oligosaccharides (HMOs), composed of galactose, N-acetylglucosamine, and glucose linked by β1-3 and β1-4 glycosidic bonds (Galβ1-3GlcNAcβ1-3Galβ1-4Glc). LNT is resistant to enzymatic degradation in the upper gastrointestinal tract and reaches the colon intact, where it is selectively utilized by the gut microbiota as a prebiotic. It has functions such as promoting the growth of Bifidobacteria and Lactobacilli, inhibiting pathogen adhesion, and regulating immune responses. However, current research has not addressed the synergistic proliferative effects of specific combinations of LNT with bovine colostrum and phosphatidylserine on AKK bacteria.

[0005] AKK bacteria are strictly anaerobic, Gram-negative bacteria belonging to the phylum Verrucomicrobia. They colonize the intestinal mucus layer and survive by using mucin as a carbon and nitrogen source. AKK bacteria are considered a "next-generation probiotic" after Bifidobacteria and Lactobacillus, and their abundance is closely related to improvements in host metabolic health, obesity, type 2 diabetes, and inflammatory bowel disease. AKK bacteria stimulate intestinal epithelial cells to secrete more mucus by degrading mucin, thus strengthening the intestinal barrier function; at the same time, their metabolites, short-chain fatty acids (such as acetic acid and propionic acid), can provide energy for intestinal epithelial cells and maintain intestinal homeostasis. Although existing technologies have confirmed a metabolic interaction between AKK bacteria and bovine colostrum, because AKK bacteria are not on the list of bacteria permitted for use in food, there is no way to increase their abundance in the human gut through exogenous direct supplementation. Instead, their growth can only be indirectly stimulated by exogenous supplementation of certain substances. Furthermore, AKK bacteria are strict anaerobic bacteria with specific nutritional requirements, making in vitro culture difficult, and there is currently a lack of safe and effective nutritional intervention methods to promote their colonization and proliferation in vivo.

[0006] In existing research, studies on the effects of bovine colostrum, PS, and LNT on gut microbiota have largely focused on the regulatory effects of single components or combinations of two components on general gut microbiota structure or traditional probiotics (such as Bifidobacterium and Lactobacillus). No studies have yet disclosed the synergistic proliferative effect of these three specific component combinations on AKK bacteria, nor have they explored the dose-response relationship and optimal ratio for promoting AKK bacteria growth. Therefore, developing a composition that can specifically promote AKK bacteria growth and is superior to the effects of single components is of great significance for regulating gut microbiota and improving metabolic health through nutritional intervention. Summary of the Invention

[0007] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies, such as the fact that AKK bacteria are strict anaerobic bacteria with specific nutritional requirements, making in vitro culture difficult, and the current lack of safe and effective strategies for promoting in vivo colonization. Furthermore, research on the effects of bovine colostrum, PS, and LNT on the intestinal flora mainly focuses on the regulatory effects of single components on Bifidobacteria, Lactobacillus, or the overall flora structure, without disclosing the synergistic proliferation effect of these three specific component combinations on AKK bacteria, nor exploring the dose-effect and optimal ratio of this combination in promoting AKK bacteria growth. This invention provides a composition and product for promoting AKK bacteria growth. In the composition of this invention, the synergistic proliferation effect of the ternary combination of bovine colostrum, PS, and LNT on AKK bacteria is superior to that of compositions with single components.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0009] The present invention provides a composition for promoting the growth of AKK bacteria, comprising the following components: phosphatidylserine (PS), bovine colostrum, and lactose-N-tetrasaccharide (LNT). The concentration ratio (g / L) of the phosphatidylserine, the bovine colostrum, and the lactose-N-tetrasaccharide is 1: (0.08-1.67): (8-122), and the concentration of the phosphatidylserine is 0.015 g / L-0.03 g / L; When the concentration of phosphatidylserine is 0.015 g / L, the concentration of lactose-N-tetrasaccharide is between 0.728 g / L and 1.092 g / L.

[0010] In this invention, the concentration ratio of phosphatidylserine, bovine colostrum and lactose-N-tetrasaccharide is preferably 1:(0.08-1.67):(16-122), more preferably 1:(0.08-1.67):(58-62).

[0011] In some preferred embodiments, the concentration ratio of the phosphatidylserine, the bovine colostrum, and the lactose-N-tetrasaccharide can be 1:1.67:16.67, 1:1.67:121.33, 1:3.33:16.67, 1:3.33:121.33, 1:0.83:8.33, 1:0.83:60.67, 1:1.67:60.67, 1:1.67:16.67, or 1:3.33:16.67, preferably 1:1.67:16.67, 1:1.67:121.33, 1:0.83:60.67, 1:1.67:60.67, or 1:1.67:16.67, and most preferably 1:0.83:60.67.

[0012] In this invention, the phosphatidylserine can be the conventional phosphatidylserine used in the dairy industry.

[0013] In some preferred embodiments, the phosphatidylserine is available from Shandong Bairui Biopharmaceutical Co., Ltd.

[0014] In some preferred embodiments, the concentration of phosphatidylserine may be 0.015 g / L, 0.02 g / L, 0.025 g / L, or 0.03 g / L.

[0015] In this invention, the bovine colostrum can be conventional bovine colostrum used in the dairy industry.

[0016] In some preferred embodiments, the bovine colostrum may be purchased from Heilongjiang Feihe Dairy Co., Ltd.

[0017] In this invention, the concentration of bovine colostrum can be 0.02 g / L - 0.1 g / L, preferably 0.025 g / L - 0.1 g / L, and more preferably 0.025 g / L - 0.05 g / L.

[0018] In some preferred embodiments, the concentration of the bovine colostrum may be 0.025 g / L, 0.05 g / L, or 0.1 g / L.

[0019] In this invention, the lactose-N-tetraose (LNT) is a conventional product in the dairy industry and is a human milk oligosaccharide. An important neutral tetrasaccharide structure in HMOs, it is composed of galactose, N-acetylglucosamine, and glucose linked by β1-3 and β1-4 glycosidic bonds (Galβ1-3GlcNAcβ1-3Galβ1-4Glc). Its chemical structure is Galβ1-4GlcNAcβ1-3Galβ1-4Glc (2-(β-D-galactosyl)-(1→4)-N-acetyl-β-D-glucosyl-(1→3)-2-(β-D-galactosyl)-(1→4)-D-glucose). It contains repeating disaccharide units (Galβ1-4GlcNAc) that are highly similar to mucin sugar chains. It can be recognized and degraded by specific glycosidic hydrolases of Akkermansia myxophilus, thereby providing a continuous source of energy and materials for the growth of the strain.

[0020] In some preferred embodiments, the lactose-N-tetrasaccharide (LNT) is available from Huang Tongxi Biotechnology Co., Ltd.

[0021] In this invention, the concentration of lactose-N-tetrasaccharide can be 0.25 g / L - 1.82 g / L, preferably 0.25 g / L - 0.546 g / L or 1.82 g / L.

[0022] In some preferred embodiments, the concentration of the lactose-N-tetrasaccharide may be 0.25 g / L, 0.364 g / L, 0.546 g / L, 0.728 g / L, 1.092 g / L, or 1.82 g / L.

[0023] In this invention, the preferred concentrations of phosphatidylserine, bovine colostrum, and lactose-N-tetrasaccharide in the composition for promoting AKK growth are (0.015 g / L - 0.06 g / L): (0.02 g / L - 0.1 g / L): (0.25 g / L - 1.82 g / L).

[0024] In some preferred embodiments, the concentration ratio of phosphatidylserine, bovine colostrum, and lactose-N-tetrasaccharide in the composition promoting AKK growth can be 0.015 g / L : 0.025 g / L : 0.25 g / L, 0.015 g / L : 0.025 g / L : 1.82 g / L, 0.015 g / L : 0.05 g / L : 0.25 g / L, 0.015 g / L : 0.05 g / L : 1.82 g / L, 0.03 g / L : 0.025 g / L : 0.25 g / L, 0.03 g / L : 0.025 g / L : 1.82 g / L, 0.03 g / L : 0.0 ... The preferred concentrations are 0.015 g / L: 0.025 g / L: 0.25 g / L, 0.015 g / L: 0.025 g / L: 1.82 g / L, 0.03 g / L: 0.025 g / L: 1.82 g / L, 0.03 g / L: 0.025 g / L: 1.82 g / L, 0.03 g / L: 0.05 g / L: 0.25 g / L, 0.03 g / L: 0.05 g / L: 1.82 g / L, or 0.03 g / L: 0.05 g / L: 0.5 g / L, with the most preferred concentration being 0.03 g / L: 0.05 g / L: 1.82 g / L.

[0025] In some preferred embodiments, when the concentration of phosphatidylserine is 0.015 g / L, the concentration of lactose-N-tetrasaccharide is 0.25-0.546 g / L or 1.82 g / L.

[0026] In some preferred embodiments, when the concentration of phosphatidylserine is 0.03 g / L, the concentration of lactose-N-tetrasaccharide is 0.25-1.82 g / L.

[0027] The present invention also provides the application of the composition for promoting the growth of AKK bacteria as described above in the culture of AKK bacteria.

[0028] In this invention, the AKK bacterium can be Akkmania, type: Akk11.

[0029] In some preferred embodiments, the AKK bacteria may be Akkmania myxophilus, model: Akk11, isolated from the intestines of healthy infants in Hongyuan Pastoral Area, Sichuan Province, and purchased from WeCon Probiotics (Suzhou) Co., Ltd.

[0030] In this invention, the inoculation amount of AKK bacteria is preferably 0.8-1.2%, for example 1%.

[0031] In this invention, the AKK bacteria are generally activated and passaged before inoculation.

[0032] In some preferred embodiments, the strain activation and subculturing are carried out by the following steps: 0.2 g of AKK (strain number: AKK11) powder stored at -28 ℃ is inoculated into 20 mL of BHI medium containing 0.4% (w / v) mucin, 0.05% (w / v) L-cysteine ​​hydrochloride and 0.0001% (w / v) resazurin, gently shaken, and placed in a 37 ℃ incubator. Activation is completed after 24 h of anaerobic culture.

[0033] In this invention, the culture medium used in the AKK culture can be conventional in the art, such as MRS medium.

[0034] In some preferred embodiments, the MRS culture medium is available from Beijing Aoboxing Biotechnology Co., Ltd.

[0035] In this invention, during the culture of AKK bacteria, the glucose concentration in the culture medium can be 0.01 g / 100 mL to 0.1 g / 100 mL, for example, 0.01 g / 100 mL, 0.025 g / 100 mL, 0.05 g / 100 mL, 0.075 g / 100 mL or 0.1 g / 100 mL.

[0036] The present invention also provides a product comprising the composition for promoting the growth of AKK bacteria as described above.

[0037] In this invention, the product may be a culture medium, food, or health product, etc.

[0038] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0039] The reagents and raw materials used in this invention are all commercially available.

[0040] The positive and progressive effects of this invention are as follows: AKK bacteria are strict anaerobic bacteria with specific nutritional requirements, making in vitro culture difficult, and currently lacking safe and effective strategies for promoting in vivo colonization. The composition of this invention contains bovine colostrum, PS, and LNT; the synergistic proliferation effect of these three specific components on AKK bacteria is superior to that of compositions containing a single component. Therefore, from a nutritional perspective, considering ease of access and broad benefits to overall health, the composition of this invention has significant application value.

[0041] In some preferred embodiments, the most preferred composition ratio is PS 1 / 2-1 times the baseline dose, bovine colostrum 1 / 4-1 / 2 times the baseline dose, and LNT 1 / 2.9-5 / 2 times the baseline dose, with concentrations of 0.015-0.03 g / L, 0.025-0.05 g / L, and 0.25-1.82 g / L, respectively. This composition effectively promotes the proliferation of AKK bacteria, and the three components have a synergistic effect. Therefore, it can increase the number of potentially beneficial AKK bacteria in the gut, improving gut health. Attached Figure Description

[0042] Figure 1 The growth curves of AKK bacteria (1% inoculum) over 36 hours at different glucose concentrations are shown.

[0043] Figure 2 The growth curves of AKK bacteria (1% inoculum) at different PS concentrations and in the control group over 36 hours are shown.

[0044] Figure 3 The growth curves of AKK bacteria (1% inoculum) in different doses of bovine colostrum and the control group over 36 hours are shown.

[0045] Figure 4 The growth curves of AKK bacteria (1% inoculum) at different doses of LNT and in the control group over 36 hours are shown.

[0046] Figure 5 The growth curves of AKK bacteria (1% inoculum) in different dosage compositions and the control group over 36 hours are shown. Detailed Implementation

[0047] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0048] In the following examples, the relevant raw material sources, equipment, strain activation and subculturing, screening of MRS culture media with different glucose contents, and other operations and conditions are as follows: 1. Raw materials: Bovine colostrum was purchased from Heilongjiang Feihe Dairy Co., Ltd.; phosphatidylserine was purchased from Shandong Baianrui Biopharmaceutical Co., Ltd.; lactose-N-tetrasaccharide (LNT) was purchased from Huang Tongxi Biotechnology Co., Ltd.

[0049] 2. Strains: Akk11, strain: Akk11, isolated from the intestines of healthy infants in Hongyuan Pastoral Area, Sichuan Province, purchased from WeCon Probiotics (Suzhou) Co., Ltd.

[0050] 3. Materials and equipment: MRS culture medium was purchased from Beijing Aoboxing Biotechnology Co., Ltd.; anaerobic culture bags were purchased from Qingdao Haibo Biotechnology Co., Ltd.; ELISA reader, model SpectraMax reg iD3, was purchased from Meigu Instruments (Shanghai) Co., Ltd.; fully automated growth curve analyzer, model BK400, was purchased from Shandong Boke Biotechnology Co., Ltd.

[0051] 4. Activation and subculturing of strain: Take 0.2 g of AKK (strain number: AKK11) powder stored at -28 ℃ and inoculate it into 20 mL of BHI medium containing 0.4% (w / v) mucin, 0.05% (w / v) L-cysteine ​​hydrochloride and 0.0001% (w / v) resazurin. Shake gently and place in an incubator at 37 ℃. Activation is completed after 24 h of anaerobic culture.

[0052] 5. Growth of AKK bacteria in MRS media with different glucose contents

[0053] The above-mentioned AKK bacteria were inoculated at a 1% inoculum into MRS medium with different glucose concentrations (0.01 g / 100 mL, 0.025 g / 100 mL, 0.05 g / 100 mL, 0.075 g / 100 mL, and 0.1 g / 100 mL, respectively). Under anaerobic conditions of 85% N2, 5% H2, and 10% CO2, the culture was incubated at 37 ℃ for 20 h. The cultured bacterial suspension was aspirated, centrifuged at 6000 r / min for 5 min (4℃), the supernatant was discarded, and the bacterial cells were resuspended in sterile PBS. The centrifugation and washing were repeated twice. The bacterial suspension resuspended in PBS was filtered through a 0.22 μm sterile filter membrane. The concentration of the filtered bacterial suspension was monitored by measuring the optical density value (OD600) at a wavelength of 600 nm, and the growth of AKK bacteria was observed.

[0054] Figure 1 This is a growth curve of AKK bacteria (1% inoculum) over 36 hours at different glucose concentrations. Figure 1 As shown, AKK bacteria grow slowly in MRS medium with 0.05 g / 100 mL glucose, and can barely maintain growth.

[0055] Therefore, the glucose concentration in the reduced-sugar MRS medium was set at 0.05 g / 100 mL for subsequent experiments on the promotion of AKK bacteria growth by PS, bovine colostrum, and LNT.

[0056] Comparative Example 1: Growth of AKK bacteria in MRS medium with different doses of PS

[0057] Using a liquid PS concentration of 0.03 g / L as the baseline dose, dosages were prepared at 1 / 3, 1 / 2, 2 / 3, 1, 5 / 3, and 50 times (where 50 times is the maximum permitted addition amount according to regulations), with concentrations of 0.01 g / L, 0.015 g / L, 0.02 g / L, 0.03 g / L, 0.050 g / L, and 1.5 g / L, respectively. (Note: The baseline dose of 0.03 g / L is the baseline dose in the reduced-sugar MRS medium; 1 / 3, 1 / 2, 2 / 3, 1, 5 / 3, and 50 times are multiples thereof; the concentration of the raw material is the concentration relative to the medium concentration. The specific preparation method calculates the PS dosage based on 0.03 g / L (e.g., 0.0003 g PS is needed for 10 mL of medium).)

[0058] The activated second-generation AKK bacterial suspension was inoculated at a volume fraction of 1% (v / v) into reduced-sugar MRS medium supplemented with the above six different doses of PS (① "reduced-sugar MRS medium" here refers to commercially available MRS medium with a glucose concentration of 0.05 g / 100 mL; ② in the "reduced-sugar MRS medium", the PS concentrations are 0.01 g / L, 0.015 g / L, 0.02 g / L, 0.03 g / L, 0.050 g / L, and 1.5 g / L, respectively). Reduced-sugar MRS medium without added PS was used as a blank control. The experiment was incubated at 37 ℃ and statically. The concentration of the filtered bacterial suspension was monitored by measuring the optical density (OD600) at a wavelength of 600 nm at 2 h intervals, and the experiment was run for 36 h. The growth curves of AKK bacteria under different doses of PS are shown below. Figure 2 As shown.

[0059] Figure 2 Growth curves of AKK bacteria (1% inoculum) over 36 hours in different PS concentrations and the control group. Figure 2 It was found that, compared with the control group, *AKK* bacteria exhibited larger OD values ​​and higher total bacterial counts during the stationary phase when culture media containing 1 / 2 times (0.015 g / L) and 1 times (0.03 g / L) of the baseline PS concentration. The OD value was particularly high when the PS concentration was 1 times the baseline (0.03 g / L). This result indicates that a certain dose of PS promotes the growth of *AKK* bacteria.

[0060] Comparative Example 2: Growth of AKK bacteria in MRS medium with different doses of bovine colostrum

[0061] Using a bovine colostrum liquid concentration of 0.1 g / L as the baseline dose, doses of 1 / 4, 1 / 2, 1, 3 / 2, 2, and 5 / 2 were set based on the baseline dose, with concentrations of 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, and 0.25 g / L bovine colostrum, respectively. The activated second-generation AKK bacterial suspension was inoculated at a volume fraction of 1% (v / v) into reduced-sugar MRS medium containing the above six different doses of bovine colostrum (① "Reduced-sugar MRS medium" here refers to commercially available MRS medium with a glucose concentration of 0.05 g / 100 mL; ② In the "reduced-sugar MRS medium", the concentrations of bovine colostrum were 0.025 g / L, 0.05 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, and 0.25 g / L, respectively). Reduced-sugar MRS medium without bovine colostrum was used as a blank control. The experimental incubation temperature was 37 ℃, and the culture was static. The concentration of the filtered bacterial suspension was monitored by measuring the optical density value (OD600) at a wavelength of 600 nm. The measurement interval was 2 h, and the culture was run for 36 h.

[0062] The growth curves of AKK bacteria (1% inoculum) in different doses of bovine colostrum and the control group over 36 hours are shown below. Figure 3 As shown. By Figure 3 The results showed that, compared with the control group, *AKK* bacteria exhibited higher OD values ​​and total bacterial counts during the stationary phase when culture media containing 1 / 4 (0.025 g / L) and 1 / 2 (0.05 g / L) of the baseline bovine colostrum dose. This indicates that bovine colostrum promotes the growth of *AKK* bacteria.

[0063] Comparative Example 3: Growth of AKK bacteria in MRS medium with different doses of LNT

[0064] Using a liquid concentration of 0.728 g / L for LNT as the baseline dose, doses of 1 / 2.9, 1 / 2, 3 / 4, 1, 3 / 2, and 5 / 2 times the baseline dose were set, with concentrations of 0.25 g / L, 0.364 g / L, 0.546 g / L, 0.728 g / L, 1.092 g / L, and 1.82 g / L LNT (the LNT addition range in the draft regulations is 0.25~1.82 g / L). The activated second-generation AKK bacterial suspension was inoculated at a volume fraction of 1% (v / v) into reduced-sugar MRS medium containing the above six different doses of LNT (① "Reduced-sugar MRS medium" here refers to commercially available MRS medium with a glucose concentration of 0.05 g / 100 mL; ② In the "reduced-sugar MRS medium", the LNT concentrations were 0.25 g / L, 0.364 g / L, 0.546 g / L, 0.728 g / L, 1.092 g / L, and 1.82 g / L LNT, respectively). Reduced-sugar MRS medium without added LNT was used as a blank control. The experimental incubation temperature was 37 ℃, and the culture was static. The concentration of the filtered bacterial suspension was monitored by measuring the optical density value (OD600) at a wavelength of 600 nm. The measurement interval was 2 h, and the culture was run for 36 h.

[0065] The growth curves of AKK bacteria (1% inoculum) at different doses of LNT and the control group over 36 hours are shown below. Figure 4 As shown. By Figure 4 The results showed that, compared with the control group, *AKK* bacteria exhibited larger OD values ​​and higher total bacterial counts during the stationary phase in media containing 1 / 2.9 (0.25 g / L), 1 / 2 (0.364 g / L), 3 / 4 (0.546 g / L), and 5 / 2 (1.82 g / L) doses of the baseline LNT. These results indicate that LNT promotes the growth of *AKK* bacteria.

[0066] Example 1: Growth of AKK bacteria in MRS medium with different dosages of the composition

[0067] Based on Comparative Example 1, the optimal dose of PS was selected as 1 / 2 to 1 times the baseline dose (concentration 0.015-0.03 g / L). Based on Comparative Example 2, the optimal dose of bovine colostrum for promoting AKK bacterial proliferation was selected as 1 / 4 to 1 / 2 times the baseline dose (concentration 0.025-0.05 g / L). Based on Comparative Example 3, the optimal dose of LNT was selected as 1 / 2.9 to 5 / 2 times the baseline dose (concentration 0.25-1.82 g / L). As shown in the table below, the endpoint values ​​of PS, bovine colostrum, and LNT raw materials were combined into a total of 8 experimental groups (1 / 2 : 1 / 4 : 1 / 2.9, 1 / 2 : 1 / 4 : 5 / 2, 1 / 2 : 1 / 2 : 1 / 2.9, 1 / 2 : 1 / 2 : 5 / 2, 1 : 1 / 4 : 1 / 2.9, 1 : 1 / 4 : 5 / 2, 1 : 1 / 2 : 1 / 2.9, 1 : 1 / 2 : 5 / 2), i.e., concentration ratios of 0.015 : 0.025 : 0.25, 0.015 : 0.025 : 1.82, 0.015 : 0.05 : 0.25, 0.015 : 0.05 : 1.82, 0.03 : 0.025 : 0.25, 0.03 : 0.025 : 1.82, 0.03 : 0.05 : 0.25, 0.03 : 0.05 : 1.82.

[0068] Table 1

[0069] These combined dosages were increased by 2 times (or cancelled if exceeding the maximum permitted addition amount), and decreased by 2 times (or cancelled if below the minimum permitted addition amount), resulting in 5 experimental groups: 1:1 / 2:2 / 2.9, 1:1:2 / 2.9, 2:1 / 2:2 / 2.9, 2:1:2 / 2.9, and 1 / 2:1 / 4:5 / 4, corresponding to concentration ratios of 0.03:0.05:0.5, 0.03:0.1:0.5, 0.06:0.05:0.5, 0.06:0.1:0.5, and 0.015:0.025:0.91, representing PS, bovine colostrum, and LNT at baseline dosage multiples. A total of 13 experimental groups were conducted, including the aforementioned 8 groups and the 5 groups.

[0070] The activated second-generation AKK bacterial suspension was inoculated at a volume fraction of 1% (v / v) into reduced-sugar MRS medium containing different doses of PS, bovine colostrum, and LNT combination. Reduced-sugar MRS medium without the combination was used as a blank control. The experimental incubation temperature was 37 ℃, and the culture was static. The concentration of the filtered bacterial suspension was monitored by measuring the optical density value (OD600) at a wavelength of 600 nm. The measurement interval was 2 h, and the operation lasted for 36 h.

[0071] The growth curves of AKK bacteria (1% inoculum) at different dosages of the composition and the control group over 36 hours are shown below. Figure 5 As shown. By Figure 5 It can be seen that during the stable period, compositions of 0.015:0.025:0.25, 0.015:0.05:1.82, 0.03:0.025:1.82, 0.03:0.05:1.82, and 0.03:0.05:0.5 all promoted the growth of AKK and increased OD. 600 Values. In particular, the 0.03:0.05:1.82 composition promoted AKK growth most significantly, approximately equal to the OD value of the control group. 600 1.56 times.

[0072] Figure 5 The corresponding data is shown in Table 2-3 below.

[0073] Table 2

[0074] Table 3

[0075] Among them, for the 0.06:0.1:0.5 group (high PS, low LNT type): PS 0.06 g / L is twice the baseline dose, exceeding the tolerance threshold of AKK bacteria cell membrane, resulting in toxic inhibition; LNT 0.5 g / L is only 0.69 times the baseline dose, indicating a severe carbon source deficiency; this group exhibits the characteristic of "initial inhibition followed by slow recovery" (severe inhibition in the first 12 hours, followed by slow recovery), which is consistent with the biological law of high concentration PS toxicity + low energy repair.

[0076] For the 0.015:0.025:0.91 group (low PS, low bovine colostrum type): the concentrations of both PS and bovine colostrum were too low (1 / 2 and 1 / 4 of the baseline dose, respectively), failing to reach the concentration threshold for synergistic stimulation; LNT 0.91 g / L was in the "low-effect zone" near the baseline dose of 0.728 g / L and 1.092 g / L (from...). Figure 4 (As shown in the single-factor experiment). This group consistently had lower concentrations than the control group and showed no recovery, indicating that ternary synergy requires each component to reach its minimum effective concentration.

[0077] In this invention, the ternary synergistic effect of PS, bovine colostrum, and LNT exists within a strict concentration window: (1) PS upper limit effect: When the PS concentration exceeds 0.03 g / L (1 times the baseline dose), it produces cell membrane toxicity to AKK bacteria, and the synergistic effect turns into inhibition (e.g., 0.06:0.1:0.5 group).

[0078] (2) LNT trough effect: When the PS concentration is low (0.015 g / L), the LNT concentration is in the "effect trough zone" between 0.728 and 1.092. At this time, the ternary synergy cannot be established and the effect is not as good as the control group (such as the 0.015:0.025:0.91 group).

[0079] Example 2: Growth of AKK bacteria in MRS medium containing appropriate doses of PS, bovine colostrum, LNT, and the combination thereof

[0080] According to Comparative Example 1, the optimal dose of PS to promote the proliferation of AKK bacteria was selected as 1 / 2 times the baseline dose (0.015 g / L). According to Comparative Example 2, the optimal dose of bovine colostrum was selected as 1 / 2 times the baseline dose (0.05 g / L). According to Comparative Example 3, the optimal dose of LNT was selected as 5 / 2 times the baseline dose (1.82 g / L). According to Example 1, the suitable dose of the composition was a combination of the three in the following ratios: 0.015:0.025:0.25, 0.015:0.05:1.82, 0.03:0.025:1.82, 0.03:0.05:1.82, and 0.03:0.05:0.5, respectively.

[0081] The activated second-generation AKK bacterial suspension was inoculated at a 1% (v / v) volume fraction into reduced-sugar MRS medium supplemented with the three optimal doses of PS, bovine colostrum, LNT, and five suitable combinations, respectively. Reduced-sugar MRS medium without PS, bovine colostrum, LNT, or any combination served as a blank control. The experiment was incubated at 37 ℃ under static conditions. The concentration of the filtered bacterial suspension was monitored by measuring the optical density (OD600) at 600 nm at 2-hour intervals, for a total run of 36 hours. The ΔOD of AKK bacteria under suitable doses of PS, bovine colostrum, LNT, and combinations was determined. 600 (Excluding blank controls) The changes over time are shown in Table 4-6.

[0082] Table 4. ΔOD of AKK bacteria under different ratios of PS, bovine colostrum, LNT combinations and 1 / 2 PS (0.015 g / L) culture conditions. 600 (Excluding blank control) Changes over time

[0083] Note: The values ​​represent the ΔOD600nm of the composition compared to that under 4 times bovine colostrum culture conditions. P <0.05, P <0.01 and P <0.001.

[0084] Table 5. ΔOD of AKK bacteria under different ratios of PS, bovine colostrum, LNT combinations and 1 / 2 times bovine colostrum (0.05 g / L) culture conditions. 600 (Excluding blank control) Changes over time

[0085] Note: The values ​​represent the ΔOD600nm of the composition compared to that under 1x PS culture conditions. P <0.05, P <0.01 and P <0.001.

[0086] Table 6. ΔOD of AKK bacteria under different ratios of PS, bovine colostrum, LNT combinations and 5 / 2 times LNT (1.82 g / L) culture conditions. 600 (Excluding blank control) Changes over time

[0087] Note: The values ​​represent the ΔOD600nm of the composition compared to that under 1 / 3.5 times the LNT culture conditions. P < 0.05 P <0.01 and P <0.001.

[0088] As shown in Table 4-6, PS (1 / 2 times the baseline dose), bovine colostrum (1 / 2 times the baseline dose), and LNT (5 / 2 times the baseline dose) all promoted the growth of AKK bacteria (ΔOD600nm was not zero). The five ratio compositions selected in this example (0.015:0.025:0.25, 0.015:0.05:1.82, 0.03:0.025:1.82, 0.03:0.05:1.82, 0.03:0.05:0.5) also promoted the growth of AKK bacteria (ΔOD600nm was not zero). The ΔOD600nm values ​​after 6 hours under the five ratio composition culture conditions were significantly higher than those under the single PS (1 / 2 times the baseline dose), bovine colostrum (1 / 2 times the baseline dose), and LNT (5 / 2 times the baseline dose) culture conditions. P <0.05、P <0.01 or P <0.001), and this significant difference lasted up to 36 hours. Meanwhile, at the corresponding time intervals of 6–36 hours, the ΔOD600nm of all these compositions was higher than the sum of the ΔOD600nm of individual PS (1 / 2 times the baseline dose), bovine colostrum (1 / 2 times the baseline dose), and LNT (5 / 2 times the baseline dose).

[0089] The above data shows that the five compositions selected in this embodiment can promote the growth of AKK bacteria to varying degrees, and PS, bovine colostrum and LNT have a synergistic effect on the proliferation of AKK bacteria.

[0090] In summary, further evidence demonstrates that the optimal composition ratio is 1 / 2-1 times the baseline dose of PS, 1 / 4-1 / 2 times the baseline dose of bovine colostrum, and 1 / 2.9-5 / 2 times the baseline dose of LNT, with concentrations of 0.015-0.03 g / L, 0.025-0.05 g / L, and 0.25-1.82 g / L, respectively. This combination effectively promotes the proliferation of AKK bacteria, exhibiting a synergistic effect. Therefore, it can increase the number of potentially beneficial AKK bacteria in the gut, improving gut health.

Claims

1. A composition for promoting the growth of AKK bacteria, characterized in that, It includes the following components: phosphatidylserine, bovine colostrum, and lactose-N-tetrasaccharide; The concentration ratio of the phosphatidylserine, the bovine colostrum, and the lactose-N-tetrasaccharide is 1: (0.08-1.67): (8-122), and the concentration of the phosphatidylserine is 0.015 g / L-0.03 g / L; When the concentration of phosphatidylserine is 0.015 g / L, the concentration of lactose-N-tetrasaccharide is between 0.728 g / L and 1.092 g / L.

2. The composition for promoting the growth of AKK bacteria as described in claim 1, characterized in that, The concentration ratio of the phosphatidylserine, the bovine colostrum, and the lactose-N-tetrasaccharide is 1: (0.08-1.67): (16-122).

3. The composition for promoting the growth of AKK bacteria as described in claim 1 or 2, characterized in that, The concentration ratio of the phosphatidylserine, the bovine colostrum, and the lactose-N-tetrasaccharide is 1: (0.08-1.67): (58-62).

4. The composition for promoting the growth of AKK bacteria as described in claim 1 or 2, characterized in that, (1) The concentration of the bovine colostrum is 0.02 g / L - 0.1 g / L; And / or, (2) the concentration of the lactose-N-tetrasaccharide is 0.25 g / L - 1.82 g / L.

5. The composition for promoting the growth of *AKK* bacteria as described in claim 1 or 2, characterized in that, (1) The concentration of the bovine colostrum is 0.025 g / L - 0.1 g / L, preferably 0.025 g / L - 0.05 g / L; And / or, (2) the concentration of the lactose-N-tetrasaccharide is 0.25 g / L - 0.546 g / L or 1.82 g / L.

6. The composition for promoting the growth of *AKK* bacteria as described in claim 1 or 2, characterized in that, In the composition for promoting the growth of AKK bacteria, the concentrations of phosphatidylserine, bovine colostrum, and lactose-N-tetrasaccharide are (0.015 g / L - 0.03 g / L): (0.025 g / L - 0.05 g / L): (0.25 g / L - 1.82 g / L).

7. The composition for promoting the growth of AKK bacteria as described in claim 1 or 2, characterized in that, In the composition for promoting the growth of AKK bacteria, the concentrations of phosphatidylserine, bovine colostrum, and lactose-N-tetrasaccharide are 0.015 g / L:0.025 g / L:0.25 g / L, 0.015 g / L:0.05 g / L:1.82 g / L, 0.03 g / L:0.025 g / L:1.82 g / L, 0.03 g / L:0.05 g / L:0.25 g / L, 0.03 g / L:0.05 g / L:1.82 g / L, or 0.03 g / L:0.05 g / L:0.5 g / L. Alternatively, when the concentration of phosphatidylserine is 0.015 g / L, the concentration of lactose-N-tetrasaccharide is 0.25-0.546 g / L or 1.82 g / L; Alternatively, when the concentration of phosphatidylserine is 0.03 g / L, the concentration of lactose-N-tetrasaccharide is 0.25-1.82 g / L.

8. The composition for promoting the growth of AKK bacteria as described in claim 1 or 2, characterized in that, When the concentration of phosphatidylserine is 0.015 g / L, the concentration of bovine colostrum is 0.025 g / L. Alternatively, when the concentration of phosphatidylserine is 0.03 g / L, the concentration of bovine colostrum is 0.025-0.05 g / L.

9. The composition for promoting the growth of AKK bacteria as described in claim 1 or 2, characterized in that, The AKK bacteria is Akkermansia myxophilus; And / or, the inoculation amount of the AKK bacteria is 0.8-1.2%.

10. A product characterized in that, It includes the composition for promoting the growth of AKK bacteria as described in any one of claims 1-9.

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