Screening and application of a synergistic synbiotic for enhancing immunity and promoting growth and development

Abstract

The present invention relates to the screening and application of a synergistic synbiotic for enhancing immunity and promoting growth and development. The present invention discloses a composition comprising Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and prebiotic 2'-FL, and the composition can be used for enhancing immunity and promoting growth and development.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotic preparations, and particularly relates to a synbiotic composition, which comprises probiotic Bifidobacterium lactis Bb-12, probiotic Bifidobacterium lactis HN019 and prebiotic 2'-FL, and the composition is used for enhancing immunity and / or promoting growth and development. Background Art

[0002] Human Milk Oligosaccharides (HMO) are the third most abundant substance in breast milk after lactose and fat. The structure of each human milk oligosaccharide has a lactose at the reducing end, and most of them have a polylactosamine as the main chain structure and contain fucose, sialic acid or both at the chain end. Human milk oligosaccharides have three main functions: (1) inhibiting the attachment and infection of specific pathogens; (2) acting as a prebiotic to promote the growth of bacteria in the intestinal symbiotic system; (3) directly reducing the inflammatory response of the mucosa under toxic stimulation.

[0003] Prebiotics are a class of carbohydrates that cannot be digested by the human body and can interact with the intestinal flora in the host intestine to produce substances or effects beneficial to the host. Currently, the prebiotics studied more include: galactooligosaccharides, fructooligosaccharides, inulin, isomaltooligosaccharides, polydextrose, xylooligosaccharides, etc.

[0004] According to the definition of synbiotics by the World Gastroenterology Organization (WGO), synbiotics are a mixture containing live microorganisms and substrates that can be selectively utilized by host microorganisms and have health benefits for the host. There are two types of synbiotics, one is complementary type (a mixture of probiotics and prebiotics); the other is synergistic type (a mixture containing specific substrates and active microorganisms that can utilize these specific substrates and have health benefits for the host). Summary of the Invention

[0005] An object of the present invention is to provide a synbiotic composition for enhancing immunity and / or promoting growth and development.

[0006] The present invention provides a synbiotic composition, which comprises Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and 2'-FL.

[0007] The synbiotic composition of the present invention is characterized in that it comprises Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and 2'-FL in a mass ratio of 0.1~1:0.17~1.66:2735~4103.

[0008] According to a specific embodiment of the present invention, the synbiotic composition of the present invention further comprises excipients, diluents and / or carriers.

[0009] According to a specific embodiment of the present invention, in the synbiotic composition of the present invention, the amount of Bifidobacterium lactis Bb-12 is 2×10 3 ~ 2×10 5 CFU / mL; the amount of Bifidobacterium lactis HN019 is 2×10 4 ~ 2×10 5 CFU / mL.

[0010] According to a specific embodiment of the present invention, the synbiotic composition of the present invention is used to enhance immunity and / or promote growth and development.

[0011] On the other hand, the present invention also provides the application of the synbiotic composition in the preparation of a product having the function of enhancing immunity and / or promoting growth and development.

[0012] According to a specific embodiment of the present invention, the present invention provides the use of the synbiotic composition in the preparation of a product for enhancing the immunity of a subject and / or promoting the growth and development of the subject.

[0013] According to a specific embodiment of the present invention, enhancing the immunity of a subject includes: enhancing the humoral immunity and / or cellular immunity of the subject, increasing the number of immune cells of the subject, or enhancing the activity of immune cells of the subject; promoting the growth and development of a subject includes: promoting an increase in the body length or height of the subject, promoting an increase in the body weight of the subject, or promoting the fat absorption ability of the subject.

[0014] In some embodiments, the subject is a zebrafish or a mammal.

[0015] 2'-Fucosyllactose (2'-FL) is one of the main neutral HMO molecules in breast milk and is a unique substance rich in human milk. 2'-FL can be used as a nutritional supplement for adults to relieve intestinal irritable syndrome or inflammatory bowel disease, and can also be used as a prebiotic to maintain the balance of intestinal flora.

[0016] Short-chain fatty acids (SCFAs) are the main metabolites produced by the fermentation of sugars or amino acids by specific gut microbiota. They are involved in the body's metabolism and play important roles in different organs and tissues. SCFAs have certain physiological functions in many organs and tissues of the body. SCFAs can promote and maintain gut health, brain function, and bone function, relieve obesity and diabetes, and also participate in immune regulation, exerting anti-tumor and antibacterial effects. In terms of gut health, SCFAs mainly enhance the intestinal barrier function by activating adenosine monophosphate (AMP), activating AMP-activated protein kinase (AMPK), and upregulating the expression of tight junction proteins; SCFAs can also enhance the intestinal barrier function by changing the consumption of O2 by epithelial cells and stabilizing the expression of the barrier protection-related transcription factor hypoxia-inducible factor (HIF). In terms of bone function, as a regulator of osteoclast metabolism and bone mass, SCFAs can bind to specific receptors to promote and maintain bone function. In terms of immune regulation, the receptor FFAR2 of SCFAs in the immune system can be expressed on eosinophils, basophils, neutrophils, monocytes, and mucosal mast cells, indicating that SCFAs play a wide role in immune responses. They can play an immune regulatory role by activating GPR, inhibiting HDAC activity, and regulating Treg cells, etc., to downregulate the expression levels of pro-inflammatory factors secreted by related immune cells and inhibit the occurrence of inflammation, allergy, etc. in the body.

[0017] In a specific embodiment of the present invention, the prebiotic 2'-fucosyllactose (2'-FL) is from SYNAURA ® , Hongmo Biotechnology (Shanghai) Co., Ltd.; Bifidobacterium animalis subsp. lactis Bb-12 is Bifidobacterium animalis subsp. lactis with the deposit number DSM15954; Bifidobacterium animalis subsp. lactis HN019 is Bifidobacterium animalis subsp. lactis with the deposit number NM97 / 09513. Description of the Drawings

[0018] Figure 1 It is a typical graph of the fluorescence intensity of zebrafish caudal vein macrophages after sample treatment. The area within the yellow dashed box is the analysis area, and the green fluorescent particles are macrophages.

[0019] Figure 2 It is the fluorescence intensity of zebrafish caudal vein macrophages after sample treatment. Compared with the model control group, *p < 0.05, **p < 0.01.

[0020] Figure 3This is a typical graph of the number of neutrophils in the caudal vein of zebrafish tails after sample treatment. The area within the yellow dashed box is the analysis region, and the green fluorescent particles are neutrophils.

[0021] Figure 4 This is the number of neutrophils in the caudal vein of zebrafish tails after sample treatment. Compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0022] Figure 5 This is a typical graph of the fluorescence intensity of T cells in zebrafish after sample treatment. The area within the yellow dashed box is the analysis region, and the red fluorescence is T cells.

[0023] Figure 6 This is the fluorescence intensity of T cells in zebrafish after sample treatment. Compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0024] Figure 7 This is a typical graph of the body length of zebrafish after sample treatment. The blue dashed line is the analysis region.

[0025] Figure 8 This is the body length of zebrafish after sample treatment. Compared with the normal control group, *p < 0.05, ***p < 0.001; compared with Experimental Example 1, △△p < 0.01; compared with Experimental Example 2, #p < 0.05, ##p < 0.01; compared with Experimental Example 3, ^p < 0.05, ^^p < 0.01, ^^^p < 0.001.

[0026] Figure 9 This is a typical graph of the staining intensity of the blood vessels from the intestine to the tail of zebrafish after sample treatment. The yellow dashed box is the analysis region.

[0027] Figure 10 This is the fluorescence intensity of the vertebral bones of zebrafish after sample treatment. Compared with the normal control group, **p < 0.01, ***p < 0.001; compared with Experimental Example 1, △p < 0.05; compared with Experimental Example 2, #p < 0.05, ##p < 0.01. Detailed implementation manners

[0028] In order to have a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solutions of the present invention will be described in detail below in combination with specific examples and the accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the relevant art.

[0029] Example 1 Screening of synbiotic combinations

[0030] Prepare the materials required for the experiment: YCFA medium (tryptone <Haibo-HB8271> 1 g / ml, yeast extract <oxoid-lp0021>0.25 g / ml, L-cysteine <Aladdin - C108238> 0.1 g / ml, hemin <Haibo - HB0310a> 5 mg / ml, NaCl <Aladdin - S433737> 0.09 g / ml, CaCl·2H2O <Aladdin C108383> 0.009 g / ml, K2HPO4 <Aladdin - P112219> 0.045 g / ml, KH2PO4 <Aladdin - P401704> 0.045 g / ml, MgSO4·7H2O <Aladdin - M433513> 0.009 g / ml, resazurin <Shanghai Yuanye - S19084> 0.1 mg / ml), vials, 2'-FL, Millex® - GP sterile filter (SLGP033R). Prepare a sufficient amount of YCFA medium before the experiment. Dispense the YCFA medium into vials at 4 mL / vial, and use a capping machine to cap each vial. Sterilize at 121°C for 15 min and set aside. Prepare the 2'-FL stock solution (40 g / L - dissolved in YCFA medium) before the experiment and filter it through a 0.22 um sterile filter head. Add the 2'-FL stock solution to the vials containing 4 mL of YCFA at 1 mL / vial, and the final concentration of 2'-FL is 8 g / L. This is used as an in vitro intestinal fermentation model. With the in vitro human intestinal fermentation model, using 2'-FL as the sole carbon source, three different strains of Bifidobacterium lactis are combined in pairs. By comparing the SCFA production rates after in vitro fermentation, the combination that can best utilize 2'-FL to produce SCFA after compounding is screened out.

[0031] Dissolve 2'-FL in YCFA medium to a concentration of 8 g / L and filter it to remove contaminants. Take 5 mL of the above - treated liquid medium containing 2'-FL and place it in a sterilized fermentation vial (vial) for later use. The newly revived and rejuvenated strains after three times of rejuvenation are first centrifuged (6000g, 4°C, 10 min) to collect the cells, then washed twice with sterile 1×PBS, and prepared into a cell suspension with OD = 1. Each strain is inoculated into the corresponding medium at a ratio of 2% (v / v). Place the fermentation vial in the corresponding growth environment and culture for 48 h. The fermented broth after fermentation is used for the subsequent detection of SCFA indicators. The detection method is as follows: Pipette 500 uL of the mixed broth into a 1.5 mL EP tube, add 100 uL of crotonic acid per tube and acidify at -80°C for 24 h. After thawing, centrifuge at 14000 rpm / min for 5 min, take the supernatant into an EP tube, filter it through a 0.22 μm aqueous filter membrane, take 150 uL into the liner of the injection vial, store at 4°C, and use gas chromatography external standard method to detect the SCFA content in the sample. The results are shown in Table 1.

[0032] Table 1. SCFA production of Bifidobacterium lactis during in vitro fermentation with 2'-FL as the sole carbon source

[0033]

[0034] Data are presented as mean ± standard error. Statistical analysis was performed using Graphpad prism version 10.1.1(270). One-way ANOVA was used for analysis and testing of multiple groups; t test was used for analysis and testing of comparisons between two groups. *p < 0.05, **p < 0.01.

[0035] As shown in Table 1, Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019, and Bifidobacterium lactis Bi-07 can all utilize 2'-FL as the sole carbon source to ferment and produce SCFA; among them, the effect of Bifidobacterium lactis HN019 and Bifidobacterium lactis Bb-12 in producing SCFA using 2'-FL is significantly better than that of Bifidobacterium lactis Bi-07. The effect of the combination of Bifidobacterium lactis HN019 and Bifidobacterium lactis Bb-12 in producing SCFA using 2'-FL is significantly better than other combinations. Therefore, the synbiotic combination of Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019, and 2′-FL has the highest SCFA production rate. Combining the results of Comparative Example 1, Comparative Example 2, and Experimental Example 1, the SCFA production of the combination of Bifidobacterium lactis Bb-12 and Bifidobacterium lactis HN019 (Experimental Example 1) is greater than the sum of the SCFA production of Bifidobacterium lactis HN019 alone (Comparative Example 2) and the SCFA production of Bifidobacterium lactis Bb-12 alone (Comparative Example 1). Therefore, the combination of Bifidobacterium lactis Bb-12 and Bifidobacterium lactis HN019 has a synergistic effect in terms of SCFA production.

[0036] Example 2. Synbiotic composition for enhancing immunity

[0037] The experimental subjects in the experiment were transgenic macrophage green fluorescent zebrafish at 3 - 5 days post-fertilization (3 - 5 dpf). The experimental subjects in the experiment on the effect of the synbiotic composition on neutrophils were 3 - 5 dpf transgenic neutrophil green fluorescent zebrafish. The experimental subjects in the experiment on the effect of the synbiotic composition on T cells were 3 - 5 dpf transgenic T cell red fluorescent zebrafish. Source of zebrafish: Fish Rearing Center of Hangzhou Huante Company; Gender: Zebrafish larvae are not differentiated by gender and can only be differentiated by gender after 2.5 - 3 months of sexual organ development; Age: 3 - 5 days; Rearing method: Zebrafish were all reared in fish rearing water at 28 °C (Water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, with a conductivity of 450 - 550 μS / cm; pH of 6.5 - 8.5; hardness of 50 - 100 mg / L CaCO3); Method of adding synbiotics: The samples were given in aqueous solution, and the fish rearing water was directly prepared according to the experimental concentration.

[0038] The specific experimental method is as follows: Randomly select the above zebrafish into 6-well plates, with 30 zebrafish treated in each well, and the volume of each well is 3 mL. The liquid is changed every day. Set up a normal control group, a model control group, and a positive control group. Except for the normal control group, the zebrafish in the other experimental groups were all intravenously injected with vinorelbine tartrate injection (Jiangsu Hansoh Pharmaceutical Co., Ltd., batch number 600211003) to establish a zebrafish immunosuppression model. For the zebrafish in the positive control group, Bailing Capsules (Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd., batch number 2307029D) were given in aqueous solution, and the dosage was 15.0 μg / mL. Lactobacillus paracasei Bb-12, Lactobacillus paracasei HN019, and prebiotic 2'-FL were dissolved in water and added to each well so that the concentrations of Lactobacillus paracasei Bb-12, Lactobacillus paracasei HN019, and prebiotic 2'-FL in the well were the concentrations shown in Table 2. After treatment at 28 °C for 2 days, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The NIS-Elements D 3.20 advanced image processing software was used to analyze and collect data, and the fluorescence intensities of the caudal vein macrophages, neutrophils, and T cells of the zebrafish were analyzed respectively. The immunopotentiating effect of the synbiotic composition was evaluated based on the statistical analysis results of the above indicators.

[0039] The statistical treatment results were expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis. p < 0.05 indicated that the difference was statistically significant. The specific statistical method is as follows: Statistical analysis between two groups: Explore the data, and the exploration results were obtained by the K-S test. If the p value of the normality test > 0.05 (i.e., following a normal distribution), then the independent samples T test in parametric tests was used for statistical analysis; if the p value of the normality test < 0.05 (i.e., not following a normal distribution), then two independent samples in non-parametric tests were used for statistical analysis.

[0040] Table 2. Results of the efficacy of synbiotic composition in enhancing immunity (n = 10)

[0041]

[0042] Compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001;

[0043] Compared with Experimental Example 3, ^ p < 0.05.

[0044] (1) Effect of synbiotic composition on macrophages

[0045] As can be seen from Table 2, the fluorescence intensities of macrophages in the normal control group and the model control group were 109988 ± 9604 pixels and 80282 ± 3924 pixels respectively, and the fluorescence intensity of macrophages in the model control group decreased significantly. The fluorescence intensities of macrophages in Comparative Examples 1, 2, and 3 were 87340 ± 4844 pixels, 89874 ± 4465 pixels, and 90738 ± 6219 pixels respectively. Compared with the model group, p > 0.05 in all cases. Therefore, Comparative Examples 1-3 had no obvious effect on the recovery of macrophage fluorescence intensity. The fluorescence intensity of macrophages in Comparative Example 4 was 92578 ± 5504 pixels, and compared with the model group, p > 0.05, and it also had no obvious effect on the recovery of macrophage fluorescence intensity. The fluorescence intensities of macrophages in Experimental Examples 1, 2, 3, and Comparative Example 5 were 100238 ± 7691 pixels, 106694 ± 6628 pixels, 100118 ± 6698 pixels, and 96057 ± 4483 pixels respectively. Compared with the model group, p < 0.05 & p < 0.05 & p <0.001 & p < 0.05. Therefore, Experimental Examples 1-3 and Comparative Example 5 had statistical significance for the recovery of macrophage fluorescence intensity. See details in Figure 1 and Figure 2 .

[0046] The above results indicate that there are statistical significances in the restoration of macrophage fluorescence intensity in Experimental Examples 1-3 and Comparative Example 5. Compared with the insignificant restoration of macrophage fluorescence intensity in the groups of Comparative Examples 1, 2, 3, and 4, it shows advantages, suggesting that the combined use group of three components of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL, and prebiotic 2'-FL 250 μg / mL (Experimental Example 1), the combined use group of three components of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL, and prebiotic 2'-FL 375 μg / mL (Experimental Example 2), the combined use group of three components of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL, and prebiotic 2'-FL 375 μg / mL (Experimental Example 3), and the combined use group of three components of Bifidobacterium lactis Bb-12 0.000914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL, and prebiotic 2'-FL 375 μg / mL (Comparative Example 5) have a synergistic effect compared with the single use of each component. The above results also suggest that the groups of Experimental Examples 1, 2, 3, 4, and Comparative Example 5 have advantages over the group of Comparative Example 4.

[0047] In terms of absolute values, the higher the absolute value of the fluorescence intensity of macrophages, the better the effect of enhancing immunity. The absolute values of the fluorescence intensity of macrophages in Experimental Examples 1, 2, and 3 were 100238, 106694, and 100118 respectively, which were all higher than that of the 96057 in Comparative Example 5, indicating that Experimental Examples 1, 2, and 3 had advantages over Comparative Example 5. It is suggested that the combined group of three components of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL, and prebiotic 2'-FL 250 μg / mL (Experimental Example 1), the combined group of three components of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL, and prebiotic 2'-FL 375 μg / mL (Experimental Example 2), and the combined group of three components of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL, and prebiotic 2'-FL 375 μg / mL (Experimental Example 3) were all superior to the combined group of three components of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 0.0076 μg / mL, and prebiotic 2'-FL 500 μg / mL (Comparative Example 4) and the combined group of three components of Bifidobacterium lactis Bb-12 0.000914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL, and prebiotic 2'-FL 375 μg / mL (Comparative Example 5).

[0048] (2)Effect of synbiotic composition on neutrophils

[0049] As can be seen from Table 2, the numbers of neutrophils in the normal control group and the model control group were 64.4 ± 6.07 and 40.0 ± 2.39 respectively, and the number of neutrophils in the model control group decreased significantly. The numbers of neutrophils in Comparative Examples 1, 2, and 3 were 51.8 ± 3.50, 54.6 ± 4.66, and 52.6 ± 5.01 respectively, and p < 0.05 when compared with the model group. Therefore, the effects of Comparative Examples 1-3 on the recovery of neutrophil numbers were all statistically significant. The numbers of neutrophils in Comparative Example 4, Experimental Examples 1, 2, 3, and Comparative Example 5 were 52.4 ± 4.32, 55.3 ± 4.41, 61.5 ± 2.81, 55.1 ± 4.16, and 53.0 ± 4.94 respectively, and p < 0.05 & p < 0.01 & p < 0.001 & p < 0.01 & p < 0.05 when compared with the model group. Therefore, Comparative Example 4, Experimental Examples 1, 2, 3, and Comparative Example 5 all had statistically significant effects on the recovery of neutrophil numbers. For details, see Figure 3 and Figure 4 。

[0050] The above results indicate that the neutrophil count recovery in Experimental Examples 1, 2, and 3 groups was statistically significant, with p < 0.01 & p < 0.001 & p < 0.01. Compared with the neutrophil count recovery in Comparative Examples 1, 2, and 3 groups, where p < 0.05, it shows an advantage, suggesting that the combined use of three components of Bifidobacterium lactis Bb-12 at 0.0914 μg / mL, Bifidobacterium lactis HN019 at 0.152 μg / mL, and prebiotic 2'-FL at 250 μg / mL (Experimental Example 1), the combined use of three components of Bifidobacterium lactis Bb-12 at 0.0914 μg / mL, Bifidobacterium lactis HN019 at 0.0152 μg / mL, and prebiotic 2'-FL at 375 μg / mL (Experimental Example 2), and the combined use of three components of Bifidobacterium lactis Bb-12 at 0.00914 μg / mL, Bifidobacterium lactis HN019 at 0.152 μg / mL, and prebiotic 2'-FL at 375 μg / mL (Experimental Example 3) had a synergistic effect compared with the single use of each component.

[0051] From the absolute values, the higher the absolute value of the neutrophil count, the better the immune-enhancing effect. The neutrophil count in Experimental Example 2 group was 61.5, which was higher than that in Comparative Example 4 group (52.4) and Comparative Example 5 group (53.0), indicating that Experimental Example 2 group had an advantage over Comparative Examples 4 and 5 groups. In terms of significance, Experimental Example 2 group had a significant effect of three stars, and Experimental Examples 1 and 3 groups had a significant effect of two stars, both of which had advantages compared with the one-star significant effect in Comparative Examples 4 and 5 groups. It is suggested that the combined use of three components of Bifidobacterium lactis Bb-12 at 0.0914 μg / mL, Bifidobacterium lactis HN019 at 0.152 μg / mL, and prebiotic 2'-FL at 250 μg / mL (Experimental Example 1), the combined use of three components of Bifidobacterium lactis Bb-12 at 0.0914 μg / mL, Bifidobacterium lactis HN019 at 0.0152 μg / mL, and prebiotic 2'-FL at 375 μg / mL (Experimental Example 2), and the combined use of three components of Bifidobacterium lactis Bb-12 at 0.00914 μg / mL, Bifidobacterium lactis HN019 at 0.152 μg / mL, and prebiotic 2'-FL at 375 μg / mL (Experimental Example 3) all had advantages compared with the combined use of three components of Bifidobacterium lactis Bb-12 at 0.00914 μg / mL, Bifidobacterium lactis HN019 at 0.0076 μg / mL, and prebiotic 2'-FL at 500 μg / mL (Comparative Example 4) and the combined use of three components of Bifidobacterium lactis Bb-12 at 0.000914 μg / mL, Bifidobacterium lactis HN019 at 0.0152 μg / mL, and prebiotic 2'-FL at 375 μg / mL (Comparative Example 5).

[0052] (3)Effect of synbiotic composition on T cells

[0053] As shown in Table 2, the fluorescence intensities of T cells in the normal control group and the model control group were 27032±2396 pixels and 14717±1539 pixels respectively, and the fluorescence intensity of T cells in the model control group was significantly decreased. The fluorescence intensities of T cells in Comparative Examples 1, 2, and 3 were 23669±3671 pixels, 22582±2749 pixels, and 21345±2285 pixels respectively. Compared with the model group, p < 0.05 for all, so the restoration effects of Comparative Examples 1-3 on the fluorescence intensity of T cells were all statistically significant. The fluorescence intensities of T cells in Comparative Example 4, Experimental Examples 1, 2, 3, and Comparative Example 5 were 23438±3178 pixels, 26110±1249 pixels, 25045±2238 pixels, 25163±2583 pixels, and 23820±2683 pixels respectively. Compared with the model group, p < 0.05 & p < 0.001 & p < 0.01 & p <0.01 & p < 0.01, so Comparative Example 4, Experimental Examples 1, 2, 3, and Comparative Example 5 all had statistically significant effects on the restoration of the fluorescence intensity of T cells. See details in Figure 5 and Figure 6 .

[0054] The above results showed that Experimental Examples 1-3 and Comparative Example 5 all had statistically significant effects on the restoration of the fluorescence intensity of T cells, and p < 0.001 & p < 0.01 & p < 0.01 & p < 0.01. Compared with the p< 0.05 for the fluorescence intensities of T cells in Comparative Examples 1, 2, and 3, it indicated an advantage, suggesting that the combined use group of three components of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL, and prebiotic 2′-FL 250 μg / mL (Experimental Example 1), the combined use group of three components of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL, and prebiotic 2'-FL 375 μg / mL (Experimental Example 2), the combined use group of three components of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL, and prebiotic 2'-FL 375 μg / mL (Experimental Example 3), and the combined use group of three components of Bifidobacterium lactis Bb-12 0.000914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL, and prebiotic 2'-FL 375 μg / mL (Comparative Example 5) had a synergistic effect compared with the single use of each component.

[0055] In terms of absolute values, the higher the absolute value of the fluorescence intensity of T cells, the better the effect of enhancing immunity. The fluorescence intensities of T cells in Experimental Examples 1, 2, and 3 were 26,110, 25,045, and 25,163 pixels respectively, which were higher than those of 23,438 pixels in Comparative Example 4 and 23,820 pixels in Comparative Example 5, indicating that Experimental Examples 1, 2, and 3 all had advantages over Comparative Example 4 and Comparative Example 5. In terms of significance, Experimental Example 1 had a significant effect of three stars, Experimental Examples 2, 3, and Comparative Example 5 had a significant effect of two stars, and they had advantages compared with Comparative Example 4 which had a significant effect of one star. It is suggested that the combined use group of three components of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL and prebiotic 2'-FL 250 μg / mL (Experimental Example 1), the combined use group of three components of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL and prebiotic 2'-FL 375 μg / mL (Experimental Example 2), the combined use group of three components of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL and prebiotic 2'-FL 375 μg / mL (Experimental Example 3), were all superior to the combined use group of three components of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 0.0076 μg / mL and prebiotic 2'-FL 500 μg / mL (Comparative Example 4) and the combined use group of three components of Bifidobacterium lactis Bb-12 0.000914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL and prebiotic 2'-FL 375 μg / mL (Comparative Example 5).

[0056] In summary, in terms of enhancing immunity, the synbiotic composition has a synergistic effect compared with the single use of each component. Its characteristics are that, by mass, the content ratio of Bifidobacterium lactis Bb-12 probiotic, Bifidobacterium lactis HN019 probiotic and prebiotic 2'-FL is 0.1~1:0.17~1.66:2735~4103. It was measured that the amount of Bifidobacterium lactis Bb-12 in the synbiotic composition was 2×10 3 ~ 2×10 5 CFU / mL, and the amount of Bifidobacterium lactis HN019 was 2×10 4 ~ 2×10 5 CFU / mL.

[0057] Example 3 The synbiotic composition is used for promoting growth and development

[0058] The experimental subjects in the experiment were wild-type AB strain zebrafish at 6 hours post-fertilization to 4 days post-fertilization (6 hpf - 4 dpf). The experimental subjects in the experiment on the effect of the synbiotic composition on fat absorption were zebrafish with a melanin allele gene mutation (albino) at 5 - 6 dpf. Source of zebrafish: Fish Rearing Center of Hangzhou Huante Company; Gender: Zebrafish larvae are not divided into male and female, and they can be divided into male and female only after the sexual organs develop in 2.5 - 3 months; Rearing method: Zebrafish were all reared in fish-raising water at 28 °C (Water quality: Add 200 mg of instant sea salt to every 1 L of reverse osmosis water, the conductivity is 450 - 550 μS / cm; pH is 6.5 - 8.5; Hardness is 50 - 100 mg / L CaCO3); Method of adding synbiotics: The samples were given in water solution, and the fish-raising water was directly prepared according to the experimental concentration.

[0059] The specific experiments are as follows: (1) Experiment on the effect on body length: Randomly select the above zebrafish in 6-well plates, with 30 zebrafish in each well, and the volume of each well is 3 mL. The liquid is changed every day. A normal control group and a positive control group are set. For the zebrafish in the positive control group, Swisse "Calcium&VitaminD” (Swisse, batch number 33152A) was given in water solution, and the test dosage was 500 μg / mL. Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and prebiotic 2'-FL were given in water solution and added to each well to make the concentrations of Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and prebiotic 2'-FL in the well the concentrations shown in Table 2. After treatment at 28 °C for the corresponding number of days, 10 zebrafish were randomly selected from each experimental group and placed under a dissection microscope for photographing. The data were analyzed and collected using Image J advanced image processing software, and the body length of the zebrafish was analyzed. The effect of the sample on the body length was evaluated based on the statistical analysis results of this index. (2) Experiment on the effect on fat absorption: Randomly select the above zebrafish in beakers, with 30 zebrafish in each beaker. After eluting for 3 h after giving egg yolk powder in water solution, the synbiotic composition (concentration shown in Table 3) and the positive control group were given in water solution, and at the same time, a normal control group (normal feeding group) was set, and the volume of each beaker was 20 mL. After treatment at 28 °C for 16 h, egg yolk powder was added to all groups, and after continuing to treat for 8 h, Oil Red O was given for whole-body fat staining. After the staining was completed, 10 zebrafish were randomly selected from each experimental group and placed under a dissection microscope for photographing. The data were collected using NIS-Elements D 3.20 advanced image processing software, and the staining intensity of blood vessels from the intestine to the tail was analyzed. The effect of the sample on fat absorption was evaluated based on the statistical analysis results of this index.

[0060] The results of statistical analysis were expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. A p < 0.05 indicated a statistically significant difference. The specific statistical methods were as follows: Statistical analysis between two groups: Explore the data, and the exploration results were obtained by the K-S test. If the p value of the normality test > 0.05 (i.e., conforming to the normal distribution), then the independent samples T-test in parametric tests was used for statistical analysis; if the p value of the normality test < 0.05 (i.e., not conforming to the normal distribution), then two independent samples in non-parametric tests were used for statistical analysis.

[0061] Table 3. Results of the efficacy of the synbiotic composition in promoting growth and development (n = 10)

[0062]

[0063] Compared with the normal control group, *p < 0.05, **p < 0.01, ***p < 0.001;

[0064] Compared with Experimental Example 1, △ p < 0.05, △△ p < 0.01;

[0065] Compared with Experimental Example 2, # p < 0.05, ## p < 0.01, ### p < 0.001;

[0066] Compared with Experimental Example 3, ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001.

[0067] (1) Effect of the synbiotic composition on body length

[0068] As can be seen from Table 3, the body length of the normal control group was 3.52 ± 0.025 mm, and the body lengths of Comparative Examples 1, 6, and 7 were 3.50 ± 0.029 mm, 3.47 ± 0.039 mm, and 3.55 ± 0.041 mm, respectively. Compared with the normal group, p > 0.05 in all cases. Therefore, Comparative Examples 1, 6, and 7 had no obvious effect on increasing body length. The body lengths of Comparative Examples 4 and 5 were 3.57 ± 0.034 mm and 3.51 ± 0.039 mm, respectively. Compared with the normal group, p > 0.05, and they also had no obvious effect on increasing body length. The body lengths of Experimental Examples 1, 2, and 3 were 3.60 ± 0.014 mm, 3.64 ± 0.023 mm, and 3.66 ± 0.019 mm, respectively. Compared with the normal group, p < 0.05 & p < 0.01 & p < 0.001. Therefore, Experimental Examples 1, 2, and 3 were all statistically significant for increasing body length. In addition, compared with Experimental Example 3, p < 0.001 & p < 0.001 & p < 0.05 for Comparative Examples 1, 6, and 7. See details in Figure 7 and Figure 8 。

[0069] The above results indicate that there are statistically significant effects on body length increase in Experimental Examples 1, 2, and 3 groups. Compared with the insignificant effects on body length increase in Comparative Examples 1, 4, 5, 6, and 7 groups, it shows an advantage. It is suggested that the combined use group of three components of Bifidobacterium lactis Bb-12 at 0.0914 μg / mL, Bifidobacterium lactis HN019 at 0.152 μg / mL, and prebiotic 2'-FL at 250 μg / mL (Experimental Example 1), the combined use group of three components of Bifidobacterium lactis Bb-12 at 0.0914 μg / mL, Bifidobacterium lactis HN019 at 0.0152 μg / mL, and prebiotic 2'-FL at 375 μg / mL (Experimental Example 2), and the combined use group of three components of Bifidobacterium lactis Bb-12 at 0.00914 μg / mL, Bifidobacterium lactis HN019 at 0.152 μg / mL, and prebiotic 2'-FL at 375 μg / mL (Experimental Example 3) have a synergistic effect compared with the single use of each component. The above results also suggest that Experimental Examples 1, 2, and 3 groups have an advantage over Comparative Examples 4 and 5 groups. From the comparison between groups, p < 0.05 & p < 0.05 & p < 0.01 when comparing Comparative Examples 4 and 5 and Experimental Example 1 group with Experimental Example 3 group. It is suggested that the combined use group of three components of Bifidobacterium lactis Bb-12 at 0.0914 μg / mL, Bifidobacterium lactis HN019 at 0.152 μg / mL, and prebiotic 2'-FL at 250 μg / mL (Experimental Example 1), the combined use group of three components of Bifidobacterium lactis Bb-12 at 0.0914 μg / mL, Bifidobacterium lactis HN019 at 0.0152 μg / mL, and prebiotic 2'-FL at 375 μg / mL (Experimental Example 2), and the combined use group of three components of Bifidobacterium lactis Bb-12 at 0.00914 μg / mL, Bifidobacterium lactis HN019 at 0.152 μg / mL, and prebiotic 2'-FL at 375 μg / mL (Experimental Example 3) all have an advantage over the combined use group of three components of Bifidobacterium lactis Bb-12 at 0.00914 μg / mL, Bifidobacterium lactis HN019 at 0.0076 μg / mL, and prebiotic 2'-FL at 500 μg / mL (Comparative Example 4) and the combined use group of three components of Bifidobacterium lactis Bb-12 at 0.000914 μg / mL, Bifidobacterium lactis HN019 at 0.0152 μg / mL, and prebiotic 2'-FL at 375 μg / mL (Comparative Example 5).

[0070] (2)Effect of synbiotic composition on fat absorption

[0071] As can be seen from Table 3, the staining intensity of blood vessels from the intestine to the tail in the normal control group was 49065±2165 pixels. The staining intensities of blood vessels from the intestine to the tail in Comparative Examples 1 and 6 were 64507±2991 pixels and 64005±3010 pixels respectively. Compared with the normal group, p < 0.001 & p < 0.001. Therefore, the effects of promoting fat absorption in Comparative Examples 1 and 6 were both statistically significant. The staining intensity of blood vessels from the intestine to the tail in Comparative Example 7 was 55338±3197 pixels. Compared with the normal group, p > 0.05, and the effect of promoting fat absorption was not obvious. The staining intensities of blood vessels from the intestine to the tail in Comparative Example 4, Experimental Examples 1-3, and Comparative Example 5 were 62081±2058 pixels, 62906±1788 pixels, 70393±3218 pixels, 67676±2686 pixels, and 60633±3118 pixels respectively. Compared with the normal group, p < 0.05 & p < 0.01 & p < 0.001. Therefore, the effects of promoting fat absorption in Comparative Example 4, Experimental Examples 1-3, and Comparative Example 5 were all statistically significant. See details in Figure 9 and Figure 10 。

[0072] In terms of absolute values, the higher the absolute value of the intestinal-to-tail vascular staining intensity, the better the growth promotion effect. The intestinal-to-tail vascular staining intensities of experimental examples 2 and 3 were 70393 and 67676 pixels respectively, which were higher than those of comparative examples 1, 6, and 7 (64507, 64005, and 55338 pixels) and also higher than those of comparative examples 4, experimental example 1, and comparative example 5 (62081, 62906, and 60633 pixels), indicating that experimental examples 2 and 3 had advantages over comparative example 5. It is suggested that the combined use of the three components of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL, and prebiotic 2'-FL 375 μg / mL in the composition (experimental example 2), and the combined use of the three components of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL, and prebiotic 2'-FL 375 μg / mL in the composition (experimental example 3) had a synergistic effect compared with the single use of each component. The above results also suggest that the combined use of the three components of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL, and prebiotic 2'-FL 375 μg / mL in the composition (experimental example 2) and the combined use of the three components of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL, and prebiotic 2'-FL 375 μg / mL in the composition (experimental example 3) were both superior to the combined use of the three components of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 0.0076 μg / mL, and prebiotic 2'-FL 500 μg / mL in the composition (comparative example 1), the combined use of the three components of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL, and prebiotic 2'-FL 250 μg / mL in the composition (experimental example 1), and the combined use of the three components of Bifidobacterium lactis Bb-12 0.000914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL, and prebiotic 2'-FL 375 μg / mL in the composition (comparative example 5).

[0073] In summary, in terms of promoting growth and development, the synbiotic composition had a synergistic effect compared with the single use of each component. Its characteristics were that, by mass, the content ratio of Bifidobacterium lactis Bb-12 probiotic, Bifidobacterium lactis HN019 probiotic, and prebiotic 2'-FL was 0.1~1:0.17~1.66:2735~4103. It was measured that the amount of Bifidobacterium lactis Bb-12 in the synbiotic composition was 2×10 3 ~ 2×10 5 CFU / mL, and the amount of Bifidobacterium lactis HN019 was 2×10 4 ~ 2×10 5 CFU / mL。

[0074] In the specific embodiments described above, the objectives, technical solutions, and beneficial effects of the present invention have been further described in detail. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a synbiotic composition in the preparation of a product for promoting the growth and development of a subject, wherein the synbiotic composition consists of Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and 2'-FL, and the mass ratio of Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and 2'-FL is 0.1-1:0.17-1.66:2735-4103, and the promotion of the growth and development of the subject is to promote the weight gain of the subject or promote the fat absorption of the subject.

2. Use of a synbiotic composition in the preparation of a product for promoting the growth and development of a subject, wherein the synbiotic composition consists of (1) Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and 2'-FL, and (2) excipients, diluents and / or carriers, and the mass ratio of Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and 2'-FL is 0.1-1:0.17-1.66:2735-4103, and the promotion of the growth and development of the subject is to promote the weight gain of the subject or promote the fat absorption of the subject.

3. The use according to claim 1 or 2, wherein the amount of Bifidobacterium lactis Bb-12 is 2×10 3 ~ 2×10 5 CFU / mL; the amount of Bifidobacterium lactis HN019 is 2×10 4 ~ 2×10 5 CFU / mL.

4. The use according to claim 1 or 2, wherein the subject is a zebrafish or a mammal.

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