Screening and application of synergistic synbiotics for enhancing immunity and promoting growth and development

Abstract

The invention relates to screening and application of synergistic synbiotics for enhancing immunity and promoting growth and development. The invention discloses a pharmaceutical composition which comprises bifidobacterium lactis Bb-12, bifidobacterium lactis HN019 and prebiotics 2 '-FL, and the pharmaceutical 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 specifically relates to a synbiotic composition, which comprises probiotic Bifidobacterium lactis Bb-12, probiotic Bifidobacterium lactis HN019 and prebiotic 2'-FL. The composition is used for enhancing immunity and / or promoting growth and development, wherein the synbiotic composition is a medicine. Background Art

[0002] Human milk oligosaccharides (HMOs) are the third most abundant substance in breast milk after lactose and fat. Each type of HMO has a lactose at the reducing end, most of which have polylactosamine as the main structural chain and contain fucose, sialic acid, or both at the chain end. HMOs have three main functions: (1) inhibiting the attachment and infection of specific pathogens; (2) acting as prebiotics to promote the growth of bacteria in the intestinal symbiotic system; and (3) directly alleviating the inflammatory response of the mucosa to toxic stimuli.

[0003] Prebiotics are a type of carbohydrate that cannot be digested by the human body. They can interact with the intestinal flora in the host's intestines to produce substances or effects that are beneficial to the host. Currently, the most studied prebiotics include: galacto-oligosaccharides, fructo-oligosaccharides, inulin, isomerized lactose, polydextrose, xylulose oligosaccharides, etc.

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

[0005] There is a need for solutions to enhance immunity and / or promote growth and development in the areas of infant formula, complementary foods, nutritional supplements and pharmaceuticals, and it would be beneficial if new synbiotic compositions could be provided. Summary of the invention

[0006] One object of the present invention is to provide a synbiotic composition for enhancing immunity and / or promoting growth and development.

[0007] The present invention provides a synbiotic composition comprising Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and 2'-FL.

[0008] 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.

[0009] According to a specific embodiment of the present invention, the synbiotic composition of the present invention further comprises an excipient, a diluent and / or a carrier.

[0010] 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.

[0011] The synbiotic composition of the present invention is a food, an additive or a medicine.

[0012] In the synbiotic composition of the present invention, the food includes dairy products, beverages and health products; and the additives include food additives and feed additives.

[0013] 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.

[0014] On the other hand, the present invention also provides the use of the synbiotic composition in preparing products having the functions of enhancing immunity and / or promoting growth and development.

[0015] According to a specific embodiment of the present invention, the present invention provides use of the synbiotic composition in preparing a product for enhancing the immunity of a subject and / or promoting the growth and development of a subject.

[0016] In some embodiments, the product is a food ingredient, a food additive, a feed additive, a medicine, a liquid drink, a food or a health product.

[0017] According to a specific embodiment of the present invention, enhancing the immunity of the subject includes: enhancing the humoral immunity and / or cellular immunity of the subject, increasing the number of immune cells of the subject, or improving the activity of immune cells of the subject; promoting the growth and development of the subject includes: promoting the increase of the subject's body length or height, promoting the subject's weight gain, or promoting one or more of the subject's fat absorption capacity.

[0018] In some embodiments, the subject is a zebrafish or a mammal.

[0019] In some embodiments, the mammal is a mouse or a human.

[0020] 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 irritable bowel syndrome or inflammatory bowel disease, and can also be used as a prebiotic to maintain the balance of intestinal flora.

[0021] Short-chain fatty acids (SCFA) are the main metabolites produced by the fermentation of sugars or amino acids by specific intestinal flora. They participate in the body's metabolism and play an important role in different organs and tissues. SCFA has certain physiological functions for many organs and tissues of the body. SCFA can promote and maintain intestinal health, brain function and bone function, relieve obesity and diabetes, and participate in immune regulation, and exert anti-tumor and antibacterial effects. In terms of intestinal health, SCFA mainly enhances intestinal barrier function by activating adenosine monophosphate (AMP) and activating adenosine monophosphate-activated protein kinase (AMPK) to upregulate the expression of tight junction proteins; SCFA can also enhance intestinal barrier function by changing the expression of epithelial cells O 2 The consumption of SCFA stabilizes the expression of the barrier protection-related transcription factor hypoxia-inducible factor (HIF), thereby enhancing the intestinal barrier function. In terms of bone function, SCFA, as a regulator of osteoclast metabolism and bone mass, binds to specific receptors to promote and maintain bone function. In terms of immune regulation, the receptor FFAR2 of SCFA in the immune system can be expressed on eosinophils, basophils, neutrophils, monocytes and mucosal mast cells, indicating that SCFA has a wide range of roles in immune responses. It can downregulate the expression level of proinflammatory factors secreted by related immune cells by activating GPR, inhibiting HDAC activity and regulating Treg cells, thereby exerting an immunomodulatory effect and inhibiting the occurrence of inflammation and allergies in the body.

[0022] In a specific embodiment of the present invention, the prebiotic 2'-fucosyllactose (2'-Fucosyllactose, 2'-FL) comes from Hongmo Biotechnology (Shanghai) Co., Ltd.; Bifidobacterium animals subsp. lactis Bb-12 is Bifidobacterium animals subsp. lactis with the deposit number DSM15954; Bifidobacterium animals subsp. lactis HN019 is Bifidobacterium animals subsp. lactis with the deposit number NM97 / 09513. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1This is a typical graph of the fluorescence intensity of macrophages in the tail vein of zebrafish after sample treatment. The yellow dotted box is the analysis area, and the green fluorescent particles are macrophages.

[0024] Figure 2 Fluorescence intensity of zebrafish tail vein macrophages after sample treatment, compared with the model control group, *p<0.05, **p<0.01.

[0025] Figure 3 This is a typical graph of the number of neutrophils in the tail vein of zebrafish after sample treatment. The yellow dotted box is the analysis area, and the green fluorescent particles are neutrophils.

[0026] Figure 4 The number of neutrophils in the caudal vein of zebrafish after sample treatment, compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0027] Figure 5 This is a typical graph of zebrafish T cell fluorescence intensity after sample treatment. The yellow dotted box is the analysis area, and the red fluorescence is T cells.

[0028] Figure 6 Fluorescence intensity of zebrafish T cells after sample treatment, compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0029] Figure 7 This is a typical diagram of zebrafish body length after sample treatment, and the blue dotted line is the analysis area.

[0030] Figure 8 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.

[0031] Fig. 9 This is a typical image of the staining intensity of blood vessels from the intestine to the tail of zebrafish after sample treatment. The yellow dotted box is the analysis area.

[0032] Fig.10 is the fluorescence intensity of zebrafish vertebrae 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 DESCRIPTION

[0033] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below in conjunction with specific examples and drawings. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as those commonly understood by ordinary technicians in the relevant fields.

[0034] Example 1 Screening of synbiotic combinations

[0035] Prepare the materials needed for the experiment: YCFA medium (casein peptone <Haibo-HB8271> 1g / ml, yeast extract <oxoid-lp0021>0.25g / ml, L-cysteine ​​<Aladdin-C108238> 0.1g / ml, hemoglobin <Haibo-HB0310a> 5mg / ml, NaCl <Aladdin-S433737> 0.09g / ml, CaCl·2H 2 O<Aladdin C108383> 0.009g / ml, K 2 HPO 4 <Aladdin-P112219> 0.045g / ml, KH 2 PO 4 <Aladdin-P401704> 0.045g / ml, MgSO 4 7H 2 O<Aladdin-M433513>0.009g / ml, Resazurin<Shanghai Yuanye-S19084>0.1mg / ml), vials, 2'-FL, Sterile filter (SLGP033R). Prepare enough YCFA medium before the experiment, and fill the YCFA medium into vials at 4 mL / bottle. Use a capping instrument to cap each vial, sterilize at 121°C for 15 min, and set aside. Prepare each 2'-FL mother liquor (40 g / L-dissolved in YCFA medium) before the experiment, and use a 0.22um sterile filter head for membrane treatment. Add 2'-FL mother liquor to the vial containing 4 mL YCFA at 1 mL / bottle, and the final concentration of 2'-FL is 8 g / L, which is used as an in vitro intestinal fermentation model. With the help of the human intestinal in vitro fermentation model, 2'-FL is used as the single carbon source, and three different strains of Bifidobacterium lactis are used for two-to-two combination compounding. By comparing the yield of SCFA after in vitro fermentation, the combination that can best utilize 2'-FL to produce SCFA after compounding is screened out.

[0036] Dissolve 2'-FL in YCFA medium to a concentration of 8 g / L, and pass through a membrane to remove foreign bacteria. Take 5 mL of the above-treated liquid medium containing 2'-FL and place it in a fermentation bottle (cillin bottle) after high-temperature sterilization for use. The newly revived strains that have been rejuvenated after three rejuvenations are first centrifuged (6000g, 4°C, 10min) to collect the bacteria, and then wash twice with sterile 1×PBS to prepare a bacterial suspension with OD=1, and inoculate each strain in the corresponding culture medium at a ratio of 2% (v / v). The fermentation vial was placed in the corresponding growth environment for 48 hours. The bacterial solution after fermentation was used for the subsequent detection of SCFA indicators. The detection method was as follows: 500uL of the mixed bacterial solution was drawn into a 1.5mL EP tube, 100uL / tube was added with crotonic acid and acidified at -80℃ for 24 hours, and after thawing, it was centrifuged at 14000rpm / min for 5 minutes, the supernatant was taken into an EP tube, filtered with a 0.22μm aqueous filter membrane, 150uL was taken into the liner tube of the injection bottle, and stored at 4℃, and the SCFA content in the sample was detected using the gas chromatography external standard method. The results are shown in Table 1.

[0037] Table 1. SCFA production by in vitro fermentation of Bifidobacterium lactis using 2'-FL as the sole carbon source

[0038]

[0039] Data are presented as mean ± standard error. Graphpad prism version 10.1.1 (270) was used for statistical analysis. One-way ANOVA was used for multiple group comparisons and t test was used for comparisons between two groups. *p<0.05, **p<0.01.

[0040] As shown in Table 1, Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019, and Bifidobacterium lactis Bi-07 can all use 2'-FL as the sole carbon source to ferment and produce SCFA; among them, Bifidobacterium lactis HN019 and Bifidobacterium lactis Bb-12 are significantly better than Bifidobacterium lactis Bi-07 in producing SCFA using 2'-FL. The effect 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 yield. 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 SCFA production.

[0041] Example 2 Synbiotic composition for enhancing immunity

[0042] The experimental subjects in the experiment were transgenic macrophage green fluorescent zebrafish 3-5 days after fertilization (3-5dpf), the experimental subjects in the experiment of the effect of the synbiotic combination on neutrophils were 3-5dpf transgenic neutrophil green fluorescent zebrafish, and the experimental subjects in the experiment of the effect of the synbiotic combination on T cells were 3-5dpf transgenic T cell red fluorescent zebrafish. Zebrafish source: Hangzhou Huante Company Fish Farming Center; Gender: Zebrafish fry are not divided into males and females, and they can only be divided into males and females after completing the development of sexual organs at 2.5-3 months; Age: 3-5 days; Feeding method: Zebrafish are all raised in 28℃ fish farming water (water quality: 200mg instant sea salt is added to each 1L reverse osmosis water, the conductivity is 450-550μS / cm; pH is 6.5-8.5; hardness is 50-100mg / L CaCO 3 ); Synbiotics addition method: Dissolve in water and give to samples, and directly prepare fish water according to the experimental concentration.

[0043] The specific experimental method is as follows: randomly select the above zebrafish in a 6-well plate, process 30 zebrafish in each well, and the capacity of each well is 3mL, and the liquid is changed every day. Set a normal control group, a model control group and a positive control group. Except the normal control group, the zebrafish of the remaining experimental groups are intravenously injected with vinorelbine tartrate injection (Jiangsu Hausen Pharmaceutical Co., Ltd., batch number 600211003) to establish a zebrafish immunodeficiency model. For the zebrafish of the positive control group, water-soluble Bailing capsules (Hangzhou Sino-US Huadong Pharmaceutical Co., Ltd., batch number 2307029D) are given, and the dosage is 15.0 μg / mL. Water-soluble Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and prebiotics 2'-FL, and add them to each well so that the concentrations of Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and prebiotics 2'-FL in the well are the concentrations shown in Table 2. After 2 days of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The fluorescence intensity of macrophages, neutrophils and T cells in the tail vein of zebrafish were analyzed respectively. The statistical analysis results of the above indicators were used to evaluate the immune-enhancing efficacy of the synbiotic combination.

[0044] The statistical results were expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant. The specific statistical methods were as follows: Statistical analysis between the two groups: The data were explored and the exploration results were obtained by KS test. If the normality test p > 0.05 (i.e., it obeys the normal distribution), the independent sample T test in the parameter test was used for statistical analysis; if the normality test p < 0.05 (i.e., it does not obey the normal distribution), two independent samples in the non-parametric test were used for statistical analysis.

[0045] Table 2. Results of the efficacy of synbiotic composition in enhancing immunity (n=10)

[0046]

[0047] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001;

[0048] Compared with Experimental Example 3, ^ p<0.05.

[0049] (1) Effects of synbiotic composition on macrophages

[0050] As can be seen from Table 2, the fluorescence intensity 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 was significantly reduced. The fluorescence intensity of macrophages in groups 1, 2, and 3 of the comparative examples were 87340±4844 pixels, 89874±4465 pixels, and 90738±6219 pixels, respectively, compared with the model group, p>0.05, so comparative examples 1-3 had no obvious effect on the recovery of macrophage fluorescence intensity. The fluorescence intensity of macrophages in group 4 of the comparative example was 92578±5504 pixels, compared with the model group, p>0.05, and the recovery of macrophage fluorescence intensity was also not obvious. The macrophage fluorescence intensity of 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, so experimental examples 1-3 and comparative example 5 have statistical significance in the recovery of macrophage fluorescence intensity. Figure 1 and Figure 2 .

[0051] The above results show that Experimental Examples 1-3 and Comparative Example 5 have statistically significant effects on the recovery of macrophage fluorescence intensity, which is not obvious compared with the recovery of macrophage fluorescence intensity in Comparative Examples 1, 2, 3, and 4, indicating that there is an advantage, suggesting that the three-component combination group of the composition 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 three-component combination group of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN0190.0152 μg / mL and prebiotic 2'-FL 375 μg / mL (Experimental Example 2), the three-component combination group of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL and prebiotic 2'-FL The three-component combination group (Experimental Example 3) and the three-component combination group (Comparative Example 5) of Bifidobacterium lactis Bb-12 0.000914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL and prebiotic 2'-FL 375 μg / mL have synergistic effects compared with the single use of each component. The above results also suggest that Experimental Examples 1, 2, 3, 4 and Comparative Example 5 groups have advantages over Comparative Example 4 group.

[0052] From the absolute value point of view, the higher the absolute value of macrophage fluorescence intensity, the better the effect of enhancing immunity. The absolute values ​​of macrophage fluorescence intensity in Experimental Examples 1, 2, and 3 were 100238, 106694, and 100118, respectively, which were all higher than 96057 in Comparative Example 5, indicating that Experimental Examples 1, 2, and 3 have advantages over Comparative Example 5. The combination group of the three components of the composition 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 combination group 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 combination group 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) are all better than the combination of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 The three-component combination group of 0.0076 μg / mL and prebiotic 2'-FL 500 μg / mL (Comparative Example 4) and the three-component combination group 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 advantages.

[0053] (2) Effects of synbiotic combination on neutrophils

[0054] As shown in Table 2, the number 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 was significantly reduced. The number of neutrophils in the control groups 1, 2, and 3 was 51.8±3.50, 54.6±4.66, and 52.6±5.01, respectively, and compared with the model group, p<0.05, so the recovery effect of the number of neutrophils in the control groups 1-3 was statistically significant. The number of neutrophils in the comparative example 4, experimental examples 1, 2, 3 and comparative example 5 groups were 52.4±4.32, 55.3±4.41, 61.5±2.81, 55.1±4.16 and 53.0±4.94, respectively. Compared with the model group, p<0.05&p<0.01&p<0.001&p<0.01&p<0.05, so the recovery of the number of neutrophils in the comparative example 4, experimental examples 1, 2, 3 and comparative example 5 groups was statistically significant. See details Figure 3 and Figure 4 .

[0055] The above results show that the recovery of the number of neutrophils in Experimental Examples 1, 2, and 3 groups is statistically significant, and p<0.01&p<0.001&p<0.01, compared with the recovery of the number of neutrophils in Comparative Examples 1, 2, and 3 groups, all p<0.05, indicating that it has advantages, suggesting that the combination of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL and prebiotic 2'-FL 250 μg / mL three-component combination group (Experimental Example 1), the combination of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL and prebiotic 2'-FL 375 μg / mL three-component combination group (Experimental Example 2), the combination of Bifidobacterium lactis Bb-12 The combination group of three components (Experimental Example 3) of 0.00914 μg / mL, 0.152 μg / mL of Bifidobacterium lactis HN019 and 375 μg / mL of prebiotic 2'-FL has a synergistic effect compared with the use of each component alone.

[0056] From the absolute value, the higher the absolute value of neutrophil count, the better the effect of enhancing immunity. The number of neutrophils in Experimental Example 2 was 61.5, which was higher than 52.4 in Comparative Example 4 and 53.0 in Comparative Example 5, indicating that Experimental Example 2 had an advantage over Comparative Example 4 and 5. From the significance level, Experimental Example 2 had a significant effect of three stars, and Experimental Examples 1 and 3 had a significant effect of two stars, which were both superior to the significant effect of one star in Comparative Example 4 and 5. The combination group of the three components of the composition 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 combination group 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 (Experimental Example 2), and the combination group 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 (Experimental Example 3) are all better than the combination group of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 The three-component combination group of 0.0076 μg / mL and prebiotic 2'-FL 500 μg / mL (Comparative Example 4) and the three-component combination group 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 advantages.

[0057] (3) Effects of synbiotic composition on T cells

[0058] As shown in Table 2, the fluorescence intensity 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 reduced. The fluorescence intensity of T cells in the comparison groups 1, 2, and 3 were 23669±3671 pixels, 22582±2749 pixels, and 21345±2285 pixels, respectively, and compared with the model group, p<0.05, so the recovery effect of comparison groups 1-3 on the fluorescence intensity of T cells was statistically significant. The T cell fluorescence intensity of the comparative example 4, experimental examples 1, 2, 3 and comparative example 5 groups was 23438±3178 pixels, 26110±1249 pixels, 25045±2238 pixels, 25163±2583 pixels and 23820±2683 pixels, compared with the model group, p<0.05&p<0.001&p<0.01&p<0.01&p<0.01, so the recovery of T cell fluorescence intensity in comparative example 4, experimental examples 1, 2, 3 and comparative example 5 groups was statistically significant. See for details Figure 5 and Figure 6 .

[0059] The above results show that the recovery of T cell fluorescence intensity in Experimental Examples 1-3 and Comparative Example 5 groups is statistically significant, and p<0.001&p<0.01&p<0.01&p<0.01, compared with the T cell fluorescence intensity in Comparative Examples 1, 2, and 3 groups p<0.05, indicating that it has advantages, suggesting that the combination of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL and prebiotic 2′-FL 250 μg / mL three-component combination group (Experimental Example 1), the combination of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL and prebiotic 2′-FL 375 μg / mL three-component combination group (Experimental Example 2), the combination of Bifidobacterium lactis Bb-12 The three-component combination group of 0.00914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL and prebiotic 2'-FL 375 μg / mL (Experimental Example 3) and the three-component combination group 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 synergistic effects compared with the use of each component alone.

[0060] From the absolute value, the higher the absolute value of T cell fluorescence intensity, the better the effect of enhancing immunity. The fluorescence intensity of T cells in Experimental Examples 1, 2, and 3 were 26110, 25045, and 25163 pixels, respectively, which were higher than 23438 pixels in Comparative Example 4 and 23820 pixels in Comparative Example 5, indicating that Experimental Examples 1, 2, and 3 were superior to Comparative Example 4 and Comparative Example 5. From the significance level, Experimental Example 1 had a significant effect of three stars, and Experimental Examples 2, 3, and Comparative Example 5 had a significant effect of two stars, which was superior to Comparative Example 4, which had a significant effect of one star. The combination group of the three components of the composition 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 combination group 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 (Experimental Example 2), and the combination group 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 (Experimental Example 3) are all better than the combination of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 The three-component combination group of 0.0076 μg / mL and prebiotic 2'-FL 500 μg / mL (Comparative Example 4) and the three-component combination group 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 advantages.

[0061] In summary, in terms of enhancing immunity, the synbiotic composition has a synergistic effect compared with each component alone, characterized in that, by mass, the content ratio of Bifidobacterium lactis Bb-12 probiotics, Bifidobacterium lactis HN019 probiotics and prebiotic 2'-FL is 0.1-1:0.17-1.66:2735-4103. It was determined that the amount of Bifidobacterium lactis Bb-12 in the synbiotic composition is 2×10 3 ~2×10 5 CFU / mL, the amount of Bifidobacterium lactis HN019 was 2×10 4 ~2×10 5 CFU / mL.

[0062] Example 3 Synbiotic composition for promoting growth and development

[0063] The experimental subjects in the experiment were wild-type AB strain zebrafish 6 hours to 4 days after fertilization (6hpf-4dpf), and the experimental subjects in the experiment on the effect of the synbiotic composition on fat absorption were 5-6dpf melanin allele mutant zebrafish (albino). Zebrafish source: Hangzhou Huante Company Fish Farming Center; Gender: Zebrafish fry are not divided into males and females, and they can only be divided into males and females after completing the development of sexual organs at 2.5-3 months; Feeding method: Zebrafish are all raised in 28℃ fish farming water (water quality: 200mg instant sea salt is added to each 1L reverse osmosis water, the conductivity is 450-550μS / cm; pH is 6.5-8.5; hardness is 50-100mg / L CaCO 3 ); Synbiotics addition method: Dissolve in water and give to samples, and directly prepare fish water according to the experimental concentration.

[0064] The specific experiments are as follows: (1) Experiment on the effect on body length: The above zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well. The volume of each well was 3 mL, and the liquid was changed every day. A normal control group and a positive control group were set up. For the zebrafish in the positive control group, Swisse "Calcium & Vitamin D" (Swisse, batch number 33152A) was given in water, and the experimental dose was 500 μg / mL. Water-soluble Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and prebiotic 2'-FL were added to each well so that the concentrations of Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and prebiotic 2'-FL in the well were 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 dissecting 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 statistical analysis results of this indicator were used to evaluate the effect of the sample on body length. (2) Experiment on the effect on fat absorption: The above zebrafish were randomly selected in beakers, and 30 zebrafish were treated in each beaker. The water-soluble was given egg yolk powder for 3 hours and then washed off. The water-soluble was given a synbiotic composition (concentrations shown in Table 3) and a positive control group. A normal control group (normal feeding group) was also set up. The capacity of each beaker was 20 mL. After treatment at 28°C for 16 hours, egg yolk powder was added to all groups. After continuing to treat for 8 hours, Oil Red O was given for overall fat staining. After staining, 10 zebrafish were randomly selected from each experimental group and placed under a dissecting microscope for photography. NIS-Elements D 3.20 advanced image processing software was used to collect data, and the staining intensity of the blood vessels from the intestine to the tail was analyzed. The statistical analysis results of this indicator were used to evaluate the effect of the sample on fat absorption.

[0065] The statistical results were expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant. The specific statistical methods were as follows: Statistical analysis between the two groups: The data were explored and the exploration results were obtained by KS test. If the normality test p > 0.05 (i.e., it obeys the normal distribution), the independent sample T test in the parameter test was used for statistical analysis; if the normality test p < 0.05 (i.e., it does not obey the normal distribution), two independent samples in the non-parametric test were used for statistical analysis.

[0066] Table 3. Results of the efficacy of synbiotic composition in promoting growth and development (n=10)

[0067]

[0068] Compared with the normal control group, *p<0.05, **p<0.01, ***p<0.001;

[0069] Compared with Experimental Example 1, △ p<0.05, △△ p<0.01;

[0070] Compared with Experimental Example 2, # p<0.05, ## p<0.01, ### p<0.001;

[0071] Compared with Experimental Example 3, ^ p<0.05, ^^ p<0.01, ^^^ p<0.001.

[0072] (1) Effect of synbiotic composition on body length

[0073] As can be seen from Table 3, the body length of the normal control group is 3.52±0.025mm, and the body lengths of the comparison groups 1, 6, and 7 are 3.50±0.029mm, 3.47±0.039mm, and 3.55±0.041mm, respectively. Compared with the normal group, p>0.05, so the increase in body length of comparison groups 1, 6, and 7 is not obvious. The body lengths of comparison groups 4 and 5 are 3.57±0.034mm and 3.51±0.039mm, respectively, compared with the normal group, p>0.05, and the increase in body length is not obvious. The body lengths of experimental examples 1, 2, and 3 are 3.60±0.014mm, 3.64±0.023mm, and 3.66±0.019mm, respectively, compared with the normal group, p<0.05&p<0.01&p<0.001, so the increase in body length of experimental examples 1, 2, and 3 is statistically significant. In addition, compared with the experimental example 3 group, the comparison examples 1, 6, and 7 groups had p<0.001&p<0.001&p<0.05. Figure 7 and Figure 8 .

[0074] The above results show that the increase in body length in Experimental Examples 1, 2, and 3 groups is statistically significant, compared with the insignificant increase in body length in Comparative Examples 1, 4, 5, 6, and 7 groups, which indicate an advantage, suggesting that the combination of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.152 μg / mL and prebiotic 2'-FL 250 μg / mL three-component combination group (Experimental Example 1), the combination of Bifidobacterium lactis Bb-12 0.0914 μg / mL, Bifidobacterium lactis HN019 0.0152 μg / mL and prebiotic 2'-FL 375 μg / mL three-component combination group (Experimental Example 2), the combination of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN0190.152 μg / mL and prebiotic 2'-FL The 375 μg / mL three-component combination group (Experimental Example 3) has a synergistic effect compared with the use of each component alone. The above results also suggest that Experimental Examples 1, 2, and 3 have advantages over Comparative Examples 4 and 5. From the comparison between the groups, Comparative Examples 4, 5 and Experimental Example 1 compared with Experimental Example 3, p<0.05&p<0.05&p<0.01. The combination group of the three components of the composition 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 combination group 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 (Experimental Example 2), and the combination group 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) are all better than the combination of Bifidobacterium lactis Bb-12 0.00914 μg / mL, Bifidobacterium lactis HN019 The three-component combination group of 0.0076 μg / mL and prebiotic 2'-FL 500 μg / mL (Comparative Example 4) and the three-component combination group 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 advantages.

[0075] (2) Effect of synbiotic composition on fat absorption

[0076] As shown in Table 3, the staining intensity of the blood vessels from the intestine to the tail in the normal control group was 49065±2165 pixels, and the staining intensity of the blood vessels from the intestine to the tail in the comparison groups 1 and 6 was 64507±2991 pixels and 64005±3010 pixels, respectively, compared with the normal group p<0.001&p<0.001, so the fat absorption promoting effects of comparison groups 1 and 6 were statistically significant. The staining intensity of the blood vessels from the intestine to the tail in the comparison group 7 was 55338±3197 pixels, compared with the normal group p>0.05, and the fat absorption promoting effect was not obvious. The staining intensity 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, so the effects of promoting fat absorption in Comparative Example 4, Experimental Examples 1-3 and Comparative Example 5 were statistically significant. Fig. 9 and Fig.10 .

[0077] From the absolute value point of view, the higher the absolute value of the staining intensity of the blood vessels from the intestine to the tail, the better the effect of promoting growth and development. The staining intensity of the blood vessels from the intestine to the tail in Experimental Examples 2 and 3 were 70393 and 67676 pixels, respectively, which were higher than 64507, 64005, and 55338 pixels in Comparative Examples 1, 6, and 7, and 62081, 62906, and 60633 pixels in Comparative Example 4, Experimental Example 1, and Comparative Example 5, indicating that Experimental Examples 2 and 3 have advantages over Comparative Example 5. The results show that the three-component combination group 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 three-component combination group 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) have a synergistic effect compared with the use of each component alone. The above results also suggest that the three-component combination group of the composition 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 three-component combination group 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) are both better than the three-component combination group 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 1). 0.0914μg / mL, Bifidobacterium lactis HN019 0.152μg / mL and prebiotic 2'-FL 250μg / mL three-component combination group (Experimental Example 1), the combination of Bifidobacterium lactis Bb-12 0.000914μg / mL, Bifidobacterium lactis HN019 0.0152μg / mL and prebiotic 2'-FL 375μg / mL three-component combination group (Comparative Example 5) has advantages.

[0078] In summary, in terms of promoting growth and development, the synbiotic composition has a synergistic effect compared with each component alone, characterized in that, by mass, the content ratio of Bifidobacterium lactis Bb-12 probiotics, Bifidobacterium lactis HN019 probiotics and prebiotic 2'-FL is 0.1-1:0.17-1.66:2735-4103. It was determined that the amount of Bifidobacterium lactis Bb-12 in the synbiotic composition is 2×10 3 ~2×10 5 CFU / mL, the amount of Bifidobacterium lactis HN019 was 2×10 4 ~2×10 5 CFU / mL.

[0079] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A pharmaceutical composition comprising Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and 2'-FL, wherein the mass ratio of Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and 2'-FL is 0.1-1:0.17-1.66:2735-4103.

2. The pharmaceutical composition of claim 1, further comprising an excipient, a diluent and / or a carrier.

3. The pharmaceutical composition of claim 1, 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. Use of Bifidobacterium lactis Bb-12, Bifidobacterium lactis HN019 and 2'-FL as defined in any one of claims 1 to 3 in the preparation of a medicament for enhancing immunity of a subject and promoting growth and development of a subject, as well as promoting growth and development of a subject.

5. The method of claim 4, wherein the enhancing the immunity of the subject comprises: One or more of enhancing the humoral immunity and / or cellular immunity of the subject, increasing the number of immune cells of the subject, or improving the activity of immune cells of the subject; The promoting the growth and development of the subject includes: promoting one or more of the following: increasing the length or height of the subject, increasing the weight of the subject, or promoting fat absorption.

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

7. The use according to claim 5, wherein the subject is a zebrafish or a mammal.

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