Probiotic agent for enhancing immunity and improving intestinal flora and application thereof

Abstract

According to the probiotic agent for enhancing the immunity and improving the intestinal flora and the application of the probiotic agent, five specific different types of bifidobacterium strains are compounded, potential interaction exists among the five strains, and the five strains can cooperate with one another, so that the effect of enhancing the immunity and improving the intestinal flora is achieved. The bifidobacteria has the advantages that the bifidobacteria has synergistic interaction in the aspects of promoting the growth of immune organs and increasing the total quantity and diversity of intestinal flora, the bifidobacteria is matched with basic fermentation strains for yogurt fermentation, meanwhile, the composite prebiotics are added for synergistic yogurt fermentation, the quantity of the bifidobacteria at the end of a shelf life after the yogurt fermentation is finished is still not less than 1 * 10 < 8 > CFU / mL or 1 * 10 < 8 > CFU / g, and the content of the bifidobacteria is not less than the stability of lactic acid bacteria in the yoghourt is remarkably improved, so that the number of viable bacteria which enter the intestinal tract of a human body and play roles is remarkably increased, and the yoghourt has relatively good tolerance in the intestinal tract; the prepared yoghurt is fine and smooth in texture, rich in taste and rich in fragrance; in addition, the immune decline and intestinal flora disorder caused by cyclophosphamide (CTX) can be remarkably improved, and a positive effect on immunological enhancement is achieved.

Description

Background Art

[0002] As the pace of modern life accelerates, more and more young people are in sub-health conditions, and people are paying more and more attention to health issues, especially the improvement of immunity. Probiotics are active microorganisms that are beneficial to the host. When they reach the intestine in a certain number, they can change the composition of the host's intestinal flora in specific parts, playing an important role in maintaining the balance of intestinal ecology and enhancing immunity. In recent years, more and more studies have shown that probiotics have a positive effect on improving intestinal flora and enhancing host immunity.

[0003] As a good carrier for probiotics, milk matrix is ​​considered to be one of the best ways to supplement high-dose probiotics. Common probiotic yogurts on the market usually only contain the basic fermentation strains of Streptococcus salivarius thermophilus subsp. and Lactobacillus delbrueckii subsp. bulgaricus. Although these basic fermentation strains are mature in yogurt fermentation, their probiotic effects are very limited. This is mainly because Streptococcus salivarius thermophilus subsp. and Lactobacillus delbrueckii subsp. bulgaricus subsp. are difficult to tolerate the low pH gastric acid environment, resulting in an extremely limited number of live bacteria that enter the intestines and play a role. Therefore, in order to give full play to the live bacteria function of yogurt, it is necessary to add other highly active probiotics to prepare highly active fermented milk products.

[0004] Bifidobacterium is a common functional probiotic strain that has good tolerance to the gastrointestinal environment and can play a probiotic role in the intestine. Our previous studies have shown that the 100 billion active bifidobacterium yogurt fermented with a composite bifidobacterium agent (including BLa36, BL21, BBr16, BAC30, BI45) can tolerate the stress environment in the gastrointestinal tract better than ordinary yogurt, and exhibits excellent probiotic properties for improving constipation (CN118652824A). In addition, bifidobacteria are also considered to be immunomodulators or biomarkers of human diseases, which can enhance immunity by improving the phagocytic function of macrophages. Our team has disclosed two strains of bifidobacteria that may have a positive effect on immunomodulation, BLa80 (CN117959343A) and BBr16 (CN118185842A). However, it is not yet known whether milk-based bifidobacteria can also play an immunomodulatory role in the body. Summary of the invention

[0005] The invention discloses a probiotic agent for increasing immunity and improving intestinal flora and application thereof, aiming to solve the technical problems existing in the prior art.

[0006] The present invention adopts the following technical solutions: On the one hand, the present invention provides a probiotic for increasing immunity and improving intestinal flora, the probiotic comprising a composite bifidobacterium agent and a basic agent; the strain in the composite bifidobacterium agent is a strain of animal Bifidobacterium lactis subsp. Bifidobacterium animalis subsp. lactis BLa80 strain, Bifidobacterium longum subsp. longum with a deposit number of CGMCC No. 10452 Bifidobacterium longum subsp. longum BL21 strain, Bifidobacterium breve with a deposit number of CGMCC No. 24471 Bifidobacterium breve BBr16 strain, Bifidobacterium adolescentis with a deposit number of CGMCC No. 19884 Bifidobacterium adolescentis BAC30 strain, Bifidobacterium longum subsp. infantis with a deposit number of CGMCC No. 15134 Bifidobacterium longum subsp. infantile BI45 strain; the strain in the basic bacterial agent is Streptococcus salivarius thermophilic subspecies with a preservation number of CGMCC No. 15752 Streptococcus thermophilus ST81 strain and Lactobacillus delbrueckii subsp. bulgaricus with a deposit number of CGMCC No. 15751 Lactobacillus delbrueckii subsp. bulgaricus LB42 strain composition; wherein the total viable count of the composite bifidobacterium inoculum is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0007] Preferably, the ratio of the viable cell count of the BLa80 strain, BL21 strain, BBr16 strain, BAC30 strain, and BI45 strain is (1-10):(1-10):(1-10):(1-5):(1-5).

[0008] Preferably, the ratio of the viable counts of the ST81 strain to the LB42 strain is 30:(0.001-10); the mass ratio of the composite bifidobacterium agent to the basic agent is (10-300):(10-40).

[0009] Preferably, the dosage form of the probiotic includes solution, lyophilized powder, capsule, tablet or granule.

[0010] When the probiotic is a freeze-dried powder, it is prepared by the following preparation method: the relevant strains are inoculated into the culture medium for activation and fermentation to obtain fermentation liquid; the fermentation liquid is centrifuged, mixed with a freeze-dried protective agent and then freeze-dried to obtain bacterial powder of each strain; the bacterial powder of each strain is mixed according to the ratio of the number of live bacteria to obtain.

[0011] In a second aspect, the present invention provides an application of the above-mentioned probiotic agent in yogurt fermentation, and the yogurt fermentation method comprises: mixing raw cow's milk with a composite prebiotic and auxiliary additives, sterilizing and cooling, and using the mixture as a fermentation base; inoculating a composite bifidobacterium agent and a basic agent into the fermentation base for fermentation.

[0012] Preferably, according to the composite bifidobacterium agent provided in the first aspect, the basic fermentation strains are compounded, and composite prebiotics are added to synergistically ferment yogurt. The composite prebiotics can effectively promote the proliferation of bifidobacteria. After the yogurt is fermented, the number of bifidobacteria is 1×10 8 CFU / mL or 1×10 8 CFU / g, the yogurt has a delicate texture, rich taste and strong aroma.

[0013] Preferably, the inoculation amount of the composite bifidobacterium agent is 10-300 g / T; and / or, the inoculation amount of the basic agent is 10-40 g / T; and / or, the inoculation amount of the composite prebiotic is 0.1-5 kg / T.

[0014] Preferably, the compound prebiotics include fructooligosaccharides, xylooligosaccharides or isomaltooligosaccharides; and / or the auxiliary additives include white sugar, cream or whey protein powder.

[0015] Preferably, the raw milk accounts for 80-95wt% of the fermentation base, the compound prebiotics accounts for 0.01-1wt% of the fermentation base; the white sugar accounts for 0.1-8wt% of the fermentation base; the light cream accounts for 0.1-5wt% of the fermentation base, and the whey protein powder accounts for 1-10wt% of the fermentation base.

[0016] Preferably, the homogenization pressure of the raw milk, white sugar, cream, whey protein powder and compound prebiotics is 180-220 bar, and the raw milk, white sugar, cream, whey protein powder and compound prebiotics are sterilized at 90-99° C. for 5-15 min after being mixed, and then placed in cold water to cool to 30-40° C.; and / or, the fermentation temperature is 37-43° C., the fermentation time is 5-10 h, and the fermentation endpoint is when the acidity reaches 70-80° T or pH=4.3-4.5.

[0017] In a third aspect, the present invention provides the use of the above-mentioned probiotics in the preparation of products for increasing immunity and regulating intestinal flora, wherein the products include foods, health products or medicines.

[0018] Preferably, the product for increasing immunity and regulating intestinal flora prepared by the probiotics described above, when the body ingests cyclophosphamide, a drug used to treat various malignant tumors and autoimmune diseases, which causes low immunity and intestinal mucosal damage to the body, can significantly improve body weight, immune organ index and immunoglobulin content in serum through the probiotics described above or yogurt prepared by the probiotics or food, health products or medicines prepared by the probiotics, and has an immune-enhancing effect, while also having a positive regulatory effect on intestinal flora imbalance.

[0019] The technical solution adopted by the present invention can achieve the following beneficial effects: The present invention provides a probiotic agent for increasing immunity and improving intestinal flora and its application. Five specific different types of bifidobacterium strains are compounded to increase immunity and regulate intestinal flora. There is potential interaction between the five strains, which can cooperate with each other to promote the growth of immune organs and increase the total number and diversity of intestinal flora. The five strains are used in yogurt fermentation with basic fermentation strains, and composite prebiotics are added to ferment yogurt. The number of bifidobacteria at the end of the shelf life after the fermentation of the yogurt is still not less than 1×10 8 CFU / mL or 1×10 8 CFU / g, significantly improving the stability of lactic acid bacteria in yogurt, significantly increasing the number of live bacteria that enter the human intestine and play a role, and having better tolerance in the intestine; the yogurt produced has a delicate texture, rich taste, and strong aroma; and can significantly improve the immune decline and intestinal flora disorder caused by cyclophosphamide (CTX), and has a positive effect on immune enhancement.

[0020] The BLa80 strain involved in the present invention is classified and named as Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. lactis The depository is the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is March 5, 2018. The deposit number is CGMCC No. 15410. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0021] The BL21 strain involved in the present invention is classified as Bifidobacterium longum subsp. Bifidobacterium longum subsp. longumThe depository is the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is January 27, 2015. The deposit number is CGMCC No. 10452. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0022] The BBr16 strain involved in the present invention is classified and named Bifidobacterium breve Bifidobacterium breve The depository is the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is March 7, 2022. The deposit number is CGMCC No. 24471. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0023] The BAC30 strain involved in the present invention is classified and named Bifidobacterium adolescentis Bifidobacterium adolescentis The depository is the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is May 28, 2020. The deposit number is CGMCC No. 19884. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0024] The BI45 strain involved in the present invention is classified as Bifidobacterium longum subsp. infantis Bifidobacterium longum subsp. infantile The depository is the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is December 27, 2017. The deposit number is CGMCC No. 15134. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0025] The ST81 strain involved in the present invention is classified and named Streptococcus salivarius subsp. thermophilus Streptococcus salivarius subsp . thermophilus The depository is the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is May 11, 2018. The deposit number is CGMCC No. 15752. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0026] The LB42 strain involved in the present invention is classified and named as Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus delbrueckii subsp. bulgaricus The depository is the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is May 11, 2018. The deposit number is CGMCC No. 15751. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure shows the statistical graph of the weight change of mice provided in Experimental Example 1 of the present invention. Figure 2 Shown is a statistical graph of the results of immune organ index changes provided in Experimental Example 2 of the present invention; Figure 3 The figure shows the statistical graph of the mouse immune serum index results provided in Experimental Example 3 of the present invention; Figure 4 Shown is a statistical diagram of mouse α diversity results provided by Experimental Example 4 of the present invention; Figure 5 Shown is a statistical graph of the abundance results of the mouse phylum level provided in Experimental Example 5 of the present invention; Figure 6 Shown is a statistical graph of the abundance results of the genus Mouse provided in Experimental Example 5 of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention and the corresponding drawings. In the description of the present invention, it should be noted that the term "or" is usually used in the sense of including "and / or", unless the content clearly indicates otherwise.

[0029] Example 1 Preparation of probiotic powder: The BLa80, BL21, BBr16, BAC30, BI45, ST81 and LB42 strains were inoculated into MRS liquid medium, cultured at 37°C for 24 h for activation, and activated twice to obtain activation solution. The activation solution was inoculated into MRS liquid medium at an inoculum of 3% (v / v), cultured at 37°C for 18 h to obtain bacterial solution. The bacterial solution was centrifuged at 4000 rpm for 5 min at 4°C, filtered to obtain bacterial bodies, mixed with 5 times the mass of protective agent, and freeze-dried to obtain BLa80 bacterial powder, BL21 bacterial powder, BBr16 bacterial powder, BAC30 bacterial powder, BI45 bacterial powder, ST81 bacterial powder and LB42 bacterial powder. The number of viable bacteria was 1×10 12 CFU / g, 5×10 11 CFU / g, 1×10 11 CFU / g, 1×10 12 CFU / g, 6×10 11 CFU / g, 1×10 11 CFU / g, 1×10 11 CFU / g.

[0030] Preparation of composite bifidobacterium agent: BLa80 bacterial powder, BL21 bacterial powder, BBr16 bacterial powder, BAC30 bacterial powder and BI45 bacterial powder in a mass ratio of 1:1:1:1:1 were evenly mixed to obtain a composite bifidobacterium agent.

[0031] Preparation of basic fermentation agent: The basic fermentation agent was obtained by mixing ST81 bacterial powder and LB42 bacterial powder in a mass ratio of 30:0.001.

[0032] Preparation of yogurt containing the above-mentioned probiotics: fermenting yogurt with basic fermentation bacteria and compound bifidobacterium bacteria, and adding compound prebiotics to promote the rapid proliferation of bifidobacteria. The specific operation steps are as follows: (1) Preheat the raw milk to 60℃, then add white sugar, cream, whey protein powder, and compound prebiotics and homogenize to fully dissolve, wherein the raw milk accounts for 90% of the total weight, the cream accounts for 2% of the total weight, the whey protein powder accounts for 2%, the white sugar accounts for 5%, and the compound prebiotics accounts for 1%. The homogenization pressure is 180 bar, sterilized at 95℃ for 15min, and then quickly cooled to 40℃ in cold water. The compound prebiotic formula is 3kg / T of xylooligosaccharide inoculation amount, 0.2kg / T of fructooligosaccharide inoculation amount, and 0.2kg / T of isomaltooligosaccharide inoculation amount. The compound prebiotics can effectively promote the proliferation of bifidobacteria.

[0033] (2) The basic fermentation agent is inoculated at an inoculation rate of 40 g / T and the composite bifidobacterium agent is inoculated at an inoculation rate of 300 g / T into the above sterilized fermentation base, the mass ratio of the composite bifidobacterium agent to the basic agent is 10:1, the fermentation temperature is 37-43°C, the fermentation time is 5-10 hours, and the fermentation is stopped when the acidity reaches 70-80°T and the pH is 4.3-4.5. After refrigeration at 4-10°C overnight, the finished product is packaged at 200 g / bottle.

[0034] (3) The number of live bifidobacteria in the yogurt was counted. The number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 6×10 8 CFU / g, the number of viable bacteria after refrigeration at 4°C for 28 days is 2.5×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0035] Example 2 The implementation method of Example 2 is the same as that of Example 1, except that: BLa80 bacterial powder, BL21 bacterial powder, BBr16 bacterial powder, BAC30 bacterial powder and BI45 bacterial powder in a mass ratio of 2:2:2:1:1 were evenly mixed to obtain a composite bifidobacterium agent.

[0036] The number of live bifidobacteria in the yogurt was counted, and the number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 8×10 8 CFU / g, the number of viable bacteria after refrigeration at 4°C for 28 days is 4×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0037] Example 3 The implementation method of Example 3 is the same as that of Example 1, except that: BLa80 bacterial powder, BL21 bacterial powder, BBr16 bacterial powder, BAC30 bacterial powder and BI45 bacterial powder in a mass ratio of 10:10:10:1:1 were evenly mixed to obtain a composite bifidobacterium agent.

[0038] The number of live bifidobacteria in the yogurt was counted, and the number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 3×10 8 CFU / g, the number of viable bacteria after refrigeration at 4°C for 28 days is 1.5×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0039] Example 4 The implementation method of Example 4 is the same as that of Example 1, except that: BLa80 bacterial powder, BL21 bacterial powder, BBr16 bacterial powder, BAC30 bacterial powder and BI45 bacterial powder in a mass ratio of 1:1:1:5:5 were evenly mixed to obtain a composite bifidobacterium agent.

[0040] The number of live bifidobacteria in the yogurt was counted. The number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 2×10 8 CFU / g, the number of viable bacteria after refrigeration at 4℃ for 28 days is 1×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0041] Example 5 The implementation method of Example 5 is the same as that of Example 2, except that: The preparation ratio of the basic fermentation bacterial agent is 3:1, and the ST81 bacterial powder and LB42 bacterial powder are mixed evenly.

[0042] The number of live bifidobacteria in the yogurt was counted, and the number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 6.5×10 8 CFU / g, the number of viable bacteria after refrigeration at 4°C for 28 days is 3.5×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0043] Example 6 The implementation method of Example 6 is the same as that of Example 2, except that: The preparation ratio of the basic starter is 10:1, and the ST81 bacterial powder and LB42 bacterial powder are mixed evenly.

[0044] The number of live bifidobacteria in the yogurt was counted, and the number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 9×10 8 CFU / g, the number of viable bacteria after refrigeration at 4℃ for 28 days is 5×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0045] Example 7 The implementation method of Example 7 is the same as that of Example 6, except that: The mass ratio of the composite bifidobacterium agent to the basic agent is 30:1.

[0046] The number of live bifidobacteria in the yogurt was counted, and the number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 7×10 8 CFU / g, the number of viable bacteria after refrigeration at 4°C for 28 days is 3×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0047] Example 8 The implementation method of Example 8 is the same as that of Example 6, except that: The inoculation amount of the basic fermentation bacteria was changed to 10g / T, and the inoculation amount of the composite bifidobacterial agent remained at 300g / T.

[0048] The number of live bifidobacteria in the yogurt was counted, and the number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 7×10 8 CFU / g, the number of viable bacteria after refrigeration at 4°C for 28 days is 2×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0049] Example 9 The implementation method of Example 9 is the same as that of Example 6, except that: The inoculation amount of the basic fermentation bacteria was changed to 10g / T, and the inoculation amount of the composite bifidobacterial agent was changed to 100g / T.

[0050] The number of live bifidobacteria in the yogurt was counted, and the number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 3×10 8 CFU / g, the number of viable bacteria after refrigeration at 4°C for 28 days is 1.5×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0051] Example 10 The implementation method of Example 10 is the same as that of Example 6, except that: The inoculation amount of the basic fermentation bacteria was changed to 20g / T, and the inoculation amount of the composite bifidobacterial agent was changed to 200g / T.

[0052] The number of live bifidobacteria in the yogurt was counted, and the number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 4.5×10 8 CFU / g, the number of viable bacteria after refrigeration at 4℃ for 28 days is 1×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0053] Embodiment 11 The implementation method of Example 11 is the same as that of Example 6, except that: The inoculation amount of the basic fermentation bacteria remained unchanged at 40g / T, and the inoculation amount of the composite bifidobacterial agent was changed to 200g / T.

[0054] The number of live bifidobacteria in the yogurt was counted, and the number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 5.5×10 8 CFU / g, the number of viable bacteria after refrigeration at 4°C for 28 days is 1.5×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0055] Example 12 The implementation method of Example 12 is the same as that of Example 6, except that: The preparation of the composite bifidobacterium agent was changed to a four-bacteria mixture, specifically, BL21 bacterial powder, BBr16 bacterial powder, BAC30 bacterial powder, and BI45 bacterial powder in a mass ratio of 2:2:1:1 were evenly mixed to obtain the composite bifidobacterium agent.

[0056] The number of live bifidobacteria in the yogurt was counted, and the number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 4.5×10 8 CFU / g, the number of viable bacteria after refrigeration at 4℃ for 28 days is 1×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0057] Embodiment 13 The implementation method of Example 13 is the same as that of Example 6, except that: The preparation of the composite bifidobacterium agent was changed to a four-bacteria mixture, specifically, BLa80 bacterial powder, BL21 bacterial powder, BAC30 bacterial powder, and BI45 bacterial powder in a mass ratio of 2:2:1:1 were evenly mixed to obtain the composite bifidobacterium agent.

[0058] The number of live bifidobacteria in the yogurt was counted, and the number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 6×108 CFU / g, the number of viable bacteria after refrigeration at 4°C for 28 days is 2.5×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0059] Embodiment 14 The implementation method of Example 14 is the same as that of Example 6, except that: The preparation of the composite bifidobacterium agent was changed to a three-bacteria mixture, specifically, BL21 bacterial powder, BAC30 bacterial powder, and BI45 bacterial powder in a mass ratio of 2:1:1 were evenly mixed to obtain the composite bifidobacterium agent.

[0060] The number of live bifidobacteria in the yogurt was counted, and the number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration was 2.5×10 8 CFU / g, the number of viable bacteria after refrigeration at 4℃ for 28 days is 1×10 8 CFU / g. The counting method refers to GB / T4789.35.

[0061] ; From the data results in Table 1, it can be seen that the yogurt product obtained by fermenting the composite bifidobacterium inoculant with the basic fermentation inoculant involved in the present invention counts the number of live bifidobacteria in the yogurt. The number of live bifidobacteria in the composite bifidobacterium yogurt before refrigeration is 180 billion CFU / bottle. As the refrigeration time increases, the number of live bifidobacteria decreases, but after refrigeration (0-4°C) for 28 days, the number of live bifidobacteria is still 100 billion CFU / bottle, which indicates that the composite bifidobacterium yogurt has good stability and can still maintain a high number of live bacteria after refrigeration for a period of time.

[0062] Experimental Example 1: Establishment of animal model (1) Experimental animals Fifty SPF-grade male ICR mice (20–25 g, 4 weeks old) were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. with license number SCXK (Beijing) 2019-0010. All animal experiments were approved by the Ethics Committee of Tianjin Agricultural College (license number No. 2023LLSC08). During the study, the animals were housed in standard cages under laboratory conditions (temperature 22–25 °C, relative humidity 40–60%), with a 12-h light and dark cycle per day, and free access to food and water for one week.

[0063] (2) Experimental groups After a week of adaptation, the mice were divided into 5 groups (10 in each group). As shown in Table 2, they are normal control group (NC), model group (MC), low-dose composite probiotic yogurt group (LPYC, probiotic concentration: CFU / 200g), medium-dose composite probiotic yogurt group (MPYC, probiotic concentration: CFU / 200g), high-dose composite probiotic yogurt group (HPYC, probiotic concentration: CFU / 200g). The control group and the model group were gavaged with 200 μL of normal saline, and each yogurt group was gavaged with 200 μL of probiotic yogurt for 14 days.

[0064] .

[0065] (3) Establishment of animal model The 7th, 8th and 9th days were the modeling period. The NC group was still gavaged with 200 μL of normal saline; the MC group received intraperitoneal injection of cyclophosphamide (CTX) once a day during this period (at a dose of 80 mg / kg) to induce immunosuppression, and supplemented with 200 μL of normal saline 4 hours after CTX intervention; the LPYC, MPYC and HPYC groups received intraperitoneal injection of cyclophosphamide (CTX) once a day during this period (at a dose of 80 mg / kg) to induce immunosuppression, and supplemented with 200 μL of compound probiotic yogurt 4 hours after CTX intervention. After three consecutive days of cyclophosphamide injection, the weight of the mice decreased significantly, indicating that the model was successfully established.

[0066] At the end of the experiment, mice were sacrificed by cervical dislocation after eye blood sampling and serum collection. Spleen and thymus were collected and weighed, and small intestinal tissue and colon contents were removed for further analysis.

[0067] (4) Evaluation of weight changes under different dietary interventions Cyclophosphamide is a widely used drug for the treatment of various malignancies and autoimmune diseases, however, it can also cause toxic damage to the body. Intraperitoneal injection of cyclophosphamide (CTX) can produce immunosuppression and intestinal mucosal damage.

[0068] The body weight of mice was recorded regularly during the experimental period of animal model establishment. Figure 1 The figure shows the statistical graph of the changes in the weight and immune organ index of mice provided in Experimental Example 1 of the present invention. Figure 1As shown in Table 3, compared with the normal control (NC) group, after intraperitoneal injection of CTX for three consecutive days (days 7, 8, and 9) during the modeling period, the body weight of mice in the model control (MC) group decreased significantly by 2.22 g, while the supplementation of compound probiotic yogurt could significantly alleviate this downward trend. The body weight of mice in the LPYC group, MPYC group, and HPYC group only decreased by 0.56 g, 0.35 g, and 0.61 g, respectively, which alleviated the weight loss trend by 74.8%, 84.2%, and 72.5%, respectively, compared with the MC group. During the entire 14-day experimental period, the body weight of mice in the MC group decreased by 48.3% compared with the NC group, while the supplementation of compound probiotic yogurt could significantly delay the weight loss caused by CTX. Compared with the MC group, the weight gain of the LPYC group, the MPYC group, and the HPYC group increased by 47.4%, 52.6%, and 52.9%, respectively.

[0069]

[0070] Experimental Example 2: Evaluation of organ index under different dietary interventions The spleen and thymus are two important immune organs in the human body and play a key role in immune regulation. The spleen is the site of T cells and B cells’ settlement and immune response, and can synthesize biologically active substances. The thymus can regulate peripheral immune organs and immune cells, and provide a place for the differentiation and maturation of T cells. Therefore, the indexes of the spleen and thymus are considered to be important indicators reflecting immune function.

[0071] Organ index determination: The collected immune organs spleen and thymus were rinsed with physiological saline, dried with filter paper and weighed. The ratio of the weight to the final weight of the animal was the organ index.

[0072]

[0073] Figure 2 The figure shows the statistical graph of the changes in the immune organ index provided in Experimental Example 2 of the present invention ( Figure 2 Middle a is a statistical chart showing the changes in spleen index of mice in different experimental groups; Figure 2 (b) is a statistical chart showing the changes in thymus index of mice in different experimental groups); Figure 2As shown in Table 4, compared with the NC group, the spleen index and thymus index of the mice in the MC group were significantly reduced, by 9.3% and 22.1, respectively. The compound probiotic yogurt groups (LPYC, MPYC, HPYC) can significantly delay the decrease of spleen index and thymus index caused by CTX. Compared with the MC group, the LPYC group can increase the spleen index and thymus index by 15.5% and 11.9%, the MPYC group can increase the spleen index and thymus index by 20.7% and 27.7%, and the HPYC group can increase the spleen index and thymus index by 21.9% and 10.7%, respectively, indicating that the compound probiotic yogurt may enhance the host's immune system by promoting the growth of immune organs.

[0074] Experimental Example 3: Analysis of immune serum indicators under different dietary interventions Enzyme-linked immunosorbent assay (ELISA) was used to further explore the effects of CTX and compound probiotic yogurt supplementation on the secretion levels of cytokines (TNF-α, IL-6, IL-12) and immunoglobulins (IgA, IgG) in serum, and then quantitatively evaluate the effects on immune effects.

[0075]

[0076] Figure 3 The figure shows the statistical graph of the mouse immune serum index results provided in Experimental Example 3 of the present invention ( Figure 3 a is a statistical graph of TNF-α in the serum concentration of mice in different experimental groups. Figure 3 Middle b is the statistical chart of IL-6 in the serum concentration of mice in different experimental groups. Figure 3 Middle c is the statistical chart of IL-6 in the serum concentration of mice in different experimental groups. Figure 3 Middle d is the statistical chart of IgA concentration in the serum of mice in different experimental groups. Figure 3 (e) is a statistical graph of the IgG results in the serum concentration of mice in different experimental groups. Figure 3As shown in Table 5, compared with the NC group, the levels of TNF-α, IL-6, IL-12, IgA and IgG in the serum of the mice in the MC group were significantly reduced, by 76.7%, 39%, 64.4%, 30.7% and 38.6%, respectively, indicating that the supplementation of CTX significantly promoted the decline of the body's immune effect. The supplementation of compound probiotic yogurt can significantly improve the decline of the immune effect caused by CTX. Compared with the MC group, the serum levels of TNF-α, IL-6, IL-12, IgA and IgG in the LPYC group increased by 85.8%, 47.5%, 2.3 times, 36.3% and 45.8%, respectively, in the MPYC group increased by 92.3%, 42.6%, 83.3%, 39.5% and 39.6%, respectively, in the HPYC group increased by 80.8%, 36.1%, 1 times, 28.9% and 21.6%, respectively.

[0077] Experimental Example 4: Analysis of intestinal flora under different dietary interventions Mouse fecal DNA was extracted according to the kit TIANamp Stool DNA Kit (purchased from Tiangen Biochemical Technology Co., Ltd.). The V3-V4 region of the 16S rRNA gene was amplified, and the amplified product was sent to Tianjin Jinke Biotechnology Co., Ltd. for library construction and sequencing. The original fastq file was quality filtered using Trimmomatic software, and the obtained double-end sequences were denoised, merged, and clustered into amplicon sequence variants (ASVs) using the DADA2 program in QIIME2 (v2021.11). Based on the Silva 138 99% OUTs database, the obtained ASV sequences were classified and annotated using the naive Bayes classifier trained by QIIME2, and the alpha diversity index was calculated using QIIME2 to analyze the composition of the intestinal flora.

[0078] Figure 4 The figure shows the statistical graph of mouse α diversity results provided in Experimental Example 4 of the present invention ( Figure 4 Figure a is a statistical chart of the Chao1 index of intestinal microorganisms in different experimental groups of mice. Figure 4 Middle b is the statistical chart of observed_species index of intestinal microorganisms in different experimental groups of mice. Figure 4 (c) is a statistical chart of the Shannon index of intestinal microorganisms in different experimental groups of mice. Figure 4It can be seen that the Chao1 index, observed_species index and Shannon index of the intestinal microorganisms of the MC group mice were lower than those of the other four groups, indicating that CTX supplementation significantly reduced the species richness and diversity of the mouse intestine. The compound probiotic yogurt intervention increased the α-diversity of the intestinal flora, that is, the compound probiotic yogurt reversed the CTX-induced decrease in the number of species by increasing the total number and diversity of the mouse intestinal flora.

[0079] Experimental Example 5: Analysis of intestinal flora composition under different dietary interventions The obtained ASV sequences were classified and annotated using the naive Bayes classifier trained with QIIME2, and the alpha diversity index was calculated using QIIME2 as well as the composition of the intestinal flora was analyzed.

[0080] Figure 5 The figure shows the statistical graph of the abundance results of mouse phylum level provided in Experimental Example 5 of the present invention ( Figure 5 Middle a is the statistical chart of the relative abundance of the phylum level in mice of different experimental groups. Figure 5 (b) is a statistical graph of the F / B values ​​of mice in different experimental groups); Figure 5 As shown in a, combined with Table 6, Figure 5 The value in a is the integer of the value in Table 6. At the phylum level, Firmicutes and Bacteroidetes are the two most important phyla, accounting for nearly 80% of the total bacteria. Compared with the NC group, the supplementation of CTX significantly increased the abundance of Firmicutes and significantly reduced the abundance of Bacteroidetes. The supplementation of compound probiotic yogurt reversed this trend. Figure 5 As shown in b, the F / B (Firmicutes / Bacteroidetes) value is usually used to evaluate the degree of intestinal flora disorder. The larger the F / B value, the greater the degree of intestinal flora disorder. Figure 5 As shown in Figure b, from the intestinal flora improvement index F / B (Firmicutes / Bacteroidetes) value, it can be seen that the F / B value after compound probiotic yogurt treatment is lower than that of the MC group and closer to the NC group, indicating that compound probiotic yogurt treatment has a positive effect on regulating intestinal flora.

[0081]

[0082] Figure 6 The figure shows the statistical graph of the abundance results of the mouse genus provided in Experimental Example 5 of the present invention ( Figure 6 Middle a is the statistical chart of the relative abundance of mouse genus in different experimental groups. Figure 6 Middle b is a statistical chart of the relative abundance of Lactobacillus in the intestines of mice in different experimental groups. Figure 6 Figure c is a statistical chart of the relative abundance of Akkermansia in the intestines of mice in different experimental groups. Figure 6 Figure d in the middle is a statistical chart of the relative abundance of Dunnella in the intestines of mice in different experimental groups. Figure 6 Figure e is a statistical chart of the relative abundance of Bacteroides in the intestines of mice in different experimental groups. Figure 6 As shown in Table 7, at the genus level, the supplementation of CTX will cause the relative abundance of beneficial intestinal bacteria such as Akkermansia, Dunnella, and Bacteroides to decrease significantly, while the supplementation of compound probiotic yogurt will significantly restore the abundance of these beneficial intestinal bacteria. In addition, CTX treatment will significantly increase the relative abundance of Lactobacillus. Although Lactobacillus is generally considered to be a beneficial genus in the intestine, when its abundance is too high, it will become an opportunistic pathogen and a potentially harmful genus. The supplementation of compound probiotic yogurt will significantly reduce the abundance of Lactobacillus, making it closer to the NC group. These results all show that the treatment of compound probiotic yogurt has a positive effect on the improvement of intestinal flora.

[0083] In summary, this 100 billion active bifidobacteria yogurt has good stability during its shelf life, good tolerance in the intestine, can effectively enhance immunity and improve the imbalance of intestinal flora, and is a functional yogurt that is beneficial to human health.

[0084] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented.

[0085] Those skilled in the art should understand that any improvement to the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention. The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, and these simple modifications all belong to the protection scope of the present invention.

[0086] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A probiotic for increasing immunity and improving intestinal flora, characterized in that: The probiotics include a composite bifidobacterium agent and a basic agent; The strain in the composite bifidobacterium agent is Bifidobacterium animalis subsp. lactis with a deposit number of CGMCC No. 22547. Bifidobacterium animalis subsp. lactis BLa80 strain, Bifidobacterium longum subsp. longum with a deposit number of CGMCC No. 10452 Bifidobacterium longum subsp. longum BL21 strain, Bifidobacterium breve with a deposit number of CGMCC No. 24471 Bifidobacterium breve BBr16 strain, Bifidobacterium adolescentis with a preservation number of CGMCC No. 19884 Bifidobacterium adolescentis BAC30 strain, Bifidobacterium longum subsp. infantis with a deposit number of CGMCC No. 15134 Bifidobacterium longum subsp. infantile BI45 strain composition; The strain in the basic bacterial agent is Streptococcus salivarius thermophilus subsp. Streptococcus thermophilus ST81 strain and Lactobacillus delbrueckii subsp. bulgaricus with a deposit number of CGMCC No. 15751 Lactobacillus delbrueckii subsp. bulgaricus LB42 strain composition; The total number of viable bacteria in the composite bifidobacterium agent is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

2. The probiotic according to claim 1, characterized in that The ratio of the number of viable bacteria of the BLa80 strain, BL21 strain, BBr16 strain, BAC30 strain and BI45 strain is (1-10):(1-10):(1-10):(1-5):(1-5).

3. The probiotic according to claim 1, characterized in that The ratio of the viable counts of the ST81 strain to the LB42 strain is 30:(0.001-10); the mass ratio of the composite bifidobacterium bacterial agent to the basic bacterial agent is (10-300):(10-40).

4. The probiotic according to claim 1, characterized in that The dosage form of the probiotics includes solution, lyophilized powder, capsule, tablet or granule.

5. The use of the probiotic according to any one of claims 1 to 4 in yogurt fermentation, characterized in that: The method for fermenting yogurt comprises: The raw milk is mixed with the compound prebiotics and auxiliary additives, sterilized and then cooled to serve as a fermentation base; The compound bifidobacterium bacterial agent and the basic bacterial agent are inoculated into the fermentation base material for fermentation.

6. The use according to claim 5, characterized in that: The inoculation amount of the composite bifidobacterium agent is 10-300 g / T; and / or, the inoculation amount of the basic agent is 10-40 g / T; and / or, the inoculation amount of the composite prebiotic is 0.1-5 kg / T.

7. The use according to claim 6, characterized in that: The compound prebiotics include fructooligosaccharides, xylooligosaccharides or isomaltooligosaccharides; and / or the auxiliary additives include white sugar, cream or whey protein powder.

8. The use according to claim 7, characterized in that: The raw milk accounts for 80-95wt% of the fermentation base, the compound prebiotics accounts for 0.01-1wt% of the fermentation base, the white sugar accounts for 0.1-8wt% of the fermentation base, the light cream accounts for 0.1-5wt% of the fermentation base, and the whey protein powder accounts for 1-10wt% of the fermentation base.

9. The use according to claim 8, characterized in that: The homogenization pressure of the raw milk, white sugar, cream, whey protein powder and compound prebiotics is 180-220 bar. The raw milk, white sugar, cream, whey protein powder and compound prebiotics are sterilized at 90-99° C. for 5-15 minutes and then placed in cold water to cool to 30-40° C.; And / or, the fermentation temperature is 37-43°C, the fermentation time is 5-10 hours, and the fermentation endpoint is that the acidity reaches 70-80°T or pH=4.3-4.

5.

10. Use of the probiotic according to any one of claims 1 to 4 in the preparation of a product for increasing immunity and improving intestinal flora, characterized in that: The products include food, health products or medicines.

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