A compound probiotic for enhancing immunity and a functional yogurt prepared therefrom

Abstract

The present invention provides a compound probiotic for enhancing immunity and a functional yogurt prepared therefrom. The compound probiotic is composed of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024. In the present invention, three strains with different properties, namely Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024, are combined in a certain proportion, and a synergistic effect is generated among the three strains, which has significant technical advantages in enhancing immunity. The product of the present invention has high safety, is not prone to generating resistance, and has good safety and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and particularly relates to a compound probiotic for enhancing immunity and a functional yogurt prepared therefrom. Background Art

[0002] Probiotics are very closely related to humans. They are the most important beneficial bacteria in the human intestine and play important roles in human nutrition, immunity, health care, and even anti-aging and longevity. The higher the proportion of probiotics in the human intestine, the better the microecological environment and the younger and more energetic the intestine will be.

[0003] Currently, the probiotics that have been discovered and used mainly include the following three categories: Lactobacillus spp., such as Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus acidophilus, etc.; Bifidobacterium spp., such as Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, etc.; other genera, such as Streptococcus thermophilus, Pediococcus acidilactici, yeasts, etc.

[0004] Chinese Patent CN116970530A discloses a Bifidobacterium breve FPHC4024, which can cause excessive water absorption in the colon and has a good effect on preventing, alleviating and treating infantile diarrhea. Chinese Patent CN117018043A discloses a probiotic strain and its application, and a composition for preventing and treating infantile diarrhea, and the probiotic strain is Lactobacillus reuteri FPH2951.

[0005] Immunity is an active defense mechanism of the human body itself and is of great significance for maintaining physiological and life activities. The immune cells that maintain the immune function in the human body include T cells, B cells, neutrophils, and macrophages. Insufficient or excessive immunity will bring adverse effects to oneself, mainly manifested as loss of appetite, malnutrition, mental trance, physical weakness, general fatigue, sleep disorders, etc. In modern society, with the improvement of health awareness, people also have higher requirements for preventing and treating "sub-health". However, due to the fast pace of modern society, most people cannot exercise every day to enhance their immunity. Therefore, seeking a safer and more effective method to enhance immunity is particularly important for human health. Summary of the Invention

[0006] In view of the above-mentioned prior art, the present invention provides a compound probiotic for enhancing immunity and a functional yogurt prepared therefrom.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] One aspect of the present invention provides a compound probiotic for enhancing immunity, which is composed of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024.

[0009] Furthermore, Lactobacillus helveticus LZ-R-5 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on June 24, 2021. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO. 22776;

[0010] Lactobacillus reuteri FPHC2951 was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on November 3, 2022. The deposit address is the 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Guangdong Province, China. The deposit number is GDMCC NO. 62946;

[0011] Bifidobacterium breve FPHC4024 was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on November 3, 2022. The deposit address is the 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Guangdong Province, China. The deposit number is GDMCC NO. 62945.

[0012] Furthermore, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 is 1-3:1-3:3-6.

[0013] Furthermore, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 is 2:2:3-6.

[0014] Furthermore, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 is 2:2:5.

[0015] Furthermore, in the compound probiotics, the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 is 5-9×10 9 cfu / mL.

[0016] The preparation method of the composite probiotics of the present invention is as follows: After thawing and activating Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 respectively, they are statically cultured at 37°C for 24 h in MRS medium according to an inoculation amount of 2%. After the obtained bacterial cultures are centrifuged at 6000 rpm for 10 min, three kinds of single bacterial cells are obtained respectively; after freeze-drying the three kinds of bacterial cells respectively, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 1-3:1-3:3-6, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 8×10 9 cfu / mL to prepare the composite probiotics.

[0017] In the second aspect, the present invention also provides the application of the composite probiotics in the preparation of health products for improving immunity.

[0018] In the third aspect, the present invention also provides a functional yogurt, which is obtained by inoculating the above composite probiotics in sweetened fresh raw milk and fermenting and ripening.

[0019] Further, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 1-3:1-3:3-6; the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 5-9×10 9 cfu / mL; the fermentation temperature is 33-38°C, the fermentation time is 12-18 h; the ripening temperature is 4-8°C, and the ripening time is 30-40 h.

[0020] Further, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 1-3:1-3:3-6; the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 6-8×10 9 cfu / mL; the fermentation temperature is 35°C, the fermentation time is 15 h; the ripening temperature is 6°C, and the ripening time is 35 h.

[0021] Even further, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 2:2:5; the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 8×10 9cfu / mL; the fermentation temperature is 35°C and the fermentation time is 15 h; the after-ripening temperature is 6°C and the after-ripening time is 35 h.

[0022] Further, the sugar is white granulated sugar, and the ratio of the white granulated sugar to the fresh raw milk is 6 - 10 g: 100 mL.

[0023] Further, the ratio of the white granulated sugar to the fresh raw milk is 8.6 g: 100 mL.

[0024] The composite probiotic of the present invention is composed of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024, which produce a synergistic effect during the fermentation of milk. The functional yogurt prepared with the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 being 1 - 3: 1 - 3: 3 - 6 has a significant immune-enhancing effect.

[0025] Fourthly, the present invention also provides a functional acid milk powder, which is prepared by spray-drying the above-mentioned functional yogurt.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The composite probiotic for enhancing immunity of the present invention significantly increases body weight, improves the spleen index and thymus index, increases the phagocytosis rate and phagocytosis index, and significantly improves immunity. The functional yogurt fermented by the composite probiotic of the present invention significantly improves the spleen index and thymus index, increases the contents of IgG, IgA, and IgM in the serum, and enhances humoral immunity.

[0028] Through a large number of analyses and tests, the present invention combines three strains with different properties, Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024, in a certain proportion, and a synergistic effect is produced among the three strains. It has significant technical advantages in enhancing immunity. Compared with single strains, the composite probiotic of the present invention has a better effect in enhancing immunity. The three strains of the present invention are all probiotics, the product has high safety, and is not prone to generating resistance, and has good safety and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the specific embodiments.

[0030] Figure 1 It is a schematic diagram of the effect of the composite probiotic of the present invention on body weight; among them, compared with the blank control group, # p < 0.05, ## p < 0.01; compared with the Example 1 group, **p < 0.01.

[0031] Figure 2 Schematic diagram of the effect of the compound probiotics of the present invention on the spleen index; wherein, compared with the blank control group, # p < 0.05, ## p < 0.01; compared with the Example 1 group, **p < 0.01.

[0032] Figure 3 Schematic diagram of the effect of the compound probiotics of the present invention on the thymus index; wherein, compared with the blank control group, # p < 0.05, ## p < 0.01; compared with the Example 1 group, **p < 0.01.

[0033] Figure 4 Schematic diagram of the effect of the compound probiotics of the present invention on the phagocytosis rate; wherein, compared with the blank control group, # p < 0.05, ## p < 0.01; compared with the Example 1 group, **p < 0.01.

[0034] Figure 5 Schematic diagram of the effect of the compound probiotics of the present invention on the phagocytosis index; wherein, compared with the blank control group, # p < 0.05, ## p < 0.01; compared with the Example 1 group, **p < 0.01.

[0035] Figure 6 Schematic diagram of the effect of the functional yogurt of the present invention on the IgG content; wherein, compared with the blank control group, # p < 0.05, ## p < 0.01; compared with the Example 5 group, **p < 0.01.

[0036] Figure 7 Schematic diagram of the effect of the functional yogurt of the present invention on the IgA content; wherein, compared with the blank control group, # p < 0.05, ## p < 0.01; compared with the Example 5 group, **p < 0.01.

[0037] Figure 8 Schematic diagram of the effect of the functional yogurt of the present invention on the IgM content; wherein, compared with the blank control group, # p < 0.05, ## p < 0.01; compared with the Example 5 group, *p < 0.05. Detailed implementation manners

[0038] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0039] In the following embodiments of the present invention, the experimental methods are all conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0040] The sources of the strains used in the following embodiments of the present invention are as follows:

[0041] Lactobacillus helveticus LZ-R-5 was deposited on June 24, 2021 at the China General Microbiological Culture Collection Center, with the deposit address being No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO. 22776;

[0042] Limosilactobacillus reuteri FPHC2951 was deposited on November 3, 2022 at the Guangdong Provincial Microbiological Culture Collection Center, with the deposit address being the 5th floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Guangdong Province, China, and the deposit number being GDMCC NO. 62946. Limosilactobacillus reuteri FPHC2951 has been disclosed in Patent CN117018043A.

[0043] Bifidobacterium breve FPHC4024 was deposited on November 3, 2022 at the Guangdong Provincial Microbiological Culture Collection Center, with the deposit address being the 5th floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Guangdong Province, China, and the deposit number being GDMCC NO. 62945. Bifidobacterium breve FPHC4024 has been disclosed in Patent CN116970530A.

[0044] Limosilactobacillus reuteri LR06 was deposited on June 24, 2021 at the China General Microbiological Culture Collection Center, with the deposit address being: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO. 22775. Limosilactobacillus reuteri LR06 has been disclosed in Patent CN114468305A.

[0045] The components of MRS medium (g / L) are as follows: peptone 10 g / L, beef extract 10 g / L, glucose 15 g / L, lactose 15 g / L, yeast powder 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, cysteine hydrochloride 0.5 g / L.

[0046] Example 1 A compound probiotic for enhancing immunity and its preparation method

[0047] A compound probiotic for enhancing immunity is composed of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024. Among them, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 2:2:5, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 8×10 9 cfu / mL.

[0048] The preparation method of the compound probiotic of the present invention is as follows: After thawing and activating Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 respectively, they are statically cultured in MRS medium at 37°C for 24 h with an inoculation amount of 2%. After the obtained bacterial culture is centrifuged at 6000 rpm for 10 min, three single bacterial cells are obtained respectively; after freeze-drying the three bacterial cells respectively, freeze-dried bacterial powder is obtained. The viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 2:2:5, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 8×10 9 cfu / mL, and they are mixed to obtain the compound probiotic.

[0049] Example 2 A compound probiotic for enhancing immunity and its preparation method

[0050] A compound probiotic for enhancing immunity is composed of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024. Among them, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 1:3:3, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 9×10 9 cfu / mL.

[0051] The preparation method of the compound probiotics of the present invention is as follows: After thawing and activating Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 respectively, they are statically cultured in MRS medium at 37°C for 24 h with an inoculation amount of 2%. After the obtained bacterial cultures are centrifuged at 6000 rpm for 10 min, three kinds of single bacterial cells are obtained respectively; after freeze-drying the three kinds of bacterial cells respectively, lyophilized bacterial powder is obtained. The viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 1:3:3, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 9×10 9 cfu / mL, and they are mixed to obtain compound probiotics.

[0052] Example 3 A compound probiotic for enhancing immunity and its preparation method

[0053] A compound probiotic for enhancing immunity is composed of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024. Among them, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 3:3:6, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 6×10 9 cfu / mL.

[0054] The preparation method of the compound probiotics of the present invention is as follows: After thawing and activating Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 respectively, they are statically cultured in MRS medium at 37°C for 24 h with an inoculation amount of 2%. After the obtained bacterial cultures are centrifuged at 6000 rpm for 10 min, three kinds of single bacterial cells are obtained respectively; after freeze-drying the three kinds of bacterial cells respectively, lyophilized bacterial powder is obtained. The viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 3:3:6, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 6×10 9 cfu / mL, and they are mixed to obtain compound probiotics.

[0055] Example 4 A compound probiotic for enhancing immunity and its preparation method

[0056] A compound probiotic for enhancing immunity is composed of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024. Among them, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 2:3:4, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 5×10 9 cfu / mL.

[0057] The preparation method of the compound probiotic of the present invention is as follows: After thawing and activating Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 respectively, they are statically cultured in MRS medium at 37°C for 24 hours according to an inoculation amount of 2%. After the obtained bacterial culture is centrifuged at 6000 rpm for 10 minutes, three single bacterial cells are obtained respectively; after freeze-drying the three bacterial cells respectively, the obtained freeze-dried bacterial powder has a viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 of 2:3:4, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 5×10 9 cfu / mL, and they are mixed to obtain the compound probiotic.

[0058] Example 5 A functional yogurt and its preparation method

[0059] (1) Preparation of the compound probiotic suspension

[0060] After thawing and activating Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024, they are statically cultured in MRS medium at 37°C for 24 hours according to an inoculation amount of 2%. After the obtained bacterial culture is centrifuged at 6000 rpm for 10 minutes, the bacterial cells are resuspended with PBS to obtain the compound probiotic suspension.

[0061] Among them, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 2:2:5, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 8×10 9 cfu / mL.

[0062] (2) Fermentation and ripening

[0063] Add granulated sugar to fresh raw milk for blending, with the dosage ratio of granulated sugar to fresh raw milk being 8.6 g: 100 mL. Perform pasteurization at 75 °C for 20 min to obtain a mixture of granulated sugar and fresh raw milk. After the mixture of granulated sugar and fresh raw milk cools to room temperature, inoculate the compound probiotic suspension prepared in step (1) into it, stir gently and evenly, then ferment at 35 °C for 15 h, and place the fermented yogurt at 6 °C for after-ripening for 35 h to obtain the functional yogurt of the present invention.

[0064] Example 6 A functional yogurt and its preparation method

[0065] (1) Preparation of compound probiotic suspension

[0066] After thawing and activating Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024, cultivate them statically at 37 °C in MRS medium for 24 h according to an inoculation amount of 2%. After the obtained bacterial culture is centrifuged at 6000 rpm for 10 min, resuspend the cells with PBS to obtain a compound probiotic suspension.

[0067] Among them, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 1:3:3, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 6×10 9 cfu / mL.

[0068] (2) Fermentation and after-ripening

[0069] Add granulated sugar to fresh raw milk for blending, with the dosage ratio of granulated sugar to fresh raw milk being 6 g: 100 mL. Perform pasteurization at 72 °C for 20 min to obtain a mixture of granulated sugar and fresh raw milk. After the mixture of granulated sugar and fresh raw milk cools to room temperature, inoculate the compound probiotic suspension prepared in step (1) into it, stir gently and evenly, then ferment at 33 °C for 18 h, and place the fermented yogurt at 4 °C for after-ripening for 30 h to obtain the functional yogurt of the present invention.

[0070] Example 7 A functional yogurt and its preparation method

[0071] (1) Preparation of compound probiotic suspension

[0072] After thawing and activating Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024, cultivate them statically at 37 °C in MRS medium for 24 h according to an inoculation amount of 2%. After the obtained bacterial culture is centrifuged at 6000 rpm for 10 min, resuspend the cells with PBS to obtain a compound probiotic suspension.

[0073] Among them, the viable cell count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 3:3:6, and the total viable cell count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 9×10 9 cfu / mL.

[0074] (2) Fermentation and ripening

[0075] Add granulated sugar to fresh raw milk for blending. The dosage ratio of granulated sugar to fresh raw milk is 10 g:100 mL. Perform pasteurization at 75°C for 20 min to obtain a mixture of granulated sugar and fresh raw milk. After the mixture of granulated sugar and fresh raw milk is cooled to room temperature, inoculate the compound probiotic suspension prepared in step (1) into it, stir gently and evenly, then ferment at 38°C for 12 h, and place the fermented yogurt at 8°C for ripening for 30 h to obtain the functional yogurt of the present invention.

[0076] Comparative Example 1 A compound probiotic

[0077] Compared with Example 1, the difference is that the compound probiotic does not contain Bifidobacterium breve FPHC4024 and is composed of Lactobacillus helveticus LZ-R-5 and Lactobacillus reuteri FPHC2951. Specifically as follows:

[0078] A compound probiotic is composed of Lactobacillus helveticus LZ-R-5 and Lactobacillus reuteri FPHC2951. Among them, the viable cell count ratio of Lactobacillus helveticus LZ-R-5 and Lactobacillus reuteri FPHC2951 is 2:2, and the total viable cell count of Lactobacillus helveticus LZ-R-5 and Lactobacillus reuteri FPHC2951 is 8×10 9 cfu / mL.

[0079] The preparation method of the compound probiotic is as follows: After thawing and activating Lactobacillus helveticus LZ-R-5 and Lactobacillus reuteri FPHC2951 respectively, cultivate them statically at 37°C in MRS medium for 24 h according to an inoculation amount of 2%. After the obtained bacterial culture is centrifuged at 6000 rpm for 10 min, single bacterial cells are obtained respectively. After freeze-drying the bacterial cells respectively, the viable cell count ratio of Lactobacillus helveticus LZ-R-5 and Lactobacillus reuteri FPHC2951 is 2:2, and the total viable cell count of Lactobacillus helveticus LZ-R-5 and Lactobacillus reuteri FPHC2951 is 8×10 9 cfu / mL, and they are mixed to obtain the compound probiotic.

[0080] Comparative Example 2 A compound probiotic

[0081] Compared with Example 1, the difference is that Lactobacillus reuteri LR06 is used to replace Lactobacillus reuteri FPHC2951, and the compound probiotic is composed of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri LR06 and Bifidobacterium breve FPHC4024. Specifically as follows:

[0082] A compound probiotic is composed of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri LR06 and Bifidobacterium breve FPHC4024. Among them, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri LR06 and Bifidobacterium breve FPHC4024 is 2:2:5, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri LR06 and Bifidobacterium breve FPHC4024 is 8×10 9 cfu / mL.

[0083] The preparation method of the compound probiotic is as follows: After thawing and activating Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri LR06 and Bifidobacterium breve FPHC4024 respectively, they are statically cultured in MRS medium at 37°C for 24 h according to an inoculation amount of 2%. After the obtained bacterial culture is centrifuged at 6000 rpm for 10 min, three kinds of single bacteria are obtained respectively. After freeze-drying the three kinds of bacteria respectively, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri LR06 and Bifidobacterium breve FPHC4024 is 2:2:5, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri LR06 and Bifidobacterium breve FPHC4024 is 8×10 9 cfu / mL, and they are mixed to obtain the compound probiotic.

[0084] Comparative Example 3 A compound probiotic

[0085] Compared with Example 1, the difference is that the compounding ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri LR06 and Bifidobacterium breve FPHC4024 is different, and the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 is 1:4:2. Specifically as follows:

[0086] A compound probiotic for improving immunity is composed of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024. Among them, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 is 1:4:2, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 is 8×10 9 cfu / mL.

[0087] The preparation method of the compound probiotics is as follows: After thawing and activating Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 respectively, they are statically cultured in MRS medium at 37°C for 24 h with an inoculation amount of 2%. After the obtained bacterial culture is centrifuged at 6000 rpm for 10 min, three single bacterial cells are obtained respectively. After freeze-drying the three kinds of bacterial cells respectively, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 1:4:2, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 8×10 9 cfu / mL, and they are mixed to obtain the compound probiotics.

[0088] Comparative Example 4 A kind of yogurt is prepared by the following method:

[0089] (1)Preparation of the compound probiotic suspension

[0090] After thawing and activating Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri LR06, and Bifidobacterium breve FPHC4024 respectively, they are statically cultured in MRS medium at 37°C for 24 h with an inoculation amount of 2%. After the obtained bacterial culture is centrifuged at 6000 rpm for 10 min, the bacterial cells are resuspended with PBS to obtain the compound probiotic suspension.

[0091] Among them, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri LR06, and Bifidobacterium breve FPHC4024 is 2:2:5, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri LR06, and Bifidobacterium breve FPHC4024 is 8×10 9 cfu / mL.

[0092] (2)Fermentation and after-ripening

[0093] Sucrose is added to raw fresh milk for preparation, and the dosage ratio of sucrose to raw fresh milk is 8.6 g:100 mL. It is pasteurized at 75°C for 20 min to obtain the sucrose and raw fresh milk mixture; after the sucrose and raw fresh milk mixture is cooled to room temperature, the compound probiotic suspension prepared in step (1) is inoculated into it, and gently stirred evenly, and then fermented at 35°C for 15 h. The fermented yogurt is placed at 6°C for after-ripening for 35 h to obtain it.

[0094] Comparative Example 5 A kind of yogurt is prepared by the following method:

[0095] (1)Preparation of the compound probiotic suspension

[0096] After thawing and activating Lactobacillus helveticus LZ-R-5 and Lactobacillus reuteri FPHC2951, they were statically cultured in MRS medium at 37°C for 24 h with an inoculation amount of 2%. After the obtained bacterial culture was centrifuged at 6000 rpm for 10 min, the cells were resuspended with PBS to obtain a compound probiotic suspension.

[0097] Among them, the viable count ratio of Lactobacillus helveticus LZ-R-5 to Lactobacillus reuteri FPHC2951 is 2:2, and the total viable count of Lactobacillus helveticus LZ-R-5 and Lactobacillus reuteri FPHC2951 is 8×10 9 cfu / mL.

[0098] (2) Fermentation and ripening

[0099] Sucrose was added to fresh raw milk for preparation, and the dosage ratio of sucrose to fresh raw milk was 8.6 g:100 mL. Pasteurization was carried out at 75°C for 20 min to obtain a mixture of sucrose and fresh raw milk. After the mixture of sucrose and fresh raw milk was cooled to room temperature, the compound probiotic suspension prepared in step (1) was inoculated into it, and gently stirred evenly. Then it was fermented at 35°C for 15 h, and the fermented yogurt was ripened at 6°C for 35 h to obtain the product.

[0100] Comparative Example 6 A yogurt was prepared by the following method:

[0101] (1) Preparation of compound probiotic suspension

[0102] After thawing and activating Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024, they were statically cultured in MRS medium at 37°C for 24 h with an inoculation amount of 0.5%. After the obtained bacterial culture was centrifuged at 5000 rpm for 20 min, the cells were resuspended with PBS to obtain a compound probiotic suspension.

[0103] Among them, the viable count ratio of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 2:2:5, and the total viable count of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 8×10 9 cfu / mL.

[0104] (2) Fermentation and ripening

[0105] Mix fresh raw milk with granulated sugar in a ratio of 5 g:100 mL, and perform pasteurization at 75°C for 20 min to obtain a mixture of granulated sugar and fresh raw milk. After the mixture of granulated sugar and fresh raw milk cools to room temperature, add the compound probiotic suspension prepared in step (1), stir gently and evenly, and then ferment at 35°C for 22 h to obtain the product.

[0106] Effect Example 1 Study on the Effect of the Compound Probiotic of the Invention on the Immune Function of Mice

[0107] 1. Experimental Animals and Grouping

[0108] 96 SPF-grade Kunming mice, weighing 18 - 22 g. The 96 mice were divided into 2 batches for experiments. The first batch of mice was used for the experiment of determining the ratio of organ weight to body weight; the second batch of mice was used for the experiment of phagocytosis of chicken red blood cells by mouse peritoneal macrophages. Each batch of mice was randomly divided into 6 groups according to body weight, namely blank control group, Example 1 group, Example 2 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group, with 8 mice in each group.

[0109] The Example 1 group, Example 2 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group were respectively given intragastric administration of the compound probiotics prepared in Example 1, Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3 at a concentration of 50 mg / mL, 0.2 mL / (mouse·d); the blank control group was given intragastric administration of normal saline, 0.2 mL / (mouse·d); intragastric administration was performed once a day, and continuous intragastric administration was carried out for 28 d according to the experimental needs. During the experiment, feed and drinking water were added to the mice in time every day, and except for regular intragastric administration, the mice were allowed to eat and drink freely. The bedding wood chips were replaced regularly to maintain environmental hygiene and cleanliness. The general state of the mice was observed every day.

[0110] 2 Test Methods

[0111] 2.1 Determination of Organ-Weight Ratio

[0112] After continuous intragastric administration of the mice in each group for 28 d, the mice were fasted for 12 h before being sacrificed. Weigh the mice and record their body weights, then sacrifice the mice by cervical dislocation, dissect their spleens and thymuses, and remove the fat and connective tissues around the organs. Weigh the wet weights of the spleens and thymuses with an electronic balance and calculate the organ indices.

[0113] Thymus index = thymus weight (mg) / body weight (10 g).

[0114] Spleen index = spleen weight (mg) / body weight (10 g).

[0115] 2.2 Test for Phagocytosis of Chicken Red Blood Cells by Mouse Peritoneal Macrophages

[0116] Mice in each group were continuously intragastrically administered for 28 days. 30 minutes after the last intragastric administration, 1 mL of 5% (v / v) packed chicken red blood cell suspension was injected into the abdominal cavity of each mouse for immunization. The macrophage phagocytosis percentage and macrophage phagocytosis index were determined by the drop method.

[0117] Phagocytosis percentage = (number of macrophages phagocytosing chicken red blood cells among 100 macrophages / 100 macrophages) × 100%.

[0118] Phagocytosis index = total number of chicken red blood cells phagocytosed by 100 macrophages / 100 macrophages.

[0119] 3. Statistical method

[0120] SPSS 22.0 software was used for data processing. The obtained data were expressed as mean ± standard deviation (`x±s). Normal distribution test and homogeneity of variance test were performed on the data of each group. One-way ANOVA was used for inter-group comparison, and P < 0.05 was considered statistically significant.

[0121] 4. Results and discussion

[0122] 4.1 Effects of the compound probiotic of the present invention on the body weight of mice

[0123] After continuously intragastrically administering mice with the compound probiotic of the present invention and normal saline for 28 days respectively, the body weights of the mice were weighed, and the results are shown in Figure 1 .

[0124] Compared with the blank control group, the average body weights of the compound probiotic mice in Example 1 group, Example 2 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group of the present invention were significantly increased, with significant differences, indicating that the compound probiotic of the present invention has a promoting effect on the increase of the body mass of mice.

[0125] 4.2 Effects of the compound probiotic of the present invention on the immune indexes of mice

[0126] Compared with the blank control group, the spleen indexes and thymus indexes of the mice in Example 1 group, Example 2 group, and Comparative Example 1-3 groups were significantly increased, as shown in Figure 2 and Figure 3 . The effect of Example 1 in increasing the spleen index and thymus index was better than that of Comparative Example 1-3.

[0127] Compared with the blank control group, the phagocytosis rate and phagocytosis index of the mice in Example 1 group, Example 2 group, and Comparative Example 1-3 groups were significantly increased, as shown in Figure 4 and Figure 5 . The effect of Example 1 in increasing the phagocytosis rate and phagocytosis index was better than that of Comparative Example 1-3.

[0128] Through the determination test of organ-body weight ratio and the test of phagocytosis of chicken red blood cells by mouse peritoneal macrophages, it is shown that the compound probiotics of the present invention significantly improve immunity. The effects of improving immunity of the compound probiotics in Example 1 group and Example 2 group of the present invention are significantly better than those in Comparative Example 1-3 groups. Without Bifidobacterium breve FPHC4024 under the same dose, or replacing Lactobacillus reuteri FPHC2951 with Lactobacillus reuteri LR06, or changing the ratio of three strains of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024, the immunopotentiating effect of the prepared compound probiotics is significantly weakened. It shows that three specific strains of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 play a significant synergistic effect in enhancing immunity according to a specific ratio, and have significant technical advantages.

[0129] Effect Example 2 Study on the effect of the functional yogurt of the present invention on immune function

[0130] 1. Experimental animals and grouping

[0131] Forty SPF-grade Kunming mice, weighing 18-22 g. They were randomly divided into a blank control group, an Example 5 group, a Comparative Example 4 group, a Comparative Example 5 group, and a Comparative Example 6 group according to body weight, with 8 mice in each group.

[0132] The Example 5 group, the Comparative Example 4 group, the Comparative Example 5 group, and the Comparative Example 6 group were respectively intragastrically administered with the yogurt prepared in Example 5, Comparative Example 4, Comparative Example 5, and Comparative Example 6 at 60 mg / mL, 0.5 mL / (mouse·day); the blank control group was intragastrically administered with normal saline, 0.5 mL / (mouse·day); intragastric administration was performed once a day, with normal diet and drinking water. Intragastric administration was continued for 10 d, then fasting for 1 d. On the 2nd day after fasting, blood was collected by eye socket enucleation, and then the mice were sacrificed by cervical dislocation.

[0133] 2. Test method

[0134] Effect of the functional yogurt of the present invention on mouse immunoglobulins (IgA, IgM, IgG)

[0135] The blood obtained by eye socket enucleation was placed in a 4°C refrigerator overnight, then centrifuged at 3000 r / min for 15 min, and the supernatant was separated to obtain serum. The contents of IgA, IgM, and IgG were measured by indirect enzyme-linked immunosorbent assay.

[0136] 3. Statistical method

[0137] SPSS 22.0 software was used for data processing. The obtained data were expressed as mean ± standard deviation (`x±s). Normal distribution test and homogeneity of variance test were performed on the data of each group. One-way analysis of variance was used for comparison between groups, and P < 0.05 was considered statistically significant.

[0138] 4. Results and Discussion

[0139] 4.1 Effect of the functional yogurt of the present invention on the content of serum immunoglobulins in mice

[0140] Immunoglobulins are the main antibodies mediating humoral immunity, and the level of serum antibodies reflects to a certain extent the disease resistance of the body. The determination of serum immunoglobulins is a common method for examining the level of humoral immunity. Usually, detecting these three types of immunoglobulins, IgG, IgA, and IgM, can represent the overall level of serum immunoglobulins.

[0141] As can be seen from Figures 6 - 8 it, the functional yogurt of the present invention can significantly increase the contents of IgG, IgA, and IgM in the serum of mice and improve the immune function of mice. The effect of the functional yogurt prepared in Example 5 of the present invention in enhancing immunity is significantly better than that of the yogurt prepared in Comparative Example 4, Comparative Example 5, and Comparative Example 6.

[0142] The above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description, and the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A composite probiotic for improving immunity, characterized in that: The composite probiotics are composed of Lactobacillus helveticus LZ-R-5, Limosilactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024; The Lactobacillus helveticus LZ-R-5 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 24, 2021, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.22776; The Lactobacillus reuteri FPHC2951 was deposited in Guangdong Microbiological Culture Collection Center on November 3, 2022, with the deposit address being 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, China, and the deposit number is GDMCC NO.62946; The Bifidobacterium breve FPHC4024 was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 3, 2022, with the deposit address being 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, China, and the deposit number is GDMCC NO.62945; The ratio of the live counts of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 is 1-3:1-3:3-6, and the total live counts of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 are (5-9)×10 9 cfu / mL.

2. The composite probiotic according to claim 1, characterized in that The ratio of the live bacteria counts of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 is 2:2:3-6.

3. The composite probiotic according to claim 2, characterized in that The ratio of the live bacteria counts of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 is 2:2:

5.

4. The composite probiotic according to claim 1, characterized in that The preparation method of the composite probiotics is as follows: Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 are thawed and activated respectively, and then statically cultured in an MRS medium at 37°C for 24 hours according to an inoculation amount of 2%, and the obtained bacterial culture is centrifuged at 6000rpm for 10 minutes to obtain three single bacterial bodies respectively; the three bacterial bodies are freeze-dried to obtain freeze-dried bacterial powder, and the ratio of the live bacterial counts of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 is 1-3:1-3:3-6, and the total live bacterial counts of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 are 8×10 9 cfu / mL, and mixed to obtain composite probiotics.

5. Use of the composite probiotic according to claim 1 in preparing a health product for improving immunity.

6. A functional yogurt, characterized in that: The functional yogurt is obtained by inoculating the composite probiotics described in claim 1 into fresh milk with added sugar, and fermenting and ripening the milk; the ratio of the live bacteria counts of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 is 1-3:1-3:3-6; the total live bacteria counts of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951 and Bifidobacterium breve FPHC4024 are (6-8)×10 9 cfu / mL; the fermentation temperature is 33-38°C, and the fermentation time is 12-18h; the ripening temperature is 4-8°C, and the ripening time is 30-40h; the sugar is white sugar, and the ratio of the white sugar to fresh milk is 6-10g:100mL.

7. The functional yogurt according to claim 6, characterized in that The ratio of the live bacteria counts of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 is 2:2:5; the total live bacteria counts of Lactobacillus helveticus LZ-R-5, Lactobacillus reuteri FPHC2951, and Bifidobacterium breve FPHC4024 are 8×10 9 cfu / mL; the fermentation temperature is 35°C, and the fermentation time is 15h; the ripening temperature is 6°C, and the ripening time is 35h; the ratio of the white sugar to the fresh milk is 8.6g:100mL.

8. A functional yogurt powder, prepared by spray-drying the functional yogurt according to claim 6 or 7.

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