Fermentation method for bifidobacterium longum BBMN68, fermentation product thereof, postbiotic thereof, and use thereof

Abstract

The present invention relates to the technical field of microorganisms. Particularly disclosed are a fermentation method for Bifidobacterium longum BBMN68, a fermentation product thereof, a postbiotic thereof, and use thereof. In the fermentation method for Bifidobacterium longum BBMN68 of the present invention, a fermentation substrate comprises alkaline protease-hydrolyzed milk and an shaddock peel extract; the hydrolysis rate of the alkaline protease-hydrolyzed milk is 9-11%, and the water-soluble dietary fiber content of the shaddock peel extract is 75-80%; the mass ratio of the alkaline protease-hydrolyzed milk to the shaddock peel extract is (10-15):(1-3). The fermentation product prepared by the described method has a high active ingredient content. When the fermentation product is prepared into a postbiotic and administered, the immune function of the organism can be restored, the damage to immune organs can be reduced, inflammation can be relieved, and gut microbiota can be regulated.

Description

Fermentation method of Bifidobacterium longum BBMN68, fermentation product, postbiotic and application thereof

[0001] The present application claims priority to the Chinese application with the application number CN202410531798.6, the application date of 2024 / 04 / 29 and the invention name of "Fermentation method of Bifidobacterium longum BBMN68, fermentation product, postbiotic and application thereof". TECHNICAL FIELD

[0002] The present application relates to the field of microbial technology, in particular, to the fermentation method of Bifidobacterium longum BBMN68, fermentation product, postbiotic and application thereof. BACKGROUND

[0003] The organism with low immunity is prone to have the performance of weak constitution, malnutrition, mental debilitation, fatigue, decreased appetite, sleep disorders and so on, and the diseases such as cold, asthma and inflammation often recur. The existing immune enhancers include biological factors (thymus factor, transfer factor, etc.), chemical synthesis (liposomes, polynucleic acid and surface active molecules, etc.), trace elements (vitamin E, vitamin A and selenium, etc.), natural food and medicine (propolis, ginseng, donkey-hide gelatin, radix codonopsis, ganoderma lucidum, angelica sinensis, radix rehmanniae preparata, radix paeoniae alba, etc.) and microorganisms (probiotics, yeast, fungi). Although the medicine has good curative effect, it has certain drug resistance and side effects, the food has poor effect and significant individual differences. The existing probiotic products are mainly concentrated in live bacteria products, which have many shortcomings such as single product form, high storage condition requirement and single mechanism of action, which affect the efficacy of the products. Therefore, how to safely and stably improve the immunity of the organism is a problem to be solved by people.

[0004] In recent years, the field of postbiotics has attracted more and more attention. Postbiotics are defined as bioactive substances (including inactivated bacteria, bacterial lysates, bacterial intracellular components, cell membrane and cell wall components and metabolic products) that are produced during fermentation and are beneficial to health. Compared with probiotics, postbiotics have advantages in antibacterial, antioxidant and immunomodulatory effects; and it is convenient to transport and store, and can be used with other preparations. While exerting similar effects of probiotics, it avoids the problems of low bioavailability of live bacteria, difficulty in controlling dosage, unstable effect and easy transmission of drug resistance genes, and has strong application potential and economic value.

[0005] Bifidobacterium longum BBMN68 is a kind of probiotics with good physiological function isolated from the intestinal tract of Bama longevity. Its probiotic function mainly manifests in improving intestinal digestive function and enhancing the body's immunity. Yang Haiying et al. improved the secretion of immunoglobulin A in the duodenum of mice by gavage with Bifidobacterium longum BBMN68, which confirmed that Bifidobacterium longum BBMN68 can enhance the immune barrier function of the intestinal tract. At the same time, it was found that oral administration of active Bifidobacterium longum BBMN68 can improve the phagocytosis of mouse macrophages, the activity of NK cells, the level of serum hemolysin and the proliferation of spleen lymphocytes. This research result confirmed that Bifidobacterium BBMN68 can improve the innate and acquired immune regulation ability of the body. Zhu Jinfeng et al. found that BBMN68 can regulate immunity by improving cell activity after contacting mouse macrophages with Bifidobacterium longum BBMN68 complete cells, cell walls and cell contents. Bifidobacterium longum BBMN68 live bacteria and different cell components have been confirmed to have the effect of enhancing the immune function of the body, but the efficacy, immune regulation and functional factors after inactivation are not known, and how to improve its efficacy needs further research. SUMMARY

[0006] One of the purposes of the present application is to provide a method for improving the active ingredients and efficacy of Bifidobacterium longum BBMN68 fermentation and its application in the preparation of postbiotic products.

[0007] In order to achieve this purpose, the technical scheme of the present application is as follows:

[0008] A method for fermenting Bifidobacterium longum BBMN68, the fermentation substrate comprises alkali protease hydrolyzed milk and grapefruit peel extract; the hydrolysis rate of the alkali protease hydrolyzed milk is 9-11%, the water-soluble dietary fiber content of the grapefruit peel extract is 75-80%; and the mass ratio of the alkali protease hydrolyzed milk to the grapefruit peel extract is (10-15):(1-3).

[0009] The Bifidobacterium longum BBMN68 of the present application has a preservation number of CGMCC No. 2265 and is disclosed in Chinese patent CN101649303B. The present application found that when Bifidobacterium longum BBMN68 is fermented, the addition of grapefruit peel extract in the substrate can produce immune-regulating substances vanillin and linalyl acetate, and when alkali protease hydrolyzed milk and grapefruit peel extract are compounded at a specific ratio as a substrate, the content of vanillin and linalyl acetate in the fermentation product can be significantly improved, and the content of various active ingredients (lactic acid, acetic acid, propionic acid, hexanoic acid, butyric acid, exopolysaccharide and total short-chain fatty acid) in the fermentation product can also be improved, so that the product obtained by fermentation can improve the immunity and obtain better biological activity.

[0010] In the present application, the main component of the pomelo peel extract is water-soluble dietary fiber extracted from pomelo peel, which is prepared by using pomelo peel as raw material and water-soluble dietary fiber as extraction target by a method known in the art.

[0011] The pomelo peel extract of the present application can specifically include 75-80% water-soluble dietary fiber, 10-15% starch, 5-7% fat, and 5-9% protein. Preferably, it includes 75-80% water-soluble dietary fiber, 8-10% starch, 5-7% fat, and 6-8.5% protein.

[0012] The extraction method can refer to: Pan Wenjie, Xue Zhenglian, Zhou Haili. Ultrasonic-assisted extraction of water-soluble dietary fiber from pomelo peel [J]. Anhui Agricultural Bulletin (first half of the month), 2011, 17 (15): 198-200.

[0013] The alkali protease hydrolyzed milk of the present application is a substance obtained by hydrolyzing animal milk with alkali protease by a method known in the art. The specific preparation method can be: adding alkali protease at 25 U / mL in animal milk with pH of 8.5, hydrolyzing at 55°C until the hydrolysis rate is 9-11%, and then inactivating the enzyme at 95°C for 5 min to obtain the hydrolyzed milk.

[0014] The preparation raw material of the alkali protease hydrolyzed milk of the present application can be directly animal raw milk, or can be skimmed milk after animal milk is skimmed, or can be an aqueous solution of animal milk powder or skimmed milk powder with a concentration of 10-15%. Preferably, skimmed milk is used to improve the enzymatic efficiency.

[0015] In addition, it is found that when the alkali protease hydrolyzed milk and the pomelo peel extract are used as substrates for fermentation of other bifidobacterium (such as BB12), vanillin and linalyl acetate cannot be produced, although the production of other beneficial ingredients is promoted to a certain extent, but the promotion effect is not as good as the combination of the substrate of the present application and Bifidobacterium longum BBMN68.

[0016] The method for fermenting Bifidobacterium longum BBMN68 of the present application, wherein the fermentation substrate further comprises a carbon source, a nitrogen source, an antioxidant, and water, or further comprises a surfactant.

[0017] Preferably, the fermentation substrate comprises 10-15 parts of alkali protease hydrolyzed milk, 1.5-2.0 parts of yeast extract powder or corn steep liquor, 0.8-2.5 parts of glucose or sucrose, 0.03-0.09 parts of ascorbic acid or cysteine hydrochloride, 1.0-3.0 parts of pomelo peel extract, 78-85 parts of water, or further comprises 0.08-0.15 parts of Tween-80.

[0018] More preferably, the fermentation substrate comprises 10 parts of alkaline protease hydrolyzed milk, 1.8 parts of yeast extract powder, 2 parts of glucose, 0.06 parts of ascorbic acid, 0.1 parts of Tween-80, 2 parts of grapefruit peel extract, and 84.04 parts of water.

[0019] The inoculation concentration of the Bifidobacterium longum BBMN68 in the fermentation substrate is 1-5x10 7 CFU / mL.

[0020] The fermentation condition of the present application is anaerobic, 37±1℃, and static culture.

[0021] The present application also provides a Bifidobacterium longum BBMN68 fermentation product prepared by the above-mentioned method for fermenting Bifidobacterium longum BBMN68.

[0022] The Bifidobacterium longum BBMN68 fermentation product of the present application has high content of various active ingredients, and can have better use effect.

[0023] The present application further provides a composition as a fermentation substrate for improving the content of one or more of vanillin, linalyl acetate, lactic acid, acetic acid, propionic acid, hexanoic acid, butyric acid, exopolysaccharide, and total short-chain fatty acid in the Bifidobacterium longum BBMN68 fermentation product; the composition comprises alkaline protease hydrolyzed milk and grapefruit peel extract; the hydrolysis rate of the alkaline protease hydrolyzed milk is 9-11%, the water-soluble dietary fiber content of the grapefruit peel extract is 75-80%; and the mass ratio of the alkaline protease hydrolyzed milk to the grapefruit peel extract is (10-15):(1-3).

[0024] The present application further provides a method for preparing a postbiotic by inactivating the above-mentioned Bifidobacterium longum BBMN68 fermentation product.

[0025] The postbiotic of the present application is a postbiotic preparation with the fermentation product of inactivated Bifidobacterium longum BBMN68 as the active ingredient, and the fermentation product is at least one of fermentation culture, fermentation concentrate, fermentation diluent, and broken product of Bifidobacterium longum BBMN68, which can be liquid or solid.

[0026] The postbiotic of the present application can be prepared by conventional methods in the art, and the specific preparation method can comprise:

[0027] (1) Activating and culturing the Bifidobacterium longum BBMN68 strain with MRSC liquid medium, and anaerobically and statically culturing to the logarithmic phase;

[0028] (2) using the culture, a concentrate obtained by concentrating the culture, or a dry product, a liquid product, or a diluent obtained by adding an auxiliary to the wet bacterial body collected by centrifugation as an active ingredient, to prepare a metagenomic preparation with inactivated Bifidobacterium longum BBMN68; the number of inactivated Bifidobacterium longum BBMN68 in the metagenomic preparation is ≥ 500 billion / g.

[0029] Preferably, the method for preparing the metagenomic preparation comprises:

[0030] (1) heating the bacterial body in the above-mentioned Bifidobacterium longum BBMN68 fermentation product at 42-45℃ for 5-15 minutes (preferably, 45℃ for 10 minutes);

[0031] (2) mixing with an inactivation protective agent and adjusting the pH value to 4.3-4.8;

[0032] (3) inactivating at 65-120℃ for 1-30 minutes.

[0033] In the present application, the bacterial body is first pretreated at a lower temperature, then mixed with a protective agent and adjusted to a specific pH value, and then inactivated by heating (the higher the inactivation temperature, the shorter the heating time in general), which can better maintain the integrity of the bacterial body and thus improve the shelf life of the metagenomic product. If the pH value in step (2) is changed to > 6, the integrity of the bacterial body will decrease even if subsequent low-temperature heating is performed. Directly performing the subsequent steps without the low-temperature treatment in step (1) is also not conducive to the maintenance of the integrity of the bacterial body.

[0034] More preferably, the method further comprises: (4) vacuum drying at 40-60℃.

[0035] In the present application, the metagenomic preparation in powder form can be obtained by vacuum drying, which is more conducive to storage and transportation.

[0036] The inactivation protective agent used in the preparation of the metagenomic preparation in the present application mainly serves to facilitate transportation and long-term storage, and can be routinely selected by those skilled in the art according to common sense in the field.

[0037] Preferably, the inactivation protective agent comprises: 8-12 parts of skimmed milk powder, 1-3 parts of resistant dextrin, 4-8 parts of lactose, 4-6 parts of trehalose, 1-3 parts of inulin, 1-3 parts of malt dextrin, and 0.2-0.6 parts of sodium glutamate; or the inactivation protective agent comprises: 8-12 parts of skimmed milk powder, 2.5-3.5 parts of L-cysteine, and 8-12 parts of xylan.

[0038] In the method of the present application, the bacterial body in the Bifidobacterium longum BBMN68 fermentation product is obtained by centrifuging the Bifidobacterium longum BBMN68 fermentation product, and the volume-to-mass ratio of the bacterial body to the inactivation protective agent is (3.8-4.2): 1L / kg.

[0039] The pH value in the method step (2) of the present application is adjusted by one or more of lactic acid, acetic acid or citric acid.

[0040] The postbiotic prepared by the above method is mainly inactivated Bifidobacterium longum BBMN68 powder, which can improve the body's immunity and can be taken orally, and the recommended dosage is not less than 20 billion bacteria per day.

[0041] The present application also provides a postbiotic prepared by the above method.

[0042] The postbiotic of the present application not only restores the body's immune function, but also is convenient to eat, convenient to store, simple to treat, high in remission rate, and has no toxic side effects.

[0043] The postbiotic of the present application can also include at least one suitable adjuvant for postbiotic preparation, such as a protective agent, an excipient, etc.; the dosage form of the postbiotic can be powder, pill, capsule, granule, tablet, liquid preparation or gel.

[0044] The present application also provides the use of the above postbiotic in the preparation of a drug, health product or food.

[0045] The present application also provides a product, which is a drug, health product or food, comprising the above postbiotic.

[0046] The product of the present application can improve the immune suppression of immune organs, repair the medulla and cortex of the spleen, form complete lymphoid follicles, reduce the decrease of humoral immune function and the production of inflammation, regulate the composition of intestinal flora (the relative abundance of Lachnospira is significantly reduced, and the relative abundance of Lactobacillus, Selenomonas, Ruminococcus, Clostridium-UGG-014, Bifidobacterium, Dubosiella and Adlercreutzia is significantly increased, especially Dubosiella, which can improve intestinal metabolism, immune capacity and reduce the occurrence of diseases such as bacterial infection), and promote the gradual recovery of immune function, thereby improving the body's immunity through a comprehensive mechanism.

[0047] The present application further provides a fermented milk, which comprises animal milk, sugar, protein powder, starch, pectin, agar, inactivated starter and the above postbiotic, or further comprises prebiotics.

[0048] Preferably, the fermented milk comprises 90-95 parts of animal milk, 5.5-6 parts of sugar, 0.6-0.7 parts of protein powder, 0.38-0.42 parts of starch, 0.08-0.12 parts of pectin, 0.28-0.32 parts of agar, 0.008-0.012 parts of inactivated starter and 0.08-0.12 parts of the postbiotic of claim 6, or further comprises 0.28-0.32 parts of prebiotics.

[0049] More preferably, the prebiotic is inulin, and the fermenting agent comprises Lactobacillus bulgaricus and Streptococcus thermophilus.

[0050] The animal milk of the present application can be the milk of a cow, a sheep, a camel, a deer, a horse, etc.

[0051] The present application adds the above-mentioned postbiotic to the preparation of fermented milk, thereby improving the efficacy of the fermented milk, and further adding a prebiotic to obtain a fermented milk product with improved immune effect and stronger efficacy.

[0052] The fermented milk (100 g / bottle) of the present application contains more than 10 billion postbiotic Bifidobacterium longum BBMN68 per bottle of final product, and it is recommended to take 2 bottles per day to achieve better immune-boosting effect.

[0053] The fermented milk of the present application can be prepared by methods known in the art, preferably by the following method:

[0054] (1) Preheat the animal milk to 50-55℃, add white sugar, egg white powder, starch, pectin, agar, inulin, and maintain the temperature during preheating for 15-18 min;

[0055] (2) Homogenize at 65℃, 25MPa, after homogenization, sterilize at 135℃ for 8s, cool to 42℃ to obtain the fermentation substrate;

[0056] (3) Add the fermenting agent and Bifidobacterium longum BBMN68 inactivated bacteria powder (postbiotic) to the fermentation substrate, and ferment at 42℃ until the acidity reaches 70-72°T, stop fermentation, sterilize at 76-78℃ for 30s, and cool to 20℃ to obtain the fermentation product;

[0057] (4) Homogenize the fermentation product and aseptically fill it.

[0058] The present application further provides the use of the above-mentioned postbiotic or product or fermented milk in improving the immune function of the body, reducing the damage to immune organs, reducing inflammation, and / or regulating the intestinal flora.

[0059] The present application has at least the following beneficial effects:

[0060] The present application improves the content of various active ingredients in the fermentation product by specific setting of the fermentation substrate of Bifidobacterium longum BBMN68, and the obtained fermentation product can improve the immune function through a comprehensive mechanism. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 is an electron micrograph of the bacteria in the inactivated bacteria powder of Example 5.

[0062] Figure 2 is an electron micrograph of the bacteria in the inactivated bacteria powder of Comparative Example 5.

[0063] Figure 3 is a microscopic view of HE staining of the spleen of a mouse in the blank control group (C) to observe histological changes (x400).

[0064] Figure 4 is a microscopic view of HE staining of the spleen of a mouse in the model group (M) to observe histological changes (x400).

[0065] Figure 5 is a microscopic view of HE staining of the spleen of a mouse in the levamisole positive drug group (LMS) to observe histological changes (x400).

[0066] Figure 6 is a microscopic view of HE staining of the spleen of a mouse in the postbiotic low-dose group (68L) to observe histological changes (x400).

[0067] Figure 7 is a microscopic view of HE staining of the spleen of a mouse in the postbiotic high-dose group (68H) to observe histological changes (x400).

[0068] Figure 8 is a microscopic view of HE staining of the spleen of a mouse in the postbiotic yogurt group (68Y) to observe histological changes (x400).

[0069] Figure 9 is a microscopic view of HE staining of the spleen of a mouse in the postbiotic + prebiotic yogurt group (68JY) to observe histological changes (x400).

[0070] The color arrows in Figures 3 to 9, if any, represent the following meanings: black arrow: necrotic cell fragments, red arrow: granulocytes, green arrow: red blood cells, yellow arrow: megakaryocytes. DETAILED DESCRIPTION

[0071] Item 1. A method for fermenting Bifidobacterium longum BBMN68, characterized in that the fermentation substrate comprises alkaline protease hydrolyzed milk and grapefruit peel extract; the hydrolysis rate of the alkaline protease hydrolyzed milk is 9-11%, the water-soluble dietary fiber content of the grapefruit peel extract is 75-80%; and the mass ratio of the alkaline protease hydrolyzed milk to the grapefruit peel extract is (10-15):(1-3).

[0072] Item 2. The method for fermenting Bifidobacterium longum BBMN68 according to item 1, characterized in that the fermentation substrate further comprises a carbon source, a nitrogen source, an antioxidant, and water, or further comprises a surfactant.

[0073] Preferably, the fermentation substrate comprises 10-15 parts of alkaline protease hydrolyzed milk, 1.5-2.0 parts of yeast extract powder or corn steep liquor, 0.8-2.5 parts of glucose or sucrose, 0.03-0.09 parts of ascorbic acid or cysteine hydrochloride, 1.0-3.0 parts of grapefruit peel extract, 78-85 parts of water, or further comprises 0.08-0.15 parts of Tween-80.

[0074] and / or, the inoculation concentration of Bifidobacterium longum BBMN68 in the fermentation substrate is 1-5×107 CFU / mL;

[0075] and / or, the fermentation condition is: anaerobic, 37±1℃, static culture.

[0076] Item 3. A Bifidobacterium longum BBMN68 fermentation product, which is prepared by the method for fermenting Bifidobacterium longum BBMN68 according to any one of items 1-2.

[0077] Item 4. A composition for use as a fermentation substrate for improving the content of one or more of vanillin, linalyl acetate, lactic acid, acetic acid, propionic acid, hexanoic acid, butyric acid, exopolysaccharide and total short-chain fatty acid in a Bifidobacterium longum BBMN68 fermentation product; the composition comprises alkali protease hydrolyzed milk and grapefruit peel extract; the hydrolysis rate of the alkali protease hydrolyzed milk is 9-11%, the water-soluble dietary fiber content of the grapefruit peel extract is 75-80%; and the mass ratio of the alkali protease hydrolyzed milk to the grapefruit peel extract is (10-15):(1-3).

[0078] Item 5. A method for preparing a postbiotic, which is characterized by inactivating the Bifidobacterium longum BBMN68 fermentation product according to item 3.

[0079] Preferably, the method comprises:

[0080] (1) heating the bacterial cells in the Bifidobacterium longum BBMN68 fermentation product according to item 3 at 42-45℃ for 5-15 minutes;

[0081] (2) mixing with an inactivation protective agent and adjusting the pH value to 4.3-4.8;

[0082] (3) inactivating at 65-120℃ for 1-30 minutes;

[0083] More preferably, the method further comprises: (4) vacuum drying at 40-60℃;

[0084] and / or, the inactivation protective agent comprises: 8-12 parts of skimmed milk powder, 1-3 parts of resistant dextrin, 4-8 parts of lactose, 4-6 parts of trehalose, 1-3 parts of inulin, 1-3 parts of malt dextrin and 0.2-0.6 parts of sodium glutamate; or the inactivation protective agent comprises: 8-12 parts of skimmed milk powder, 2.5-3.5 parts of L-cysteine and 8-12 parts of xylan;

[0085] and / or, the bacterial cells in the Bifidobacterium longum BBMN68 fermentation product are obtained by centrifuging the Bifidobacterium longum BBMN68 fermentation product, and the volume-to-mass ratio of the bacterial cells to the inactivation protective agent is (3.8-4.2):1 L / kg;

[0086] and / or, the pH value in step (2) is adjusted with one or more of lactic acid, acetic acid or citric acid.

[0087] Item 6. A postbiotic, which is prepared by the method of item 5.

[0088] Item 7. Use of the postbiotic of item 6 in the preparation of a pharmaceutical product, a health product or a food product.

[0089] Item 8. A product, which is a pharmaceutical product, a health product or a food product, comprising the postbiotic of item 6.

[0090] Item 9. A fermented milk, which comprises animal milk, sugar, protein powder, starch, pectin, agar, inactivated starter culture and the postbiotic of item 6, or further comprises prebiotics.

[0091] Preferably, the fermented milk comprises 90-95 parts of animal milk, 5.5-6 parts of sugar, 0.6-0.7 parts of protein powder, 0.38-0.42 parts of starch, 0.08-0.12 parts of pectin, 0.28-0.32 parts of agar, 0.008-0.012 parts of inactivated starter culture and 0.08-0.12 parts of the postbiotic of item 6, or further comprises 0.28-0.32 parts of prebiotics, more preferably the prebiotics are inulin, and the starter culture comprises Lactobacillus bulgaricus and Streptococcus thermophilus.

[0092] Item 10. Use of the postbiotic of item 6 or the product of item 8 or the fermented milk of item 9 in improving immunity, reducing immune organ damage, reducing inflammation and / or regulating intestinal flora.

[0093] The preferred embodiments of the present application will be described in detail below with reference to the following examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and substitutions to the present application without departing from the spirit and principles of the present application.

[0094] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0095] The grapefruit peel extract used in the specific embodiments of the present application comprises 75.2% water-soluble dietary fiber, 9.8% starch, 6.7% fat and 8.3% protein. The hydrolysis rate of the alkaline protease hydrolyzed milk is 10%. The protective agent comprises 10 parts of skimmed milk powder, 3 parts of L-cysteine and 10 parts of xylan.

[0096] The extract of pomelo peel used in Example 3-5 includes water-soluble dietary fiber 79.9%, starch 8.2%, fat 5.5%, and protein 6.4%. The hydrolysis rate of the alkaline protease hydrolyzed skim milk is 10%. The protective agent includes 8 parts of milk powder, 1.5 parts of resistant dextrin, 2 parts of lactose, 1 part of trehalose, 1 part of inulin, 1 part of malt dextrin, and 0.5 part of sodium glutamate.

[0097] Example 1

[0098] In this example, an inactivated bacteria powder is provided, which includes inactivated Bifidobacterium longum BBMN68, and the specific preparation process is as follows (each part represents 10 g):

[0099] 1. Raw materials:

[0100] Water 84.89 parts, alkaline protease hydrolyzed milk 10.0 parts, corn syrup 1.5 parts, sucrose 2.5 parts, cysteine hydrochloride 0.06 parts, Tween-80 0.08 parts, and pomelo peel extract 1.0 part.

[0101] 2. Preparation method:

[0102] ① Preheat the water to 55°C, and then add the alkaline protease hydrolyzed milk, sucrose, pomelo peel extract, corn syrup, cysteine hydrochloride, and Tween-80, and maintain the temperature and stir for 15 min;

[0103] ② Heat the material obtained in step ① to 65°C, and then homogenize under the conditions of 65°C and 20 MPa. After homogenization, sterilize at 125°C for 300 s, and then cool to 37°C to obtain a fermentation substrate;

[0104] ③ Add Bifidobacterium longum BBMN68 (the fermentation strain Bifidobacterium longum BBMN68 is activated and cultured in MRSC liquid medium) to the fermentation substrate to make the concentration 1×10 7 CFU / mL, and then incubate at 37°C under anaerobic conditions for 16 h. The culture is incubated to the logarithmic phase, and the viable bacterial count reaches more than 5×10 9 CFU / mL;

[0105] ④ Centrifuge the fermentation material obtained in step ③ to collect the wet bacteria, and then pretreat at 45°C for 5 min. Add the protective agent (wet bacteria: protective agent = 4:1 L / kg) at a volume-to-mass ratio of 4:1, adjust the pH value to 4.3 with lactic acid, inactivate at 120°C for 1 min, and then vacuum dry at 50°C to obtain the inactivated bacteria powder of Bifidobacterium longum BBMN68.

[0106] Example 2

[0107] In this example, an inactivated bacteria powder is provided, and the specific preparation process is as follows:

[0108] 1. Raw materials:

[0109] Water 83.08 parts, alkaline protease hydrolyzed milk 12.0 parts, corn syrup 1.8 parts, sucrose 1.5 parts, cysteine hydrochloride 0.03 parts, grapefruit peel extract 1.5 parts.

[0110] 2. Preparation method:

[0111] ①Preheat water to 55℃, add alkaline protease hydrolyzed milk, sucrose, grapefruit peel extract, corn syrup, cysteine hydrochloride, and maintain temperature stirring for 15 min;

[0112] ②Heat the material obtained in step ① to 65℃, and homogenize under the condition of 65℃, 20MPa, after homogenization, sterilize at 125℃ for 300s, and cool to 37℃ to obtain a fermentation substrate;

[0113] ③Add Bifidobacterium longum BBMN68 to the fermentation substrate to make its concentration 1×10 7 CFU / mL, and incubate at 37℃ under anaerobic conditions for 16h, culture to logarithmic phase, and the number of viable bacteria reaches more than 5×10 9 CFU / mL;

[0114] ④Centrifuge the fermentation material obtained in step ③ to collect wet bacteria, pretreat at 42℃ for 15 min, add protective agent (wet bacteria: protective agent = 4:1 L / kg) at a volume to mass ratio of 4:1, adjust pH to 4.5 by adding acetic acid, inactivate at 100℃ for 3 min, and vacuum dry at 50℃ to obtain Bifidobacterium longum BBMN68 inactivated bacteria powder.

[0115] Example 3

[0116] An inactivated bacteria powder is provided in this example, as follows:

[0117] 1. Raw materials

[0118] Water 78.96 parts, alkaline protease hydrolyzed skim milk 15.0 parts, yeast extract powder 2.0 parts, glucose 0.8 parts, ascorbic acid 0.09 parts, Tween-80 0.15 parts, grapefruit peel extract 3.0 parts.

[0119] 2. Preparation method:

[0120] ①Preheat water to 55℃, add alkaline protease hydrolyzed skim milk, glucose, grapefruit peel extract, yeast extract powder, ascorbic acid, and Tween-80, and maintain temperature stirring for 15 min;

[0121] ②Heat the material obtained in step ① to 65℃, and homogenize under the condition of 65℃, 20MPa, after homogenization, sterilize at 125℃ for 300s, and cool to 37℃ to obtain a fermentation substrate;

[0122] III. Adding Bifidobacterium longum BBMN68 to the fermentation medium to a concentration of 1 x 10 7 CFU / mL, and incubating at 37°C under anaerobic conditions for 16 h until the logarithmic phase and the viable bacterial count reaches 5 x 10 9 CFU / mL or more;

[0123] IV. Centrifuging the fermentation product obtained in step III to collect the wet bacterial cells, pretreating at 45°C for 10 min, adding the protective agent (wet bacterial cells: protective agent = 4: 1 L / kg) at a volume-to-mass ratio of 4: 1, adding citric acid to adjust the pH value to 4.8, inactivating at 85°C for 10 min, and vacuum drying at 50°C to obtain Bifidobacterium longum BBMN68 inactivated bacterial powder.

[0124] Example 4

[0125] In this example, an inactivated bacterial powder is provided, as follows:

[0126] 1. Raw materials

[0127] Water 83.86 parts, alkaline protease hydrolyzed skim milk 12.0 parts, yeast extract 1.5 parts, glucose 1.3 parts, ascorbic acid 0.09 parts, Tween-80 0.08 parts, and grapefruit peel extract 1.0 part.

[0128] 2. Preparation method:

[0129] I. Preheating water to 55°C, adding alkaline protease hydrolyzed skim milk, glucose, grapefruit peel extract, yeast extract, ascorbic acid, and Tween-80, and stirring at the temperature for 15 min;

[0130] II. Heating the material obtained in step I to 65°C, homogenizing at 65°C and 20 MPa, sterilizing at 125°C for 300 s after homogenization, and cooling to 37°C to obtain a fermentation medium;

[0131] III. Adding Bifidobacterium longum BBMN68 to the fermentation medium to a concentration of 5 x 10 7 CFU / mL, and incubating at 37°C under anaerobic conditions for 16 h until the logarithmic phase and the viable bacterial count reaches 5 x 10 9 CFU / mL or more;

[0132] IV. Centrifuging the fermentation product obtained in step III to collect the wet bacterial cells, pretreating at 45°C for 10 min, adding the protective agent (wet bacterial cells: protective agent = 4: 1 L / kg) at a volume-to-mass ratio of 4: 1, adding lactic acid to adjust the pH value to 4.5, inactivating at 65°C for 30 min, and vacuum drying at 50°C to obtain Bifidobacterium longum BBMN68 inactivated bacterial powder.

[0133] Example 5

[0134] An inactivated bacteria powder is provided in this embodiment, which is specifically as follows:

[0135] 1. Raw materials

[0136] Water 84.04 parts, alkaline protease hydrolyzed skim milk 10.0 parts, yeast extract powder 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 part, and grapefruit peel extract 2.0 parts.

[0137] 2. Preparation method

[0138] ① The water was preheated to 55℃, and the alkaline protease hydrolyzed skim milk, glucose, grapefruit peel extract, yeast extract powder, ascorbic acid, and Tween-80 were added, and stirred at the temperature for 15 min;

[0139] ② The material obtained in step ① was heated to 65℃, and homogenized at 65℃ and 20 MPa, and then sterilized at 125℃ for 300 s, and cooled to 37℃ to obtain a fermentation substrate;

[0140] ③ Bifidobacterium longum BBMN68 was added to the fermentation substrate to a concentration of 5×10 7 CFU / mL, and incubated at 37℃ under anaerobic conditions for 16 h, and the viable cell count reached more than 5×10 9 CFU / mL in the logarithmic phase;

[0141] ④ The fermentation product obtained in step ③ was centrifuged to collect the wet bacteria, which was pretreated at 45℃ for 10 min, and then added to the protective agent (wet bacteria: protective agent = 4:1 L / kg) at a volume-to-mass ratio of 4:1, and the pH value was adjusted to 4.5 by adding lactic acid, and then inactivated at 75℃ for 1 min, and vacuum dried at 50℃ to obtain the Bifidobacterium longum BBMN68 inactivated bacteria powder.

[0142] Example 6

[0143] An inactivated bacteria powder is provided in this embodiment, which is specifically as follows:

[0144] 1. Raw materials

[0145] Water 85.54 parts, alkaline protease hydrolyzed skim milk 11.5 parts, yeast extract powder 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 part, and grapefruit peel extract 0.5 part.

[0146] 2. The preparation method is the same as that in Example 5.

[0147] Example 7

[0148] In this example, a kind is provided to inactivate bacteria powder, compared with example 5, the proportion of grapefruit skin extract and alkaline protease hydrolysis skim milk in fermentation substrate is changed, as follows:

[0149] 1, raw materials:

[0150] Water 82.54 parts, alkaline protease hydrolysis skim milk 8.5 parts, yeast extract powder 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 parts, grapefruit skin extract 3.5 parts.

[0151] 2, the preparation method is same with example 5.

[0152] Comparative example 1

[0153] In this comparative example, a kind of inactivated bacteria powder is provided, compared with example 5, there is no grapefruit skin extract in fermentation substrate, as follows:

[0154] 1, raw materials:

[0155] Water 86.04 parts, alkaline protease hydrolysis skim milk 10.0 parts, yeast extract powder 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 parts.

[0156] 2, the preparation method is same with example 5.

[0157] Comparative example 2

[0158] In this comparative example, a kind of inactivated bacteria powder is provided, compared with example 5, ordinary skim milk is used instead of alkaline protease hydrolysis skim milk, as follows:

[0159] 1, raw materials:

[0160] Water 84.04 parts, skim milk 10.0 parts, yeast extract powder 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 parts, grapefruit skin extract 2.0 parts.

[0161] 2, the preparation method is same with example 5.

[0162] Comparative example 3

[0163] In this comparative example, a kind of inactivated bacteria powder is provided, compared with example 5, other water-soluble dietary fiber is used instead of grapefruit skin extract, as follows:

[0164] 1, raw materials:

[0165] Water 84.04 parts, alkaline protease hydrolysis skim milk 10.0 parts, yeast extract powder 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 parts, soybean polysaccharide 2.0 parts.

[0166] 2. The preparation method is the same as that of Example 5.

[0167] Comparative Example 4

[0168] In this comparative example, an inactivated bacteria powder is provided, which replaces the grapefruit peel extract with other water-soluble dietary fibers compared to Example 5, as follows:

[0169] 1. Raw materials:

[0170] Water 84.04 parts, alkaline protease hydrolyzed skim milk 10.0 parts, yeast extract 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 parts, β-polyglucose 2.0 parts.

[0171] 2. The preparation method is the same as that of Example 5.

[0172] Comparative Example 5

[0173] In this comparative example, an inactivated bacteria powder is provided, which has the same preparation method as Example 5, except that in step ④ of the preparation, the step of pretreating at 45°C for 10 min is removed, and the subsequent steps are performed directly. It is found that the cell breakage rate increases (about 10%), and the cells are severely shrunk (see Figure 2), which is more prone to rupture in the subsequent steps.

[0174] Experimental Example 1

[0175] In this experimental example, the inactivated bacteria powder prepared in the above examples is observed under an electron microscope and the number of live bacteria is detected. It is found that the cells are intact and not ruptured (see Figure 1 for a representative electron micrograph of Example 5), and no live bacteria are detected by plate counting.

[0176] Method for observing bacteria strains under a scanning electron microscope:

[0177] Take 0.1 g of bacteria powder and add 10 mL of PBS, shake and mix evenly, centrifuge at 8000 rpm for 3-5 min, discard the supernatant, and pour into 2.5% glutaraldehyde fixing solution. Dehydrate with alcohol gradient, the gradient is 30%, 50%, 60%, 70%, 80%, 90%, 95%, and 100%, 15 min per gradient. Use a critical point dryer for critical point drying. After drying, the bacteria powder is fixed on the sample stage with carbon conductive double-sided tape, ensuring uniform dispersion and firm adhesion. Use a suction ball to remove the excess bacteria on the sample stage, spray gold and observe under a scanning electron microscope.

[0178] Method for detecting live bacteria by plate counting:

[0179] Refer to "GB 4789.35-2023 National Food Safety Standard Food Microbiological Examination Lactic Acid Bacteria Examination" for bifidobacterium counting.

[0180] According to the estimation of the content of Bifidobacterium in the sample to be detected, 2-3 consecutive appropriate dilutions are selected, 1 mL of sample homogenate is taken in a sterile flat dish for each dilution, and two dishes are prepared for each dilution. After the dilution is transferred to the dish, 15 mL-20 mL of mupirocin lithium salt and cysteine hydrochloride modified MRS agar medium cooled to 48℃-50℃ is poured into the dish, and the dish is rotated to mix evenly. After the medium is solidified, it is inverted and cultured at 36℃±1℃ in an anaerobic environment. According to the growth characteristics of Bifidobacterium, the culture is generally selected for 48 h, and if the colonies do not grow or grow smaller, the culture can be selected for 72 h. After culture, count all the colonies on the plate. The sample dilution to the plate pouring is required to be completed within 15 min.

[0181] Experimental Example 2

[0182] In this experimental example, the active metabolites in the inactivated bacterial powder prepared in the above examples and comparative examples are detected, and the specific results are shown in Tables 1 and 2.

[0183] Table 1 Active metabolites in the fermentation product of the example

[0184] Table 2 Active metabolites in the fermentation product of the comparative example

[0185] The content detection method of linalyl acetate and vanillin is referred to GB 1886.126-2015 Food Safety National Standard Food Additive Linalyl Acetate, GB 5009.284-2021 Food Safety National Standard Determination of Vanillin, Methyl Vanillin, Ethyl Vanillin and Coumarin in Food Third Method.

[0186] The total short-chain fatty acid is a fatty acid with a chain length of 1-6 carbon atoms, specifically including acetic acid, propionic acid, isobutyric acid, valeric acid, isovaleric acid, n-butyric acid, caproic acid, etc.

[0187] From the test results, compared with the comparative example, the fermentation product of the example of the application contains a large amount of active metabolites, including natural vanillin, linalyl acetate, lactic acid, acetic acid, propionic acid, caproic acid, butyric acid, exopolysaccharide, etc. which are helpful to regulate immunity. Linalyl acetate belongs to terpenoids, and studies have shown that it has anti-inflammatory effects in mouse allergic asthma models, rat inflammatory edema models, and endothelial cell and epithelial cell models. Vanillin has antibacterial effect, certain antioxidant property and cancer prevention effect, and can participate in signal transmission between bacterial cells. Exopolysaccharide has antioxidant, immunocompetent, anti-tumor and anti-ulcer biological activity.

[0188] Experimental Example 3

[0189] The inactivated bacteria powder prepared by the present application and the yogurt containing the bacteria powder are verified for the effect of immunity recovery.

[0190] 1.1 Experimental animals

[0191] 120 SPF level healthy Balb / c male mice, 5-6 weeks old, weighing 20±2 g, were purchased and raised in standard mouse cages in the animal room. During the adaptation period, they were allowed to eat and drink freely. During the experiment, they were numbered and labeled, and their food intake, mental state, etc. were observed and recorded. Other management measures refer to the mouse feeding and management manual.

[0192] 1.1.1 Establishment and grouping of animal models

[0193] The SPF level Balb / c male mice (weighting 20±2 g) were randomly divided into 10 groups, 12 in each group, as follows:

[0194] (1) C: blank control group;

[0195] (2) M: CTX cyclophosphamide modeling group;

[0196] (3) LMS: levamisole positive drug group;

[0197] (4) 68L: low-dose probiotic group of Example 5 (BBMN68: 1×10 8 individuals / (kg·d));

[0198] (5) 68H: high-dose probiotic group of Example 5 (BBMN68: 1×10 10 individuals / (kg·d));

[0199] (6) 68Y: probiotic yogurt group (BBMN68: 5×10 8 individuals / (kg·d));

[0200] (7) 68JY: probiotic + prebiotic yogurt group (BBMN68: 5×10 8 individuals / (kg·d) + inulin 25 g / (kg·d)).

[0201] Among them, the 68Y group is a fermented dairy product containing the probiotic Bifidobacterium longum BBMN68 prepared in Example 5, and the preparation method is as follows:

[0202] (1) Preheat 92.55 kg of raw milk to 50℃, add 6.0 kg of white sugar, 0.65 kg of egg white powder, 0.4 kg of starch, 0.1 kg of pectin, and 0.3 kg of agar, and maintain the temperature during preheating for 15 min;

[0203] (2) homogenized at 65℃, 25MPa, sterilized at 135℃ for 8s after homogenization, and cooled to 42℃ to obtain a fermentation base;

[0204] (3) 0.01kg of a fermenting agent (containing Lactobacillus bulgaricus and Streptococcus thermophilus, with a live bacteria number ratio of 1:2) and 0.1kg of the inactivated Bifidobacterium longum BBMN68 powder (postbiotic) prepared in Example 5 were added to the fermentation base, and fermentation was performed at 42℃ until the acidity reached 70°T, the fermentation was stopped, sterilized at 76℃ for 30s, and cooled to 20℃ to obtain a fermentation product;

[0205] (4) The fermentation product was homogenized and aseptically filled, and the protein content in the final product was 3.2%.

[0206] Among them, the 68JY group is a fermented dairy product containing the postbiotic Bifidobacterium longum BBMN68 and prebiotics prepared in Example 5, and the preparation method is as follows:

[0207] (1) 92.5kg of raw milk was preheated to 55℃, 5.8kg of white granulated sugar, 0.6kg of protein powder, 0.4kg of starch, 0.1kg of pectin, 0.3kg of agar, and 0.3kg of inulin were added, and the temperature during preheating was maintained for 18min;

[0208] (2) homogenized at 65℃, 25MPa, sterilized at 135℃ for 8s after homogenization, and cooled to 42℃ to obtain a fermentation base;

[0209] (3) 0.01kg of a fermenting agent (containing Lactobacillus bulgaricus and Streptococcus thermophilus, with a live bacteria number ratio of 1:2) and 0.1kg of the inactivated Bifidobacterium longum BBMN68 powder (postbiotic) prepared in Example 5 were added to the fermentation base, and fermentation was performed at 42℃ until the acidity reached 72°T, the fermentation was stopped, sterilized at 78℃ for 30s, and cooled to 20℃ to obtain a fermentation product;

[0210] (4) The fermentation product was homogenized and aseptically filled, and the protein content in the final product was 3.1%.

[0211] In addition to the blank control group, the remaining 9 groups were injected with cyclophosphamide 50mg / kg / d, continuously for 5d, to establish an immunosuppression (hypofunction) model. Except for the blank control group and the model group (CTX cyclophosphamide modeling group), physiological saline 0.1mL / 10g was used for gavage, and the positive drug group of the drug levamisole was used for gavage with levamisole 50mg / (kg·d), and the remaining 7 groups were gavaged according to the respective target substance administration amount, continuously for 15d.

[0212] The health status of each group of mice was observed during the experiment, including development, diet, mood, fur color, and mental state; the body weight of each group of mice was measured between 8-10 am every day. The food intake of each group of mice was measured every three days, and the cage bedding was changed to ensure a clean and tidy environment. The data were recorded and statistically analyzed. The mice were fasted and deprived of water for 12 h before being sacrificed to ensure that the recovered intestinal tract was suitable for subsequent experiments.

[0213] 1.1.2 Blood and tissue, intestinal content sample collection

[0214] After the mice were enucleated and blood was taken, 70 μL of the whole liquid was drawn into a 1.5 mL anticoagulant tube using a pipette for whole blood leukocyte count analysis. The remaining blood was left at room temperature until the serum separated at 4500 r / min for 15 min, and then was aliquoted for subsequent experimental analysis.

[0215] After the mice were enucleated and decapitated, the mouse body was disinfected with 75% ethanol, and the spleen and thymus, mesenteric lymph nodes, and inguinal lymph nodes were removed, washed with PBS, weighed, and the organ index was calculated. The spleen tissue was removed, washed with ice PBS, and divided into two parts. One part was placed in a 2 mL centrifuge tube containing 4% paraformaldehyde solution and fixed at room temperature for 20 h before paraffin embedding; the other part was washed with ice PBS, quickly frozen in liquid nitrogen, and stored in a -80°C ultra-low temperature freezer for subsequent analysis.

[0216] The proximal cecal contents were removed, washed with ice PBS, and placed in a 1.5 mL cryogenic tube for 16S rDNA analysis of intestinal flora diversity changes.

[0217] 1.2 Animal experiment detection indexes

[0218] 1.2.1 Mouse organ index detection

[0219] After the last administration, the mice were fasted for 12 h, enucleated and blood was taken, decapitated, and then the thymus and spleen were removed and weighed to calculate the thymus index and spleen index of the mice.

[0220] Thymus index = thymus weight (mg) / body weight (10 g);

[0221] Spleen index = spleen weight (mg) / body weight (10 g).

[0222] 1.2.2 Mouse peripheral immune organ lymph node determination

[0223] After the last administration, the mice were fasted for 12 h, enucleated and blood was taken, decapitated, and the mesenteric lymph nodes and inguinal lymph nodes were removed and weighed.

[0224] 1.2.3 Effect on immune cells in whole blood of immunosuppressed mice caused by cyclophosphamide

[0225] After the last administration, the mice were fasted for 12 h, and then the eyeballs were removed to obtain 70 μL of whole blood. The white blood cell, neutrophil, lymphocyte, eosinophil, and monocyte contents in the blood of the mice were detected by a fully automatic blood cell analyzer.

[0226] 1.2.4 Pathological observation of the spleen of mice

[0227] The tissues were paraffin-embedded, and the paraffin blocks were cut into 4-μm-thick sections by a microtome, followed by hematoxylin / eosin (HE) staining. A Pannoramic 250 digital slide scanner produced by 3DHISTECH (Hungary) was used for image acquisition of the sections. Each section was first observed at a low magnification to observe the general lesions of the whole tissue, and then a 400-fold image of the region to be observed was collected to observe the specific lesions.

[0228] 1.2.5 Determination of physiological and biochemical indexes

[0229] The contents of IL-2 and TNF-α in the serum, as well as the contents of serum immunoglobulins IgA, IgM, and IgG, were determined according to the instructions of the kit.

[0230] 1.2.6 Analysis of intestinal microbial flora

[0231] The cecal contents were collected for high-throughput sequencing of intestinal microorganisms. After DNA extraction, PCR amplification, fluorescence quantification, and Miseq library construction, the samples were subjected to Miseq sequencing. The data obtained by Miseq sequencing were subjected to bioinformatics analysis. First, the PE reads were spliced according to the overlap relationship, and the sequence quality was controlled and filtered. After the samples were distinguished, OTU clustering analysis and taxonomic analysis were performed. Based on the results of OTU clustering analysis, various diversity index analysis and sequencing depth detection of OTU can be performed; based on the taxonomic information, statistical analysis of the community structure can be performed at each classification level. On the basis of the above analysis, a series of in-depth statistical and visualization analyses such as multivariate analysis and difference significance test of the community composition and phylogenetic information of multiple samples can be performed.

[0232] 2 Results

[0233] 2.1 Effect on the immune organs of mice

[0234] The immune system is a biological defense mechanism, which is composed of immune organs, immune cells and immune molecules. Spleen, thymus and lymph node tissue are the main immune organs of animal body. Thymus directly regulates the cellular immune function of the body and has an indirect effect on humoral immunity. The spleen contains a large number of lymphocytes and macrophages, which are closely related to the specific immunity of the body. Therefore, the thymus and spleen indexes can reflect the immune function of the body to a certain extent.

[0235] The experimental results are shown in Table 3. Compared with the blank control group, the thymus index and the wet weight of lymph nodes of the model group mice were significantly reduced, showing immunosuppression, and the spleen index was significantly increased. This finding may be due to the compensatory enlargement of the spleen caused by the postbiotic and its products under the condition of immune deficiency. This compensatory effect may improve the immunity of the host (P<0.05). The intervention of the postbiotic and its products can improve the thymus index and the wet weight of lymph nodes of the immunosuppressed mice and reduce the spleen index (P<0.05). This shows that the intervention of the postbiotic and its products can alleviate the damage to the immune organs caused by cyclophosphamide and improve the non-specific immunity of mice.

[0236] Table 3 Effects of peripheral immune organs of immunosuppressed mice

[0237] The small letters on the numbers in the table represent significant differences, P<0.05.

[0238] 2.2 Effects on the histopathological characteristics of immune organ tissues of immunosuppressed mice

[0239] The microscopic images of the histological changes of the spleen of mice in each group observed by HE staining are shown in Figures 3 to 9. As can be seen from the figures, compared with the blank control group, the white pulp and red pulp of the model group mice are not clear, the white pulp is atrophied to varying degrees, the lymph node volume is small or even disappears, the extramedullary hematopoiesis in the red pulp is obvious, and there are visible sheet-like distribution of red blood cells, granulocytes, and scattered distribution of megakaryocytes and cell fragments, and inflammatory cell infiltration. In the postbiotic and its product intervention group, the capsule of the mouse spleen tissue is complete, the capsule and the fibrous connective tissue of the splenic trabecula are not proliferated, the boundary between the white pulp and the red pulp is clear, the structure of the periarterial lymph sheath and the lymph node in the white pulp is relatively complete and clear, and the white pulp is not obviously atrophied or proliferated. The results show that the intervention of the postbiotic and its products can alleviate the damage to the immune organ spleen tissue caused by cyclophosphamide.

[0240] 2.3 Effects on the whole blood immune cells of immunosuppressed mice

[0241] The lymphocytes and leukocytes in the blood reflect the overall immune response level of the body. The experimental results are shown in Table 4. Compared with the blank control group, the model group significantly increased the proportion of mouse leukocytes and eosinophils, and reduced the proportion of neutrophils, lymphocytes and monocytes. Compared with the model group, the invented probiotics and their products significantly reduced the proportion of mouse leukocytes and peripheral blood eosinophils, and increased the proportion of neutrophils, lymphocytes and monocytes (P<0.05). Studies have shown that the increase in the proportion of eosinophils is often associated with the production of inflammation, which is caused by the use of the drug myelosuppressive agent cyclophosphamide, resulting in an increase in the proportion of allergy-related eosinophils. The invented probiotics and their products alleviate the inhibitory effect of cyclophosphamide on the humoral immune function of mice, and reduce the production of inflammation to promote the gradual recovery of the immune function of mice.

[0242] Table 4 Effect on immune suppression peripheral blood cells

[0243] The lower case letters on the numbers in the table represent significant differences, P<0.05.

[0244] 2.4 Effect on serum cytokines of immunosuppressed mice

[0245] Cytokines are potential therapeutic targets for regulating immune responses. Th cells are initial CD4 + T cells, which are divided into different subtypes due to different functions, the most common of which are Th1 and Th2 types. To ensure normal immunity and body health, the secretion of cytokines can maintain Th1 and Th2 cells in a balanced state. IL-2 is a T cell growth factor that has a great influence on the proliferation of T cells and the differentiation of effector cells and memory cells, and can promote the occurrence of T cell immune response. TNF-α plays an important role in innate immune response, acquired humoral immune response and cellular immune response, and is one of the most important cytokines in the body, which is a prototype cytokine for maintaining immune balance in the body.

[0246] The experimental results are shown in Table 5. Compared with the blank control group, the model group significantly reduced the levels of TNF-α and IL-2 in the serum of mice. Compared with the model group, the invented probiotics and their products increased the content of cytokines in the serum (P<0.05). It is shown that the invented probiotics and their products alleviate the inhibitory effect of cyclophosphamide on the humoral immune function of mice, and in the aspect of immune cells, the invented probiotics and their products exhibit enhanced Th1 cell immune regulation ability.

[0247] Table 5 Effect on serum cytokines

[0248] The lower case letters on the numbers in the table represent significant differences, P<0.05.

[0249] 2.5 Effect on serum immunoglobulin of immunosuppressed mice

[0250] Immunoglobulin (Ig) refers to an animal protein with antibody activity, mainly existing in plasma, body fluid, tissue and some secretions. In the human body, the structure of IgA mainly exists in the form of monomer and dimer, and is divided into serum type and secretory type. Secretory IgA is the main component of the body's mucosal defense system, and is the first line of defense against pathogens and harmful substances in the respiratory tract, digestive tract, urinary and genital tract, etc. It is the most important antibody in the body's mucosal immune process. The experimental results are shown in Table 6. It can be seen from the table that the IgA level of the mice treated with cyclophosphamide is significantly lower than that of the blank control group (P<0.05). Compared with the model group, the postbiotic and its products of the application can increase the secretion of serum IgA (P<0.05), indicating that they can enhance the humoral immune response of immunosuppressed mice.

[0251] At present, most of the antibacterial and antiviral antibodies belong to IgG type, which plays a major role in the cellular immune process of the body, has the effects of promoting the phagocytosis of mononuclear macrophages, neutralizing the toxicity of bacterial toxins, and combining with viral antigens to make the virus lose the ability to infect host cells. The experimental results are shown in Table 6. Compared with the model group, the IgG content in the serum of the immunosuppressed mice after being given the postbiotic and its products by gavage was significantly up-regulated (P<0.05), indicating that the postbiotic and its products of the application have a positive regulation effect on the IgG level in intestinal tissue, and the postbiotic and its products of the application have a certain improvement effect on the immune function of the immunosuppressed mice treated with cyclophosphamide.

[0252] Immunoglobulin M (IgM) is the largest immunoglobulin in molecular weight, which is mainly synthesized and secreted by plasma cells in the spleen and lymph nodes, and mainly distributed in serum, accounting for 5% to 10% of total serum Ig. IgM has strong bactericidal, complement-activating, immune opsonization and agglutination effects, and also participates in the pathological process of some autoimmune diseases and hypersensitivity reactions. The IgM level of the mice treated with cyclophosphamide was significantly lower than that of the blank control group (P<0.05). Compared with the model group, the postbiotic and its products of the application can increase the secretion of serum IgM (P<0.05), indicating that they can enhance the humoral immune response of immunosuppressed mice.

[0253] Table 6 Effect on serum immunoglobulin

[0254] The small letters on the numbers in the table represent significant differences, P<0.05.

[0255] 2.6 Effect on intestinal flora of immunosuppressed mice

[0256] The results of the study on the influence of the postbiotic on the characteristic flora of the immunocompromised mice show (the experimental results are shown in Table 7) that 7 beneficial bacteria and 1 harmful bacteria of the 8 bacteria related to immunity are changed. The abundance of the 7 beneficial bacteria related to immunity and metabolic conditions is increased, and the abundance of the 1 harmful bacteria related to intestinal diseases and inflammatory conditions is reduced; the results show that the postbiotic has the effect of regulating intestinal flora.

[0257] Table 7 Influence of the product on the characteristic flora of the immunocompromised mice

[0258] The inactivated bacteria powder (postbiotic) prepared in the remaining examples also has similar effects to the inactivated bacteria powder of Example 5, and can improve the immunity of mice, reduce the damage to immune organs, reduce inflammation, and regulate intestinal flora. In each example, the overall effect of Example 5 is the best.

[0259] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application are within the scope of the present application.

Claims

1. A method of fermenting Bifidobacterium longum BBMN68, characterized by, The fermentation substrate comprises alkaline protease hydrolyzed milk and grapefruit peel extract; the hydrolysis rate of the alkaline protease hydrolyzed milk is 9-11%, the water-soluble dietary fiber content of the grapefruit peel extract is 75-80%; and the mass ratio of the alkaline protease hydrolyzed milk to the grapefruit peel extract is (10-15):(1-3).

2. The method of fermenting Bifidobacterium longum BBMN68 according to claim 1, characterized in that, The fermentation substrate further comprises a carbon source, a nitrogen source, an antioxidant and water, or further comprises a surfactant; Preferably, the fermentation substrate comprises 10-15 parts of alkaline protease hydrolyzed milk, 1.5-2.0 parts of yeast extract or corn steep liquor, 0.8-2.5 parts of glucose or sucrose, 0.03-0.09 parts of ascorbic acid or cysteine hydrochloride, 1.0-3.0 parts of grapefruit peel extract, 78-85 parts of water, or further comprises 0.08-0.15 parts of Tween-80; and / or the inoculation concentration of Bifidobacterium longum BBMN68 in the fermentation substrate is 1-5 x 10 7 CFU / mL; And / or, the fermentation conditions are: anaerobic, 37±1℃, static culture.

3. A Bifidobacterium longum BBMN68 ferment, characterized in that, Prepared by the method of fermenting Bifidobacterium longum BBMN68 according to any one of claims 1-2.

4. A composition as a fermentation substrate for improving the content of one or more of vanillin, linalyl acetate, lactic acid, acetic acid, propionic acid, hexanoic acid, butyric acid, exopolysaccharide and total short-chain fatty acid in Bifidobacterium longum BBMN68 fermentation product; the composition comprises alkaline protease hydrolyzed milk and grapefruit peel extract; the hydrolysis rate of the alkaline protease hydrolyzed milk is 9-11%, the water-soluble dietary fiber content of the grapefruit peel extract is 75-80%; and the mass ratio of the alkaline protease hydrolyzed milk to the grapefruit peel extract is (10-15):(1-3).

5. A method of preparing a postbiotic, characterized in that, Obtained by inactivating the Bifidobacterium longum BBMN68 fermentation product according to claim 3; Preferably, the method comprises: (1) heating the bacteria in the Bifidobacterium longum BBMN68 fermentation product according to claim 3 at 42-45℃ for 5-15 minutes; (2) mixing with an inactivation protective agent and adjusting the pH value to 4.3-4.8; (3) inactivating at 65-120℃ for 1-30 minutes; More preferably, the method further comprises: (4) vacuum drying at 40-60℃; And / or, the inactivation protective agent comprises: 8-12 parts of skimmed milk powder, 1-3 parts of resistant dextrin, 4-8 parts of lactose, 4-6 parts of trehalose, 1-3 parts of inulin, 1-3 parts of malt dextrin and 0.2-0.6 parts of sodium glutamate; or the inactivation protective agent comprises: 8-12 parts of skimmed milk powder, 2.5-3.5 parts of L-cysteine and 8-12 parts of xylan; And / or, the bacteria in the Bifidobacterium longum BBMN68 fermentation product are obtained by centrifuging the Bifidobacterium longum BBMN68 fermentation product, and the volume-to-mass ratio of the bacteria to the inactivation protective agent is (3.8-4.2):1L / kg; And / or, the pH value in step (2) is adjusted with one or more of lactic acid, acetic acid or citric acid.

6. A postbiotic, characterized in that, Prepared by the method according to claim 5.

7. Use of the postbiotic according to claim 6 in the preparation of a pharmaceutical, nutraceutical or food product.

8. A product which is a pharmaceutical, nutraceutical or food product, characterised in that, Comprising the postbiotic according to claim 6.

9. A fermented milk, characterized in that, The fermented milk comprises animal milk, sugar, protein powder, starch, pectin, agar, inactivated fermenting agent and the postbiotic of claim 6, or further comprises prebiotic; Preferably, the fermented milk comprises 90-95 parts of animal milk, 5.5-6 parts of sugar, 0.6-0.7 parts of protein powder, 0.38-0.42 parts of starch, 0.08-0.12 parts of pectin, 0.28-0.32 parts of agar, 0.008-0.012 parts of inactivated fermenting agent and 0.08-0.12 parts of the postbiotic of claim 6, or further comprises 0.28-0.32 parts of prebiotic, more preferably, the prebiotic is inulin, and the fermenting agent comprises Lactobacillus bulgaricus and Streptococcus thermophilus.

10. Use of the postbiotic of claim 6 or the product of claim 8 or the fermented milk of claim 9 in improving immunity, reducing immune organ damage, reducing inflammation and / or regulating intestinal flora.

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