Probiotic compositions and uses thereof

Abstract

The present application belongs to the technical field of microbial research, and particularly relates to a probiotic composition and use thereof, the probiotic composition comprising two Bifidobacterium animalis lactis strains, or one Bifidobacterium animalis lactis strain and one Bifidobacterium longum longum subsp, or two Bifidobacterium longum longum subsp; and the number of viable bacteria contained in the composition is 1x10 8 CFU / 100g~1x10 12 CFU / 100g, based on the total mass of the composition. Experimental results show that the compound Bifidobacterium can regulate intestinal flora, and has a regulating effect on intestinal flora disorder caused by Enterobacter sakazakii infection, can further intervene in the intestinal colonization process of Enterobacter sakazakii, thereby reducing the colonization level of Enterobacter sakazakii in the intestine of mice, and can inhibit the viability of Enterobacter sakazakii in the intestine of mice. And the probiotic composition can relieve inflammation and tissue damage caused by Enterobacter sakazakii infection.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 202310813974.0, filed on July 5, 2023, entitled "Probiotic Composition and Use Thereof". Technical Field

[0002] This invention belongs to the field of microbial research technology, specifically relating to probiotic compositions and their uses. Background Technology

[0003] The intestine is the largest immune organ in vertebrates. Maintaining good intestinal mucosal immune homeostasis requires a robust mucosal epithelial barrier to enhance absorption and resist pathogen invasion. Disruption of the intestinal epithelial barrier function can lead to various intestinal diseases. The intestinal epithelial barrier comprises various intestinal epithelial cells, which, in addition to their digestive function, also regulate the intestinal mucosal immune response. Simultaneously, the intestine contains a large number of symbiotic microbiota, which also play a regulatory role in nutrient uptake, metabolism, and immune homeostasis. In recent years, the relationship between gut microbiota dysbiosis and inflammatory bowel disease has received increasing attention.

[0004] Cronobacter (formerly known as Cronobacter sakazakii) is a foodborne opportunistic pathogen, resistant to high temperatures (54-62℃), acids (pH < 3.9), and dryness, exhibiting strong environmental resistance and widespread distribution in food and the natural environment. Even in harsh environments such as dryness, Cronobacter sakazakii can survive for extended periods, forming a protective biofilm and demonstrating high toxicity and pathogenicity. Cronobacter sakazakii infection primarily harms infants and young children, causing meningitis, necrotizing enterocolitis, and bacteremia through contaminated infant formula. Infants infected with this bacteria experience rapid onset of illness, with a mortality rate exceeding 50%, and survivors often suffer severe neurological sequelae. Simultaneously, Cronobacter sakazakii can also infect other age groups, such as the elderly and those with weakened immune systems, causing sepsis, neuritis, encephalomyelitis, brain abscess, hydrocephalus, and necrotizing enterocolitis.

[0005] Many scholars have dedicated themselves to the study of this bacterium and have established accurate, sensitive, and specific detection methods. However, research on the pathogenic mechanism of Cronobacter sakazakii contamination is still insufficient, and prevention and control mainly rely on drugs and antibiotics. However, newborns, especially premature infants, are a special population and usually cannot take medication directly. Furthermore, the intestinal flora of infants has not yet established a relatively stable normal ecosystem at birth, and the acidity of their stomachs is higher than that of adults, making it more suitable for Cronobacter sakazakii to survive, thus increasing the risk of pathogenicity after infection. Secondly, the blood-brain barrier is not fully developed in infants; after entering the intestinal epithelial cells, Cronobacter sakazakii can further enter the brain via the bloodstream, causing brain infection and ultimately leading to death or severe neurological sequelae. Summary of the Invention

[0006] The problem the invention aims to solve

[0007] The existing technologies have limitations, such as insufficient research on the relationship between gut microbiota dysbiosis and inflammatory bowel disease, and the fact that the treatment of symptoms caused by pathogenic bacterial infections is mainly based on drugs and antibiotics, without taking into account the special characteristics of key populations such as infants and premature infants, who cannot directly take drugs for prevention and treatment.

[0008] Through extensive screening, this invention has yielded a probiotic composition that helps regulate the gut microbiota and is suitable for relieving symptoms such as inflammatory response and tissue damage caused by Cronobacter sakazakii infection. It can also be used for special populations such as infants.

[0009] Solution for solving the problem

[0010] To address the problems in the prior art, the present invention provides the following technical solution:

[0011] [1]. A probiotic composition, wherein the probiotic composition comprises two strains of Bifidobacterium animalis subsp. lactis, or comprises one strain of Bifidobacterium animalis subsp. lactis and one strain of Bifidobacterium longum subsp. longum, or two strains of Bifidobacterium longum subsp. longum; and, based on the total mass of the composition, the number of viable bacteria contained in the composition is 1 × 10⁻⁶. 8 CFU / 100g ~ 1×10 12 CFU / 100g.

[0012] [2]. According to the probiotic composition of [1], wherein, in the composition, the ratio between the two strains of Bifidobacterium lactis, or between the one strain of Bifidobacterium lactis and the one strain of Bifidobacterium longum, or between the two strains of Bifidobacterium longum, is 4:1 to 1:4, based on the number of live bacteria.

[0013] [3]. The method for preparing the probiotic composition according to [1] or [2], wherein the method includes the step of mixing the two strains of Bifidobacterium lactis, or the one strain of Bifidobacterium lactis and the one strain of Bifidobacterium longum, or the two strains of Bifidobacterium longum.

[0014] [4]. Use of the probiotic composition according to [1] or [2] in the preparation of products that help regulate the gut microbiota.

[0015] [5]. Use of the probiotic composition according to [1] or [2] in the preparation of a product having the function of relieving symptoms caused by Cronobacter sakazakii infection.

[0016] [6]. According to the use described in [5], the symptoms caused by the Cronobacter sakazakii infection include inflammatory response, tissue damage and / or changes in gut microbiota.

[0017] [7]. According to the use described in [6], wherein,

[0018] The inflammatory response includes any one or more of the following symptoms: elevated levels of pro-inflammatory cytokines in intestinal tissue, elevated levels of anti-inflammatory cytokines in intestinal tissue, elevated levels of nuclear transcription factors in intestinal tissue, thrombocytosis in whole blood, and elevated blood uric acid.

[0019] The tissue damage includes any one or more of the following symptoms: destruction of intestinal epithelial structure, edema, loss of villi and crypt structures, inflammatory cell infiltration, destruction of goblet cells, loss of mucus layer, and decreased expression of intestinal barrier-related genes.

[0020] [8]. A product comprising a probiotic composition according to [1] or [2].

[0021] [9]. The product according to [8] further contains plant or extract thereof, milk-containing ingredients, animal-derived ingredients, functional additives, trace element supplements and / or any acceptable excipients.

[0022]

[10] . The products described in [8] or [9] are intended to help regulate gut microbiota or relieve symptoms caused by Cronobacter sakazakii infection.

[0023] The effects of the invention

[0024] By implementing the above technical solutions, the present invention achieves the following technical effects:

[0025] Through extensive screening, this invention has developed a probiotic composition that helps regulate the gut microbiota and can be used to alleviate symptoms such as inflammatory response and tissue damage caused by Cronobacter sakazakii infection.

[0026] Experimental results show that the compound Bifidobacterium can regulate the intestinal flora and has a regulatory effect on the intestinal flora disorder caused by Cronobacter sakazakii infection. Furthermore, it can intervene in the intestinal colonization process of Cronobacter sakazakii, thereby reducing the colonization level of Cronobacter sakazakii in the mouse intestine and inhibiting the activity of Cronobacter sakazakii in the mouse intestine. Moreover, the probiotic composition can alleviate the inflammatory response and tissue damage caused by Cronobacter sakazakii infection. In addition, the probiotic composition provided by this invention can be added to food or health products for use by special populations such as infants and premature infants, helping to regulate their intestinal flora. Attached Figure Description

[0027] Figure 1 The effects of different probiotic groups on the changes in the transcriptional levels of intestinal cytokines (IL-10, IL-1β, and TNF-α) and nuclear transcription factor (PPARγ) in mice; among them, CG was the control group, MG was the model group, TG1 was test group 1 (a combination of two strains of Bifidobacterium lactis), TG2 was test group 2 (a combination of Bifidobacterium lactis and Lactobacillus acidophilus), TG3 was test group 3 (a combination of Bifidobacterium lactis and Lactobacillus helveticus), and TG4 was test group 4 (a combination of Bifidobacterium lactis and Bifidobacterium longum).

[0028] Figure 2 The effects of two animal Bifidobacterium lactis subsp. lactis combinations on the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), anti-inflammatory cytokines (IL-10), and nuclear transcription factor (PPARγ) in mouse intestinal tissue were investigated. Among them, CG was the control group, MG was the model group, PG was the Bifidobacterium combination prevention group, and TG was the Bifidobacterium combination intervention group.

[0029] Figure 3 H&E staining results of intestinal tissues of mice in each treatment group; where CG is the control group, MG is the model group, PG is the Bifidobacterium combination prevention group, and TG is the Bifidobacterium combination intervention group.

[0030] Figure 4 Intestinal tissue damage scores for different groups; where CG was the control group, MG was the model group, PG was the bifidobacteria combination prevention group, and TG was the bifidobacteria combination intervention group.

[0031] Figure 5 Effects of different treatment groups on the intestinal mucus layer of mice; where CG was the control group, MG was the model group, PG was the bifidobacteria combination prevention group, and TG was the bifidobacteria combination intervention group.

[0032] Figure 6 The effects of different treatment groups on the transcriptional levels of Claudin 3, ZO-1 and E-cadherin 1; where CG was the control group, MG was the model group, PG was the Bifidobacterium combination prevention group, and TG was the Bifidobacterium combination intervention group.

[0033] Figure 7 Changes in key indicators related to immune response or inflammation (RBC, HGB, PLT, WBC, GRAN, and LYMF) in whole blood of mice; where CG was the control group, MG was the model group, PG was the bifidobacteria combination prevention group, and TG was the bifidobacteria combination intervention group.

[0034] Figure 8The changes in serum indicators related to liver and kidney function (BUN, AST, ALT, and UA) in mice were observed. Among them, CG was the control group, MG was the model group, PG was the bifidobacteria combination prevention group, and TG was the bifidobacteria combination intervention group.

[0035] Figure 9 The abundance changes of intestinal microbiota at the phylum level in mice; where CG was the control group, MG was the model group, PG was the Bifidobacterium combination prevention group, and TG was the Bifidobacterium combination intervention group.

[0036] Figure 10 The abundance of intestinal microbiota in mice at the genus level; where CG was the control group, MG was the model group, PG was the Bifidobacterium combination prevention group, and TG was the Bifidobacterium combination intervention group.

[0037] Figure 11 Heatmap analysis was used to analyze the relative abundance of mouse gut microbiota after intervention; CG was the control group, MG was the model group, PG was the Bifidobacterium combination prevention group, and TG was the Bifidobacterium combination intervention group. Detailed Implementation

[0038] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0039] (definition)

[0040] Unless otherwise stated, the terminology used in this invention has the following meanings:

[0041] In this specification, the numerical range referred to as "value A to value B" refers to the range that includes the endpoint values ​​A and B. The numerical range referred to as "above" or "below" refers to the numerical range that includes the stated value. The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0042] In this specification, the terms "about," "substantially," or "essentially" can mean that a value includes the standard deviation of the error of the apparatus or method used to determine that value. The numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains a standard deviation due to the aforementioned testing apparatus or method. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in the present invention are modified with "about." Here, "about" generally means that the standard deviation of the actual value from the theoretical model or theoretical data is within the range of 3%, preferably 2%, and more preferably 1%.

[0043] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0044] In this specification, the terms "optional" or "optional" are used to indicate the use or omission of certain substances, components, procedures, application conditions, etc.

[0045] Unless otherwise specified, “room temperature” in this instruction manual usually refers to a temperature of 23±2℃.

[0046] In this specification, "water" includes any feasible type of water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0047] All unit names used in this manual are international standard unit names, and unless otherwise stated, the "%" indicates weight or mass percentage.

[0048] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0049] In addition, unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0050] The technical solution of the present invention will be further described in detail below:

[0051] (Probiotic composition)

[0052] The present invention provides a probiotic composition comprising two strains of Bifidobacterium animalis subsp. lactis, or comprising one strain of Bifidobacterium animalis subsp. lactis and one strain of Bifidobacterium longum subsp. longum, or comprising two strains of Bifidobacterium longum subsp. longum.

[0053] In some specific embodiments, the *Bifidobacterium animalis* subsp. *lactamase* includes any one or more of strains Bb-12, Bi-07, and HN019. In some specific embodiments, the *Bifidobacterium longum* subsp. *longum* includes strain BB536.

[0054] The present invention does not impose any particular limitation on the source of Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. longis contained in the probiotic composition. For example, finished bacterial cells can be obtained by purchasing them through commercial channels, or bacterial cells can be obtained by fermentation culture.

[0055] To achieve better effects in regulating the gut microbiota and / or alleviating symptoms such as inflammation and tissue damage caused by Cronobacter sakazakii infection, the number of live bacteria in the composition can be controlled. In some embodiments, the composition contains 1 × 10⁻⁶ live bacteria per unit mass of the total composition. 8 CFU / 100g ~ 1×10 12 CFU / 100g, for example, could be approximately 1×10⁻⁶. 8 CFU / 100g, 5×10 8 CFU / 100g, 1×10 9 CFU / 100g, 5×10 9 CFU / 100g, 1×10 10 CFU / 100g, 5×10 10 CFU / 100g, 1×10 11 CFU / 100g, 5×10 11 CFU / 100g or 1×10 12 CFU / 100g, etc.; preferably 1×10 8 CFU / 100g ~ 1×10 11 CFU / 100g; more preferably 1×10 8 CFU / 100g ~ 1×10 10 CFU / 100g.

[0056] Furthermore, in some specific embodiments, in the composition, the ratio of the two *Bifidobacterium lactis* strains, or the ratio of one *Bifidobacterium lactis* strain to one *Bifidobacterium longum* strain, or the ratio of the two *Bifidobacterium longum* strains, based on the number of viable bacteria, is 4:1 to 1:4. Exemplarily, it can be about 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, or 1:4, etc.; preferably 3:1 to 1:3; more preferably 2:1 to 1:2.

[0057] Furthermore, the present invention does not particularly limit the form of the probiotic composition. In some embodiments, the composition is a solid, for example, it may be in the form of a lyophilized powder. In other embodiments, the composition is a solid-liquid mixture, for example, it may be a bacterial sludge produced by fermentation and removal of the culture medium, or it may be a bacterial suspension containing the culture medium after fermentation, or a suspension of bacterial sludge and solvent diluted with a solvent.

[0058] The present invention does not particularly limit the solvents mentioned above; for example, they can be water or physiological saline. To facilitate the use of the probiotic composition and maintain bacterial activity, in some embodiments, the probiotic composition comprises Bifidobacterium bacteria and sterile physiological saline, wherein the Bifidobacterium bacteria are selected from one of the following combinations: two strains of Bifidobacterium animalis subsp. lactis, one strain of Bifidobacterium animalis subsp. lactis and one strain of Bifidobacterium longum subsp. longum, or two strains of Bifidobacterium longum subsp. longum.

[0059] For the sake of consideration regarding the method of use of the probiotic composition and to meet the requirements of relevant laws and regulations, in some embodiments, the probiotic composition is a non-food composition. In some embodiments, the probiotic composition is a non-food and non-health product composition.

[0060] (Preparation method of probiotic composition)

[0061] The present invention also provides a method for preparing the probiotic composition, the method comprising the step of mixing the two strains of Bifidobacterium animalis lactis, or the one strain of Bifidobacterium animalis lactis and a strain of Bifidobacterium longum, or the two strains of Bifidobacterium longum.

[0062] In some specific embodiments, the preparation method of the probiotic composition includes the following steps: fermenting and culturing the two strains of Bifidobacterium animalis subsp. lactis, or fermenting and culturing the one strain of Bifidobacterium animalis subsp. lactis and one strain of Bifidobacterium longum subsp. longum, or the two strains of Bifidobacterium longum subsp. longum, centrifuging to collect the bacterial cells, washing with sterile physiological saline and resuspending and mixing, to obtain the composition; preferably, collecting bacterial cells cultured for 24 hours.

[0063] (product)

[0064] The present invention further provides a product comprising the above-described probiotic composition.

[0065] In addition to the essential components described above, the products of this invention may also include other optional components as needed for the final product, such as:

[0066] The product contains milk or protein components. The milk components include dairy products such as fresh milk, milk powder, whey protein, or cheese derived from raw cow (sheep) milk. The protein components may be derived from plant proteins, such as soy protein or peanut protein.

[0067] Plants or plant extracts, including fruits such as fig, pomegranate, kiwi, orange, tangerine, pineapple, strawberry, apple, rubber, grape, pear, cherry, blueberry, blackberry, blackcurrant, cranberry, raspberry, melon, amla, and bilberry, or their extracts; fruits and vegetables such as onion, cucumber, tomato, cauliflower, carrot, spinach, kale, Brussels sprouts, garlic, basil, and oregano, or their extracts; grains such as rice (indica, japonica, glutinous rice), cereals (wheat, barley, oats, rye), corn, sorghum, millet, foxtail millet, yellow millet, buckwheat, soybean, broad bean, pea, mung bean, red bean, and kidney bean, or their extracts; nuts such as walnut, pistachio, cashew, hazelnut, almond, apricot kernel, pine nut, peanut, sunflower seed, chestnut, macadamia nut, and ginkgo, or their extracts; and coffee or its extracts.

[0068] Animal-derived ingredients, including meat products from cattle, sheep, fish, or poultry.

[0069] The fat component may include at least one of saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, OPO structured lipids, DHA, EPA, ARA, and phospholipids. More specifically, the fat includes safflower seed oil, walnut oil, peanut oil, corn oil, soybean oil, argan oil, olive oil, tea oil, sacha inchi oil, coconut oil, perilla oil, deep-sea fish oil, cocoa butter, palm oil, tallow, cream, lard, medium-chain triglycerides, or lecithin, etc.

[0070] Functional additives include vitamins (one or more of vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, niacin, choline, inositol, and biotin), starch, modified starch, amino acids (L-lysine-L-glutamic acid, L-glutamic acid, L-arginine, L-tryptophan, L-glutamine, taurine, L-valine, L-isoleucine, or L-leucine, etc.), traditional Chinese medicine or its extracts, and dietary fiber (inulin, konjac flour, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin, or soybean fiber, etc.).

[0071] Micronutrient supplements may include metal ion salts of organic acids, such as one or more of the following: calcium citrate, L-calcium lactate, calcium hydrogen phosphate, potassium gluconate, sodium citrate, ferrous gluconate, potassium iodide, zinc gluconate, sodium selenite, copper gluconate, chromium sulfate, manganese gluconate, and magnesium gluconate.

[0072] Any acceptable excipients, including but not limited to solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, food flavorings, food colorings, etc.

[0073] The present invention does not specifically limit the type of the aforementioned product. In some embodiments, the product is a food. In some embodiments, the product is a health food. In some embodiments, the product is a medicine. In other embodiments, the product is a non-food. In other embodiments, the product is a non-health food. In other embodiments, the product is a non-medicine.

[0074] For different product categories, the present invention does not particularly limit the specific form of the product. For example, it can be in powder form or liquid form.

[0075] The present invention does not specifically limit the target population of the product. For example, the product can be used for infants, children, teenagers or adults.

[0076] In some specific implementations, the products described in this invention may be infant formula, baby food, children's formula, children's snacks, formula milk powder for pregnant women, milk powder for middle-aged and elderly people, or nutritional or dietary supplements.

[0077] In other specific embodiments, the products of the present invention may be powdered reconstituteable foods (solid beverages, instant coffee, grain powder, nut powder or lotus root powder, etc.), baked goods (bread, cake or biscuit baked goods, etc.), beverages (carbonated beverages, fruit and vegetable juice beverages, functional beverages, tea beverages, milk beverages or alcoholic beverages, etc.), candies (gel candies, hard candies, compressed candies, etc.), milk and dairy products (fresh milk, milk powder, whey powder, fermented milk, cheese or condensed milk, etc. derived from fresh cow (sheep) milk), pasta products (noodles, instant noodles, steamed buns, dumplings or wontons, etc.), etc.

[0078] In some other specific embodiments, the product of the present invention is an oral preparation, which includes, but is not limited to, tablets, pills, granules, powders, teas, capsules, or oral liquids.

[0079] The product provided by this invention is suitable in principle for all population groups, especially those who need to regulate their gut microbiota or are prone to Cronobacter sakazakii infection, such as infants, young children, the elderly, and people with weakened immune systems. The components of the product can also be adjusted accordingly for different population groups.

[0080] (Uses that help regulate gut microbiota)

[0081] This invention proposes using specific combinations of Bifidobacterium bacteria to regulate the intestinal flora. Furthermore, in some embodiments, the regulation of the intestinal flora is not intended to prevent and / or treat diseases.

[0082] Therefore, the probiotic composition provided by this invention can be used to prepare products that help regulate the gut microbiota. The products described in this invention also help regulate the gut microbiota.

[0083] This invention does not specifically limit the category of products that help regulate gut microbiota. In some embodiments, the products that help regulate gut microbiota are food products. In some embodiments, the products that help regulate gut microbiota are health food products.

[0084] (Uses to relieve symptoms caused by Cronobacter sakazakii infection)

[0085] This invention proposes the use of specific Bifidobacterium bacteria in combination to alleviate symptoms caused by Cronobacter sakazakii infection, such as post-infection inflammatory responses and tissue damage. Therefore, the probiotic composition provided by this invention can be used to prepare products that alleviate symptoms caused by Cronobacter sakazakii infection, and the products described in this invention also have the function of alleviating symptoms caused by Cronobacter sakazakii infection.

[0086] The present invention also provides a method for alleviating symptoms caused by Cronobacter sakazakii infection, the method comprising administering an appropriate amount of the probiotic composition or the product to a person infected with Cronobacter sakazakii. The administration includes the person infected with Cronobacter sakazakii consuming the probiotic composition or the product.

[0087] Symptoms of Cronobacter sakazakii infection primarily include inflammatory response, tissue damage, and / or changes in gut microbiota. In some specific embodiments, the inflammatory response includes any one or more of the following symptoms: elevated levels of pro-inflammatory cytokines, elevated levels of anti-inflammatory cytokines, and elevated levels of nuclear transcription factors in intestinal tissue; increased platelet count in whole blood; and elevated serum uric acid. In some specific embodiments, the tissue damage includes any one or more of the following symptoms: destruction of intestinal epithelial structure, edema, loss of villi and crypt structures, inflammatory cell infiltration, goblet cell destruction, loss of the mucus layer, and decreased expression of intestinal barrier-related genes.

[0088] Consuming the above-mentioned probiotic composition or product can improve the symptoms caused by Cronobacter sakazakii infection and can be used as an adjunct treatment for Cronobacter sakazakii infection.

[0089] This invention does not specifically limit the category of products for relieving symptoms caused by Cronobacter sakazakii infection. In some embodiments, the product for relieving symptoms caused by Cronobacter sakazakii infection is a pharmaceutical product.

[0090] Example

[0091] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional in the art.

[0092] I. Experimental Methods

[0093] 1. Strains and laboratory animals

[0094] The experimental probiotics included *Bifidobacterium animalis* subsp. *lactamase*, *Bifidobacterium longum* subsp. *longum*, *Lactobacillus acidophilus*, and *Lactobacillus helveticus*. *Bifidobacterium animalis* subsp. *lactamase* included *Bifidobacterium animalis* subsp. *lactamase* Bb-12 from Chr. Hansen, and *Bifidobacterium animalis* subsp. *lactamase* Bi-07 and HN019 from Danisco. *Bifidobacterium longum* subsp. *longum* included *Bifidobacterium longum* subsp. *longum* BB536 from Morinaga. *Lactobacillus acidophilus* and *Lactobacillus helveticus* were obtained from the laboratory. The experimental animals were SPF-grade male C57BL / 6 mice, 6 weeks old, purchased from Shanghai Silex Laboratory Animal Co., Ltd.

[0095] 2. Culture of Bifidobacteria and Lactobacillus

[0096] Collect Bifidobacterium or Lactobacillus cells cultured for 24 hours daily by centrifugation, wash twice with sterile physiological saline, and resuspend until the viable cell count is 5 × 10⁻⁶. 9 CFU / mL bacterial suspension.

[0097] The probiotic combination used for gavage in animal experiments consisted of two strains of Bifidobacterium lactis (Bb-12 combined with Bi-07) (live count 1×10⁻⁶). 9 CFU / mL bacterial suspension, viable count ratio 1:1); Bifidobacterium animalis subsp. lactis Bb-12 compound with Lactobacillus acidophilus (viable count 1×10⁻⁶). 9 CFU / mL bacterial suspension, viable count ratio 1:1); Bifidobacterium animalis subsp. lactis Bb-12 compound with Lactobacillus helveticus (viable count 1×10⁻⁶). 9 CFU / mL bacterial suspension, viable count ratio 1:1); Bifidobacterium animalis subsp. lactis Bb-12 compounded with Bifidobacterium longum subsp. BB536 (viable count 1×10⁻⁶). 9 (CFU / mL bacterial suspension, viable cell ratio 1:1).

[0098] 3. Animal Experiment Design

[0099] 3.1 Probiotic combination screening experiment

[0100] Animal experiments used male C57BL / 6J mice (SPF grade, 6 weeks old, 18-20 g) who were randomly divided into three groups: a normal control group (CG), a model group (MG), and test groups 1-4 (compound probiotic test groups), with 10 mice in each group. The environment was maintained at 22℃ and 55% humidity, with a 12-hour day-night cycle, and free access to food and water. After purchase, the animals underwent a one-week acclimatization period.

[0101] The control group (CG) consisted of male C57BL / 6J mice. The CG group was gavaged with physiological saline, 200 μL / mouse / day.

[0102] The model group (MG) mice were first administered 5% NaHCO3, 200 μL / mouse / day, followed by another 200 μL / mouse 30 minutes later. 10 A mouse model of foodborne pathogen Cronobacter sakazakii infection was established using CFU / mL Cronobacter sakazakii. During the modeling period, the mice were weighed daily, fecal occult blood was detected, and the presence of diarrhea, bloody stools, and rectal bleeding was recorded. The disease activity index (DAI) was also calculated.

[0103] Test Group 1 (TG1) mice were administered a combined suspension of two strains of Bifidobacterium lactis, 200 μL / mouse / day, at a concentration of 1.0 × 10⁻⁶. 9 After continuous gavage administration of CFU / mL for 14 days, Cronobacter sakazakii was administered via gavage. Before administering Cronobacter sakazakii, animals were gavaged with 5% NaHCO3 (200 μL / animal / day), followed by another 200 μL / animal 30 minutes later. 10 CFU / mL Cronobacter sakazakii

[0104] Test Group 2 (TG2) mice were administered a combined suspension of *Bifidobacterium lactis* subsp. *lactospirum* and *Lactobacillus acidophilus* by gavage, 200 μL / mouse / day, 1.0 × 10⁻⁶. 9 After continuous gavage administration of CFU / mL for 14 days, Cronobacter sakazakii was administered via gavage. Before administering Cronobacter sakazakii, animals were gavaged with 5% NaHCO3 (200 μL / animal / day), followed by another 200 μL / animal 30 minutes later. 10 CFU / mL Cronobacter sakazakii

[0105] Test Group 3 (TG3) mice were administered a combined suspension of Bifidobacterium lactis subsp. lactis and Lactobacillus helveticus via gavage, 200 μL / mouse / day, 1.0 × 10⁻⁶. 9 After continuous gavage administration of CFU / mL for 14 days, Cronobacter sakazakii was administered via gavage. Before administering Cronobacter sakazakii, animals were gavaged with 5% NaHCO3 (200 μL / animal / day), followed by another 200 μL / animal 30 minutes later. 10 CFU / mL Cronobacter sakazakii

[0106] Test Group 4 (TG4) involved gavage administration of a combined bacterial suspension of *Bifidobacterium lactis* subsp. *semi-Lactobacillus* and *Bifidobacterium longum* subsp. *semi-Lactobacillus*, 200 μL / mouse / day, 1.0 × 10⁻⁶. 9 After continuous gavage administration of CFU / mL for 14 days, Cronobacter sakazakii was administered via gavage. Before administering Cronobacter sakazakii, animals were gavaged with 5% NaHCO3 (200 μL / animal / day), followed by another 200 μL / animal 30 minutes later. 10 CFU / mL Cronobacter sakazakii

[0107] 3.2 Experiment on the reduction of inflammatory response and tissue damage caused by Cronobacter sakazakii infection by Bifidobacterium combination

[0108] Animal experiments used male C57BL / 6J mice (SPF grade, 6 weeks old, 18-20 g) which were randomly divided into four groups: a control group, a model group, a bifidobacteria prevention group, and a therapeutic group, with 10 mice in each group. The environment was maintained at 22°C and 55% humidity, with a 12-hour day-night cycle, and free access to food and water. After purchase, the animals underwent a one-week acclimatization period.

[0109] The control group (CG) consisted of male C57BL / 6J mice. The CG group was gavaged with physiological saline, 200 μL / mouse / day.

[0110] The model group (MG) mice were first administered 5% NaHCO3, 200 μL / mouse / day, followed by another 200 μL / mouse 30 minutes later. 10 A mouse model of foodborne pathogen Cronobacter sakazakii infection was established using CFU / mL Cronobacter sakazakii. During the modeling period, the mice were weighed daily, fecal occult blood was detected, and the presence of diarrhea, bloody stools, and rectal bleeding was recorded. The disease activity index (DAI) was also calculated.

[0111] Prevention Group (PG) mice were administered Bifidobacterium bacterial suspension by gavage, 200 μL / mouse / day, 1.0 × 10⁻⁶. 9 After continuous gavage administration of CFU / mL for 14 days, Cronobacter sakazakii was administered via gavage. Before administering Cronobacter sakazakii, animals were gavaged with 5% NaHCO3 (200 μL / animal / day), followed by another 200 μL / animal 30 minutes later. 10 CFU / mL Cronobacter sakazakii

[0112] The intervention group (Therapeutic Group, TG) consisted of mice infected with Cronobacter sakazakii and administered Bifidobacterium bacterial suspension by gavage, 200 μL / mouse / day, 1.0 × 10⁻⁶. 9 The concentration of CFU / mL was measured. Colonic tissue, colonic contents, and serum were collected from mice, and the collected samples were rapidly frozen in an ultra-low temperature freezer for subsequent analysis. Simultaneously, the expression levels of inflammatory factors and changes in immune cells were detected, and pathological examinations were performed on intestinal tissues from different locations in mice. Hematologic and immunohistochemical staining and immunohistochemistry were used to analyze the repair of intestinal tissue damage after infection.

[0113] II. Experimental Results

[0114] 1. Research on screening of probiotic combinations

[0115] The experiment established a foodborne pathogen Cronobacter sakazakii infection model in C57 mice, and conducted a study on the antagonistic effect of probiotic combinations on Cronobacter sakazakii infection in mice. The study investigated the changes in the transcriptional levels of intestinal cytokines and peroxisome proliferator-activated receptor γ (PPARγ) in mice by different probiotic combinations.

[0116] Animal experiments were conducted to analyze the antagonistic effects of different treatments on *Cronobacter sakazakii*. The groups were divided into a control group (CG), a model group (MG), a prevention group (PG), test group 1 (TG1) (a combination of two *Bifidobacterium lactis* strains), test group 2 (TG2) (a combination of *Bifidobacterium lactis* and *Lactobacillus acidophilus*), test group 3 (TG3) (a combination of *Bifidobacterium lactis* and *Lactobacillus helveticus*), and test group 4 (TG4) (a combination of *Bifidobacterium lactis* and *Bifidobacterium longum*). Changes in the mRAN levels of pro-inflammatory cytokines (TNF-α, IL-1β), anti-inflammatory cytokines (IL-10), and the nuclear transcription factor PPARγ in mouse intestinal tissue were detected. The results showed that the combination of two *Bifidobacterium lactis* strains, and the combination of *Bifidobacterium lactis* and *Bifidobacterium longum*, had a good antagonistic effect on *Cronobacter sakazakii*. The results are shown in [Figure 1]. Figure 1 .

[0117] 2. Experiment on the use of a combination of bacteria to alleviate inflammatory responses and tissue damage caused by Cronobacter sakazakii infection.

[0118] This experiment established a foodborne pathogen Cronobacter sakazakii infection model in C57 mice and conducted an antagonistic study on the effect of Bifidobacterium synergistically (a combination of two animal Bifidobacterium subsp. lactis) on Cronobacter sakazakii infection in mice. The results showed that Bifidobacterium could alleviate the inflammatory response and tissue damage caused by Cronobacter sakazakii infection, intervene in the colonization process of Cronobacter sakazakii, thereby reducing the colonization level of Cronobacter sakazakii in the mouse intestine, and inhibiting the activity of Cronobacter sakazakii in the mouse intestine. The correlation between Cronobacter sakazakii infection and changes in intestinal flora structure was analyzed, and the mechanism by which Bifidobacterium intervenes in Cronobacter sakazakii infection by regulating the intestinal microecology was analyzed.

[0119] (1) Changes in the transcriptional levels of mouse intestinal cytokines and PPARγ

[0120] Animal experiments were conducted to analyze the antagonistic effects of different treatments on *Cronobacter sakazakii*. The mice were divided into a control group (CG), a model group (MG), a prevention group (PG), and a therapeutic group (TG). Changes in the mRAN levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), anti-inflammatory cytokines (IL-10), and the nuclear transcription factor PPARγ in mouse intestinal tissue were measured. The results are shown below. Figure 2 .

[0121] Cronobacter sakazakii significantly upregulated the expression of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the mouse colon, leading to colonic immune dysregulation, exacerbating inflammation and damage to colonic tissue. In contrast, Bifidobacterium downregulated the expression of pro-inflammatory cytokines to varying degrees, promoting colonic immune homeostasis in mice. Therefore, Bifidobacterium plays a role in regulating the host immune response. Compared to the normal group, the MG group showed a significantly increased transcription level of the pro-inflammatory cytokine TNF-α in the colon, while the PG and TG groups showed varying degrees of increased relative transcription levels of PPARγ. Bifidobacterium significantly downregulated the expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), bringing the immune response towards equilibrium.

[0122] (2) Observation of H&E pathological staining of mouse intestinal tissue

[0123] H&E staining can reflect histopathological changes and is used to assess tissue damage. H&E staining was used to observe the effects of different treatment groups on the pathological changes of mouse intestinal tissue. Figure 3 This is the result of H&E staining of paraffin sections. Figure 4 The results are based on inflammation scores for each group of slides according to the tissue damage scoring criteria.

[0124] Analysis showed that the CG group had an intact intestinal epithelial structure, with tightly and neatly arranged villi containing abundant goblet cells, intact crypt and gland structures, and no inflammatory cell infiltration. In the MG group, most of the epithelial structure was destroyed, with severe edema, disappearance of villi and crypt structures, and extensive inflammatory cell infiltration. The colon tissue damage score of the model group was 14.9±0.35, which was significantly higher than that of the CG group. Bifidobacterium significantly reduced intestinal tissue damage, with most of the intestinal epithelial structure intact, intact crypt structures, and a large number of goblet cells preserved.

[0125] (3) Effects on the intestinal barrier of mice

[0126] The intestine is covered by a layer of mucus, which acts as a physical barrier to protect it. The main component of this mucus layer is mucin secreted by goblet cells. Alcian blue staining was used to assess the integrity of the colonic mucus layer. After mice were sacrificed, intestinal tissue was harvested, sectioned in paraffin, and then stained with alcian blue. The results are as follows: Figure 5 As shown, the CG group contains a large number of goblet cells, which are neatly arranged and have a normal shape. The goblet cells contain mucus. Compared with the normal group, the goblet cells in the model group are almost completely destroyed and the mucus layer is completely lost. This may lead to pathogens coming into contact with the intestinal epithelium, which will exacerbate colon inflammation and damage the colon tissue. The PG and TG groups contain a large number of normal goblet cells and a relatively intact mucus layer.

[0127] (4) RT-qPCR detection of changes in the transcriptional levels of intestinal barrier-related genes

[0128] RT-qPCR was used to detect changes in the transcriptional levels of intestinal barrier-related genes to investigate the effect of Bifidobacterium on antagonizing the intestinal barrier in mice damaged by Cronobacter sakazakii. The protective effect of Bifidobacterium on the mucus layer of mouse intestinal tissue is correlated with the regulation of tight junction-related genes. The TG group significantly upregulated the transcriptional levels of intestinal barrier-related genes such as tight junction proteins (Claudin 3 and ZO-1) and cadherin (E-cadherin 1), indicating that Bifidobacterium participates in the protection of the intestinal barrier. Results are shown in [Figure number missing]. Figure 6 .

[0129] (5) Effects of Cronobacter sakazakii on blood parameters in mice

[0130] To analyze the changes in the immune system or blood inflammatory response induced by gavage administration of Cronobacter sakazakii suspension in mice, as well as liver and kidney function damage, the main indicators related to immune response or inflammation in whole blood (RBC, HGB, PLT, WBC, GRAN, and LYMF) were measured. The differences in serum indicators related to liver and kidney function (BUN, UA, AST, and ALT) among the four groups of mice were analyzed. The results are as follows: Figure 7 and 8 As shown.

[0131] In the biochemical parameters of whole blood in mice, there were no significant differences in RBC, HGB, WBC, GRAN, and LYMF among the four groups of mice (P>0.05). The PLT content in the MG group was significantly higher than that in the CG group, indicating that Cronobacter sakazakii induced an increase in PLT in whole blood of mice, while Bifidobacterium showed an inhibitory effect on this increase in PLT. In terms of indicators related to liver and kidney function in mouse serum, BUN, AST, and ALT did not show significant differences among the four groups of mice administered the samples by gavage (P>0.05), while the UA index showed significant differences among the different groups.

[0132] (6) High-throughput sequencing analysis of changes in mouse gut microbiota

[0133] Based on the quantitative results of PicoGreen fluorescent dye, the KAPA Biosystems Library Quantification Kit was used according to its instructions. The molar concentration (nM) of the library was calculated as: A×10⁶ / (650*B), where A represents the mass concentration (ng / μL), B represents the fragment size, and 650 represents the average molecular weight per bp. The constructed libraries were diluted to 2 nM and mixed according to the required data volume. A 2 N NaOH solution was diluted to 0.1 N, and 10 μL of the mixed library was taken and denatured at room temperature (25℃) for 5 min. The reaction was terminated by adding 980 μL of pre-chilled HT1 (a reagent from the reagent kit, at which point the sample concentration was 20 pM). 500 μL of the mixture was mixed with 500 μL of HT1 (at which point the sample concentration was 10 pM). 1 mL of the mixture was added to a melted Reagent Kit Cartridge and sequenced using a MiSeq sequencer. Analysis revealed changes in gut microbiota in mice infected with Cronobacter sakazakii and after intervention with Bifidobacterium. Changes in gut microbiota structure at the phylum and genus levels were observed, as follows: Figure 9 and 10 .

[0134] Sequencing data were used to study the changes in the gut microbiota after Bifidobacterium antagonizes Cronobacter sakazakii. The changes in the species and abundance of the gut microbiota in each group were analyzed, and the results are as follows: Figure 11 As shown, compared with the control group, the intestinal flora of mice in the Cronobacter sakazakii group showed that some flora increased in abundance while others decreased in abundance. After intervention with Bifidobacterium, the abundance of the intestinal flora of the intervention group mice generally showed a trend of recovery to the abundance distribution of the control group.

[0135] Industrial availability

[0136] The probiotic composition provided by this invention can be widely used in the food, health food, and pharmaceutical industries.

Claims

1. Use of a probiotic composition in the preparation of a product having the function of helping to regulate the intestinal flora and / or alleviate symptoms caused by Cronobacter sakazakii infection; The probiotic composition contains probiotics, specifically the components that help regulate the intestinal flora and / or alleviate symptoms caused by Cronobacter sakazakii infection. These probiotics consist of two Bifidobacterium lactis strains, Bb-12 and Bi-07. Furthermore, the composition contains 1 × 10⁻⁶ live bacteria by total mass. 8 CFU / 100g ~ 1×10 12 CFU / 100g; Furthermore, in the composition, the ratio between the two animal Bifidobacterium lactis strains is 2:1 to 1:2, based on the number of viable bacteria. Symptoms caused by Cronobacter sakazakii infection include inflammatory response, tissue damage, and changes in gut microbiota.

2. The use according to claim 1, characterized in that, The inflammatory response includes any one or more of the following symptoms: elevated levels of pro-inflammatory cytokines in intestinal tissue, elevated levels of anti-inflammatory cytokines in intestinal tissue, elevated levels of nuclear transcription factors in intestinal tissue, thrombocytosis in whole blood, and elevated blood uric acid. The tissue damage includes any one or more of the following symptoms: destruction of intestinal epithelial structure, edema, loss of villi and crypt structures, inflammatory cell infiltration, destruction of goblet cells, loss of mucus layer, and decreased expression of intestinal barrier-related genes.

3. The use of a product to help regulate gut microbiota and / or alleviate symptoms caused by Cronobacter sakazakii infection, characterized in that, The product contains a probiotic composition as defined in claim 1 or 2; the symptoms caused by the Cronobacter sakazakii infection include inflammatory response, tissue damage, and changes in gut microbiota.

4. The use according to claim 3, characterized in that, The product also contains plants or their extracts, milk-containing ingredients, animal-derived ingredients, functional additives, trace element supplements and / or any acceptable excipients.

5. The use according to claim 3 or 4, characterized in that, The inflammatory response includes any one or more of the following symptoms: elevated levels of pro-inflammatory cytokines in intestinal tissue, elevated levels of anti-inflammatory cytokines in intestinal tissue, elevated levels of nuclear transcription factors in intestinal tissue, thrombocytosis in whole blood, and elevated blood uric acid. The tissue damage includes any one or more of the following symptoms: destruction of intestinal epithelial structure, edema, loss of villi and crypt structures, inflammatory cell infiltration, destruction of goblet cells, loss of mucus layer, and decreased expression of intestinal barrier-related genes.

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