CN113005067A

CN113005067A - Multifunctional composite probiotic preparation and preparation method thereof

Info

Publication number: CN113005067A
Application number: CN202110347197.6A
Authority: CN
Inventors: 陈大伟; 童颖佳; 童群义; 陈金林; 吉峰
Original assignee: Yancheng Weikang Biotechnology Co ltd
Current assignee: Jiangsu Lanze Biotechnology Co ltd
Priority date: 2021-03-31
Filing date: 2021-03-31
Publication date: 2021-06-22
Anticipated expiration: 2041-03-31
Also published as: CN113005067B

Abstract

The invention relates to a multifunctional composite probiotic preparation and a preparation method and application thereof, and the multifunctional composite probiotic preparation is prepared by adopting lactobacillus plantarum MiuCul-01, lactobacillus acidophilus MiuLac-01, lactobacillus casei MiaoTech-01, lactobacillus rhamnosus MiaoTech-11, streptococcus thermophilus MiaoPro-01, bifidobacterium bifidum Miuyo-01, bifidobacterium lactis Miuyo-11, bifidobacterium infantis Miuyo-21 and bifidobacterium breve Miuyo-31 and a new process combining mixed bacteria inoculation, anaerobic liquid culture, solid-liquid separation, solid-state culture and the like.

Description

Multifunctional composite probiotic preparation and preparation method thereof

Technical Field

The invention belongs to the technical field of probiotic preparations, and particularly relates to a multifunctional composite probiotic preparation and a preparation method thereof.

Background

Helicobacter pylori is a spiral, micro-anaerobic bacterium that has very stringent growth conditions. Helicobacter pylori infection first causes chronic gastritis and results in gastric ulcers and gastric atrophy, and in severe cases gastric cancer. The common symptoms of patients with chronic gastritis and peptic ulcer are: after eating, the upper abdomen is full, uncomfortable or painful, often accompanied by other adverse symptoms such as belching, abdominal distension, acid regurgitation, anorexia, etc. Some patients may also have recurrent severe abdominal pain, minor upper gastrointestinal bleeding, etc. According to statistics: about 1% of patients with chronic gastritis and gastric ulcer develop gastric cancer, which is one of the most common malignant tumors worldwide and the second cause of cancer death. Therefore, timely and effective use of antibiotics to kill helicobacter pylori has great significance for preventing and controlling gastric cancer.

Allergy is the phenomenon of an abnormal increase in the susceptibility of an organism to certain external stimuli (antigens), i.e. an abnormal, too high immune response, simply an oversensitivity to a substance (antigen). The occurrence and development of allergic diseases are related to the immune system of the body, wherein a type 2 immune response mechanism plays an important role in the allergic diseases and can generate specific IgE antibodies aiming at allergens to enable the body to be in a sensitized state. When the sensitized organism contacts the same allergen again, IgE antibodies adsorbed on the surfaces of mast cells and eosinophilic granulocytes are combined with corresponding antigens to cause degranulation of the mast cells and release allergic mediators such as histamine and the like, so that allergic reaction is caused, the organism is subjected to tissue injury or dysfunction, common symptoms comprise abdominal pain, diarrhea and vomiting, or skin is extremely itchy and difficult to boil, and the severe people can suffer shock. For people with allergic constitution, a method of avoiding contact with allergen is adopted to reduce the occurrence of allergy, but the effect is very little, and the avoidance of allergen can only relieve the allergic symptoms temporarily but cannot radically cure the allergy. Thus, methods have emerged to induce immune tolerance, which, while feasible, are difficult to completely desensitize under current sanitary conditions.

The common immune organ in humans is the lymphoid organ, and T cells expressing CD4 in lymph are called helper T cells (Th). Depending on the function of helper T cells, or on their response to different cytokines and their ability to secrete cytokines, T helper cells can be subdivided into two major components, i.e. T helper cells can be differentiated into two types (Th1 and Th 2). Generally, Th1 is beneficial for enhancing cellular immunity, Th2 is beneficial for enhancing humoral immunity, and there is a dynamic balance between Th1 and Th2 under normal conditions. And the two can balance each other: the immune response of Th1 is vigorous, so that the activity and function of Th2 are greatly reduced by inhibition, and vice versa. The main effect of Th1 is to resist the stimulation of various external microbes, so the infection is more and more, and the stimulation of microbes to Th1 is more and more, and the activated number is more, and as a result, the immune response of Th2 is inhibited. Or the following steps: stimulation of a Th 1-type immune response reduces the Th 2-type immune response, which can lead to allergies. The research shows that allergic diseases are related to T helper cell imbalance, and the excessive Th2 is easy to cause allergy. Thus, stimulation of a Th1 type immune response, and thus a reduction of a Th2 type immune response, reduces the incidence of allergic reactions. Since the 80's of the 20 th century, there has been a clear upward trend in allergic diseases, especially in western countries or most modern countries. Scholars have found that urban children growing up in a very clean and hygienic environment suffer from allergic diseases at a much higher rate than rural children growing up in a dirty environment, and thus have found a relationship between the human living environment and allergic diseases, thereby proposing a "hygienic hypothesis". "hygienic assumption" holds that: too little exposure to pathogens such as bacteria and viruses during the juvenile period results in insufficient stimulation of Th1 cells, failing to balance Th2 cells, and eventually leading to an allergic predisposition. From the viewpoint of "hygiene hypothesis", it is considered that proper microbial stimulation is necessary for the development of normal immune function and oral tolerance of the body, and is beneficial for inducing the differentiation of Th0 cells to Th1 cells and inhibiting the Th2 cell response, thereby playing a role in immune balance. The more microorganisms a child is exposed to at an early stage, the less chance of developing allergic disease later on. However, the presence of pathogenic microorganisms in the environment can cause serious epidemic diseases in humans, and thus there is a substantial risk of exposing children directly to environmental microorganisms.

Diabetes is a series of metabolic disorder syndromes of sugar, protein, fat, water, electrolyte and the like, which are caused by hypofunction of pancreatic islets, insulin resistance and the like due to the action of various pathogenic factors such as genetic factors, immune dysfunction, microbial infection and toxins thereof, free radical toxins, mental factors and the like on the body. Diabetes is one of the most serious chronic diseases in the world, and chronic hyperglycemia causes chronic complications in the human body, thereby causing chronic damage to blood vessels and nerves, and finally possibly affecting the heart, the kidney, the eyes and the nerves and causing serious complications. The human body must hydrolyze polysaccharides in the food to glucose by key digestive enzymes (alpha-amylase and alpha-glucosidase) in the intestine to be absorbed by the body, and thus inhibition of alpha-glucosidase reduces glucose intake by the body. Alpha-glucosidase is an oligosaccharide hydrolase existing at the brush border of the small intestine mucous membrane, and can hydrolyze oligosaccharide, maltose and the like into glucose, thereby promoting the absorption of the glucose and increasing the blood sugar content. Therefore, lowering postprandial blood glucose by inhibiting alpha-glucosidase activity has become an effective target for clinical intervention in diabetes. The drugs currently used clinically mainly include acarbose, voglibose and miglitol. There are reports of data: the lactic acid bacteria can also produce a plurality of alpha-glucosidase inhibitors in the culture process, so that the probiotics can consume glucose through the growth and the propagation of the probiotics in the intestinal tract, and the metabolite (the alpha-glucosidase inhibitor) produced in the culture process can reduce the glucose absorption rate in the intestinal tract, thereby reducing the blood sugar of a human body.

Obesity refers to a condition in which the body is significantly overweight and has an excessively thick fat layer, and an excess of fat tissue is accumulated in the body. The main causes of obesity are: excessive intake of high calorie food, lack of exercise, etc. When the energy intake of the body exceeds the energy consumption, the body energy is excessive to promote the accumulation of fat, and obesity is formed; in addition, diseases, environmental, psychological and toxic factors cause nutritional imbalance, energy metabolism disorder, and obesity. Obesity not only affects body beauty, but also causes various diseases such as: hypertension, coronary heart disease, angina pectoris, cerebrovascular diseases, diabetes, hyperlipidemia, hyperuricemia, female menoxenia, etc., and can increase the probability of malignant tumor, and influence the functions of digestive system and endocrine system.

There are two main sources of human fat: one is the direct intake of fat in food; the other is the conversion into fat in vivo by using excess sugar. Fat in food is decomposed into fatty acid, cholesterol and other fat digestion products in small intestine under the action of various enzymes and bile acid salts, and then absorbed into blood stream through small intestine wall, so that the fat is digested and absorbed by human body, and can be re-synthesized into fat in human body; on the other hand, when the sugar intake of human body is excessive, the sugar (such as glucose, etc.) can be converted into fat by tricarboxylic acid cycle. Therefore, the inhibition of citrate lyase, a key enzyme of the tricarboxylic acid cycle, can reduce the synthesis of human cell fat, thereby reducing the fat content of the human body. After the intestinal tract is colonized, in the process of growth and reproduction of the intestinal tract, substances such as lipids (such as triglyceride, cholesterol and the like), micromolecular saccharides (such as glucose), amino acids or small peptides and the like in the intestinal tract are utilized to synthesize thallus components, participate in the digestion, absorption and metabolism functions of nutrient substances, and fully play the roles of reducing body fat and cholesterol, thereby reducing the body fat rate, reducing the weight, reducing the food utilization rate, promoting the metabolism, improving the obesity and the like. Multiple studies have shown that: the probiotics can reduce the synthesis of fat and decompose redundant grease, so that the fat of a human body can not be excessively accumulated.

The probiotics are non-pathogenic microorganisms, but can stimulate the immune system of the intestinal tract, change the balance of Th1/Th2, enable the immune response to progress towards the direction of Th1 cells, inhibit the reaction of Th2 cells and play the role of immunoregulation, thereby reducing the occurrence of allergic diseases. In addition, the probiotics and the metabolites thereof also have the function of enhancing the intestinal barrier function, reduce the food antigens from penetrating the intestinal mucosa and avoid the food antigens from being exposed to the immune system. Most probiotic bacteria are non-pathogenic gram-positive bacteria, the major components of their cell walls being Peptidoglycan (PG), bacterial Polysaccharides (PS) and lipoteichoic acid (LTA), all of which have immunostimulatory properties.

The invention patent with application number 201780023391.0 discloses bifidobacterium for reducing food, energy and/or fat intake, which discloses animal bifidobacterium with the functions of reducing fat and losing weight; the invention patent with application number 201680007471.2, "immunomodulating composition comprising bifidobacterium", discloses a composition for modulating immunity; the invention patent with the application number of 202010455121.0 discloses an animal bifidobacterium capable of improving the immunity, namely a bifidobacterium animalis BZ11 compound microbial inoculum with the function of regulating the intestinal tract; the invention patent with application number 201510962576.0 discloses a bifidobacterium composition for enhancing immunity, which discloses a bifidobacterium composition for enhancing immunity; the invention patent with application number 201610280825.2, namely application of bifidobacterium infantis in food or medicine for preventing and treating food allergy, discloses bifidobacterium infantis capable of preventing and treating allergy, and the bifidobacterium infantis can be applied to food or medicine; the invention patent with the application number of 202010699202.5 discloses bifidobacterium animalis with the function of losing weight, namely a bifidobacterium animalis NX-6 and application thereof in preparing medicaments for reducing fat and losing weight; the invention patent with application number 201811068444.3 discloses a preparation method of probiotic powder, and the probiotic preparation comprises composite probiotic powder of lactobacillus acidophilus, bifidobacterium lactis, lactobacillus rhamnosus, lactobacillus paracasei and the like, and some auxiliary materials, and has the function of improving the immunity of human bodies. The patents provide a new idea for the application of the composite probiotic preparation in the aspects of reducing fat and losing weight, regulating immunity, resisting allergy and the like.

At present, in the process of producing various probiotic powder, liquid fermentation is almost completely adopted in the probiotic production industry, after the liquid fermentation is finished, moisture is removed through centrifugal separation or other solid-liquid separation methods to obtain wet thalli, and then a drying protective agent or a carrier is added for uniformly mixing, freeze drying, crushing, granulating, coating, packaging and the like. In the solid-liquid separation process, substances with bacteriostatic action such as bacteriocin, organic acid and the like generated by probiotics in the liquid fermentation process are removed, and the bacteriostatic effect of the metabolic product of the composite probiotic preparation cannot be reflected in the product, so that the probiotic product produced by the method only utilizes the anti-allergic and lipid-lowering weight-losing effects of the probiotics and cannot utilize the anti-allergic and lipid-lowering weight-losing effects of the bacteriostatic substances; in addition, the wet thallus separated from the liquid fermentation liquor has high moisture content, so the stress death rate of the thallus is high during drying, and the proper amount of a drying protective agent or a carrier is added into the wet thallus, so that the quantity of the thallus actually obtained after the liquid fermentation product is dried is also reduced.

The invention patent with application number 201310187056.8 discloses a method for preparing lactobacillus acidophilus by combining liquid fermentation and solid fermentation, namely, firstly performing liquid fermentation, then using the product of the liquid fermentation as a strain to be mixed with a solid culture medium, and then performing solid fermentation, wherein the product contains the live lactobacillus acidophilus and the metabolite thereof, for example: bifidobacteria, n-butyric acid, acetic acid, formic acid, etc.; the invention patent with application number 201510559188.8, a culture medium and a method for high-density solid state fermentation culture of lactobacillus, discloses a method for solid state fermentation culture of lactobacillus, which comprises the following steps: inoculating lactobacillus strain into culture medium at 3-5% v/v, mixing, and fermenting at 30-37 deg.C under sealed condition. The strains adopted in the two patents are liquid strains prepared by a triangular flask or a small fermentation tank, or can be a combined process of liquid culture and solid culture, namely the liquid strains obtained by liquid culture are added into a solid culture medium, and solid fermentation is carried out after uniform mixing, so as to obtain a solid fermentation product. However, since the solid content in the liquid fermentation broth is very low (2-5%), even if the amount of the added liquid culture broth reaches 50%, only a small amount of liquid strains (1-2.5%) can be added into the solid culture medium as strains for solid fermentation, so that the initial strain concentration in the solid fermentation medium is not high, the solid fermentation time is long, and the viable bacteria concentration in the product is small.

In conclusion, the traditional practical production process of probiotics, namely liquid culture and centrifugal separation, or the liquid culture and solid culture process disclosed by the invention has the defects of low strain content, poor fermentation effect, high death rate of thalli during drying, low strain activity in a finished product and the like.

In view of this, this patent is filed.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a multifunctional compound probiotic preparation and a preparation method thereof, and a novel preparation process ensures that the product has large quantity of thalli and high activity, contains metabolites generated by strains during solid culture, and can enhance the effects of resisting helicobacter pylori, reducing blood sugar, conditioning intestines and stomach, increasing immunity, resisting allergy, reducing fat and losing weight and the like of the compound probiotic preparation during use.

The invention aims to provide a multifunctional compound probiotic preparation.

The invention further aims to provide a preparation method of the multifunctional composite probiotic preparation.

According to a specific embodiment of the present invention, the multifunctional complex probiotic preparation comprises the following components:

lactobacillus plantarum MiuCul-01, Lactobacillus acidophilus MiuLac-01, Lactobacillus casei MiaoTech-01, Lactobacillus rhamnosus MiaoTech-11, Streptococcus thermophilus MiaoPro-01, Bifidobacterium bifidum Miuyo-01, Bifidobacterium lactis Miuyo-11, Bifidobacterium infantis Miuyo-21 and Bifidobacterium breve Miuyo-31.

The multifunctional compound probiotic preparation comprises the following components in parts by weight:

10-15 parts of lactobacillus plantarum MiuCul-01, 5-10 parts of lactobacillus acidophilus MiuLac-01, 3-7 parts of lactobacillus casei MiaoTech-01, 5-10 parts of lactobacillus rhamnosus MiaoTech-11, 3-7 parts of streptococcus thermophilus MiaoPro-01, 3-7 parts of bifidobacterium bifidum Miuyo-01, 2-6 parts of bifidobacterium lactis Miuyo-11, 10-15 parts of bifidobacterium infantis Miuyo-21 and 3-7 parts of bifidobacterium breve Miuyo-31.

12.5 parts of Lactobacillus plantarum MiuCul-01, 7.5 parts of Lactobacillus acidophilus MiuLac-01, 5 parts of Lactobacillus casei MiaoTech-01, 7.5 parts of Lactobacillus rhamnosus MiaoTech-11, 5 parts of Streptococcus thermophilus MiaoPro-01, 5 parts of Bifidobacterium bifidum Miuyo-01, 4 parts of Bifidobacterium lactis Miuyo-11, 12.5 parts of Bifidobacterium infantis Miuyo-21 and 5 parts of Bifidobacterium breve Miuyo-31.

The multifunctional composite probiotic preparation according to the embodiment of the invention, wherein the viable count of the composite probiotic preparation is (8-10) x10¹¹CFU/g。

The multifunctional complex probiotic preparation according to the embodiment of the invention, wherein the bifidobacterium bifidum Miuyo-01, bifidobacterium infantis Miuyo-21, bifidobacterium breve Miuyo-31 and bifidobacterium lactis Miuyo-11 are all derived from infant feces, and are obtained by separating and culturing the infant feces

Further, the isolated culture conditions of the bifidobacterium bifidum Miuyo-01, the bifidobacterium infantis Miuyo-21, the bifidobacterium breve Miuyo-31 and the bifidobacterium lactis Miuyo-11 are as follows: MRS culture medium, pH6.0-7.5, temperature 34-40 deg.C.

The multifunctional composite probiotic preparation according to the specific embodiment of the invention, wherein the lactobacillus plantarum MiuCul-01 is obtained by isolated culture from kimchi.

Further, the culture conditions of the lactobacillus plantarum MiuCul-01 are as follows: MRS culture medium, pH6.2-6.4, temperature 37 ℃.

Further, the lactobacillus acidophilus MiuLac-01 is obtained by separating and culturing from fermented milk products.

Further, the culture conditions of the lactobacillus acidophilus MiuLac-01 are as follows: MRS culture medium, pH6.2-6.4, temperature 37 ℃.

Further, the lactobacillus casei MiaoTech-01 is obtained by separating and culturing from a fermented milk product.

Further, the culture conditions of the lactobacillus casei MiaoTech-01 are as follows: MRS culture medium, pH6.2-6.4, temperature 37 ℃.

Further, the lactobacillus rhamnosus MiaoTech-11 is obtained by separating and culturing from a probiotic preparation.

Further, the culture conditions of the lactobacillus rhamnosus MiaoTech-11 are as follows: MRS culture medium, pH6.2-6.4, temperature 37 ℃.

Further, the streptococcus thermophilus MiaoPro-01 is obtained by separating and culturing from a yoghurt starter.

Further, the culture conditions of the streptococcus thermophilus MiaoPro-01 are as follows: MRS culture medium, pH6.2-6.4, temperature 37 ℃.

The preparation method of the multifunctional composite probiotic preparation according to the embodiment of the invention comprises the following steps:

(1) uniformly mixing lactobacillus plantarum MiuCul-01, lactobacillus acidophilus MiuLac-01, lactobacillus casei MiaoTech-01, lactobacillus rhamnosus MiaoTech-11, streptococcus thermophilus MiaoPro-01, bifidobacterium bifidum Miuyo-01, bifidobacterium lactis Miuyo-11, bifidobacterium infantis Miuyo-21 and bifidobacterium breve Miuyo-31 according to a ratio to form a composite probiotic preparation, putting the composite probiotic preparation into a liquid culture medium for first anaerobic culture, and centrifuging the produced culture solution to obtain wet thalli of the composite probiotic preparation for later use;

(2) mixing the wet thalli of the compound probiotic preparation obtained in the step (1) with a solid culture medium, carrying out anaerobic culture for the second time in a sealed state, and carrying out low-temperature drying, freeze drying or vacuum drying after the culture is finished to form a compound probiotic preparation solid;

(3) and (3) crushing the solid of the composite probiotic preparation obtained in the step (2), carrying out anaerobic granulation and carrying out anaerobic coating to obtain the multifunctional composite probiotic preparation.

According to the preparation method of the multifunctional composite probiotic preparation, the wet thalli of the composite probiotic preparation are obtained after centrifugal separation is carried out by adopting a disc centrifuge or a tubular centrifuge under the aseptic condition in the step (1); the liquid culture medium comprises sodium acetate, peptone, yeast extract and tomato sauce; the temperature of the first anaerobic culture is 36-38 ℃, and the time is 48-72 hours.

According to the preparation method of the multifunctional composite probiotic preparation, in the step (2), the solid culture medium comprises sodium acetate, soybean peptide, yeast extract and food-grade calcium carbonate, and is subjected to high-temperature dry heat sterilization and aseptic cooling to below 38 ℃ for later use before use; and before the second anaerobic culture, the water content of the mixture of the wet thalli of the compound probiotic preparation and the solid culture medium is adjusted to 45-55 percent.

The preparation method of the multifunctional composite probiotic preparation according to the specific embodiment of the invention, wherein in the step (2), the temperature of the second anaerobic culture is 36-38 ℃ and the time is 72-120 hours; the low-temperature drying or vacuum drying temperature is 30-40 ℃; the water content of the solid of the compound probiotic preparation is 4-6%.

After the intestinal tract is colonized, in the process of growth and reproduction of the intestinal tract, substances such as lipids (such as triglyceride, cholesterol and the like), micromolecular saccharides (such as glucose), amino acids or small peptides and the like in the intestinal tract are utilized to synthesize thallus components, participate in the digestion, absorption and metabolism functions of nutrient substances, and fully play the roles of reducing body fat and cholesterol, thereby reducing the body fat rate, reducing the weight, reducing the food utilization rate, promoting the metabolism, improving the obesity and the like. Several studies have shown that: the probiotics can reduce the synthesis of fat and decompose redundant grease, so that the fat of a human body is not excessively accumulated. The probiotics can effectively regulate the physique of obese patients, and the action mechanism is as follows:

firstly, the appetite is inhibited and the satiety is increased

The probiotics and the metabolites thereof can stimulate the release of satiety factors such as cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1) and the like, reduce the secretion of stomach somatotropin, thereby reducing the food intake and reducing the weight and fat accumulation. There are studies showing that: the short chain fatty acid (such as acetic acid, butyric acid and propionic acid) can provide energy required by growth for intestinal epithelial cells, can promote intestinal peristalsis and relieve intestinal inflammation, can regulate appetite center and increase satiety.

② reducing cholesterol and fat

The probiotics can assimilate and absorb cholesterol in the growth process, promote the precipitation of the cholesterol and cholic acid by generating metabolites such as bile salt hydrolase and the like, reduce the chance of the cholesterol entering blood and interfere the absorption of the cholesterol by intestinal tracts. The probiotics and the metabolites thereof can also produce substances such as a citrate lyase inhibitor, an alpha-glucosidase inhibitor, short chain fatty acid and the like, thereby influencing fat metabolism and carbohydrate metabolism and playing the roles of reducing fat and losing weight.

Regulating intestinal flora

The probiotics can play a synergistic role in the digestive process and relieve partial intestinal malabsorption symptoms by releasing digestive enzymes in the intestinal tract. And after the probiotics enter the intestinal tract, the unbalanced intestinal flora can be normalized by the modes of flora occupation, bacterial proliferation, harmful bacterium killing and the like, and the normal function of the intestinal tract is recovered.

Fourthly, the probiotics can generate important nutrient substances

Probiotics are capable of producing important nutrients such as: pantothenic acid, nicotinic acid, B1, B2, B6, vitamin K, and other vitamins, and can produce short chain fatty acids, antioxidants, amino acids, and the like. The metabolites can accelerate the decomposition and transformation of fat, and improve the metabolism of human body, thereby achieving the purpose of fast slimming.

In conclusion, the probiotics and the metabolites thereof participate in sugar-lipid metabolism and cholesterol-fat metabolism of human bodies in intestinal tracts, block the formation of fat, accelerate the oxidative decomposition of fat accumulated in the bodies and play a role in long-acting weight loss.

At present, the species with probiotic effect are more, but mainly lactobacillus and bifidobacterium, such as: lactobacillus plantarum, lactobacillus acidophilus, lactobacillus casei, lactobacillus rhamnosus, lactobacillus gasseri, bifidobacterium longum, bifidobacterium breve, bifidobacterium lactis, bifidobacterium bifidum, bifidobacterium infantis, bifidobacterium adolescentis, etc., and also small amounts of other types of lactic acid bacteria, such as: streptococcus thermophilus, and the like.

The composite probiotics used by the invention comprise a plurality of facultative anaerobes and obligate anaerobes with similar culture conditions, and mainly comprise lactobacillus and bifidobacterium. The various species used in the present invention are characterized as follows:

lactobacillus plantarum is a straight or bent rod, a round-ended rod, anaerobic or facultative anaerobic, usually lacking in flagellar hair, but capable of movement. Gram positive, no sporulation. The lactobacillus plantarum is usually present in fermented vegetables and fruit juice, has the optimal growth temperature of 30-35 ℃, the optimal pH value of about 6.5, and can resist gastric acid and bile salt environments with the pH value of about 2.0. Belongs to homotype lactobacillus fermentum, can produce DL-lactic acid, has the activity of 1, 6-fructose diphosphate aldolase and hexose monophosphate pathway, can grow in gluconate, and produces C02. Fermentation of 1 molecule of ribose or other pentose sugar produces 1 molecule of lactic acid and 1 molecule of acetic acid. The bacterium can produce special lactobacillus during the propagation process, and the lactobacillus is a biological preservative. The lactobacillus plantarum is used as a probiotic flora of the gastrointestinal tract of a human body, and has multiple functions of maintaining the balance of flora in the intestinal tract, improving the immunity of the organism, promoting the absorption of nutrient substances and the like. Lactobacillus plantarum preparations are very widely used in the food industry. Because of the characteristics of good tolerance to salt and nitrite, no obvious and direct decomposition effect on protein and fat, and the like, the method is widely applied to fermented meat products and fermented vegetable foods.

Lactobacillus acidophilus belongs to gram-positive bacilli, Lactobacillus, and is in the shape of short rod, the end of the rod is round, without flagella and capsule, and the rod appears in single, double and short chain, facultative anaerobe or microaerophilic bacteria. Mainly exists in intestinal tracts of human beings and some animals, and has the optimal growth temperature of 35-38 ℃ and the optimal pH value of 5.5-6.2. Obvious colonies can be formed on an MRS culture medium, the colonies are round, white and convex, and the surface is smooth. The lactobacillus acidophilus has the characteristics of acid resistance and bile acid salt resistance, releases lactic acid, acetic acid and antibiotic substances (acidophil, lactein and the like) with bacteriostatic action in small intestine, and has certain antagonistic action on pathogenic bacteria in intestinal tract. Lactobacillus acidophilus is one of the probiotics widely researched and developed in the lactobacillus family at present, is an important microorganism in human intestinal tracts, and has the effects of balancing intestinal microflora, regulating intestinal dysfunction, enhancing intestinal mucosa barrier of organisms, regulating immune systems in the intestinal tracts, resisting cancers and tumors. The lactobacillus acidophilus colonizes in intestinal tract, can activate macrophage activity of organism, induce in vivo specific and nonspecific immunoreaction, and improve anti-infection ability of human and animal. Since the main metabolite of lactobacillus acidophilus is organic acid, it can reduce the oxidation-reduction potential and pH in the environment, and inhibit the growth of acidic bacteria and aerobic bacteria.

The lactobacillus casei belongs to lactobacillus, is gram-positive bacteria, does not produce spores, flagellum and motion, performs facultative heterotypic fermentation on lactose, and does not liquefy gelatin; the optimal growth temperature is 37 ℃, the thalli are different in length, and the two ends of the thalli are square and are often chained; the colony is coarse, grey white and sometimes yellowish, and can ferment various sugars. Lactobacillus casei is present in the human mouth, intestinal tract, and is also commonly found in milk and cheese, dairy products, feed, dough, and garbage. After entering the human body, lactobacillus casei can survive in the intestinal tract in a large amount, and has the effects of regulating the intestinal flora balance, promoting the digestion and absorption of the human body and the like. Meanwhile, the lactobacillus casei has the functions of efficiently reducing blood pressure and cholesterol, promoting cell division, generating antibody immunity, enhancing human immunity, preventing cancer, inhibiting tumor growth and the like; also has the beneficial health effects of relieving lactose intolerance and allergy.

Lactobacillus rhamnosus is mostly present in the intestinal tracts of humans and animals, and the taxonomy of bacteria belongs to the genus Lactobacillus, the subsp. The lactobacillus rhamnosus can not utilize lactose, can ferment various monosaccharides (glucose, arabinose, maltose and the like), most strains can produce a small amount of soluble ammonia, but can not produce indole and hydrogen sulfide, and has the biological characteristics of acid resistance, bile salt resistance, multiple antibiotic resistance and the like. Has outstanding gastric acid and bile resistance, and can enter human intestinal tracts in vivo.

Streptococcus thermophilus is a commonly used strain for making yoghurt. Studies have shown that this bacterium can only reach the upper half of the small intestine, unlike bifidobacteria which can reach the large intestine. Streptococcus thermophilus is lactase and helps lactose intolerant people digest lactose.

Bacteria of the genus bifidobacterium are one of the important members of the human and animal intestinal flora. Some strains of bifidobacteria can be used as probiotics in the food, pharmaceutical and feed sectors. Bifidobacteria are an important beneficial intestinal microorganism. The bifidobacterium is used as a physiological beneficial bacterium, and has a plurality of important physiological functions of biological barrier, nutrition, anti-tumor, immunity enhancement, gastrointestinal tract function improvement, aging resistance and the like on human health. The bifidobacteria used in the present invention include the following species: 4 species of bifidobacterium bifidum, bifidobacterium lactis, bifidobacterium infantis, bifidobacterium breve and the like are obligate anaerobes.

Through the mixed bacteria inoculation and mixed bacteria culture of the lactobacillus and the bifidobacterium, the mixture of the 5 kinds of lactobacillus and the 4 kinds of bifidobacterium can be obtained through simultaneous culture, and the obtained probiotic preparation product has the functions and effects of resisting helicobacter pylori, reducing blood sugar, regulating intestines and stomach, increasing immunity, resisting allergy, reducing fat and losing weight and the like through the synergistic effect of the lactobacillus and the bifidobacterium.

The invention does not carry out the separation operation of strains or metabolites after the solid culture is finished, but directly carries out low-temperature drying (freeze drying or vacuum drying) on the cultured strains, the metabolites and the like together to obtain the high-activity composite probiotic preparation solid. Therefore, the product also contains metabolites with bacteriostatic ability, such as organic acids, bacteriocins and the like, which are generated by strains during solid state culture, and also can generate metabolites for reducing blood sugar and increasing immunity, and can enhance the function effects of the composite probiotic preparation in various aspects, such as antianaphylaxis, blood sugar reduction, lipid reduction, weight reduction, immunity increase and the like during use.

The complex probiotic preparation used in the invention comprises several bifidobacteria with similar culture conditions, and the bifidobacteria have an antiallergic function in the growth process in the intestinal tract. The synergistic antiallergic effect among the strains can be increased through the synergistic effect of the strains.

The immunity is measured more frequently, for example: various immunity detection methods such as NK cell activity measurement, cellular immunity measurement, body fluid immunity measurement, monocyte-macrophage function measurement and the like. The invention adopts two methods of 'NK cell activity determination' or 'cell immune function determination' to determine immune function. The result shows that the multifunctional composite probiotic preparation has better effects of reducing blood sugar and increasing immunity.

Bifidobacterium bifidum Miuyo-01 (Latin name: Bifidobacterium bifidum), Bifidobacterium infantis Miuyo-21 (Latin name: Bifidobacterium infantis), Bifidobacterium breve-31 (Latin name: Bifidobacterium breve), Bifidobacterium lactis Miuyo-11 (Latin name: Bifidobacterium lactis), Lactobacillus plantarum MiuCul-01 (Latin name: Lactobacillus plantarum), Lactobacillus acidophilus MiuLac-01 (Latin name: Lactobacillus acidophilus), Lactobacillus casei MiaTech-01 (Latin name: Lactobacillus casei), Lactobacillus rhamnosus MiaTech-11 (Latin name: Lactobacillus sanctus rhynogenus), Lactobacillus pro-01 (Strausella typus) and Lactobacillus rhamnosus MiaTech-11 (Latin name: Lactobacillus sanctus), Streptococcus faecalis Miyami-12, and the northern microbiological culture center of the northern western society of the research center, 3, the preservation numbers are respectively CGMCC 20865, CGMCC 20864, CGMCC 20863, CGMCC20866, CGMCC 20867, CGMCC 20868, CGMCC 20862, CGMCC 20861 and CGMCC 20869.

The invention adopts the new processes of 'mixed bacteria inoculation', 'anaerobic liquid culture + solid-liquid separation + solid culture' to carry out the composite probiotic preparation thallus culture, which can enhance the anti-allergic, lipid-lowering and weight-losing effects: the compound probiotic preparation thallus with higher activity is obtained, antibacterial substances such as bacteriocin, organic acid and the like generated in the solid culture process can be retained, helicobacter pylori and bacteria causing intestinal infection can be inhibited or killed, and the compound probiotic preparation thallus has the functions of resisting allergy, reducing fat and losing weight; meanwhile, various metabolites with the functions of reducing blood sugar and increasing immunity can be produced. In addition, the solid culture has low water content and is easy to dry, the stress death of the composite probiotic preparation in the drying period can be reduced, and the activity of the composite probiotic preparation in the product can be greatly improved by combining proper anaerobic granulation and anaerobic coating procedures. The composite probiotic preparation produced by comprehensively adopting the measures can improve the functional effects of the product in various aspects such as helicobacter pylori resistance, blood sugar reduction, gastrointestinal conditioning, immunity enhancement, allergy resistance, fat reduction, weight reduction and the like.

Compared with the prior art, the invention has the following beneficial effects:

(1) the multifunctional composite probiotic preparation obtained by the invention has large thallus quantity and high activity, and contains metabolites generated by probiotics; the produced composite probiotic preparation has better effects in various aspects such as resisting helicobacter pylori, reducing blood sugar, conditioning intestines and stomach, increasing immunity, resisting allergy, reducing fat and losing weight and the like in the gastrointestinal tract of a human body;

(2) in the preparation process, liquid anaerobic culture is adopted, and the produced culture solution is centrifugally separated to obtain wet thalli of the composite probiotic preparation; mixing the wet thalli with a sterile solid culture medium, and carrying out secondary solid anaerobic culture in a sealed state; and after the solid culture is finished, drying at low temperature to obtain the high-activity composite probiotic preparation solid.

(3) The composite probiotic preparation obtained by the invention has the following advantages:

high viable count

Wet thalli centrifugally separated after liquid fermentation is subjected to solid anaerobic culture in a solid culture medium for a period of time, so that the number of thalli is greatly increased, and the activity is obviously increased; the probiotic with high activity is easier to be planted in the intestinal tract, and shows better physiological function in the intestinal tract;

② small drying stress and small strain drying loss

The water content of the solid culture is only 45-55%, which is far lower than that of the wet thallus obtained by the liquid culture, therefore, the damage and death rate caused by the dry solid culture are far lower than those of the dry liquid culture, and the viable count of the product obtained by the invention can reach 10¹¹cfu/g or more;

③ the product contains various metabolites produced in the process of strain propagation

The invention adopts a plurality of strains (including 5 kinds of lactobacillus and 4 kinds of bifidobacterium) for mixed culture, so that a plurality of metabolites can be generated in the solid growth and reproduction process of the strains, and the prepared composite probiotic preparation containing the plurality of metabolites has better functions and effects in various aspects such as helicobacter pylori resistance, blood sugar reduction, gastrointestinal tract conditioning, immunity enhancement, allergy resistance, fat reduction, weight reduction and the like in the gastrointestinal tract of a human body.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without making any creative effort, shall fall within the protection scope of the present invention.

In some more specific embodiments, the multifunctional composite probiotic preparation comprises the following components in parts by weight:

In other specific embodiments, the multifunctional composite probiotic preparation comprises the following components in parts by weight:

A method for preparing a multifunctional composite probiotic preparation, comprising the steps of:

In the step (1), a disc centrifuge or a tubular centrifuge is adopted to carry out centrifugal separation under the aseptic condition to obtain the wet thalli of the composite probiotic preparation; the liquid culture medium comprises sodium acetate, peptone, yeast extract and tomato sauce; the temperature of the first anaerobic culture is 36-38 ℃, and the time is 48-72 hours.

In the step (2), the solid culture medium comprises sodium acetate, soybean peptide, yeast extract and food-grade calcium carbonate, and is subjected to high-temperature dry heat sterilization and aseptic cooling to below 38 ℃ for later use before use; before the second anaerobic culture, the water content of the mixture of the wet thalli of the compound probiotic preparation and the solid culture medium is adjusted to 45-55 percent; in the step (2), the temperature of the second anaerobic culture is 36-38 ℃, and the time is 72-120 hours; the low-temperature drying or vacuum drying temperature is 30-40 ℃; the water content of the solid of the composite probiotic preparation is 4-6%.

The following are more specific examples:

example 1 isolation of strains, Strain characterization and identification

1.1 sample collection:

collecting and separating lactobacillus plantarum samples from pickled vegetables;

collecting and separating lactobacillus acidophilus samples from naturally fermented dairy products;

collecting and separating a lactobacillus casei sample from a naturally fermented dairy product;

a sample of lactobacillus rhamnosus was isolated from a probiotic preparation sold by this company;

streptococcus thermophilus was isolated from a sample of yoghurt starter sold by this company.

The bifidobacterium sample is selected from healthy infants 3 months to 1 year after birth and not using antibacterial drugs recently, and the sample is collected from the feces and separated, namely the separated strain comes from the intestinal tract of the healthy infants.

1.2 isolation of the Strain

1.2.1 isolation of Lactobacillus plantarum MiuCul-01:

the pickle samples were from Sichuan farmer made pickles. Taking 5-10g of pickle sample, cutting, sufficiently crushing by using a mortar, placing in a sterile 50mL centrifuge tube, adding 20mL of sterile normal saline, shaking, uniformly mixing, placing in a 30 ℃ water-proof incubator, standing and culturing overnight, sucking 1mL of sample liquid, and sequentially performing gradient dilution by 10 times to 10 times by using the sterile normal saline^-1、10^-2、10^-3、10^-4、10^-5、10^-6And respectively taking 100 mu L of bacterial suspension from each gradient, coating the bacterial suspension on an MRS plate, placing the MRS plate in an anaerobic incubator at 37 ℃ for inverted culture, and selecting bacterial colonies from the coated culture plates with different gradients for transfer culture after 48 hours. On the basis, the breast stems conforming to the plants are pickedThe bacterial colony with typical characteristic of bacteria is subjected to MRS plate repeated streak culture and separation, and a single bacterial colony which accords with the growth form of the lactobacillus plantarum bacterial colony is selected to be inoculated into an MRS liquid culture medium and cultured in an anaerobic culture box at 37 ℃. Finally, a strain with good growth performance is obtained.

1.2.2 isolation of Lactobacillus acidophilus MiuLac-01:

the fermented milk sample was derived from naturally fermented milk. Taking 5mL of fermented and deteriorated yoghurt of a milk company, placing the fermented and deteriorated yoghurt in a sterile 50mL centrifuge tube, adding 20mL of fresh milk, shaking and uniformly mixing, placing the mixture in a 30 ℃ water-proof incubator, standing and culturing the mixture overnight, sucking 1mL of sample solution, and sequentially performing gradient dilution on the sample solution by 10 times to 10 times by using sterile normal saline^-1、10^-2、10^-3、10^-4、10^-5、10^-6And respectively taking 100 mu L of the bacterial suspension for each gradient, coating the suspension on an MRS plate, placing the MRS plate in an anaerobic incubator at 37 ℃ for inverted culture, and selecting bacterial colonies from the coated culture plates with different gradients for transfer culture after 48 hours. On the basis, selecting a bacterial colony conforming to the typical characteristics of lactobacillus acidophilus to perform MRS plate repeated streaking culture separation, selecting a single bacterial colony conforming to the growth form of the lactobacillus acidophilus bacterial colony to be inoculated into an MRS liquid culture medium, and culturing in an anaerobic culture box at 37 ℃. Finally, a strain with good growth performance is obtained.

1.2.3 isolation of Lactobacillus casei MiaoTech-01:

the fermented milk sample was derived from naturally fermented milk. Taking 5mL of fermented and deteriorated yoghurt of a milk company, placing the fermented and deteriorated yoghurt in a sterile 50mL centrifuge tube, adding 20mL of fresh milk, shaking and uniformly mixing, placing the mixture in a 30 ℃ water-proof incubator, standing and culturing the mixture overnight, sucking 1mL of sample solution, and sequentially performing gradient dilution on the sample solution by 10 times to 10 times by using sterile normal saline^-1、10^-2、10^-3、10^-4、10^-5、10^-6And respectively taking 100 mu L of the bacterial suspension liquid from each gradient, coating the bacterial suspension liquid on an MRS plate added with calcium carbonate, placing the MRS plate in an anaerobic incubator at 37 ℃ for inverted culture, and selecting a single bacterial colony with a typical calcium-dissolving ring from the coated culture plates with different gradients for transfer culture after 48 hours. On the basis, the lactobacillus casei dictionary is selectedAnd carrying out MRS plate repeated streak culture and separation on the bacterial colony with the type characteristic, selecting a single bacterial colony which accords with the growth form of the lactobacillus casei bacterial colony, inoculating the single bacterial colony into an MRS liquid culture medium, and culturing in an anaerobic incubator at 37 ℃. Finally, a strain with good growth performance is obtained.

1.2.4 isolation of Lactobacillus rhamnosus MiaoTech-11:

the probiotic preparation samples sold by the company are separated and purified. Taking 4g of probiotic preparation, placing in a sterile 50mL centrifuge tube, adding 20mL of sterile normal saline, shaking, mixing uniformly, placing in a 30 ℃ water-proof incubator, standing for overnight culture, sucking 1mL of sample solution, and sequentially diluting with sterile normal saline with gradient of 10 times to 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6And respectively coating 100 mu L of bacterial suspension on an MRS plate for each gradient, placing the MRS plate in an anaerobic incubator at 37 ℃ for inverted culture, and selecting bacterial colonies from the coated culture plates with different gradients for transfer culture after 48 hours. On the basis, selecting a bacterial colony conforming to the typical characteristic of the lactobacillus rhamnosus to perform MRS plate repeated streaking culture separation, selecting a single bacterial colony conforming to the growth form of the bacterial colony of the lactobacillus rhamnosus to be inoculated into an MRS liquid culture medium, and culturing in an anaerobic culture box at 37 ℃. Finally, a strain with good growth performance is obtained.

1.2.5 isolation of Streptococcus thermophilus MiaoPro-01:

the sample of the yoghurt starter sold by the company is separated and purified. Taking 4g of a yogurt starter sample, placing the yogurt starter sample in a sterile 50mL centrifuge tube, adding 20mL of sterile normal saline, shaking, mixing uniformly, placing in a 30 ℃ water-proof incubator, standing for overnight culture, sucking 1mL of sample liquid, and sequentially performing gradient dilution by 10 times to 10 times with the sterile normal saline^-1、10^-2、10^-3、10^-4、10^-5、10^-6And respectively taking 100 mu L of the bacterial suspension for each gradient, coating the suspension on an MRS plate, placing the MRS plate in an anaerobic incubator at 37 ℃ for inverted culture, and selecting bacterial colonies from the coated culture plates with different gradients for transfer culture after 48 hours. On the basis, selecting colonies meeting the typical characteristics of streptococcus thermophilus to repeatedly scratch MRS platesAnd (3) performing linear culture and separation, selecting a single colony which accords with the growth form of the streptococcus thermophilus colony, inoculating the single colony into an MRS liquid culture medium, and culturing in an anaerobic incubator at 37 ℃. Finally, a strain with good growth performance is obtained.

1.2.5 isolation of Bifidobacterium strains

Taking 5-10g of infant feces sample, placing in a sterile 50ml centrifuge tube, adding 20ml of sterile normal saline, shaking, mixing uniformly, placing in an anaerobic incubator at 37 ℃, standing overnight, sucking 1ml of sample liquid, and sequentially performing gradient dilution with 10 times of sterile normal saline to 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6100 mu l of bacterial suspension is respectively taken from each gradient and coated on an improved MRS plate, the improved MRS plate is placed in an anaerobic incubator at 37 ℃ for inverted culture, after 24 hours, bacterial colonies are selected from the coated culture plates with different gradients for transfer culture, on the basis, the bacterial colonies meeting the typical characteristics of bifidobacterium are selected to be repeatedly subjected to streak culture and separation on the improved MRS plate, single bacterial colonies meeting the growth morphology of the bacterial colonies of the bifidobacterium are selected to be inoculated into an improved MRS liquid culture medium, and the improved MRS liquid culture medium is cultured in the anaerobic incubator at 37 ℃. Finally, 4 strains of bifidobacterium with good growth performance are obtained.

1.3 identification of the strains

1.3.1 colony morphology and physiological and biochemical experimental analysis

The methods of references (1), (2), (3) and (4) such as colony morphology and cell morphology observation experiments and biochemical analysis experiments of microorganisms were carried out:

(1) the classification, identification and experimental method of lactic acid bacteria [ M ] Beijing: china light industrial press, 1999;

(2) yufeng, tang jie, engineering microbiology experiment [ M ]. sichuan: southwest university of transportation publishers, 2007;

(3) the isolation and culture of bifidobacteria, such as Qilingling, the university of Qiqiihale, 2008 (3);

(4) zhao laugh, et al, isolation of bifidobacteria from infant feces and study of strain characteristics thereof, industrial microorganisms 2019 (2);

the results are shown in tables 1, 2 and 3:

TABLE 1 morphological characteristics of colonies and cells

Lactobacillus is a gram-positive bacillus, belongs to anaerobic bacteria, and has rod-shaped and slender cells without branches. No spore, no clamping film, round colony, white (sometimes yellow), semitransparent, smooth and convex, and neat edge.

Bifidobacteria are gram-positive bacilli and belong to the group of strictly anaerobic bacteria. The cells are rod-shaped with a bifurcation at one end without movement. The colony is smooth, convex, complete in edge, white in cream, shiny and soft in texture.

TABLE 2 Biochemical analysis results of the strains

Note: "+" is positive; "-" is negative.

TABLE 3 sugar fermentation test

Note: "+" is positive; "-" is negative.

Most of the lactobacillus and the bifidobacterium can ferment glucose, sucrose, fructose, maltose, lactose and the like; the acid and the gas are not generated when the glucose is fermented; most of the lactobacillus can ferment raffinose, xylose, trehalose, cellobiose, sorbitol, mannitol and the like; most bifidobacteria are unable to ferment raffinose, xylose, trehalose, cellobiose, sorbitol, mannitol, and the like.

1.3.216S rRNA partial sequence analysis

The genomic DNA of the selected strain was amplified by PCR and detected by gel electrophoresis using 1% agarose. Bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R (5 'GGTTACCTTGTTCGACTT-3') were used for PCR amplification, purification and sequencing to obtain PCR product sequences, and species identification was performed.

By integrating the results of colony morphology analysis, physiological and biochemical experiment analysis and 16S rRNA partial sequence analysis, 9 strains obtained by separation and purification of the invention are respectively identified as: lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus, Streptococcus thermophilus, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium breve, and are respectively named as "Lactobacillus plantarum MiuCul-01", "Lactobacillus acidophilus MiuLac-01", "Lactobacillus casei MiaoTech-01", "Lactobacillus rhamnosus MiaoTech-11", "Streptococcus thermophilus MiaoPro-01", "Bifidobacterium bifidum Miuyo-01", "Bifidobacterium lactis Miuyo-11", "Bifidobacterium infantis-21", "Bifidobacterium breve Miuyo-31".

EXAMPLE 2 characterization of the strains

2.1 method for determining viable count in product

The total number of the used bacterial strains is 9, the bacterial strains are divided into two categories of lactobacillus and bifidobacterium, after the bacterial strains are diluted to a certain multiple by sterilized normal saline in a gradient manner, the lactobacillus and the bifidobacterium respectively adopt an MRS agar culture medium pouring method and an improved MRS agar culture medium plate pouring method, and the total number of bacterial colonies is counted after the bacterial strains are cultured for 48 hours at 37 ℃.

2.2 growth at different pH

Respectively inoculating the activated 9 strains in an MRS liquid culture medium and an improved MRS liquid culture medium, culturing to obtain strain seed liquid, inoculating 1mL of the seed liquid into 19mL of the MRS liquid culture medium and the improved MRS liquid culture medium with the pH values of 6.0, 6.5, 7.0 and 7.5 respectively, performing anaerobic culture at 37 ℃ for 24h, and measuring initial and final OD600 values (namely the absorbance value at a wavelength of 600nm, which can be generally used for comparing the cell density or the growth condition of thalli in the culture liquid). The OD600 values were determined by comparing the pH at which the growth rate was the maximum (pH 6.0 for Lactobacillus and pH7.0 for Bifidobacterium), i.e., the growth of the cells in the culture media with different pH values was calculated according to the following formula, based on 100% of the number of cells cultured at pH6.0 and pH7.0, respectively:

the cell growth (%) - (OD cultured at other pH)/OD cultured at optimum pH X100%.

The results are shown in Table 4:

TABLE 4 growth of various strains in media of different pH

The results are shown in Table 4: the lactobacillus obtained by culturing in the culture medium with the initial pH value of 6.0, 6.5 and 7.5 is reduced to a certain extent compared with the OD value of the lactobacillus cultured in the culture medium with the optimal pH value, but still has higher growth rate compared with a blank control group which is not cultured; for bifidobacteria, the optimum pH is pH7.0,

each of the above-mentioned strains cultured under the optimum pH condition was subjected to centrifugal separation, washed 2 times with Phosphate Buffer Solution (PBS), and then resuspended in PBS. 1mL of the resuspension solution is mixed with 9mL of artificial gastric juice with pH2.0, 2.5 and 3.0 respectively, the mixture is treated for 3 hours at 37 ℃, and then the total number of probiotics before and after the treatment is respectively determined.

The survival rate before and after the artificial gastric juice treatment is calculated according to the following formula by taking the number of bacteria which are not treated by the artificial gastric juice as a reference:

survival (%) (number of bacteria before treatment-number of bacteria after treatment)/number of bacteria before treatment × 100%.

The results are shown in Table 5:

TABLE 5 survival rates of various strains after treatment with artificial gastric juice at different pH

The results are shown in Table 5:

the lactobacillus thallus cultured at pH6.0 has survival rate of over 75% and high survival rate after being treated by artificial gastric juice with different pH values, so that the lactobacillus thallus can enter intestinal tract in the form of live bacteria under normal eating conditions.

The bifidobacterium thallus cultured at pH7.0 has survival rate of 30-40% and low survival rate after being treated by artificial gastric juice with different pH values. Therefore, it is often necessary to take the form of a coating to allow entry into the intestine as viable bacteria under normal consumption conditions.

The Bifidobacterium strains grow well in the environment with pH of 6.0-7.5, and the optimum pH is 7.0. The survival rate of the thallus cultured at pH7.0 in artificial gastric juice at pH2.0 for 3 hr is lower than 40%.

2.3 growth at different temperatures

Respectively inoculating the activated strains in an MRS liquid culture medium and an improved MRS liquid culture medium, culturing to obtain strain seed liquid, respectively inoculating 1mL of each strain seed liquid in 19mL of the MRS liquid culture medium and the improved MRS liquid culture medium with the pH value of 7.0, respectively carrying out anaerobic culture at the temperature of 31 ℃, 34 ℃, 37 ℃ and 40 ℃ for 24 hours, and determining the initial and finished OD600 values.

The growth of the cells at different temperatures was calculated according to the following formula, using the OD600 value at the optimum growth temperature as a control (i.e., the number of cells cultured at the maximum growth rate was taken as 100%):

the cell growth (%) is the number of cells cultured at other temperatures/the number of cells cultured at the optimum growth temperature × 100%.

The growth conditions of the strains at different temperatures are shown in Table 6:

TABLE 6 growth of various strains at different temperatures

From the experimental results of table 6, the following conclusions can be drawn:

according to experiments, the optimal growth temperature of the lactobacillus plantarum is 34 ℃, and the optimal growth temperature of the streptococcus thermophilus is 45 ℃; the optimal growth temperature of the rest lactobacilli and various bifidobacteria is 37 ℃, and the optimal growth temperature is similar to the body temperature of a human body. Most strains grow well at the temperature of 31-40 ℃ and have good temperature resistance;

in summary, the following steps: the majority of the 9 strains separated and purified by the invention grow well at pH7.0, and still have a certain growth rate between pH6.0-7.5; the optimum growth temperature of most strains is 37 ℃, and a certain growth rate is still kept between 31 ℃ and 40 ℃. Some thermophilic or acidophilic strains, such as streptococcus thermophilus, lactobacillus plantarum, lactobacillus acidophilus, etc., have high growth speed at pH7.0 and 37 ℃ although the optimum growth pH and optimum growth temperature may not be at pH7.0 and 37 ℃, and have certain growth speed when the optimum growth conditions (pH7.0 and 37 ℃) of most strains are adopted during multi-strain mixed culture. Therefore, the fermentation conditions of the strains are pH7.0 and 37 ℃, so that most of the strains can reach the optimal growth conditions, and a small amount of strains which do not reach the optimal growth conditions can also have better growth speed.

2.4 functional Effect of Complex strains

The compound strain of the invention is prepared from the following components: 5 strains of lactobacillus such as lactobacillus acidophilus MiuLac-01, lactobacillus casei MiaoTech-01, lactobacillus rhamnosus MiaoTech-11, lactobacillus plantarum MiuCul-01, streptococcus thermophilus MiaoPro-01, and the like; the bifidobacterium bifidum Miuyo-01, the bifidobacterium lactis Miuyo-11, the bifidobacterium infantis Miuyo-21, the bifidobacterium breve Miuyo-31 and other 4 strains of bifidobacterium and the like, and the total 9 probiotics, so that the composite strain also has the functional effects of various lactobacilli and bifidobacteria. The invention confirms the functional effect of the compound strain through various functional experiments.

2.4.1 anti-helicobacter pylori Effect of Lactobacillus plantarum

The method of the reference (steel, etc., research on the method for antagonizing helicobacter pylori bacteria in vitro. J. drug analysis 2014(10)) adopts an in vitro bacteria inhibition test (perforation method) to perform the experiment of lactobacillus plantarum against helicobacter pylori.

2.4.2 hypoglycemic Effect of Lactobacillus acidophilus

The method of the reference (screening of strains producing alpha-glucosidase inhibitor, such as Zhu Yu Ping, and optimization of solid state fermentation conditions. science and technology in food industry: 2014(3)) measures the alpha-glucosidase inhibition rate of Lactobacillus acidophilus, thereby presuming that the Lactobacillus acidophilus has the blood sugar reducing effect, and then, the human body test is further adopted to confirm the blood sugar reducing effect.

2.4.3 gastrointestinal function-regulating Effect of Lactobacillus casei

The method of the reference (research on the immunity enhancing function of scorpion health care wine, such as Van Zhi Yun, food research and development, 2017(19)) adopts a human body test method to confirm the effect of regulating the gastrointestinal function of lactobacillus casei.

2.4.4 functional Effect of Lactobacillus rhamnosus and Streptococcus thermophilus on enhancing human Immunity

The method of the reference literature (Van Zhi Yun, etc., research on the immunity enhancing function of scorpion health care wine, food research and development, 2017(19)) adopts the activity of mouse NK cells and the conversion reaction of mouse spleen lymphocytes induced by ConA to confirm that the lactobacillus rhamnosus and the streptococcus thermophilus have the effect of enhancing the immunity of human bodies.

2.4.5 antiallergic Effect of Bifidobacterium complex

According to the method of the reference (Liu Shi just et al Balb/c mouse peanut allergy model establishment and pathogenesis. Shenzhen university academic project. 2012(2)), the mouse test is adopted to determine the anti-allergic effect of the compound bifidobacterium.

2.4.6 Immunity-enhancing Effect of Bifidobacterium complex

The method of the reference (research on the immunity enhancing function of scorpion health care wine, such as Van Zhi Yun, food research and development, 2017(19)) adopts a human body test method to confirm the functional effect of the composite bifidobacterium on enhancing the immunity.

2.4.7 blood sugar lowering effect of Bifidobacterium complex

The alpha-glucosidase inhibition rate is determined by referring to a method of Zhusalping (such as Zhusalping, screening strains producing alpha-glucosidase inhibitors, optimizing solid state fermentation conditions, science and technology in food industry: 2014(3)), and the blood sugar reducing effect is confirmed by adopting a human body test.

2.4.4 lipid-lowering and weight-losing effects of Bifidobacterium complex

According to the method of the reference (Lijiangxin, etc., research on weight and fat reducing effects of apple polyphenol, food science 2008(8)), the fat and weight reducing effects of the apple polyphenol are determined by a mouse test.

The results are shown in Table 7:

TABLE 7 efficacy test methods and results for the respective strains (see examples 7, 8, 9, 10 for specific data)

Example 3 composite probiotic raw powder prepared by liquid method and preparation thereof

The strains used in the invention comprise:

5 kinds of lactobacillus such as lactobacillus plantarum MiuCul-01, lactobacillus acidophilus MiuLac-01, lactobacillus casei MiaoTech-01, lactobacillus rhamnosus MiaoTech-11, streptococcus thermophilus MiaoPro-01, etc.;

4 kinds of bifidobacteria, such as bifidobacterium bifidum Miuyo-01, bifidobacterium lactis Miuyo-11, bifidobacterium infantis Miuyo-21, bifidobacterium breve Miuyo-31, and the like.

The 9 strains can be divided into two types of lactobacillus and bifidobacterium, and the optimal growth conditions of the two types of strains are slightly different: lactobacilli can grow in a slightly acidic medium, while bifidobacteria can grow well in a neutral medium; the optimal growth conditions for most strains are 37 ℃, but the optimal growth temperature for lactobacillus plantarum is slightly lower, about 33-34 ℃, while the optimal growth temperature for streptococcus thermophilus is higher, about 45 ℃.

In the mixed culture process of the composite probiotics, in order to obtain the total number of the probiotics to the maximum extent, the invention adopts a culture medium with neutral pH, at the culture temperature of 37 ℃, and adds a proper amount of pH regulators such as sodium acetate, calcium carbonate and the like into the culture medium, thereby ensuring that most strains can normally grow. Although the culture temperature at 37 ℃ may not be the optimum temperature for Lactobacillus plantarum and Streptococcus thermophilus, these two bacteria still have a certain growth at 37 ℃. These 9 species of bacteria are required to be cultured under anaerobic conditions, but the tolerance to oxygen is different, and the lactobacilli are relatively more tolerant to trace amounts of oxygen than the bifidobacteria, so that when they are cultured in a mixed culture, the lactobacilli may grow better in the early stage of the culture, but the bifidobacteria may grow faster in the later stage of the culture as the oxygen is gradually consumed. In order to reduce oxygen in the culture medium as much as possible, the culture medium adopted by the invention is an improved MRS culture medium more suitable for the growth of bifidobacteria, namely 0.5g/L of L-cysteine hydrochloride is added into the MRS culture medium.

3.1 culture Medium

(1) Modified MRS solid medium (for slant and plate culture, used in isolation and preservation of species):

20g/L of glucose, 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract, 15g/L of agar, 2g/L of K042 g, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 801 g/L of Tween, 200.5g/L of MgS04.7H200.05g/L, 0.5g/L of L-cysteine hydrochloride.

Adjusting pH to 7.0 with 1mol/L NaOH, sterilizing at 121 deg.C for 20min, cooling to about 40 deg.C, and chamfering or flattening.

(2) Modified MRS liquid medium (for triangle flask culture and fermentor culture):

20g/L of glucose powder, 10g/L of peptone powder, 10g/L of beef extract, 5g/L of yeast extract, 10g/L of whey powder, 10g/L of tomato sauce, K2HP 042 g/L, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, Tween-801 g/L, MgS04.7H200.5g/L, MnS04.4H200.05g/L and 0.5g/L of L-cysteine hydrochloride.

Adjusting pH to 7.0 with 1mol/L NaOH, and sterilizing at 121 deg.C for 20 min.

3.2 preparation method of composite probiotics by liquid method

The preparation method belongs to the traditional probiotic preparation method, is used as a contrast of the patent, is a product obtained by directly drying after liquid culture and centrifugal separation, does not undergo solid culture, and comprises the following steps:

(1) strain culture

And (3) performing slant culture or plate culture on the composite probiotic strain by using the improved MRS solid culture medium, wherein the strain is the composite probiotic consisting of the 9 probiotic strains.

(2) Triangular flask culture or strain tank culture:

respectively inoculating each strain obtained by slant or plate culture in a triangular flask modified MRS liquid culture medium, and culturing in an anaerobic incubator at 37 deg.C for 24h to obtain triangular flask liquid strain.

(3) Liquid state culture:

the above 5 strains were inoculated in a ratio of "a certain strain solution to a liquid medium of 1: 100", and all were inoculated in a fermenter so that the total inoculum size was 5:100, and the resultant was subjected to anaerobic liquid culture at 37 ℃ for 24 hours in the fermenter to obtain a strain culture solution.

(4) Centrifugal separation:

and (3) separating the liquid culture solution by using a tubular centrifuge or a disc centrifuge, wherein the rotating speed of the centrifuge is 10000r/min, and the centrifugation time is 20min, so as to obtain wet thalli, wherein the water content is 80%.

(5) Drying

Placing the wet thalli in a freeze dryer, and freeze-drying for 20 hours at the temperature of minus 40 ℃ to obtain liquid-method composite probiotic raw powder; through determination: the total bacteria number of the composite probiotic raw powder product prepared by the invention is 3.63 multiplied by 10¹¹CFU/g。

(6) Granulating, coating and packaging.

Adding appropriate amount of adjuvants (such as trehalose, skimmed milk powder, etc.) into the probiotic raw powder to obtain composite probiotic preparation product prepared by liquid method;

through determination: the total bacteria count of the composite probiotic product prepared in the example is 3.15 multiplied by 10¹¹CFU/g。

EXAMPLE 4 Complex probiotic raw powder prepared by solid State method and preparation thereof (solid State method 1)

4.1 Medium

(1) Modified MRS solid medium (for slant and plate culture):

20g/L of glucose, 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract, 15g/L, K2HP 042 g/L of agar, 2g/L of diammonium hydrogen citrate, 5g/L, Tween-801 g/L, MgS04.7 H200.5g/L, MnS04.4 H200.05g/L of sodium acetate and 0.5g/L of L-cysteine hydrochloride. Adjusting pH to 7.0 with 1mol/L NaOH, and sterilizing at 121 deg.C for 20 min.

20g/L glucose, 10g/L peptone, 10g/L beef extract, 5g/L yeast extract, 10g/L whey powder, 10g/L, K2HP 042 g/L tomato sauce, 2g/L diammonium hydrogen citrate, 5g/L, Tween-801 g/L, MgS04.7 H200.5g/L, MnS04.4 H200.05g/L sodium acetate and 0.5 g/L-cysteine hydrochloride. Adjusting pH to 7.0 with 1mol/L NaOH, and sterilizing at 121 deg.C for 20 min.

(3) Culture medium for solid culture (for solid culture):

raw materials for solid state culture were prepared according to the following formulation ratios:

100g of wet thallus, 20g of glucose, 10g of peptone powder, 10g of yeast extract powder, 10g of whey powder, 10g of tomato powder, K2HP 042 g, 0.5g/L of L-cysteine hydrochloride and 2g of food-grade calcium carbonate.

Except for wet bacteria, the other raw materials are respectively sterilized (respectively sterilized by high-temperature instantaneous sterilization equipment for 10s at 150 ℃), and are uniformly mixed in an aseptic manner after being cooled to be used as the raw material of the solid culture medium for later use.

4.2 preparation method of microbial inoculum

The preparation method of the composite probiotic preparation comprises the following steps:

(1) and (3) strain culture:

the modified MRS solid culture medium is used for slant culture or plate culture of strains.

(2) Triangular flask culture or strain tank culture:

inoculating strain obtained by slant or plate culture in improved MRS liquid culture medium, culturing in anaerobic incubator at 37 deg.C for 24 hr to obtain liquid strain in triangular flask,

(3) liquid state culture:

inoculating the triangular flask liquid strain or the seed tank liquid strain into a fermentation tank, and performing anaerobic liquid state culture at 37 ℃ for 24 hours in the fermentation tank to obtain a thallus culture solution;

(4) centrifugal separation:

separating the liquid culture solution with a tubular centrifuge or a disc centrifuge at 10000r/min for 20min to obtain wet thallus with water content of 80%; immediately transferring the wet bacteria into a solid culture medium for culture;

(5) solid-state culture:

mixing the wet thallus with a sterile solid culture medium in a sterile room according to a formula, wherein the formula comprises the following components:

100g of wet thallus, 20g of glucose, 10g of peptone powder, 10g of yeast extract powder, 10g of whey powder, 10g of tomato powder, K2HP 042 g, 0.5g/L of L-cysteine hydrochloride and 2g of food-grade calcium carbonate, wherein the moisture content of the solid culture medium is controlled to be 50-55%. Then placing in an anaerobic solid state fermentation tank, and carrying out sealed anaerobic solid state culture at 37 ℃ for 72 h.

(6) And (3) drying:

freeze drying at-40 deg.C for 20 hr to obtain composite probiotic raw powder.

Through determination: the total bacteria number of the composite probiotic raw powder product prepared by the invention is 5.82 multiplied by 10¹¹CFU/g。

(7) Granulating, coating and packaging.

And (4) granulating and coating the composite probiotic raw powder to obtain a composite probiotic preparation product.

Through determination: the total bacteria number of the composite probiotic preparation product prepared by the example is 5.25 multiplied by 10¹¹CFU/g。

EXAMPLE 5 Complex probiotic preparation by solid State Process (solid State Process 2)

The process operation of example 5 is essentially the same as example 4, with only a portion of the operating parameters being modified to illustrate the effect of solid cultured complex probiotic products produced under different operating conditions.

5.1 Medium

The solid medium of example 4 was identical to example 4 except that whey powder was not added.

5.2 preparation method of microbial inoculum

Example 5 the solid medium was controlled to have a water content of 52% and cultured in a sealed anaerobic solid state at 37 ℃ for 96 hours. The solid culture was dried by freeze-drying, and the other steps were the same as in example 4, to obtain a crude powder product and a preparation product, respectively.

Through determination: the total bacteria number of the composite probiotic raw powder product prepared by the embodiment is 6.76 multiplied by 10¹¹CFU/g; the total bacteria number of the preparation product prepared from the strain is 5.86 multiplied by 10¹¹CFU/g。

EXAMPLE 6 Complex probiotic preparation by solid State Process (solid State Process 3)

6.1 Medium

Example 6 the medium was the same as in example 4.

6.2 preparation method of microbial inoculum

The temperature of triangular flask culture, liquid culture and solid culture is 37 ℃, the water content of the solid culture medium is controlled to be 55%, the solid culture medium is sealed and anaerobically cultured for 120h at 37 ℃, other parameters are the same as example 4, and the raw powder product and the preparation product are respectively prepared.

Through determination: the total bacteria number of the composite probiotic raw powder product prepared by the embodiment is 6.21 multiplied by 10¹¹CFU/g; the total number of bacteria in the preparation product is 6.35 × 10¹¹CFU/g。

In the preparation method, if the culture medium of the fermentation tank is large, the triangular flask liquid spawn can be inoculated in the 1-3-level spawn tank for enlarged culture, so that the liquid spawn used for the fermentation tank is obtained, and the spawn cultured by the triangular flask is directly inoculated in small-scale culture.

In addition, to compare the differences between the liquid and solid processes, example 4 also produced a liquid process product without formulation, which was called raw powder after drying.

Example 7 functional Effect of Complex Probiotics against helicobacter pylori

7.1.1 comparison of the anti-helicobacter pylori Effect of the products at the same weight concentration

Weighing 1g of each sample of the groups respectively, adding 10mL of sterile water to prepare 0.1g/mL of probiotic solution for later use.

The diameter of the antibacterial ring is measured to judge the antibacterial effect of the composite probiotics on HP, the result is expressed by the mean value plus or minus standard deviation, and the result is shown in the table 8:

TABLE 8 anti-helicobacter pylori effect of the composite probiotic products produced by different processes at the same weight concentration

As shown in table 8:

for the composite probiotic raw powder product, the inhibition zone of the composite probiotic raw powder prepared by adopting the solid state method in the embodiment 4 of the invention is larger than that of the probiotic raw powder prepared by adopting the liquid state method in the embodiment 3.

For probiotic preparation products, the inhibition zone of the probiotic preparation prepared by adopting a solid-state method in the embodiments 4, 5 and 6 of the invention is larger than that of the probiotic preparation prepared by adopting a liquid-state method; the SPSS 24.0 software is adopted to carry out one-factor analysis of variance (ANOVA) on experimental data, an LSD method is used for carrying out multiple comparison, and P <0.05 is taken as a difference significance judgment standard, so that the result shows that the anti-pylorus bacteria effect of the microbial inoculum prepared by the preparation method is significantly different from that of a liquid method, and the effect of the anti-pylorus-spirobacterium probiotic microbial inoculum prepared by the preparation method is more obvious.

7.1.2 comparison of the anti-helicobacter pylori Effect of the composite probiotic product at the same bacterial count

Respectively weighing 1g of composite probiotic finished product, adding a proper amount of sterile water, measuring the number of bacteria, and adding sterile water to adjust the total concentration of the composite probiotic to be 1x10¹⁰CFU/ml, 10ml for each administration, so the total number of the compound probiotics for each administration is 10x10¹⁰CFU, i.e. 1x10¹¹CFU。

The experimental method for resisting helicobacter pylori is shown in 7.1.1.

And (3) measuring the diameter of the antibacterial ring, judging the antibacterial effect of the composite probiotics on the HP, and expressing the result by 'average value plus or minus standard deviation'. The results are shown in Table 9:

TABLE 9 anti-helicobacter pylori effect of the composite probiotic products at the same bacterial count

As shown in table 9, when the preparation contains the same number of bacteria, the inhibition zones of the microbial inoculum prepared by the methods 4, 5 and 6 are larger than that of the microbial inoculum prepared by the liquid method; the test of t is adopted, P is less than 0.05, and the anti-helicobacter pylori effect of the microbial inoculum prepared by the preparation method is obviously different from that of a liquid method.

The inhibitory effect of complex probiotics on H.pylori should have approximately the same inhibitory effect when the same number of bacteria is used if it is the effect of only live bacteria, but the results of Table 10 show that: compared with the composite probiotic preparation prepared by the liquid method, the composite probiotic preparation prepared by the solid method in the embodiments 4, 5 and 6 of the invention has a larger inhibition zone, which shows that the effect of resisting helicobacter pylori is more obvious. In the liquid method, metabolites generated in the liquid culture process are removed by centrifugal separation in the preparation process, so the antibacterial effect mainly comes from the strains; the microbial inoculum prepared by the invention contains the bacterial agent, in addition to the composite probiotics, also contains metabolites such as bacteriocin, organic acid and the like generated in the solid culture process, and the composite probiotics and the metabolites are synergistic, so that the anti-helicobacter pylori effect of the whole microbial inoculum is improved.

7.1.3 Effect of Complex probiotic preparations on the treatment of helicobacter pylori infection

180 patients diagnosed with H.pylori infection were randomly divided into 6 groups of 30.

Placebo or placebo groups were treated with non-medicated blank starch tablets 2 times a day, 1 bag each time, 2 grams each for 4 weeks.

The positive group was treated with standard triple combination therapy with omeprazole (0.8mg/kg.d) capsule, amoxicillin (50mg/kg.d), clindamycin (15mg/kg.d) for 2 weeks, two weeks later with no antibiotic, followed by omeprazole for 2 weeks for a total treatment period of 4 weeks.

The liquid culture method is adopted for preparing composite probiotic bacteria, and the composite probiotic bacteria preparation is prepared by centrifugal separation and drying for treatment, wherein the treatment is carried out 3 times a day, 1 bag each time and 2g each bag, and the treatment lasts for 4 weeks.

Example groups a total of 3 groups were treated with the formulations of examples 1-3 orally 3 times a day for 4 weeks, 1 bag each time, 2 grams each.

During the administration period, the patient is asked to determine whether adverse reactions occur. Two weeks after discontinuation, the cure was assessed by the 14C-urea breath test. The results are shown in Table 10:

TABLE 10 treatment of H.pylori infection with probiotic combinations produced by different processes

Description of the drawings: the symptoms of the patient completely disappear to be cured; the symptoms are improved and effective; symptoms were not improved or were not effective.

As can be seen from table 10, compared with the microbial inoculum prepared by the conventional liquid method, the complex probiotic microbial inoculum of examples 4, 5 and 6 of the present invention has higher total effective rate on helicobacter pylori, which indicates that the microbial inoculum prepared by the present invention has more significant effect on resisting helicobacter pylori.

EXAMPLE 8 anti-allergic Effect of Complex Probiotics

According to the method of the reference (Liu Shi gang et al, Balb/c mouse peanut allergy model establishment and pathogenesis, Shenzhen university academic project edition 2012(2)), the mouse test is adopted to determine the anti-allergic effect of the compound bifidobacterium, and the method comprises the following steps:

8.1 Experimental methods

Firstly, experimental materials:

test animals: SPF-grade Balb/c female mice are selected, and are 5-6 weeks old (the body weight is 18-20g), 120 mice are selected, and are randomly divided into 8 groups, and each group comprises 15 mice.

Peanut Crude Protein (CPE) is prepared by a literature method.

Phosphate Buffered Saline (PBS), pH 7.2-7.4.

II, a test method:

1. dose and group:

each group of mice is respectively sensitized by a blank control group (without sensitization and without probiotic treatment), a model group or a positive control group (with peanut crude protein and without composite probiotic preparation treatment), and the rest groups are sensitized by peanut histone, and the composite bifidobacterium preparation prepared by the invention is used for gastric perfusion treatment, wherein the dosage of the composite bifidobacterium is respectively as follows: the liquid method control group (example 3) and the solid method experimental group of the invention, example 4, example 5 and example 6, all have the dosage of 0.1g/ml, and the details are shown in Table 8. All mice were taken 2 times daily for 10 consecutive days.

2. Sensitization and challenge in mice:

firstly, 10 mice are separated out to be used as a blank control group, and the blank control group is injected by PBS buffer solution without sensitization; all the other mice are sensitized by peanut crude protein solution injection, then the allergic mice are selected and randomly grouped into 10 mice each group, and the redundant mice are not used. And (3) performing intragastric administration treatment on the allergic mice with the probiotic solution according to the samples and the dosage in the table 6 every day, continuously performing intragastric administration for 10 days, and performing intragastric administration on the negative control group and the positive control group by using sterile purified water.

The Balb/c mice are divided into a blank control group and a peanut sensitization model group (a model group for short, and a positive control group), 10 mice in each group are sensitized in a way that: the model group is injected with 100 mug peanut crude protein and 1mg aluminum hydroxide adjuvant every 3 weeks for sensitization twice by subcutaneous injection; after 1 week of the 2 nd immunization, 200. mu.g of CPE was injected subcutaneously for challenge, and the crude peanut protein was replaced with PBS solution of the same volume as the PBS control group, which was otherwise identical to the model group.

3. Detection of total IgE antibodies in serum:

after the feeding experiment of the compound bifidobacterium preparation is finished, blood is taken from mice of each dosage group, the mice are stood for 2 hours at room temperature, the centrifugation is carried out for 10 minutes at 4000r/min, and the upper serum is carefully taken out. The content of total IgE antibodies in serum is detected by adopting an ELISA reagent, and the detection is carried out according to the product specification.

8.2 comparison of the antiallergic Effect of the products at the same weight concentration

The content of total IgE antibodies in serum was measured, and the measurement results are expressed as "mean. + -. standard deviation". In the experiment, SPSS 24.0 software is adopted to carry out one-factor analysis of variance (ANOVA) on experimental data, multiple comparisons are carried out by an LSD method, P <0.05 is taken as a judgment standard of difference significance, and the result is shown in a table 11:

TABLE 11 antiallergic effect of different process-produced complex probiotic products at the same weight concentration

The results of the experiment are shown in table 11:

IgE-mediated allergic reactions are the main cause of allergy, and IgE antibodies in serum are key factors for the development of allergic reactions. From the total content of IgE antibodies in the serum of the blank control group and the model group, the IgE change of the mouse corresponds to the allergic symptoms, namely the content of the serum IgE antibodies of the blank mouse is lower, and the content of the serum IgE antibodies of the model group is higher. After the probiotic intragastric administration treatment, each probiotic treatment group shows a good effect of treating allergy.

When the preparations contain probiotics with the same weight concentration, the IgE values of the inocula prepared in the probiotics examples 3, 4, 5 and 6 are larger than that of a blank control group, and the IgE values are obviously different; the anti-allergic effect of the microbial inoculum prepared by the solid-state method (examples 4, 5 and 6) is better than that of the microbial inoculum prepared by the liquid-state method (example 3), and the anti-allergic effect is very different. Therefore, the complex bifidobacterium preparations produced in examples 4, 5 and 6 prepared by the solid state method have particularly remarkable effect of reducing allergic symptoms in mice.

Compared with the probiotic raw powder product, the probiotic preparation product has slightly poor antiallergic effect, probably because the probiotics are slightly lost in the preparation process, but the preparation product is more convenient for people to take.

8.3 comparison of antiallergic Effect of products with the same number of bacteria

Respectively weighing 1g of probiotic finished product, adding a proper amount of sterile water, measuring the number of bacteria, and adding sterile water to adjust the total probiotic concentration to be 1x10¹⁰CFU/ml, 10ml for each administration, so the total number of composite probiotics for each administration is 10x10¹⁰CFU, i.e. 1x10¹¹CFU。

The experimental method for resisting allergy is shown in 8.1.

The content of total IgE antibodies in serum was measured, and the measurement results are expressed as "mean. + -. standard deviation". In the experiment, SPSS 24.0 software is adopted to carry out one-factor analysis of variance (ANOVA) on experimental data, an LSD method is used for carrying out multiple comparison, P <0.05 is taken as a judgment standard of difference significance, and the result is shown in a table 12:

TABLE 12 antiallergic effect of the composite probiotic product at the same number of bacteria

As shown in table 12, when the preparations contain the same number of bacteria, the IgE values of the inocula prepared in the composite probiotics examples 3, 4, 5 and 6 are larger than the IgE value of the blank control group, and have significant difference; the anti-allergic effect of the microbial inoculum prepared by the solid-state method (examples 4, 5 and 6) is better than that of the microbial inoculum prepared by the liquid-state method (example 3), and the anti-allergic effect is very different. Therefore, the complex bifidobacterium preparations produced in examples 4, 5 and 6 prepared by the solid state method have particularly remarkable effect of reducing allergic symptoms in mice. Compared with the composite probiotic raw powder product, the composite probiotic preparation product has slightly poor antiallergic effect.

The anti-allergic effect of the complex probiotic, if only the effect of the live bacteria, should be about the same when the same number of bacteria is used, but the results of table 9 show that: compared with the composite probiotic preparation prepared by the liquid method, the IgE value produced by the composite probiotic preparation prepared by the solid method in the embodiments 4, 5 and 6 of the invention is larger, which shows that the antiallergic effect is more obvious. In the liquid method, metabolites generated in the liquid culture process are removed by centrifugal separation in the preparation process, so that the antiallergic effect of the liquid method mainly comes from the strains; the microbial inoculum prepared by the invention contains the bacterial agent, in addition to the composite probiotics, also contains metabolites such as bacteriocin, organic acid and the like generated in the solid culture process, and the composite probiotics and the metabolites are synergistic, so that the overall anti-allergic effect of the microbial inoculum is improved.

Example 9: comparison of the Immunity-enhancing Effect of Complex Probiotics

9.1 Effect of Complex probiotic formulations on NK cell Activity

9.1.1 Experimental methods

Firstly, experimental materials:

test animals: SPF-level Kunming female mice are selected, and are 6-8 weeks old (the body weight is 18-22g), 120 mice are selected, and are randomly divided into 8 groups, and each group comprises 15 mice.

Other materials: mouse lymphoma cells (YAC-1), Hank's solution, RPMI1640 complete culture solution, LDH matrix solution, etc.

II, a test method:

1. dose and group:

the negative control group (pure water), the liquid control group (example 3), and the solid experimental group of the invention (example 4, example 5, and example 6) were used for each group of mice, and the dosage was 0.1 g/ml. All mice were taken 2 times daily for 30 consecutive days.

2. The feeding method comprises the following steps:

during experiments, samples are respectively taken and prepared into sample liquid with 20ml by purified water, and the sample liquid is respectively used for animal gavage. Animals were acclimated for 3 days in the laboratory, randomized into groups, gavaged daily at the samples and doses given in table 4 for 30 consecutive days, and the control group was gavaged with sterile purified water.

3. Preparation of effector cells (mouse splenocytes):

after the feeding experiment of the compound probiotic preparation is finished, the spleen of each dose of mice is taken aseptically and placed in a small plate containing a proper amount of aseptic Hank's solution, and the spleen is gently ground by forceps to prepare single cell suspension. The spleens were filtered through a 200 mesh screen, or the spleens were triturated with 4 layers of gauze, or washed 2 times with Hank's solution, and centrifuged for 10min (1000r/min) each time. Discarding supernatant to bounce the cell pulp, adding 0.5mL of sterilized water for 20 seconds, lysing erythrocytes, adding 0.5mL of 2-fold Hank's solution and 8mL of Hank's solution, centrifuging at 1000rpm for 10min, resuspending with 1mL of RPMI1640 complete culture solution containing 10% calf serum, diluting with 1% glacial acetic acid, counting (viable cell number should be above 95%), staining with talofen blue to count viable cell number (should be above 95%), and adjusting cell concentration to 2 × 10 with RPMI complete culture solution⁷one/mL.

4. Passage of target cells (YAC-1 cells)

The target cells were subcultured 24h before the experiment. The cells were washed 3 times with Hank's solution and adjusted to 4X 10 concentration in RPMI1640⁵one/mL.

5. NK cell Activity assay (LDH method)

Taking the concentration of 4 × 10⁵Each 100 mul of target cells (YAC-1) and effector cells (mouse spleen cells) per ml (effective target ratio 50:1) were added to a U-shaped 96-well culture plate, each 100 mul of target cells and culture medium were added to natural target cell release holes, and each 100 mul of target cells and 1% NP40 were added to maximum target cell release holes;each of the above was prepared by repeating 3 wells, incubating for 4 hours at 37 ℃ in a 5% CO2 incubator, centrifuging the 96-well plate for 5 minutes at 1500r/min, aspirating 100. mu.l of the supernatant per well, placing the plate in a flat-bottomed 96-well plate, adding 100. mu.l of LDH matrix solution, reacting for 3 minutes, adding 30. mu.l of 1mol/L HCl per well, and measuring the optical density at 492nm in an enzyme-linked immunosorbent.

NK cell activity was calculated as follows:

NK cell activity (%) - (reaction well OD-natural release well OD)/(maximum release well OD-natural release well OD) × 100%

The NK cell activity of the test sample group is obviously higher than that of the negative control group, and the result of the experiment can be judged to be positive.

9.1.2 comparison of the Immunity-enhancing Effect of the products at the same weight concentration (Effect on NK cell Activity in mice)

Respectively weighing 1g of each sample of the groups, adding 10mL of sterile water to prepare 0.1g/mL of probiotic solution for later use; the negative control group used only sterile water and no probiotic.

The effect of complex probiotics on mouse NK cell activity was measured and the assay results are expressed as "mean ± standard deviation". In the experiment, SPSS 24.0 software is adopted to carry out one-factor analysis of variance (ANOVA) on experimental data, multiple comparisons are carried out by an LSD method, P <0.05 is taken as a judgment standard of difference significance, and the result is shown in a table 13:

TABLE 13 Effect of different processed probiotic products on NK cell Activity in mice at the same weight concentration

Note: NK cell Activity conversion value ═ sin-1 (A)^1/2)

As shown in table 13:

for probiotic raw powder products, the NK cell activity in the serum of mice when the probiotic raw powder prepared by the solid-state method is fed in example 4 of the invention is greater than that of the probiotic raw powder prepared by the liquid-state method fed in example 3.

For probiotic preparation products, the NK cell activity in the serum of mice fed with the probiotic preparation prepared by the solid-state method of examples 4, 5 and 6 of the present invention was greater than that fed with the probiotic preparation prepared by the liquid-state method (example 3); compared with a liquid method, the solid feeding method has a significant difference in the effect of enhancing the immunity of the probiotic preparation, which shows that the probiotic preparation prepared by the preparation method has a more obvious effect of enhancing the immunity.

9.1.3 comparison of the Immunity-enhancing Effect of the product with the same number of bacteria (influence on NK cell Activity of mice)

The experimental method for enhancing the immunity is shown in 9.1.1.

The effect of the complex probiotic preparation in mouse serum on mouse NK cell activity was measured and the assay results are expressed as "mean ± standard deviation". In the experiment, SPSS 24.0 software is adopted to carry out one-factor analysis of variance (ANOVA) on experimental data, multiple comparisons are carried out by an LSD method, P <0.05 is taken as a judgment standard of difference significance, and the result is shown in a table 14:

TABLE 14 Complex probiotic products (Effect on NK cell Activity in mice) at the same number of bacteria

Note: NK cell Activity conversion value ═ sin-1 (A)^1/2)

As shown in Table 14, when the preparation contains the same number of bacteria, the microbial inoculum prepared by the preparation method 4, 5 and 6 has larger influence on the activity of NK cells of the mice; the microbial inoculum prepared by the solid-state method has the effect of enhancing the immunity, and has significant difference compared with the liquid-state method.

The boosting effect of probiotics, if only live, should be about the same when the same number of bacteria is used, but the results in table 14 show that: compared with the compound probiotic preparation prepared by the liquid method, the compound probiotic preparation prepared by the solid method in the embodiments 4, 5 and 6 of the invention has larger influence on the activity of NK cells of mice, which shows that the effect of enhancing immunity is more obvious. In the preparation process of the liquid method, metabolites generated in the liquid culture process are removed by centrifugal separation, so the immunity enhancing effect of the liquid method mainly comes from the strains; the microbial inoculum prepared by the invention contains the composite probiotics and also contains metabolites generated in the solid culture process, and the composite probiotics and the metabolites are synergistic, so that the overall immunity increasing effect of the microbial inoculum is improved.

9.2 Effect of Complex probiotic preparations on ConA-induced splenic lymphocyte transformation responses in mice

9.2.1 Experimental methods

Firstly, experimental materials:

test animals: SPF-level Kunming female mice are selected, and are 6-8 weeks old (the weight is 18-22g), 105 mice are selected, and are randomly divided into 7 groups, and each group comprises 15 mice.

Other materials: hank's solution, RPMI1640 complete culture solution, etc.

II, a test method:

1. dose and group:

2. The feeding method comprises the following steps:

during experiments, samples are respectively taken and prepared into sample liquid with 20ml by purified water, and the sample liquid is respectively used for animal gavage. Animals were acclimated for 3 days in the laboratory, randomized into groups, gavaged daily at the samples and doses in table 12 for 30 consecutive days, and the control group was gavaged with sterile purified water.

Third, ConA-induced mouse spleen lymphocyte transformation experiment (MTT method):

1. lymphocyte proliferation reaction:

after the feeding experiment of the compound probiotic preparation is finished, the spleen of each dose group of mice is aseptically taken out, the mice are placed in aseptic Hank's solution for grinding, after centrifugal washing at 1000r/min, the cells are suspended in 1ml of complete culture solution, the viable cells are counted and the concentration is adjusted to be 3 multiplied by 10⁶One/ml, the cell suspension was added to a 24-well plate in two wells, 1ml per well, 75. mu.l ConA (equivalent to 7.5. mu.l/ml) in 1 well, and the other well was used as a control. Culturing at 37 deg.C for 72h in 5% CO 2.4 hours before the end of the culture, 0.7ml of the supernatant was aspirated from each well, and 0.7ml of serum-free RPMI1640 medium was added to each well together with 50. mu.l/ml of MTT (5mg/ml), and the culture was continued for 4 hours. After the culture, 1ml of isopropanol was added to each well, and the mixture was blown and beaten uniformly to dissolve the purple crystals, and the purple crystals were added to a 96-well plate, and the plate was used as a replicate of 3 wells, and the optical density was measured at a wavelength of 570nm, and the proliferative transformation ability of lymphocytes was calculated according to the following formula.

Proliferation potency of lymphocytes ═ OD (ConA well) -OD (control well)

2. The experimental results are as follows:

for the ConA-induced splenic lymphocyte transformation reaction of mice, the difference of the optical density of the high-dose group is higher than that of the control group, and the difference has significant significance (see table 12). The results show that the spleen lymphocyte transformation test of the sample of example 1 is positive, and the tested sample enhances the cellular immunity.

The effect of the complex probiotic formulation on ConA-induced splenic lymphocyte transformation in mice is shown in table 15:

TABLE 15 Effect of different Probiotics on ConA-induced splenic lymphocyte transformation in mice

From the results of table 15, it can be seen that: the complex probiotic preparations produced by the solid state method from examples 4, 5 and 6 had a particularly significant effect on ConA-induced splenic lymphocyte transformation in mice compared to the negative control and liquid state methods.

According to the experimental result of example 8, the complex probiotic preparation can improve the NK cell activity of mice, increase the spleen lymphocyte transformation reaction of mice induced by ConA, and show that it has the effect of enhancing immunity, wherein the effect is more obvious in example 4, example 5 and example 6 which adopt the solid state method.

Example 10 comparison test of hypoglycemic Effect of Complex Probiotics (measurement of content of. alpha. -glucosidase inhibitor)

10.1 determination of the content of alpha-glucosidase inhibitor of Complex Probiotics

10.1.1 test method

The product obtained in each example is selected, and the content of the alpha-glucosidase inhibitor is determined by referring to a cinnabar method. The test method is as follows:

materials and reagents:

4-nitrophenyl-D-glucopyranoside (PNPG), alpha-glucosidase; sterile 0.1mol/L phosphate buffer solution (PBS, pH 6.8); the other reagents are analytically pure.

Preparing a sample extracting solution:

the cells were suspended in sterile 0.1mol/L phosphate buffer (PBS, pH6.8) and the concentration of the cells was adjusted to 1X10⁹CFU/mL, incubation at 37 deg.C for 12h, centrifugation at 4 deg.C and 8000r/min for 15min, filtering the supernatant through 0.22 μm water-based microfiltration membrane to obtain sample extract, and storing at-80 deg.C.

Determination of α -glucosidase inhibition:

the alpha-glucosidase inhibitory activity was determined by PNPG method.

Principle of PNPG method:

because PNP released by PNPG catalyzed and hydrolyzed by alpha-glucosidase has certain absorbance at 405nm, the activity change of the alpha-glucosidase can be determined by detecting the amount of the PNP, and the inhibition rate of a sample on the alpha-glucosidase can be determined.

To the diluted sample, 50. mu.L of an α -glucosidase solution (25 mg/mL), 50. mu.L of 4-PNPG (0.9133mg/mL) and 120. mu.L of 0.5mol/L phosphate buffer (pH 6.8) were added to a 96-well plate. After mixing, the mixture was reacted in an incubator at 37 ℃ for 45 min. The reaction was stopped by adding 50. mu.L of sodium carbonate solution (0.67mol/L), and the absorbance was measured at 405nm with a microplate reader. The absorbance values obtained with the same volume of buffer instead of sample served as a control. The inhibition rate of alpha-glucosidase was calculated by the following formula.

Inhibition ratio of α -glucosidase%_Contrast-OD_{Sample (I)})/OD_Control×100

In the formula:

OD_controlIs the absorbance value not added to the sample well, i.e. the blank.

OD_{Sample (I)}Is the absorbance value of the wells to which the sample extract (i.e., containing the alpha-glucosidase inhibitor) was added.

10.1.2 comparison of hypoglycemic Effect of the product at the same weight concentration (measurement of. alpha. -glucosidase inhibition)

Weighing 1g of each sample of the groups respectively, adding 10mL of sterile water to prepare 0.1g/mL of probiotic solution for later use. The PNPG method measures the inhibitory effect of each group of samples on α -glucosidase to determine the hypoglycemic effect of the complex bifidobacterium, and the results are expressed as "mean ± standard deviation", as detailed in table 16:

the experimental results are as follows:

TABLE 16 alpha-glucosidase inhibition (%)

Sample (I)	Dosage form	Alpha-glucosidase inhibition (%)
			Negative control group	0.1g/ml	0
Example 3 raw powder	0.1g/ml	68.6
			Example 4 raw powder	0.1g/ml	82.8
Example 3 formulations	0.1g/ml	61.5
			Example 4 formulations	0.1g/ml	75.8
Example 5 formulations	0.1g/ml	88.4
			Example 6 formulations	0.1g/ml	82.6

10.1.3 comparison of hypoglycemic Effect of the product with the same number of bacteria (measurement of. alpha. -glucosidase inhibition)

Respectively weighing 1g of probiotic finished product, adding a proper amount of sterile water, measuring the number of bacteria, and adding sterile water to adjust the total probiotic concentration to be 1x10¹⁰CFU/ml, spare.

The inhibitory effect on α -glucosidase of the samples of the examples of the same number of bacteria was measured according to the similar test method as in example 9.1.1, and the results are expressed as "mean ± standard deviation", and are shown in table 17:

TABLE 17 inhibition ratio (%) of alpha-glucosidase of Bifidobacterium complex produced by different processes with the same number of cells

Sample (I)	Dosage form	Alpha-glucosidase inhibition (%)
			Negative control group	1x10¹⁰CFU/ml	0
Example 3 raw powder	1x10¹⁰CFU/ml	62.2
			Example 4 raw powder	1x10¹⁰CFU/ml	78.3
Example 3 formulations	1x10¹⁰CFU/ml	56.6
			Example 4 formulations	1x10¹⁰CFU/ml	65.3
Example 5 formulations	1x10¹⁰CFU/ml	82.6
			Example 6 formulations	1x10¹⁰CFU/ml	76.2

The above experimental results actually determined the difference between the α -glucosidase activity of the sample and the activity of the α -glucosidase standard, i.e., defined the α -glucosidase inhibition ratio of 0 for the buffer without the sample, and defined the α -glucosidase inhibition ratio of the sample as the percentage difference in absorbance between the sample and the control. It should be noted that: in order to simplify the determination procedure, the present invention does not set a positive control, i.e. does not use positive α -glucosidase inhibitor acarbose or DNJ as a control, and does not subtract the sample background, so the determined inhibition rate may be slightly different from the actual one, but since the difference is not large, the results in tables 16 and 17 still have good reference value for understanding the inhibition effect of the probiotic samples on α -glucosidase.

According to the experimental results of tables 16 and 17, the probiotic product of the present invention has a better α -glucosidase inhibition rate than the conventional liquid process probiotic product, in addition to the higher probiotic count of the probiotic product of the present invention. Thus, it is expected that the products of the invention will generally have a better hypoglycemic effect than probiotic preparations produced by liquid processes.

10.2 comparison test of hypoglycemic Effect of Complex Probiotics (therapeutic Effect of hyperglycemia)

According to the products obtained in the embodiments, the blood sugar reduction experiment of the products is carried out, and the experimental scheme and the experimental result are as follows:

experimental materials and methods:

a subject: two type diabetes patients with fasting blood glucose of more than or equal to 7.8mmol/L are selected as experimental objects, 210 patients diagnosed with adult type II diabetes are randomly divided into 7 groups, and each group contains 30 patients.

Blank control or placebo group: the treatment was performed 3 times a day with 1 bag per time and 2g per bag for 4 weeks using an empty starch formulation without dosing.

Positive experimental group: the medicine is taken by acarbose at a dose of 75mg/d in three times, 25mg each time, for 4 weeks.

Liquid method control (example 3): the compound probiotics is cultured by adopting a traditional liquid culture method, and the compound probiotics preparation prepared by drying after centrifugal separation is used for treatment, 3 times a day, 1 bag each time and 2 grams each bag lasts for 4 weeks.

Example set: a total of 3 groups (examples 4-6) were treated with the probiotic complexes orally, 3 times a day, 1 bag each time, 2 grams each, for 4 weeks, according to table 15.

During the administration period, the patient is asked to determine whether adverse reactions occur.

The experimental results are as follows:

TABLE 18 hypoglycemic Effect (fasting blood glucose level, mmol/L) of Complex Probiotics produced by different Processes

TABLE 19 hypoglycemic Effect of Complex Probiotics produced by different Processes (postprandial 2h blood sugar value, mmol/L)

In conclusion, the composite probiotic preparation prepared by the invention has good hypoglycemic effect, the effect is better than that of the composite probiotic preparation prepared by the liquid method, although the composite probiotic preparation is slightly lower than that of a commonly used hypoglycemic drug, namely the carbopol, the composite probiotic preparation has small side effect, and the composite probiotic preparation also has other effects of regulating intestinal tracts, enhancing immunity and the like, and is not limited by the dose of the drug. Therefore, the composite probiotics can be used as an auxiliary conditioning product for the diabetes patients to assist other hypoglycemic drugs to play a role in reducing blood sugar.

Example 11: contrast test of lipid-lowering and weight-losing effects of composite probiotics

The fat-reducing and weight-losing effect of the compound probiotics on mice can be characterized by measuring the body weight, Lee's index, blood fat and other indexes of the mice fed with the compound probiotics.

11.1 Effect of bacterial agents on mouse lipid-lowering and weight-losing effects at the same weight concentration

The products obtained in the above examples 3-6 are selected, the compound probiotics with the same weight are fed according to the dosage of table 3, the lipid-lowering and weight-losing effects of the compound probiotics on mice are determined after the experimental period is finished, and the experimental scheme and the experimental results are as follows:

11.1.1 materials and reagents:

kunming mouse, male and female half, 4 weeks old, weight 18-22 g.

11.1.2 test method:

animal grouping: after adaptive feeding for 1 week, the weight of the bought mice was measured as the initial weight, and the mice with too large and too small weight were removed, and 80 mice were selected and randomly divided into 8 groups of 10 mice each with half of males and females.

Blank control group (distilled water), model control group (high fat diet 3g), positive control group (orlistat 400 μ g), example 3 (liquid method control group) raw flour, example 3 (liquid method control group) preparation, example 4 raw flour, example 4 preparation, example 5 preparation, example 6 preparation.

Mice were fed basal diet normally with 3 meals a day. After each meal of basic feed is fed for about 30 minutes, 10ml of solution is prepared by using the corresponding dosage of each group in the table 3 for intragastric administration, then the weight is measured for 1 time per week, feeding experiments are carried out for 4 weeks, and the final weight is measured and blood is collected to measure the blood fat content at the end of the experiments.

11.1.3 Observation indicators and methods:

general observation:

activity level, hair color, body shape, diet, drinking water and feces status.

Detecting growth indexes:

the growth index mainly detects body weight (g) and obesity index (Lee's index), wherein the Lee's index is calculated by the following method:

lee's index ═ body weight (g)]^1/3Length of body (cm) × 1000

③ detecting blood fat:

blood lipid determination (total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein) by use of full-automatic biochemical analyzer.

11.1.4 results of the experiment:

general conditions:

the hair color of the six groups of mice is unchanged.

The appetite of the model control group is enhanced, the body form becomes round, the activity is slower than that of the normal control group, and the stool is slightly increased;

the positive control group mice have weak stool, and the compound probiotic group mice have no diarrhea phenomenon.

Compared with a model control group, the mice of the compound probiotic group have less food intake, more flexible activity and obviously increased stool volume.

② weight change:

each group is randomly grouped after 1 week of adaptive feeding, and the initial weight difference of mice among the groups has no statistical significance (P is more than 0.05); when the feeding experiment of 4 weeks is finished, the weight of the model control group is obviously increased compared with that of the blank control group; the weight of the positive control group is obviously reduced compared with that of the blank control group; the probiotic groups of the different examples had significantly reduced body weight compared to the placebo group; the body weight was significantly reduced compared to the model control group, and the results are shown in table 20:

TABLE 20 Effect of Complex Probiotics on mouse body weight

After the feeding experiment of 4 weeks is finished, blood is collected to determine the blood fat content, and the result is shown in a table 21:

TABLE 21 Effect of Complex Probiotics on mouse blood lipids

As can be seen from the results in tables 20 and 21:

compared with the blank control, the composite probiotic raw powder produced by the example 3 and the example 4 has particularly remarkable effect on reducing fat and losing weight of mice.

Compared with the blank control, the complex probiotic preparation produced by example 3, example 4, example 5, example 6 and the like has particularly remarkable effect on the effects of reducing fat and losing weight of mice.

Compared with the compound probiotic preparation prepared by the liquid culture method (example 3), the compound probiotic preparation prepared by the solid culture method (examples 4-6) has particularly remarkable effect on reducing fat and losing weight of mice.

The above experimental results show that: the composite probiotics has a certain effect of reducing the weight of a mouse, and the composite probiotics utilizes micromolecular nutrients such as glucose, fatty acid and the like to synthesize intestinal thallus components in the intestinal tract, so that the excrement discharge is increased, and the fat removal is facilitated; in addition, the metabolite produced by the composite probiotics is also beneficial to reducing the synthesis of fat in the body. Therefore, the composite probiotics have the effect of inhibiting the weight gain of mice. The experimental result of the influence of the composite probiotics on the blood fat of the mouse shows that: the compound probiotics can reduce TG, CHO and LDL-C and increase HDL-C, so that the compound probiotics is beneficial to improving fat metabolism in vivo and has good lipid-lowering function. From the experimental results of tables 20 and 21, it was also found that: compared with the traditional liquid probiotic product, the solid probiotic product has better lipid-lowering and weight-losing effects.

In conclusion, the solid-state composite probiotic preparation prepared by the invention has good lipid-lowering and weight-losing effects, the effect is better than that of the liquid-state composite probiotic preparation, although the solid-state composite probiotic preparation is slightly lower than that of a common lipid-lowering and weight-losing medicament-orlistat, the composite probiotic preparation has other effects of adjusting intestinal tracts, increasing immunity and the like due to small side effect, and is not limited by medicament dose. Therefore, the composite probiotics can be used as an auxiliary conditioning product for obese patients, and can assist other lipid-lowering and weight-losing medicines to play the role of lowering lipid and losing weight.

11.2 Effect of the same bacterial count on the lipid-lowering and weight-losing effects of mice

The test procedure is as in example 11.1.

Respectively weighing 1g of composite probiotic finished product, adding a proper amount of sterile water, measuring the number of bacteria, and adding sterile water to adjust the total concentration of the composite probiotic to be 1x10¹⁰CFU/ml, 10ml for each drench, so the total number of the compound probiotics for each drench is 10x10¹⁰CFU, i.e. 1x10¹¹CFU。

The effect of the same number of bacteria of the example samples on the lipid-lowering and weight-reducing effect of mice was determined according to a similar test method as in example 11.1, and the results are expressed as "mean ± standard deviation" and are shown in table 22:

TABLE 22 Effect of the preparation products on the fat and weight reducing effects of mice with the same number of bacteria

After the feeding experiment of 4 weeks is finished, blood is collected to determine the blood fat content, and the result is shown in a table 23:

TABLE 23 Effect of the preparation products on mouse blood lipid with the same bacterial count

As shown in tables 22 and 23, example 3 is the effect of the liquid preparation on the fat and weight reducing effect of mice, while examples 4, 5 and 6 are the effect of the 3 solid preparations on the fat and weight reducing effect of mice.

The experimental results of tables 22 and 23 show that: the preparation product with the same bacteria number has approximately the same effect of reducing fat and losing weight. The results of the experiments combining the above tables 20, 21, 22 and 23 also found that: compared with the composite probiotic product prepared by the traditional liquid method, the composite probiotic product prepared by the solid culture method has better lipid-lowering and weight-losing effects.

The effect of probiotics on the lipid-lowering and weight-losing effect of mice, if only the effect of viable bacteria, should have about the same effect when the same number of bacteria is used, but the above results indicate that: compared with the composite probiotic product prepared by the liquid method (example 3), the composite probiotic products prepared by the solid method in examples 4, 5 and 6 of the invention have better lipid-lowering and weight-losing effects. In the preparation process of the liquid preparation, metabolites generated in the liquid culture process are removed by centrifugal separation, so the effect of reducing fat and losing weight mainly comes from the strains; the solid preparation prepared by the invention contains the citric acid lyase inhibitor, the alpha-glucosidase inhibitor, the short chain fatty acid and other metabolites with the lipid-lowering and weight-losing capabilities, which are generated in the solid culture process, in addition to the composite probiotics, and the composite probiotics and the metabolites are synergistic, so that the influence of the whole microbial inoculum on the lipid-lowering and weight-losing effects of mice is improved.

The above experimental results show that: the composite probiotics has a certain effect of reducing the weight of a mouse, and the composite probiotics utilizes micromolecular nutrients such as glucose, fatty acid and the like to synthesize intestinal thallus components in the intestinal tract, so that the excrement discharge is increased, and the fat removal is facilitated; in addition, the metabolites produced by the probiotics are also beneficial to reducing the synthesis of fat in the body. Therefore, the probiotics have the effect of inhibiting the weight gain of the mice. The experimental result of the influence of the composite probiotics on the blood fat of the mouse shows that: the compound probiotics can reduce TG, CHO and LDL-C and increase HDL-C, so that the compound probiotics are beneficial to improving fat metabolism in vivo and have good lipid-lowering function. From the experimental results of tables 22 and 23, it was also found that: compared with the traditional liquid method composite probiotic product, the composite probiotic product has better lipid-lowering and weight-losing effects. The composite probiotic preparation prepared by the invention has good lipid-lowering and weight-losing effects, the effect is superior to that of a composite probiotic preparation prepared by a liquid method, although the composite probiotic preparation is slightly lower than a commonly used lipid-lowering and weight-losing medicament-orlistat, the composite probiotic preparation has other effects of adjusting intestinal tracts, increasing immunity and the like due to small side effect, and is not limited by medicament dosage. Therefore, the composite probiotics can be used as an auxiliary conditioning product for obese patients to assist other lipid-lowering and weight-reducing medicines to play the role of lowering lipid and reducing weight.

By combining the experimental results of the above examples, the composite probiotic preparation prepared by the processes of mixed bacteria inoculation of composite bifidobacteria, liquid culture, centrifugal separation, solid culture and the like is found to have the functional effects of high strain content, rich probiotic metabolite, good anti-helicobacter pylori effect, blood sugar reduction, gastrointestinal conditioning effect, immunity enhancement effect, anti-allergy effect, lipid reduction and weight reduction effect and the like.

The above description is only for the specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. The multifunctional compound probiotic preparation is characterized by comprising the following components:

2. The multifunctional composite probiotic preparation according to claim 1, characterized in that it comprises the following components in parts by weight:

3. The multifunctional composite probiotic preparation according to claim 1, characterized in that it comprises the following components in parts by weight:

4. The multifunctional composite probiotic preparation according to claim 1, characterized in that the viable count of the composite probiotic preparation is (8-10) x10¹¹CFU/g。

5. The multifunctional composite probiotic preparation according to any one of claims 1 to 4, characterized in that the Bifidobacterium bifidum Miuyo-01, Bifidobacterium infantis Miuyo-21, Bifidobacterium breve Miuyo-31 and Bifidobacterium lactis Miuyo-11 are derived from infant feces and are obtained by separating and culturing from the infant feces; the isolated culture conditions of the bifidobacterium bifidum Miuyo-01, the bifidobacterium infantis Miuyo-21, the bifidobacterium breve Miuyo-31 and the bifidobacterium lactis Miuyo-11 are as follows: MRS culture medium, pH6.0-7.5, temperature 34-40 deg.C.

6. The multifunctional composite probiotic preparation according to any one of claims 1 to 4, characterized in that the Lactobacillus plantarum MiuCul-01 is obtained by isolated culture from kimchi; the lactobacillus acidophilus MiuLac-01 is obtained by separating and culturing fermented milk products; the lactobacillus casei MiaoTech-01 is obtained by separating and culturing from fermented dairy products; the lactobacillus rhamnosus MiaoTech-11 is obtained by separating and culturing a probiotic preparation; the streptococcus thermophilus MiaoPro-01 is obtained by separating and culturing from a yoghurt starter.

7. Method for preparing the multifunctional complex probiotic formulation according to any of claims 1 to 6, characterized in that it comprises the following steps:

(2) mixing the wet thalli of the compound probiotic preparation obtained in the step (1) with a solid culture medium, carrying out anaerobic culture for the second time in a sealed state, and carrying out low-temperature drying or freeze drying or vacuum drying after the culture is finished to form a compound probiotic preparation solid;

8. The preparation method of the multifunctional composite probiotic preparation according to claim 7, wherein in the step (1), a disc centrifuge or a tube centrifuge is adopted to perform centrifugal separation under aseptic conditions to obtain wet thalli of the composite probiotic preparation; the liquid culture medium comprises sodium acetate, peptone, yeast extract and tomato sauce; the temperature of the first anaerobic culture is 36-38 ℃, and the time is 48-72 hours.

9. The preparation method of the multifunctional composite probiotic preparation according to claim 7, wherein in the step (2), the solid culture medium comprises sodium acetate, soybean peptide, yeast extract and food-grade calcium carbonate, and the solid culture medium is subjected to high-temperature dry heat sterilization and aseptic cooling to below 38 ℃ for later use before use; and before the second anaerobic culture, the water content of the mixture of the wet thalli of the compound probiotic preparation and the solid culture medium is adjusted to 45-55 percent.

10. The preparation method of the multifunctional composite probiotic preparation according to claim 7, wherein in the step (2), the temperature of the second anaerobic culture is 36-38 ℃ and the time is 72-120 hours; the low-temperature drying or vacuum drying temperature is 30-40 ℃; the water content of the solid of the composite probiotic preparation is 4-6%.