CN113005066B

CN113005066B - Compound bifidobacterium preparation for resisting allergy, increasing immunity, reducing blood sugar and fat and losing weight and preparation method thereof

Info

Publication number: CN113005066B
Application number: CN202110347193.8A
Authority: CN
Inventors: 陈大伟; 童颖佳; 童群义; 陈金林; 吉峰
Original assignee: Jiangsu Lanze Biotechnology Co ltd
Current assignee: Jiangsu Lanze Biotechnology Co ltd
Priority date: 2021-03-31
Filing date: 2021-03-31
Publication date: 2023-07-21
Anticipated expiration: 2041-03-31
Also published as: CN113005066A

Abstract

The invention relates to a compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar, reducing blood fat and losing weight, a preparation method and application thereof, which are prepared by adopting novel processes of combination of bifidobacterium bifidum Miuyo-01, bifidobacterium infantis Miuyo-21, bifidobacterium breve Miuyo-31 and bifidobacterium lactis Miuyo-11, such as mixed bacteria inoculation, anaerobic liquid culture, solid-liquid separation, solid-state culture and the like, and the produced compound bifidobacterium preparation has large bacterial quantity and high activity and contains a plurality of metabolites such as alpha-glucosidase inhibitor, bacterial polysaccharide, peptidoglycan, lipoteichoic acid and the like produced by the compound bifidobacterium; has better antiallergic, immunity enhancing, blood sugar reducing, blood lipid reducing and weight reducing effects; liquid anaerobic culture is adopted in the preparation process, and the produced culture solution is subjected to centrifugal separation to obtain wet thalli of the composite bifidobacterium; mixing the wet thalli with a sterile solid culture medium, and performing a second solid anaerobic culture in a sealed state; and (5) drying at low temperature after the solid culture is finished to obtain the high-activity composite bifidobacterium solid.

Description

Compound bifidobacterium preparation for resisting allergy, increasing immunity, reducing blood sugar and fat and losing weight and preparation method thereof

Technical Field

The invention belongs to the technical field of probiotic preparations, and in particular relates to a compound bifidobacterium preparation for resisting allergy, increasing immunity, reducing blood sugar and fat and losing weight and a preparation method thereof.

Background

Allergy is a phenomenon in which an organism's susceptibility to some external stimulus (antigen) is abnormally increased, i.e. an abnormal, too high immune response, simply being oversensitive to some substance (antigen). The occurrence and development of allergic diseases are related to the immune system of the organism, wherein the type 2 immune response mechanism plays an important role in the allergic diseases, and can generate specific IgE antibodies aiming at allergens so as to lead the organism to be in a sensitized state. When the sensitized organism contacts the same allergen again, igE antibody adsorbed on the surfaces of the mast cells and the eosinophils is combined with the corresponding antigen to cause degranulation of the mast cells and release allergic mediums such as histamine and the like, so that allergic reaction is caused, the organism is subjected to tissue injury or dysfunction, common symptoms are abdominal pain, diarrhea and vomiting, or skin itching and difficult decoction are caused, and shock can be caused for more serious people. For people with allergic constitution, the method of avoiding contact with allergen is adopted to reduce the occurrence of allergy, but the effect is very weak, and the allergen is avoided, so that the allergic symptoms can be temporarily reduced, but the allergic symptoms can not be radically cured. Thus, methods of inducing immune tolerance have emerged, which, although somewhat viable, are difficult to fully desensitize under current hygienic conditions.

A common immune organ in humans is a lymphoid organ, and T cells expressing CD4 in the gonorrhea are called helper T cells (Th). T helper cells can be subdivided into two major parts, depending on their function, or on their response to different cytokines and their ability to secrete cytokines, i.e. T helper cells can be divided into two types (Th 1 and Th 2). In general, th1 is beneficial for enhancing cellular immunity, while Th2 is beneficial for enhancing humoral immunity, with a dynamic balance between Th1 and Th2 normally present. And the two can mutually balance: th1 immune response is vigorous, th2 activity and function is inhibited and greatly reduced, and vice versa. The main effect of Th1 is to resist the stimulation of various microbes outside, and the more the external infection is, the more the Th1 is stimulated by microbes, and the more the Th1 is activated, the more the Th2 immune response is inhibited. Or the following: stimulation of a Th1 type immune response reduces the Th2 type immune response that can lead to allergy. Studies have shown that allergic diseases are associated with T helper cell imbalance and Th2 is too strong to cause allergy. Thus, stimulating a Th1 type immune response and thus reducing a Th2 type immune response will reduce the incidence of allergic reactions. Allergic diseases have a significant trend to rise in the 80 s of the 20 th century, especially in western or mostly modern countries. Students find that long urban children in very clean and sanitary environments suffer from a much higher proportion of allergic diseases than long rural children in dirty environments, and thus find the relationship between the living environment of the person and the allergic diseases, thereby proposing a "sanitary hypothesis". "hygienic hypothesis" considers: too little exposure to pathogens such as bacteria and viruses during childhood results in insufficient stimulation of Th1 cells and thus failure to equilibrate with Th2 cells, ultimately leading to an allergic predisposition. From the "hygienic hypothesis" point of view, moderate microbial stimulation is considered necessary for the formation of normal immune functions and oral tolerance of the body, which is advantageous for inducing differentiation of Th0 cells into Th1 cells while suppressing Th2 cell responses, thereby playing an immune balance role. The more microorganisms that children are exposed to early, the lower the chance of developing allergic diseases later on. However, the presence of pathogenic microorganisms in the environment can cause serious epidemic diseases, and thus, there is a substantial risk of exposing children directly to the environmental microorganisms.

Probiotics are non-pathogenic microorganisms, but can stimulate the immune system of intestinal tracts, change the balance of Th1/Th2, enable immune response to be carried out towards the direction of Th1 cells, inhibit the reaction of Th2 cells and play a role in regulating immunity, thereby reducing the occurrence of allergic diseases. In addition, probiotics and their metabolites also have the effect of enhancing intestinal barrier function, reducing the penetration of food antigens through the intestinal mucosa, avoiding their exposure to the immune system. Most probiotics are nonpathogenic gram-positive bacteria, and the cell walls of the probiotics are mainly composed of Peptidoglycan (PG), bacterial Polysaccharide (PS) and lipoteichoic acid (LTA), and all have immunostimulating properties.

Diabetes is a series of metabolic disorder syndromes of sugar, protein, fat, water, electrolyte and the like caused by islet hypofunction, insulin resistance and the like due to the action of various pathogenic factors such as genetic factors, immune dysfunction, microbial infection, toxins thereof, free radical toxins, mental factors and the like on organisms. Diabetes is one of the most serious chronic diseases worldwide, and long-term hyperglycemia can lead to chronic complications in the human body, thereby causing chronic damage to blood vessels and nerves, and finally possibly affecting the heart, kidneys, eyes and nerves, and causing serious complications. The human body must hydrolyze the polysaccharide substances in the food into glucose by the intestinal key digestive enzymes (alpha-amylase and alpha-glucosidase) to be absorbed by the human body, so that inhibiting the alpha-glucosidase can reduce the glucose intake by the body. The alpha-glucosidase is an oligosaccharide hydrolase existing at the brush-shaped edge of the small intestine mucosa, and can hydrolyze oligosaccharide, maltose and the like into glucose, thereby promoting the absorption of 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 data reports: the lactobacillus can also produce some alpha-glucosidase inhibitors in the culture process, so that the probiotics can be taken to consume glucose through the growth and propagation of the probiotics in the intestinal tract, and the glucose absorption rate in the intestinal tract can be reduced through the metabolite (alpha-glucosidase inhibitor) produced in the culture process, so that the blood sugar of a human body is reduced.

Obesity refers to a state where the body is significantly overweight and fat layers are too thick, and fat tissue in the body accumulates excessively. The causes of obesity are mainly: excessive intake of food with high calories, hypoexercise, etc. Obesity is formed when the body's energy intake exceeds the energy consumption, causing the body's energy to be excessive and promoting fat accumulation; in addition, obesity can also be formed due to unbalanced nutrition of the body, and energy metabolism and disorder caused by diseases, environment, psychology, and excessive toxins of the body. Obesity affects not only body beauty, but also causes various diseases such as: hypertension, coronary heart disease, angina pectoris, cerebrovascular disease, diabetes, hyperlipidemia, hyperuricemia, female menoxenia, etc., and can increase probability of malignant tumor, and influence digestive system function and endocrine system function.

The sources of human fat are mainly two: one is fat that is directly ingested into food; the other is the conversion to fat in vivo using excess sugar. Fat in the food is decomposed into fatty acid, cholesterol and other fat digestion products through the action of various enzymes and bile acid salts in the small intestine, and then is absorbed into blood flow through the small intestine wall, so that the fat is digested and absorbed by a human body, and the fat can be possibly synthesized again in the human body; on the other hand, when the sugar is taken up by the human body in excess, the sugar (e.g., glucose, etc.) can also be converted into fat through the tricarboxylic acid cycle system. Therefore, inhibiting citrate lyase, a key enzyme of the tricarboxylic acid cycle system, can reduce the synthesis of human cell fat, thereby reducing the fat content of the human body. After the probiotics are colonised in the intestinal tract, substances such as lipid (such as triglyceride, cholesterol and the like), micromolecular sugar (such as glucose), amino acids or small peptide and the like in the intestinal tract are utilized to synthesize thallus components in the growth and reproduction process of the probiotics, and the probiotics participate in digestion, absorption and metabolism functions of nutrient substances, so that the effects of reducing body fat and cholesterol in the body are fully exerted, and the body fat rate, the weight and the food utilization rate are reduced, the metabolism is promoted, the obesity constitution is improved and the like. Several studies showed that: the probiotics can reduce the synthesis of fat and decompose redundant grease, so that the fat of a human body cannot be excessively accumulated.

The patent of the invention with the application number of 201780023391.0, namely bifidobacterium for reducing food, energy and/or fat intake, discloses bifidobacterium animalis with the functions of reducing fat and losing weight; the patent application 201680007471.2, "immunoregulatory composition comprising bifidobacteria" discloses a composition for regulating immunity; the invention patent with application number 202010455121.0 discloses an animal bifidobacterium capable of improving immunity, namely an animal bifidobacterium BZ11 composite microbial inoculum with the functions of improving immunity and regulating intestinal tracts; the invention patent application number 201510962576.0 discloses a bifidobacterium composition capable of enhancing immunity; the invention patent application number 201610280825.2 'application of bifidobacterium infantis in food or medicine for preventing and treating food allergy' discloses bifidobacterium infantis capable of preventing and treating allergy and application of bifidobacterium infantis in food or medicine; the invention patent with application number 202010699202.5 discloses an animal bifidobacterium NX-6 and application thereof in preparing lipid-lowering and weight-losing medicaments; 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 a plurality of auxiliary materials, and has the effect of improving the immunity of a human body. 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 production process of various probiotics powder in the probiotics production industry, liquid fermentation is almost adopted, moisture is removed through centrifugal separation or other solid-liquid separation methods after the liquid fermentation is completed to obtain wet thalli, and then a drying protective agent or a carrier is added for uniform mixing, freeze drying, crushing, granulating, coating, packaging and the like. In the solid-liquid separation process, substances with bacteriostasis such as bacteriocin and organic acid generated by probiotics in the liquid fermentation process are removed, and the bacteriostasis effect of the composite bacterial metabolites cannot be reflected in the product, so that the probiotic product produced by the method only utilizes the antiallergic and lipid-lowering and weight-losing effects of the probiotics, but fails to utilize the antiallergic and lipid-lowering and weight-losing effects of the bacteriostasis substances; in addition, the wet thalli obtained by separating from the liquid fermentation liquid has high moisture content, high thalli stress death rate during drying, and the quantity of thalli obtained by the liquid fermentation product after drying is reduced due to the addition of a proper amount of drying protective agent or carrier into the wet thalli.

The patent 201310187056.8 discloses a process for producing lactobacillus acidophilus by combining liquid fermentation and solid fermentation, namely, firstly, carrying out liquid fermentation, then mixing the liquid fermentation product serving as a strain with a solid culture medium, and then carrying out solid fermentation, wherein the product contains lactobacillus acidophilus viable bacteria and metabolites thereof, such as: bifidobacteria, n-butyric acid, acetic acid, formic acid, etc.; the invention patent application number 201510559188.8 discloses a method for culturing lactobacillus by solid-state fermentation, which is characterized in that: inoculating lactobacillus strain into culture medium according to inoculum size of 3-5% v/v, mixing, and sealing and fermenting at 30-37deg.C. The strains adopted in the two patents are liquid strains prepared by using a triangular flask or a small-sized fermentation tank, and can also be calculated as a process combining liquid culture and solid culture, namely, adding the liquid strains obtained by liquid culture into a solid culture medium, and carrying out solid fermentation after uniformly 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 fermentation broth reaches 50%, only a small amount of liquid strain (1-2.5%) can be added into the solid fermentation broth as strain for solid fermentation, so that the initial concentration of the strain in the solid fermentation broth is low, the solid fermentation time is long, and the viable concentration in the product is small.

In summary, the conventional actual production process of probiotics, namely the liquid culture and centrifugal separation process and the process of liquid culture and solid culture of the invention have the defects of low strain content, poor fermentation effect, high bacterial death rate during drying, low strain activity in finished products and the like.

For this reason, this patent is filed.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar, reducing blood fat and losing weight and a preparation method thereof, and the novel preparation process ensures that the product has large bacterial quantity and high activity, contains organic acid generated by strains during solid culture, bacteriocin citrate lyase inhibitor, alpha-glucosidase inhibitor and other metabolites with the functions of inhibiting bacteria, reducing blood sugar, reducing blood fat and reducing blood sugar, and can also generate blood sugar reducing; contains bacterial polysaccharide and other metabolites with the function of enhancing the immunity of human body; and metabolites with antiallergic effects such as Peptidoglycan (PG), bacterial Polysaccharide (PS) and lipoteichoic acid (LTA), and the like, can enhance the effects of the composite bifidobacterium preparation in various aspects such as anti-helicobacter pylori immunity enhancement, antiallergic, blood sugar reduction, gastrointestinal conditioning, immunity enhancement and the like when in use.

The invention aims to provide a compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar, reducing blood fat and losing weight.

Still another object of the present invention is to provide a method for preparing the above-mentioned compound bifidobacterium preparation for antiallergic, increasing immunity, reducing blood sugar, reducing blood lipid and losing weight.

The compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar and reducing fat and losing weight comprises the following components:

bifidobacterium bifidum Miuyo-01, bifidobacterium infantis Miuyo-21, bifidobacterium breve Miuyo-31 and bifidobacterium lactis Miuyo-11.

The compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar and reducing fat and losing weight comprises the following components in parts by weight:

10-15 parts of bifidobacterium bifidum Miuyo-01, 5-10 parts of bifidobacterium infantis Miuyo-21, 3-7 parts of bifidobacterium breve Miuyo-31 and 2-6 parts of bifidobacterium lactis Miuyo-11.

12-13 parts of bifidobacterium bifidum Miuyo-01, 7-8 parts of bifidobacterium infantis Miuyo-21, 4-6 parts of bifidobacterium breve Miuyo-31 and 3-5 parts of bifidobacterium lactis Miuyo-11.

12.5 parts of bifidobacterium bifidum Miuyo-01, 7.5 parts of bifidobacterium infantis Miuyo-21, 5 parts of bifidobacterium breve Miuyo-31 and 4 parts of bifidobacterium lactis Miuyo-11; preferably, the viable count of the compound bifidobacterium preparation is (4-5) multiplied by 1011CFU/g.

According to the compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar, reducing blood fat and losing weight, which is provided by the specific embodiment of the invention, further, bifidobacterium bifidum Miuyo-01, bifidobacterium infantis Miuyo-21, bifidobacterium breve Miuyo-31 and bifidobacterium lactis Miuyo-11 are all from infant feces, and are obtained by separating and culturing the infant feces.

The compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar, reducing blood fat and losing weight according to the specific embodiment of the invention is further characterized in that the isolated culture conditions of bifidobacterium bifidum Miuyo-01, bifidobacterium infantis Miuyo-21, bifidobacterium breve Miuyo-31 and bifidobacterium lactis Miuyo-11 are as follows: MRS medium; the pH is 6.0-7.5, and the temperature is 34-40 ℃.

A method for preparing a compound bifidobacterium preparation for antiallergic, immunity enhancing, blood sugar reducing, lipid lowering and weight losing according to a specific embodiment of the present invention comprises the steps of:

(1) Uniformly mixing bifidobacterium bifidum Miuyo-01, bifidobacterium infantis Miuyo-21, bifidobacterium breve Miuyo-31 and bifidobacterium lactis Miuyo-11 according to a proportion to form composite bifidobacterium, placing the composite bifidobacterium into a liquid culture medium for primary anaerobic culture, and obtaining composite bifidobacterium wet thalli after centrifugal separation of the generated culture solution for later use;

(2) Mixing the wet bacterial cells of the composite bifidobacterium obtained in the step (1) with a solid culture medium, performing anaerobic culture for the second time in a sealed state, and performing low-temperature drying or freeze drying or vacuum drying after the culture is completed to form a composite bifidobacterium solid;

(3) Crushing the composite bifidobacterium solid obtained in the step (2), carrying out anaerobic granulation and anaerobic coating to obtain the composite bifidobacterium preparation for resisting allergy, increasing immunity, reducing blood sugar and blood fat and losing weight.

According to the preparation method of the compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar, reducing blood fat and losing weight, in the step (1), a dish type centrifuge or a tube type centrifuge is adopted for centrifugal separation under the aseptic condition to obtain the compound bifidobacterium wet thalli.

Further, the liquid medium comprises oligosaccharide, peptone, yeast extract and tomato sauce.

Further, the temperature of the first anaerobic culture is 36-38 ℃ and the time is 48-72 hours.

According to the preparation method of the compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar, reducing blood fat and losing weight, in the step (2), the solid culture medium comprises oligosaccharide, soybean peptide, yeast extract and food-grade calcium carbonate, and is subjected to high-temperature dry heat sterilization before use and is subjected to aseptic cooling to below 38 ℃ for standby; the water content of the mixture of the wet bacterial cells of the composite bifidobacterium and the solid culture medium is adjusted to 45-55% before the second anaerobic culture.

According to the preparation method of the compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar and reducing fat and losing weight, 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 temperature is 30-40 ℃.

Further, the water content of the composite bifidobacterium solid is 4-6%.

The invention does not separate the strain or metabolite after the solid culture is completed, but directly dries the cultured strain and metabolite at low temperature (freeze drying or vacuum drying) together to obtain the high-activity compound bifidobacterium solid. Therefore, the product also contains organic acid, bacteriocin and other metabolites with antibacterial capability generated by the strain during solid state culture, can also generate metabolites for reducing blood sugar and increasing immunity, and can enhance the various functional effects of the composite bifidobacterium preparation such as antiallergic, blood sugar reducing, lipid reducing, weight losing, immunity increasing and the like during use.

The compound bifidobacterium preparation used in the invention comprises a plurality of bifidobacteria with similar culture conditions, and the bifidobacteria have antiallergic function in the growth process in intestinal tracts. Through the synergistic effect of multiple strains, the synergistic antiallergic effect among the strains can be improved.

The bifidobacterium strain used in the invention is characterized by the following:

bifidobacteria are gram-positive, motionless, rod-shaped cells, sometimes bifurcated at one end, strictly anaerobic bacteria that are widely found in the digestive tract, vagina, and mouth of humans and animals. Bacteria of the genus bifidobacterium are one of the important constituent members of the intestinal flora of humans and animals. Some strains of bifidobacteria may be used as probiotics in food, pharmaceutical and feed applications. Bifidobacteria are an important intestinal beneficial microorganism. The bifidobacterium is used as a physiologically beneficial bacterium, and has various important physiological functions such as biological barrier, nutrition effect, anti-tumor effect, lipid-lowering and weight-losing effect, immunity enhancing effect, gastrointestinal tract function improvement, allergy resistance, aging resistance and the like for human health. The bifidobacterium complex used in the present invention includes several bifidobacteria having similar culture conditions, for example: bifidobacterium bifidum, bifidobacterium lactis, bifidobacterium infantis, bifidobacterium breve, and the like. The mixture of the 4 bifidobacteria can be obtained by simultaneous culture through mixed bacteria inoculation and mixed bacteria culture of the bifidobacteria, and the obtained compound bifidobacteria preparation product has various effects of reducing fat and reducing blood sugar, enhancing immunity, resisting allergy and the like through the synergistic effect of the bifidobacteria.

Immunity is the body's own defense mechanism, which is the body's ability to recognize and eliminate any foreign bodies (viruses, bacteria, etc.) that invade, treat aging, injury, death, degenerated own cells, and recognize and treat mutant cells and virus-infected cells in the body. When the immune function of human body is disregulated or the immune system is not healthy, the human body diseases are easy to be caused, especially the repeated attacks of cold, tonsillitis, asthma, bronchitis, pneumonia, diarrhea and the like are caused. Medical research has shown that: the intestinal tract is the most densely concentrated part of the human body microorganisms and is also the largest immune organ of the human body. 60% of the immune cells of the human body are positioned on intestinal mucosa, and intestinal flora can promote the maturation of intestinal lymphoid tissues and the establishment of immune homeostasis, and plays an important role in immunoregulation, namely: maintaining the microecological balance and intestinal flora health are important factors in increasing the immunocompetence. The probiotics are beneficial microorganisms which can normally grow and reproduce in the gastrointestinal tract, and the supplementing probiotics are beneficial to restoring intestinal microecological balance, repairing intestinal bacterial membrane barrier, regulating systemic immune function and the like, and are effective measures for enhancing resistance and resisting viruses.

More methods for measuring immunity are available, for example: NK cell activity assay, cellular immune function assay, humoral immune function assay, mononuclear-macrophage function assay, and the like. The invention selects two methods of NK cell activity measurement or cell immunity function measurement to measure immunity function. The results show that the compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar and reducing blood fat and losing weight has better effects of reducing blood sugar and enhancing immunity.

The bifidobacterium bifidum Miuyo-01 (Latin name: bifidobacterium bifidum), the bifidobacterium lactis Miuyo-11 (Latin name: bifidobacterium lactis), the bifidobacterium infantis Miuyo-21 (Latin name: bifidobacterium infantis) and the bifidobacterium breve Miuyo-31 (Latin name: bifidobacterium breve) are stored in the North Chen West road No. 1, no. 3 in the North YangKorea of Beijing city on the 12 th month of 2020, and the storage numbers are CGMCC 20866, CGMCC 20867, CGMCC 20868 and CGMCC 20869 respectively.

The invention adopts the novel processes of mixed bacteria inoculation, anaerobic liquid culture, solid-liquid separation and solid culture to culture the composite bifidobacterium thallus, which can strengthen the effects of antiallergic, immunity increasing, blood sugar reducing, lipid reducing and weight losing: the composite bifidobacterium thallus with higher activity is obtained, meanwhile, bacteriocin, organic acid and other antibacterial substances generated in the solid state culture process can be reserved, helicobacter pylori and bacteria causing intestinal tract infection can be inhibited or killed, and the composite bifidobacterium 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 bifidobacterium during the drying period can be reduced, and the activity of the composite bifidobacterium in the product can be greatly improved by combining proper anaerobic granulation and anaerobic coating procedures. The composite bifidobacterium preparation produced by comprehensively adopting the measures can increase the functional effects of the product in various aspects such as antiallergic, blood sugar reducing, lipid reducing and weight losing, immunity increasing and the like.

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

(1) The compound bifidobacterium preparation obtained by the invention has large bacterial quantity and high activity, and contains metabolic products generated by the compound bifidobacterium; the produced compound bifidobacterium preparation has better antiallergic and lipid-lowering weight-losing effects in human gastrointestinal tracts;

(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 bifidobacterium; mixing the wet thalli with a sterile solid culture medium, and performing a second solid anaerobic culture in a sealed state; and (5) drying at low temperature after the solid culture is finished to obtain the high-activity composite bifidobacterium solid.

(3) The composite bifidobacterium preparation bacteria obtained by the invention have the following advantages:

(1) high viable count

Inoculating a plurality of bifidobacteria, mixing and anaerobically culturing, and carrying out anaerobic solid-state culture on the wet thalli centrifugally separated after liquid anaerobic culture in a solid-state culture medium for a period of time, so that the quantity of the thalli is greatly increased, and the activity is also obviously increased;

(2) the drying stress is small, and the strain drying loss is small

The water content of the solid culture is only 45-55%, which is far lower than that of wet thallus obtained by liquid culture, so that the damage and death rate caused by the dry solid culture are far lower than those of the liquid culture, and the viable count of the product obtained by the invention can reach 10 ¹¹ cfu/g or more;

(3) the product contains various metabolites produced in the solid culture process of the strain

In the solid culture process of the strain, various metabolites such as alpha-glucosidase inhibitor, bacterial polysaccharide, peptidoglycan, lipoteichoic acid and the like can be produced. Compared with the composite bifidobacterium preparation prepared by the traditional method, the composite bifidobacterium preparation containing the metabolite has better antiallergic, immunity enhancing, blood sugar reducing, lipid reducing and weight losing effects in human gastrointestinal tracts.

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 will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.

In some more specific embodiments, the antiallergic, immunity-enhancing, blood sugar-reducing, lipid-lowering and weight-losing compound bifidobacterium preparation comprises the following components in parts by weight: 10-15 parts of bifidobacterium bifidum Miuyo-01, 5-10 parts of bifidobacterium infantis Miuyo-21, 3-7 parts of bifidobacterium breve Miuyo-31 and 2-6 parts of bifidobacterium lactis Miuyo-11.

In other specific embodiments, the antiallergic, immunity-enhancing, blood glucose-reducing, lipid-lowering and weight-losing compound bifidobacterium preparation comprises the following components in parts by weight:

12-13 parts of bifidobacterium bifidum Miuyo-01, 7-8 parts of bifidobacterium infantis Miuyo-21, 4-6 parts of bifidobacterium breve Miuyo-31 and 3-5 parts of bifidobacterium lactis Miuyo-11;

the preparation method comprises the following steps:

(2) Mixing the wet bacterial cells of the composite bifidobacterium obtained in the step (1) with a solid culture medium, performing anaerobic culture for the second time in a sealed state, and drying at a low temperature after the culture is completed to form a composite bifidobacterium solid;

In the step (1), a disc centrifuge or a tube centrifuge is adopted to carry out centrifugal separation under the aseptic condition to obtain the composite bifidobacterium wet thalli; the liquid culture medium comprises oligosaccharide, 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 oligosaccharide, 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 standby before use; before the second anaerobic culture, the moisture content of the mixture of the wet bacterial cells of the composite bifidobacterium and the solid culture medium is regulated to 45% -55%; 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 temperature is 30-40 ℃; the water content of the composite bifidobacterium solid is 4-6%.

The following are more specific examples:

example 1 isolation and characterization of Strain

1.1 sample collection:

healthy infants, which have not recently used antibacterial drugs 3 months to 1 year after birth, are selected, and samples are collected from their faeces for isolation, i.e. the isolated strain is from the intestinal tract of the healthy infant.

1.2 isolation of strains

Taking infant feces sample 5-10g, placing into a sterile 50ml centrifuge tube, adding 20ml sterile physiological saline, shaking, mixing, placing into an anaerobic incubator at 37deg.C, standing for culturing overnight, sucking 1ml sample solution, sequentially diluting with 10 times of sterile physiological saline to 10 times ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 Each gradient is respectively coated with 100 mu l of bacterial suspension on an improved MRS flat plate, placed in an anaerobic incubator at 37 ℃ for inversion culture, colonies are selected from coated culture flat plates with different gradients for transfer culture after 24 hours, on the basis, colonies conforming to the typical characteristics of bifidobacteria are selected, repeated streak culture separation is carried out on the improved MRS flat plate, and single colonies conforming to the growth form of the bifidobacteria are selected and inoculated into an improved MRS liquid culture medium for culture in the anaerobic incubator at 37 ℃. Finally obtaining 4 strains with good growth performance.

1.3 identification of strains

1.3.1 colony morphology and physiological Biochemical Experimental analysis

Colony morphology and cell morphology observation experiments, microbial biochemical analysis experiments, and the like were performed by the methods of references (1) and (2):

(1) Qin Lingling separation and culture of bifidobacteria, zizihaer university journal 2008 (3);

(2) Zhao Xiaoxiao separation of bifidobacteria in infant feces and research on strain characteristics thereof, industrial microorganisms 2019 (2).

The experimental results are shown in tables 1, 2 and 3:

TABLE 1 morphological characterization of colonies and cells

The bifidobacterium is obligately anaerobic, and the optimal growth temperature is 37-41 ℃, and the bifidobacterium can also grow at 25-28 ℃ or 43-45 ℃. The optimal growth pH value is 6.7-7.0, and the growth is not carried out at pH5.0 or above 8.0. The drug sensitivity test is sensitive to various antibiotics such as chloramphenicol, tetracycline, erythromycin, etc., but is resistant to antibiotics such as ampicillin, gentamicin, furazolidone, amikacin, neomycin, ciprofloxacin, etc.

TABLE 2 Biochemical analysis results of strains

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

TABLE 3 saccharide fermentation test

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

Most of the fermentation products can ferment glucose, sucrose, fructose, maltose, lactose and the like; raffinose, xylose, trehalose, cellobiose, sorbitol, mannitol, etc. cannot be fermented. Acid and no gas are produced when glucose is fermented.

1.3.2 16S rRNA partial sequence analysis

Genomic DNA of the selected strain was amplified by PCR and detected by gel electrophoresis using 1% agarose. PCR amplification was performed using bacterial universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R (5 'GGTTACCTTGTTCGACTT-3'), and after purification, the sequence was sequenced, and strain identification was performed.

And (3) combining the results of colony morphology analysis, physiological and biochemical experiment analysis and 16S rRNA partial sequence analysis, and respectively identifying 4 strains obtained by separation and purification of the invention as follows: bifidobacterium bifidum, bifidobacterium lactis, bifidobacterium infantis, bifidobacterium breve and named respectively: "Bifidobacterium bifidum Miuyo-01", "Bifidobacterium lactis Miuyo-11", "Bifidobacterium infantis Miuyo-21", "Bifidobacterium breve Miuyo-31".

Example 2 Properties of Strain

2.1 method for measuring the number of viable bacteria in a product

After dilution with sterile physiological saline to a certain multiple, the total number of colonies was counted after culturing for 48 hours at 37℃by using a modified MRS agar medium plate pouring method.

2.2 growth at different pH

Inoculating the activated 5 strains into an improved MRS liquid culture medium, culturing to obtain strain seed liquid, inoculating 1mL of seed liquid into 19mL of improved MRS liquid culture medium with pH of 6.0, 6.5, 7.0 and 7.5 respectively, performing anaerobic culture at 37 ℃ for 24 hours, determining the OD600 value (namely the absorbance value at 600nm wavelength, which can be used for comparing the cell density or the cell growth condition in the culture solution) after the initial and the end, and calculating the cell growth condition in different pH culture media according to the following formula by taking the OD600 value of pH7.0 as a reference (namely the number of cultured cells of pH7.0 is 100 percent):

Cell growth (%) = (OD of other pH culture)/OD of pH7.0 culture×100%.

The results are shown in Table 4:

TABLE 4 growth of various species in different pH Medium

As shown in Table 4, the bacterial solutions obtained by culturing in the media of pH6.0, 6.5 and 7.5 were somewhat lower than the OD value of the bacterial solution cultured in the media of pH7.0, but still had a higher growth rate than the control group without culturing. Therefore, each strain still has certain growth capacity under the pH environments of pH6.0 and 7.5.

Each strain cultured at pH7.0 was subjected to centrifugation, washed 2 times with Phosphate Buffer (PBS), and resuspended in PBS. 1mL of the heavy suspension was mixed with 9mL of artificial gastric juice at pH2.0, 2.5 and 3.0, and treated at 37℃for 3 hours, and then the total number of bifidobacteria complex before and after the treatment was measured, respectively.

The number of bacteria not treated by artificial gastric juice is used as a control, and the survival rate before and after the artificial gastric juice treatment is calculated according to the following formula:

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 values

As shown in Table 5, the cells cultured at pH7.0 had a survival rate of 30 to 40% and a low survival rate after artificial gastric juice treatment at different pH values. Thus, it is often necessary to take the form of a coating to gain entry into the gut in the form of a living bacterium under normal eating conditions.

Each strain grows well under the environment with pH value between 6.0 and 7.5, and the optimal pH value is 7.0. The survival rate of the bacterial cells obtained by culturing at pH7.0 in artificial gastric juice at pH2.0 for 3h is lower than 40%.

2.3 growth at different temperatures

Inoculating each strain after activation into an improved MRS liquid culture medium, culturing to obtain strain seed liquid, inoculating 1mL of each strain seed liquid into 19mL of the improved MRS liquid culture medium with pH of 7.0, performing anaerobic culture at 31 ℃, 34 ℃, 37 ℃ and 40 ℃ for 24 hours, and determining OD600 values after initiation and termination.

The cell growth at different temperatures was calculated by the following formula, with reference to the OD600 value at the optimum growth temperature (i.e., with 100% of the number of cells cultured at the maximum growth rate):

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

According to the experiment, the optimum growth temperature of each bifidobacterium is 37 ℃, and the growth conditions 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:

the optimal growth temperature of each bifidobacterium is 37 ℃, and the bifidobacterium has the optimal growth temperature similar to the human body temperature. The growth is good under the temperature environment of 31-40 ℃, and the heat resistance is good;

To sum up: the above 5 strains separated and purified by the invention grow well at pH7.0, and still have a certain growth speed between pH6.0 and 7.5; the optimal growth temperature is 37 ℃ and a certain growth speed is still maintained between 31 ℃ and 40 ℃.

2.4 functional Effect of composite Strain

The composite strain of the invention is composed of 4 bifidobacteria such as bifidobacterium bifidum Miuyo-01, bifidobacterium lactis Miuyo-11, bifidobacterium infantis Miuyo-21, bifidobacterium breve Miuyo-31 and the like, thus the composite strain also has the functional effects of various bifidobacteria. The invention confirms the functional effect of the composite strain through various functional experiments.

2.4.1 antiallergic Effect of bifidobacterium Complex

The antiallergic effect of bifidobacterium complex was determined by the mouse test, by the method of reference (Liu Zhigang et al, balb/c mouse peanut allergy model establishment and pathogenesis, shenzhen university journal of academy of technology, 2012 (2)).

2.4.2 effects of Compound bifidobacteria on increasing immunity

The method of the reference (Fan Zhiyun, etc. the functional research of the scorpion health care wine for enhancing the immunity, the food research and development, 2017 (19)) adopts the method of human body test to confirm the functional effect of the compound bifidobacterium for enhancing the immunity.

2.4.3 hypoglycemic Effect of Compound bifidobacteria

The method of Zhu Yunping (Zhu Yunping et al. Screening of strain producing alpha-glucosidase inhibitor and optimization of conditions for solid state fermentation. Food industry science and technology. 2014 (3)), the alpha-glucosidase inhibition rate was measured, and the effect of lowering blood sugar was confirmed by using a human body test.

2.4.4 lipid-lowering and weight-reducing effects of composite bifidobacteria

The method of the reference (Li Jianxin, et al, apple polyphenol weight-reducing and lipid-lowering effect research, food science, 2008 (8)), and the lipid-lowering and lipid-lowering effects were measured by using a mouse test.

The experimental results are shown in table 7:

TABLE 7 efficacy experiments on the individual strains and results (see examples 7, 8, 9, 10 for specific data)

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

3.1 Medium

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

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 K2HP04, 2g/L of diammonium citrate, 5g/L of sodium acetate, 80 g/L of Tween-80, 04.7H200.5g/L of MgS, 04.4H20.05 g/L of MnS and 0.5g/L of L-cysteine hydrochloride.

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

(2) Improved MRS liquid medium (for delta flask culture and fermenter 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, 2g/L of K2HP04, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 80 g/L of Tween-80, 0.5g/L of MgS04.7H20, 0.05g/L of MnS 04.4H20.5 g/L of L-cysteine hydrochloride.

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

3.2 preparation method of composite bifidobacterium by liquid method

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

(1) Strain culture

Slant culture or plate culture of composite bifidobacterium strain is performed using the improved MRS solid culture medium, and the strain comprises:

4 bifidobacterium bifidum Miuyo-01, bifidobacterium lactis Miuyo-11, bifidobacterium infantis Miuyo-21, bifidobacterium breve Miuyo-31 and the like.

(2) Triangular flask culture or strain tank culture:

inoculating each strain obtained by slant or plate culture into a triangle bottle modified MRS liquid culture medium, and culturing in an anaerobic incubator at 37deg.C for 24 hr to obtain triangle bottle liquid strain.

(3) Liquid culture:

inoculating the 5 strains according to the ratio of a certain strain liquid to a liquid culture medium=1:100, and fully inoculating the strains into a fermentation tank, wherein the total inoculum size is 5:100, and performing anaerobic liquid culture for 24 hours at 37 ℃ in the fermentation tank to obtain a bacterial culture liquid.

(4) And (3) centrifugal separation:

separating the liquid culture solution with a tube centrifuge or a disk centrifuge at a rotation speed of 10000r/min for 20min to obtain wet thallus with water content of 80%.

(5) Drying

Freeze-drying wet thalli in a freeze dryer at-40 ℃ for 20 hours to obtain liquid-state composite bifidobacterium raw powder; the measurement is as follows: the total bacterial count of the composite bifidobacterium raw powder product prepared by the invention is 2.89 multiplied by 10 ¹¹ CFU/g。

(6) Granulating, coating and packaging.

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

the measurement is as follows: the total bacterial count of the composite bifidobacterium product prepared in the example is 2.18 multiplied by 10 ¹¹ CFU/g。

EXAMPLE 4 solid state method of preparation of composite Bifidobacterium raw powder and preparation thereof (solid state method 1)

4.1 Medium

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

20g/L of glucose, 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract, 15g/L, K2HP04 g/L of agar, 2g/L of diammonium citrate, 5g/L, tween-80 g/L, mgS 04.7H200.5 g/L, mnS 04.4H20.05 g/L-cysteine hydrochloride, and 0.5 g/L-cysteine hydrochloride. Adjusting pH to 7.0 with 1mol/L NaOH, and sterilizing at 121deg.C for 20min.

(2) Improved MRS liquid medium (for delta flask culture and fermenter culture):

20g/L of glucose, 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract, 10g/L of whey powder, 10g/L, K HP04 2g/L of tomato sauce, 2g/L of diammonium hydrogen citrate, 5g/L, tween-801g/L, mgS 04.7H20.5 g/L, mnS 04.4H20.05 g/L of sodium acetate and 0.5 g/L-cysteine hydrochloride. Adjusting pH to 7.0 with 1mol/L NaOH, and sterilizing at 121deg.C for 20min.

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

the solid-state culture materials were prepared according to the following formulation ratio:

100g of wet thalli, 20g of glucose, 10g of peptone powder, 10g of yeast extract powder, 10g of whey powder, 10g of tomato powder, 2g of K2HP04, 0.5g/L of L-cysteine hydrochloride and 2g of food grade calcium carbonate.

Except for wet bacteria, the other raw materials are sterilized respectively (sterilized respectively by high-temperature instantaneous sterilization equipment at 150 ℃ for 10 s), and are mixed uniformly after cooling, and are used as raw materials of a solid culture medium for standby.

4.2 preparation method of microbial inoculum

The preparation method of the composite bifidobacterium bacterial agent comprises the following steps:

(1) Culturing strains:

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

(2) Triangular flask culture or strain tank culture:

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

(3) Liquid culture:

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

(4) And (3) centrifugal separation:

separating the liquid culture solution by a tube centrifuge or a disc centrifuge, wherein the rotation speed of the centrifuge is 10000r/min, the centrifugation time is 20min, and wet thalli with the moisture content of 80 percent is obtained; immediately transferring wet thalli into a solid culture medium for culture;

(5) Solid state culture:

mixing the wet thalli with a sterile solid culture medium in a sterile room according to the formula:

100g of wet thalli, 20g of glucose, 10g of peptone powder, 10g of yeast extract powder, 10g of whey powder, 10g of tomato powder, 2g of K2HP04, 0.5g/L of L-cysteine hydrochloride, 2g of food grade calcium carbonate and controlling the moisture content of a solid culture medium to be 50% -55%. Then placing the mixture in an anaerobic solid state fermentation tank, and sealing the anaerobic solid state fermentation tank at 37 ℃ for 72 hours.

(6) And (3) drying:

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

The measurement is as follows: the total bacterial count of the composite bifidobacterium raw powder product prepared by the invention is 4.13 multiplied by 10 ¹¹ CFU/g。

(7) Granulating, coating and packaging.

Granulating and coating the composite bifidobacterium raw powder to obtain a composite bifidobacterium preparation product.

The measurement is as follows: the total bacterial count of the composite bifidobacterium preparation product prepared in the example is 3.25 multiplied by 10 ¹¹ CFU/g。

EXAMPLE 5 preparation of Compound Bifidobacterium by solid State method (solid State method 2)

The process operation of example 5 is essentially the same as that of example 4, with only a portion of the operating parameters being modified to demonstrate the effect of producing a solid state culture bifidobacterium complex product under different operating conditions.

5.1 Medium

The solid medium of example 4 was not supplemented with whey powder, otherwise the same as in example 4.

5.2 preparation method of microbial inoculum

Example 5 the moisture content of the solid medium was controlled to 52%, and anaerobic solid-state culture was performed at 37℃for 96 hours under sealing. The solid culture was dried by freeze-drying, and the other steps were the same as in example 4, to prepare a raw powder product and a formulated product, respectively.

The measurement is as follows: the total bacterial count of the composite bifidobacterium raw powder product prepared in the embodiment is 4.92 multiplied by 10 ¹¹ CFU/g; making it into a productThe total bacterial count of the preparation product obtained by the preparation is 4.38X10 ¹¹ CFU/g。

EXAMPLE 6 Compound Bifidobacterium preparation prepared by solid State method (solid State method 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 the triangular flask culture, the liquid culture and the solid culture is 37 ℃, the moisture content of the solid culture medium is controlled to be 55%, the anaerobic solid culture is sealed for 120 hours at 37 ℃, and other parameters are the same as those of the example 4, so that the raw powder product and the preparation product are respectively prepared.

The measurement is as follows: the total bacterial count of the composite bifidobacterium raw powder product prepared in the embodiment is 4.67 multiplied by 10 ¹¹ CFU/g; the total bacterial count of the preparation product obtained by preparing the microbial inoculum into the preparation is 4.05X10 ¹¹ CFU/g。

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

EXAMPLE 7 anti-sensitization Effect of Bifidobacterium Complex

The antiallergic effect of bifidobacterium complex was determined by the mouse test, by the method of reference (Liu Zhigang et al, establishment of a peanut allergy model in Balb/c mice, pathogenesis, university of Shenzhen journal of academy of technology, 2012 (2)), as follows:

7.1 Experimental methods

1. Experimental materials:

test animals: SPF-class Balb/c female mice were selected and 5-6 weeks old (body weight 18-20 g) and were randomly divided into 8 groups of 15 animals each.

Crude peanut protein (crude peanut extract protein, CPE) was prepared by literature.

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

2. The test method comprises the following steps:

1. dose and grouping:

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

2. Sensitization and challenge of 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 and is not sensitized; all other mice were sensitized by injection with a crude peanut protein solution, and then allergic mice were selected and randomly grouped into 10 mice each, and the excess mice were discarded. The allergic mice were subjected to intragastric treatment with the compound bifidobacterium solution daily according to the samples and doses of table 6, and the negative control group and the positive control group were perfused with sterile purified water for 10 days.

Balb/c mice are divided into a blank control group and a peanut sensitization model group (a model group for short, a positive control group), and each group comprises 10 sensitization modes: the model group was sensitized twice by subcutaneous injection, 3 weeks apart, with 100 μg of crude peanut protein and 1mg of aluminum hydroxide adjuvant per injection; after 1 week of immunization 2, 200 μg CPE was injected subcutaneously for challenge, and the PBS control group replaced the peanut crude protein with the same volume of PBS solution, the others being identical to the model group.

3. Detection of total IgE antibodies in serum:

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

7.2 comparison of antiallergic effects of the products at the same weight concentration

1g of each group of samples is weighed and added with 10mL of sterile water to prepare 0.1g/mL of compound bifidobacterium solution for later use.

The content of total IgE antibodies in serum was measured, and the measurement results were expressed as "mean ± standard deviation". In the experiment, SPSS 24.0 software is adopted to carry out single-factor analysis of variance (ANOVA) on experimental data, an LSD method is adopted to carry out multiple comparison, P <0.05 is used as a difference significance judgment standard, and the result is shown in Table 8:

TABLE 8 antiallergic effects of Complex Bifidobacterium products produced by different technologies at the same weight concentration

IgE-mediated allergic reactions are the main cause of allergy, and IgE antibodies in serum are key contributing factors to the development of allergic reactions. From the total IgE antibody content in the serum of mice in the blank group and the model group, it can be seen that the change of the IgE of the mice corresponds to the allergic symptoms, i.e. the serum IgE antibody content of the blank mice is lower, while the serum IgE antibody content of the mice in the model group is higher. After the gastric lavage treatment of the compound bifidobacteria, each compound bifidobacteria treatment group shows good allergy treatment effect.

When the preparation contains the same weight concentration of the compound bifidobacterium, the IgE values of the microbial inoculum prepared by the compound bifidobacterium examples 3, 4, 5 and 6 are larger than those of the blank control group, and the difference is obvious; the antiallergic effect of the microbial agents prepared by the solid state method (examples 4, 5 and 6) is better than that of the microbial agents prepared by the liquid state method (example 3), and the antiallergic effect of the microbial agents prepared by the solid state method has very remarkable difference. Thus, the complex bifidobacterium formulations produced by examples 4, 5, 6 prepared by the solid state method have a particularly remarkable effect on reducing allergic symptoms in mice.

Compared with the original powder of the compound bifidobacterium, the antiallergic effect of the compound bifidobacterium preparation product is slightly poorer, the reason is probably that the compound bifidobacterium can be slightly lost in the preparation process, but the preparation product is more convenient for people to take.

7.3 comparison of antiallergic effects of products with the same bacterial count

Respectively weighing and compoundingAdding a proper amount of sterile water into 1g of the finished bifidobacterium product, and after the bacterial count is determined, adding the sterile water to adjust the total concentration of the composite bifidobacterium to be 1x10 ¹⁰ CFU/ml, 10ml each time, so the total number of composite bifidobacteria per time is 10x10 ¹⁰ CFU, i.e. 1X10 ¹¹ CFU。

The antiallergic test method is shown in 7.1.

The content of total IgE antibodies in serum was measured, and the measurement results were expressed as "mean ± standard deviation". In the experiment, SPSS 24.0 software is adopted to carry out single-factor analysis of variance (ANOVA) on experimental data, an LSD method is adopted to carry out multiple comparison, P <0.05 is used as a difference significance judgment standard, and the result is shown in Table 9:

TABLE 9 antiallergic Effect of Complex Bifidobacterium products at the same bacterial count

As shown in table 9, when the preparation contains the same bacterial count, the IgE values of the bacterial agents prepared by the bifidobacterium complex examples 3, 4, 5 and 6 are larger than those of the blank control group, and the difference is obvious; the antiallergic effect of the microbial agents prepared by the solid state method (examples 4, 5 and 6) is better than that of the microbial agents prepared by the liquid state method (example 3), and the antiallergic effect of the microbial agents prepared by the solid state method has very remarkable difference. Thus, the complex bifidobacterium formulations produced by examples 4, 5, 6 prepared by the solid state method have a particularly remarkable effect on reducing allergic symptoms in mice. Compared with the composite bifidobacterium raw powder product, the composite bifidobacterium preparation product has slightly poorer antiallergic effect.

Antiallergic effect of Complex bifidobacteria, if only the effect of live bacteria, should have approximately the same antiallergic effect when the same number of bacteria is used, but the results of Table 9 show that: compared with the compound bifidobacterium microbial inoculum prepared by a liquid method, the compound bifidobacterium microbial inoculum prepared by the solid method in examples 4, 5 and 6 has larger IgE value, which shows that the antiallergic effect is more obvious. In the liquid state method, the metabolic products generated in the liquid state culture process are separated by centrifugation, so that the antiallergic effect is mainly derived from the strain; besides the compound bifidobacterium, the microbial inoculum prepared by the invention also contains metabolites such as bacteriocin, organic acid and the like generated in the solid state culture process, and the compound bifidobacterium and the metabolites are synergistic, so that the whole antiallergic effect of the microbial inoculum is improved.

Example 8: contrast of immunity-increasing effects of composite bifidobacteria

8.1 Effect of Complex Bifidobacterium preparation on NK cell Activity

8.1.1 Experimental methods

1. Experimental materials:

test animals: SPF-grade Kunming female mice were selected and 6-8 weeks old (body weight 18-22 g) and were randomly divided into 8 groups of 15 animals each.

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

2. The test method comprises the following steps:

1. dose and grouping:

the dosages of each group of mice were 0.1g/ml in each of the negative control group (pure water), the liquid control group (example 3), and the solid state test groups according to the present invention, examples 4, 5, and 6. All mice were taken for 30 days 2 times daily.

2. The feeding method comprises the following steps:

in the experiment, samples are respectively taken and prepared into 20ml of sample liquid by purified water, and the sample liquid is respectively used for animal stomach irrigation. Animals were subjected to intragastric administration for 30 days on a daily basis according to the samples and doses of table 4, and the control group was administered sterile purified water, at random, after 3 days of adaptation under laboratory conditions.

3. Preparation of effector cells (mouse spleen cells):

after the feeding experiment of the compound bifidobacterium preparation is finished, the mice in each dosage group are aseptically taken out of the spleen, placed in a small plate containing a proper amount of asepsis Hank's liquid, and lightly grinded by forceps to prepare single cell suspension. The spleen was either ground with a 200 mesh screen or with 4 layers of gauze or washed 2 times with Hank's solution and centrifuged for 10min (1000 r/min) each. The supernatant was discarded to bounce the cell slurry, and 0.5mL of sterilized water was added for 20 seconds to split After erythrocyte lysis, 0.5mL of 2-fold Hank's solution and 8mL of Hank's solution are added, centrifugation is performed at 1000rpm for 10min, 1mL of RPMI1640 complete culture solution containing 10% calf serum is used for resuspension, 1% glacial acetic acid is used for dilution and counting (the number of living cells is more than 95%), the number of living cells is counted by using the dyeing of the typhoid blue (more than 95%), and finally the cell concentration is adjusted to be 2 multiplied by 10 by using the RPMI1640 complete culture solution ⁷ And each mL.

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

Target cells were subcultured 24h prior to the experiment. Washing with Hank's solution 3 times before application, and adjusting cell concentration to 4×10 with RPMI1640 complete culture solution ⁵ And each mL.

5. NK cell Activity assay (LDH method)

The concentration is 4 multiplied by 10 ⁵ 100 μl (effective target ratio 50:1) of each of target cells (YAC-1) and effector cells (mouse spleen cells) per ml, adding into a U-shaped 96-well culture plate, naturally releasing Kong Jiaba cells and 100 μl of culture solution from each of target cells, and maximally releasing Kong Jiaba cells and 100 μl of 1% NP40 from each of target cells; each of the above was subjected to 3 multiplex wells, cultured in a 5% CO2 incubator at 37℃for 4 hours, and then the 96-well plates were centrifuged at 1500r/min for 5 minutes, 100. Mu.l of the supernatant was aspirated from each well and placed in a flat-bottom 96-well plate, 100. Mu.l of LDH matrix solution was simultaneously added thereto, and reacted for 3 minutes, 30. Mu.l of 1mol/L HCl was added to each well, and the optical density value was measured at 492nm in an ELISA.

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). Times.100%

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

8.1.2 comparison of the effects of increasing the immunity of the products at the same weight concentration (influence on the NK cell Activity of mice)

Weighing 1g of each group of samples respectively, adding 10mL of sterile water, and preparing 0.1g/mL of compound bifidobacterium solution for later use; the negative control group used only sterile water and no bifidobacterium complex.

The effect of bifidobacterium complex on the NK cell activity of mice was measured and the measurement results were expressed as "mean ± standard deviation". In the experiment, SPSS 24.0 software is adopted to carry out single-factor analysis of variance (ANOVA) on experimental data, an LSD method is adopted to carry out multiple comparison, P <0.05 is used as a difference significance judgment standard, and the result is shown in Table 10:

TABLE 10 Effect of Complex bifidobacterium products produced by different technologies at the same weight concentration on NK cell Activity in mice

Note that: NK cell activity conversion value=sin-1 (a ^1/2 )

As shown in table 10:

for the composite bifidobacterium raw powder product, NK cell activity in serum of mice fed with the composite bifidobacterium raw powder prepared by the solid state method in the embodiment 4 of the invention is larger than that of the composite bifidobacterium raw powder prepared by the liquid state method in the embodiment 3.

For the compound bifidobacterium preparation products, NK cell activity in serum of mice fed with the compound bifidobacterium preparation prepared by the solid state method is larger than that of the compound bifidobacterium preparation prepared by the liquid state method (example 3); the immunity increasing effect of the microbial inoculum prepared by the feeding solid method is obviously different from that of the microbial inoculum prepared by the liquid method, which shows that the immunity increasing effect of the composite bifidobacterium microbial inoculum prepared by the preparation method is more obvious.

8.1.3 comparison of the effects of increasing immunity of the products at the same bacterial count (influence on NK cell Activity of mice)

Respectively weighing 1g of composite bifidobacterium finished product, adding a proper amount of sterile water, measuring the bacterial count, and adding the sterile water to adjust the total concentration of the composite bifidobacterium to 1x10 ¹⁰ CFU/ml, 10ml each time, so the total number of composite bifidobacteria per time is 10x10 ¹⁰ CFU, i.e. 1X10 ¹¹ CFU。

The experimental method for increasing immunity is shown in 7.1.1.

The effect of the complex bifidobacterium preparation in the serum of the mice on the NK cell activity of the mice was measured, and the measurement results are expressed as "mean value.+ -. Standard deviation". In the experiment, SPSS 24.0 software is adopted to carry out single-factor analysis of variance (ANOVA) on experimental data, an LSD method is adopted to carry out multiple comparison, P <0.05 is used as a difference significance judgment standard, and the result is shown in Table 11:

TABLE 11 Complex bifidobacterium products at the same bacterial count (Effect on NK cell Activity in mice)

Note that: NK cell activity conversion value=sin-1 (a ^1/2 )

As shown in table 11, when the preparation contains the same bacterial count, the bacterial agents prepared by 4, 5 and 6 have greater influence on the activity of the NK cells of the mice; the microbial inoculum prepared by the solid-state method has an immunity enhancing effect which is obviously different from that of the microbial inoculum prepared by the liquid-state method.

The immunity enhancing effect of the bifidobacterium complex should have about the same immunity enhancing effect when the same bacterial count is used if it is only the effect of live bacteria, but the results of table 10 show that: compared with the compound bifidobacterium microbial inoculum prepared by a liquid method, the compound bifidobacterium microbial inoculum prepared by the solid method in examples 4, 5 and 6 has larger influence on the activity of NK cells of mice, which shows that the effect of increasing the immunity is more obvious. In the liquid state method, the metabolites generated in the liquid state culture process are separated by centrifugation, so that the immunity increasing effect is mainly derived from the strain; besides the complex bifidobacterium, the microbial inoculum prepared by the invention also contains metabolites generated in the solid state culture process, and the complex bifidobacterium and the metabolites are synergistic, so that the overall immunity increasing effect of the microbial inoculum is improved. 8.2 Effect of Complex bifidobacterium preparation on ConA-induced mouse spleen lymphocyte transformation response

8.2.1 Experimental methods

1. Experimental materials:

test animals: SPF-grade Kunming female mice were selected and 6-8 weeks old (body weight 18-22 g), and 105 animals were randomly divided into 7 groups of 15 animals each.

Other materials: hank's solution, RPMI1640 complete culture solution, and the like.

2. The test method comprises the following steps:

1. dose and grouping:

2. The feeding method comprises the following steps:

in the experiment, samples are respectively taken and prepared into 20ml of sample liquid by purified water, and the sample liquid is respectively used for animal stomach irrigation. Animals were conditioned for 3 days under laboratory conditions, randomly grouped, and perfused daily with samples and doses of table 12 for 30 consecutive days, and control perfused with sterile purified water.

3. ConA-induced mouse spleen lymphocyte transformation assay (MTT method):

1. lymphocyte proliferation response:

after the feeding experiment of the compound bifidobacterium preparation is finished, the mice of each dose group are aseptically taken out of spleen, put into asepsis Hank's liquid for grinding, centrifugally washed by 1000r/min, and then the cells are suspended in 1ml of complete culture liquid, living cells are counted and the concentration is adjusted to 3 multiplied by 10 ⁶ The cell suspension was added to 24-well plates in two wells per ml,1 well with 75. Mu.l ConA solution (equivalent to 7.5. Mu.l/ml) per well and the other well served as a control. Placed in 5% CO2 and incubated at 37℃for 72h. 4 hours before the end of the culture, 0.7ml of supernatant was aspirated from each well, 0.7ml of serum-free RPMI 1640 medium was added to each well, and 50. Mu.l/ml/well of MTT (5 mg/ml) was added thereto, and the culture was continued for 4 hours. After the completion of the culture, 1ml of isopropyl alcohol was added to each well, and the mixture was blown to be uniform to dissolve the purple crystals, and the mixture was added to a 96-well culture plate to prepare a parallel sample of 3 wells, and the optical density was measured at a wavelength of 570nm, whereby the proliferation and transformation capacity of lymphocytes was calculated as follows.

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

2. Experimental results:

the difference in optical density was significantly greater in the high dose group than in the control group in response to ConA-induced transformation of spleen lymphocytes in mice (see table 12). The results indicate that the spleen lymphocyte transformation test of the sample in example 1 is positive, and the tested sample enhances the cell immunity.

The effect of the complex bifidobacterium preparation on ConA-induced mouse spleen lymphocyte transformation responses is shown in table 12: TABLE 12 influence of Complex bifidobacteria produced by different technologies on ConA-induced transformation of mouse spleen lymphocytes

From the results in table 12, it can be seen that: the complex bifidobacterium formulations produced by the solid state method of example 4, example 5 and example 6 have a particularly pronounced effect on ConA-induced mouse spleen lymphocyte transformation responses compared to negative controls and liquid state methods.

According to the experimental results of example 8, the compound bifidobacterium preparation can improve NK cell activity of mice and increase ConA-induced spleen lymphocyte transformation reaction of mice, which shows that the compound bifidobacterium preparation has an immunity enhancing effect, wherein the effect is more obvious in examples 4, 5 and 6 adopting a solid state method.

Example 9 comparative test of hypoglycemic Effect of Bifidobacterium Complex (alpha-glucosidase inhibitor content measurement)

9.1 determination of the content of alpha-glucosidase inhibitor of bifidobacterium complex

9.1.1 test methods

The products obtained in the examples above were selected and the α -glucosidase inhibitor content was determined by the method of reference Zhu Yunping. The test method is as follows:

materials and reagents:

4-nitrophenyl-D-glucopyranoside (PNPG), α -glucosidase; sterile 0.1mol/L phosphate buffer (PBS, pH 6.8); the remaining reagents were all analytically pure.

Preparing a sample extracting solution:

the bacteria were suspended in sterile 0.1mol/L phosphate buffer (PBS, pH 6.8) and the bacterial concentration was adjusted to 1X 10 ⁹ CFU/mL, incubation at 37deg.C for 12h, centrifugation at 4deg.C at 8000r/min for 15min, and filtering the supernatant with 0.22 μm water-based microfiltration membrane to obtain sample extract, and preservation at-80deg.C.

Determination of the inhibition ratio of α -glucosidase:

the PNPG method was used to determine the alpha-glucosidase inhibitory activity.

Principle of PNPG method:

since PNPs released by the catalytic hydrolysis of PNPG by alpha-glucosidase have a certain absorbance at 405nm, the change of the activity of alpha-glucosidase can be measured by detecting the quantity of PNPs, thereby measuring the inhibition rate of the sample on the alpha-glucosidase.

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

Inhibition of α -glucosidase% = (OD _Control- OD _{Sample of} )/OD _Control ×100

Wherein:

OD _control Is the absorbance value without addition of sample wells, i.e., the blank.

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

9.1.2 comparison of the hypoglycemic Effect of the products at the same weight concentration (determination of the inhibition ratio of alpha-glucosidase)

1g of each group of samples is weighed and added with 10mL of sterile water to prepare 0.1g/mL of compound bifidobacterium solution for later use. The PNPG method determines the inhibition effect of each group of samples on alpha-glucosidase, so as to determine the hypoglycemic effect of bifidobacterium complex, and the results are expressed as an average value ± standard deviation, and are shown in table 13:

experimental results:

TABLE 13 alpha-glucosidase inhibition Rate (%)

Sample of	Dosage of	Alpha-glucosidase inhibition Rate (%)
			Negative control group	0.1g/ml	0
EXAMPLE 3 raw powder	0.1g/ml	60.2
			EXAMPLE 4 raw powder	0.1g/ml	78.8
Example 3 formulation	0.1g/ml	55.5
			Example 4 formulation	0.1g/ml	71.3
Example 5 formulation	0.1g/ml	85.6
			Example 6 formulation	0.1g/ml	76.2

9.1.3 comparison of hypoglycemic Effect of products with the same bacterial count (determination of alpha-glucosidase inhibition Rate)

Respectively weighing 1g of composite bifidobacterium finished product, adding a proper amount of sterile water, measuring the bacterial count, and adding the sterile water to adjust the total concentration of the composite bifidobacterium to 1x10 ¹⁰ CFU/ml, ready for use.

The inhibition effect of the same bacterial count of the example samples on alpha-glucosidase was measured according to a similar test method as in example 9.1.1, and the results are expressed as "mean ± standard deviation" and are shown in table 14:

TABLE 14 alpha-glucosidase inhibition Rate (%)

Sample of	Dosage of	Alpha-glucosidase inhibition Rate (%)
			Negative control group	1x10 ¹⁰ CFU/ml	0
EXAMPLE 3 raw powder	1x10 ¹⁰ CFU/ml	55.2
			EXAMPLE 4 raw powder	1x10 ¹⁰ CFU/ml	72.3
Example 3 formulation	1x10 ¹⁰ CFU/ml	49.5
			Example 4 formulation	1x10 ¹⁰ CFU/ml	56.3
Example 5 formulation	1x10 ¹⁰ CFU/ml	76.6
			Example 6 formulation	1x10 ¹⁰ CFU/ml	71.2

The above experimental results actually measure the difference between the α -glucosidase activity of the sample and the activity of the α -glucosidase standard, i.e., the α -glucosidase inhibition rate of the buffer solution without sample is defined as 0, while the α -glucosidase inhibition rate of the sample is defined as the percentage difference in absorbance between the sample and the control. It should be noted that: in order to simplify the measurement procedure, the invention does not set a positive control, namely does not use positive alpha-glucosidase inhibitor acarbose or DNJ as a control, and does not deduct the background of the sample, so the measured inhibition rate can be slightly different from the actual one, but the results in the table 13 and the table 14 still have good reference value for knowing the inhibition effect of the composite bifidobacterium sample on the alpha-glucosidase.

According to the experimental results of tables 13 and 14, the composite bifidobacterium product of the present invention has a better alpha-glucosidase inhibition rate than the conventional liquid-process composite bifidobacterium product, and the composite bifidobacterium product of the present invention has a higher quantity of composite bifidobacterium. Thus, it is expected that the product of the present invention will generally have a better hypoglycemic effect than the liquid-state produced bifidobacterium complex formulation.

9.2 comparative test for hypoglycemic Effect of Complex bifidobacteria (therapeutic effect of hyperglycemia)

According to the products obtained in the above embodiments, the hypoglycemic experiments of the products are performed, and the experimental schemes and experimental results are as follows:

experimental materials and methods:

a subject: type II diabetics with fasting blood glucose of 7.8mmol/L or more are selected as experimental subjects, and 210 patients diagnosed with adult type II diabetes are randomly divided into 7 groups of 30 patients.

Blank or placebo group: treatment was performed with a blank starch preparation without drug 3 times per day, 1 bag per time, 2 grams per bag, for 4 weeks.

Positive experimental group: acarbose is used for treatment, 75mg/d, and the medicine is taken three times, 25mg each time, and the treatment is carried out for 4 weeks.

Liquid method control group (example 3): culturing the bifidobacterium complex by adopting a traditional liquid culture method, centrifuging and drying to prepare the bifidobacterium complex bacterial preparation for treatment, wherein the preparation is treated by 1 bag per time for 3 times per day, and each bag is 2 g for 4 weeks.

Group of examples: the treatment was carried out orally with the compound bifidobacteria according to table 15, 3 times per day, 1 bag per bag, 2 g per bag for 4 weeks, totally divided into 3 groups (examples 4-6).

The adverse reaction is determined by inquiry during the administration period.

Experimental results:

TABLE 15 hypoglycemic Effect of Compound bifidobacteria produced by different technologies (fasting blood glucose value, mmol/L)

Table 16 hypoglycemic effects of bifidobacterium complex produced by different processes (postprandial 2h blood glucose values,

mmol/L)

in conclusion, the composite bifidobacterium preparation prepared by the method has good hypoglycemic effect, has better effect than the composite bifidobacterium preparation prepared by a liquid method, is slightly lower than the commonly used hypoglycemic drug, namely acarbose, but has small side effect, has other effects of regulating intestinal tract, increasing immunity and the like, and is not limited by the dosage of the drug. Therefore, the compound bifidobacterium can be used as an auxiliary conditioning product for diabetics to assist other hypoglycemic drugs to play a role in reducing blood sugar.

Example 10: lipid-lowering and weight-losing effect comparison test of composite bifidobacteria

The lipid-lowering and weight-losing effects of the bifidobacterium complex on mice can be characterized by measuring the indexes such as the body weight, the Lee's index, the blood lipid and the like of the mice fed with the bifidobacterium complex.

10.1 Effect of microbial inoculum on lipid-lowering and weight-reducing effects in mice at the same weight concentration

The products obtained in examples 3-6 were selected, and the same weight of bifidobacterium complex was fed each time at the dose of table 3, and the lipid-lowering and weight-reducing effects of bifidobacterium complex on mice were measured after the experimental period was ended, and the experimental scheme and experimental results were as follows:

10.1.1 materials and reagents:

kunming mice, male and female halves, 4 weeks of age, and body weight 18-22g.

10.1.2 test method:

grouping animals: the purchased mice were fed with basal feed for 1 week, and after the initial weight was measured, the oversized mice were removed, 80 mice were selected and randomly divided into 8 groups of 10 mice each, and the male and female were half.

Blank (distilled water), model (high fat feed 3 g), positive (orlistat 400 μg), example 3 (liquid control) base powder, example 3 (liquid control) formulation, example 4 base powder, example 4 formulation, example 5 formulation, example 6 formulation.

Mice were fed basal feed normally 3 meals daily. After about 30 minutes of feeding the basal feed per meal, 10ml of solution was prepared for stomach infusion with the corresponding doses of each group of table 3, and then the body weight was measured 1 time per week, and a total of 4 weeks of feeding experiments were performed, and the final body weight was measured at the end of the experiment and blood was taken to determine the blood lipid content.

10.1.3 observations index and method:

(1) general observations:

degree of activity, body hair color, body shape, diet, drinking water and fecal condition.

(2) And (3) detecting a growth index:

the growth index mainly detects the body weight (g) and the obesity index (Lee's index), wherein the calculation method of the Lee's index is as follows:

Lee's index= [ body weight (g) ]] ^1/3 Body length (cm) ×1000

(3) Blood lipid detection:

blood lipid measurement (total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein) was performed using a fully automatic biochemical analyzer.

10.1.4 experimental results:

(1) general conditions:

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

The model control group has the advantages of appetite enhancement, rounding of body shape, dullness of activity compared with the normal control group and slightly increased stool;

the positive control group mice have loose stool, and the compound bifidobacterium group mice have no diarrhea phenomenon.

Compared with the model control group, the mice with the compound bifidobacterium have less food intake, more flexible movement and obviously increased stool volume.

(2) Weight change:

the groups are randomly grouped after 1 week of adaptive feeding, and the initial weight difference of the mice among the groups has no statistical significance (P is more than 0.05); at the end of the feeding experiment for 4 weeks, the weight of the model control group is obviously increased compared with that of the blank control group; the positive control group and the blank control group are compared, so that the weight is obviously reduced; the compound bifidobacteria group of the different examples showed a significant decrease in body weight compared to the blank group; body weight was significantly reduced compared to the model control group, and the results are shown in table 17:

TABLE 17 influence of Complex bifidobacteria on mouse body weight

After the 4-week feeding experiment is finished, blood is collected to measure the blood fat content, and the results are shown in Table 18:

table 18 Effect of Compound bifidobacteria on blood lipid in mice

From the results of tables 17 and 18, it can be seen that:

the composite bifidobacterium raw powder produced by example 3 and example 4 has a particularly remarkable effect on lipid-lowering and weight-losing of mice compared with a blank control.

The compound bifidobacterium preparations produced by example 3, example 4, example 5, example 6, and the like have particularly remarkable effects on lipid-lowering and weight-losing effects in mice, compared with the blank control.

The compound bifidobacterium preparation prepared by the solid state culture method (examples 4-6) has a particularly remarkable effect on lipid-lowering and weight-losing effects on mice compared with the compound bifidobacterium preparation prepared by the liquid state culture method (example 3).

The experimental results show that: the compound bifidobacteria have a certain effect of reducing the weight of mice, and the compound bifidobacteria synthesize intestinal thallus components in intestinal tracts by utilizing small molecular nutrient substances such as glucose, fatty acid and the like, so that the discharge of stool is increased, and the fat is removed; in addition, the metabolites produced by the bifidobacterium complex are also beneficial to reducing the synthesis of fat in the body. Thus, bifidobacterium complex has an effect of inhibiting weight gain in mice. The experimental result of the influence of the compound bifidobacterium on the blood fat of the mice shows that: the compound bifidobacterium can reduce TG, CHO, LDL-C and increase HDL-C, so that the compound bifidobacterium is beneficial to improving fat metabolism in vivo and has good lipid-lowering function. The experimental results according to tables 17 and 18 also found that: the solid-state method composite bifidobacterium product has better lipid-lowering and weight-losing effects than the traditional liquid-state method composite bifidobacterium product.

In conclusion, the solid-state method composite bifidobacterium preparation prepared by the method has good lipid-lowering and weight-losing effects, is better than the liquid-state method composite bifidobacterium preparation in effect, is slightly lower than the common lipid-lowering and weight-losing drug-orlistat, has small side effect, has other effects of regulating intestinal tracts, increasing immunity and the like, and is not limited by drug dosage. Therefore, the compound bifidobacterium can be used as an auxiliary conditioning product for obese patients to assist other lipid-lowering and weight-losing medicaments to play the role of lipid-lowering and weight-losing.

10.2 Effect of products on lipid-lowering and weight-reducing effects of mice with the same bacterial count

The test procedure is as in example 10.1.

Respectively weighing 1g of composite bifidobacterium finished product, adding a proper amount of sterile water, measuring the bacterial count, and adding the sterile water to adjust the total concentration of the composite bifidobacterium to 1x10 ¹⁰ CFU/ml and 10ml is filled each time, so the total number of the composite bifidobacteria is 10x10 ¹⁰ CFU, i.e. 1X10 ¹¹ CFU。

The effect of the same bacterial count of the example samples on the lipid-lowering and weight-losing effects of the mice were determined according to a similar test method as in example 10.1, and the results are expressed as "mean ± standard deviation", as shown in table 19:

table 19 effects of products on lipid-lowering and weight-reducing effects of mice for 4 weeks after the end of feeding experiment, blood was collected and the lipid content was measured, and the results are shown in table 20:

TABLE 20 Effect of products on blood lipid in mice with the same bacterial count

As shown in table 19 and table 20, example 3 is the effect of the products prepared by the liquid method on the lipid-lowering and weight-losing effect of mice, and examples 4, 5 and 6 are the effect of the products prepared by the 3 solid methods on the lipid-lowering and weight-losing effect of mice, respectively.

The experimental results in tables 19 and 20 show that: products with the same bacterial count have approximately the same lipid-lowering and weight-losing effects. The experimental results of tables 17, 18, 19 and 20 above were summarized as follows: the composite bifidobacterium product prepared by the solid state culture method has better lipid-lowering and weight-losing effects than the composite bifidobacterium product prepared by the traditional liquid state method.

Effect of bifidobacterium complex on lipid-lowering and weight-losing effects in mice, if only live bacteria are used, the same effect should be obtained when the same number of bacteria is used, but the above results indicate that: compared with the composite bifidobacterium product prepared by the liquid method (example 3), the composite bifidobacterium product prepared by the solid method in examples 4, 5 and 6 of the invention has better lipid-lowering and weight-losing effects. During the preparation process of the liquid preparation, the metabolic products generated in the liquid culture process are separated by centrifugation, so that the lipid-lowering and weight-losing effects mainly come from the strain; besides the compound bifidobacterium bacteria, the solid preparation prepared by the invention also contains metabolites with lipid-lowering and weight-losing capabilities such as a citrate lyase inhibitor, an alpha-glucosidase inhibitor, short-chain fatty acid and the like which are generated in the solid culture process, and the compound bifidobacterium and the metabolites are synergistic, so that the influence of the whole bacterial preparation on the lipid-lowering and weight-losing effects of mice is improved.

The experimental results show that: the compound bifidobacteria have a certain effect of reducing the weight of mice, and the compound bifidobacteria synthesize intestinal thallus components in intestinal tracts by utilizing small molecular nutrient substances such as glucose, fatty acid and the like, so that the discharge of stool is increased, and the fat is removed; in addition, the metabolites produced by the bifidobacterium complex are also beneficial to reducing the synthesis of fat in the body. Thus, bifidobacterium complex has an effect of inhibiting weight gain in mice. The experimental result of the influence of the compound bifidobacterium on the blood fat of the mice shows that: the compound bifidobacterium can reduce TG, CHO, LDL-C and increase HDL-C, so that the compound bifidobacterium is beneficial to improving fat metabolism in vivo and has good lipid-lowering function. From the experimental results of tables 7 and 8 it was also found that: the composite bifidobacterium product has better lipid-lowering and weight-losing effects than the traditional liquid composite bifidobacterium product. The composite bifidobacterium preparation prepared by the invention has good lipid-lowering and weight-losing effects, has better effect than the liquid composite bifidobacterium preparation, is slightly lower than the common lipid-lowering and weight-losing drug-orlistat, has small side effect, has other effects of regulating intestinal tract, increasing immunity and the like, and is not limited by the dosage of the drug. Therefore, the compound bifidobacterium can be used as an auxiliary conditioning product for obese patients to assist other lipid-lowering and weight-losing medicaments to play the role of lipid-lowering and weight-losing.

The experimental results of the examples are combined, and the composite bifidobacterium preparation prepared by adopting the processes of mixed bacteria inoculation of composite bifidobacterium, liquid culture, centrifugal separation, solid culture and the like is found to have the functional effects of high strain content, rich bifidobacterium metabolite, good antiallergic effect, immunity enhancement, blood sugar reduction, lipid reduction, weight reduction and the like.

Aiming at the defects of the traditional process, the invention adopts a novel process combining mixed bacteria inoculation, liquid culture, solid-liquid separation, solid culture and the like, and adds a solid culture procedure on the basis of the traditional liquid culture and solid-liquid separation; or it can be said that: the invention is based on the traditional combination process of liquid culture and solid culture, and adds a solid-liquid separation process in the middle. The improved fermentation process makes full use of the advantages of high liquid fermentation speed, high number of wet strain thalli after solid-liquid separation, and capability of greatly recovering and increasing the strain vitality of the compound bifidobacterium through solid-state fermentation, and can also generate bacteriocin, organic acid and other antiallergic substances in the solid-state fermentation process, can also generate metabolic products for reducing blood sugar and increasing immunity, and can enhance the functional effects of the compound bifidobacterium preparation in multiple aspects of antiallergic, blood sugar reducing, gastrointestinal conditioning, immunity increasing, lipid reducing, weight losing and the like when in use. In addition, the solid culture medium added by solid fermentation and the culture product thereof also have the function of a drying protective agent, and thalli generated by the solid fermentation have small stress during drying and low death rate during the drying of the thalli, so that the finally obtained product has high bacterial activity, contains a proper amount of bacteriocin, organic acid and a plurality of metabolites with the functions of reducing blood sugar and increasing immunity, and has better effects of resisting allergy, reducing blood sugar, regulating intestines and stomach, increasing immunity and reducing blood fat and losing weight in the actual use process. The composite bacteria used in the invention are bifidobacteria, the growth environments are similar, the produced metabolites are similar, but the requirements on the growth environments are slightly different, so that the composite bacteria can fully exert the adaptability of the composite bacteria to the growth environments when the composite bacteria are mixed and cultivated, the total bacterial count at the end of cultivation is higher than that of the composite bacteria cultivated by a single strain, and the functional effect of the composite bacteria is more comprehensive.

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. The method for preparing the compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar and reducing fat is characterized in that the compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar and reducing fat is prepared from the following raw materials:

12-13 parts of bifidobacterium bifidum Miuyo-01 (Latin name: bifidobacterium bifidum), 3-5 parts of bifidobacterium lactis Miuyo-11 (Latin name: bifidobacterium lactis), 7-8 parts of bifidobacterium infantis Miuyo-21 (Latin name: bifidobacterium infantis) and 4-6 parts of bifidobacterium breve Miuyo-31 (Latin name: bifidobacterium breve) and; the preservation numbers of bifidobacterium bifidum Miuyo-01, bifidobacterium breve Miuyo-31, bifidobacterium infantis Miuyo-21 and bifidobacterium lactis Miuyo-11 are CGMCC 20866, CGMCC 20867, CGMCC 20868 and CGMCC 20869 respectively;

The method comprises the following steps:

adopting a disk centrifuge or a tube centrifuge to carry out centrifugal separation under the aseptic condition to obtain composite bifidobacterium wet thalli; the liquid culture medium comprises oligosaccharide, peptone, yeast extract and tomato sauce; the temperature of the first anaerobic culture is 36-38 ℃ and the time is 48-72 hours;

the solid culture medium comprises oligosaccharide, 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 standby before use; before the second anaerobic culture, the moisture content of the mixture of the wet bacterial cells of the composite bifidobacterium and the solid culture medium is regulated to 45% -55%;

The temperature of the second anaerobic culture is 36-38 ℃ and the time is 72-120 hours; the low-temperature drying temperature is 30-40 ℃; the water content of the composite bifidobacterium solid is 4-6%;

2. The preparation method of the compound bifidobacterium preparation for resisting allergy, increasing immunity, reducing blood sugar and reducing blood fat and losing weight according to claim 1, wherein the compound bifidobacterium preparation for resisting allergy, increasing immunity, reducing blood sugar and reducing blood fat and losing weight is prepared from the following raw materials:

12.5 parts of bifidobacterium bifidum Miuyo-01, 7.5 parts of bifidobacterium infantis Miuyo-21, 5 parts of bifidobacterium breve Miuyo-31 and 4 parts of bifidobacterium lactis Miuyo-11; the viable count of the composite bifidobacterium preparation is (4-5) multiplied by 10 ¹¹ CFU/g。

3. The method for preparing the compound bifidobacterium preparation for resisting allergy, increasing immunity, reducing blood sugar, reducing blood fat and losing weight according to claim 1 or 2, wherein 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.

4. The preparation method of the compound bifidobacterium preparation for resisting allergy, enhancing immunity, reducing blood sugar and reducing blood fat and losing weight according to claim 1, wherein the separation culture conditions of bifidobacterium bifidum Miuyo-01, bifidobacterium infantis Miuyo-21, bifidobacterium breve Miuyo-31 and bifidobacterium lactis Miuyo-11 are as follows: MRS medium; the pH is 6.0-7.5, and the temperature is 34-40 ℃.