WO2001085186A1

WO2001085186A1 - Preparation that contains the intestinal beneficial bacterium fermentation culture

Info

Publication number: WO2001085186A1
Application number: PCT/CN2001/000617
Authority: WO
Inventors: Bingxin Wu; Lishan Dong; Xiaolin Sun; Hong Wang
Original assignee: Bingxin Wu; Lishan Dong; Xiaolin Sun; Hong Wang
Priority date: 2000-04-26
Filing date: 2001-04-26
Publication date: 2001-11-15
Also published as: AU6373501A; CN1273838A

Abstract

The present application relates to beneficial microorganisms preparation that contains various kinds of probiotics that benefit to the intestine and the producing method. The application further disclosed producing said preparation by fractional inoculation and culturing the probiotics under the same fermentation condition. The said preparation can not only be used to regulate the micro-ecological balance in enteron but also to improve the mouse's immunological functioning by stimulating the specific or non-specific immunological response. The preparation can be used to improve the function of the gastrointestinal tract.

Description

Within one month from the date, the applicant can submit opinions to the unit.

Invention Field

The present invention generally relates to a nutritional composition containing inactivated or dead probiotics and its metabolites for restoring and maintaining the health and related physiological functions of the intestinal tract, its production method, and its ability to improve the intestinal flora balance And stimulate specific or non-specific immune functions. The present invention particularly relates to a method for culturing and multiplying a variety of probiotics in the same medium and under the same fermentation conditions by staged inoculation and symbiotic fermentation, and the preparation of the probiotic mixed culture for the purpose of restoring and maintaining the It is used in nutrition composition for normal flora balance, improvement of body specific or non-specific immune function and antitumor activity. Background of the invention

All factors that interfere with the host, whether physical, chemical, or biological, can cause microecological balance imbalances. For healthy people, changes in the internal or external environment, such as surgery (especially abdominal surgery), trauma, mental stress, tumors, bile salt stimulation, and abuse of antibiotics, often lead to microbial flora in the intestine, which is beneficial and harmful. Imbalance between intestinal microecologies. Among them, the most important factor leading to the imbalance of the normal intestinal microflora is the long-term abuse of antibiotics. Once the fermentation process of the normal flora is disrupted, it can lead to the reduction of many beneficial bacteria, which in turn causes colonic mucosal failure. At the same time, colonic mucosal function is severely impaired due to the rapid growth of potentially malignant bacteria. Harmful bacteria penetrate the dysfunctional colon wall and infect organs, cause abscesses in organs or weaken or even lose most organ functions. In the past, people often used various antibiotics to prevent or treat intestinal flora imbalances, especially secondary infections caused by normal intestinal flora imbalances after abdominal surgery. However, the large-scale use of antibiotics for a long time is not only expensive, but also difficult to correct the normal intestinal flora imbalance, especially because of the excessive growth of pathogenic bacteria often leading to further deterioration of the disease. In order to adjust the normal intestinal micro-ecological balance, a generally accepted method is to screen and use known non-pathogenic non-toxic bacteria that are dominant in the normal gastrointestinal tract and can colonize the small intestinal mucosa, with or without addition Dietary fiber, oligosaccharides or immunococci proteins, sulfur-containing amino acids and other auxiliary ingredients are made into pharmaceutical compositions or nutritional supplements and administered to patients who need to undergo treatment for normal intestinal microecological disorders. A large number of laboratory studies and clinical observations have proved that micro-ecological balance therapy can play an irreplaceable role in improving and maintaining the balance of the gastrointestinal flora, improving the body's specific or non-specific immunity, and reducing blood cholesterol.

There are many prior art documents describing the therapeutic effects of probiotics and their applications. For example, Fernandes reviewed in detail the probiotic effects of Lactobacillus introduced into the body through food or fermented dairy products (especially Fernandes et al., "Therapeutic role of dietary lactobacilli and lactobacillic fermented diary products", FEMS Microbiology Reviews 46: 343-356, 1987). Nobuo Saegara's research proves that oral administration of Streptococcus faecalis can significantly improve lipid metabolism in humans and animals (Microecology and therapy, vol. 15, 271-280, 1985). European patent EP 0199535 describes a pure culture of Lactobacillus acidic acid isolated from feces and tested in vitro to demonstrate adhesion to the intestinal mucosa. International patent WO 89/05849 describes lactic acid bacteria isolated from porcine gastrointestinal tract that can adhere to porcine gastrointestinal epithelial cells and are resistant to strong acids and bile. The bacteria can be used to make fermented dairy products and used to prevent or treat E. coli diarrhea in piglets. On the other hand, U.S. Patent No. 4,805,368 discloses a dietary fiber-based composition containing Lactobacillus acidophilus and / or Bifidobacterium bifidum, Leuconostoc citrovorum, and Propionibacterium shelving ( Propionibacterium shermanii) and its preparation method. U.S. Patent No. 4,913,913 describes a method of mixing or separately cultivating L. casei and B. longum to prepare a lactic acid bacteria-fermented dairy product containing Bifidobacterium. U.S. Patent No. 5,716,615 discloses a pharmaceutical composition for treating gastrointestinal diseases and hypercholesterolemia including Streptococcus thermophilus, Lactobacillus and Bifidobacterium. U.S. Patent 5,744,134 Compositions for promoting gastrointestinal health are also described, which contain immunoglobulins and soluble dietary fiber, polyvalent iron chelating molecules and gluconic acid, and one or more intestinal probiotics. Finally, Chinese patent 93101172.0 filed and issued by the present inventor in 1993 discloses a heat-killed Lactobacillus acidoilus, Bifidobacterium breve, and Enterococcus faecium ) Nutritional supplements (the so-called "Sanmo oral solution"). Unlike other existing technologies, although the suspended liquid nutritional supplement does not contain live probiotics, it is believed that the suspension contains a variety of beneficial bacterial enzymes and other bacterial metabolites, and is used in the production process It is rich in soluble fiber and soy isoflavones, so it not only utilizes the survival and growth of bacteria, but also provides these and other beneficial ingredients for the nutritional mixture.

However, whether it is a composition containing a plurality of probiotics, whether live or dead, and whether it contains only one or more bacteria or a composition containing other auxiliary ingredients, the above-mentioned prior arts are not described in the same medium. A method for cultivating and multiplying more than two intestinal probiotics under the same fermentation conditions by using stage culture and symbiotic fermentation technology. Summary of invention

Accordingly, it is an object of the present invention to provide a suspended liquid nutritional composition containing inactivated or heat-killed one or more probiotic bacteria and containing soluble dietary fiber and soy isoflavones.

According to a preferred embodiment of this object of the present invention, the probiotic bacteria escape from Bifidobacterium, Lactobacillus, Streptococcus, Enterococcus, Propionibacterium, Leuconostoc and Bacillus One or more non-pathogenic bacteria in the human intestine.

According to a preferred embodiment of the present invention, the probiotic is selected from the group consisting of Bifidobacterium bifidus, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus plantarum, Streptococcus thermophilus, Enterococcus faecalis, Enterococcus faecium, Lectobacillus citric acid, Bacillus subtilis, and Licheniformis. According to one preferred embodiment of this object of the present invention, wherein said soluble fiber per ml of containing an amount of about 20-40 μ g, and wherein said soy isoflavone content per ml of 30- 100 μ g _a '

Another object of the present invention is to provide a method for producing a suspension liquid nutrition composition as defined above, the method comprising:

(1) providing a liquid culture medium consisting essentially of soybean sprout cooking solution, proteolytic beef broth, yeast extract, sugar and minerals;

(2) One or more bacteria are inoculated into the culture medium in sequence according to the ratio of the biomass of the wet bacteria to the total weight of the culture medium of 0.5-1.0%, and after each kind of bacteria is inserted, the Anaerobic culture at a temperature of 38-41 X and H6.6 for 4-10 hours;

(3) When the total concentration of the bacterial cells in the fermentation culture reaches about 7 × lOVml or more, the temperature is reduced to about 32 ° C and maintained for 2-4 hours to complete the biological metabolic transformation;

(4) When the pH of the fermentation culture is reduced to approximately 3.5-3.9, the temperature is increased to 48-50 X and maintained for 4-6 hours to complete the heat inactivation of the bacteria and stabilization of the fermentation system .

According to a preferred embodiment of this object of the present invention, wherein said liquid culture medium is made of about 25% soybean sprout decoction, about 2.5% trypsin hydrolyzed beef broth, and about 1.3% yeast extract , About 1.3% glucose, about 1.3% sucrose and a suitable amount of minerals including ¾, Ca ", K +, Na ⁺ .

According to a preferred embodiment of the present invention, the soy bean sprout cooking solution is a germinated soybean mixed with 30% (W / V) in water and cooked at 105-121 ° C for 30 minutes, and then conventionally The obtained filtrate was filtered.

It is still another object of the present invention to provide the use of the nutritional composition in correcting and maintaining the normal microecological balance of the intestine.

It is still another object of the present invention to provide the use of the nutritional composition in regulating non-specific immune response of the body.

It is still another object of the present invention to provide the application of the nutritional composition described above in biotransformation or modification of active ingredients of one or more natural medicines, and to exert or improve their biological activities. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the growth curve of the biomass of each of the three bacteria over time when the Bifidobacterium breve, Enterococcus faecium, and Lactobacillus acidophilus were cultured separately by fermentation.

Fig. 2 shows the growth curve of total biomass over time when the bifidobacterium brevis, Enterococcus faecium, and Lactobacillus acidophilus are mixed and cultured in the same medium and under the same culture conditions. Detailed description of the invention

The present invention relates to a nutritional composition containing heat-killed intestinal probiotics, and containing soluble fiber and soybean isoflavones provided by a microbial culture medium. The invention further relates to a method for cultivating two or more probiotics in the same medium and under the same conditions by using the techniques of hierarchical inoculation and symbiotic fermentation. After the nutritional composition is administered orally, it can effectively restore and maintain the normal flora of the gastrointestinal tract, improve specific or non-specific immune functions, and reduce blood cholesterol levels.

At present, most commercial probiotic preparations claim to use one or more strains that can tolerate strong acidity in the stomach (1.0) and the upper intestine 1-1. 5% bile salt environment, and can be on the surface of the small intestinal mucosa Settled probiotics. However, the developers of these formulations have relatively ignored the probiotic effects of a large number of metabolites of probiotics. In fact, at present, the field of microecology generally accepts such a view: In order to maintain the microecological balance of the body, in addition to the normal intestinal bacterial population, there is a type of so-called "biomass", including microbial surface-active substances and a series of Metabolites, such as vitamins, antibodies, peptides, peptidoglycans, and amino acids. These substances can effectively inhibit the growth of certain toxic bacteria in the gastrointestinal tract and in fermentable foods. Many experiments have demonstrated the beneficial effects of certain dead bacteria and their fragments such as cell walls, intestinal materials, and bacterial metabolites on humans and animals. For example, it has been shown that killed lactic acid bacteria can adhere to intestinal mucosal epithelial cells, and through competitive repulsion, spatially inhibit the contact between pathogenic bacteria and the brush-shaped edge binding site of intestinal cells, thereby preventing pathogenic bacteria from Colonization of the intestine is the first critical step in cutting off bacterial infections. Coconnier Studies by et al. Showed that heat-killed Lactobacillus acidophilus and Lactobacillus bulgaricus on Caco-2 cells (a cell line with typical characteristics of intestinal cell differentiation used to study the organization and function of human intestinal cells ) And mucosal-secreting HT29- MTX cells are highly adherent, which can prevent the intestinal pathogens from adhering and invading CaCO-2 and HT29- MTX cells by blocking the pathogenic receptors of human intestinal cells ( Coconnier MH, J. Diarrhoeal Des. Res. 11 (4): 235-242, 1993; Coconnier, FEMS Microbiol, lett. 110 (3): 299-305, 1993). In addition, Quwehand and Conway (J. Appl. Bacteriol. 80 (3): 311-318, 1996) found that after the death of Lactobacillus 104r cell line, an inhibitor of enterotoxin-type E. coli K88 on the small intestinal mucosa was released. It is also proved that this substance which can be inactivated by lysozyme and contains glucose and galactose can exhibit such inhibitory activity only when the dead bacteria are continuously put into the bacterial culture during the cultivation process.

Many studies have also been conducted on the tumor suppressive activity of dead probiotics. Intestinal bacteria such as E. coli and Enterococcus faecalis are known to produce a variety of enzymes that convert pre-carcinogens to carcinogens, such as β-glucuronidase and nitroreductase. In addition, lactate and short-chain fatty acids (SCFA) produced by probiotic metabolism can cause a decrease in the intestinal pH value, inhibit the activity of these metabolic enzymes, and prevent the formation of carcinogens in the intestine. Furthermore, the special binding ability of probiotics such as lactic acid bacteria to mutagens also exerts certain anti-mutagenic and tumorigenic inhibition effects. Studies by Zhang Xuebin (J. Dairy Sci. 73 (3): 1477-1481, 1991) found that certain lactic acid bacteria such as Streptococcus lactis and Lactobacillus acidophilus, as well as bacterial components of Bifidobacterium (including cytoplasm and bacteria) Somatic cell wall skeleton component) has strong binding ability to mutagenic agents TRP-P-1 and N-nitrosodimethylamine. Sekine et al. (Caneer Res., 45: 1300-1310, 1985) demonstrated that peptidoglycan (WPG) isolated from the cell wall of Bifidobacterium infantis, as a biological response modifier, has a significant inhibitory effect on tumor growth. Further research has shown that the antitumor activity of probiotic bacteria wall preparations (WPG) is caused by PG-induced cytokine expression and activation of macrophages to produce large amounts of nitric oxide.

Finally, in terms of the immunomodulatory effects of dead probiotics, heat-killed Lactic acid bacteria KVS20 can activate resting and active macrophages (Kitazawa, H. et al., J. Dairy Sci., 74: 2082- 2088, 1991). In addition, some people have found that heat-killed Streptococcus thermophilus can stimulate macrophages and T helper cells to produce cytokines, increase TNF-α levels by about 135-176 times, and increase IL-6 levels by about 31-192 times ( Marin ML, J. Food Prot. 61C7): 859-864, 1998).

It can be seen from the previous research results of the fans that the probiotic dead bacteria preparation has proved to be effective both in theory and in practice. In addition, a problem that should not be ignored here is that many conventional probiotics, including Enterococcus faecium, have been found to carry antibiotic-resistant plasmids, and these plasmids may be foreign or Transfer between probiotics or pathogenic bacteria inherent in the intestine. Once such antibiotic-resistant plasmids are transferred into intestinal pathogenic bacteria, it may cause serious adverse consequences, thereby leaving potential hidden dangers for patients using live probiotic preparations. This is also the inventor's determination to use inactivated probiotics. The bacterial preparation is one of the reasons for the nutritional composition of the present invention.

In order to prepare a probiotic preparation containing a dead probiotic symbiotic fermentation culture as a nutritional composition or supplement, any two or more human normal intestinal probiotics known in the art may be selected. The probiotics include, but are not limited to, one or more human intestines selected from the group consisting of Bifidobacterium, Lactobacillus, Streptococcus, Enterococcus, Propionibacterium, Leuconostoc and Bacillus Non-pathogenic bacteria, including but not limited to Bifidobacterium, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus delbrueckii, plants Lactobacillus, Streptococcus thermophilus, Enterococcus faecalis, Enterococcus faecium, Leuconostoc citric acid, Bacillus subtilis and Bacillus licheniformis.

The medium used for the fermentation culture of the probiotics used in the present invention is basically composed of soybean sprout cooking solution, proteolytic beef broth, yeast extract, sugar and minerals. The medium used in the present invention is different from the conventional bacterial fermentation medium, and is mainly characterized in that it contains a basic carbon source (glucose and sucrose), a nitrogen source (enzymatic broth and yeast extract) and minerals. Soybean sprout cooking solution with a large weight ratio (about 25%) of the liquid culture medium was added. A typical medium composition used in the present invention is as follows: 2. 5% enzymatic beef broth, 1.3% yeast extract, 25% soybean sprouts, 1.3% glucose, 1.3% sucrose, 0.05% MgS0 ₄ , 0.05% CaC0 ₃ , 0.03% K ₂ HP0 ₄ , 0.03% KH ₂ P0 ₄ , 0.03% NaCI „The above ratios are weight percentages (W / W).,

It should be particularly noted here that the soybean sprout cooking solution in the medium is not mainly added as a nitrogen source and / or a carbon source. The purpose of adding soybean sprout cooking solution to the medium is to provide or supplement soy isoflavones, soluble fiber, and soybean oligosaccharides (such as stachyose and raffinose) to the probiotic culture broth. Soy isoflavones (including daidzein, genistein and daidzein) as phytoestrogens are known to have anti-free radicals, lower blood lipids, reduce the risk of cardiovascular disease, and prevent endocrine disorders caused by menopausal women Osteoporosis and other effects. The soluble food fiber contained in the soybean sprout extract can prevent the absorption of neutral fat and cholesterol in the intestine, promote gastrointestinal motility, prevent constipation, and delay or inhibit the absorption of carbohydrates. In addition, the soybean oligosaccharide contained in the soybean sprout extract can be decomposed and used by the intestinal lactic acid bacteria, but is hardly used by the intestinal harmful bacteria, which is beneficial to correct the intestinal flora imbalance. In short, the use of soybean sprout cooking liquid as a probiotic culture medium component and one of the main components of the nutritional composition of the present invention represents an important aspect of the technical features of the present invention. After testing, the concentrations of soy isoflavones and soluble fibers in the probiotic liquid culture prepared according to the method of the present invention were 20-40 μg ml and 30-100 μg / ml, respectively.

In order to prepare a soybean sprout cooking solution as a main component of the culture medium, freshly ripened soybeans are first immersed in about 2 times the volume of warm water for 5 to 6 hours, so that the soybean tissue swells with water absorption. It is then kept at 25-27 ° C for 60-72 hours to allow soybeans to grow germs that are about 3-8 cm long. Collect germinated and water-swelled soybeans, add tap water to the germinated soybeans at a rate of about 3 liters of water per liter of germinated soybeans, and heat in a high-pressure cooking vessel at a controlled temperature of about 105-121 for 30 minutes. After heating, the germinated soybean cooking liquor thus obtained was filtered through a double filter cloth and collected.

Another major component in the medium used in the present invention is protease-treated fresh beef and beef liver tissue hydrolysate. Different from the conventional broth used in the prior art, the broth of the present invention not only uses beef tissue in the selection of materials, but also adds a rich variety of Enzyme-like bovine liver tissues, and crude broth trypsin extracts from pig or bovine pancreas are also used in the preparation of broth. Therefore, in order to prepare an enzymatic beef soup as a culture ingredient used in the present invention, carefully selected beef and beef liver tissues are mixed at a weight ratio of 5: 2 and ground into minced meat. Add 1.5 volumes of water to it and stir well, and heat at about 100 ° C for about 60 minutes. Then the temperature was lowered to 38-41 ° C, and about 1% (V / V) of crude trypsin preparation prepared according to the method was added thereto. Digestion treatment at pH 7-9 for about 1 hour. After enzymatic hydrolysis, the supernatant was collected by centrifugation at 2000 rmp to obtain protease hydrolyzed beef broth. The broth produced in this way greatly increases the nutrients required for bacterial growth, reduces waste of raw materials, and significantly improves the taste and mouth feel of the culture medium and the bacterial culture obtained after fermentation.

In order to prepare crude trypsin extracts for hydrolyzing beef and beef liver tissues, pancreatic tissue homogenates of animals such as cattle, horses, sheep or pigs can be prepared first, and the resulting homogenates and anhydrous can be used in a ratio of 1: 1: 3. Ethanol and distilled water were mixed and stirring was continued for 72 hours. After stirring, centrifuge (500 rpm, 10 minutes) to collect the supernatant and adjust the pH of the resulting supernatant to 5.5 with concentrated hydrochloric acid to obtain the desired crude trypsin preparation.

As a specific example, the present invention specifically selects normal intestinal probiotics including Lactobacillus acidophilus, Bifidobacterium breve, and Streptococcus faecium, which are distributed in different sections of the normal human intestinal tract and have certain complementarities in physiological functions with each other. To prepare the nutritional composition or nutritional supplement containing the probiotic symbiotic fermentation culture of the present invention. Therefore, without departing from the basic ideas and principles of the present invention, the selection of two or more other probiotics of any genus or species to prepare a nutritional composition or nutritional supplement with a certain health function will all fall It is within the scope of the claims pending.

Generally speaking, the methods of preparing one or more probiotic preparations in the prior art are mostly the fermentation of bacteria of each species or strain, and when the bacteria multiply to the desired concentration, the live and / or dead bacteria are collected. Biomass, mixed bacterial suspension or lyophilized bacterial powder obtained after lyophilization, and adding one or more nutrients or matrix components to make a medicine or nutritional composition or supplement with certain therapeutic or preventive effects . However, from the perspective of industrialized large-scale production, a common disadvantage of these methods is that the production process is complicated, The cost is high, and it is difficult to find the optimum ratio of bacterial concentration when mixing various strains or strains.

The inventors found in the practice of using traditional fermentation cultures of Enterococcus faecium, Bifidobacterium, and Lactobacillus acidophilus to produce oral nutritional solutions containing these three probiotics, using the same medium and similar In the case of the culture conditions, the Enterococcus faecium (o) used to prepare the nutrient solution had a rapid proliferation rate at the beginning of fermentation, that is, the bacteria began to rapidly proliferate at the 3rd hour, but the number of bacteria reached only 6.5 hours. Reached about 1/2 of the peak. After the 13th hour, the bacterial proliferation rate began to increase slowly, and at 23 to 25 hours, it was relatively stable at the level of 2.7 x lOVml (see Figure 1). It can be seen from FIG. 1 that Lactobacillus acidophilus (oral) and Bifidobacterium breve (▲), which are separately fermented, have different proliferation curves from Enterococcus faecium. L. acidophilus proliferates slowly and enters a plateau soon after reaching a high peak, followed by a relatively gentle rising phase. The proliferation rate of Bifidobacterium breve is also relatively gentle, reaching a peak at about 15 hours, but it quickly decreases after 16-17 hours and tends to a stable proliferation level similar to that of Streptococcus faecium (about 3.0 x 10 OVml). As shown in FIG. 1, when the respective three kinds of fermentation alone, although the peak levels at different times, but into a stable state, each of which does not exceed the number of cell proliferation 3. 0 χ 10 ⁸ cells / ml. Therefore, the method of separately fermenting the three bacteria is not only cumbersome in terms of process operation, but also the number of bacteria proliferation is limited when the medium is used for fermentation production.

In view of the shortcomings of the prior art, the inventor tried to properly adjust and control the pH value and temperature of the culture during the fermentation without changing the culture medium, and explored and established three kinds of cells to be graded and seeded in the same culture medium. Symbiotic fermentation method. The detailed steps of this method are described as follows:

First provide sterilized, basically 25% soybean sprout cooking liquor, 2.5 warm white enzyme hydrolyzed beef broth, 1.3% yeast extract, 1.3% glucose, 1.3 sucrose, A liquid medium consisting of 0.03% NaCI, 0.05% MgS0 ₄ , 0.05% CaC0 ₃ > 0.03% K ₂ HP0 ₄ and 0.03% KH ₂ P0 ₄ .

Under the conditions of about pH 7.0 and temperature ⁴⁰ ° C, Bifidobacterium breve is inoculated into the medium at a ratio of 0.5 to 1.0% of the total weight of the medium, and fermented at a temperature of about 38-40 ° C for 6 to 8 hours. After fermentation for about 6 to 8 hours, the concentration of Bifidobacterium breve in the culture reached about 3.5 x 10 ⁸ / ml. Then, the culture medium is inoculated with about 0.5-1.0% of the wet biomass of Lactobacillus acidophilus, and the fermentation is continued for 4-8 hours after stirring. When the concentration of Lactobacillus acidophilus in the fermentation culture reaches approximately 3.0 X 10Vml, or the total number of bacteria of Enterococcus faecium and L. acidophilus reaches approximately 6. 5 X lOVml, and the pH of the fermentation culture drops to 5.0 -5.5 hours, adjust the pH to about 5.8-6. 3 with a 0.5N NaOH solution, and then enter Enterococcus faecium into the above culture again at a ratio of 0.5 to 1.0%, and Incubate at the same temperature for 4-8 hours.

When the fermentation process is carried out for about 24 hours and the total bacterial concentration reaches about 6 × 10 OVml or more, the temperature of the fermentation system is reduced to about 30-34 by controlling the temperature of the circulating water, and maintained at this temperature for 2-4 hours to The metabolic transformation of the inherent components of the culture medium by the living microorganisms in the reaction system is completed. When the pH of the culture is reduced to about 3.8-4.2, the temperature of the system is raised to about 49 3, killing all living bacterial cells to obtain heat-killed Lactobacillus acidophilus, Bifidobacterium Bacillus and Enterococcus faecium and their metabolites, and a total fermentation culture containing soluble 'raw dietary fiber, soy isoflavones and soy oligosaccharides provided by a fermentation medium.

It is particularly surprising that using the graded inoculation and mixed symbiotic fermentation method of the present invention not only greatly reduces the operation steps, but also significantly increases the proliferation rate of the three probiotics. As mentioned earlier, when three bacteria were fermented and cultured in the same medium, although they each showed different proliferation curves, the bacterial concentration of each bacteria did not exceed 3.0 when cultured for about 24-25 hours. x lOVml (see Figure 1). However, according to the method of the present invention for gradual inoculation and symbiotic fermentation, three kinds of bacteria are cultured at the same time. These bacteria not only do not interfere with each other and inhibit symbiosis, but it seems that there may be some mutual synergy and growth promotion effect. From the curve shown in Figure 2, it can be seen that three probiotics are inoculated sequentially in the same medium, and the three bacteria are symbiotically fermented under the same fermentation conditions, and cultured to about 24-25 hours of culture The total bacterial concentration is as high as 11 X lOVml, which obviously exceeds the total bacterial concentration when the three bacteria are fermented separately. Tube single-phase addition. Although not constrained by theory, we speculate that the reason for this phenomenon of in vitro symbiotic proliferation may be due to the metabolic complementarity of the three probiotics, that is, the metabolites of one of the bacteria (such as Bifidobacterium) and acetic acid and Lactic acid) will be more conducive to the growth of another bacteria (such as Lactobacillus acidophilus) inoculated later. In addition, proper pH adjustment during fermentation is also one of the important factors that promote the symbiosis of three bacteria with different tolerances to acids in the same environment. It is reasonable to believe that the multi-stage probiotic inoculation and symbiotic fermentation method of the present invention is also applicable to the symbiotic cultivation of more species (such as five, seven, or more) probiotics under simulated intestinal survival conditions to prepare A variety of live or dead probiotics and other nutritional medicines or nutritional compositions.

Fermented cultures containing heat-killed Lactobacillus acidophilus, Bifidobacterium breve and Enterococcus faecium can be directly prepared according to the method of the present invention as a nutritional supplement for restoring and maintaining the normal flora of the gastrointestinal tract and / or Improve body specific or non-specific immune function (including anti-tumor immune function).

Alternatively, after the fermentation is complete (about 24 hours) and before the temperature of the fermentation system is increased to kill the bacteria, a total amount of bacterial culture suitable for processing or transformation by the bacteria or its metabolites may be added to the total bacterial culture. One or more drugs or mixtures of any natural origin of known or unknown structure. Under fermentation conditions close to physiological conditions (37, pH 6.0), one or more components in these natural drug extracts will undergo structural changes or metabolic transformation under the action of anaerobic probiotics or their metabolites, Transform the original inactive natural drug compound into an active compound, or modify some originally weak molecules into molecules or aggregates with stronger biological activity, or make some originally have strong physiological toxicity Compounds (especially natural compounds of animal origin) become less or non-toxic compounds or aggregates thereof.

Furthermore, as known to those skilled in the art, after completing the fermentation step according to the stepwise inoculation and symbiotic fermentation method of the present invention, the live bacteria can be centrifuged and subjected to freeze-drying treatment according to conventional methods to prepare live probiotics. Thousands of dry powder. Such powders can be mixed with appropriate carriers or excipients to prepare tablets, emulsions, suspensions, capsules, suppositories, syrups and other live formulations in different dosage forms. For example, in the case of tablet preparations In the following, natural mineral powders such as kaolin and talc, or synthetic mineral powders such as silicates, and sugars such as sucrose, lactose, and glucose can be used as solid carriers. Alkyl sulfonates or aryl sulfonic acids can be added according to different dosage forms. Emulsifiers such as salt, dispersants such as lignin, carboxymethyl cellulose, starch, and polyvinylpyrrolidone, and lubricants such as magnesium stearate, talc, stearic acid, and sodium dodecanoate. The preferred dosage form of the probiotic live bacteria preparation is a solid preparation such as a tablet or a powder or a suppository, especially a tablet. In the case of solid preparations, preferred excipients are maltodextrin, microcrystalline cellulose, corn starch, fructose, lactose and dextrose. In addition to commonly used excipients, additives such as sodium citrate, calcium carbonate, and calcium dihydrogen phosphate can also be added. In the case of preparing a tablet pharmaceutical composition, it may further contain an anticholinergic, an antihistamine, an analgesic, an adrenergic, an anti-inflammatory agent, a liver-protective agent, an anti-lipid agent, and an anti-tumor agent.

In order to improve the survival rate of Lactobacillus acidophilus and other probiotics, L-cystine, vitamins (such as vitamins A, C, D, and E), minerals (such as calcium carbonate, magnesium oxide, and carbonic acid) can be added to the tablet Magnesium), lactose, whey protein concentrate, autolyzed yeast extract, chitosan, lecithin, and soluble or non-soluble dietary fiber (such as apple fiber or cereal fiber). The addition of L-cystine is mainly to reduce the oxygen in the solid tablets to extend the survival time of bacterial cells. Vitamins are added primarily to take advantage of their antioxidant properties. It is known that dietary fiber has excellent protective and stimulating effects on the growth of Lactobacillus acidophilus in the presence of minerals. Autolyzed yeast extracts are not only used to stimulate the growth of bacteria in the gastrointestinal tract, but are also a major source of vitamin B. It has been reported that vitamin D and lactose play an important role in the absorption of calcium in the human gastrointestinal tract. In addition, lactose also utilizes the growth of Lactobacillus acidophilus in the intestine. Evidence has shown that chitosan as a dietary fiber of animal origin has the effect of promoting calcium absorption in the intestine. Lecithin is mainly used as an emulsifier and lubricant to facilitate the preparation of oral tablets.

Generally speaking, oral tablets containing lyophilized live lactic acid bacteria, bifidobacteria and enterococcus faecium each weigh about 500-1500 mg, preferably 750 mg, and the weight of the bacteria in each tablet is about 30- 80%. The following examples are intended to further illustrate the advantages and applications of probiotic preparations prepared according to the method of the present invention, and these examples do not in any way limit the scope of the claims to be issued of the invention. Examples

Example 1: Preparation of a probiotic symbiotic fermentation culture

This example is intended to exemplify a method for producing a mixed symbiotic fermentation culture of Lactobacillus acidophilus, Bifidobacterium breve, and Enterococcus faecium in the same culture medium by using a step inoculation and a symbiotic fermentation method.

1. Selection of probiotic strains

Bifidobacteria are the most important physiological bacteria in humans and mammals, and are mainly distributed in the lower intestine and colon. It is known that Bifidobacteria play an important role in human nutrition, growth, development and immune function. L. acidophilus is the most widely distributed probiotic in the human body. In the intestinal tract, the bacteria are mainly distributed in the peripheral intestine. Lactobacillus acidophilus participates in the biosynthesis of vitamins, especially B vitamins, and has the functions of assisting food digestion, promoting nutrient metabolism, and improving host tolerance to lactose. Enterococcus faecium is mainly distributed in the cecum of the small intestine, and has the function of helping cholesterol catabolism and lowering blood cholesterol. Therefore, the present invention selects three common probiotic strains such as Bifidobacterium breve, Lactobacillus acidophilus, and Enterococcus faecium, mainly based on their overall widespread distribution in the human intestinal tract and the complementarity of their physiological activities.

These bacteria can be purchased from bacteria collection agencies such as China Type Culture Collection (CCTCC) (Wuhan, China), or isolated from normal human feces or intestinal mucosa.

2.Media composition and preparation

Medium composition: Each liter of medium contains 25ml protease hydrolyzed beef broth, 15g yeast extract, 250ml soybean sprout cooking solution, 12g glucose, 13g steamed sugar, 5g MgS0 ₄ , 3g CaC0 ₃ , 3g NaCI, 3g K ₂ HP0 ₄ And 3g KH ₂ P0 ₄ .

Prepared for use in formulating this product, essentially following the methods outlined in the detailed description of this description Proteolytically hydrolyzed beef broth and soybean sprouts (or germinated soybeans) cooking fluids for specific media. After the ingredients of the culture medium are mixed uniformly, they are autoclaved at 121Ό for 30 minutes for use.

3. Grading inoculation and symbiotic fermentation method

100 liters of the sterilized medium was heated to about 40 ° C, and the wet weight of the biomass accounted for about 0.8% of the total weight of the medium / W)) inoculated the activated Bifidobacterium 5 小时。 1000g (wet weight), and anaerobic fermentation at 39 ° C for 7.5 hours. When the pH dropped to 5.0 during the fermentation, an appropriate amount of 0.5N NaOH was added dropwise to the medium under continuous stirring to adjust the pH to approximately 6.2, and the medium was again inoculated with activated Enterococcus faecium The biomass was about 950 g (wet weight) and stirred for 20 minutes. The fermentation was continued at the same temperature for 6 hours. When the pH in the fermentor dropped to about 5.5, 0.5N NaOH solution was added to the medium again to adjust to 6.2, and then inoculated with about 850 g (wet weight) of Lactobacillus acidophilus biomass, and continued Fermented at the same temperature. When the total bacterial concentration in the fermentation tank reaches more than 6.5 X 108 / ml, and the pH is lowered to about 3.5-3.8, the temperature of the fermentation culture is reduced to about 32 ° C by controlling the temperature of the circulating water, and Hold for about 2 hours to complete the biotransformation of the fermentation product. After the fermentation is completed, according to the purpose of using the probiotic symbiotic fermentation culture thus obtained, (1) centrifugally collect the live probiotics of the symbiotic fermentation and freeze-dry them to prepare a solid medicine or nutrition containing lyophilized bacteria powder Supplementary composition; (2) adding a natural drug extract to be transformed to a culture including a culture medium and live bacteria and their metabolites to prepare a pharmaceutical composition having specific biological activity and function; (3) allowing fermentation The temperature of the culture was raised to about 39 ° C, and the proliferated viable bacteria were killed by heat. After the culture supernatant was centrifuged or a nutritional supplement composition containing probiotic symbiotic fermentation products and bacterial fragments was directly prepared.

The biochemical analysis of the supernatant of the symbiotic fermentation culture showed that the content of hydrolyzed amino acids in the culture supernatant reached 23. 322 mg / ml, which can meet the requirements for the growth of bifidobacteria and lactobacillus and contains disulfide-bonded cysts. The amino acid content is 1.8 mg / ml (hydrolyzed) and 0.33 mg / ml (free). The total sugar content in the supernatant of the symbiotic fermentation culture was about 0.04 mg / ml, in addition to reducing sugars, there were also oligosaccharides and dietary fiber required for the growth and reproduction of probiotics such as bifidobacteria. The latter has an effect on inhibiting the rise of blood sugar in the intestine and preventing neutrality Fat and cholesterol absorption and relief of constipation. In addition, it was extracted with ethyl acetate and used

Analysis by HPLC (255nm), the bacterial transformation product of soybean isoflavones in the culture supernatant, namely daidzein, was measured up to 80 μg / mL. Furthermore, the culture supernatant also contained a variety of vitamins and a certain amount of minerals. Among them are the B vitamins (about 40 mg / l), minerals such as P, Fe, Zn, Mg, K and Na (respectively 5. 04, 5. 36, 8. 29, 88. 83, 783, 283 mg / l). These test results further indicate that the supernatant of the symbiotic fermentation culture can be used as a medium to provide all the nutrients required for bacterial growth. After proliferation of the bacteria to a certain extent, due to the large amount of cell metabolism acidic products (organic acid), it led to a value _P H drop culture system (pH 4. 0 or lower), and further stop the growth of the bacteria. Example 2: In vitro bacteriostatic and bactericidal activity of probiotic cultures against intestinal pathogenic bacteria This example aims to illustrate the symbiotic fermentation culture of bifidobacteria, Lactobacillus acidophilus and Enterococcus faecium prepared by the method of the present invention In vitro bacteriostatic and bactericidal effects of certain substances on certain known intestinal pathogenic bacteria.

1. Using the D. dysenteriae, D15 dysentery carrying a drug-resistant plasmid, multi-resistant E. coli and Salmonella typhimurium as target bacteria, the probiotic symbiotic fermentation culture of the present invention (sample No. IV) and A single probiotic fermentation culture prepared in a similar way (samples I, Π, and ffl) has in vitro bacteriostatic activity against these pathogenic bacteria. First, use a hole punch to punch holes on a nutrient agar plate (diameter 6 μm), and add an 18-hour broth culture solution of the above pathogenic bacteria diluted 105 times with physiological saline to each experimental well. After incubating the plate 37 for 2 hours, 0.1 ml of a probiotic culture supernatant of single fermentation or mixed symbiotic fermentation was added to each experimental well. 37 anaerobic incubation for 24 hours, then take out the agar plate to observe and record the diameter (face) of the bacteriostatic ring around the well. The results are shown in Listing 1 below. Table 1 In vitro bacteriostatic activity of symbiotic fermentation culture and single bacteria fermentation culture on intestinal pathogenic bacteria

Sample

Pathogenic bacteria # 1 #ffl #IV

Pestridium freundii 8. 0 *-― 13. 0

D15 dysenteriae--― 11. 5 Multidrug-resistant Escherichia coli-― 8. 5 Typhoid bacteria--1 10. 5

* The numbers in the table are the diameter of millimeters (mm) of the bacteriostatic ring of the culture supernatant. From the data given in Table 1, it can be seen that under the experimental conditions that simulate the intestinal environment (the pathogenic bacteria dilution solution contains 0.1% bile salt), a plurality of strains of probiotics symbiotic fermentation culture liquid prepared according to the method of the present invention The diameter of the bacteriostatic ring of the supernatant against the four common intestinal pathogenic bacteria reached 8. 5-13. 0 let. However, under the same conditions, the culture supernatant of each of the three bacteria hardly showed such a ring of inhibition. The experimental results show that the probiotic symbiotic fermentation culture prepared according to the method of the present invention has very significant bacteriostatic activity in vitro.

2. The common intestinal pathogenic bacillus dysenteriae, multidrug-resistant Escherichia coli, Salmonella typhimurium and Staphylococcus aureus are used as target bacteria, and the probiotic symbiotic fermentation culture of the present invention is used to detect these pathogens by the pouring plate method. In vitro bactericidal activity of microorganisms.

First, about 4 μl of the pathogenic bacteria suspension 105 bacteria / ml was added to a test tube containing 4 ml of the supernatant of the probiotic symbiotic fermentation culture prepared according to the method of the present invention, and incubated at 37 ° C. for about 18 hours. After pre-cultivation, at 0, 2, and 6 hours of the test, 1 ml of the test sample was added to 10 ml of molten nutrient agar, mixed and poured onto a glass plate. Place the plate in a 37 incubator for 24 hours and observe and record the number of bacterial colonies formed. As a control, physiological saline (NS) was used. Table 2 In vitro bactericidal activity of probiotic symbiotic fermentation cultures against intestinal pathogens, typhoid bacillus, dysentery, escherichia coli, staphylococcus aureus

+ Culture + NS + culture + NS + culture + S + culture + brine

0 288 * 890 1080 1200 1248 1800 1302 1460

2 0 1120 0 1140 62 1950 1050 1540

6 0 1320 0 1260 0 1920 620 1488

* Values are the corresponding pathogenic bacteria plus probiotic symbiotic fermentation culture (experimental group) or physiological saline

(Control group) The number of pathogenic bacteria colonies formed on the nutrient agar plate after incubation for different time (0.2, 6 hours). It can be seen from the results shown that after contacting the gastrointestinal pathogens with the probiotic symbiotic fermentation culture prepared according to the method of the present invention for about 2 hours, all Salmonella typhimurium and Escherichia typhimurium dispersed in a nutrient agar medium Almost all of the dysentery dysentery and drug-resistant E. coli were killed. After about 6 hours of contact, Staphylococcus aureus, which was less sensitive to most of the co-fermented cultures, was also killed. The experimental results show that the probiotic symbiotic fermentation culture prepared according to the method of the present invention has very significant in vitro bactericidal activity. Example 3: Probiotics co-fermented culture supernatant promotes in vitro growth of intestinal probiotics

This example is intended to exemplify the effect of a probiotic co-fermentation culture prepared according to the method of the present invention on B. longum, B. bifidum, and L acidcilus. Growth promotion in vitro.

Lml (the concentration of bacteria in the culture medium after the inoculation was 106 cells / ml) was used as the experimental bacteria using Bifidobacterium longum, Bifidobacterium bifidus, and Lactobacillus acidophilus as experimental bacteria. Do not inoculate (1) conventional PTYG medium (5 _{g of} tryptone, 5 _g of soy peptone, 1 _g of yeast extract, 10 g of glucose, 40 ml of saline solution, 0.5 g of cysteic acid hydrochloride per liter of medium) 80 0.1ml, 0.1% resazurin 1ml); (2) lack of yeast extract or (3) lack of tryptone and soy peptone, or (4) lack of peptone and yeast extract, but both A probiotic symbiotic fermentation culture supernatant replaces these components in the PTYG medium and a pure symbiotic fermentation culture supernatant (as a medium). After inoculation, test tubes were incubated at 37 ° C for 24 hours. After the culture, the culture solution was collected, and a 1/100 dilution of 0.1 ml was used to make a Hungate anaerobic roller, and the culture was continued at 37 ° C for 48 hours, and then the colony was counted. The results are shown in Listing 3 below.

Medium Bifidobacterium longum Bifidobacterium longum Lactobacillus acidophilus probiotic symbiotic culture 1. 53 X 10 ⁸ 1. 03 X 10 1. 04 10 'Full-component PTYG medium 1. 39 X 10 ⁸ 1. 22 X 10 8 x 10 ⁸

Lack of yeast extract

PTYG symbiosis 2. 0 X 10 ⁸ 24 X 10 10 1.2 X 10 ^! PTYG + lacking peptone

Symbiotic culture 1. 37 X 10 ⁸ 6. 4 X 10 ⁹ 4. 5 X 10 ^! Lack of peptone and yeast extraction

PTYD + symbiotic culture 1.67 X 10 ⁸ 9 · 7 X 10 ^! 3. 0 X 10 ⁸ It can be seen from the data shown in Table 3 that known probiotics such as Bifidobacterium and Lactobacillus acidophilus are known. After inoculating the bacteria in the supernatant of the probiotic symbiotic culture of the present invention without any conventional culture medium components, the number of inoculated bacteria grew to more than 10 ⁸ cfu / ml after being cultured for 48 hours. On the other hand, the probiotic symbiotic fermentation culture supernatant of the present invention is used instead of conventional PTYD culture. The carbon source, nitrogen source, and mineral source material in the nutrient base, and the number of cells of the three tested bacteria after the culture were also above 10 ⁸ cfu / ml. Therefore, it can be considered that the supernatants of various probiotic bacterial symbiotic fermentation cultures prepared according to the method of the present invention have obvious growth promotion effects on the main probiotics in the normal human intestine. Although the relevant mechanism is not clear, it is speculated that this probiotic growth promotion activity and the culture contain oligosaccharides (including fructooligosaccharides, galactooligosaccharides, and soybean oligosaccharides) as carbon sources (the so-called " Bifido factor "), hydrolyzed amino acids (including amino acids containing disulfide bonds such as cystine), minerals (calcium, magnesium, phosphorus, potassium, etc.), B vitamins (Bl, B2, B6) and other nitrogen source substances related. Example 4: Effects of probiotic symbiotic fermentation cultures on non-specific immune function in mice This example aims to observe and evaluate the present invention by means of a mouse macrophage phagocytosis test (in vivo method) and a mouse delayed-type footpad response test Of Probiotic Symbiotic Fermentation Cultures on Nonspecific Immune Function in Mammals.

1. Mice! ^ Macrophage phagocytosis test (in vivo method)

A healthy Balb / c mouse weighing about 20-25 g was used as an animal model, and chicken red blood cells were used as phagocytic cells. The cell phagocytosis test was basically performed according to the conventional methods described in the literature. To put it simply, the animals were randomly divided into an experimental group and a control group (10 animals in each group). After routine group raising for 3 days, the animals of the experimental group were administrated with a probiotic symbiotic fermentation culture prepared in accordance with the present invention every day. The stock solution (supernatant) was 0.5 ml, which was continuously administered for 15 days. Animals in the control group were fed the same volume of saline. Fifteen days later, the animals were treated by neck dissection and peritoneal phagocytic cells were collected. Chicken erythrocytes washed with physiological saline were used as target cells. The erythrocyte phagocytic numbers of peritoneal macrophages in the experimental and control animals were counted and recorded under a high-power microscope. Percentage phagocytosis (%) and phagocytic index of macrophages. The results are shown in Listing 4 below. Group (n = 10) Percentage phagocytosis rate (%) Phagocytic index (X ± SD) Experimental group 88.2 ± 5. 51 5. 27 ± 0.556

Control group 67.7 ± 8. 08 24. 2 ± 0.62

2. Delayed footpad response in mice

A healthy Kunming mouse weighing about 20-25 g was used as an animal model, and a delayed-type footpad response test was performed according to a conventional method. To put it simply, the caged group animals were continuously fed with the probiotic symbiotic fermentation culture prepared according to the method of the present invention for a total of 12 days (0.5ml stock solution / piece / day), and on the left hind foot of the mouse on the 13th day 0.1ml chicken red blood cells (103 cells / ml) were injected into the pad. After 96 hours, animals were challenged with the same amount of chicken red blood cells on the right hind footpad. Before and 24 hours after the challenge, the thickness of the right hind footpad of the mouse was measured under a stereo microscope, and the thickness of the footpad (mm) was calculated therefrom. The same volume of physiological saline was used as a control. The results are shown in Listing 5 below. Table 5.Effects of probiotic symbiotic fermentation cultures on delayed footpad response in mice

Group (n = 10) Foot pad thickness (mm) Experimental group 1. 89 soil 0. 43 *

Control group 0.98 ± 0.40

* The values given are the mean ± standard deviation of 10 animals. P <0.01. The ability of macrophages to engulf foreign bodies represents one of mammalian non-specific immune functions, and the delayed footpad allergy reflects mammalian cellular immune status. From the data shown in Tables 3 and 4, it can be seen that the probiotic symbiotic fermentation culture of the present invention shows good non-specific immune function enhancing activity, so it can be used clinically as an immune enhancer.

Claims

Rights request

1. A suspension liquid nutrition composition containing inactivated or heat-killed one or more intestinal probiotics, and containing soluble dietary fiber and soy isoflavones.

2. The nutritional composition according to claim 1, wherein said probiotic is selected from the group consisting of Lactobacillus, Bifidobacterium, Streptococcus, Enterococcus, Propionibacterium, Leuconostoc, Bacillus One or more non-pathogenic bacteria in the human intestine.

3. The nutritional composition according to claim 1, wherein said probiotic is selected from the group consisting of Bifidobacterium bifidus, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus bulgaricus, cheese Lactobacillus, Lactobacillus delbrueckii, Lactobacillus plantarum, Streptococcus thermophilus, Enterococcus faecalis, Enterococcus faecium, Lectobacillus citric acid, Bacillus subtilis and Bacillus licheniformis.

4. The nutritional composition according to claim 1, wherein said soluble fiber content is about 20-40 μg per liter, and wherein said soybean isoflavone content is 30-100 μg per liter.

5. A method for producing a suspended liquid nutritional composition as defined in any one of claims 1-4, the method comprising:

(2) According to the proportion of the biomass of the wet bacteria in the total weight of the culture medium of about 0.5 to 1.0%, two or more intestinal probiotics to be cultured are sequentially inoculated into the culture medium, and each time After injecting a bacterium, the anaerobic culture is performed at a temperature of 38-41 ° 0 and a temperature of 116.6 for 4-10 hours;

(3) When the fermentation culture of the total bacterial concentration of 7 x l0 ⁸ / ml or about the temperature to about 32 ° C and maintained for 2-4 hours, to complete the metabolic transformation of the microorganism;

(4) When the pH of the fermentation culture is reduced to approximately 3.5-3.9, the temperature is increased to 48-50 ° C and maintained for 4-6 hours to complete the heat inactivation of the bacteria and stabilization of the fermentation system .

6- The method according to claim 5, wherein said liquid culture medium is made up of about 25% Bean sprouts decoction, about 2.5% trypsin hydrolyzed beef broth, about 1.3% yeast extract, about 1.3% glucose, about 1.3% sucrose and appropriate amounts including Mg Ca ^, K +, fe ⁺ Consisting of minerals.

7. The method according to claim 5, wherein said intestinal probiotics are selected from the group consisting of Bifidobacterium bifidus, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus bulgaricus, cheese Lactobacillus, Lactobacillus delbrueckii, Lactobacillus plantarum, Streptococcus thermophilus, Enterococcus faecalis, Enterococcus faecium, Leuconostoc citric acid, Bacillus subtilis and Bacillus licheniformis.

8. Use of a nutritional composition as defined in any one of claims 1-4 in correcting and maintaining the normal microecological balance of the intestine.

9. Use of a nutritional composition as defined in any one of claims 1-4 in regulating a body's non-specific immune response.

10. The use of the nutritional composition as defined in any one of claims 1 to 4 for biotransforming or modifying the active ingredients of one or more natural medicines and to make them exert or improve their biological activity.