CN1273838A - Nutritive composition containing symbiotic fermentation culture medium of dead probiotics - Google Patents
Nutritive composition containing symbiotic fermentation culture medium of dead probiotics Download PDFInfo
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- CN1273838A CN1273838A CN00111082A CN00111082A CN1273838A CN 1273838 A CN1273838 A CN 1273838A CN 00111082 A CN00111082 A CN 00111082A CN 00111082 A CN00111082 A CN 00111082A CN 1273838 A CN1273838 A CN 1273838A
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- lactobacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
A nutritive composition containing deactivated or dead probiotics and its metabolism resultant for recovering and maintaining the health and relative phisiological functions of gastrointestinal tract is disclosed. The process for preparing it features that in same culture medium and under same fermenting condition, a classified inoculation and symbiotic fermentation method is used to culture and reproduce more probiotics. Said mixed probiotic culture resultant can be used to prepare the nutritive composition for recovering and maintaining the balance of normal microbial pools in gastrointestinaltract, improving specific or non-specific immunity and resisting against tumor.
Description
The present invention generally relates to and is used to recover and keeps gastrointestinal tract health and relevant physiological function, contains the alimentation composition, its production method of probiotic bacteria deactivation or dead and metabolite thereof and is improving the intestinal microbial population balance and stimulating application in specificity or the non-specific immunity.The present invention be more particularly directed in same culture medium and same fermentation condition under, cultivate and breed the method for multiple probiotic bacteria with classification inoculation, symbiotic fermentation method, and said probiotic bacteria mixed culture preparation be used for recovering and keep gastrointestinal tract normal flora balance, the alimentation composition that improves body specificity or non-specific immunity and anti-tumor activity uses.
All disturb hosts' factor, and though be physics, chemistry, still biological capital causes the dysequilibrium of little ecology.Human body for health, usually may be because the variation of organismic internal environment such as operation (particularly abdominal operation), wound, stress, tumor, bile salt-stimulated and abuse of antibiotics or external environment causes microorganism species disorder in the intestinal, the dysequilibrium between promptly useful and harmful intestinal microecology.Wherein, causing the most important factor of the normal microorganism species imbalance of intestinal is long-term abuse of antibiotics.In case the sweat of normal flora is damaged, can causes the minimizing of many beneficial bacterias, and then make mucous membrane of colon depletion.Simultaneously, because of the ramp grievous injury mucous membrane of colon function of potential malignant bacteria.Noxious bacteria penetrates the colon wall of malfunction and infects organ, causes the abscess of organ or most organ dysfunctions are weakened even loses.In the past, people usually used the secondary infection due to various antibiotic prophylaxis or the normal group of treatment alteration of intestinal flora, the particularly abdominal operation rear intestinal dysbacteriosis.Yet long-time a large amount of to use antibiotic not only to spend very high, and be difficult to correct normal alteration of intestinal flora, particularly often causes the state of an illness further to worsen because of the undue growth of pathogenic bacterium.
In order to adjust normal intestinal microecological balance, a kind of generally accepted method be screening and utilize people known, in normal gastrointestinal tract, preponderate and non-pathogenic avirulence antibacterial that can field planting on mucous membrane of small intestine, add or do not add dietary fiber, oligosaccharide or auxiliary compositions such as immune coccus albumen, sulfur-containing amino acid, make pharmaceutical composition or supplementary, come into operation and carry out the patient of the little ecological disturbance treatment of normal intestinal in needs.A large amount of laboratory research and clinical observations prove, the microecological balance therapy can be for improving and keeping the gastrointestinal bacterial flora balance, improve body specificity or nonspecific immunity and reduce cholesterolemia and bring into play irreplaceable effect.
Existing many prior art document descriptions the therapeutical effect and the application thereof of probiotic bacteria.For example, Fernandes has commented in detail with food or fermented dairy product mode and has introduced intravital lactobacillus, the prebiotic effect of lactobacillus acidophilus (Fernandeset al. particularly, " Therapeutic role of dietary lactobacilli and lactobacillic fermented diary products ", FEMSMicrobiology Reviews 46:343-356,1987).Nobuo Saegara studies have shown that, oral streptococcus faecalis can be improved the lipid metabolism (Microecology and therapy, vol.15,271-280,1985) of humans and animals significantly.European patent EP 0199535 has been described from feces isolating, in vitro tests proof can be on intestinal mucosa the pure culture of adherent acid lactobacillus.International monopoly WO89/05849 has described from the Gaster Sus domestica intestinal isolating, can adhere on the Gaster Sus domestica intestinal epithelial cell, and strong acid and bile be had the lactobacillus of toleration.Can use said antibacterial to make fermented milk products, and in order to prevent or to treat the coliform diarrhea of piglets.On the other hand, United States Patent (USP) 4,805,368 disclose a kind of probiotic composition that contains lactobacillus acidophilus and/or bifidobacterium bifidum, leuconostoc cremoris (Leuconostoc citrovorum) and Xie Shi propionibacterium (Propionibacterium shermanii) based on dietary fiber and preparation method thereof.United States Patent (USP) 4,913,913 have described mixing or have cultivated cheese lactobacillus (L.casei) and bifidobacterium longum (B.longum) respectively, contain the method for the lactic acid bacteria fermentation milk of bacillus bifidus with preparation.United States Patent (USP) 5,716,615 disclose and have comprised streptococcus thermophilus, lactobacillus and bacillus bifidus, are used for the treatment of the pharmaceutical composition of gastroenteropathy and hypercholesterolemia.United States Patent (USP) 5,744,134 also described and contained immunoglobulin and water soluble dietary fiber, multivalence ferrum chelating molecule and gluconic acid, and one or more beneficial bacteria of intestinal tract, is used to promote the compositions of gastrointestinal tract health.At last, the inventor submitted to the Chinese patent 93101172.0 of also having authorized to disclose a kind of supplementary (i.e. so-called " SANZHU KOUFUYE ") that contains the dead lactobacillus acidoilus of heat kill (Lactobacillus acidoilus), bifidobacterium breve (Bifidobacterium breve) and enterococcus faecalis (Enterococcus faecium) in 1993.Different with other prior aries, though this suspension attitude supplementary does not contain probiotic bacteria alive, but it is believed that the bacterial enzyme and other opsonigenous substances that contain multiple beneficial in the said suspension, and owing to used the medium component that is rich in soluble fiber and soybean isoflavone in its production process, so the survival and the growth that utilize antibacterial are not only arranged, and provide these and other beneficiating ingredient for said nutritional blend.
Yet, no matter be to produce the compositions that contains alive or dead multiple probiotic bacteria, and no matter only contain the compositions that one or more antibacterials still contain other auxiliary elements in addition, above-mentioned prior art all be not described in the same culture medium and same fermentation condition under, cultivate and breed the method for two or more beneficial bacteria of intestinal tract with classification cultivation and symbiotic fermentation technology.
Therefore, an object of the present invention is to provide and a kind ofly contain one or more probiotic bacterias deactivation or that heat kill is dead, and contain the suspension attitude alimentation composition of water soluble dietary fiber and soybean isoflavone.
This purpose preferred embodiment according to the present invention, wherein said probiotic bacteria are selected from one or more people's intestinal non-pathogenic bacterias of Bifidobacterium, Lactobacillus, Streptococcus, Enterococcus, propionibacterium, Leuconostoc and bacillus.
This purpose preferred embodiment according to the present invention, wherein said probiotic bacteria are selected from bifidobacterium bifidum, bifidobacterium breve, bifidobacteria infantis, bifidobacterium longum, lactobacillus acidophilus, Lactobacillus bulgaricus, cheese lactobacillus, Deshi Lactobacillus, Lactobacillus plantarum, streptococcus thermophilus, enterococcus faecalis, dung intestinal ball coccus, have a liking for citric acid leukonid, bacillus subtilis and bacillus licheniformis.
This purpose preferred embodiment according to the present invention, wherein said soluble fiber content is about every milliliter and contains 20-40 μ g, and wherein said isoflavone content is every milliliter of 30-100 μ g.
Another object of the present invention provides the method for the suspension attitude alimentation composition that production above limits, and this method comprises:
(1) provides the fluid medium of forming by Semen sojae atricolor or big bean sprout cooking liquor, proteolysis beef meat soup, yeast extract, sugar and mineral basically;
(2) account for the ratio of culture medium gross weight 0.5-1.0% according to the wet thallus Biomass, one or more antibacterials of inoculation in culture medium successively, and behind a kind of antibacterial of every access, under 38-41 ℃ temperature and pH6.6 condition, anaerobism was cultivated 4-10 hour;
(3) the thalline total concentration reaches 7 * 10 approximately in fermentation culture medium
8When/ml is above, make temperature reduce to about 32 ℃ and kept 2-4 hour, transform to finish biological metabolism;
(4) when the pH of fermentation culture medium reduces to about 3.5--3.9, elevate the temperature to 48-50 ℃ and kept 4-6 hour, to finish hot deactivation of thalline and fermentation system stabilisation.
This purpose preferred embodiment according to the present invention, wherein said fluid medium are to comprise Mg by about 25% big bean sprout decoction liquor, about 2.5% trypsin hydrolyzing beef meat soup, about 1.3% yeast extract, about 1.3% glucose, about 1.3% sucrose and appropriate amount
++, Ca
++, K
+, Na
+Mineral form.
This purpose preferred embodiment according to the present invention, wherein said Semen sojae atricolor or big bean sprout cooking liquor be germinated soybean in water by 30% (W/V) mixed and in 105-121 ℃ of following steaming and decocting 30 minutes, filter the filtrate that obtains through routine then.
A further object of the present invention provides above-mentioned alimentation composition in the application of correcting and keeping in the normal microecological balance of intestinal.
A further object of the present invention provides the application of above-mentioned alimentation composition in adjusting the body nonspecific immune reaction.
A further object of the present invention provides above-mentioned alimentation composition at biotransformation or modify the effective ingredient of one or more natural drugs and make it to bring into play or improve application in its biologic activity.
When accompanying drawing 1 shows independent fermentation culture bifidobacterium breve, enterococcus faecalis and lactobacillus acidophilus, the time dependent growth curve of three kinds of antibacterials Biomass separately.
Accompanying drawing 2 be presented in the same culture medium and same condition of culture under, when bifidobacterium breve, enterococcus faecalis and lactobacillus acidophilus mixobiosis are cultivated, the time dependent growth curve of total biomass.
The present invention relates to a kind ofly contain the dead beneficial bacteria of intestinal tract of heat kill, and the Soluble Fiber that is provided by microbiological culture media is provided And the alimentation composition of isoflavones. The invention further relates to classification inoculation and symbiotic fermentation technology, in same cultivation Cultivate the method for two or more probio under base and the similarity condition. After said alimentation composition oral administration comes into operation, can Effectively recover and keep the composition of gut flora balance, improve specificity or non-specific immune function and reduce the blood courage solid The alcohol level.
Present commercial probiotics preparation, what mostly declare to use is that a strain or many strains can tolerate highly acid (1.0) in the stomach With upper part of small intestine 1-1.5% cholate environment, and the probio that can settle down on the mucous membrane of small intestine surface. Yet, the grinding of these preparations Personnel processed have but despised the prebiotic effect of a large amount of metabolites of probio relatively. In fact, the microecology field is general at present Accept such viewpoint: for keeping the microecological balance of body, except enteron aisle normal bacteria colony, also have a class what is called " prebiotic substance ", comprise microorganism surface reactive material and a series of metabolite thereof, for example vitamin, antibody, peptide, Peptide glycan and amino acid etc. But these materials can suppress to reach in the intestines and stomach life of some poisonous bacterium in the fermented foodstuff effectively Long. Existing many some dead bacterium and its fragment such as cell membrane, enteral prods and opsonigenous substances pair of having experimental results show that The beneficial effect of humans and animals. For example the someone proves, killed lactic acid bacteria can adhere on the intestinal epithelial cell, and is logical Cross competitive exclusion and spatially suppress contacting of pathogenic bacteria and intestinal cell hair brush border binding site, thereby stop pathogenic bacteria First key step that bacterium infects is cut off in field planting in enteron aisle. The people such as Coconnier studies show that, heat kill is dead Lactobacillus acidophilus and lactobacillus bulgaricus to the Caco-2 cell (a kind of tissue and function for research people intestinal cell, Clone with typical intestinal cell differentiating characteristic) and mucous membrane secretion property HT29-MTX cell have the height adhesiveness, Thereby can stop by the cause of disease acceptor of blocking-up people intestinal cell pathogenic entero becteria gluing CaCO-2 and HT29-MTX cell Echo invasion and attack (Coconnier MH, J.DiarrhoealDes.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) find, discharges after the death of lactobacillus 104r cell line a kind ofly can suppress the intestines poison The material that plain type Escherichia coli K88 adheres at mucous membrane of small intestine, and prove this can be under the lysozyme effect inactivation and containing The material of glucose and galactolipin has just showed this when only constantly being discharged in the bacterial cultures by dead bacterium in incubation The inhibition activity of sample.
Also many researchs have been carried out with regard to the tumors inhibition activity of dead probio. Known enteric bacteria such as Escherichia coli and excrement intestines Coccus can produce the multiple precarcinogen that makes and be converted into carcinogenic enzyme, such as beta-Glucuronidase and nitroreductase etc. In addition, The lactic acid that the probio metabolism produces and SCFA (SCFA) can cause pH value reduction in the enteron aisle, makes these metabolic enzymes Activity is suppressed, thereby stops the formation of carcinogen in the enteron aisle. Moreover the probios such as lactic acid bacteria are to mutagenic matter The special combination ability is also brought into play certain anti-sudden change and tumour and is generated inhibitory action. Zhang Xuebin (J.Dairy Sci.73 (3): 1477-1481,1991) research is found, some lactic acid bacteria such as streptococcus cremoris and lactobacillus acidophilus, and The thalline composition of bifidobacterium bifidum (comprising endochylema and somatic cells wall framework ingredient) is to mutagens TRP-P-1 and N-nitrous The base dimethylamine has very strong binding ability. The people such as Sekine (Caneer Res., 45:1300-1310,1985) prove the baby The peptide glycan that separates in the bifidobacterium cells wall (WPG) has obviously tumor growth as a kind of BRM The ground inhibitory action. And studies show that further the antitumor activity of probiotics bacterial wall preparation (WPG) is because WPG Inducing cell factor expression and activating macrophage produce that a large amount of nitric oxides cause.
At last, with regard to the immunoregulation effect of dead probio, existing people finds that the dead lactic acid bacteria KVS20 of heat kill can activate The macrophage (Kitazawa, H.et al., J.Dairy Sci., 74:2082-2088,1991) of static and active stage. In addition, somebody Find, but dead streptococcus thermophilus stimulating expression of macrophage and the t helper cell of heat kill produces cell factor, and the TNF-alpha levels is increased Add about 135-176 doubly, the IL-6 level increases about 31-192 doubly (Marin ML, J.Food Prot.61C7): 859-864,1998).
Result of study before above-mentioned can find out, the dead bacteria preparation of probio be in theory or in practice all Prove effective. In addition, a problem that can not be ignored also should mentioning here is to have now found that to comprise that VREF exists Interior many traditional probio all may be carried the antibiotic resistance plasmid, and these plasmids may be under proper condition external Or enteron aisle in shift between intrinsic probio or the pathogenic bacteria. In case such antibiotic resistance plasmid changes pathogenic entero becteria over to In, just might cause serious adverse consequences, use the patient of live probiotics preparation to stay potential risk thereby give, this is also Inventor's probiotics preparation of determine using deactivation as one of reason of alimentation composition of the present invention.
In order to prepare the probiotics preparation as dead probio symbiotic fermentation culture medium of containing of alimentation composition or replenishers, Can select any two or more normal beneficial bacteria of intestinal tract of people known in the art. Said probio comprises but not Be only limited to and be selected from Bifidobacterium, lactobacillus, streptococcus, enterococcus spp, propionibacterium, Leuconostoc and gemma One or more people's enteron aisle non-pathogenic bacterias of Bacillus, particularly comprise but be not only limited to bifidobacterium bifidum, bifidobacterium breve, Bifidobacterium infantis, bifidobacterium longum, lactobacillus acidophilus, lactobacillus bulgaricus, cheese lactobacillus, Lactobacillus delbrueckii, Lactobacillus plantarum, streptococcus thermophilus, enterococcus faecalis, dung intestines ball coccus, have a liking for citric acid leukonid, bacillus subtilis and ground Clothing shape bacillus.
The culture medium that is used for the probio that fermented and cultured the present invention uses is by soybean or big bean sprouts cooking liquor, albumen basically Enzymolysis beef meat soup, yeast extract, sugar and mineral matter form. The culture medium that uses among the present invention is different from conventional bacterium and sends out The ferment culture medium is primarily characterized in that wherein except containing basic carbon source (dextrose plus saccharose), nitrogenous source (enzymolysis meat soup and ferment Female medicinal extract) and outside the mineral matter, the soybean or the big bean sprouts that account for the big weight ratio (about 25%) of fluid nutrient medium have also been added especially Cooking liquor. It is composed as follows to be used for typical culture medium of the present invention:
2.5% enzymolysis beef meat soup, 1.3% yeast extract, 25% big bean sprouts decoction liquor, 1.3% glucose, 1.3% sucrose, 0.05%MgSO4、0.05％CaCO
3、0.03％K
2HPO
4、0.03％KH
2PO
4, 0.03%NaCI. Above ratio is equal (W/W) is weight percentage.
What should particularly point out here is that the soybean in the culture medium or big bean sprouts cooking liquor mainly are not as nitrogenous source and/or carbon source Add. The purpose that adds soybean or big bean sprouts cooking liquor in the culture medium mainly is to provide or replenish big for probiotic's culture liquid Beans isoflavones, Soluble Fiber and soy oligosaccharide (such as stachyose and raffinose). Known isoflavones compounds (bag Draw together daidzein, genistein and Daidezin) as phytoestrogen, have Green Tea Extract, reduce blood fat, reduce The danger of angiocardiopathy, prevent the effects such as osteoporosis that climacteric, woman's endocrinopathy caused. Big bean sprouts leachate In contained solubility food fiber can stop neutral fat and cholesterol in the enteron aisle absorption, promote gastrointestinal peristalsis, prevent Constipation also delays or suppresses the absorption of carbohydrate. In addition, contained soy oligosaccharide can be by enteron aisle in the leachate of big bean sprouts Lactic acid bacteria is decomposed utilization, but is utilized by harmful intestinal tract bacteria hardly, thereby is conducive to correct intestinal bacilli illness. In a word, with Soybean or big bean sprouts cooking liquor have represented as one of main component of probiotic's culture based component and alimentation composition of the present invention An importance of the technology of the present invention feature. After testing, contained in the probio liquid culture that makes by the inventive method The concentration of isoflavones and Soluble Fiber is respectively 20-40 μ g/ml and 30-100 μ g/ml.
In order to prepare as the soybean of culture medium key component or big bean sprouts cooking liquor, at first with fresh ripe soybean about 2 Soaked 5-6 hour in the warm water of times volume, so that soyabean tissue's imbibition. Transposition is incubated 60-72 under 25-27 ℃ of environment then Hour, so that soybeans they grow goes out to be about 3-8 centimetre plumule. Collect the also soybean of imbibition that germinates or do not germinate, according to whenever The ratio that liter jin germinated soybean adds about 3 premium on currency adds running water in soybean or germinated soybean, and at the about 105-121 of control temperature ℃ the autoclaving container in the heating 30 minutes. After the heating, filter and collect the germinated soybean that so obtains by double-layer filter cloth Cooking liquor.
Another key component that is used for culture medium of the present invention is fresh beef and the hydrolysis of beef liver tissue of Protease Treatment Thing. Different from employed conventional meat soup in the prior art is: meat soup of the present invention is being selected not only to use beef by material Tissue also adds and has used the cattle liver tissue that is rich in various enzymes, has also used from pig or ox pancreas in the meat soup preparation in addition and has carried The rough trypsin inhibitor of getting. Therefore, in order to prepare the enzymolysis steamed beef soup as the used cultivation composition of the present invention, at first will The careful beef of selecting and beef liver tissue were by 5: 2 weight ratio mixing and rub into meat gruel. To wherein adding 1-1.5 times of volume Water stirs, and in about 60 minutes of about 100 ℃ of lower heating. Make then drop in temperature to 38-41 ℃, to wherein adding about 1% (V/V) the thick trypsin inhibitor for preparing as follows. Digestion process is about 1 hour under the pH7-9 condition. After the enzyme hydrolysis, With the centrifugal collection supernatant of 2000rmp, obtain the steamed beef soup of protease hydrolytic. The meat soup that so makes has increased bacterium greatly The required nutritional labeling of growing has reduced the waste of raw material, and has improved significantly gained bacterium training after culture medium and the fermentation Support taste and the mouthfeel of thing.
For the thick pancreatin extract for the preparation of hydrolysis beef and beef liver tissue, can at first prepare animal such as ox, horse, sheep or The pancreas tissue homogenate of pig, and in 1: 1: 3 ratio gained homogenate is mixed with absolute ethyl alcohol and distilled water, continue then to stir Mixed 72 hours. Centrifugal (500rpm, 10 minutes) are collected supernatant and with concentrated hydrochloric acid the pH of gained supernatant are transferred to 5.5 after stirring, Namely obtain required rough trypsin inhibitor.
As instantiation, the special different sections of selecting to be distributed in Normal Human Intestinal of the present invention, and each other in the physiology merit Can on have certain complementarity the normal beneficial bacteria of intestinal tract that comprises lactobacillus acidophilus, bifidobacterium breve and streptococcus faecalis, with Prepare alimentation composition or the nutritious supplementary pharmaceutical that contains the profitable probliotics symbiotic fermentation culture medium of the present invention. Therefore, do not deviating from this Under the prerequisite of fundamental idea of the invention and principle, select the probio of two or more other any genus or kind, the preparation tool Alimentation composition or nutritious supplementary pharmaceutical that certain health care is arranged all will fall into the await the reply range of claim of the present invention.
In general, the method that preparation comprises one or two or more kinds probiotics preparation in the prior art mostly be with each kind or The bacterium of strain is fermented respectively, when bacterial multiplication to the desired concentration, collect that live and/or dead biomass, mixed cell Suspension or the freeze-dried vaccine powder that obtains behind frozen dried, and add one or more nutrients or matrix components are made and are had one Medicine or alimentation composition or the replenishers of fixed treatment or prevention effect. Yet, on large-scale industrialization production angle, A common shortcoming of these methods is that production process is numerous and diverse, and the cost that spends is higher, and when mixing each bacterial classification or bacterial strain Be difficult to find the suitableeest bacterial concentration ratio.
Difference fermentation culture enterococcus faecalis, bacillus bifidus and lactobacillus acidophilus that the former use of the inventor is traditional, contain with production in the practice of nutrient oral liquor of these three kinds of probiotic bacterias and find, under the situation of using identical culture medium and similar condition of culture, the enterococcus faecalis (zero) that is used to prepare said nutritional solution is very fast at the growth rate of fermentation incipient stage, antibacterial began rapid propagation in promptly the 3rd hour, but only reached about 1/2 of peak value to the 6.5th hour bacterial population.After the 13rd hour, bacterial multiplication speed begins slow increase again, and is relatively stable 2.7 * 10 by 23-25 hour
8The level of/ml (referring to accompanying drawing 1).As can be seen from Figure 1, performance has the growth curve different with enterococcus faecalis to the lactobacillus acidophilus () that ferments separately respectively with bifidobacterium breve (▲).Acidophilia Lactococcus growth rate is slower, and promptly enters a platform after reaching high peaks very soon, is mild relatively ascent stage then.The growth rate of bifidobacterium breve is also comparatively mild, reaches peak value at about the 15th hour, but very fast decline and be tending towards the stable propagation level (about 3.0 * 10 similar to streptococcus faecalis after 16-17 hour
8/ ml).As shown in Figure 1, three kinds of bacterium when fermenting separately separately, though reach peak level at different time, enter steady statue after, their cell proliferation number averages separately are no more than 3.0 * 10
8Cell/ml.Therefore, the method that three kinds of antibacterials are fermented respectively is not only pretty troublesome in the technological operation, and when utilizing culture medium fermenting and producing of the present invention, the bacterial multiplication number also is limited.
Defective at prior art, the inventor attempts under the situation that does not change culture medium, the pH value and the cultivation temperature of culture in the sweat suitably adjusted and control, three kinds of cells classification inoculation, symbiotically fermented method in same culture medium groped and set up.Now be described in detail as follows with regard to this method operating procedure:
At first provide through sterilization treatment, basically by weight ratio 25% Semen sojae atricolor or big bean sprout cooking liquor, 2.5% protease hydrolysis beef meat soup, 1.3% yeast extract, 1.3% glucose, 1.3 sucrose, 0.03%NaCI, 0.05%MgSO
4, 0.05%CaCO
3, 0.03%K
2HPO
4And 0.03%KH
2PO
4The fluid medium of forming.
Under approximately pH7.0 and the about 40 ℃ of conditions of temperature, at first the ratio that accounts for culture medium gross weight 0.5-1.0% according to the wet thallus Biomass is to the inoculation of medium bifidobacterium breve, and in about 38-40 ℃ temperature bottom fermentation 6-8 hour.After fermenting about 6-8 hour, bifidobacterium breve concentration reaches 3.5 * 10 approximately in the culture
8/ ml.Account for the wet Biomass of lactobacillus acidophilus of culture medium gross weight 0.5-1.0% then to inoculation of medium, the back that stirs is continued fermentation and was cultivated 4-8 hour.Lactobacillus acidophilus concentration reaches 3.0 * 10 approximately in fermentation culture medium
8/ ml, or total bacterial population of enterococcus faecalis and acidophilia Lactococcus reaches 6.5 * 10 approximately
8/ ml, and the pH of fermentation culture medium transfers to about 5.8-6.3 with 0.5N NaOH solution with pH, and then inserts enterococcus faecalis according to the ratio of 0.5-1.0% in above-mentioned culture when reducing to 5.0-5.5, and under same temperature, continue to cultivate 4-8 hour.
When sweat carried out about 24 hours, and total bacterial concentration reaches 6 * 10 approximately
8When/ml was above, the temperature by control recirculated water made the temperature of fermentation system be reduced to about 30-34 ℃, and keeps under this temperature 2-4 hour, to finish in the reaction system viable microbial to the metabolic conversion of culture medium proper constituent.When the pH of culture is reduced to about 3.8-4.2, the temperature of system is increased to about 49 ℃, kill the bacterial cell that all are lived, obtaining the containing dead lactobacillus acidophilus of heat kill, bacillus bifidus and enterococcus faecalis and their metabolite, and contain total fermentation culture medium of the solubility dietary fiber, soybean isoflavone and the soy oligosaccharide that provide by fermentation medium.
Amazing especially is to use classification inoculation mixobiosis fermentation process of the present invention not only to simplify operating procedure greatly, and improved the multiplication rate of three kinds of probiotic bacterias significantly.As previously mentioned, when using three kinds of bacterium of same culture medium difference fermentation culture, although they show different growth curves separately, when cultivating about 24-25 hour, the bacterial concentration of every kind of antibacterial all is no more than 3.0 * 10
8/ ml (referring to accompanying drawing 1).Yet, cultivate three kinds of antibacterials simultaneously according to classification inoculation symbiotic fermentation method of the present invention, as if phase mutual interference and symbiosis not only do not take place and suppress phenomenon in these antibacterials to each other, and also may have certain collaborative mutually and growth-promoting effect.From curve shown in Figure 2 as can be seen, three kinds of probiotic bacterias of classification inoculation successively in same culture medium, and under same fermentation condition, make three kinds of bacteria paragenesis fermentations, the antibacterial total concentration of fermentation culture to about 24-25 hour culture medium is up to 11 * 10
8/ ml, this concentration has obviously substantially exceeded the simple additive value of the antibacterial total concentration when three kinds of antibacterials are fermented respectively.Though be not limited to theory, but we infer that the reason that this external symbiosis propagation phenomenon occurs may be because three kinds of probiotic bacterias have the metabolism complementarity, and the another kind of antibacterial (as lactobacillus acidophilus) that the metabolite of promptly wherein a kind of antibacterial (as bacillus bifidus) (as acetic acid and lactic acid) will more help inoculating afterwards grows.In addition, suitably adjust pH in the sweat and facilitate three kinds of antibacterials one of the symbiotic key factor in same environment that acid is had different tolerance.Have reason to believe, multiple probiotic bacteria classification inoculation symbiotic fermentation method of the present invention also is applicable in mimic intestinal under the conditions of existence symbiosis culture more kinds of (for example five kinds, more than seven kinds or seven kinds) probiotic bacteria, contains the medicine or the alimentation composition of multiple work or dead probiotic bacteria and other nutritional labelings with preparation.
Can be directly with the fermentation culture medium that contains the dead lactobacillus acidophilus of heat kill, bifidobacterium breve and enterococcus faecalis by the inventive method preparation as supplementary, be used for recovering and keeping gastrointestinal tract normal flora balance and/or improve the body specificity or non-specific immunity (comprising the antineoplastic immune function).
Perhaps, can be after fermentation be finished the temperature of (about 24 hours) and rising fermentation system with before the dead antibacterial of heat kill, in total bacterial cultures, add one or more medicines or its mixture that are suitable for by said antibacterial or the processing of its metabolite or any natural origin that transform, known structure or unknown structure.Near (37 ℃ of the fermentation conditions of physiological status, pH6.0) under, structural change or metabolic conversion will take place in one or more compositions in these natural drug extracts under the effect of anaerobism probiotic bacteria or its metabolite, make the natural drug chemical compound of original non-activity change into activated chemical compound, perhaps some original active more weak molecular modification is become to have strong biology of active molecule or its aggregation, some was had originally manage toxic chemical compound (the particularly native compound of animal origin) than Johnson ﹠ Johnson and become less toxicity or avirulent chemical compound or its aggregation are arranged.
Moreover, as is known to persons skilled in the art, also can be after finishing fermentation step by classification inoculation symbiotic fermentation method of the present invention, the antibacterial that centrifugalize is lived, and carry out lyophilization according to a conventional method and handle, to prepare probiotic bacteria xeraphium art alive.Such powder and appropriate carriers or mixed with excipients can be made the active bacteria formulation of different dosage forms such as tablet, Emulsion, suspending agent, capsule, suppository, syrup.For example, under the situation of preparation tablet formulation, can use sugar such as synthetic mineral dust such as natural minerals powder such as Kaolin, Pulvis Talci or silicate and sucrose, lactose, glucose as solid carrier, and can add emulsifying agents such as alkylsulfonate or arylsulphonate according to the difference of dosage form, dispersants such as lignin, carboxymethyl cellulose, starch and polyvinylpyrrolidone, and lubricant such as magnesium stearate, Pulvis Talci, stearic acid, dodecyl sodium sulfate.The preferred dosage form of probiotics viable bacteria preparation is tablet or solid formulations such as powder agent or suppository, particularly tablet.Under the situation of preparation solid formulation, preferred excipient is maltodextrin, microcrystalline Cellulose, corn starch, fructose, lactose and dextrose.Except excipient commonly used, also can add additives such as sodium citrate, calcium carbonate, dalcium biphosphate.Under the situation of preparation tablet medicine compositions, wherein also can contain anticholinergic, antihistaminic, analgesic, beta adrenergic agent, antiinflammatory, liver protectant, lipotropism agent and antitumor agent.
In order to improve the survival rate of lactobacillus acidophilus and other probiotic bacterias, can in tablet, add L-cystine, vitamin (as vitamin A, C, D and E), mineral (as calcium carbonate, magnesium oxide and magnesium carbonate), lactose, Lactalbumin concentrate, autolysing yeast extract, chitosan, lecithin and solubility or non-solubility dietary fiber (as apple fiber or corn fiber).Wherein adding the L-cystine mainly is in order to reduce the oxygen in the solid tablet, to prolong the time-to-live of bacterial cell.Adding vitamin mainly is the anti-oxidant properties of utilizing them.Known in the presence of mineral, dietary fiber has fabulous protection and stimulation to the growth of lactobacillus acidophilus.The yeast extract of self-dissolving not only is used to stimulate the growth of antibacterial in gastrointestinal tract, and is the main source of vitamin B.There is report to think that vitamin D and lactose have important function for the absorption of calcium among the human gastrointestinal tract.In addition, lactose also has the growth that utilizes lactobacillus acidophilus in the intestinal.Show that on evidence chitosan is as the dietary fiber of animal origin, it has the effect that promotes calcium absorption in the intestinal.Lecithin is convenient to the preparation of oral tablet then mainly as emulsifying agent and lubricant.
In general, contain every heavily about 500-1500mg of oral tablet of freeze dried live lactobacillus, bacillus bifidus and enterococcus faecalis, be preferably 750mg, and every middle antibacterial weight accounts for the 30-80% of composition total weight.
The following example is intended to further to illustrate advantage and the application by the probiotics preparation of the inventive method preparation, and these embodiment also do not limit the present invention in any way the scope of the claim that awaits the reply.
Embodiment 1: the preparation of probiotic bacteria symbiotic fermentation culture medium
Present embodiment is intended to illustrate with classification inoculation, symbiotic fermentation method, produces the method for the symbiotic fermentation culture medium of blended lactobacillus acidophilus, bifidobacterium breve and enterococcus faecalis in same culture medium.
1, the selection of probiotic bacteria strain
Bacillus bifidus is that people or breast are fed most important physiological antibacterial in the animal body, mainly is distributed in small intestinal bottom and the colon.Known bacillus bifidus all has important function for nutrition, growth, growth and the immunologic function of human body.Lactobacillus acidophilus is the probiotic bacteria the most widely that distributes in the human body.In gastrointestinal tract, this antibacterial mainly is distributed in widely different intestinal portion.Lactobacillus acidophilus is participated in vitamin, and particularly the biosynthesis of vitamin B group has supplementary food digestion, promotes the nutrient metabolism and improves the functions such as toleration of host to lactose.Enterococcus faecalis mainly is distributed in the caecum part of small intestinal, has the metabolism of the cholesterol decomposition of help, reduces the effect of cholesterolemia.Therefore, the present invention selects three common probiotics strains such as bifidobacterium breve, lactobacillus acidophilus and enterococcus faecalis for use, mainly is based on the overall popularity that they distribute in people's intestinal and the complementarity of physiologically active thereof.
Can buy from China typical culture collection center (CCTCC) culture presevation mechanisms such as (China, Wuhan), perhaps from normal person's feces or intestinal submucosa tissue, separate obtaining these bacterial isolateses.
2, culture medium is formed and preparation
Culture medium is formed: every liter of culture medium contains 25ml protease hydrolysis beef meat soup, 15g yeast extract, 250ml Semen sojae atricolor or big bean sprout cooking liquor, 12g glucose, 13g steaming sugar, 5g MgSO
4, 3g CaCO
3, 3g NaCI, 3g K
2HPO
4And 3gKH
2PO
4
Basically be used to prepare the protease hydrolysis beef meat soup and big bean sprout (or Semen sojae atricolor or germinated soybean) cooking liquor of defined medium of the present invention according to the described method preparation of this description detailed part.Behind each composition mix homogeneously of culture medium, standby in 121 ℃ of autoclavings 30 minutes.
3, classification inoculation symbiotic fermentation method
To be heated to about 40 ℃ for 100 liters through the above-mentioned culture medium of sterilization treatment, the ratio (W/W) that accounts for culture medium gross weight about 0.8% in the Biomass weight in wet base is at the activatory bacillus bifidus 1000g of inoculation of medium (weight in wet base), and in 39 ℃ of following anaerobic fermentations 7.5 hours.When pH reduced to 5.0 in the sweat, it was about 6.2 so that pH is transferred to continue to stir the 0.5NNaOH that drips appropriate amount down in culture medium, and once more to the inoculation of medium about 950g of activatory enterococcus faecalis Biomass (weight in wet base), and stirred 20 minutes.Under same temperature, continued fermentation culture 6 hours.When pH in the fermentation tank reduces to approximately 5.5 the time, in culture medium, add 0.5NNaOH solution once more will transfer to 6.2, inoculate the about 850g of lactobacillus acidophilus Biomass (weight in wet base) then, and continue at temperature bottom fermentation equally.When total bacterial concentration in the fermentation tank reaches more than 6.5 * 108/ml, and pH makes the temperature of fermentation culture medium be reduced to about 32 ℃ by the control circulating water temperature, and kept about 2 hours, to finish the biotransformation of tunning when being reduced to about 3.5-3.8.After fermentation is finished, according to the difference of the application target of the probiotic bacteria symbiotic fermentation culture medium that so obtains, probiotic bacteria that can the symbiotically fermented work of (1) centrifugal collection and lyophilizing it, contain the solid drugs or the nutritional supplement composition of lyophilizing mycopowder with preparation; (2) in the culture that comprises culture medium and viable bacteria and metabolite thereof, add natural drug extract to be transformed, the pharmaceutical composition that has particular biological activity and function with preparation; (3) make the temperature of fermentation culture medium be increased to about 39 ℃, the viable bacteria of the dead propagation of heat kill, behind the centrifugalize culture supernatant or directly preparation contains the nutritional supplement composition of profitable probliotics symbiotic fermentation product and bacterial debris.
Biochemical analysis result to the symbiotic fermentation culture medium supernatant shows, hydrolysising amino acid content reaches 23.322mg/ml in the culture supernatant, wherein bacillus bifidus can be satisfied and growth of lactobacillus is required, and the cystine that contains disulfide bond is 1.8mg/ml (hydrolysis) and 0.33mg/ml (free).Total sugar content is about 0.04mg/ml in the symbiotic fermentation culture medium supernatant, wherein except that reducing sugar, also has required oligosaccharide and the dietary fibers of probiotic bacteria growth and breeding such as bacillus bifidus.The latter has the inhibition blood sugar increasing in intestinal, stop the effect of neutral fat and cholesterol absorption and constipation relieving.In addition, analyze (255nm) with ethyl acetate extraction and with the HPLC method, the antibacterial converted product that records soybean isoflavone in the culture supernatant is that daidzein is up to 80 μ g/ml.Moreover, also contain multivitamin and a certain amount of mineral in the culture supernatant, wherein mainly be vitamin B group (about 40mg/l) and mineral such as P, Fe, Zn, Mg, K and Na (be respectively 5.04,5.36,8.29,88.83,783,283mg/l).These testing results show that further the symbiotic fermentation culture medium supernatant can provide bacterial growth required whole nutrients as culture medium completely.After bacterial multiplication arrives to a certain degree, because bacterial metabolism produces a large amount of acid products (organic acid), thus cause the pH value of cultivating system to descend (pH4.0 is following), and then antibacterial is stopped growing.
Embodiment 2: probiotic bacterial cultures is to the external antibacterial and bactericidal activity of pathogenic entero becteria
Present embodiment is intended to illustrate the external antibacterial and bactericidal action of the symbiotic fermentation culture medium of the bacillus bifidus, bacillus acidophilus and the enterococcus faecalis that prepare by the inventive method to some known pathogenic entero becteria.
1, with bacterium flexneri, the D15 dysentery bacterium of carrying R-plasmid, many drug resistance escherichia coli and Salmonella typhimurium detect probiotic bacteria symbiotic fermentation culture medium of the present invention (IV sample) and press the external bacteriostatic activity of the single probiotic fermenting cultures (I, II and III sample) of similarity method preparation to these pathogen with the The Small Well method as target bacteria.At first on the Nutrient agar plate, punch (diameter 6mm), and in each experimental port, add 18 hours cultured solution of broth with the above-mentioned pathogen of 105 times of dilutions of normal saline with card punch.37 ℃ of insulations of flat board after 2 hours, are added the probiotic bacterial cultures supernatant that 0.1ml ferments separately or mixobiosis is fermented respectively in each experimental port.37 ℃ of following anaerobic heat-preservations 24 hours, take out then that agar plate is observed and the record well around inhibition zone diameter (mm).Shown in the following tabulation 1 of result.Table 1 symbiotic fermentation culture medium and single strain fermentation culture are to the external bacteriostatic activity of pathogenic entero becteria
The test specimen pathogenic bacterium
#I #II #III #IV bacterium flexneri 8.0*--the 13.0D15 dysentery bacterium--escherichia coli of drug resistance more than-11.5---8.5 Bacillus typhi---10.5
*Numeral is inhibition zone diameter millimeter (mm) number of culture supernatant in the table.
Given data as can be seen from table 1, under the experiment condition (the pathogen diluent contains 0.1% cholate) of simulation intestinal environment, the inhibition zone diameters of four kinds of common pathogenic entero becterias is reached 8.5-13.0mm by many probiotics symbiotic fermentation culture fluid supernatant of the inventive method preparation.Yet under similarity condition, so antibacterial ring does not almost appear in three kinds of antibacterials single culture thing supernatant separately.Experimental result shows that the probiotic bacteria symbiotic fermentation culture medium for preparing by the inventive method has very remarkable vitro bacteriostatic activity.
2, with common pathogenic entero becteria bacterium flexneri, many drug resistances escherichia coli, Salmonella typhimurium and staphylococcus aureus as target bacteria, detect the body outer disinfecting activity of probiotic bacteria symbiotic fermentation culture medium of the present invention with pour plate method to these pathogenic microorganisms.
At first, at the about 4 μ l pathogenic bacterium suspensions of the adding in vitro 105 antibacterials/ml that contains the probiotic bacteria symbiotic fermentation culture medium supernatant that 4ml makes by the inventive method, and in about 18 hours of 37 ℃ of insulations.After pre-the cultivation, get the 1ml test specimen respectively in 0,2,6 hour of test and be added in the Nutrient agar that 10ml dissolves, be poured on the glass plate after the mixing.Place 37 ℃ of incubator insulations to observe and write down formed bacteria colony count after 24 hours flat board.Use normal saline (NS) in contrast.
Table 2 probiotic bacteria symbiotic fermentation culture medium is to the extracorporeal disinfecting living-article of pathogenic entero becteria
Bacillus typhi Dysentery bacterium Escherichia coli Staphylococcus aureusTime of contact
+ culture+NS+culture+NS+culture+S+culture+saline 0 288
*890 1,080 1,200 1,248 1,800 1,302 14,602 0 1,120 0 1,140 62 1,950 1,050 15,406 0 1,320 0 1,260 0 1,920 620 1488
*Numerical value is after corresponding pathogenic bacterium add probiotic bacteria symbiotic fermentation culture medium (experimental group) or normal saline (matched group) insulation different time (0.2,6 hour), the pathogenic bacterium clump count that forms on nutrient agar panel.
From the result shown in the table 2 as can be seen, gastrointestinal tract pathogen is with after probiotic bacteria symbiotic fermentation culture medium by the inventive method preparation contacts about 2 hours, and all the Salmonella mouse typhuss Du bacterium, flexner's dysentery Du bacterium, the drug resistance large intestine Du bacterium that are scattered in the nutrient agar almost all are killed.After contacting about 6 hours, the overwhelming majority also is killed to the said lower staphylococcus aureus of fermentation culture medium sensitivity altogether.Experimental result shows that the probiotic bacteria symbiotic fermentation culture medium performance for preparing by the inventive method has very remarkable vitro bactericidal activity.
Embodiment 3: probiotic bacteria is total to the growth in vitro facilitation of fermentation culture medium supernatant to beneficial bacteria of intestinal tract
The probiotic bacteria that present embodiment is intended to illustrate by the inventive method preparation is total to the growth in vitro facilitation of fermentation culture medium to bifidobacterium longum (B.longum), bifidobacterium bifidum (B.bifidum) and lactobacillus acidophilus (L.acidcilus).
Use bifidobacterium longum, two bacillus bifiduss and lactobacillus acidophilus as experimental bacteria, the activated back of experimental bacteria by every pipe 0.1ml (inoculation afterwards the culture medium bacterial concentration be 106/ml) be inoculated in (1) conventional PTYG culture medium (every liter of culture medium contains tryptone 5g, soy peptone 5g, yeast extract 10g, glucose 10g, saline solution 40ml, cysteine hydrochloride 0.5g Tween 80 0.1ml, 0.1% "diazoresorcinol" 1ml) respectively; (2) lack yeast extract or (3) lack tryptone and soy peptone, or (4) lack peptone and yeast extract, but all replace in the PTYG culture medium and simple symbiotic fermentation culture medium supernatant (as culture medium) of these compositions with probiotic bacteria symbiotic fermentation culture medium supernatant of the present invention.After the inoculation test tube was placed 37 ℃ of following constant temperature culture 24 hours.Cultivate the back and collect culture fluid, get 1/100 diluent 0.1ml making Hungate anaerobism and roll pipe, and continue down to cultivate 48 hours, carry out colony counting then in 37 ℃.Shown in the following tabulation 3 of result.Table 3 probiotic bacteria symbiotic fermentation culture medium supernatant influences culture medium bacillus bifidus bifidobacterium longum lactobacillus acidophilus probiotics symbiotic culture product 1.53 * 10 to the growth of bacillus bifidus and lactobacillus acidophilus
81.03 * 10
101.04 * 10
8Whole part PTYG culture medium 1.39 * 10
81.22 * 10
108 * 10
8The PTYG symbiosis culture thing 2.0 * 10 that lacks yeast extract
81.24 * 10
101.2 * 10
9The PTYG+ symbiosis culture thing 1.37 * 10 that lacks peptone
86.4 * 10
94.5 * 10
8Its living culture 1.67 * 10 of PTYD+ that lacks peptone and yeast extract
89.7 * 10
93.0 * 10
8
From the data shown in the table 3 as can be seen, known probiotic bacterias such as bacillus bifidus and lactobacillus acidophilus are inoculated in cultivate in the probiotics symbiotic culture product supernatant of the present invention that does not contain any conventional medium component after 48 hours, the antibacterial number average of being inoculated is bred to 10
8More than the cfu/ml.On the other hand, replace carbon source, nitrogenous source and mineral origin material in the conventional PTYD culture medium with probiotic bacteria symbiotic fermentation culture medium supernatant of the present invention, the cell number of cultivating back three kinds of tested antibacterials is also 10
8More than the cfu/ml.Therefore can think that multiple (strain) probiotic bacteria symbiotic fermentation culture medium supernatant for preparing by the inventive method has the obvious growth facilitation to the main probiotic bacteria in normal person's intestinal.Though relevant mechanism is still indeterminate, infer that the oligosaccharide (comprising fruit oligose, galactooligosacchari(es and soy oligosaccharide) (i.e. so-called " bifidus factor ") that contains in this probiotic bacteria growth-promoting activity and the said culture as carbon source, hydrolysis amino acid (comprising that cystine etc. contains the aminoacid of disulfide bond), mineral (calcium, magnesium, phosphorus, potassium etc.), vitamin B group (B1, B2, B6) and other nitrogen sources are relevant.Embodiment 4 probiotic bacteria symbiotic fermentation culture mediums are to the influence of mice non-specific immunity
Present embodiment is intended to by Turnover of Mouse Peritoneal Macrophages phagocytosis test (intracorporal method) and mice delayed footpad reaction experimental observation and estimates the influence of probiotic bacteria symbiotic fermentation culture medium of the present invention to the mammal non-specific immunity.
1, Turnover of Mouse Peritoneal Macrophages phagocytosis test (intracorporal method) as animal model, as by phagocyte, carries out the cytophagy test according to the conventional method of having described in the document with chicken red blood cell with the healthy Balb/c mice of the about 20-25g of body weight basically.Briefly, animal is divided into experimental group and matched group (10 every group) at random, conventional stable breeding came into operation by probiotic bacteria symbiotic fermentation culture medium stock solution (supernatant) 0.5ml of the inventive method preparation to experimental group animal every filling every day stomach after 3 days, came into operation continuously 15 days.Control animals is then raised with the normal saline with volume.After 15 days, disconnected neck is handled animal and is collected abdominal cavity phagocytic, the chicken red blood cell of washing with normal saline is as target cell, the high power microscope down erythrocyte of counting and record experimental group and control animals peritoneal macrophage is engulfed number, and by the percentage phagocytic rate (%) and the phagocytic index of calculating macrophage.Shown in the following tabulation 4 of result.Table 4 probiotic bacteria symbiotic fermentation culture medium is to the influence of Turnover of Mouse Peritoneal Macrophages phagocytic activity
Group (n=10) percentage phagocytic rate (%) phagocytic index (X ± SD)
Experimental group 88.2 ± 5.51 5.27 ± 0.56
Matched group 67.7 ± 8.08 24.2 ± 0.62
2, mice delayed footpad reaction
As animal model, carry out the test of delayed footpad reaction with the healthy Kunming mouse of the about 20-25g of body weight according to a conventional method.Briefly, minute continuous feed of the animal of cage stable breeding by the probiotic bacteria symbiotic fermentation culture medium of the inventive method preparation totally 12 days (0.5ml stock solution/only/day), was injected 0.1ml chicken red blood (103 cells/ml) on the 13rd day in the mice left hind foot pad.Attack the right back foot pad of animal with the chicken red blood cell of same amount after 96 hours.Under stereomicroscope, measured the thickness of the right back foot pad of mice before attacking with attack in back 24 hours respectively, and calculate the sufficient thickened degree (mm) of filling up by it.The normal saline of the same volume of use in contrast in the test.Shown in the following tabulation 5 of result.
Table 5 probiotic bacteria symbiotic fermentation culture medium is to the influence of mice delayed footpad reaction
The sufficient mat thickness of group (n=10) (mm)
Experimental group 1.89 ± 0.43
*
Matched group 0.98 ± 0.40
*Given numerical value is average ± standard deviation of 10 animals.P＜0.01。
The ability of macrophage phagocytic foreign body is represented one of mammal non-specific immunity, and sexual type foot pad allergy then reflects mammiferous cellular immunity late.From the data shown in table 3 and 4 as can be seen, probiotic bacteria symbiotic fermentation culture medium of the present invention, performance has good non-specific immunity enhanced activity, can use as immunostimulant so have clinically.
Claims (10)
1, a kind ofly contains more than one beneficial bacteria of intestinal tract deactivation or that heat kill is dead, and contain the suspension attitude alimentation composition of water soluble dietary fiber and soybean isoflavone.
2, according to the alimentation composition of claim 1, wherein said probiotic bacteria is selected from one or more people's intestinal non-pathogenic bacterias of Lactobacillus, Bifidobacterium, Streptococcus, Enterococcus, propionibacterium, Leuconostoc, bacillus.
3, according to the alimentation composition of claim 1, wherein said probiotic bacteria is selected from bifidobacterium bifidum, bifidobacterium breve, bifidobacteria infantis, bifidobacterium longum, lactobacillus acidophilus, Lactobacillus bulgaricus, cheese lactobacillus, Deshi Lactobacillus, Lactobacillus plantarum, streptococcus thermophilus, enterococcus faecalis, dung intestinal ball coccus, has a liking for citric acid leukonid, bacillus subtilis and Bacillus licheniformis.
4, according to the alimentation composition of claim 1, wherein said soluble fiber content is about every milliliter and contains 20-40 μ g, and wherein said isoflavone content is every milliliter of 30-100 μ g.
The method of the suspension attitude alimentation composition that each limited among 5, the production claim 1-4, this method comprises:
(1) provides the fluid medium of forming by Semen sojae atricolor or big bean sprout cooking liquor, proteolysis beef meat soup, yeast extract, sugar and mineral basically;
(2) account for the ratio of culture medium gross weight 0.5-1.0% according to the wet thallus Biomass, in culture medium, inoculate two or more beneficial bacteria of intestinal tract to be cultivated successively, and behind a kind of antibacterial of every access, under 38-41 ℃ temperature and pH6.6 condition, anaerobism was cultivated 4-10 hour;
(3) the thalline total concentration reaches 7 * 10 approximately in fermentation culture medium
8When/ml is above, make temperature reduce to about 32 ℃ and kept 2-4 hour, to finish the metabolic conversion of microbial body;
(4) reduce to about 3.5--3.9 as the pH of fermentation culture medium, elevate the temperature to 48-50 ℃ and kept 4-6 hour, to finish hot deactivation of thalline and fermentation system stabilisation.
6, according to the method for claim 5, wherein said fluid medium is to comprise Mg by about 25% big bean sprout decoction liquor, about 2.5% trypsin hydrolyzing beef meat soup, about 1.3% yeast extract, about 1.3% glucose, about 1.3% sucrose and appropriate amount
++, Ca
++, K
+, Na
+Mineral form.
7, according to the method for claim 5, wherein said beneficial bacteria of intestinal tract is selected from bifidobacterium bifidum, bifidobacterium breve, baby's bifid bar castor, bifidobacterium longum, lactobacillus acidophilus, Lactobacillus bulgaricus, cheese lactobacillus, Deshi Lactobacillus, Lactobacillus plantarum, streptococcus thermophilus, enterococcus faecalis, dung intestinal ball coccus, has a liking for citric acid leukonid, bacillus subtilis and Bacillus licheniformis.
8, the alimentation composition that each limited among the claim 1-4 is in the application of correcting and keeping in the normal microecological balance of intestinal.
9, the application of the alimentation composition that each limited among the claim 1-4 in adjusting the body nonspecific immune reaction.
10, the alimentation composition that each limited among the claim 1-4 is at biotransformation or modify the effective ingredient of one or more natural drugs and make it to bring into play or improve application in its biologic activity.
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| ES2952438A1 (en) * | 2022-03-23 | 2023-10-31 | Igen Biolab Group Ag | POSTBIOTIC FOOD ADDITIVE FOR PRODUCTS OBTAINED FROM CEREAL FLOUR, FOOD COMPOSITION BASED ON CEREAL FLOUR FOR PREPARING BAKERY AND PASTRY PRODUCTS WHICH INCLUDES THE ADDITIVE, AND PRODUCT BASED ON CEREAL FLOUR FROM BAKERY, BAKERY AND PASTRY WHICH INCLUDES THE FOOD COMPOSITION (Machine-translation by Google Translate, not legally binding) |
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Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1056750C (en) * | 1993-12-10 | 2000-09-27 | 济南三株药业有限公司 | Sanzhu oral liquid and its preparing method |
| CN1099289C (en) * | 1997-08-01 | 2003-01-22 | 北京东方百信生物技术有限公司 | Medicinal prepn. contg. beneficial bacteria named 'Junyikang' and method for preparing same |
| SE510813C2 (en) * | 1997-12-08 | 1999-06-28 | Arla Ekonomisk Foerening | Bacterial strain of the species Lactobacillus Paracasei subsp. paracasei, its composition for use in food, and product containing the strain |
| CN1119154C (en) * | 1998-01-20 | 2003-08-27 | 上海信谊药业有限公司 | Live triple bifidobacteria preparation and preparing method thereof |
-
2000
- 2000-04-26 CN CN00111082A patent/CN1273838A/en active Pending
-
2001
- 2001-04-26 WO PCT/CN2001/000617 patent/WO2001085186A1/en active Application Filing
- 2001-04-26 AU AU63735/01A patent/AU6373501A/en not_active Abandoned
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| KR102445903B1 (en) * | 2021-07-06 | 2022-09-22 | 롯데제과 주식회사 | Lactobacillus plantarum LRCC5314 having an effect of blood glucose lowering-activity and controlling stress and its application |
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Also Published As
| Publication number | Publication date |
|---|---|
| AU6373501A (en) | 2001-11-20 |
| WO2001085186A1 (en) | 2001-11-15 |
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