CN101486987B - Preparation of freeze-dried bifidobacteria powder - Google Patents
Preparation of freeze-dried bifidobacteria powder Download PDFInfo
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- CN101486987B CN101486987B CN2009100568729A CN200910056872A CN101486987B CN 101486987 B CN101486987 B CN 101486987B CN 2009100568729 A CN2009100568729 A CN 2009100568729A CN 200910056872 A CN200910056872 A CN 200910056872A CN 101486987 B CN101486987 B CN 101486987B
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a preparation method of freeze dried microbial powder of bifidobacteria, which comprises the technical processes of strain fermented cultivation, separation of a fermented fluid, emulsification of microbial soil and vacuum freeze drying of an emulsified solution. The preparation method is characterized in that a bifidobacteria strain is inoculated into a fluid culture medium in a triangular flask and cultivated at the temperature of 30 to 40 DEG C for 10 to 30 hours, sterilized at the temperature of 115 DEG C for 15 minutes, inoculated into a large fermentation flask and fermentation cultivated at the temperature of 35 to 40 DEG C for 10 to 30 hours, and sterilized at the temperature of 115 DEG C for 15 minutes; the pH value of the fermented fluid is 5.5 to 6.5, and the OD600 is larger than 2.0; the vacuum freeze drying of the emulsified solution comprises the following steps: the emulsified solution is pre-frozen at the temperature of minus 60 to minus 40 DEG C for 1 to 5 hours, and then vacuum frozen and dried at the temperature of minus 60 DEG C and with the vacuum degree of 1 to 8 pa for 10 to 30 hours. The freeze dried microbial powder of bifidobacteria can be preserved for 18 months at the temperature of minus 18 DEG C with unaffected liveness and stability.
Description
Technical field
The present invention discloses a kind of preparation method of freeze-dried bifidobacteria powder, is applied to biological technical field, for bifidobacteria viable bacteria preparation Products Development and the application at aspects such as food, healthcare products, medicine thereof provide freeze-dried vaccine powder manufacture craft efficiently.
Background technology
1899, Tisser was at first by separating a kind of anaerobic gram-positive bacillus in breast milk nutrition youngster's the ight soil, the thalline hettocyrtosis, and terminal common bifurcated is so a bifidus bacillus.Bifidus bacillus is the polymorphic bacillus of Gram-positive, is forms such as Y font, V font, bending, spatula shape, and its typical characteristic is forked bacillus, thereby is named as bifidus, and the Latin derivation is the meaning of splitting, separating.Bifidus bacillus does not form gemma, and Yamamoto Methylene Blue ZF dyeing thalline is painted irregular, no mobility, and obligate anaerobic, peroxidase is negative, does not reduce nitrate salt, and indole reaction is negative, and gelatine liquefication is negative, the benzidine reaction feminine gender; 37～41 ℃ of optimum growth temperatures, 35～40 ℃ of the righttest leavening temperatures, 25～28 ℃ of minimum growth temperatures, 43～45 ℃ of maximum growth temperatures; Initial growth pH value is 6.7～7.0, is below 4.5～5.0 or the pH value is not grow in the environment more than 8.0～8.5 in the pH value.Bifidus bacillus is different from homotype or the heterofermentation of milk-acid bacteria to the catabolic pathway of sugary carbohydrates, but via special bifid branch road, wherein the fructose-6-phosphate ketolase is its critical enzyme.To glucose metabolism is by the hydrolysis of fructose-6-phosphate ketolase approach, generates acetate and lactic acid at last, and the two mol ratio is 1.5: 1, does not generate CO
2, butyric acid, propionic acid; DNA based composition molar percentage is 57～68.Bifidus bacillus belongs to Propionibacteriaceae, has 11 kinds, 30 subspecies and type, like bifurcated bifidus bacillus, bifidus longum bb, JCM 1192T, youth bifidus longum bb, bifidobacteria infantis etc.Different types of bifidus bacillus is different to the fermentation capacity of sugar, produces not aerogenesis of acid.How positive catalase test is, and most ability decomposing ureas do not produce indole and hydrogen sulfide.There is not inside and outside toxin.Bifidus bacillus is generally to tolerances such as kantlex, Xin Meisu, Streptomycin sulphate, polymyxin, qingfengmeisu qiong, Metronidazole plain BP.98 99s.To Romicil, lincomycin, vancomyein, penicillin G, penbritin, Oxacyclotetradecane,erythromycin deriv, Zoofurin sensitivity.
In 20 beginnings of the century, once attempted to be transplanted in the baby intestinal of comparatively small amt, in order to improve the resistibility of baby to intestinal tract disease with the bifidus bacillus broth culture.After the 1950's; Repeatedly the beneficial effect of bifidus bacillus to body discussed in international conference; Understanding to its physiological significance deepens continuously, and is being used for prevention and is treating little ecologic disturbance and some disease has obtained certain achievement, and bifidus bacillus causes extensive concern in the world.
New birth youngster gave birth to the back several hours, and bifidus bacillus can field planting in enteron aisle, after this accompanies throughout one's life.Newborn infant's meconium is aseptic, and the birth back can detect intestinal bacilli, micrococcus, suis in several hours, and its quantity constantly increases, and the 24h intestinal bacteria are preponderated.Being born can detect a small amount of bifidus bacillus on the 2nd day, but increased very rapidly, and the 4th～5 day begins to preponderate; The quantity such as intestinal bacilli that occur this moment at first descended gradually, had then set up the flora that has comparative advantage with bifidus bacillus in the 6th～8 day, and obligatory anaerobic bacteria particularly bifidobacterium fermentation produces a large amount of acetic acid and lactic acid; And the surge capability of breast milk a little less than; Enteron aisle pH descends rapidly, and breast milk nutrition youngster ight soil pH is 5.1 the 7th day the time after birth, thereby facultative anaerobe is suppressed.Bifidus bacillus by its surperficial teichoic acid secure adhesion in the intestinal mucosa epithelial cell, and field planting propagation there.
The enteron aisle normal microflora is one group of huge ecological organization; Population is above 400 kinds; Wherein a fairly large number of genus bifidobacterium gram-positive anaerobism sporeless bacterium is one of dominant bacteria in people and the many Mammals enteron aisles, participates in a series of physiological processs such as immunity, nutrition, digestion and protection.
Bifidus bacillus has different physiological roles, is the probiotic bacterium of generally acknowledging at present.The physiological function that can improve human intestinal disease resistance this respect about bifidus bacillus has many research and report, and bifidus bacillus is inhibited to spoilage organism and pathogenic bacteria, pathogenicity bo dust Xi Shi intestinal bacteria, streptococcus aureus, shigella dysenteriae, salmonella typhi, mycetozoan, Candida albicans etc.Bifidus bacillus not only produces various VITAMINss such as VB1, VB2, VB6, VB12, nicotinic acid and folic acid etc. for needed by human body, can also be through suppressing the supply that some VITAMINs decomposer ensures VITAMINs.Bifidus bacillus can promote the intestinal tube wriggling, and osmotic pressure increases in the intestinal tube because the organic acid that bifidus bacillus produces can make again, the moisture hypersecretion, and moisture increases and relief of constipation in the ight soil.Bifidus bacillus can also can absorb harmful meta-bolitess such as a large amount of indoles that saprophytic microorganism in some enteron aisles produces, hydrogen sulfide, amine in enteron aisle; Suppress to produce the spoilage organism of amine; Reduce the pH value in the enteron aisle, the ionic that ammonia is become be difficult to absorb reaches the effect of reduction blood ammonia and liver function protecting.Bifidus bacillus passes through the former increased response human immunologic function of induction of immunity in enteron aisle.Bifidus bacillus through suppress saprophytic microorganism growth play the effect that prevents enteron aisle cancer and intestinal canal tumour with decompose carcinogenic substances.
The bifidus bacillus goods are to utilize advantage physiological bacteria-bifidus bacillus of host self to make starter, enlarge to produce through industrial fermentation and process active bacteria formulation, return the original habitat of host again, reach the goods of regulating or keeping body normal physiological state.At present the commodity of relevant bifidus bacillus can be divided into bifidus bacillus food, contain specific health food, the bifidus bacillus medicine of bifidus bacillus and growth helping matter thereof according to its function on the domestic and international market.According to the difference of employed strain number, can be divided into the single strain preparation again and join the bacterial strain preparation.China's " regulation (trying) is declared and evaluated to probiotic bacterium class protective foods " has listed the probiotic bacterium bacterial classification list (enforcement on July 1st, 2005) that can be used for protective foods, that is: bifidobacterium bifidum Bifidobacteriumbifidum; Bifidobacterium infantis Bifidobacterium infantis; Long bifidus bacillus Bifidobacterium longum; Short bifidus bacillus Bifidobacterium breve; Bifidobacterium adolescentis Bifidobacterium adolescentis; Lactobacillus delbrueckii subsp.Bulgaricus plants in lactobacillus delbruckii Bulgaria; Lactobacterium acidophilum Lactobacillus acidophilus; Lactobacterium casei cheese subspecies Lactobacillus casei subsp.Casei; Thermophilus streptococcus Streptococcus thermophilus; Lactobacillus reuteri Lactobacillus reuteri.Wherein bifidumbacterium bifidum, bifidobacteria infantis, JCM 1192T, bifidus longum bb, bifidobacterium adolescentis all belong to bifidus bacillus.Wherein bifidobacteria infantis and JCM 1192T only exist in baby's enteron aisle and preponderate, along with people's age increases and then reduces gradually and disappear; Be main with long bifid then in adult's the enteron aisle,, thereby use less because there is certain objection in the glucuroide that long bifid had with the youth bifid.And the activity of the immuno-stimulating of bifidobacterium adolescentis is superior to other bifidus bacillus, uses to become more and more widely.
After the sixties in 20th century, China has begun the research in this field, attempts to utilize little ecological system active bacteria formulation such as bifidus bacillus to reach ill to cure the disease, not sick diseases prevention, anosis health care triple effect, has shown gratifying prospect.Domestic have many enterprises to utilize the bifidus bacillus lyophilized powder to be the raw material production biological products, and as its unit number of viable of medicine major control, number of viable directly influences the function of medicine.Therefore, the raising number of viable is particularly important in the lyophilized powder production process.Active bacteria formulation should be kept higher viable bacteria level before consumption, and current many products all can not prevent the decay of viable count effectively.How strain screening adapts in the amplification cultivation technological operation, and it is very important optimizing principal elements such as influence in number of viable quality such as leavening temperature, cultivation pH value and the substratum bifidus factor in the process of fermentation.In addition; Utilizing Vacuum Freezing & Drying Technology to produce active bacteria formulation is that comparatively ideal is a kind of in the multiple method for preserving; But freezing and drying process can cause the passivation of damage, death and some zymoprotein molecule of part microorganism cells; If the protective material selection is improper in the freeze-dry process, then fermentative activity descends thereupon.So, obtain the quality and quantity viable bacteria of enough survivals and prolong the viable bacteria preservation term,, and reduce cost the key problem in technology that has become bifidobacteria viable bacteria preparation product research exploitation and used so that give full play to its physiological function.
Summary of the invention
The objective of the invention is to disclose a kind of preparation method of freeze-dried bifidobacteria powder; Be a kind of preparation method of high-density high stability high reactivity freeze-dried vaccine powder; Make the fermented liquid cell concentration reach 1.0 * 108cfu/g through 30～40 ℃ of 10～30 hours high-density culture; Make bacterium mud concentration reach 1.0 * 109cfu/g through efficient fermented liquid stripping technique; After optimization technologies such as high reactivity emulsification and vacuum lyophilization, obtaining 1.0 * 1010cfu/g can be at 18 months its active and stable impregnable freeze-dried bifidobacteria powder of-18 degree storages.
The present invention is on the basis to the bifidobacterium strains characteristic research; Through adding bifidus factor and optimizing technology to its high density and activity fermentation culture; Carry out technology and technologies such as the emulsification of high stability high reactivity, freeze-drying through special protective preparation; Freezeproof protectant in the hope of fermentation culture and the activity stability defencive function that obtains greater activity to greatest extent, reduces the death of thalline in the preparation process; The survival rate that improves cell also prolongs the viable bacteria preservation term, for the preparation and the application of freeze-dried bifidobacteria powder provides technology and technology.
The present invention is achieved in that a kind of preparation method of freeze-dried bifidobacteria powder; Its technical process comprises strain fermentation cultivation, fermented liquid separation, the emulsification of bacterium mud and emulsion vacuum lyophilization; It is characterized in that: the strain fermentation of described freeze-dried bifidobacteria powder is cultivated, and bifidobacterium species is inserted in the triangular flask liquid nutrient medium cultivate, and the bacterial classification weight percentage that inserts in the substratum is 0.1%～0.01%; Cultivated 10～30 hours down at 30～40 ℃; Insert in the substratum of big fermentor tank again and carry out fermentation culture, the inoculation weight percentage in the substratum is 1%～5%, 35～40 ℃ and cultivated 10～30 hours; The weight percent of said substratum consists of: bifidus factor 1～5%, glucose 1～2%, peptone 0.5～2%, yeast extract paste 0.5～2%, dibasic ammonium citrate 0.1～0.2%, potassium hydrogenphosphate 0.1～0.2%, anhydrous sodium acetate 0.1～1%, sal epsom 0.05～0.1%, tween-80 0.0001%, all the other are water; 115 ℃ of sterilizations 15 minutes, and cultivate acidity to make fermented liquid pH value be 5.5～6.5 to add sodium hydroxide solution control.
The big fermentor tank of said bacterial classification carries out fermentating culturing process, and fermented liquid pH value is 5.5～6.5, OD600 is more than 2.0, and microscopy bifidumbacterium bifidum form is normal, does not have assorted bacterium, stops fermentation.
Said fermented liquid separating technology adopts ceramic membrane pore size 0.01～1 μ m; Filtering separation thalline under 0.05MPa～0.5MPa hydraulic condition; Or carrying out under 4000～10000rpm condition, and, obtain bacterium mud with sterilized water washing 1～2 time with tubular type or disc centrifuge separating thallus.
Said bacterium mud emulsifying process; According to viable bacteria bacterium mud: the ratio of bacterium mud protective material=0.1: 1～1: 0.1 is with bacterium mud and bacterium mud protective material mixing and emulsifying; Bacterium mud protective material prescription each component weight percent is: skim-milk 1～10%, lactose 1～10%, W-Gum 1～10%, inhibitor 0.1～1%, tween-80 0.01～0.05%; All the other are water, and under 105 ℃～115 ℃ conditions, sterilize 15 minutes postcooling to 5～20 of bacterium mud protective material are ℃ subsequent use; Described inhibitor is selected a kind of in isoascorbic acid, L-glutamic acid and glycocoll.
Said emulsion vacuum freeze-drying technique is: in the final vacuum lyophilize in 1～5 hour of-60～-40 ℃ of pre-freezes, the lyophilize temperature is-60 ℃, and vacuum tightness is 1～8pa, vacuum lyophilization 10～30 hours; Obtain viable count after the drying greater than 1.0 * 10
11The high reactivity bifidumbacterium bifidum freeze-dried vaccine powder of cfu/g.
Said strain fermentation cultivation, fermented liquid separation and bacterium mud emulsifying process operating environment conditional request are for carrying out in the clean environment that is lower than 25 degree, humidity RH＜35% in temperature.
Described bifidus bacillus is selected one or more in bifidumbacterium bifidum Bifidobacterium bifidum, bifidobacterium adolescentis Bifidobacteriumadolescentis, JCM 1192T Bifidobacterium breve, bifidobacteria infantis Bifidobacterium infantis and bifidus longum bb Bifidobacterium longum.
Described bifidus bacillus; Its screening strain excellent is all through following test: 1. to the resistibility of sour, bile and osmotic pressure; 2. to the sticking of human intestinal cell, the 3. digestion security of taking the photograph the people is 4. to the inhibition of pathogenic bacterium such as streptococcus aureus, pathogenic colon bacillus, Candida albicans and Gardnerella; 5. in the of short duration field planting of colon, 6. confirm the clinical study of health effect etc.
The invention has the beneficial effects as follows: the inventive method prepare greater than 1.0 * 1010cfu/g freeze-dried bifidobacteria powder; Can store 18 months at-18 degree; It is active impregnable with stability, for bifidobacteria viable bacteria preparation Products Development and the application at aspects such as food, healthcare products, medicine thereof provide freeze-dried vaccine powder manufacturing technology efficiently.
Embodiment
Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the scope of restriction the present invention protection that the scope of the present invention's protection is said being as the criterion of elaboration with claims of application of the present invention.The following experimental technique of unreceipted actual conditions among the routine embodiment, usually according to normal condition, or the condition of advising according to manufacturer.Each ratio and per-cent are all based on weight unit, unless stated otherwise.
Embodiment 1: the preparation of bifidumbacterium bifidum freeze-dried vaccine powder
The bifidumbacterium bifidum bacterial classification that filters out is inserted the triangular flask liquid nutrient medium earlier; The back is inserted to cultivate down at 30～40 ℃ respectively in the fermentor tank and was carried out amplification cultivation step by step in 10～30 hours; The culture medium prescription weight percent consists of: bifidus factor 2%, glucose 2%, peptone 1.5%, yeast extract paste 1%, dibasic ammonium citrate 0.1%, potassium hydrogenphosphate 0.2%, anhydrous sodium acetate 0.1%, sal epsom 0.05%, tween-80 0.0001%, all the other are water, 115 ℃ of sterilizations 15 minutes, and cultivate acidity to make fermented liquid pH value be 5.5～6.5 to add the control of NaOH solution; The strain fermentation amplification cultivation; Cultivated 10～30 hours, OD600 is more than 2.0, and microscopy bifidumbacterium bifidum form is normal; Do not have assorted bacterium, stop fermentation.Carrying out with the disc centrifuge separating thallus under the 8000rpm condition, and, obtain bacterium mud with sterilized water washing 2 times.
Bacterium mud protective material prescription each component weight percent is: skim-milk 5%, lactose 10%, W-Gum 5%, isoascorbic acid 0.1%, tween-80 0.01%; All the other are water, and under 105 ℃～115 ℃ conditions, sterilize 15 minutes postcooling to 5～20 of bacterium mud protective material are ℃ subsequent use.According to viable bacteria bacterium mud: the ratio of bacterium mud protective material=1: 1 is with bacterium mud and bacterium mud protective material mixing and emulsifying.In the final vacuum lyophilize in 1～5 hour of-60～-40 ℃ of pre-freezes, the lyophilize temperature is-60 ℃, and vacuum tightness is 1～8pa, obtains high reactivity bifidumbacterium bifidum freeze-dried vaccine powder after the drying under 10～30 hours conditions of vacuum lyophilization.
Strain fermentation cultivation, fermented liquid separation and bacterium mud emulsifying process operating environment conditional request are for carrying out in the clean environment that is lower than 25 degree, humidity RH＜35% in temperature.
Viable count is greater than 1.0 * 10 in the product for preparing
11Cfu/g, vacuum-packed with two aluminium film phonograph seals, every bag of 50g or pack according to customer requirement.
Embodiment 2: the preparation of bifidobacterium adolescentis freeze-dried vaccine powder
The bifidobacterium adolescentis bacterial classification that filters out is inserted the triangular flask liquid nutrient medium earlier; The back is inserted to cultivate down at 30～40 ℃ respectively in the fermentor tank and was carried out amplification cultivation step by step in 10～30 hours, and the culture medium prescription weight percent consists of: bifidus factor 1%, glucose 1%, peptone 0.5%, yeast extract paste 2%, dibasic ammonium citrate 0.2%, potassium hydrogenphosphate 0.1%, anhydrous sodium acetate 1%, sal epsom 0.1%, tween-80 0.0001%, and all the other are water; Sterilized 15 minutes for 115 ℃; And cultivate acidity to make fermented liquid pH value be 5.5～6.5 to add NaOH solution control, the strain fermentation amplification cultivation was cultivated 10～30 hours; OD600 is more than 2.0; Microscopy bifidobacterium adolescentis form is normal, does not have assorted bacterium, stops fermentation.Under 0.05MPa～0.5MPa hydraulic condition, carry out, and, obtain bacterium mud with sterilized water washing 1～2 time with tubular type or disc centrifuge separating thallus.
Bacterium mud protective material prescription each component weight percent is: skim-milk 8%, lactose 8%, W-Gum 5%, isoascorbic acid 0.1%, tween-80 0.05%; All the other are water, and under 105 ℃～115 ℃ conditions, sterilize 15 minutes postcooling to 5～20 of bacterium mud protective material are ℃ subsequent use.According to viable bacteria bacterium mud: the ratio of bacterium mud protective material=1: 2 is with bacterium mud and bacterium mud protective material mixing and emulsifying.
Other are with embodiment 1.
Viable count is greater than 1.0 * 10 in the product for preparing
11Cfu/g, vacuum-packed with two aluminium film phonograph seals, every bag of 50g or pack according to customer requirement.
Embodiment 3: the preparation of bifidobacteria infantis freeze-dried vaccine powder
The bifidobacteria infantis bacterial classification that filters out is inserted the triangular flask liquid nutrient medium earlier; The back is inserted to cultivate down at 30～40 ℃ respectively in the fermentor tank and was carried out amplification cultivation step by step in 10～30 hours, and the culture medium prescription weight percent consists of: bifidus factor 5%, glucose 2%, peptone 1.5%, yeast extract paste 2%, dibasic ammonium citrate 0.1%, potassium hydrogenphosphate 0.2%, anhydrous sodium acetate 0.5%, sal epsom 0.08%, tween-80 0.0001%, and all the other are water; Sterilized 15 minutes for 115 ℃; And cultivate acidity to make fermented liquid pH value be 5.5～6.5 to add NaOH solution control, the strain fermentation amplification cultivation was cultivated 10～30 hours; OD600 is more than 2.0; Microscopy bifidobacteria infantis form is normal, does not have assorted bacterium, stops fermentation.Carrying out with the disc centrifuge separating thallus under the 8000rpm condition, and, obtain bacterium mud with sterilized water washing 2 times.
Bacterium mud protective material prescription each component weight percent is: skim-milk 1%, lactose 10%, W-Gum 10%, isoascorbic acid 0.8%, tween-80 0.06%; All the other are water, and under 105 ℃～115 ℃ conditions, sterilize 15 minutes postcooling to 5～20 of bacterium mud protective material are ℃ subsequent use.According to viable bacteria bacterium mud: bacterium mud protective material=2: 1 is with bacterium mud and bacterium mud protective material mixing and emulsifying.
Other are with embodiment 1.
Viable count is greater than 1.0 * 10 in the product for preparing
11Cfu/g, vacuum-packed with two aluminium film phonograph seals, every bag of 50g or pack according to customer requirement.
Embodiment 4: the preparation of bifidus longum bb freeze-dried vaccine powder
The bifidus longum bb bacterial classification that filters out is inserted the triangular flask liquid nutrient medium earlier; The back is inserted to cultivate down at 30～40 ℃ respectively in the fermentor tank and was carried out amplification cultivation step by step in 10～30 hours, bifidus factor 3%, glucose 1.5%, peptone 1.2%, yeast extract paste 1.5%, dibasic ammonium citrate 0.15%, potassium hydrogenphosphate 0.12%, anhydrous sodium acetate 0.6%, sal epsom 0.08%, tween-80 0.0001%, and all the other are water; Sterilized 15 minutes for 115 ℃; And cultivate acidity to make fermented liquid pH value be 5.5～6.5 to add NaOH solution control, the strain fermentation amplification cultivation was cultivated 10～30 hours; OD600 is more than 2.0; Microscopy bifidus longum bb form is normal, does not have assorted bacterium, stops fermentation.Under 0.05MPa～0.5MPa hydraulic condition, carry out, and, obtain bacterium mud with sterilized water washing 1～2 time with tubular type or disc centrifuge separating thallus.
Bacterium mud protective material prescription each component weight percent is: skim-milk 4%, lactose 6%, W-Gum 7%, isoascorbic acid 0.8%, tween-80 0.04%; All the other are water, and under 105 ℃～115 ℃ conditions, sterilize 15 minutes postcooling to 5～20 of bacterium mud protective material are ℃ subsequent use.According to viable bacteria bacterium mud: the ratio of bacterium mud protective material=3: 1 is with bacterium mud and bacterium mud protective material mixing and emulsifying.
Other are with embodiment 1.
Viable count is greater than 1.0 * 10 in the product for preparing
11Cfu/g, vacuum-packed with two aluminium film phonograph seals, every bag of 50g or pack according to customer requirement.
Embodiment 5: the preparation of JCM 1192T freeze-dried vaccine powder
The JCM 1192T bacterial classification that filters out is inserted the triangular flask liquid nutrient medium earlier; The back is inserted to cultivate down at 30～40 ℃ respectively in the fermentor tank and was carried out amplification cultivation step by step in 10～30 hours; The culture medium prescription weight ratio consists of: bifidus factor 3%, glucose 1.8%, peptone 2%, yeast extract paste 1.5%, dibasic ammonium citrate 0.2%, potassium hydrogenphosphate 0.2%, anhydrous sodium acetate 0.8%, sal epsom 0.06%, tween-80 0.0001%, all the other are water.115 ℃ of sterilizations 15 minutes, and cultivate acidity to make fermented liquid pH value be 5.5～6.5 to add NaOH solution control, the strain fermentation amplification cultivation was cultivated 10～30 hours, and OD600 is more than 2.0, and microscopy JCM 1192T form is normal, does not have assorted bacterium, and termination is fermented.Carrying out with the disc centrifuge separating thallus under the 8000rpm condition, and, obtain bacterium mud with sterilized water washing 2 times.
Bacterium mud protective material prescription each component weight percent is: skim-milk 2%, lactose 2%, W-Gum 3%, isoascorbic acid 0.15%, tween-80 0.02%; All the other are water, and under 105 ℃～115 ℃ conditions, sterilize 15 minutes postcooling to 5～20 of bacterium mud protective material are ℃ subsequent use.According to viable bacteria bacterium mud: the ratio of bacterium mud protective material=1: 10 is with bacterium mud and bacterium mud protective material mixing and emulsifying.
Other are with embodiment 1.
Viable count is greater than 1.0 * 10 in the product for preparing
11Cfu/g, vacuum-packed with two aluminium film phonograph seals, every bag of 50g or pack according to customer requirement.
The scope that the present invention requires to protect does not receive the restriction of said specific embodiments; Specific embodiment provided by the invention is only as further specifying example of the present invention; Those skilled in the art can be easy to the present invention is made the replacement of multiple improvement and component with reference to the description of this case specification sheets, and these improvement and replacements that need not creative work also should fall within the protection domain of appended claims of the present invention.
Claims (5)
1. the preparation method of a freeze-dried bifidobacteria powder; Its technical process comprises strain fermentation cultivation, fermented liquid separation, the emulsification of bacterium mud and emulsion vacuum lyophilization; It is characterized in that: said strain fermentation culture process: bifidobacterium species is inserted in the triangular flask liquid nutrient medium cultivate; Cultivated 10～30 hours down at 30～40 ℃, inserting big fermentor tank again carries out fermentation culture, cultivates 10～30 hours for 35～40 ℃; The weight percent of said substratum consists of: bifidus factor 1～5%, glucose 1～2%, peptone 0.5～2%, yeast extract paste 0.5～2%, dibasic ammonium citrate 0.1～0.2%, potassium hydrogenphosphate 0.1～0.2%, anhydrous sodium acetate 0.1～1%, sal epsom 0.05～0.1%, tween-80 0.0001%, all the other are water; 115 ℃ of sterilizations 15 minutes, and cultivate acidity to make fermented liquid pH value be 5.5～6.5 to add sodium hydroxide solution control; Said bacterium mud emulsifying process; According to viable bacteria bacterium mud: the ratio of bacterium mud protective material=0.1: 1～1: 0.1 is with bacterium mud and protective material mixing and emulsifying; Bacterium mud protective material prescription each component weight percent is: skim-milk 1～10%, lactose 1～10%, W-Gum 1～10%, inhibitor 0.1～1%, tween-80 0.01～0.05%; All the other are water, and under 105 ℃～115 ℃ conditions, sterilize 15 minutes postcooling to 5～20 of bacterium mud protective material are ℃ subsequent use; Described inhibitor is selected a kind of in isoascorbic acid, L-glutamic acid and glycocoll.
2. the preparation method of a kind of freeze-dried bifidobacteria powder according to claim 1; It is characterized in that: said fermented liquid separating technology adopts ceramic membrane pore size 0.01～1 μ m; Filtering separation thalline under 0.05MPa～0.5MPa hydraulic condition; Or carrying out under 4000～10000rpm condition, and, obtain bacterium mud with sterilized water washing 1～2 time with tubular type or disc centrifuge separating thallus.
3. the preparation method of a kind of freeze-dried bifidobacteria powder according to claim 1; It is characterized in that: said emulsion vacuum freeze-drying technique is: in the final vacuum lyophilize in 1～5 hour of-60～-40 ℃ of pre-freezes; The lyophilize temperature is-60 ℃; Vacuum tightness is 1～8pa, vacuum lyophilization 10～30 hours; Obtain viable count after the drying greater than 1.0 * 10
11The freeze-dried bifidobacteria powder of cfu/g.
4. according to the preparation method of claim 1 or 3 described a kind of freeze-dried bifidobacteria powders, it is characterized in that: said strain fermentation cultivation, fermented liquid separation and bacterium mud emulsifying process operating environment conditional request are for carrying out in the clean environment that is lower than 25 degree, humidity RH＜35% in temperature.
5. the preparation method of a kind of freeze-dried bifidobacteria powder according to claim 1, it is characterized in that: described bifidus bacillus is selected one or more in bifidumbacterium bifidum Bifidobacterium bifidum, bifidobacterium adolescentis Bifidobacterium adolescentis, JCM 1192T Bifidobacterium breve, bifidobacteria infantis Bifidobacterium infantis and bifidus longum bb Bifidobacterium longum.
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| CN107868769A (en) * | 2017-12-18 | 2018-04-03 | 江南大学 | A kind of high activity bifidobacteria freeze-dried vaccine powder producing method and application |
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| CN113005066B (en) * | 2021-03-31 | 2023-07-21 | 江苏蓝泽生物科技有限公司 | Compound bifidobacterium preparation for resisting allergy, increasing immunity, reducing blood sugar and fat and losing weight and preparation method thereof |
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