CN102021162A - High-activity bifidobacterium powder and preparation method thereof - Google Patents
High-activity bifidobacterium powder and preparation method thereof Download PDFInfo
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Abstract
The invention provides a high-activity bifidobacterium powder and a preparation method thereof, belonging to the technical field of preparation of bifidobacterium products. The high-activity bifidobacterium powder is characterized by being prepared from bifidobacteria by adopting a high-density culture technology and a double-layer embedment technology. The prepared bifidobacterium powder has high viable bacteria content reaching more than 1011cfu/g and high gastric acid resistant capacity and cholate resistant capacity, effectively colonizes intestinal tracts to exert therapeutic effect and solves the problem of reduced viable bacteria content of the traditional bifidobacterium products in the storage and consumption process. The invention has easiness and convenience for operation, low cost and suitability for industrialized production, can be prepared into various dosage forms as required, can not only be directly taken, but also be used for researching functional foods and pharmaceutical preparations as an additive.
Description
Technical field
The present invention relates to a kind of bifidus bacillus goods, particularly relate to a kind of high activity bifidobacteria bacterium powder and preparation method thereof, belong to bifidus bacillus goods preparing technical field, the high activity bifidobacteria bacterium powder that the present invention makes can be made all kinds of formulations as required, both can directly take, and can be used as additive again and be used to develop functional foodstuff and pharmaceutical preparation.
Background technology
Bifidus bacillus is to be found from the breast feeding baby's of health ight soil first and separated by doctor Tisser.H in Pasteur Institut in 1899, it is important physical bacterium in the human intestinal, the symbiotic relationship that is remaining harmony all one's life with the people, be the dominant bacteria in the healthy individual enteron aisle, its beneficial effect that promotes health is considerably beyond other milk-acid bacteria.But, owing to reasons such as body aging, environmental pollution, metatrophia, abuses of antibiotics, the bifidus bacillus quantity of human intestinal is descended, cause the intestinal microecology imbalance, cause the flora transposition, bring out endogenous infection, cause body disease.Therefore, bifidobacteria based biological chemistry barrier, suppress pathogenic bacterium and spoilage organism, nutrition is provided, improves affirming of immunizing power, anti-tumor function and clinical efficacy thereof, domestic and international investigator develops multiple bifidus bacillus food in succession and little ecological medicine is used for nourishing function and treats the alteration of intestinal flora that various factors causes, increase bifidus bacillus quantity in the human intestinal by the external source mode, promote health.
Though the exploitation of bifidus bacillus goods emerges in an endless stream, the most key problem that exists in numerous goods is that product viable bacteria content in storage and process of consumption descends.Discover that bifidus bacillus has material impact in intravital survival of people and multiplication capacity to its prebiotic effect, obtain desired result of treatment, bifidus bacillus must reach enough quantity, after guaranteeing entering human body enough viable bacteria performance nourishing functions is arranged.Yet because bifidus bacillus is had relatively high expectations to nutritional condition, very responsive to oxygen, poor to the resistibility of low pH value, the quantity of bifidus bacillus sharply descends in the product that causes coming into the market.In addition, bifidus bacillus produces beneficial effect to human body, must still have a large amount of survival bacterium arrival enteron aisles also to grow on intestinal mucosa surely after by gastric environment.But in hydrochloric acid in gastric juice and cholate environment, bifidus bacillus bacterium number can very fast decline, and making does not finally have enough bifidus bacilluss to play a role in human body.Therefore, how to improve bifidobacteria viable bacteria quantity in the product, guarantee that it brings into play curative effect decided at the higher level but not officially announced the growing of human intestinal and become our problem demanding prompt solution.
Domestic researchist has done big quantity research at above actual application problem, is mainly reflected in aspects such as bacterial classification genetic modification, high-density culture, microcapsule technology, protective material research.A kind of bifid bacterium microcapsule and manufacture method thereof are disclosed among the patent CN 1056877C; the compound active bacteria formulation of a kind of bifidus bacillus is disclosed among the CN 1063051C; a kind of acid resistant enteric coated capsule of active binding bifidobacterium strain is disclosed among the CN 1149092C; three patents all adopt microcapsulary; the ability that makes bifidus bacillus can resist external environment strengthens; can keep out the erosion of hydrochloric acid in gastric juice and cholate; under liquid state or normal temperature state, store for a long time; still can effectively protect its activity; but; generally but need could dissolve more than six hours after microcapsule embedded bifidus bacillus arrives enteron aisle and disengage, effect is limited.Patent CN 1116410C discloses active bacteria formulation of a kind of high survival probability and preparation method thereof, and this patent is carried out the deep liquid high-density culture with bifidus bacillus, and fermented liquid contains viable count 5 * 10
8Cfu/mL; thalline adopts lyophilized powder, microcapsule, the canned capsule of inflated with nitrogen, the two aluminium packing of inflated with nitrogen quadruple sfgd.; make active bacteria formulation; make the thalline survival rate significantly improve; can reach 2 years under the preparation preservation period normal temperature, still, said preparation technology is more loaded down with trivial details, time-consuming; viable count is still lower after high-density culture, and the later stage adopts microcapsulary to have beneficial effect performance problem equally.A kind of bifidobacteria viable bacteria preparation and special-purpose protecting agent thereof are disclosed among the patent CN 100519738C; a kind of composite protectant of high activity bifidobacteria dry powder formulation is disclosed among the CN 100497594C; two patents adopt special protective material; with its lyophilisate of making, viable bacteria content all reaches 1.0 * 10
11More than the cfu/g, solved bifidus bacillus goods normal temperature simultaneously and preserved difficult problem, still, the validity problem of the beneficial fruit of coming into force of effective field planting enteron aisle of bifidus bacillus and performance is also unresolved.
Summary of the invention
The objective of the invention is to descend and the effective field planting enteron aisle of bifidus bacillus and the performance benefit deficiency really that comes into force, a kind of high activity bifidobacteria bacterium powder and preparation method thereof is provided at the product that exists in present bifidus bacillus goods viable bacteria content in storage and process of consumption.The bifidus bacillus bacterium powder viable bacteria content height that the present invention makes can reach 10
11More than the cfu/g, stomach juice-resistant and cholate ability are strong, effective intestinal canal colonization performance curative effect, solved exist in the present bifidus bacillus goods problem.
The technical solution that the present invention provides is: this high activity bifidobacteria bacterium powder is characterized in that described bacterium powder adopts high-density culture and double-layer embedment technique to make by bifidus bacillus.
For better finishing purpose of the present invention, described bifidus bacillus is at least a kind of among bifidobacterium infantis AS1.2202, bifidobacterium breve AS1.2213, bifidus longum bb CICC 6186, bifidumbacterium bifidum CICC 6071, the bifidobacterium adolescentis CICC 6070.
Bifidobacterium infantis AS 1.2202 in the above-mentioned bifidus bacillus, bifidobacterium breve AS 1.2213 are by the preservation of Chinese common micro-organisms culture presevation administrative center, bifidus longum bb CICC 6186, bifidumbacterium bifidum CICC6071, bifidobacterium adolescentis CICC 6070 belong to existing bifidobacterium species by the preservation of China Committee for Culture Collection of Microorganisms Research for Industrial Microbial Germ preservation administrative center.
The preparation method of this high activity bifidobacteria bacterium powder that the present invention provides includes the following step:
A, high-density culture
(1) actication of culture: under the aseptic condition, thalline is inoculated on liquid nutrient medium and the agar plate in the picking ampoul tube, and anaerobism was cultivated 24～48 hours under 36～38 ℃ of conditions, activated for 2～3 generations continuously, promptly got activated spawn;
Wherein, the compound method that is used for bifidus bacillus liquid nutrient medium of the present invention is as follows: Tryptones 5.0g, yeast extract 10.0g, soy peptone 5.0g, glucose 10.0g, L～cysteine hydrochloride 0.5g, tween 80 1.0mL, salts solution 40mL, Jia Shui are settled to 1000mL, regulate pH6.8～7.0,121 ℃ sterilization 20min.Wherein the composition of salts solution is: calcium chloride 0.2g, potassium primary phosphate 1.0g, sal epsom 0.48g, sodium bicarbonate 10.0g, dipotassium hydrogen phosphate 1.0g, sodium-chlor 2.0g, Jia Shui are settled to 1000mL.
(2) seed culture: by in 5%～10% the inoculum size access liquid nutrient medium, anaerobism was cultivated 20～28 hours under 36～38 ℃ of conditions, as inoculum with activated spawn.
(3) fermentation culture: inoculum by in 5%～10% the inoculum size access liquid nutrient medium, is carried out the fermentor tank high-density culture under 36～38 ℃ of conditions.
(4) fermentation stops: stop fermentation when medium pH and glucose content all no longer reduce, obtain bifidus bacillus high density fermentation liquid.
B, double-layer embedment
(1) centrifugal concentrate: with bifidus bacillus high density fermentation liquid centrifugal 10～20min under 4 ℃, the condition of 6000～8000r/min, sterilized water washing 2～3 times is taken off layer precipitation and is active bacterium mud.
(2) add protective material: by 1: 4～1: 10 (V
Protective material/ V
Fermented liquid) ratio in active bacterium mud, add the thalline freezing drying protective agent, 150～250r/min stirs 15～30min to evenly under the aseptic condition, the preparation bacteria suspension.
Wherein, the compound method that is used for freezing drying protective agent of the present invention is as follows: skim-milk 8～12g, sucrose 8～12g, Sodium Glutamate 0.5～1.5g, sorbyl alcohol 0.5～1.5g; add water and be settled to 100mL, heated and stirred makes its dissolving, then 115 ℃ of sterilization 15min.
(3) I layer embedding: by 1: 1～1: 5 (V
I layer embedding liquid/ V
Protective material) ratio I layer embedding liquid is added in the bacteria suspension, 150～250r/min stirs 15～30min to evenly under the aseptic condition.
Wherein, being used for I layer embedding liquid of the present invention is protein soln, comprises in casein, opalescin, Protalbinic acid, vitellin, myoprotein, soybean protein isolate, the herbal medicine albumin etc. one or more.
(4) II layer embedding: by 1: 1～1: 5 (V
II layer embedding liquid/ V
Protective material) ratio II layer embedding liquid is added in the above-mentioned mixed solution, 150～250r/min stirs 15～30min to evenly under the aseptic condition.
Wherein, be used for II layer embedding liquid of the present invention, comprise in carrageenin, xanthan gum, guar gum, pectin, tapioca (flour), agar, sodium alginate, Viscogum BE, the konjak gum etc. one or more for being hydrosol.
C, lyophilize
After will placing-70 ℃ refrigerator pre-freeze 2～6h through the bifidus bacillus of double-layer embedment, in condenser temperature-45～-55 ℃, vacuum lyophilization under vacuum tightness 0.1～10 handkerchief promptly gets bifidus bacillus bacterium powder.
Compared with prior art, the invention has the beneficial effects as follows:
(1) high-density culture is significantly increased the more traditional training method of cell density, thereby reach the minimizing volume of culture, can also shorten the production cycle, finally improve the specific production rate of specific product, reduce production costs thereby reduce facility investment, improve the competitive power on market.
(2) the outer colloid of double-layer embedment does not dissolve in sour environment, and protection thalline safety is by stomach, the destruction that is not subjected to hydrochloric acid in gastric juice and cholate.Arrive outer colloid dissolving behind the enteron aisle, expose internal layer protein, assist thalline to be attached on the intestines wall fine hair and grow in that enteron aisle is decided at the higher level but not officially announced, nutrition is provided simultaneously, promote growing microorganism.
(3) Vacuum Freezing ﹠ Drying Technology is carried out under low temperature, low pressure, and basic secluding air effectively suppresses product generation physico-chemical property and changes, and can preserve active substance preferably, and give the product satisfactory stability, is convenient to long storage.
Specific embodiments
Test shows that the technology that the present invention provides is fit to the preparation of any bifidus bacillus high reactivity bacterium powder, and the embodiment that below provides only is described further technical scheme of the present invention, rather than restriction protection scope of the present invention.
Embodiment 1: the preparation of bifidobacteria infantis bacterium powder
1, high-density culture
(1) actication of culture: under the aseptic condition, thalline is inoculated on liquid nutrient medium and the agar plate in the picking ampoul tube, and anaerobism was cultivated 48 hours under 37 ℃ of conditions, activated for 3 generations continuously, promptly got activated spawn.
Wherein, it is as follows to be used for bifidus bacillus culture medium preparation method of the present invention: Tryptones 5.0g, yeast extract 10.0g, soy peptone 5.0g, glucose 10.0g, L～cysteine hydrochloride 0.5g, tween 80 1.0mL, salts solution 40mL, Jia Shui are settled to 1000mL, regulate pH6.8～7.0,121 ℃ sterilization 20min.Wherein the composition of salts solution is: calcium chloride 0.2g, potassium primary phosphate 1.0g, sal epsom 0.48g, sodium bicarbonate 10.0g, dipotassium hydrogen phosphate 1.0g, sodium-chlor 2.0g, Jia Shui are settled to 1000mL.
(2) seed culture: by in 5% the inoculum size access liquid nutrient medium, anaerobism was cultivated 24 hours under 37 ℃ of conditions, as inoculum with activated spawn.
(3) fermentation culture: inoculum by in 5% the inoculum size access liquid nutrient medium, is carried out the fermentor tank high-density culture under 37 ℃ of conditions.The fermenting process significant parameter is controlled to be:
PH control: inoculation back NaOH regulates the initial pH to 7.0 of substratum, and stream adds NaOH and keeps pH 〉=5.0 between yeast phase;
Glucose control: glucose concn in the monitoring substratum between yeast phase, stream adds glucose and keeps glucose content 〉=4g/L.
(4) fermentation stops: stop fermentation when medium pH and glucose content all no longer reduce, the viable count of the bifidus bacillus high density fermentation liquid of acquisition can reach 5 * 10
9More than the cfu/mL.
2, double-layer embedment
(1) centrifugal concentrate: with bifidus bacillus high density fermentation liquid centrifugal 15min under 4 ℃, the condition of 6000r/min, sterilized water washing 2 times is taken off layer precipitation and is active bacterium mud.
(2) add protective material: press V
Protective material/ V
Fermented liquid=1: 8 ratio adds the thalline freezing drying protective agent in active bacterium mud, 200r/min stirs 15min to evenly under the aseptic condition, prepares bacteria suspension.
Wherein, the compound method that is used for freezing drying protective agent of the present invention is as follows: skim-milk 10g, sucrose 10g, Sodium Glutamate 1.0g, sorbyl alcohol 1.0g, and add water and be settled to 100mL, heated and stirred makes its dissolving, then 115 ℃ of sterilization 15min.
(3) I layer embedding: press V
I layer embedding liquid/ V
Protective material=1: 2 ratio adds I layer embedding liquid in the bacteria suspension, and 200r/min stirs 15min to even under the aseptic condition.
Wherein, being used for I layer embedding liquid of the present invention is 5% soybean protein isolate solution.
(4) II layer embedding: press V
II layer embedding liquid: V
Protective material=1: 2 ratio adds II layer embedding liquid in the above-mentioned mixed solution, and 200r/min stirs 15min to even under the aseptic condition.
Wherein, be used for the colloid mixing solutions that II layer embedding liquid of the present invention is the konjak gum of 0.04% kappa-carrageenan and 0.06%.
3, lyophilize
After will placing-70 ℃ refrigerator pre-freeze 4h through the bifidus bacillus of double-layer embedment, at condenser temperature-50 ℃, vacuum lyophilization under vacuum tightness 0.1～10 handkerchief promptly gets bifidus bacillus bacterium powder, and viable count can reach 1.0 * 10
11More than the cfu/g.
Embodiment 2: the preparation of bifidobacterium adolescentis bacterium powder
1, high-density culture
(1) actication of culture: under the aseptic condition, thalline is inoculated on liquid nutrient medium and the agar plate in the picking ampoul tube, and anaerobism was cultivated 48 hours under 37 ℃ of conditions, activated for 3 generations continuously, promptly got activated spawn.
(2) seed culture: by in 5% the inoculum size access liquid nutrient medium, anaerobism was cultivated 24 hours under 37 ℃ of conditions, as inoculum with activated spawn.
(3) fermentation culture: inoculum by in 5% the inoculum size access liquid nutrient medium, is carried out the fermentor tank high-density culture under 37 ℃ of conditions.The fermenting process significant parameter is controlled to be:
PH control: inoculation back NaOH regulates the initial pH to 7.0 of substratum, and stream adds NaOH and keeps pH 〉=5.0 between yeast phase;
Glucose control: glucose concn in the monitoring substratum between yeast phase, stream adds glucose and keeps glucose content 〉=4g/L.
(4) fermentation stops: stop fermentation when medium pH and glucose content all no longer reduce, the viable count of the bifidus bacillus high density fermentation liquid of acquisition can reach 1 * 10
10More than the cfu/mL.
2, double-layer embedment
(1) centrifugal concentrate: with bifidus bacillus high density fermentation liquid centrifugal 15min under 4 ℃, the condition of 6000r/min, sterilized water washing 2 times is taken off layer precipitation and is active bacterium mud.
(2) add protective material: press V
Protective material/ V
Fermented liquid=1: 8 ratio adds the thalline freezing drying protective agent in active bacterium mud, 200r/min stirs 15min to evenly under the aseptic condition, prepares bacteria suspension.
(3) I layer embedding: press V
I layer embedding liquid/ V
Protective material=1: 2 ratio adds I layer embedding liquid in the bacteria suspension, and 200r/min stirs 15min to even under the aseptic condition.
(4) II layer embedding: press V
II layer embedding liquid: V
Protective material=1: 2 ratio adds II layer embedding liquid in the above-mentioned mixed solution, and 200r/min stirs 15min to even under the aseptic condition.
3, lyophilize
After will placing-70 ℃ refrigerator pre-freeze 4h through the bifidus bacillus of double-layer embedment, at condenser temperature-50 ℃, vacuum lyophilization under vacuum tightness 0.1～10 handkerchief promptly gets bifidus bacillus bacterium powder, and viable count can reach 2.0 * 10
11More than the cfu/g.
Embodiment 3: the preparation of bifidumbacterium bifidum bacterium powder
1, high-density culture
(1) actication of culture: under the aseptic condition, thalline is inoculated on liquid nutrient medium and the agar plate in the picking ampoul tube, and anaerobism was cultivated 48 hours under 37 ℃ of conditions, activated for 3 generations continuously, promptly got activated spawn.
(2) seed culture: by in 5% the inoculum size access liquid nutrient medium, anaerobism was cultivated 24 hours under 37 ℃ of conditions, as inoculum with activated spawn.
(3) fermentation culture: inoculum by in 5% the inoculum size access liquid nutrient medium, is carried out the fermentor tank high-density culture under 37 ℃ of conditions.The fermenting process significant parameter is controlled to be:
PH control: inoculation back NaOH regulates the initial pH to 7.0 of substratum, and stream adds NaOH and keeps pH 〉=5.0 between yeast phase;
Glucose control: glucose concn in the monitoring substratum between yeast phase, stream adds glucose and keeps glucose content 〉=4g/L.
(4) fermentation stops: stop fermentation when medium pH and glucose content all no longer reduce, the viable count of the bifidus bacillus high density fermentation liquid of acquisition can reach 5 * 10
9More than the cfu/mL.
2, double-layer embedment
(1) centrifugal concentrate: with bifidus bacillus high density fermentation liquid centrifugal 15min under 4 ℃, the condition of 6000r/min, sterilized water washing 2 times is taken off layer precipitation and is active bacterium mud.
(2) add protective material: press V
Protective material/ V
Fermented liquid=1: 8 ratio adds the thalline freezing drying protective agent in active bacterium mud, 200r/min stirs 15min to evenly under the aseptic condition, prepares bacteria suspension.
(3) I layer embedding: press V
I layer embedding liquid/ V
Protective material=1: 2 ratio adds I layer embedding liquid in the bacteria suspension, and 200r/min stirs 15min to even under the aseptic condition.
Wherein, being used for I layer embedding liquid of the present invention is 5% soybean protein isolate solution.
(4) II layer embedding: press V
II layer embedding liquid: V
Protective material=1: 2 ratio adds II layer embedding liquid in the above-mentioned mixed solution, and 200r/min stirs 15min to even under the aseptic condition.
Wherein, be used for the colloid mixing solutions that II layer embedding liquid of the present invention is the konjak gum of 0.04% kappa-carrageenan and 0.06%.
3, lyophilize
After will placing-70 ℃ refrigerator pre-freeze 4h through the bifidus bacillus of double-layer embedment, at condenser temperature-50 ℃, vacuum lyophilization under vacuum tightness 0.1～10 handkerchief promptly gets bifidus bacillus bacterium powder, and viable count can reach 1.0 * 10
11More than the cfu/g.
Embodiment 4: the preparation of bifidus longum bb bacterium powder
1, high-density culture
(1) actication of culture: under the aseptic condition, thalline is inoculated on liquid nutrient medium and the agar plate in the picking ampoul tube, and anaerobism was cultivated 48 hours under 37 ℃ of conditions, activated for 3 generations continuously, promptly got activated spawn.
(2) seed culture: by in 5% the inoculum size access liquid nutrient medium, anaerobism was cultivated 24 hours under 37 ℃ of conditions, as inoculum with activated spawn.
(3) fermentation culture: inoculum by in 5% the inoculum size access liquid nutrient medium, is carried out the fermentor tank high-density culture under 37 ℃ of conditions.The fermenting process significant parameter is controlled to be:
PH control: inoculation back NaOH regulates the initial pH to 7.0 of substratum, and stream adds NaOH and keeps pH 〉=5.0 between yeast phase;
Glucose control: glucose concn in the monitoring substratum between yeast phase, stream adds glucose and keeps glucose content 〉=4g/L.
(4) fermentation stops: stop fermentation when medium pH and glucose content all no longer reduce, the viable count of the bifidus bacillus high density fermentation liquid of acquisition can reach 1.5 * 10
10More than the cfu/mL.
2, double-layer embedment
(1) centrifugal concentrate: with bifidus bacillus high density fermentation liquid centrifugal 15min under 4 ℃, the condition of 6000r/min, sterilized water washing 2 times is taken off layer precipitation and is active bacterium mud.
(2) add protective material: press V
Protective material/ V
Fermented liquid=1: 8 ratio adds the thalline freezing drying protective agent in active bacterium mud, 200r/min stirs 15min to evenly under the aseptic condition, prepares bacteria suspension.
(3) I layer embedding: press V
I layer embedding liquid/ V
Protective material=1: 2 ratio adds I layer embedding liquid in the bacteria suspension, and 200r/min stirs 15min to even under the aseptic condition.
Wherein, being used for I layer embedding liquid of the present invention is 5% soybean protein isolate solution.
(4) II layer embedding: press V
II layer embedding liquid: V
Protective material=1: 2 ratio adds II layer embedding liquid in the above-mentioned mixed solution, and 200r/min stirs 15min to even under the aseptic condition.
Wherein, be used for the colloid mixing solutions that II layer embedding liquid of the present invention is the konjak gum of 0.04% kappa-carrageenan and 0.06%.
3, lyophilize
After will placing-70 ℃ refrigerator pre-freeze 4h through the bifidus bacillus of double-layer embedment, at condenser temperature-50 ℃, vacuum lyophilization under vacuum tightness 0.1～10 handkerchief promptly gets bifidus bacillus bacterium powder, and viable count can reach 3.0 * 10
11More than the cfu/g.
Embodiment 5: bifidus bacillus hybrid bacterial strain fermentative preparation bacterium powder (is example with bifidobacteria infantis and bifidobacterium adolescentis)
1, high-density culture
(1) actication of culture: under the aseptic condition, bifidobacteria infantis and bifidobacterium adolescentis thalline are inoculated on liquid nutrient medium and the agar plate in the picking ampoul tube respectively, and anaerobism was cultivated 48 hours under 37 ℃ of conditions, activated for 3 generations continuously, promptly got activated spawn.
Wherein, it is as follows to be used for bifidus bacillus culture medium preparation method of the present invention: Tryptones 5.0g, yeast extract 10.0g, soy peptone 5.0g, glucose 10.0g, L～cysteine hydrochloride 0.5g, tween 80 1.0mL, salts solution 40mL, Jia Shui are settled to 1000mL, regulate pH6.8～7.0,121 ℃ sterilization 20min.Wherein the composition of salts solution is: calcium chloride 0.2g, potassium primary phosphate 1.0g, sal epsom 0.48g, sodium bicarbonate 10.0g, dipotassium hydrogen phosphate 1.0g, sodium-chlor 2.0g, Jia Shui are settled to 1000mL.
(2) seed culture: respectively activatory bifidobacteria infantis and bifidobacterium adolescentis bacterial classification are inserted in the liquid nutrient medium by 5% inoculum size, anaerobism was cultivated 24 hours under 37 ℃ of conditions, as inoculum.
(3) fermentation culture: simultaneously bifidobacteria infantis and bifidobacterium adolescentis inoculum are inserted in the liquid nutrient medium by 5% inoculum size, under 37 ℃ of conditions, carry out the fermentor tank high-density culture.The fermenting process significant parameter is controlled to be:
PH control: inoculation back NaOH regulates the initial pH to 7.0 of substratum, and stream adds NaOH and keeps pH 〉=5.0 between yeast phase;
Glucose control: glucose concn in the monitoring substratum between yeast phase, stream adds glucose and keeps glucose content 〉=4g/L.
(4) fermentation stops: stop fermentation when medium pH and glucose content all no longer reduce, the viable bacteria number average of bifidobacteria infantis and bifidobacterium adolescentis can reach 2.5 * 10 in the bifidus bacillus high density fermentation liquid of acquisition
9More than the cfu/mL.
2, double-layer embedment
(1) centrifugal concentrate: with bifidobacteria infantis and bifidobacterium adolescentis high density fermentation liquid centrifugal 15min under 4 ℃, the condition of 6000r/min, sterilized water washing 2 times is taken off layer precipitation and is active bacterium mud.
(2) add protective material: press V
Protective material/ V
Fermented liquid=1: 8 ratio adds the thalline freezing drying protective agent in active bacterium mud, 200r/min stirs 15min to evenly under the aseptic condition, prepares bacteria suspension.
Wherein, the compound method that is used for freezing drying protective agent of the present invention is as follows: skim-milk 10g, sucrose 10g, Sodium Glutamate 1.0g, sorbyl alcohol 1.0g, and add water and be settled to 100mL, heated and stirred makes its dissolving, then 115 ℃ of sterilization 15min.
(3) I layer embedding: press V
I layer embedding liquid/ V
Protective material=1: 2 ratio adds I layer embedding liquid in the bacteria suspension, and 200r/min stirs 15min to even under the aseptic condition.
Wherein, being used for I layer embedding liquid of the present invention is 5% soybean protein isolate solution.
(4) II layer embedding: press V
II layer embedding liquid: V
Protective material=1: 2 ratio adds II layer embedding liquid in the above-mentioned mixed solution, and 200r/min stirs 15min to even under the aseptic condition.
Wherein, be used for the colloid mixing solutions that II layer embedding liquid of the present invention is the konjak gum of 0.04% kappa-carrageenan and 0.06%.
3, lyophilize
After will placing-70 ℃ of refrigerator pre-freeze 4h through the bifidobacteria infantis and the bifidobacterium adolescentis of double-layer embedment, at condenser temperature-50 ℃, vacuum lyophilization under vacuum tightness 0.1～10 handkerchief promptly gets and mixes bifidus bacillus bacterium powder, and viable count can reach 1.0 * 10
11More than the cfu/g.
Embodiment 6: prescription is formed
Each component is mixed by last table consumption, makes capsule, 0.25g/ grain, each 4, every day 2 times; Make electuary, 1g/ bag, each 1 bag, every day 2 times; Make tablet, 0.5g/ grain, each 2, every day 2 times.
Embodiment 7: the yogurt (is example with the bifidobacterium adolescentis) that contains bifidus bacillus bacterium powder
Earlier sweet milk being heated to 85 ℃ kept 15 seconds or was heated to 135 ℃ to keep carrying out disinfection in 2 seconds, be cooled to 37 ℃, then add 5% bifidobacterium adolescentis bacterium powder therein, cultivate 16h for 37 ℃, make acidity reach 2.0%, stir cooling then, the cooling and fermentation breast is carried out homogenize handle canned preservation in container.
Embodiment 8: the milk powder (is example with bifidobacteria infantis, bifidumbacterium bifidum) that contains bifidus bacillus bacterium powder
Bifidobacteria infantis bacterium powder, each 5g of bifidumbacterium bifidum bacterium powder and 990g milk powder are mixed packing after the weighing.
Embodiment 9: the vegetables juice (is example with the Radix Dauci Sativae juice) that contains bifidus bacillus bacterium powder
With bifidobacteria infantis bacterium powder 1g with mix at 85 ℃ of Radix Dauci Sativae juice 1000mL after keeping down carrying out disinfection in 15 seconds, add acesulfame potassium 0.1g, the back that stirs is canned.
Embodiment 10: the fruit juice (is example with the Sucus Mali pumilae) that contains bifidus bacillus bacterium powder
With bifidobacteria infantis bacterium powder 1g with mix at 85 ℃ of Sucus Mali pumilae 1000mL after keeping down carrying out disinfection in 15 seconds, add sucrose 0.1g, the back that stirs is canned.
Claims (8)
1. a high activity bifidobacteria bacterium powder is characterized in that described bacterium powder adopts high-density culture and double-layer embedment technique to make by bifidus bacillus.
2. high activity bifidobacteria bacterium powder according to claim 1 is characterized in that described bifidus bacillus is at least a kind of among bifidobacterium infantis AS 1.2202, bifidobacterium breve AS 1.2213, bifidus longum bb CICC 6186, bifidumbacterium bifidum CICC 6071, the bifidobacterium adolescentis CICC 6070.
3. the preparation method of the described high activity bifidobacteria bacterium of claim 1 powder is characterized in that including the following step:
A, high-density culture
(1) actication of culture: under the aseptic condition, thalline is inoculated on liquid nutrient medium and the agar plate in the picking ampoul tube, and anaerobism was cultivated 24～48 hours under 36～38 ℃ of conditions, activated for 2～3 generations continuously, promptly got activated spawn;
(2) seed culture: by in 5%～10% the inoculum size access liquid nutrient medium, anaerobism was cultivated 20～28 hours under 36～38 ℃ of conditions, as inoculum with activated spawn.
(3) fermentation culture: inoculum by in 5%～10% the inoculum size access liquid nutrient medium, is carried out the fermentor tank high-density culture under 36～38 ℃ of conditions.
(4) fermentation stops: stop fermentation when medium pH and glucose content all no longer reduce, obtain bifidus bacillus high density fermentation liquid.
B, double-layer embedment
(1) centrifugal concentrate: with bifidus bacillus high density fermentation liquid centrifugal 10～20min under 4 ℃, the condition of 6000～8000r/min, sterilized water washing 2～3 times is taken off layer precipitation and is active bacterium mud.
(2) add protective material: by 1: 4～1: 10 (V
Protective material/ V
Fermented liquid) ratio in active bacterium mud, add the thalline freezing drying protective agent, 150～250r/min stirs 15～30min to evenly under the aseptic condition, the preparation bacteria suspension.
(3) I layer embedding: by 1: 1～1: 5 (V
I layer embedding liquid/ V
Protective material) ratio I layer embedding liquid is added in the bacteria suspension, 150～250r/min stirs 15～30min to evenly under the aseptic condition.
(4) II layer embedding: by 1: 1～1: 5 (V
II layer embedding liquid/ V
Protective material) ratio II layer embedding liquid is added in the above-mentioned mixed solution, 150～250r/min stirs 15～30min to evenly under the aseptic condition;
C, lyophilize
After will placing-70 ℃ refrigerator pre-freeze 2～6h through the bifidus bacillus of double-layer embedment, in condenser temperature-45～-55 ℃, vacuum lyophilization under vacuum tightness 0.1～10 handkerchief promptly gets bifidus bacillus bacterium powder.
4. according to the preparation method of the described high activity bifidobacteria bacterium of claim 3 powder, it is characterized in that the compound method of bifidus bacillus liquid nutrient medium is as follows:
Tryptones 5.0g, yeast extract 10.0g, soy peptone 5.0g, glucose 10.0g, L～cysteine hydrochloride 0.5g, tween 80 1.0mL, salts solution 40mL, Jia Shui are settled to 1000mL, regulate pH6.8～7.0,121 ℃ of sterilization 20min, wherein the composition of salts solution is: calcium chloride 0.2g, potassium primary phosphate 1.0g, sal epsom 0.48g, sodium bicarbonate 10.0g, dipotassium hydrogen phosphate 1.0g, sodium-chlor 2.0g, Jia Shui are settled to 1000mL.
5. according to the preparation method of the described high activity bifidobacteria bacterium of claim 3 powder, it is characterized in that the compound method of freezing drying protective agent is as follows:
Skim-milk 8～12g, sucrose 8～12g, Sodium Glutamate 0.5～1.5g, sorbyl alcohol 0.5～1.5g add water and are settled to 100mL, and heated and stirred makes its dissolving, then 115 ℃ of sterilization 15min.
6. according to the preparation method of the described high activity bifidobacteria bacterium of claim 3 powder, it is characterized in that I layer embedding liquid is protein soln, comprise in casein, opalescin, Protalbinic acid, vitellin, myoprotein, soybean protein isolate, the herbal medicine albumin one or more.
7. according to the preparation method of the described high activity bifidobacteria bacterium of claim 3 powder, it is characterized in that II layer embedding liquid is hydrosol, comprise in carrageenin, xanthan gum, guar gum, pectin, tapioca (flour), agar, sodium alginate, Viscogum BE, the konjak gum etc. one or more.
8. a kind of high activity bifidobacteria bacterium powder according to claim 1 is characterized in that: described bacterium powder can be made into the capsule that is used for as food, healthcare products, medicine or foodstuff additive, electuary, granule, tablet etc.
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