CN101338295B - Bacillus bifidus freeze concentration leaven and method for preparing same - Google Patents
Bacillus bifidus freeze concentration leaven and method for preparing same Download PDFInfo
- Publication number
- CN101338295B CN101338295B CN2008101506655A CN200810150665A CN101338295B CN 101338295 B CN101338295 B CN 101338295B CN 2008101506655 A CN2008101506655 A CN 2008101506655A CN 200810150665 A CN200810150665 A CN 200810150665A CN 101338295 B CN101338295 B CN 101338295B
- Authority
- CN
- China
- Prior art keywords
- parts
- protective material
- bifidobacterium
- water
- bifidus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a bifidobacterium freezing concentration starter and a preparation method thereof. The preparation method can solve the problems of difficult culturing of the bifidobacterium, lower activity of the starter and short preservation period. The preparation method includes: culturing a strain activation tube, culturing the strain of first grade by a triangle bottle, culturing the strain of second grade with a seed pot, culturing in a fermentation pot, concentrically collecting the strain, adding a protective agent, packaging in vacuum and storing in a freezing way. The starter is mainly characterized by high live bacteria content, longer shelf life, less inoculation dosage, being capable of stabilizing each batch of fermented dairy products, preventing the strain from degradation and pollution as well as greatly improving the working production rate and the product quality of the fermented dairy product industry. Compared with the starter prepared by the vacuum freezing drying method, the freezing concentration starter prepared by the invention has the advantages of simple method, less device investment, lower cost and easy realizing.
Description
Technical field
The present invention relates to a kind of bacillus bifidus freeze concentration leaven and preparation method thereof.
Background technology
Bifidus bacillus is the intravital normal physiological bacterium of people, grows surely in enteron aisle, and be the dominant microflora of enteron aisle, what of its quantity are closely related with HUMAN HEALTH, are the class of generally acknowledging at present has promoter action to body health representative probioticss.The major physiological function of bifidus bacillus has: 1. biological barrier and biological antagonist effect; 2. enhancing immunity and antitumor action; 3. trophism; 4. suppress endotoxic generation; 5. body aging etc. is delayed in the court of a feudal ruler.With advancing age, influences such as environmental pollution and antibiotic use, the quantity of this bacterium in human intestinal descends gradually, microecological balance destroys in the enteron aisle thereby cause, and causes the generation of various diseases aging.
Replenishing the most direct approach of bifidus bacillus in enteron aisle is exactly vitro culture homology bifidus bacillus, uses as food, healthcare products or medicine with active bacteria formulation.Quantity of bifidus bacillus and the active important indicator of examining the quality of the production that is used as thereof, because bifidus bacillus is an obligatory anaerobic bacteria, very responsive to oxygen, and it is not acidproof, under the lower condition of oxygen and acidity, poor growth and dead easily causes the difficulty of cultivation big, the production cost height brings very big difficulty to bifidus bacillus production.
Summary of the invention
The purpose of this invention is to provide a kind of bacillus bifidus freeze concentration leaven and preparation method thereof, be difficult for cultivating to solve bifidus bacillus, the starter vigor is low, the problem that preservation period is short.
The preparation method of bacillus bifidus freeze concentration leaven of the present invention comprises the following steps:
A. bifidobacterium species enlarged culturing and bifidobacterium fermentation are cultivated
Bifidobacterium species is genus bifidobacterium bifidus longum bb (Bifidobacterium longum), bifidobacteria infantis (Bifidobacterium infantis), bifidobacterium adolescentis (Bifidobacterium adolescentis), bifidobacterium breve (Bifidobacterium brevis) or bifidumbacterium bifidum (Bifidobacterium bifidum); Above bacterial classification is available from Chinese common micro-organisms culture presevation administrative center, bifidus longum bb (Bifidobacteriumlongum), numbering AS 1.2186.Bifidobacteria infantis (Bifidobacterium infantis), numbering AS1.2202.Bifidobacterium adolescentis (Bifidobacterium adolescentis), numbering AS 1.2891.Bifidobacterium breve (Bifidobacterium brevis), numbering AS 1.2213.Bifidumbacterium bifidum (Bifidobacteriumbifidus) numbering AS 1.2212.
1. the actication of culture test tube is cultivated
Medium pH is transferred to 6.0～7.5, standby behind 0.05Mpa～0.14Mpa moist heat sterilization 15min～30min;
Before actication of culture, the bifidus bacillus ampoule of cryopreservation is at room temperature placed 2h～3h, at sterilisable chamber ampoule is sterilized with the cotton ball soaked in alcohol wipe surfaces of 75% concentration then, open ampoule, the substratum of drawing 0.2ml～0.3ml with aseptic straw adds in the ampoule, with fungus in the ampoule, inoculum size by 2%～5% (volume percent) is inoculated in the substratum, test tube is cultivated, and behind under anaerobic 30 ℃～42 ℃ cultivation 20h～48h, stops cultivating; Activate continuously again 2 times with identical inoculum size, culture condition, obtain 3 generation liquid culture;
2. the triangular flask first class inoculum is cultivated
Get test tube cultivate 3 generation liquid culture, by the inoculum size of 2%～5% (volume percent), insert triangular flask and carry out the first class inoculum liquid culture, under anaerobic 30 ℃～42 ℃ cultivate 20h～48h after, stop cultivating;
3. the seeding tank second class inoculum is cultivated
Get triangular flask first class inoculum liquid culture,, insert seeding tank and carry out the second class inoculum liquid culture, behind under household condition 30 ℃～42 ℃ cultivation 20h～48h, stop cultivating by the inoculum size of 2%～5% (volume percent);
4. fermentor cultivation
To by the inoculum size of 2%～5% (volume percent), insert fermentor tank through the liquid culture of seeding tank second class inoculum cultivation, 30 ℃～42 ℃ cultivations, when fermented liquid pH is lower than 5.5, regulate between fermented liquid pH to 6.0～6.5 with NaOH solution, stop to cultivate behind cultivation 20h～48h;
B. the centrifugal collection of thalline
0 ℃～8 ℃ of whizzer temperature are set, and centrifugal rotational speed 3000r/min～9000r/min, centrifugation time 10min～30min remove supernatant liquor after the centrifugal end, and the bifidus bacillus thalline of collecting precipitation is standby;
C. add protective material
The thalline of centrifugal collection is suspended in the protective material of centrifugal prefermentor nutrient solution original volume 1/10～1/2; Protectant addition preferred 1/3～1/5.
D. vacuum-packed
With the compound thin aluminum bag of sterilizing, splendid attire adds the centrifugal collection thalline after the protective material, behind vacuum tightness≤0Mpa, seals;
E. chilled storage: measure the centrifugal collection thalline eutectic point that adds after the protective material,, finally obtain bacillus bifidus freeze concentration leaven to be lower than the condition chilled storage bifidobacterium fermentation agent of 5 ℃～30 ℃ of eutectic temperatures.
The present invention fills a prescription, and 1 substratum is formed and weight proportion is: 3～15 parts of peptones, 1～10 part of extractum carnis, 3～10 parts of Tryptoness, 1～10 part of yeast extract powder, 3～15 parts of glucose, K
2HPO
40.5～5 parts, 0.5～5 part of sodium acetate, 0.5～5 part of diammonium hydrogen citrate, ZnSO
47H
20.1～0.5 part of O, MgSO
47H
20.05～0.2 part of O, 0.1～1 part of L-cysteine hydrochloride, 0.5～5 part of oligofructose, CaCO
30.5 1000 parts in～2 parts, 1～5 part of tween-80 and water.
The present invention fills a prescription, and 2 substratum are formed and weight proportion is: 3～15 parts of peptones, 1～10 part of extractum carnis, 3～10 parts of casein peptones, 1～10 part of yeast extract powder, 3～15 parts of lactose, K
2HPO
40.5～5 parts, 0.5～5 part of sodium acetate, 0.5～5 part of diammonium hydrogen citrate, ZnSO
47H
20.1～0.5 part of O, MgSO
47H
20.05～0.2 part of O, 0.1～1 part of L-cysteine hydrochloride, 0.5～5 part of oligofructose, CaCO
30.5 1000 parts in～2 parts, 1～5 part of tween-80 and water.
Protective material prescription 1 is formed and weight proportion is: 100 parts in 5～30 parts of skimmed milk or skim-milks, 0.5～5 part of glycerine and water; Protective material is at 0.05Mpa～0.14Mpa moist heat sterilization 15min～30min.
Protective material prescription 2 is formed and weight proportion is: 100 parts in 5～30 parts of skimmed milk or skim-milks, 0.5～5 part of glycerine, 3～10 parts of sucrose and water, protective material is at 0.05Mpa～0.14Mpa moist heat sterilization 15min～30min.
Protective material prescription 3 forms and weight proportion is: 100 parts in 5～30 parts of skimmed milk or skim-milks, 0.5～5 part of glycerine, L-cysteine hydrochloride or 0.1～1 part in xitix and water; skimmed milk or skim-milk and glycerine and water are at 0.05Mpa～0.14Mpa moist heat sterilization 15min～30min in the protective material, and L-cysteine hydrochloride and xitix adopt filtration sterilization in the protective material.
Protective material is filled a prescription, and 4 protective materials are formed and weight proportion is: 100 parts in 5～30 parts of skimmed milk or skim-milks, 0.5～5 part of glycerine, L-cysteine hydrochloride or 0.1～1 part in xitix, 3～10 parts of sucrose and water; Skimmed milk or skim-milk, glycerine and sucrose and water are at 0.05Mpa～0.14Mpa moist heat sterilization 15min～30min in the protective material, and L-cysteine hydrochloride and xitix adopt filtration sterilization in the protective material.
The present invention's protection is by the starter of preparation method's preparation of bacillus bifidus freeze concentration leaven.
The starter of the present invention's preparation is a kind of high density bifidobacterium fermentation agent that under freezing conditions is solid-state shape, principal feature is: the viable bacteria content height, quality guaranteed period is longer, the inoculation consumption is few, can make every batch fermentation constant product quality, also prevent the degeneration and the pollution of bacterial classification, improved the labour productivity and the quality product of cultured milk prod industry greatly.
The protective material that table 1 has shown different ingredients is the provide protection result after freezing to bifidus bacillus.
Table 1 protective material is to the influence of the freezing survival rate of bifidus bacillus
The result shows: 4 kinds of protective material prescriptions all have the better protecting effect to the bifidus bacillus chilled storage, and wherein protective material prescription 2 is better than protective material prescription 1, and protective material prescription 3 is better than protective material prescription 2, and protective material prescription 4 is better than protective material prescription 3.
The starter viable count of the present invention's preparation reaches 1 * 10
10Cfu/g～9.9 * 10
12Cfu/g ,-55 ℃～-18 ℃ the storage 3 months after survival rate can remain on more than 50%, survival rate can remain on more than 10% after 6 months.
Bifidobacteria viable bacteria of the present invention keeps the number height, can give full play to starter effect and prebiotic effect in application.Under household condition use, the fermentation activity height, easy to use.
Embodiment
The following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.
Embodiment 1:
Substratum consists of peptone 15g, extractum carnis 10g, Tryptones 3g, yeast extract powder 1g, glucose 15g, K
2HPO
45g, sodium acetate 0.5g, diammonium hydrogen citrate 0.5g, ZnSO
47H
2O 0.1g, MgSO
47H
2O 0.05g, L-cysteine hydrochloride 0.1g, oligofructose 0.5g, CaCO
30.5g, tween-80 5g and water 1000g.Medium pH is transferred to 7.5, standby behind the 0.05Mpa moist heat sterilization 30min.
Before actication of culture, bifidus longum bb (Bifidobacterium longum) ampoule of 4 ℃ of preservations is at room temperature placed 2h, at sterilisable chamber ampoule is sterilized with the cotton ball soaked in alcohol wipe surfaces of 75% concentration then, open ampoule, the substratum of drawing 0.2ml with aseptic straw adds in the ampoule, and the inoculum size of fungus in the ampoule by 5% (volume percent) is inoculated in the substratum, and test tube is cultivated, under anaerobic 37.5 ℃ cultivate 36h after, stop cultivating.Activate continuously again 2 times with identical inoculum size, culture condition, obtain 3 generation liquid culture.
With the 3rd generation culture insert triangular flask by the inoculum size of 5% (volume percent) and carry out first class inoculum and cultivate, under anaerobic 37.5 ℃ cultivate 24h after, stop cultivating.
Get triangular flask first class inoculum liquid culture,, insert seeding tank and carry out second class inoculum and cultivate by the inoculum size of 5% (volume percent), under the non-anaerobic condition 37.5 ℃ cultivate 24h after, stop cultivating.
Fermentor cultivation: will be through the liquid culture of seeding tank second class inoculum cultivation, inoculum size by 5% (volume percent), insert fermentor tank, cultivating under 37.5 ℃ of conditions under the non-anaerobic condition, when fermented liquid pH is lower than 5.5, regulate between fermented liquid pH to 6.0～6.5 with NaOH solution, stop to cultivate behind the cultivation 24h.
4 ℃ of centrifuging temperatures are set, centrifugal rotational speed 6000r/min, centrifugation time 15min.Remove supernatant liquor after the centrifugal end, the bifidus bacillus thalline of collecting precipitation is standby.
Add protective material: protect thalline, skim-milk 15g, glycerine 2g, L-cysteine hydrochloride 0.5g, sucrose 10g and water 100g as protective material with protective material prescription 4.The thalline of centrifugal collection is suspended in the protective material of centrifugal prefermentor nutrient solution original volume 1/3;
Carry out eutectic point and measure, eutectic point is-20 ℃, and-25 ℃ chilled storage is taked in vacuum packaging to bacillus bifidus freeze concentration leaven.The bacillus bifidus freeze concentration leaven storage is after 3 months, and survival rate 64% was preserved after 6 months, and survival rate is 15%.
Embodiment 2:
Substratum consists of peptone 15g, extractum carnis 10g, casein peptone 3g, yeast extract powder 1g, lactose 15g, tween-80 5g, K
2HPO
45g, sodium acetate 0.5g, diammonium hydrogen citrate 0.5g, ZnSO
47H
2O 0.1g, MgSO
47H
2O 0.05g, L-cysteine hydrochloride 0.1g, oligofructose 0.5g, CaCO
30.5g and water 1000g.Medium pH is transferred to 6.0, standby behind the 0.14Mpa moist heat sterilization 15min.
Before actication of culture, bifidobacteria infantis (Bifidobacterium infantis) ampoule of 4 ℃ of preservations is at room temperature placed 3h, at sterilisable chamber ampoule is sterilized with the cotton ball soaked in alcohol wipe surfaces of 75% concentration then, open ampoule, the substratum of drawing 0.3ml with aseptic straw adds in the ampoule, with fungus in the ampoule, inoculum size by 3% (volume percent) is inoculated in the substratum, test tube is cultivated, under anaerobic 42 ℃ cultivate 24h after, stop cultivating.Activate continuously again 2 times with identical inoculum size, culture condition, obtain 3 generation liquid culture.
With the 3rd generation culture insert triangular flask by the inoculum size of 3% (volume percent) and carry out first class inoculum and cultivate, under anaerobic 42 ℃ cultivate 20h after, stop cultivating.
Get triangular flask first class inoculum liquid culture,, insert seeding tank and carry out second class inoculum and cultivate by the inoculum size of 3% (volume percent), under the non-anaerobic condition 42 ℃ cultivate 20h after, stop cultivating.
Fermentor cultivation: will be through the liquid culture of seeding tank second class inoculum cultivation, inoculum size by 3% (volume percent), insert fermentor tank, cultivating under 42 ℃ of conditions under the non-anaerobic condition, when fermented liquid pH is lower than 5.5, regulate between fermented liquid pH to 6.0～6.5, stop to cultivate behind the cultivation 20h with NaOH solution.
8 ℃ of centrifuging temperatures are set, centrifugal rotational speed 3000r/min, centrifugation time 30min.
Add protective material: protect thalline as protective material with protective material prescription 3; skimmed milk 20.0g, glycerine 1.0g, xitix 1g and water 100g; the thalline of centrifugal collection is suspended in the protective material of centrifugal prefermentor nutrient solution original volume 1/5; carrying out eutectic point measures; eutectic point is-23 ℃, chilled storage bifidobacterium fermentation agent under-30 ℃ the condition.
Embodiment 3:
Substratum consists of peptone 5g, extractum carnis 5g, casein peptone 10g, yeast extract powder 5g, lactose 10g, tween-80 1g, K
2HPO
42g, sodium acetate 5g, diammonium hydrogen citrate 0.2g, ZnSO
47H
2O0.3g, MgSO
47H
2O 0.1g, L-cysteine hydrochloride 0.5g, oligofructose 2.5g, CaCO
31g and water 1000g.Medium pH is transferred to 7.0, standby behind the 0.1Mpa moist heat sterilization 25min.
Before actication of culture, bifidobacterium adolescentis (Bifidobacterium adolescentis) ampoule of 4 ℃ of preservations is at room temperature placed 2.5h, at sterilisable chamber ampoule is sterilized with the cotton ball soaked in alcohol wipe surfaces of 75% concentration then, open ampoule, the substratum of drawing 0.3ml with aseptic straw adds in the ampoule, with fungus in the ampoule, inoculum size by 2% (volume percent) is inoculated in the substratum, test tube is cultivated, under anaerobic 30 ℃ cultivate 48h after, stop cultivating.Activate continuously again 2 times with identical inoculum size, culture condition, obtain 3 generation liquid culture.
With the 3rd generation culture insert triangular flask by the inoculum size of 2% (volume percent) and carry out first class inoculum and cultivate, under anaerobic 30 ℃ cultivate 48h after, stop cultivating.
Get triangular flask first class inoculum liquid culture,, insert seeding tank and carry out second class inoculum and cultivate by the inoculum size of 2% (volume percent), under the non-anaerobic condition 30 ℃ cultivate 48h after, stop cultivating.
Fermentor cultivation: will be through the liquid culture of seeding tank second class inoculum cultivation, inoculum size by 2% (volume percent), insert fermentor tank, cultivating under 30 ℃ of conditions under the non-anaerobic condition, when fermented liquid pH is lower than 5.5, regulate between fermented liquid pH to 6.0～6.5, stop to cultivate behind the cultivation 48h with NaOH solution.
0 ℃ of centrifuging temperature is set, centrifugal rotational speed 9000r/min, centrifugation time 10min.
Add protective material: protect thalline as protective material with protective material prescription 2; skimmed milk 25g, glycerine 1g, sucrose 10g and water 100g; the thalline of centrifugal collection is suspended in the protective material of centrifugal prefermentor nutrient solution original volume 1/10 chilled storage bifidobacterium fermentation agent under-18 ℃ the condition.
Embodiment 4: repeat embodiment 1, following difference is arranged: substratum consists of peptone 3g, extractum carnis 1g, Tryptones 10g, yeast extract powder 10g, glucose 3g, tween-80 1g, K
2HPO
40.5g, sodium acetate 5g, diammonium hydrogen citrate 5g, ZnSO
47H
2O 0.5g, MgSO
47H
2O 0.2g, L-cysteine hydrochloride 1g, oligofructose 5g, CaCO
32g and water 1000g.Medium pH is transferred to 6.5, standby behind the 0.1Mpa moist heat sterilization sterilization 20min.
Bifidobacterium species is bifidobacterium breve (Bifidobacterium brevis), and 3 ℃ of centrifuging temperatures are set, centrifugal rotational speed 5000r/min, centrifugation time 20min.Protect thalline, skim-milk 20g, glycerine 5g and water 100g with protective material prescription 1 as protective material; As protective material protection thalline, the thalline of centrifugal collection is suspended in the protective material of centrifugal prefermentor nutrient solution original volume 1/2-55 ℃ condition chilled storage bifidobacterium fermentation agent.
Embodiment 5: repeat embodiment 2, following difference is arranged: substratum consists of peptone 3g, extractum carnis 1g, casein peptone 10g, yeast extract powder 10g, lactose 3g, K
2HPO
40.5g, sodium acetate 5g, diammonium hydrogen citrate 5g, ZnSO
47H
2O 0.5g, MgSO
47H
2O 0.2g, L-cysteine hydrochloride 1g, oligofructose 5g, CaCO
32g, tween-80 1g and water 1000g.Medium pH is transferred to 6.0, standby behind the 0.14Mpa moist heat sterilization 15min.
Bifidobacterium species is that bifidumbacterium bifidum (Bifidobacterium bifidum) is protected thalline with protective material prescription 4 as protective material, and skim-milk 30g, glycerine 0.5g, L-cysteine hydrochloride 0.1g, sucrose 3g and water 100g protect thalline as protective material.
Embodiment 6: repeat embodiment 1, following difference is arranged: substratum consists of peptone 5g, extractum carnis 5g, Tryptones 10g, yeast extract powder 5g, glucose 10g, tween-80 1g, K
2HPO
42g, sodium acetate 5g, diammonium hydrogen citrate 0.2g, ZnSO
47H
2O 0.3g, MgSO
47H
2O 0.1g, L-cysteine hydrochloride 0.5g, oligofructose 2.5g, CaCO
31g and water 1000g.Medium pH is transferred to 7.0, standby behind the 0.1Mpa moist heat sterilization 25min
Protect thalline, i.e. skim-milk 5g, glycerine 5g, L-cysteine hydrochloride 1g, sucrose 10g and water 100g with protective material prescription 4 as protective material.
Embodiment 7: repeat embodiment 1, following difference is arranged: protect thalline, i.e. skim-milk 30g, glycerine 0.5g, L-cysteine hydrochloride 0.1g and water 100g with protective material prescription 3 as protective material.
Embodiment 8: repeat embodiment 1, following difference is arranged: protect thalline, i.e. skim-milk 5g, glycerine 5g, L-cysteine hydrochloride 1g and water 100g with protective material prescription 3 as protective material.
Embodiment 9: repeat embodiment 1, following difference is arranged: protect thalline, i.e. skimmed milk 30g, glycerine 0.5g, sucrose 3g and water 100g with protective material prescription 2 as protective material.
Embodiment 10: repeat embodiment 1, following difference is arranged: protect thalline, i.e. skimmed milk 5g, glycerine 5g, sucrose 10g and water 100g with protective material prescription 2 as protective material.
Embodiment 11: repeat embodiment 1, following difference is arranged: protect thalline, i.e. skimmed milk 30g, glycerine 0.5g and water 100g with protective material prescription 1 as protective material.
Embodiment 12: repeat embodiment 1, following difference is arranged: protect thalline, i.e. skim-milk 5g, glycerine 5g and water 100g with protective material prescription 1 as protective material.
Table 2 has shown that the bacillus bifidus freeze concentration leaven for preparing by embodiment 1 is in the different storage survival results.
The package stability of table 2 bacillus bifidus freeze concentration leaven
Claims (5)
1. the preparation method of a bacillus bifidus freeze concentration leaven is characterized in that it comprises the following steps:
A. bifidobacterium species enlarged culturing and bifidobacterium fermentation are cultivated
Bifidobacterium species is genus bifidobacterium bifidus longum bb, bifidobacteria infantis, bifidobacterium adolescentis, bifidobacterium breve or bifidumbacterium bifidum;
1. the actication of culture test tube is cultivated
Medium pH is transferred to 6.0～7.5, standby behind 0.05Mpa～0.14Mpa moist heat sterilization 15min～30min;
Before actication of culture, the bifidus bacillus ampoule of cryopreservation is at room temperature placed 2h～3h, at sterilisable chamber ampoule is sterilized with the cotton ball soaked in alcohol wipe surfaces of 75% concentration then, open ampoule, the substratum of drawing 0.2ml～0.3ml with aseptic straw adds in the ampoule, with fungus in the ampoule, by volume per-cent is that 2%～5% inoculum size is inoculated in the substratum, test tube is cultivated, and behind under anaerobic 30 ℃～42 ℃ cultivation 20h～48h, stops cultivating; Activate continuously again 2 times with identical inoculum size, culture condition, obtain 3 generation liquid culture;
2. the triangular flask first class inoculum is cultivated
Get test tube cultivate 3 generation liquid culture, by volume per-cent is 2%～5% inoculum size, insert triangular flask and carry out the first class inoculum liquid culture, under anaerobic 30 ℃～42 ℃ cultivate 20h～48h after, stop cultivating;
3. the seeding tank second class inoculum is cultivated
Get triangular flask first class inoculum liquid culture, by volume per-cent is 2%～5% inoculum size, inserts seeding tank and carries out the second class inoculum liquid culture, behind under household condition 30 ℃～42 ℃ cultivation 20h～48h, stops cultivating;
4. fermentor cultivation
Will be through the liquid culture of seeding tank second class inoculum cultivation, by volume per-cent is 2%～5% inoculum size, inserts fermentor tank, 30 ℃～42 ℃ cultivations, when fermented liquid pH is lower than 5.5, regulate between fermented liquid pH to 6.0～6.5 with NaOH solution, stop to cultivate behind cultivation 20h～48h;
B. the centrifugal collection of thalline
0 ℃～8 ℃ of whizzer temperature are set, and centrifugal rotational speed 3000r/min～9000r/min, centrifugation time 10min～30min remove supernatant liquor after the centrifugal end, and the bifidus bacillus thalline of collecting precipitation is standby;
C. add protective material
The thalline of centrifugal collection is suspended in the protective material of centrifugal prefermentor nutrient solution original volume 1/10～1/2;
D. vacuum-packed
With the compound thin aluminum bag of sterilizing, splendid attire adds the centrifugal collection thalline after the protective material, behind vacuum tightness≤0Mpa, seals;
E. chilled storage: measure the centrifugal collection thalline eutectic point that adds after the protective material,, finally obtain bacillus bifidus freeze concentration leaven to be lower than the condition chilled storage bifidobacterium fermentation agent of 5 ℃～30 ℃ of eutectic temperatures;
The protective material that the C step is added is selected from a kind of in protective material prescription 1～protective material prescription 4,
The composition of protective material prescription 1 and weight proportion are: 100 parts in 5～30 parts of skimmed milk or skim-milks, 0.5～5 part of glycerine and water; Protective material is at 0.05Mpa～0.14Mpa moist heat sterilization 15min～30min,
The composition of protective material prescription 2 and weight proportion are: 100 parts in 5～30 parts of skimmed milk or skim-milks, 0.5～5 part of glycerine, 3～10 parts of sucrose and water, protective material is at 0.05Mpa～0.14Mpa moist heat sterilization 15min～30min,
The composition of protective material prescription 3 and weight proportion are: 100 parts in 5～30 parts of skimmed milk or skim-milks, 0.5～5 part of glycerine, L-cysteine hydrochloride or 0.1～1 part in xitix and water; skimmed milk or skim-milk and glycerine and water are at 0.05Mpa～0.14Mpa moist heat sterilization 15min～30min in the protective material; L-cysteine hydrochloride and xitix adopt filtration sterilization in the protective material
The composition of protective material prescription 4 and weight proportion are: 100 parts in 5～30 parts of skimmed milk or skim-milks, 0.5～5 part of glycerine, L-cysteine hydrochloride or 0.1～1 part in xitix, 3～10 parts of sucrose and water; Skimmed milk or skim-milk, glycerine and sucrose and water are at 0.05Mpa～0.14Mpa moist heat sterilization 15min～30min in the protective material, and L-cysteine hydrochloride and xitix adopt filtration sterilization in the protective material.
2. the preparation method of bacillus bifidus freeze concentration leaven according to claim 1, it is characterized in that: protectant addition is 1/3～1/5 of a centrifugal prefermentor nutrient solution original volume in the C step.
3. by the bacillus bifidus freeze concentration leaven of claim 1 or the preparation of 2 described methods.
4. the preparation method of bacillus bifidus freeze concentration leaven according to claim 1 and 2 is characterized in that: substratum is formed and weight proportion is: 3～15 parts of peptones, 1～10 part of extractum carnis, 3～10 parts of Tryptoness, 1～10 part of yeast extract powder, 3～15 parts of glucose, K
2HPO
40.5～5 parts, 0.5～5 part of sodium acetate, 0.5～5 part of diammonium hydrogen citrate, ZnSO
47H
20.1～0.5 part of O, MgSO
47H
20.05～0.2 part of O, 0.1～1 part of L-cysteine hydrochloride, 0.5～5 part of oligofructose, CaCO
30.5 1000 parts in～2 parts, 1～5 part of tween-80 and water.
5. the preparation method of bacillus bifidus freeze concentration leaven according to claim 1 and 2 is characterized in that: substratum is formed and weight proportion is: 3～15 parts of peptones, 1～10 part of extractum carnis, 3～10 parts of casein peptones, 1～10 part of yeast extract powder, 3～15 parts of lactose, K
2HPO
40.5～5 parts, 0.5～5 part of sodium acetate, 0.5～5 part of diammonium hydrogen citrate, ZnSO
47H
20.1～0.5 part of O, MgSO
47H
20.05～0.2 part of O, 0.1～1 part of L-cysteine hydrochloride, 0.5～5 part of oligofructose, CaCO
30.5 1000 parts in～2 parts, 1～5 part of tween-80 and water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101506655A CN101338295B (en) | 2008-08-09 | 2008-08-09 | Bacillus bifidus freeze concentration leaven and method for preparing same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101506655A CN101338295B (en) | 2008-08-09 | 2008-08-09 | Bacillus bifidus freeze concentration leaven and method for preparing same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101338295A CN101338295A (en) | 2009-01-07 |
| CN101338295B true CN101338295B (en) | 2010-08-11 |
Family
ID=40212465
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008101506655A Expired - Fee Related CN101338295B (en) | 2008-08-09 | 2008-08-09 | Bacillus bifidus freeze concentration leaven and method for preparing same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101338295B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635932A (en) * | 2017-02-23 | 2017-05-10 | 遵义医学院 | Probiotic group and preparation method thereof |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2013202778A1 (en) * | 2013-03-14 | 2014-10-02 | Gen-Probe Incorporated | Systems, methods, and apparatuses for performing automated reagent-based assays |
| CN103168965B (en) * | 2013-03-29 | 2014-07-16 | 重庆市畜牧科学院 | Anti-stress synbiotics traditional Chinese medicine composition applicable to chicken and preparation method of anti-stress synbiotics traditional Chinese medicine composition |
| CN103766486B (en) * | 2014-01-07 | 2015-08-19 | 甘肃省科学院生物研究所 | A kind of milky-drinks containing bifidus bacillus embedding pearl and preparation method thereof |
| CN103749672B (en) * | 2014-01-07 | 2015-08-19 | 甘肃省科学院生物研究所 | A kind of yogurt and preparation method thereof containing bifidus bacillus embedding pearl or embedded block |
| CN106318873B (en) * | 2016-10-17 | 2019-12-06 | 江苏中宜金大环保产业技术研究院有限公司 | microbial agent protective agent for high-salt organic industrial wastewater treatment and application thereof |
| CN107603908A (en) | 2017-09-29 | 2018-01-19 | 福建农林大学 | A kind of superhigh temperature aerobic fermentation microbial inoculum prepared using municipal sludge and preparation method thereof |
| CN109022322A (en) * | 2018-08-24 | 2018-12-18 | 汉臣氏（沈阳）儿童制品有限公司 | A kind of preparation method of bifidobacterium lactis freeze-dried vaccine powder |
| CN109294916A (en) * | 2018-11-06 | 2019-02-01 | 哈尔滨艾诺卫康科技发展有限公司 | A kind of culture presevation agent and the method using the preserving agent anaerobism freezing genetic engineering bacterium |
| CN111647510A (en) * | 2020-06-18 | 2020-09-11 | 科兴生物制药股份有限公司 | Bifidobacterium infantis freeze-dried powder, preparation method and composite protective agent used by same |
| CN112471372B (en) * | 2020-10-23 | 2023-06-09 | 重庆工贸职业技术学院 | Premna microphylla juice lactobacillus beverage and preparation method thereof |
| CN115094008A (en) * | 2022-07-21 | 2022-09-23 | 宜兴市天石饲料有限公司 | Research and development method of biological antibacterial preparation for preventing and treating avian clostridium perfringens |
-
2008
- 2008-08-09 CN CN2008101506655A patent/CN101338295B/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 曹永梅等.保护剂在冷冻干燥双歧杆菌中的作用.食品与发酵工业.万方,2000,26(2),第40-45页. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635932A (en) * | 2017-02-23 | 2017-05-10 | 遵义医学院 | Probiotic group and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101338295A (en) | 2009-01-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101338295B (en) | Bacillus bifidus freeze concentration leaven and method for preparing same | |
| CN100519738C (en) | Bifidobacteria viable bacteria preparation and special-purpose protective agent thereof | |
| CN106520633B (en) | A kind of preparation method of lactobacillus plantarum freeze-dried powder | |
| CN106047709B (en) | A kind of lactobacillus plantarum freeze-dried powder and preparation method thereof | |
| CN102533618A (en) | Lactobacillus plantarum CCFM8724 and application thereof | |
| CN103766486B (en) | A kind of milky-drinks containing bifidus bacillus embedding pearl and preparation method thereof | |
| CN102986869B (en) | Solid Kefir dairy product and preparation method thereof | |
| CN107287121A (en) | A kind of lactic acid bacteria freeze drying protective agent and preparation method thereof and application method | |
| CN106434463A (en) | Preparation method of lactobacillus rhamnosus lyophilized powder | |
| CN102763726A (en) | Probiotics yoghourt powder and preparation method thereof | |
| CN108244432A (en) | A kind of fermentation Cordyceps militaris probiotic beverage and preparation method thereof | |
| CN104651232B (en) | A kind of Leuconostoc mesenteroides lyophilized formulations and its preparation method and application | |
| CN104928208A (en) | Lactobacillus plantarum Lp90, and screening method and application thereof | |
| WO2017186146A1 (en) | Fruit and vegetable cultivation and probiotic fermentation of edible and medicinal fungus | |
| CN112126599B (en) | High-density culture method of lactobacillus helveticus, preparation of high-activity bacterium powder and application of high-density culture method | |
| CN101974427A (en) | Phytosterol containing active lactobacilIus plantarum viable bacterium preparation and preparation method thereof | |
| CN103380874A (en) | Preparation method for composite probiotic-fermented jam | |
| CN101731511A (en) | Probiotic active product and preparation method thereof | |
| CN103478839A (en) | Glucan beverage and preparation method thereof | |
| CN103931983B (en) | A kind of honey ferment product and preparation method thereof | |
| CN107455520A (en) | A kind of preparation method of green tea beverage | |
| CN104388333A (en) | Bacillus licheniformis and application thereof | |
| CN109699919A (en) | A kind of low ammonia taste, the preparation method of high nutrition original flavor natto | |
| CN104630096B (en) | There is bifidobacterium bifidum TMC3115 and the application thereof of suppression adipose cell | |
| CN106497842A (en) | The preparation method and application of a kind of lactic acid bacteria and aspergillus niger mixed solid fermentation preparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20100811 Termination date: 20170809 |