CN108559717A - A kind of lactobacillus acidophilus high density fermentation culture medium and its bacterium powder preparation method - Google Patents
A kind of lactobacillus acidophilus high density fermentation culture medium and its bacterium powder preparation method Download PDFInfo
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Abstract
The present invention provides a kind of lactobacillus acidophilus high density fermentation culture medium and its bacterium powder preparation methods, using lactobacillus acidophilus high density fermentation culture medium to strain high density fermentation culture：Lactobacillus acidophilus is accessed into fermentation tank after the expansion of three-level seed fermentation is numerous, ferment 18 20h, 37 DEG C of cultivation temperature, speed of agitator 150r/min, pH is automatically maintained at pH 5.7 6.0 using 50% ammonium hydroxide, and being passed through nitrogen maintains fermentation pressure tank in 0.05MPa, determines the best feed supplement time by pH change rates in culture solution, the mixed liquor of sucrose, yeast extract and whey powder is added in a manner of speed change feed supplement；Vacuum freeze drying：Elder generation is after 80 DEG C of pre-freeze 12h, 55 DEG C, and 18 30h of vacuum freeze drying under 1 7pa vacuum degrees, the survival rate of gained freeze-dried Lactobacillus acidophilus powder can reach 95% or more, and can at normal temperatures store 12 months, and viable count is not less than 1010cfu/g。
Description
Technical field
The present invention relates to biotechnology, especially a kind of lactobacillus acidophilus high density fermentation culture medium and its bacterium powder system
Preparation Method.
Background technology
At present in animal feeding process it is long-term, largely use antibiotic, make the enhancing of pathogenic bacteria drug resistance, microbial population of animal intestinal tract
Imbalance, immunity degradation.Probiotics can improve the balance of microbial population of animal intestinal tract, enhancing animal body immunocompetence, promote animal
Growth and development, the generation for improving breeding performonce fo animals, reducing harmful substance in enteron aisle, are current it has been recognized that promising
The substitute of antibiotic.
Lactobacillus acidophilus (Lactobacillus acidophilus) is one kind common in probiotics, is deposited extensively
It is in the gastrointestinal tract of host's (humans and animals) and the oral cavity and vagina of human body.Lactobacillus acidophilus belongs to lactobacillus, gram
The positive, amphimicrobian do not move, no gemma, atrichia, and form is in elongated rod-shaped, and the end of bar is rounded, and catalase is cloudy
Property, do not liquefy gelatin, does not produce indoles, hydrogen sulfide and cytochromes, does not decompose casein.Can be that 4.0-5.0 is even lower in pH value
Acidic environment in grow, optimum growth temperature be 35 DEG C -38 DEG C.Can homofermentation glucose, sucrose, lactose etc. generate DL
Type lactic acid, and the ability of decomposing protein is weaker, can only utilize the hydrolysate such as amino acid and peptides of some protein.
Lactobacillus acidophilus is one of resident flora of animal intestinal tract, can exist in gastric juice and secrete lactic acid, acetic acid
Deng the digestion power for promoting stomach；In addition, lactobacillus acidophilus can adjust intestinal flora balance, inhibit enteron aisle undesirable microorganism
Proliferation, lactobacillus acidophilus can secrete antibiotin substance (acidolin, bacillus acidophilus's element, lactein), be caused to enteron aisle
Germ generates antagonism.As probiotics, lactobacillus acidophilus equally has enhancing immunity of organisms, alleviates lactose intolerance,
Serum cholesterol content is reduced, the metabolic diseases such as diabetes, hypertension are prevented, reduce the activity of carcinogen enzyme and then is inhibited
Tumour growth generates the effects that activated material inhibits allergenic factor Reduce allergy.Due to lactobacillus acidophilus noresidue, do not generate resistance to
The advantages that pharmacological property, currently, oneself is widely used in the production of animal word material.
Invention content
Technical problem to be solved by the present invention lies in provide a kind of lactobacillus acidophilus high density fermentation training of highly-water-soluble
Support base.
Another technical problem to be solved by this invention is to provide using above-mentioned lactobacillus acidophilus high density fermentation culture
The bacterium powder preparation method of base.
In order to solve the above technical problems, the technical scheme is that：
A kind of lactobacillus acidophilus high density fermentation culture medium, by fermentation tank culture medium, supplemented medium and freeze drying protectant
Three parts form, wherein
The fermentation tank culture based formulas is：15-20g/L yeast extracts, 10-20g/L whey powders, 10-15g/L albumen
Peptone, 10-30g/L sucrose, 0.1g/L MnSO4, 5g/L sodium acetates, 0.5g/L MgSO4·7H2O, 2g/L dipotassium hydrogen phosphate, 2g/L
Diammonium hydrogen citrate, half deamination acid hydrochlorides of 0.0005-0.002g/L L-, 1mL/L Tween 80s, 0.5% antifoaming agent, surplus
For distilled water, sterilization temperature is 115 DEG C, 20min；
The feed-batch culture based formulas is：10-30g/L sucrose, 5-10g/L yeast extracts, 5-20g/L whey powders, surplus
For distilled water, sterilization temperature is 115 DEG C, 15min；
The composition of the freeze drying protectant includes：5-20% skimmed milk powers, 2-10% lactose, 1-10% trehaloses, 0.1-
1.0% ascorbic acid, 0.2-0.5% glycerine, 0.01-0.1% sorbierites, mixing obtain.
Preferably, above-mentioned lactobacillus acidophilus high density fermentation culture medium, each group is grouped as follows：
The fermentation tank culture based formulas is：20g/L yeast extracts, 15g/L whey powders, 10g/L peptones, 18g/L sugarcanes
Sugar, 0.1g/L MnSO4, 5g/L sodium acetates, 0.5g/L MgSO4·7H2O, 2g/L dipotassium hydrogen phosphate, 2g/L diammonium hydrogen citrates,
Half deamination acid hydrochlorides of 0.001g/L L-, 1mL/L Tween 80s, 0.5% antifoaming agent, surplus are distilled water, sterilization temperature
It is 115 DEG C, 20min；
The feed-batch culture based formulas is：20g/L sucrose, 5g/L yeast extracts, 15g/L whey powders, surplus are distilled water,
Sterilization temperature is 115 DEG C, 15min；
The composition of the freeze drying protectant includes：10% skimmed milk power, 4% lactose, 4% trehalose, 0.1% Vitamin C
Acid, 0.5% glycerine, 0.02% sorbierite, mixing obtain.
The bacterium powder preparation method of above-mentioned lactobacillus acidophilus high density fermentation culture medium, specific preparation process are as follows：
(1) seed culture of lactobacillus acidophilus：By the strain of lactobacillus acidophilus being stored in glycerol tube (the micro- life of Chinese industrial
Object culture presevation administrative center, CGMCC1.3221) access 5mL MRS fluid nutrient mediums in 37 DEG C of activation cultures be level-one kind
Son；Activated primary seed solution is inoculated in 3% inoculum concentration in 10mL MRS fluid nutrient mediums again, 37 DEG C of stationary cultures
It is secondary seed for 24 hours；Activated secondary seed solution is finally accessed into 150mL MRS fluid nutrient mediums with 3% inoculum concentration
In, 37 DEG C of stationary cultures are three-level seed liquor for 24 hours；
(2) fermentation tank culture：Fermenter volume is 5L, loads above-mentioned fermentation tank culture medium 3L, and sterilization temperature is 115
DEG C, 20min；The three-level seed liquor that culture obtains all is pumped into fermentation tank with 5% inoculum concentration, fermented and cultured temperature is 37
DEG C, speed of agitator 150r/min, pH are automatically maintained at pH5.7-6.0 using 50% ammonium hydroxide, and are passed through nitrogen and maintain fermentation
Pressure tank is in 0.05MPa；By the way of speed change feed supplement, 0.5h measures the change rate of pH value in one time fermentation tank, at 8-9h
It was found that pH change rates drastically decline in culture solution, illustrate that the nutriment (mainly carbon source) of culture medium at this time has been depleted
, so feed supplement should be carried out at this moment, above-mentioned feed supplement is first supplemented with the rate of 9mL/Lh by the feed supplement pump on fermentation tank and is trained
Base is supported, pH change rates change again after 12-14h, and the rate for being changed to 18mL/Lh carries out feed supplement, cultivates 18-20h；
(3) separation of zymotic fluid：By 4 DEG C of the zymotic fluid of obtained lactobacillus acidophilus, 5000-9000r/min centrifugations
10min-20min obtains bacterium mud；
(4) vacuum freeze drying of bacterium mud：According to wet thallus: above-mentioned freeze drying protectant=1:1-1:2 weight ratio will
Freeze drying protectant annex solution is added to by sterile working in the bacterial sediment object after the lactobacillus acidophilus centrifugation of collection, suspension base
Matter is the phosphate buffer of pH6.8, and stirring 30min keeps suspension uniform, and 4 DEG C of standing 5min are obtained containing the thermophilic of freeze drying protectant
Lactobacillus lactis thallus suspension liquid carries out precooling processing 12h in -80 DEG C of deep freezers, then carries out vacuum freeze drying, cold
Freeze -55 DEG C or so, vacuum degree 1-7Pa of temperature, obtaining viable count after dry 18-30h is more than 5.0 × 1011The acidophilus breast bar of cfu/g
Bacterium powder is lyophilized in bacterium.
Beneficial effects of the present invention：
Above-mentioned lactobacillus acidophilus high density fermentation culture medium enhances the freezing tolerance of lactobacillus acidophilus, improves product viable bacteria
Number can reach 95% or more using the survival rate of the bacterium powder preparation method gained freeze-dried Lactobacillus acidophilus powder of the culture medium,
It is preserved 12 months under room temperature, viable count is not less than 1010cfu/g。
Description of the drawings
Fig. 1 is process flow chart prepared by the present invention.
Specific implementation mode
Technical solution of the present invention is further described with reference to specific embodiment.
Embodiment 1
The preparation method of lactobacillus acidophilus bacterium powder, as shown in Figure 1, having, steps are as follows：
1, the seed culture of lactobacillus acidophilus：By the strain of lactobacillus acidophilus being stored in glycerol tube (the micro- life of Chinese industrial
Object culture presevation administrative center, CGMCC1.3221) access 5mL MRS fluid nutrient mediums in 37 DEG C of activation cultures be level-one kind
Son；Activated primary seed solution is inoculated in 3% inoculum concentration in 10mL MRS fluid nutrient mediums again, 37 DEG C of stationary cultures
It is secondary seed for 24 hours；Activated secondary seed solution is finally accessed into 150mL MRS fluid nutrient mediums with 3% inoculum concentration
In, 37 DEG C of stationary cultures are three-level seed liquor for 24 hours；The formula of the MRS fluid nutrient mediums is：20g/L glucose, 10g/L
Soy peptone, 5g/L yeast extracts, 10g/L beef extracts, 5g/L sodium acetates, 2g/L dipotassium hydrogen phosphates, 2g/L sodium citrates,
0.2g/L MgSO4·7H2O, 0.054g/L MnSO4,1g/L Tween-80, surplus is distilled water.
2, fermentation tank culture：Fermenter volume is 5L, loads fermentation tank culture medium 3L, and sterilization temperature is 115 DEG C,
20min；The three-level seed liquor that culture obtains all is pumped into fermentation tank with 5% inoculum concentration, fermented and cultured temperature is 37 DEG C, is stirred
Mix rotating speed 150r/min, pH are automatically maintained at pH5.7 using 50% ammonium hydroxide, and are passed through nitrogen and fermentation pressure tank is maintained to exist
0.05MPa；By the way of speed change feed supplement, 0.5h measures the change rate of pH value in one time fermentation tank, and culture solution is found at 8h
Middle pH change rates drastically decline, and illustrate that the nutriment (mainly carbon source) of culture medium at this time has been consumed and are over, so answering
Feed supplement is carried out at this moment, and sucrose, yeast extract and whey are first supplemented with the rate of 9mL/Lh by the feed supplement pump on fermentation tank
The mixed liquor of powder, pH change rates change again after 13h, and the rate for being changed to 18mL/Lh carries out feed supplement, cultivates 18h；Institute
Stating fermentation tank culture based formulas is：20g/L yeast extracts, 15g/L whey powders, 10g/L peptones, 18g/L sucrose, 0.1g/L
MnSO4, 5g/L sodium acetates, 0.5g/L MgSO4·7H2O, 2g/L dipotassium hydrogen phosphate, 2g/L diammonium hydrogen citrates, 0.001g/L
Half deamination acid hydrochlorides of L-, 1mL/L Tween 80s, 0.5% antifoaming agent, surplus are distilled water, and sterilization temperature is 115 DEG C,
20min；The feed-batch culture based formulas is：20g/L sucrose, 5g/L yeast extracts, 15g/L whey powders, surplus are distilled water, are gone out
Bacterium sterilisation temp is 115 DEG C, 15min.
Above-mentioned acquisition sample is subjected to MRS solid medium tablets and counts culture, cell concentration is up at the end of 18h
To 1.0 × 1011The cell concentration of cfu/mL.
3, the separation of zymotic fluid：By 4 DEG C of the zymotic fluid of obtained lactobacillus acidophilus, 7000r/min centrifuges 10min, obtains
Bacterium mud.
4, the vacuum freeze drying of bacterium mud：According to wet thallus: protective agent=1:1 volume ratio leads to protective agent annex solution
It crosses in the bacterial sediment object after sterile working is added to the lactobacillus acidophilus centrifugation of collection, the phosphoric acid that suspending medium is pH6.8 is slow
Fliud flushing, stirring 30min keep suspension uniform, and 4 DEG C of standing 5min are obtained containing protectant lactobacillus acidophilus thallus suspension liquid ,-
Precooling is carried out in 80 DEG C of deep freezers and handles 12h, then carries out vacuum freeze drying, -55 DEG C or so of cryogenic temperature, vacuum degree
It is 7.2 × 10 to obtain viable count after 5Pa, dry 18h11The freeze-dried Lactobacillus acidophilus powder of cfu/g；The group of the freeze drying protectant
At including：10% skimmed milk power, 4% lactose, 4% trehalose, 0.1% ascorbic acid, 0.5% glycerine, 0.02% sorbierite；Institute
The preparation method for stating the phosphate buffer of pH6.8 is：It takes the dipotassium hydrogen phosphate of 6.8g to be dissolved in the water of 250mL, adds 0.944g's
Sodium hydroxide solution 118mL, is diluted with water to 1000mL, shakes up.
After testing, the survival rate of the freeze-dried Lactobacillus acidophilus powder of preparation can reach 95.32%, the acidophilus breast bar that will be obtained
Bacterium freeze-drying bacterium powder preserves 12 months at normal temperatures, and viable count is not less than 1010cfu/g。
Embodiment 2
The preparation method of lactobacillus acidophilus bacterium powder, with embodiment 1, wherein：
The fermentation tank culture based formulas is：15g/L yeast extracts, 20g/L whey powders, 15g/L peptones, 10g/L sugarcanes
Sugar, 0.1g/L MnSO4, 5g/L sodium acetates, 0.5g/L MgSO4·7H2O, 2g/L dipotassium hydrogen phosphate, 2g/L diammonium hydrogen citrates,
Half deamination acid hydrochlorides of 0.002g/L L-, 1mL/L Tween 80s, 0.5% antifoaming agent, surplus are distilled water, sterilization temperature
It is 115 DEG C, 20min；
The feed-batch culture based formulas is：10g/L sucrose, 10g/L yeast extracts, 5g/L whey powders, surplus are distilled water,
Sterilization temperature is 115 DEG C, 15min；
The composition of the freeze drying protectant includes：20% skimmed milk power, 10% lactose, 1% trehalose, 0.1% Vitamin C
Acid, 0.2% glycerine, 0.1% sorbierite, mixing obtain.
Embodiment 3
The preparation method of lactobacillus acidophilus bacterium powder, with embodiment 1, wherein：
The fermentation tank culture based formulas is：20g/L yeast extracts, 10g/L whey powders, 10g/L peptones, 30g/L sugarcanes
Sugar, 0.1g/L MnSO4, 5g/L sodium acetates, 0.5g/L MgSO4·7H2O, 2g/L dipotassium hydrogen phosphate, 2g/L diammonium hydrogen citrates,
Half deamination acid hydrochlorides of 0.0005g/L L-, 1mL/L Tween 80s, 0.5% antifoaming agent, surplus are distilled water, sterilization temperature
Degree is 115 DEG C, 20min；
The feed-batch culture based formulas is：30g/L sucrose, 5g/L yeast extracts, 20g/L whey powders, surplus are distilled water,
Sterilization temperature is 115 DEG C, 15min；
The composition of the freeze drying protectant includes：5% skimmed milk power, 2% lactose, 10% trehalose, 1.0% Vitamin C
Acid, 0.5% glycerine, 0.01% sorbierite, mixing obtain.
It is above-mentioned that a kind of lactobacillus acidophilus high density fermentation culture medium and its bacterium powder preparation method are carried out with reference to embodiment
Detailed description, be illustrative without being restrictive, can enumerate several embodiments according to limited range, thus
The change and modification under present general inventive concept are not departed from, should be belonged within protection scope of the present invention.
Claims (3)
1. a kind of lactobacillus acidophilus high density fermentation culture medium, it is characterised in that：By fermentation tank culture medium, supplemented medium and jelly
Dry protective agent three parts composition, wherein
The fermentation tank culture based formulas is：15-20g/L yeast extracts, 10-20g/L whey powders, 10-15g/L peptones, 10-
30g/L sucrose, 0.1g/L MnSO4, 5g/L sodium acetates, 0.5g/L MgSO4·7H2O, 2g/L dipotassium hydrogen phosphate, 2g/L citric acids
Hydrogen diammonium, half deamination acid hydrochlorides of 0.0005-0.002g/L L-, 1mL/L Tween 80s, 0.5% antifoaming agent, surplus are distillation
Water, sterilization temperature are 115 DEG C, 20min；
The feed-batch culture based formulas is：10-30g/L sucrose, 5-10g/L yeast extracts, 5-20g/L whey powders, surplus are to steam
Distilled water, sterilization temperature are 115 DEG C, 15min；
The composition of the freeze drying protectant includes：5-20% skimmed milk powers, 2-10% lactose, 1-10% trehaloses, 0.1-1.0%
Ascorbic acid, 0.2-0.5% glycerine, 0.01-0.1% sorbierites, mixing obtain.
2. lactobacillus acidophilus high density fermentation culture medium according to claim 1, it is characterised in that：Each group be grouped as
Under：
The fermentation tank culture based formulas is：20g/L yeast extracts, 15g/L whey powders, 10g/L peptones, 18g/L sucrose,
0.1g/L MnSO4, 5g/L sodium acetates, 0.5g/L MgSO4·7H2O, 2g/L dipotassium hydrogen phosphate, 2g/L diammonium hydrogen citrates,
Half deamination acid hydrochlorides of 0.001g/L L-, 1mL/L Tween 80s, 0.5% antifoaming agent, surplus are distilled water, sterilization temperature
It is 115 DEG C, 20min；
The feed-batch culture based formulas is：20g/L sucrose, 5g/L yeast extracts, 15g/L whey powders, surplus are distilled water, sterilizing
Sterilisation temp is 115 DEG C, 15min；
The composition of the freeze drying protectant includes：10% skimmed milk power, 4% lactose, 4% trehalose, 0.1% ascorbic acid,
0.5% glycerine, 0.02% sorbierite, mixing obtain.
3. using the bacterium powder preparation method of lactobacillus acidophilus high density fermentation culture medium described in claim 1, it is characterised in that：Tool
Preparation step is as follows：
(1) seed culture of lactobacillus acidophilus：Strain of lactobacillus acidophilus (the Chinese industrial microbial bacteria that will be stored in glycerol tube
Kind of preservation administrative center, CGMCC1.3221) 37 DEG C of activation cultures are first order seed in access 5mL MRS fluid nutrient mediums；Again
Activated primary seed solution is inoculated in 3% inoculum concentration in 10mL MRS fluid nutrient mediums, 37 DEG C of stationary cultures are for 24 hours
For secondary seed；Finally activated secondary seed solution is accessed with 3% inoculum concentration in 150mL MRS fluid nutrient mediums, 37
DEG C stationary culture is three-level seed liquor for 24 hours；
(2) fermentation tank culture：Fermenter volume is 5L, loads fermentation tank culture medium 3L, and sterilization temperature is 115 DEG C,
20min；The three-level seed liquor that culture obtains all is pumped into fermentation tank with 5% inoculum concentration, fermented and cultured temperature is 37 DEG C, is stirred
Mix rotating speed 150r/min, pH are automatically maintained at pH5.7-6.0 using 50% ammonium hydroxide, and are passed through nitrogen and maintain fermentation pressure tank
In 0.05MPa；By the way of speed change feed supplement, 0.5h measures the change rate of pH value in one time fermentation tank, and training is found at 8-9h
PH change rates drastically decline in nutrient solution, illustrate that the nutriment (mainly carbon source) of culture medium at this time has been consumed and are over, institute
Feed supplement should be carried out at this moment, supplemented medium, 12- are first supplemented with the rate of 9mL/Lh by the feed supplement pump on fermentation tank
PH change rates change again after 14h, and the rate for being changed to 18mL/Lh carries out feed supplement, cultivates 18-20h；
(3) separation of zymotic fluid：By 4 DEG C of the zymotic fluid of obtained lactobacillus acidophilus, 5000-9000r/min centrifuges 10min-
20min obtains bacterium mud；
(4) vacuum freeze drying of bacterium mud：According to wet thallus: above-mentioned freeze drying protectant=1:1-1:2 weight ratio will be lyophilized
Protective agent annex solution is added to by sterile working in the bacterial sediment object after the lactobacillus acidophilus centrifugation of collection, and suspending medium is
The phosphate buffer of pH6.8, stirring 30min keep suspension uniform, and 4 DEG C of standing 5min obtain the acidophilus breast containing freeze drying protectant
Bacillus thallus suspension liquid carries out precooling processing 12h in -80 DEG C of deep freezers, then carries out vacuum freeze drying, freezing temperature
- 55 DEG C or so, vacuum degree 1-7Pa are spent, obtaining viable count after dry 18-30h is more than 5.0 × 1011The lactobacillus acidophilus of cfu/g is frozen
Dry bacterium powder.
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| CN109652491A (en) * | 2019-01-29 | 2019-04-19 | 润盈生物工程（上海）有限公司 | A kind of screening method for lactobacillus acidophilus amino acid requirement |
| CN110305811A (en) * | 2019-06-28 | 2019-10-08 | 天津生机集团股份有限公司 | A kind of lactobacillus plantarum microbial inoculum and its high density fermentation preparation method and application |
| CN111057668A (en) * | 2019-12-26 | 2020-04-24 | 上海源耀农业股份有限公司 | Preparation method of liquid fermentation microbial inoculum of lactobacillus acidophilus |
| CN111286471A (en) * | 2019-12-12 | 2020-06-16 | 湖北华扬科技发展有限公司 | Method for high-density culture of lactobacillus paracasei by using soybean meal hydrolysate |
| CN112063547A (en) * | 2020-08-14 | 2020-12-11 | 润盈生物工程（上海）有限公司 | Method for high-density fermentation and bacterial powder quality improvement of lactobacillus gasseri LG-G12 |
| CN112694976A (en) * | 2021-01-22 | 2021-04-23 | 武汉微康益生菌研究院有限公司 | Preparation method of lactobacillus acidophilus powder with high viable count |
| CN112725183A (en) * | 2020-12-30 | 2021-04-30 | 杭州娃哈哈科技有限公司 | Method for improving freeze drying survival rate of probiotics |
| CN112961797A (en) * | 2021-02-01 | 2021-06-15 | 武汉微康益生菌研究院有限公司 | Lactobacillus acidophilus high-density fermentation medium and application thereof |
| CN114686407A (en) * | 2022-05-13 | 2022-07-01 | 微康益生菌(苏州)股份有限公司 | Preparation method of lactobacillus acidophilus powder for improving culturable cell content |
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