CN103283973A - A method for producing a probiotic agent of live Lactobacillus acidophilus - Google Patents
A method for producing a probiotic agent of live Lactobacillus acidophilus Download PDFInfo
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- CN103283973A CN103283973A CN2013100878929A CN201310087892A CN103283973A CN 103283973 A CN103283973 A CN 103283973A CN 2013100878929 A CN2013100878929 A CN 2013100878929A CN 201310087892 A CN201310087892 A CN 201310087892A CN 103283973 A CN103283973 A CN 103283973A
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- lactobacillus acidophilus
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- active bacteria
- culture medium
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Abstract
The present invention relates to a method for producing a probiotic agent of live Lactobacillus acidophilus, belonging to the field of microbial technology and specially relates to a liquid fermentation method for producing the probiotic agent of live Lactobacillus acidophilus. The method comprises the steps of: employing the Lactobacillus acidophilus as a fermentation strain which is inoculated on an optimized liquid fermentation medium, fermenting at a temperature of 35-38 DEG C for 20-23h, drawing off and determining the number of viable cells, adsorbing fermentation broth by using vegetable starch, drying, crushing, and packaging to prepare the probiotic agent of live Lactobacillus acidophilus with stable viability. The method of the invention is advantageous in that: high density fermentation of the Lactobacillus acidophilus can be completed by using an ordinary fermentation tank, with simple operation, stable effect and low energy consumption; and centrifuges, freeze-drying machines, etc. are not required, thus reducing production costs and improving production efficiency.
Description
Technical field
The present invention relates to the microbial fermentation engineering field, specifically a kind of fluid nutrient medium prepares the method for probiotic feed additive through Lactobacillus acidophilus's anaerobic fermentation.
Background technology
Lactobacillus acidophilus (
Lactobacillus acidophilus) belong to lactobacillus (
Lactobacillus), Gram-positive bacillus, form is elongated rod-shaped, bar terminal rounded, size is 0.6 ~ 0.9 * l.5 ~ l.6 μ m, exist with single, paired or short chain form, do not move atrichia, no gemma, the catalase feminine gender, the homofermentation lactose, liquefy gelatin not, optimum growth temperature is 35
~38 ℃, do not grow below 20 ℃.Optimum pH is 5.5
~6.4 acid resistance is strong, can be in the environment that other lactic acid bacteria can not grow growth and breeding.G+C content is 36.0
~37.4%, do not produce cytochromes.Lactobacillus acidophilus is a class anaerobism or amphimicrobian microorganism, under anaerobic or the lower partial pressure of oxygen environment in solid state substrate superficial growth, 5
~10% carbon dioxide can promote the propagation of lactobacillus acidophilus.Can utilize glucose, fructose, lactose, sucrose to carry out homofermentation, fermentation produces DL type lactic acid.Lactobacillus acidophilus extensively is present in the enteron aisle of people and some animals, is beneficial microbe colony, and when having some, it can advantageously regulate the balance between useful and harmful microorganism flora in the enteron aisle, plays an important role to keeping GI physiological function.
Lactobacillus acidophilus is that a class can be utilized carbohydrate fermentation and produce the bacterium of a large amount of lactic acid, can produce SCFA, antibacterial peptide, polysaccharide, hydrogen peroxide, specific enzymes system etc. in addition.Some agricultural product castoffs through inoculating lactobacillus fermentation after, its tunning is very useful to animal intestinal health, there are some researches prove that what play a major role in fact is antibacterial peptide.Lactobacillus acidophilus mainly is to adjust the gut flora balance, suppresses the propagation of enteron aisle undesirable microorganism.Lactobacillus acidophilus has antagonism to pathogenic microorganisms, it can secrete antibiotin class material, as: acidolin (acidolin), bacillus acidophilus's element (acidophilin), lactein (1aetocidon) produce inhibitory action to pathogenic entero becteria.Lactobacillus acidophilus is one of probio of very paying attention in the lactic acid bacteria family research and development, is regarded as third generation yogurt starter culture, when it reaches some, can play the health promotion effect.
(1) biological barrier effect
The interaction of lactobacillus acidophilus and intestinal epithelial cell and close to having constituted biological containment; Simultaneously, by the interaction between himself and metabolin and other bacterium, adjust the relation between the flora, keep and guarantee the stable of the combination of flora optimal vigor and this combination, stoped pathogenic bacteria decide grow and invade, the adhesion of the growth of antagonism pathogenic bacteria and harmful microbe and toxin thereof.
(2) trophism
The lactic acid that produces behind the lactobacillus-fermented, the predigestion that can improve food in the stomach improves calcium, phosphorus, the utilization rate of iron and the absorption of vitamin D; Lactose decomposes the galactolipin that produces, and is to constitute cerebronic composition in the cerebral nervous system, with the postnatal growth of infant close relationship is arranged; Produced simultaneously a large amount of Cobastab group energy promotes the growth of body nerve cell; Improve the activity of the beta galactosidase in the body intestines, promote the absorption of lactose, thereby alleviate lactose intolerance; The energy extracellular proteinase promotes digesting and assimilating of protein.
(3) antitumaous effect
The corruptibility bacterial action can generate metabolin, the amino acid metabolite organic carcinogen matter such as (phenols, indoles, hydrogen sulfide) of nitroso compound, cholesterol and bile acid in COF and bile acid salt.The relevant intestinal bacterium enzyme of these carcinogens has GRD beta-glucuronidase, sulfydryl reductase, azo reductase etc.Lactobacillus acidophilus can be improved gut flora, suppresses the generation of carcinogen; Simultaneously, lactobacillus acidophilus and metabolite thereof have activated immunologic function, suppress formation and the propagation of cancer cell.
(4) strengthen immunity of organisms
Lactobacillus acidophilus grows in that enteron aisle is decided at the higher level but not officially announced, is equivalent to natural immunity, can activate the body macrophage activity, brings out specificity and nonspecific immune reaction, improves anti-infectious ability.
(5) delay body aging
Harmful spoilage product such as the bacteriogenic ammonia of the aging of body and intestinal toxic, amine, hydrogen sulfide, indoles, phenol, scatol have much relations.The lactic acid that the lactobacillus acidophilus metabolism produces has suppressed these putrefactions, makes the process of body aging become slow.
The Lactobacillus acidophilus is fermented through high density liquid, obtain the fermentation culture of high cell density, after plant amylum absorption, dry, pulverize, Lactobacillus acidophilus's probiotics of making is applied to livestock-raising, can regulate the microbial population of animal intestinal tract balance, strengthen immunity of organisms, improve resistance against diseases.
Mainly there is following problem and shortage in Lactobacillus acidophilus's in the past fermentation mode: (1) is adopted anaerobic jar or is kept its anaerobic environment by lasting feeding carbon dioxide or nitrogen and carry out, and raw material can not be reused, and operation is complicated, and cost is higher; (2) adopt tryptone, yeast extract, beef extract and enzymolysis product thereof etc. as the culture medium main nutrient composition in a large number, the production cost height; (3) post processing of liquid fermentation production needs high speed freezing centrifuge to carry out centrifugal and frozen drying, and complex process is with high costs, yields poorly; (4) the viable bacteria density of tunning is not high enough.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of Lactobacillus acidophilus's active bacteria formulation is to overcome preparation method's above shortcomings of existing Lactobacillus acidophilus's active bacteria formulation.
The technical scheme that realizes the object of the invention is as follows:
A kind of preparation method of Lactobacillus acidophilus's active bacteria formulation may further comprise the steps:
(1) preparation culture medium;
(2) culture medium of jar in the dissolving is loaded onto the pH electrode and is adjusted the pH value, in the fermentation tank of then culture medium being packed into;
(3) to the sterilization of heating of the culture medium in the fermentation tank;
(4) the inoculation Lactobacillus acidophilus plants daughter bacteria: add inorganic salt solution in fermentation tank cooling back, add atoleine then and insert kind of a daughter bacteria;
(5) temperature controlled fermentation; Control pH value and temperature are at the state bottom fermentation of insulation;
(6) put jar and measure zymotic fluid bacterium number;
(7) with corn flour, wheat flour or soy meal and composition adsorbs zymotic fluid thereof;
(8) mixture after will adsorbing is dry and pulverize, and namely obtains powderous preparations.
Technical advantage of the present invention:
(1) directly utilize the common fermentation jar to produce, simple to operate, steady quality, with low cost, be easy to industrialization production;
(2) with short production cycle, 72h can finish one batch production, is easy to industrialization, thereby the Lactobacillus acidophilus is widely used in livestock breeding industry;
(3) adopt anaerobic jar or keep its anaerobic environment and carry out by continuing to feed carbon dioxide or nitrogen, raw material can not be reused, and operation is complicated, and cost is higher; The atoleine that is used for secluding air can repeatedly use repeatedly, greatly reduces production cost;
(4) the present invention adopts soy meal, corn flour or wheat flour or its composition as the main component of fermentation medium, compare as main component with tryptone, yeast extract, beef extract and enzymolysis product thereof etc. that other inventions are adopted, cost reduces greatly, and tunning viable bacteria concentration can reach 1 ~ 2.0 * 10
10CFU/mL;
(5) the present invention adopts plant amylum absorption zymotic fluid, and aftertreatment technology does not need to use high speed freezing centrifuge, and the present invention is adsorbed mixture convection drying and pulverizing with plant amylum, namely makes product after the packing, and aftertreatment technology is simplified greatly, and production cost is lower;
(6) both at home and abroad about the powder Study and manufacture on starter culture, adopt vacuum freeze-drying method mostly, the advantage of the method is somatic cells survival rate height, but complex process, the cost of manufacture height, and the production cost of absorption back seasoning is low, and operating procedure is simple, is easy for large-scale industrial production and applies.
The specific embodiment
Further specify the present invention by the following examples, but not as restriction of the present invention.
The technological process of embodiment 1 Lactobacillus acidophilus's active bacteria formulation preparation may further comprise the steps:
(1) activation of bacterial classification and increment:
The present invention adopt the Lactobacillus acidophilus (
Lactobacillus acidophilus) as fermented bacterium, bacterium numbering: GIM1.268 purchases in microorganism fungus kind preservation center, Guangdong Province, but be not limited to the Lactobacillus acidophilus of this deposit number; The single bacterium colony of picking Lactobacillus acidophilus from the flat board, be inoculated into 20mL MRS culture medium (peptone 10.0 g are housed, beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, Tween 80 1.0 mL, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, distilled water 1 000 mL, pH 6.2 ~ 6.6 autoclave sterilizations) in the test tube, 37 ℃ of deep layers leave standstill cultivated about 20 hours, and bacterium liquid is inoculated in the triangular flask that proper volume MRS culture medium is housed with 0.3% inoculum concentration, 37 ℃ of deep layers leave standstill cultivate about 20 hours standby; If do not use immediately behind the medium sterilization, then need to use after heating 10min drives oxygen in boiling water bath;
(2) preparation fermentation medium:
The raw material that culture medium comprises and parts by weight are: soy meal (being fermented bean dregs) 50g, corn flour 15g, glucose 10g, yeast extract powder 2g, peptone 4g, raw material is worked as in gram, and the corresponding umber in mL of distilled water is 800 ~ 1000, dissolving, add in the fermentation tank and carry out 121 ℃, the time is 10 minutes autoclave sterilization, when fermentation tank is cooled to 35 ~ 45 ℃, add inorganic salts 1.0g/1000mL base-material, inorganic salts are formed: CaCl
20.02%, MgSO
47H
2O 0.04%, MnSO
4H
2O 0.04%, for avoiding forming precipitation, stirs in adition process, and adding atoleine thickness then is 2cm;
(3) temperature controlled fermentation: the inoculum concentration that inserts Lactobacillus acidophilus's liquid seeds is 0.3%, stir, control pH 5.5 ~ 6.4, fermentation temperature control is at 37 ℃, fermented 23 hours, putting jar, Lactobacillus acidophilus's culture is carried out colony counting, is 1.8 * 10 through the viable bacteria bulk concentration that ferments like this
10CFU/mL;
(4) adsorb zymotic fluid with plant amylum: with etc. corn flour, soya-bean cake powder and the wheat flour of quality mix, as the adsorbent of zymotic fluid;
(5) the Lactobacillus acidophilus's zymotic fluid with plant amylum absorption carries out bake drying, pulverizes and packing, makes the microbial forage additive for livestock and poultry cultivation.
What embodiment 2 and above-described embodiment 1 were different is:
(1) culture medium prescription: the raw material that culture medium comprises and parts by weight are: soy meal (being fermented bean dregs) 45g, corn flour 15g, glucose 12g, yeast extract powder 1g, peptone 3g, raw material is worked as in gram, and the corresponding umber in mL of distilled water is 800 ~ 1000, dissolving, add in the fermentation tank and carry out 115 ℃, the time is 20 minutes autoclave sterilization, when fermentation tank is cooled to 35 ~ 45 ℃, add inorganic salts 1.5g/1000mL base-material, the composition of inorganic salts: CaCl
20.02%, MgSO
47H
2O 0.04%, MnSO
4H
2O 0.04%, for avoiding forming precipitation, stirs in adition process, and adding atoleine thickness then is 1cm;
(2) temperature controlled fermentation: the inoculum concentration that inserts Lactobacillus acidophilus's liquid seeds is 1%, stir, control pH 5.5 ~ 6.4, fermentation temperature control is at 35 ℃, fermented 22 hours, putting jar, Lactobacillus acidophilus's culture is carried out colony counting, is 1.5 * 10 through the viable bacteria bulk concentration that ferments like this
10CFU/mL;
(3) adsorb zymotic fluid with plant amylum: with etc. corn flour and the wheat flour of quality mix, as the adsorbent of zymotic fluid;
(4) the Lactobacillus acidophilus's zymotic fluid with plant amylum absorption carries out bake drying, pulverizes and packing, makes the microbial forage additive for livestock and poultry cultivation.
Claims (6)
1. the preparation method of Lactobacillus acidophilus's active bacteria formulation may further comprise the steps:
(1) activation of bacterial classification and increment:
The present invention adopt the Lactobacillus acidophilus (
Lactobacillus acidophilus) as fermented bacterium, bacterium numbering: GIM1.268 purchases in microorganism fungus kind preservation center, Guangdong Province; The single bacterium colony of picking Lactobacillus acidophilus from the flat board, be inoculated into 20mL MRS culture medium (peptone 10.0 g are housed, beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, Tween 80 1.0 mL, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, distilled water 1 000 mL, pH 6.2 ~ 6.6 autoclave sterilizations) in the test tube, 37 ℃ of deep layers leave standstill cultivated about 20 hours, and bacterium liquid is inoculated in the triangular flask that proper volume MRS culture medium is housed with 0.3% inoculum concentration, 37 ℃ of deep layers leave standstill cultivate about 20 hours standby; If do not use immediately behind the medium sterilization, then need to use after heating 10min drives oxygen in boiling water bath;
(2) preparation fermentation medium:
The raw material that culture medium comprises and parts by weight are: soy meal (being fermented bean dregs) 40 ~ 50, corn flour 10 ~ 15, glucose 10 ~ 12, yeast extract powder 1 ~ 3, peptone 2 ~ 4, raw material is worked as in gram, and the corresponding umber in mL of distilled water is 800 ~ 1000, dissolving, add in the fermentation tank and carry out 100 ~ 121 ℃, the time is 10 ~ 30 minutes autoclave sterilization, when fermentation tank is cooled to 35 ~ 45 ℃, add inorganic salts 0.7 ~ 1.5g/1000mL base-material, inorganic salts are selected from CaCl
2, MgSO
47H
2O and MnSO
4H
2Among the O two kinds or three kinds for avoiding forming precipitation, stir in adition process, and adding atoleine to thickness then is 1 ~ 2cm;
(3) temperature controlled fermentation: the inoculum concentration that inserts Lactobacillus acidophilus's liquid seeds is 0.3 ~ 0.5%, stir, control pH 5.5 ~ 6.4, fermentation temperature control is at 35 ~ 38 ℃, fermented 20 ~ 23 hours, put jar, Lactobacillus acidophilus's culture is carried out colony counting, can reach 1.0 ~ 2.0 * 10 through the viable bacteria bulk concentration that ferments like this
10CFU/mL;
(4) adsorb zymotic fluid with plant amylum: with corn flour, soya-bean cake powder or wheat flour, perhaps its any combination is as the adsorbent of zymotic fluid;
(5) the Lactobacillus acidophilus's zymotic fluid with plant amylum absorption carries out bake drying, pulverizes and packing, makes the microbial forage additive for livestock and poultry cultivation.
2. the production of Lactobacillus acidophilus's active bacteria formulation according to claim 1, it is characterized in that described culture medium: soy meal (being fermented bean dregs) 40 ~ 50, corn flour 10 ~ 15, glucose 10 ~ 12, yeast extract powder 1 ~ 3, peptone 2 ~ 4, raw material is worked as in gram, the corresponding umber in mL of distilled water is 800 ~ 1000, and dissolving adds in the fermentation tank and carries out 100 ~ 121 ℃, time is 10 ~ 30 minutes autoclave sterilization, when fermentation tank is cooled to 35 ~ 45 ℃, add inorganic salts 0.7 ~ 1.5g/1000mL base-material, inorganic salts are selected from CaCl
2, MgSO
47H
2O and MnSO
4H
2Among the O two kinds or three kinds.
3. Lactobacillus acidophilus's active bacteria formulation according to claim 1, it is characterized in that: cultivation temperature is 35 ~ 38 ℃.
4. Lactobacillus acidophilus's active bacteria formulation according to claim 1 is characterized in that: plant the daughter bacteria inoculum concentration and be 0.3 ~ 0.5% inoculum concentration.
5. Lactobacillus acidophilus's active bacteria formulation according to claim 1, it is characterized in that: used bacterial classification is the Lactobacillus acidophilus of lactobacillus.
6. Lactobacillus acidophilus's active bacteria formulation according to claim 1 is characterized in that: be applied to livestock and poultry cultivation as feed addictive.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108185112A (en) * | 2017-12-27 | 2018-06-22 | 河南飞天农业开发股份有限公司 | A kind of production technology of plant protein fodder |
| CN110193030A (en) * | 2019-04-29 | 2019-09-03 | 山东苏柯汉生物工程股份有限公司 | A kind of dog probiotics and preparation method thereof |
| CN112251372A (en) * | 2020-10-10 | 2021-01-22 | 内蒙古普泽生物制品有限责任公司 | Lactobacillus acidophilus with galactosidase activity and lactose tolerance and application thereof |
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| CN101112397A (en) * | 2007-08-09 | 2008-01-30 | 天津科技大学 | Viable prebiotics bacteria microcapsule preparations for livestock and method for preparing the same |
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| CN108185112A (en) * | 2017-12-27 | 2018-06-22 | 河南飞天农业开发股份有限公司 | A kind of production technology of plant protein fodder |
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Application publication date: 20130911 |