CN103404703A - Stable and safe streptococcus thermophilus viable bacteria preparation and preparation method thereof - Google Patents
Stable and safe streptococcus thermophilus viable bacteria preparation and preparation method thereof Download PDFInfo
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- CN103404703A CN103404703A CN2013102281221A CN201310228122A CN103404703A CN 103404703 A CN103404703 A CN 103404703A CN 2013102281221 A CN2013102281221 A CN 2013102281221A CN 201310228122 A CN201310228122 A CN 201310228122A CN 103404703 A CN103404703 A CN 103404703A
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Abstract
The invention relates to a production method for a streptococcus thermophilus viable bacteria preparation, belonging to the field of microbial technology and particularly relating to a liquid anaerobic fermentation production method for a streptococcus thermophilus micro-ecological preparation. The production method comprises the following steps of taking the streptococcus thermophilus as the fermented strain, inoculating into an optimized liquid medium, cultivating for 16-20 hours at 40-42 DEG C, putting the fermentation liquid in a tank and measuring the viable bacteria quantity, adsorbing the fermentation liquid through vegetable starch and drying, after treatment by polyethylene glycol (PEG) 4000 and sodium polyacrylate, drying, smashing and packaging, and preparing the streptococcus thermophilus preparation with stable activity. The production method has the advantages of completing high density fermentation of the streptococcus thermophilus through a common fermentation tank, being simple in operation, having stable effect and low energy consumption, using no equipment such as a centrifugal machine and a freeze dryer and the like, lowering the production cost, and improving the 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 the streptococcus thermophilus anaerobic fermentation.
Background technology
Streptococcus thermophilus (Streptococcus thermophilus) amphimicrobian or micro-aerobic gram-positive bacteria, the catalase reaction feminine gender, circular or oval, paired or one-tenth chain, do not produce gemma, how not move.Streptococcus thermophilus is the Healthy People normal intestinal flora, can in human body intestinal canal, grow, breed.Can directly supplement the human normal physiological bacteria, adjust the gut flora balance, suppress and remove the bacterium that in enteron aisle, the people is had potential hazard.
Streptococcus thermophilus is considered to " generally recognized as safe (GRAS) " composition, is widely used in and produces some important fermented dairy products, comprises Yoghourt and cheese.Streptococcus thermophilus also has some functional activities, such as producing exocellular polysaccharide, bacteriocin and vitamin.In addition, streptococcus thermophilus also can be used as potential beneficial bacterium, experimental results show that it has health effect, transport activity and certain intestines and stomach adhesiveness.
Streptococcus thermophilus is applied to the function of animal feed: (1) improves intestinal microenvironment: reduce enteron aisle pH value, promote that enterocinesia prevents the pathogen field planting, the secreting bacteria element suppresses the growth of pathogen; (2) regulate blood pressure: suppress the cholesterol biosynthesis enzymatic activity, reduce cholesterol in serum content, its bacterium tunning can be regulated the control blood pressure; (3) antitumaous effect: the polysaccharide of generation, bacteriocin, lactic acid etc. have the effect of antitumor activity, and by activating body immune system, the sudden change that suppresses cell comes to antineoplastic generation; (4) delay senility: produce superoxide dismutase (SOD) and can remove in the internal metabolism process the excessive ultra-oxygen anion free radical produced, delay senility; (5) lactic acid producing: the degraded of lactose needs a kind of special enzyme, is beta galactosidase.In the person of lactose intolerance, be exactly to have lacked this a kind of enzyme, streptococcus thermophilus is a kind of bacterium that can produce beta galactosidase, so can help the digestion of lactose; (6) to the tolerance of hydrochloric acid in gastric juice, cholate: the survival rate of streptococcus thermophilus in the acid solution of PH=6.8 is 100%, and the survival rate in the acid solution of PH=4 is approximately 75%, and the survival rate in the acid solution of PH=3 is approximately 70%; Streptococcus thermophilus can be survived in the bile of higher concentration, so should avoid in vivo the infringement of bile, arrive the far-end of small intestine.
Streptococcus thermophilus is fermented through high density liquid, obtain the fermentation culture of high cell density, after plant amylum absorption, dry, pulverize, the Lactobacillus casei 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.The streptococcus thermophilus viable bacteria solid fermentation culture that process PEG-4000 and Sodium Polyacrylate were processed, bacterial activity is more stable, loss of activity is less in processing, transportation and storage, and can reach animal intestinal due to its slow releasing function, greatly improve the ability of streptococcus thermophilus viable bacteria field planting animal intestinal, greatly improved the bioactive stability of said preparation.
Mainly there is following problem and shortage in the fermentation mode of streptococcus thermophilus in the past: (1) is adopted anaerobic jar or is maintained its anaerobic environment and carry out by continuing to pass into carbon dioxide or nitrogen, and raw material can not be reused, and operates more complicatedly, and cost is higher; (2) 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; (3) 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 streptococcus thermophilus active bacteria formulation, with above shortcomings on the preparation method who overcomes existing streptococcus thermophilus active bacteria formulation.
The technical scheme that realizes the object of the invention is as follows:
A kind of preparation method of streptococcus thermophilus active bacteria formulation comprises the following steps:
(1) preparation culture medium;
(2) culture medium of tank in dissolving, load onto the pH electrode and adjust the pH value, in the fermentation tank of then culture medium being packed into;
(3) to the culture medium in fermentation tank, heat sterilizing;
(4) inoculation streptococcus thermophilus kind daughter bacteria: add inorganic salt solution after the fermentation tank cooling, then add atoleine and access kind of daughter bacteria;
(5) temperature controlled fermentation; Control pH value and temperature, at the state bottom fermentation of insulation;
(6) put tank and measure zymotic fluid bacterium number;
(7) with corn flour, wheat flour or soy meal and composition adsorbs zymotic fluid thereof, the dry and pulverizing by the mixture after absorption;
(8) by the product of solid fermentation after PEG4000 and Sodium Polyacrylate are processed, drying, make stable streptococcus thermophilus active bacteria formulation.
Compared with prior art, technical advantage of the present invention:
(1) directly utilize the common fermentation tank to produce, simple to operate, steady quality, with low cost, be easy to industrialization production;
(2) the product fermentation period obviously shortens, and reach home time of acidity or pH value of product is 16～20 hours, and traditional streptococcus thermophilus separately fermentation reach same acidity or pH value and need 48 hours even for more time; Production cycle shortens short greatly, is easy to industrialization, thereby streptococcus thermophilus can be widely used in to livestock breeding industry;
(3) product cost reduces with the fermentation pollution risk: the present invention is because the production cycle shortens, and production cost reduces, and simultaneously, the acidified milk base-material produces in earlier stage that acid is very fast, pH value descends rapidly, can better suppress living contaminants, and the product risk of failure of fermenting reduces greatly;
(4) adopt anaerobic jar or maintain its anaerobic environment and carry out by continuing to pass into carbon dioxide or nitrogen, raw material can not be reused, and operates more complicatedly, and cost is higher; For the atoleine of isolated air Reusability repeatedly, greatly reduce production cost;
(5) the present invention adopts the streptococcus thermophilus liquid fermentation medium through optimizing, and the streptococcus thermophilus bacterium is counted content and obviously improves, and tunning viable bacteria concentration can reach 2.0～3.5 * 10
10CFU/mL;
(6) 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 by plant amylum, after packing, namely makes product, and aftertreatment technology is simplified greatly, and production cost is lower;
(7) development of relevant starter powder both at home and abroad, mostly adopt vacuum freeze-drying method, the advantage of the method is that the somatic cells survival rate is high, but complex process, cost of manufacture is high, and after absorption, the production cost of seasoning is low, and operating procedure is simple, be easy for large-scale industrial production and apply, being suitable for China's Feed Enterprise and under the existing equipment condition, producing streptococcus thermophilus probiotics product.
(8) by the product of solid fermentation after PEG4000 and Sodium Polyacrylate are processed, drying, make stable streptococcus thermophilus active bacteria formulation stability better.
The specific embodiment
The technological process of the streptococcus thermophilus active bacteria formulation of embodiment 1 preparation stabilisation comprises the following steps:
(1) actication of culture:
The present invention adopts the streptococcus thermophilus viable bacteria as fermented bacterium, be purchased from Chinese common micro-organisms culture presevation administrative center, deposit number: 1.6472, by the streptococcus thermophilus viable bacteria bacterial classification of-80 ℃ of preservations at MRS culture medium (peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, diammonium hydrogen citrate 2.0g, glucose 20.0g, Tween 80 1.0mL, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.58g, manganese sulfate 0.25g, agar powder 15.0g, distilled water 1000mL, pH6.2～6.6, autoclave sterilization) on the solid inclined-plane, rule, cultivated about 16 hours for 37 ℃, general 1～3 time of actication of culture,
(2) prepare first order seed:
Streptococcus thermophilus viable bacteria by activation, with oese picking one ring, be inoculated in 20mL MRS liquid (peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, diammonium hydrogen citrate 2.0g, glucose 20.0g, Tween 80 1.0mL, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.58g, manganese sulfate 0.25g, distilled water 1000mL, pH6.2～6.6, autoclave sterilization) in test tube, 37 ℃ of standing cultivations of deep layer are about 20 hours, bacterium liquid is inoculated in the triangular flask that proper volume MRS culture medium is housed with 3% inoculum concentration, 37 ℃ of standing cultivations of deep layer are standby about 20 hours, if after medium sterilization, do not use immediately, need in boiling water bath, heat after 10min drives oxygen and use,
(3) obtaining liq fermentation medium:
Streptococcus thermophilus liquid fermentation medium formula after optimizing: yeast extract 7.5g, peptone 7.5g, glucose 10g, KH
2PO
42g, the 100mL of Tomato juice, Tween80 0.5mL, distilled water 900mL, pH6.8, stir, and through 121 ℃, 20min autoclave sterilization, when fermentation tank is cooled to 45 ℃, adds inorganic salts 1.2g/1000mL base-material, inorganic salts: CaCl
20.02%, MgSO
47H
2O 0.04%, MnSO
4H
2O 0.04%, for avoiding forming precipitation, stirs in adition process, and then adding atoleine to thickness is 2cm;
(4) liquid fermentation:
Will be through the liquid fermentation medium of sterilization treatment according to 5% inoculum concentration access first order seed bacterium, stir, keeping temperature is 40～42 ℃, pH6.6-6.8, cultivate and put tank in 20 hours, streptococcus thermophilus viable bacteria culture is carried out to colony counting, reach 3.5 * 10 through the viable bacteria bulk concentration fermented like this
10CFU/mL;
(5) with plant amylum, adsorb zymotic fluid: with corn flour, wheat flour and beancake powder, mass ratio is the mixture of 1:1:1, as the adsorbent of zymotic fluid, and the zymotic fluid mixture of plant amylum absorption is carried out to bake drying;
(6) stabilization processes:
By the streptococcus thermophilus viable bacteria solid fermentation culture of drying, adding mass ratio is that 0.25% Sodium Polyacrylate and 5% PEG4000(are dissolved in water in advance), stir and evenly mix, in 65 ℃ of lower heat treatment 60min, through super-dry, pulverizing and packing, make the microbial forage additive for livestock and poultry cultivation.
What embodiment 2 was different from above-described embodiment 1 is:
(1) culture medium prescription: yeast extract 7.5g, peptone 7.5g, glucose 10g, KH
2PO
42g, the 100mL of Tomato juice, Tween80 0.5mL, distilled water 900mL, pH6.8, stir, and through 121 ℃, 20min autoclave sterilization, when fermentation tank is cooled to 45 ℃, adds inorganic salts 1.3g/1000mL base-material, the composition of inorganic salts: MgSO
47H
2O 0.04%, MnSO
4H
2O 0.04%, for avoiding forming precipitation, stirs in adition process, and then adding atoleine thickness is 1cm;
(2) temperature controlled fermentation: the inoculum concentration of access lactobacillus bulgaricus liquid seeds is 3%, stir, initial pH6.6, control pH6.6～6.8, fermentation temperature is controlled at 40～42 ℃, ferments 16 hours, puts tank, thermophilic streptococci cultures is carried out to colony counting, is 2.6 * 10 through the viable bacteria bulk concentration fermented like this
10CFU/mL;
(3) with plant amylum, adsorb zymotic fluid: with etc. corn flour and the wheat flour of quality mix, as the adsorbent of zymotic fluid;
(4) the streptococcus thermophilus fermentation liquid of plant amylum absorption is carried out to bake drying and pulverizing, adding mass ratio is that 0.25% Sodium Polyacrylate and 5% PEG4000(are dissolved in water in advance), stir and evenly mix, in 65 ℃ of lower heat treatment 60min, through super-dry, pulverizing and packing, make the microbial forage additive for livestock and poultry cultivation.
Claims (6)
1. the preparation method of the streptococcus thermophilus active bacteria formulation of a stability and safety comprises the following steps:
(1) actication of culture:
The present invention adopts streptococcus thermophilus as fermented bacterium, be purchased from Chinese common micro-organisms culture presevation administrative center, deposit number: 1.6472, by the streptococcus thermophilus viable bacteria bacterial classification of-80 ℃ of preservations at MRS culture medium (peptone 10.0 g, 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, agar powder 15.0 g, distilled water 1 000 mL, pH 6.2 ~ 6.6, autoclave sterilization) on the solid inclined-plane, rule, cultivated about 16 hours for 37 ℃, general 1 ~ 3 time of actication of culture,
(2) prepare first order seed:
Streptococcus thermophilus by activation, with oese picking one ring, be inoculated in 20mL MRS liquid (peptone 10.0 g, 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 sterilization) in test tube, 37 ℃ of standing cultivations of deep layer are about 20 hours, bacterium liquid is inoculated in the triangular flask that proper volume MRS culture medium is housed with 3% inoculum concentration, 37 ℃ of standing cultivations of deep layer are standby about 20 hours, if after medium sterilization, do not use immediately, need in boiling water bath, heat after 10min drives oxygen and use,
(3) obtaining liq fermentation medium:
Streptococcus thermophilus liquid fermentation medium formula after optimizing: yeast extract 7.5 g, peptone 7.5g, glucose 10 g, KH
2PO
42g, 100 mL of Tomato juice, Tween 80 0.5mL, distilled water 900 mL, pH6.8, stir, and through 121 ℃, 20min autoclave sterilization, when fermentation tank is cooled to 35 ~ 45 ℃, adds inorganic salts 0.7 ~ 1.5g/1000mL base-material, and inorganic salts are selected from CaCl
2, MgSO
47H
2O and MnSO
4H
2In O two kinds or three kinds, for avoiding forming precipitation, stir in adition process, then adding atoleine to thickness is 1 ~ 2cm;
(4) liquid fermentation:
Will be through the liquid fermentation medium of sterilization treatment according to 3 ~ 5% inoculum concentration access first order seed bacterium, stir, then adding atoleine to thickness is 1 ~ 2cm, keeping temperature is 40 ~ 42 ℃, pH 6.6-6.8, cultivate and put tank in 16 ~ 20 hours, streptococcus thermophilus viable bacteria culture is carried out to colony counting, can reach 2.0 ~ 3.5 * 10 through the viable bacteria bulk concentration fermented like this
10CFU/mL;
(5) with plant amylum, adsorb zymotic fluid: with corn flour, beancake powder or wheat flour, or its any combination, as the adsorbent of zymotic fluid, and the zymotic fluid mixture of plant amylum absorption is carried out to bake drying;
(6) stabilization processes:
By the streptococcus thermophilus viable bacteria solid fermentation culture of drying, adding mass ratio is that 0.25% Sodium Polyacrylate and 5% PEG4000(are dissolved in water in advance), stir and evenly mix, in airing or 65 ℃ of lower heat treatment 60min, through super-dry, pulverizing and packing, make the microbial forage additive for livestock and poultry cultivation.
2. the streptococcus thermophilus active bacteria formulation of stability and safety according to claim 1, is characterized in that described liquid fermentation medium is: yeast extract 7.5 g, peptone 7.5g, glucose 10 g, KH
2PO
42g, 100 mL of Tomato juice, Tween 80 0.5mL, distilled water 900 mL, pH6.8, stir, through 121 ℃, 20min autoclave sterilization.
3. the streptococcus thermophilus active bacteria formulation of stability and safety according to claim 1, it is characterized in that: cultivation temperature is that temperature is 40 ~ 42 ℃, and pH 6.6 ~ 6.8.
4. the streptococcus thermophilus active bacteria formulation of stability and safety according to claim 1 is characterized in that: at different levels kinds of daughter bacteria inoculum concentrations are 3 ~ 5% inoculum concentration.
5. the streptococcus thermophilus active bacteria formulation of stability and safety according to claim 1, it is characterized in that: bacterial classification used is the streptococcus thermophilus of streptococcus.
6. the streptococcus thermophilus active bacteria formulation of stability and safety according to claim 1, it is characterized in that: the application as feed addictive is additive for farm animal feed.
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| US20200093125A1 (en) * | 2017-05-26 | 2020-03-26 | Novozymes Bioag A/S | Stable inoculant compositions comprising paraffin oils/waxes |
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Application publication date: 20131127 |