CN103146611A - Preparation method for composite-type lactobacillus microecological preparation - Google Patents
Preparation method for composite-type lactobacillus microecological preparation Download PDFInfo
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Abstract
The invention discloses a preparation method for a composite-type lactobacillus microecological preparation, and belongs to the field of lactobacillus fementation. The preparation method comprises the following steps of: separating lactobacillus strains by taking bred animals, culture water body and bottom sediment as bacterial sources; then purifying the separated lactobacillus strains to selectively breed 2-5 lactobacillus strains with large calcium-dissolving rings, fast growth speed and strong activity; then carrying out enlarging culture on the lactobacillus strains which are purified and selectively bred, and preparing composite seed solution; and finally, inoculating and fermenting the composite seed solution to obtain the composite-type lactobacillus microecological preparation. Compared with the prior art, the preparation method disclosed by the invention has the beneficial effects of simple culture equipment and operation, and stable storage time compared with a single strain fermentation water agent.
Description
Technical field
The present invention relates to the lactobacillus-fermented field, particularly a kind of preparation method of compound lactic acid bacterium probiotics.
Background technology
Along with the fast development of culture fishery, lactic acid bacteria formulation is natural, nontoxic with it, have no side effect, pollution-free, noresidue, safe and reliable superiority more and more come into one's own.As a kind of useful " bioremediation agents " that purify water, lactic acid bacteria formulation more and more comes into one's own in the application of culture fishery, it is applied to, and culture fishery concentrates on mainly that spice is thrown something and fed, fermented feed and as the leading bacterium of EM bacterium at water splashing.Wherein spice is thrown something and fed, fermented feed research concentrates on milk-acid bacteria and perch at the aquatic animal stomach, and regulates the animal intestinal microflora and to the change of feed structure.Splash regulating water quality and milk-acid bacteria can be regulated aquaculture water, be improved breeding environment, suppresses pathogenic bacteria, and disease resistance improves immunizing power, promotes that growth of animal has close relationship.These use with the following characteristic relation of milk-acid bacteria close: at first, milk-acid bacteria can fine adaptation culture environment of aquatic products.Aquaculture envrionment temperature mobility scale during cultivating is that 20-35 ℃, salinity mobility scale are that 0-38, pH value mobility scale are 6.9-8.7, and milk-acid bacteria all can better grow in this three's mobility scale.Secondly, the most bacterial classification of milk-acid bacteria belongs to the oxygen resistence anerobe, and all growths under anaerobic, hypoxemia or high oxygen condition are not only competed oxygen with cultivated animals, and can also breed by lower floor's raised growth in the lower water body of dissolved oxygen amount, suppresses pathogenic bacteria and grows.In addition, milk-acid bacteria is not subjected to time limitation in the use, can use under heavy rain, cloudy day, fine day, hot conditions.In addition, milk-acid bacteria is compared with other bacterial classification as a kind of probiotic bacterium of using the earliest, and its thalline and meta-bolites are all useful thing, without toxin, have higher security in practical application.These milk-acid bacterias these advantages in the application on aquatic products make milk-acid bacteria have a wide range of applications aspect aquatic products and study.
But, the valuable product that present most Strains of lactobacillus is coated, use cost is very high; And the Strains of lactobacillus aqua product is relatively cheap, but most products is very unstable, and viable count is lower, and storage cycle is very short, and transportation cost is higher; Solid and liquid Strains of lactobacillus product most adopt fermentor tank to ferment, and manufacturing technique requirent is strict on the one hand, needs the technical professional to operate; On the other hand, the cost of fermentor tank is higher.
Summary of the invention
The present invention proposes a kind of preparation method of compound lactic acid bacterium probiotics, solved deficiency of the prior art, be that a kind of culture device and operation are all very simple, and the storage time is compared the preparation method of very stable lactobacillus micro-ecological preparation with single strain fermentation aqua.
Technical scheme of the present invention is achieved in that
A kind of preparation method of compound lactic acid bacterium probiotics take cultivated animals, aquaculture water and bed mud as the bacterium source, isolates lactic bacterium strains by applying solid MRS substratum after enrichment in the MRS liquid nutrient medium; Then isolated lactic bacterium strains being carried out purifying, to select the molten calcium circle of 2-5 strain large, and growth is fast, energetic lactic bacterium strains; Then will carry out the lactic bacterium strains enlarged culturing that selects after the purifying seed selection, preparation compound seed liquid; At last compound seed liquid inoculation fermentation is namely got compound lactic acid bacterium probiotics.
Further, the lactic bacterium strains that selects of described purifying is four strains.
Wherein, described lactic bacterium strains carries out the MRS liquid nutrient medium that purifying adopts sterilization, carries out enrichment culture, and the bacterium liquid of enrichment is applied on the MRS inclined-plane, isolate the some strains of bacterial strain that molten calcium circle is large on MRS solid medium calciferous, through stand-by after twice separation and purification; Some strains of lactic acid bacteria bacterial strains after separation and purification are inoculated in respectively in the MRS liquid nutrient medium of sterilization, draw bacterium liquid centrifugal 10-15min under 5000rpm after cultivating 46-50 hour at 30 ℃ of-37 ℃ of temperature, abandoning supernatant keeps thalline, inject 95% physiological saline in the thalline and obtain diluting bacterium liquid, then measure dilution bacterium liquid OD with ultraviolet spectrophotometer
543mnAbsorbancy, when the absorbance of all dilution bacterium liquid is identical, when generally absorbancy is between 0.8-1.2, be inoculated in all dilution bacterium liquid respectively in the MRS liquid nutrient medium of sterilization by identical volume ratio 3-5%, then standing cultivation 24-48h under 30 ℃-37 ℃ measures with ultraviolet spectrophotometer the OD that dilutes bacterium liquid
543nm, select the OD of 8-12 strain
543nmThe bacterial strain that measured value is high, this OD
543nmMeasured value is between 0.5-1.0; Then the nutrient solution of these bacterial strains was at room temperature continued to place 10 days, carry out plate count with the MRS solid medium afterwards, incubation time is 24-48h, temperature is 30 ℃-37 ℃, select the high bacterial strain of count results, finally select four strains of lactic acid bacteria bacterial strains conservation respectively, the viable count of general count results is 10
9CFU/mL can carry out conservation.
The weight part of above-mentioned MRS liquid nutrient medium consists of: soy peptone 0.5-1.5 part, extractum carnis 0.5-1.5 part, yeast soaks powder 0.2-0.6 part, glucose 1.5-2.5 part, dipotassium hydrogen phosphate 0.1-0.3 part, sodium acetate 0.3-0.7 part, Triammonium citrate 0.1-0.3 part, sal epsom 0.05-0.06 part, manganous sulfate 0.02-0.03 part, tween 0.05-0.15 part, distilled water 99-101 part.
Above-mentioned MRS solid medium calciferous is that the weight by distilled water adds the agar powder of 1.5-1.8% and the calcium carbonate of 1-1.2% in the MRS liquid nutrient medium.
Wherein, the described four strains of lactic acid bacteria bacterial strain enlarged culturing selected after the purifying seed selection of carrying out, preparation compound seed liquid is: the four different lactic bacterium strains in strain source are carried out plate streaking, carrying out plate streaking substratum used is the MRS solid medium, being cooled to 44-46 ℃ after sterilization pours in flat board in super clean bench, carry out flat board coating counting by 10 times of gradient dilution methods after dull and stereotyped cooled and solidified, and observe miscellaneous bacteria.Carry out enlarged culturing in the MRS liquid nutrient medium of picking list bacterium colony access sterilization, cultivate 36h-48h under 30 ℃-37 ℃, then with four kinds of nutrient solutions ratio 10% by volume, 30%, 30%, 30% mixes, and then is inoculated in the MRS liquid nutrient medium of sterilization, inoculum size is 5-20%, obtains compound seed liquid at 30 ℃-37 ℃ lower mixed culture 24-48h.
Wherein, describedly with compound seed liquid inoculation fermentation be: compound seed liquid is inoculated in liquid fermentation medium, be sealed in the room temperature bottom fermentation 3-5 days after stirring, stir twice every day in the sealing and fermenting process, and release gas, sample 1-2 time during the fermentation simultaneously, observe growth and the miscellaneous bacteria rate of thalline with colony counting method, and measure the pH value, when viable count arrives 5 * 10
8CFU/mL, miscellaneous bacteria rate＜=5%, the pH value stops fermentation under the condition of 3.0-4.0, namely get compound lactic acid bacterium probiotics.
The weight part of above-mentioned liquid fermentation medium consists of: sucrose 0.5-1 part, yeast extract paste 0.2-0.5 part, sodium acetate 0.2-0.5 part, dipotassium hydrogen phosphate 0.05-1 part, Triammonium citrate 0.1-0.5 part, sal epsom 0.058-0.065 part, manganous sulfate 0.020-0.025 part, tween 0.05-0.1 part, distilled water 99-101 part.
Further, the weight part of above-mentioned liquid fermentation medium consists of: 0.8 part of sucrose, 0.4 part of yeast extract paste, 0.3 part of sodium acetate, 0.5 part of dipotassium hydrogen phosphate, 0.3 part of Triammonium citrate, 0.06 part, sal epsom, 0.023 part of manganous sulfate, 0.08 part of tween, 100 parts of distilled water.
The preparation method of aforesaid liquid fermention medium is: yeast extract paste is added boiling in tap water in proportion, opens rear continuation boiling 5 minutes until water, after add remaining composition, pour in the plastic tank of sterilization cooling stand-by after dissolve complete.
Compared with prior art, the invention has the beneficial effects as follows:
The present invention is screening lactobacillus from aquatic animal enteron aisle, aquaculture water and bed mud, by purifying constantly, screen the speed of growth high, acid producing ability is strong, and composite fermentation is carried out in the multi-strain bacteria strain of thermal adaptation wide ranges, fermented liquid contains than the single strain more meta-bolites of fermentation and flavour substances, by composite fermentation, interact between bacterial strain, the bacterium liquid stability of formation is higher, active better, the storage time is longer.Find by culture experiment, the composite fermentation liquid spice has food calling effect preferably to fish, improves simultaneously the immunizing power of cultivated animals.In addition, with this compound lactobacillus fermented liquid aquaculture water of splashing, discovery improves significantly to the water-quality guideline tool, the reduction amplitude of ammonia nitrogen of composite fermentation liquid of splashing is 35.56%, and the ammonia nitrogen of control group has increased by 45.49%, and the reduction amplitude of the nitrite of the composite fermentation liquid of splashing is 2 times of control group.
(1) adopt by different composite the forming of milk-acid bacteria of multiple different sources form, formed have diversity, complexity and stable milk-acid bacteria group, each bacterial strain can mutual benefit coexist, have complementary advantages, collaborative playing a role, powerful, use the better effects if of improving to water-quality guideline in aquaculture water, be that single lactic acid bacteria formulation can't be realized.
(2) characteristics of maximum of the present invention are to have adopted easy fermentation equipment, and easy fermentation equipment not only can be saved cost, and simple to operate.The present invention does not adopt now widely used fermentor tank, and has just used plastic tank to ferment, and there is no complicated fermentation procedure and operation instructions, not only can save the cost of buying fermentor tank, and does not need during the fermentation heating, has saved the electricity charge; In addition, this zymotechnique is simple to operate, professional operative knowledge is required lower, can well to cultivate the user used for all.
(3) product of the present invention is aqua product, but has the stability of the coated milk-acid bacteria product of solid and do not need spraying drying, complicated procedures of forming and the material such as coated, has saved to a certain extent cost.
(4) meta-bolites of product of the present invention is abundanter.Milk-acid bacteria is not a title on microbial taxonomy, division bacteria has hundreds of to belong to, do not belong to together even and with the meta-bolites between bacterial strain, larger difference is arranged, so co-fermentation can produce more abundant meta-bolites, the meta-bolitess such as abundant enzyme system, flavour substances, organic acid, lactobacillin make the taste of product better, and nutrition is abundanter.Abundant flavour substances and organic acid make when spice uses the food calling effect stronger, and the enzyme that enriches is the macro-molecular protein that cannot degrade of the single bacterial strain of degradable and produces more prebiotics, to the cultivated animals enteron aisle to improve effect better.
(5) shortcoming of the maximum of aqua lactic acid bacteria formulation is that product is unstable at present, and storing for a long time useful viable count can descend rapidly, causes result of use not obvious.And the milk-acid bacteria of this product interacts between bacterial strain through composite combined ferment, makes product more stable, compares with general single strain fermented liquid, and the storage time extends 30 days at least.
(6) function more comprehensively.According to data and studies show that, milk-acid bacteria is used in aquaculture has the raising immunity of organisms, promotes the cultivated animals growth, suppresses the harmful bacteria breeding, promote algal grown, reduce the inferior salt of ammonia nitrogen, improve water quality, reduce the water body organism accumulation, reduce the function of oxygen consumption, but in general single bacterial strain fermentation liquor can only be brought into play a kind of function wherein, and composite product bacterial strain is many, can comprehensively bring into play these functions.
Embodiment
The below is clearly and completely described the technical scheme in the present invention, and obviously, described embodiment is only several embodiment wherein of the present invention, rather than whole embodiment.Based on the embodiment in the present invention, those of ordinary skills belong to the scope of protection of the invention not making the every other embodiment that obtains under the creative work prerequisite.
Embodiment 1:
A kind of preparation method of compound lactic acid bacterium probiotics take cultivated animals, aquaculture water and bed mud as the bacterium source, isolates lactic bacterium strains by applying solid MRS substratum after enrichment in the MRS liquid nutrient medium; Then isolated lactic bacterium strains being carried out purifying, to select the three molten calcium circles of strain large, and growth is fast, energetic lactic bacterium strains; Then will carry out the lactic bacterium strains enlarged culturing that selects after the purifying seed selection, preparation compound seed liquid; At last compound seed liquid inoculation fermentation is namely got compound lactic acid bacterium probiotics:
(1) take cultivated animals, aquaculture water and bed mud as the bacterium source, isolate lactic bacterium strains by applying solid MRS substratum after enrichment in the MRS liquid nutrient medium;
(2) isolated lactic bacterium strains being carried out purifying, to select the three molten calcium circles of strain large, and growth is fast, energetic lactic bacterium strains:
Lactic bacterium strains carries out the MRS liquid nutrient medium that purifying adopts sterilization, carry out enrichment culture, the bacterium liquid of enrichment is applied on the MRS inclined-plane, isolates the large bacterial strain of molten calcium circle on MRS solid medium calciferous, stand-by through selecting 18 strains after twice separation and purification; 18 strains of lactic acid bacteria bacterial strains after separation and purification are inoculated in respectively in the MRS liquid nutrient medium of sterilization, draw 5ml bacterium liquid centrifugal 15min under 5000rpm after cultivating 46 hours at 32 ℃ of temperature, abandoning supernatant keeps thalline, inject 95% physiological saline in the thalline and obtain diluting bacterium liquid, then measure dilution bacterium liquid OD with ultraviolet spectrophotometer
543mnAbsorbancy, when the absorbance of all dilution bacterium liquid is identical, 1.1 the time, be inoculated in all dilution bacterium liquid respectively in the MRS liquid nutrient medium of sterilization by identical volume ratio 3% when absorbancy, then standing cultivation 24h under 32 ℃ measures with ultraviolet spectrophotometer the OD that dilutes bacterium liquid
543nm, select the OD of 10 strains
543nmThe bacterial strain that measured value is high, this OD
543nmMeasured value was at 0.8 o'clock; Then the nutrient solution of these bacterial strains was at room temperature continued to place 10 days, carry out plate count with the MRS solid medium afterwards, incubation time is 46h, and temperature is 30 ℃, selects the highest bacterial strain of count results, finally selects three strains of lactic acid bacteria bacterial strains conservations respectively;
Consisting of of above-mentioned MRS liquid nutrient medium used: soy peptone 1g, extractum carnis 1g, yeast soak powder 0.5g, glucose 2g, dipotassium hydrogen phosphate 0.2g, sodium acetate 0.5g, Triammonium citrate 0.2g, sal epsom 0.058g, manganous sulfate 0.025g, tween 0.1g, distilled water 100g.
Above-mentioned MRS solid medium calciferous is to add 1.5% agar powder and 1.0% calcium carbonate by the weight of distilled water in the MRS liquid nutrient medium; That is: soy peptone 1g, extractum carnis 1g, yeast soak powder 0.5g, glucose 2g, dipotassium hydrogen phosphate 0.2g, sodium acetate 0.5g, Triammonium citrate 0.2g, sal epsom 0.058g, manganous sulfate 0.025g, tween 0.1g, distilled water 100g, agar powder 1.5g, calcium carbonate 1.0g.
(3) will carry out the lactic bacterium strains enlarged culturing that selects after the purifying seed selection, preparation compound seed liquid:
The three different lactic bacterium strains in strain source are carried out plate streaking, carrying out plate streaking substratum used is the MRS solid medium, be cooled to 45 ℃ after sterilization and pour in super clean bench in flat board, carry out flat board coating counting by 10 times of gradient dilution methods after dull and stereotyped cooled and solidified, and observe miscellaneous bacteria.Carry out enlarged culturing in the MRS liquid nutrient medium of picking list bacterium colony access sterilization, cultivate 36h under 32 ℃, then with three kinds of nutrient solutions ratio 20% by volume, 40%, 40% mixes, then be inoculated in the MRS liquid nutrient medium of sterilization, inoculum size is 10%, obtains compound seed liquid at 36 ℃ of lower mixed culture 46h;
(4) compound seed liquid inoculation fermentation is namely got compound lactic acid bacterium probiotics:
Compound seed liquid is inoculated in liquid fermentation medium, be sealed in the room temperature bottom fermentation 3.5 days after stirring, stir twice every day in the sealing and fermenting process, and release gas, sample during the fermentation 2 times simultaneously, observe growth and the miscellaneous bacteria rate of thalline with colony counting method, and measure the pH value, when viable count arrives 5 * 10
8CFU/mL, miscellaneous bacteria rate＜=5%, the pH value stops fermentation under 3.5 condition, namely get compound lactic acid bacterium probiotics.
Consisting of of above-mentioned liquid fermentation medium: sucrose 0.5g, yeast extract paste 0.3g, sodium acetate 0.2g, dipotassium hydrogen phosphate 0.08g, Triammonium citrate 0.2g, sal epsom 0.06g, manganous sulfate 0.02g, tween 0.08g, distilled water 100g.
The preparation method of aforesaid liquid fermention medium is: first yeast extract paste is added boiling in tap water, opens rear continuation boiling 5 minutes until water, after add remaining composition, pour in the plastic tank of sterilization cooling stand-by after dissolve complete.
Embodiment 2:
A kind of preparation method of compound lactic acid bacterium probiotics take cultivated animals, aquaculture water and bed mud as the bacterium source, isolates lactic bacterium strains by applying solid MRS substratum after enrichment in the MRS liquid nutrient medium; Then isolated lactic bacterium strains being carried out purifying, to select the four molten calcium circles of strain large, and growth is fast, energetic lactic bacterium strains; Then will carry out the lactic bacterium strains enlarged culturing that selects after the purifying seed selection, preparation compound seed liquid; At last compound seed liquid inoculation fermentation is namely got compound lactic acid bacterium probiotics:
(1) take cultivated animals, aquaculture water and bed mud as the bacterium source, isolate lactic bacterium strains by applying solid MRS substratum after enrichment in the MRS liquid nutrient medium;
(2) isolated lactic bacterium strains being carried out purifying, to select the four molten calcium circles of strain large, and growth is fast, energetic lactic bacterium strains:
Lactic bacterium strains carries out the MRS liquid nutrient medium that purifying adopts sterilization, carry out enrichment culture, the bacterium liquid of enrichment is applied on the MRS inclined-plane, isolates the large bacterial strain of molten calcium circle on MRS solid medium calciferous, stand-by through selecting 20 strains after twice separation and purification; 20 strains of lactic acid bacteria bacterial strains after separation and purification are inoculated in respectively in the MRS liquid nutrient medium of sterilization, draw 5ml bacterium liquid centrifugal 10min under 5000rpm after cultivating 48 hours at 37 ℃ of temperature, abandoning supernatant keeps thalline, inject 95% physiological saline in the thalline and obtain diluting bacterium liquid, then measure dilution bacterium liquid OD with ultraviolet spectrophotometer
543mnAbsorbancy, when the absorbance of all dilution bacterium liquid is identical, 0.8 the time, be inoculated in all dilution bacterium liquid respectively in the MRS liquid nutrient medium of sterilization by identical volume ratio 5% when absorbancy, then standing cultivation 48h under 36 ℃ measures with ultraviolet spectrophotometer the OD that dilutes bacterium liquid
543nm, select the OD of 12 strains
543nmThe bacterial strain that measured value is high, this OD
543nmMeasured value was at 0.6 o'clock; Then the nutrient solution of these bacterial strains was at room temperature continued to place 10 days, carry out plate count with the MRS solid medium afterwards, incubation time is 28h, and temperature is 35 ℃, selects the highest bacterial strain of count results, finally selects four strains of lactic acid bacteria bacterial strains conservations respectively;
Consisting of of above-mentioned MRS liquid nutrient medium used: soy peptone 1.5g, extractum carnis 0.5g, yeast soak powder 0.3g, glucose 1.5g, dipotassium hydrogen phosphate 0.1g, sodium acetate 0.3g, Triammonium citrate 0.3g, sal epsom 0.052g, manganous sulfate 0.023g, tween 0.05g, distilled water 99g.
Above-mentioned MRS solid medium calciferous is to add 1.8% agar powder and 1.1% calcium carbonate by the weight of distilled water in the MRS liquid nutrient medium; That is: soy peptone 1.5g, extractum carnis 0.5g, yeast soak powder 0.3g, glucose 1.5g, dipotassium hydrogen phosphate 0.1g, sodium acetate 0.3g, Triammonium citrate 0.3g, sal epsom 0.052g, manganous sulfate 0.023g, tween 0.05g, distilled water 99g, agar powder 1.782g, calcium carbonate 1.089g.
(3) will carry out the lactic bacterium strains enlarged culturing that selects after the purifying seed selection, preparation compound seed liquid:
The four different lactic bacterium strains in strain source are carried out plate streaking, carrying out plate streaking substratum used is the MRS solid medium, be cooled to 44 ℃ after sterilization and pour in super clean bench in flat board, carry out flat board coating counting by 10 times of gradient dilution methods after dull and stereotyped cooled and solidified, and observe miscellaneous bacteria.Carry out enlarged culturing in the MRS liquid nutrient medium of picking list bacterium colony access sterilization, cultivate 46h under 36 ℃, then with four kinds of nutrient solutions ratio 10% by volume, 30%, 30%, 30% mixes, and then is inoculated in the MRS liquid nutrient medium of sterilization, inoculum size is 15%, obtains compound seed liquid at 35 ℃ of lower mixed culture 36h;
(4) compound seed liquid inoculation fermentation is namely got compound lactic acid bacterium probiotics:
Compound seed liquid is inoculated in liquid fermentation medium, be sealed in the room temperature bottom fermentation 4 days after stirring, stir twice every day in the sealing and fermenting process, and release gas, sample during the fermentation 2 times simultaneously, observe growth and the miscellaneous bacteria rate of thalline with colony counting method, and measure the pH value, when viable count arrives 5 * 10
8CFU/mL, miscellaneous bacteria rate＜=5%, the pH value stops fermentation under 4.0 condition, namely get compound lactic acid bacterium probiotics.
Consisting of of above-mentioned liquid fermentation medium: sucrose 0.8g, yeast extract paste 0.4g, sodium acetate 0.3g, dipotassium hydrogen phosphate 0.5g, Triammonium citrate 0.3g, sal epsom 0.06g, manganous sulfate 0.023g, tween 0.08g, distilled water 100g.
The preparation method of aforesaid liquid fermention medium is: first yeast extract paste is added boiling in tap water, opens rear continuation boiling 5 minutes until water, after add remaining composition, pour in the plastic tank of sterilization cooling stand-by after dissolve complete.
Embodiment 3:
A kind of preparation method of compound lactic acid bacterium probiotics take cultivated animals, aquaculture water and bed mud as the bacterium source, isolates lactic bacterium strains by applying solid MRS substratum after enrichment in the MRS liquid nutrient medium; Then isolated lactic bacterium strains being carried out purifying, to select the five molten calcium circles of strain large, and growth is fast, energetic lactic bacterium strains; Then will carry out the lactic bacterium strains enlarged culturing that selects after the purifying seed selection, preparation compound seed liquid; At last compound seed liquid inoculation fermentation is namely got compound lactic acid bacterium probiotics:
(1) take cultivated animals, aquaculture water and bed mud as the bacterium source, isolate lactic bacterium strains by applying solid MRS substratum after enrichment in the MRS liquid nutrient medium;
(2) isolated lactic bacterium strains being carried out purifying, to select the five molten calcium circles of strain large, and growth is fast, energetic lactic bacterium strains:
Lactic bacterium strains carries out the MRS liquid nutrient medium that purifying adopts sterilization, carry out enrichment culture, the bacterium liquid of enrichment is applied on the MRS inclined-plane, isolates the large bacterial strain of molten calcium circle on MRS solid medium calciferous, stand-by through selecting 22 strains after twice separation and purification; 22 strains of lactic acid bacteria bacterial strains after separation and purification are inoculated in respectively in the MRS liquid nutrient medium of sterilization, draw 5ml bacterium liquid centrifugal 12min under 5000rpm after cultivating 50 hours at 35 ℃ of temperature, abandoning supernatant keeps thalline, inject 95% physiological saline in the thalline and obtain diluting bacterium liquid, then measure dilution bacterium liquid OD with ultraviolet spectrophotometer
543mnAbsorbancy, when the absorbance of all dilution bacterium liquid is identical, 0.9 the time, be inoculated in all dilution bacterium liquid respectively in the MRS liquid nutrient medium of sterilization by identical volume ratio 4% when absorbancy, then standing cultivation 36h under 35 ℃ measures with ultraviolet spectrophotometer the OD that dilutes bacterium liquid
543nm, select the OD of 8 strains
543nmThe bacterial strain that measured value is high, this OD
543nmMeasured value was at 1.0 o'clock; Then the nutrient solution of these bacterial strains was at room temperature continued to place 10 days, carry out plate count with the MRS solid medium afterwards, incubation time is 28h, and temperature is 35 ℃, selects the highest bacterial strain of count results, finally selects five strains of lactic acid bacteria bacterial strains conservations respectively;
Consisting of of above-mentioned MRS liquid nutrient medium used: soy peptone 0.8g, extractum carnis 0.8g, yeast soak powder 0.4g, glucose 2.5g, dipotassium hydrogen phosphate 0.1g, sodium acetate 0.7g, Triammonium citrate 0.1g, sal epsom 0.052g, manganous sulfate 0.03g, tween 0.12g, distilled water 101g.
Above-mentioned MRS solid medium calciferous is to add 1.6% agar powder and 1.1% calcium carbonate by the weight of distilled water in the MRS liquid nutrient medium; Be soy peptone 0.8g, extractum carnis 0.8g, yeast soak powder 0.4g, glucose 2.5g, dipotassium hydrogen phosphate 0.1g, sodium acetate 0.7g, Triammonium citrate 0.1g, sal epsom 0.052g, manganous sulfate 0.03g, tween 0.12g, distilled water 101g, agar powder 1.616g, calcium carbonate 1.111g.
(3) will carry out the lactic bacterium strains enlarged culturing that selects after the purifying seed selection, preparation compound seed liquid:
The five different lactic bacterium strains in strain source are carried out plate streaking, carrying out plate streaking substratum used is the MRS solid medium, be cooled to 46 ℃ after sterilization and pour in super clean bench in flat board, carry out flat board coating counting by 10 times of gradient dilution methods after dull and stereotyped cooled and solidified, and observe miscellaneous bacteria.Carry out enlarged culturing in the MRS liquid nutrient medium of picking list bacterium colony access sterilization, cultivate 40h under 36 ℃, then with five kinds of nutrient solutions ratio 12% by volume, 22%, 22%, 22%, 22% mixes, then be inoculated in the MRS liquid nutrient medium of sterilization, inoculum size is 20%, obtains compound seed liquid at 33 ℃ of lower mixed culture 24h;
(4) compound seed liquid inoculation fermentation is namely got compound lactic acid bacterium probiotics:
Compound seed liquid is inoculated in liquid fermentation medium, be sealed in the room temperature bottom fermentation 3 days after stirring, stir twice every day in the sealing and fermenting process, and release gas, sample during the fermentation 2 times simultaneously, observe growth and the miscellaneous bacteria rate of thalline with colony counting method, and measure the pH value, when viable count arrives 5 * 10
8CFU/mL, miscellaneous bacteria rate＜=5%, the pH value stops fermentation under 3.0 condition, namely get compound lactic acid bacterium probiotics.
Consisting of of above-mentioned liquid fermentation medium: sucrose 1.0g, yeast extract paste 0.5g, sodium acetate 0.5g, dipotassium hydrogen phosphate 0.8g, Triammonium citrate 0.5g, sal epsom 0.065g, manganous sulfate 0.025g, tween 0.1g, distilled water 101g.
The preparation method of aforesaid liquid fermention medium is: first yeast extract paste is added boiling in tap water, opens rear continuation boiling 5 minutes until water, after add remaining composition, pour in the plastic tank of sterilization cooling stand-by after dissolve complete.
The above is only preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. the preparation method of a compound lactic acid bacterium probiotics is characterized in that: it isolates lactic bacterium strains take cultivated animals, aquaculture water and bed mud as the bacterium source; Then isolated lactic bacterium strains being carried out purifying, to select the molten calcium circle of 2-5 strain large, and growth is fast, energetic lactic bacterium strains; Then will carry out the lactic bacterium strains enlarged culturing that selects after the purifying seed selection, preparation compound seed liquid; At last compound seed liquid inoculation fermentation is namely got compound lactic acid bacterium probiotics.
2. the preparation method of a kind of compound lactic acid bacterium probiotics according to claim 1, it is characterized in that: the lactic bacterium strains that described purifying selects is four strains.
3. the preparation method of a kind of compound lactic acid bacterium probiotics according to claim 2, it is characterized in that: described lactic bacterium strains carries out the MRS liquid nutrient medium that purifying adopts sterilization, carry out enrichment culture, the bacterium liquid of enrichment is applied on the MRS inclined-plane, isolate the some strains of bacterial strain that molten calcium circle is large on MRS solid medium calciferous, through stand-by after twice separation and purification; Some strains of lactic acid bacteria bacterial strains after separation and purification are inoculated in respectively in the MRS liquid nutrient medium of sterilization, draw bacterium liquid centrifugal 10-15min under 5000rpm after cultivating 46-50 hour at 30 ℃ of-37 ℃ of temperature, abandoning supernatant keeps thalline, inject 95% physiological saline in the thalline and obtain diluting bacterium liquid, then measure dilution bacterium liquid OD with ultraviolet spectrophotometer
543mnAbsorbancy, when the absorbance of all dilution bacterium liquid is identical, all dilution bacterium liquid is inoculated in respectively by identical volume ratio 3-5% in the MRS liquid nutrient medium of sterilization, and then standing cultivation 24-48h under 30 ℃-37 ℃ measures the OD of dilution bacterium liquid with ultraviolet spectrophotometer
543nm, the OD that selects
543nmThe bacterial strain that measured value is high; Then the nutrient solution of these bacterial strains was at room temperature continued to place 10 days, carry out plate count with the MRS solid medium afterwards, incubation time is 24-48h, and temperature is 30 ℃-37 ℃, selects the high bacterial strain of count results, finally selects 4 strains of lactic acid bacteria bacterial strains conservations respectively.
4. the preparation method of a kind of compound lactic acid bacterium probiotics according to claim 3, it is characterized in that: the weight part of described MRS liquid nutrient medium consists of: soy peptone 0.5-1.5 part, and extractum carnis 0.5-1.5 part, yeast soaks powder 0.2-0.6 part, glucose 1.5-2.5 part, dipotassium hydrogen phosphate 0.1-0.3 part, sodium acetate 0.3-0.7 part, Triammonium citrate 0.1-0.3 part, sal epsom 0.05-0.06 part, manganous sulfate 0.02-0.03 part, tween 0.05-0.15 part, distilled water 99-101 part.
5. the preparation method of a kind of compound lactic acid bacterium probiotics according to claim 4, it is characterized in that: described MRS solid medium calciferous is that the weight by distilled water adds the agar powder of 1.5-1.8% and the calcium carbonate of 1-1.2% in the MRS liquid nutrient medium.
6. the preparation method of a kind of compound lactic acid bacterium probiotics according to claim 2, it is characterized in that: the described four strains of lactic acid bacteria bacterial strain enlarged culturing selected after the purifying seed selection of carrying out, preparation compound seed liquid is: the four different lactic bacterium strains in strain source are carried out plate streaking, carry out enlarged culturing in the MRS liquid nutrient medium of picking list bacterium colony access sterilization, cultivate 36h-48h under 30 ℃-37 ℃, then with four kinds of nutrient solutions ratio 10% by volume, 30%, 30%, 30% mixes, then be inoculated in the MRS liquid nutrient medium of sterilization, inoculum size is 5-20%, obtain compound seed liquid at 30 ℃-37 ℃ lower mixed culture 24-48h.
7. the preparation method of a kind of compound lactic acid bacterium probiotics according to claim 6, it is characterized in that: described plate streaking substratum used is the MRS solid medium.
8. the preparation method of a kind of compound lactic acid bacterium probiotics according to claim 2, it is characterized in that: describedly with compound seed liquid inoculation fermentation be: compound seed liquid is inoculated in liquid fermentation medium, be sealed in the room temperature bottom fermentation 3-5 days after stirring, stir twice every day in the sealing and fermenting process, and release gas, sample 1-2 time during the fermentation simultaneously, observe growth and the miscellaneous bacteria rate of thalline with colony counting method, and measure the pH value, when viable count arrives 5 * 10
8CFU/mL, miscellaneous bacteria rate＜=5%, the pH value stops fermentation under the condition of 3.0-4.0, namely get compound lactic acid bacterium probiotics.
9. the preparation method of a kind of compound lactic acid bacterium probiotics according to claim 8, it is characterized in that: the weight part of described liquid fermentation medium consists of: sucrose 0.5-1 part, yeast extract paste 0.2-0.5 part, sodium acetate 0.2-0.5 part, dipotassium hydrogen phosphate 0.05-1 part, Triammonium citrate 0.1-0.5 part, sal epsom 0.058-0.065 part, manganous sulfate 0.020-0.025 part, tween 0.05-0.1 part, distilled water 99-101 part.
10. the preparation method of a kind of compound lactic acid bacterium probiotics according to claim 9, it is characterized in that: the weight part of described liquid fermentation medium consists of: 0.8 part of sucrose, 0.4 part of yeast extract paste, 0.3 part of sodium acetate, 0.5 part of dipotassium hydrogen phosphate, 0.3 part of Triammonium citrate, 0.06 part, sal epsom, 0.023 part of manganous sulfate, 0.08 part of tween, 100 parts of distilled water.
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