CN104004675A - Method for production of lactic acid bacteria by utilizing ethanol industrial yellow water - Google Patents
Method for production of lactic acid bacteria by utilizing ethanol industrial yellow water Download PDFInfo
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- CN104004675A CN104004675A CN201410183608.2A CN201410183608A CN104004675A CN 104004675 A CN104004675 A CN 104004675A CN 201410183608 A CN201410183608 A CN 201410183608A CN 104004675 A CN104004675 A CN 104004675A
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 174
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 100
- 241000894006 Bacteria Species 0.000 title claims abstract description 99
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 94
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 87
- 239000004310 lactic acid Substances 0.000 title claims abstract description 26
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 claims abstract 2
- 239000000706 filtrate Substances 0.000 claims description 37
- 238000000034 method Methods 0.000 claims description 34
- 230000001954 sterilising effect Effects 0.000 claims description 23
- 239000007788 liquid Substances 0.000 claims description 20
- 239000008247 solid mixture Substances 0.000 claims description 18
- 239000000047 product Substances 0.000 claims description 15
- 235000015097 nutrients Nutrition 0.000 claims description 14
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 10
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- 235000015278 beef Nutrition 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 9
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- 238000005259 measurement Methods 0.000 claims description 9
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- 239000000843 powder Substances 0.000 claims description 6
- 230000001502 supplementing effect Effects 0.000 claims description 6
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- 235000007164 Oryza sativa Nutrition 0.000 claims description 5
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 5
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- 239000001569 carbon dioxide Substances 0.000 claims description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 5
- YXVFQADLFFNVDS-UHFFFAOYSA-N diammonium citrate Chemical compound [NH4+].[NH4+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O YXVFQADLFFNVDS-UHFFFAOYSA-N 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 5
- 235000013312 flour Nutrition 0.000 claims description 5
- 235000011187 glycerol Nutrition 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 5
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 235000009566 rice Nutrition 0.000 claims description 5
- 235000020183 skimmed milk Nutrition 0.000 claims description 5
- 239000001632 sodium acetate Substances 0.000 claims description 5
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- 235000010413 sodium alginate Nutrition 0.000 claims description 5
- 229940005550 sodium alginate Drugs 0.000 claims description 5
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- 239000012138 yeast extract Substances 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 235000012041 food component Nutrition 0.000 claims description 2
- 229920000858 Cyclodextrin Polymers 0.000 claims 1
- 239000001116 FEMA 4028 Substances 0.000 claims 1
- 240000007594 Oryza sativa Species 0.000 claims 1
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims 1
- 229960004853 betadex Drugs 0.000 claims 1
- 239000012153 distilled water Substances 0.000 claims 1
- 229940099596 manganese sulfate Drugs 0.000 claims 1
- 235000007079 manganese sulphate Nutrition 0.000 claims 1
- 239000011702 manganese sulphate Substances 0.000 claims 1
- 239000003223 protective agent Substances 0.000 claims 1
- 238000004659 sterilization and disinfection Methods 0.000 claims 1
- 235000016709 nutrition Nutrition 0.000 abstract description 15
- 230000035764 nutrition Effects 0.000 abstract 1
- 239000000758 substrate Substances 0.000 abstract 1
- 230000000050 nutritive effect Effects 0.000 description 15
- 239000004615 ingredient Substances 0.000 description 12
- 241000186660 Lactobacillus Species 0.000 description 11
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- 230000009469 supplementation Effects 0.000 description 9
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
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- WHGYBXFWUBPSRW-UHFFFAOYSA-N Cycloheptaamylose Natural products O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO WHGYBXFWUBPSRW-UHFFFAOYSA-N 0.000 description 4
- 241000209094 Oryza Species 0.000 description 4
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- 239000013589 supplement Substances 0.000 description 4
- 238000009461 vacuum packaging Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
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- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
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- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 2
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- 235000021436 nutraceutical agent Nutrition 0.000 description 2
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Dairy Products (AREA)
Abstract
A method for production of lactic acid bacteria by utilizing ethanol industrial yellow water includes collection and treatment of the ethanol industrial yellow water, cultivation of the lactic acid bacteria in the ethanol industrial yellow water, and receiving and preparation of the lactic acid bacteria. The method for production of the lactic acid bacteria by utilizing the ethanol industrial yellow water not only can make full use of the ethanol industrial yellow water, reduce the treatment cost of the yellow water, optimize comprehensive utilization of the yellow water and improve the ecological environment, but also can expand sources of a nutrition substrate in a conventional lactic acid bacteria production, lower the production costs and expand the application scope of lactic acid bacteria.
Description
Technical field
The present invention relates to comprehensive utilization field and the microorganism culturing field of alcohol industry yellow water, especially utilize the yellow water in alcohol industry to carry out microorganism culturing.
Background technology
Alcohol industry is important energy industry of China, but can produce huge by product in alcohol production process, wherein the solids component such as vinasse can continue to produce the feedstuff raw materials such as DDGS and again be utilized together with part yellow water, and the yellow water of the overwhelming majority is not well utilized.Yellow water is the liquid substance that is rich in multiple nutritional components of discharging in alcohol process producing, and contains abundant organic acid (especially lactic acid in yellow water, butyric acid, caproic acid, the carboxylic acid contents such as acetic acid are maximum), alcohol, aldehyde, esters etc. are fragrance matter, also contain amino acid simultaneously, carbohydrate, alcohol, soil ulmin, yeast thalline autolyzate, microbial cells and viable cell etc., because it is nutritious, directly enter the pollution that tends to cause serious body eutrophication in environment, for this reason, the waste water of national regulation alcohol enterprise discharge must be processed through early stage, and can discharge after reaching 4 grades of water quality standards, so, nutritive ingredient in yellow water is wasted, alcohol enterprise often increases the processing of more expense for yellow water, and most of brewery of China is all to use it for mix grain time cellar for storing things fermentation or support cellar for storing things to the processing of yellow water at present, cultivate the aspect such as artificial distiller's yeast, not only can not efficent use of resources, and cannot fundamentally solve the final outlet problem of fermentation yellow water.Therefore how to fully utilize yellow water resource, how can turn waste into wealth, the outlet problem that thoroughly solves yellow water seems particularly important, if yellow water can be turned waste into wealth, fully utilize, the cost that reduces enterprise is dropped into, also meet the needs of improvement of the ecological environment simultaneously.
Milk-acid bacteria is that a class can produce lactic acid under culture condition, and produces the general name of the probiotics of a series of enzymes simultaneously, and milk-acid bacteria has formed industrial production at present, and its finished product is used widely in the industries such as medicines and health protection, food, feed.But existing milk-acid bacteria production method adopts people for adding a large amount of high-quality nutritive substances to produce as raw material more, its production cost is higher, and then affects the application of milk-acid bacteria, and increases the cost of the related products taking milk-acid bacteria as raw material.
In alcohol industry yellow water, be rich in the nutritive substance of multiple soluble state, therefore, if can utilize alcohol industry yellow water to carry out the production of milk-acid bacteria, can solve the problem of complex utilization of alcohol industry yellow water, turn waste into wealth, can reduce again current milk-acid bacteria production cost, further expand the range of application of milk-acid bacteria.
Summary of the invention
The object of this invention is to provide a kind of technological method that utilizes alcohol industry yellow water to carry out milk-acid bacteria cultivation, reduce on the one hand link and the expense of yellow water processing in alcohol industry, the comprehensive utilization ratio that improves alcohol industry yellow water, reduces the industrial cost of milk-acid bacteria on the other hand.
The technical scheme that the present invention solves the problems of the technologies described above employing is: a kind of method of utilizing alcohol industry yellow water to produce milk-acid bacteria, and concrete steps are as follows:
(1) the general absorbent gauze for alcohol industry yellow water (bilayer) of collecting is filtered, collect filtrate as milk-acid bacteria culture medium, for subsequent use;
(2) the alcohol industry yellow water filtrate of step (1) gained is carried out to the mensuration of nutritive ingredient, and carry out nutritional supplementation according to the nutritional need of measurement result and lactobacter growth breeding, for subsequent use, concrete nutritive ingredient supplements kind and quantity is: often go up in the alcohol industry yellow water filtrate of stating and add urea 2.5g, glucose 10g, tween 80 0.5mL;
(3) the alcohol industry yellow water filtrate after nutritional supplementation in step (2) is carried out to sterilizing, and by pH regulator to 6.3 ~ 6.5 of the alcohol industry yellow water filtrate after sterilizing, for subsequent use, wherein, concrete sterilising method is preferably: by pH regulator to 3.2 ~ 3.5 of the alcohol industry yellow water after supplementing the nutrients, and to injecting carbon dioxide gas in the container of splendid attire alcohol industry yellow water, sealing room temperature are placed 24h;
(4) lactic acid bacteria culturers is inoculated on MRS liquid nutrient medium, and be cultured to the logarithmic growth middle and later periods, it is for subsequent use as kind of daughter bacteria liquid when detection viable count is not less than 1 × 108 cfu/mL, wherein the making method of MRS liquid nutrient medium is: get beef protein powder 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium-acetate 5g, dibasic ammonium citrate 2g, tween 80 0.1mL, magnesium sulfate 0.58g, manganous sulfate 0. 28g, aquae destillata 1000mL mixing, at 121 DEG C of sterilizing 20min-30min, regulate pH6.3 ~ 6.5 with high-pressure sterilizing pot;
(5) according to 5%(v/v) ratio by step (4) gained kind daughter bacteria liquid be inoculated in the alcohol industry yellow water filtrate of step (3) gained, and cultivate 24 ~ 30h under the condition of 37 ~ 37.5 DEG C;
(6) in step (5), in the milk-acid bacteria of cultivation, add 2% cooked rice flour noodles according to weightmeasurement ratio, and leave standstill 5min-10min under room temperature, then adopt large capacity whizzer, centrifugal in 3000r/min, obtain milk-acid bacteria solid mixture, for subsequent use;
(7) get the milk-acid bacteria solid mixture of step (6) gained, adopt vacuum freeze-drying method to be dried, its concrete drying means is: the milk-acid bacteria solid mixture mixing is spread out into the thickness of 0.3 ~ 0.5cm, be placed in vacuum freeze drier, temperature-45 DEG C ~-50 DEG C, vacuum tightness 10 ~ 20Pa, time of drying, 22 ~ 24h, after being dried, carried out vacuum packaging with laminated film, vacuum tightness is 0.085 ~ 0.095Mpa, can obtain lactobacillus completed product.
Further; before step (7) is carried out; according to weight ratio, in the milk-acid bacteria solid mixture obtaining to step (6), add the cycloheptaamylose of 2.5% ~ 3% sodium alginate, 4.5% ~ 5% skim-milk, 5% glycerine and 8% ~ 10% as protective material, and mix.
Milk-acid bacteria used in the present invention, purchased from Bei Nuo bio tech ltd, Shanghai, is preserved in the biological product of USS collecting center, and its deposit number is ATCC 11007, and buying the date is on October 25th, 2013.
The present invention has following beneficial effect:
First, compare with nutraceutical matrix with the raw material that current milk-acid bacteria produces, the present invention adopts alcohol industry yellow water as cultivating raw material and matrix, take full advantage of alcohol industry waste water, improve the comprehensive utilization value of alcohol industry yellow water, improve ecotope, expanded the source of milk-acid bacteria production desired nutritional matrix, reduced the production cost of milk-acid bacteria;
The second, through the cultivation of milk-acid bacteria, the nutritive substance in alcohol industry yellow water is made full use of by milk-acid bacteria, its content effectively reduces, substantially reach the emission standard of alcohol waste water, and saved link and relevant input of business processes alcohol industry yellow water, reduced the production cost of relevant enterprise;
The 3rd, the present invention directly gathers alcohol industry yellow water from the sheet frame of plate-and-frame filter press, this is because the bleeding point outbalance of alcohol industry yellow water, the yellow water nutritive ingredient that the yellow water nutritive ingredient of directly collecting from the sheet frame of plate-and-frame filter press is collected compared with water port is abundant, is more suitable as culture medium;
The 4th, the present invention, measuring under the prerequisite of alcohol industry yellow water nutrient contg, carries out nutritional supplementation to yellow water targetedly according to measurement result, to be more applicable to the Growth and reproduction of milk-acid bacteria, has further improved the output of milk-acid bacteria in alcohol industry yellow water;
The 5th, for the distinctive chemical constitution of alcohol industry yellow water, carry out the change of suitable pre-treatment and part chemical property, make its Growth and reproduction that is more suitable for milk-acid bacteria, improve the output of milk-acid bacteria;
The 6th, for the distinctive chemical property of alcohol industry yellow water, adopt the method for pH regulator and carbonic acid gas sealing to carry out sterilizing, simple to operate, sterilising effect is better, the carrying out that suitable batch production is produced;
The 7th, in milk-acid bacteria solid mixture freezing dry process, add corresponding protective material, can effectively preserve milk-acid bacteria, make its survival rate reach 85.3%.
Therefore, the method of utilizing alcohol industry yellow water to produce milk-acid bacteria provided by the present invention, can make full use of alcohol industry yellow water, reduce the processing cost of yellow water, optimize the comprehensive utilization of yellow water, improve the ecological environment, can expand again the source of nutraceutical matrix in existing milk-acid bacteria production, reduce production costs, expand the range of application of milk-acid bacteria.
Brief description of the drawings
Fig. 1 is the content of various nutritive ingredients in alcohol industry yellow water.
Fig. 2 is the impacts of different culture temperature on viable count in lactobacillus completed product.
Fig. 3 is the impacts of different incubation times on viable count in lactobacillus completed product.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated.
Embodiment 1: a kind of method of utilizing alcohol industry yellow water to produce milk-acid bacteria, what present method adopted is that corn is the yellow water of raw material production alcohol, concrete steps are as follows:
(1) the general absorbent gauze for alcohol industry yellow water (bilayer) of collecting is filtered, collect filtrate as milk-acid bacteria culture medium, for subsequent use;
(2) filtrate of step (1) gained is carried out to the mensuration of nutritive ingredient, its measurement result is shown in accompanying drawing 1, and carry out nutritional supplementation according to the nutritional need of measurement result and lactobacter growth breeding, concrete nutritive ingredient supplements kind and quantity is: often go up and in the alcohol industry yellow water of stating, add urea 2.5g, glucose 10g, tween 80 0.5mL;
(3) the alcohol industry yellow water filtrate after nutritional supplementation in step (2) is carried out to sterilizing, and by pH regulator to 6.3 ~ 6.5 of the alcohol industry yellow water filtrate after sterilizing, for subsequent use, wherein, concrete sterilising method is: by pH regulator to 3.2 ~ 3.5 of the alcohol industry yellow water filtrate after supplementing the nutrients, and to injecting carbon dioxide gas in the container of splendid attire alcohol industry yellow water filtrate, sealing room temperature are placed 24h;
(4) lactic acid bacteria culturers is inoculated on MRS liquid nutrient medium, and be cultured to the logarithmic growth middle and later periods, it is for subsequent use as kind of daughter bacteria liquid when detection viable count is not less than 1 × 108 cfu/mL, wherein the making method of MRS liquid nutrient medium is: get beef protein powder 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium-acetate 5g, dibasic ammonium citrate 2g, tween 80 0.1mL, magnesium sulfate 0.58g, manganous sulfate 0. 28g, aquae destillata 1000mL mixing, at 121 DEG C of sterilizing 20min-30min, regulate pH6.3 ~ 6.5 with high-pressure sterilizing pot;
(5) according to 5%(v/v) ratio by step (4) gained kind daughter bacteria liquid be inoculated in the alcohol industry yellow water filtrate of step (3) gained, and cultivate 24h under the condition of 37 DEG C;
(6) in step (5), in the milk-acid bacteria of cultivation, add 2% cooked rice flour noodles according to weightmeasurement ratio, and leave standstill 5min under room temperature, then adopt large capacity whizzer, centrifugal in 3000r/min, obtain milk-acid bacteria solid mixture, for subsequent use;
(7) according to the weight of the milk-acid bacteria solid mixture of step (6) gained, in milk-acid bacteria solid mixture, add 2.5% ~ 3% sodium alginate according to weight ratio, 4.5% ~ 5% skim-milk, 5% glycerine and 8% ~ 10% cycloheptaamylose are as protective material, and mix, then spread out into the thickness of 0.3 ~ 0.5cm, be placed in vacuum freeze drier, in temperature-45 DEG C ~-50 DEG C, under the condition of vacuum tightness 10 ~ 20Pa, carry out lyophilize, time of drying 22 ~ 24h, after being dried, carry out vacuum packaging with laminated film, vacuum tightness is 0.085 ~ 0.095Mpa, can obtain lactobacillus completed product.
After measured, adopting number of viable in the method gained lactobacillus completed product in the present embodiment is (95.7 ± 6) × 10
7.
Embodiment 2: a kind of method of utilizing alcohol industry yellow water to produce milk-acid bacteria, what present method adopted is that corn is the yellow water of raw material production alcohol, concrete steps are as follows:
(1) the general absorbent gauze for alcohol industry yellow water (bilayer) of collecting is filtered, collect filtrate as milk-acid bacteria culture medium, for subsequent use;
(2) filtrate of step (1) gained is carried out to the mensuration of nutritive ingredient, its measurement result is shown in accompanying drawing 1, and carry out nutritional supplementation according to the nutritional need of measurement result and lactobacter growth breeding, concrete nutritive ingredient supplements kind and quantity is: often go up in the alcohol industry yellow water filtrate of stating and add urea 2.5g, glucose 10g, tween 80 0.5mL;
(3) the alcohol industry yellow water filtrate after nutritional supplementation in step (2) is carried out to sterilizing, and by pH regulator to 6.3 ~ 6.5 of the alcohol industry yellow water filtrate after sterilizing, for subsequent use, wherein, concrete sterilising method is: by pH regulator to 3.2 ~ 3.5 of the alcohol industry yellow water filtrate after supplementing the nutrients, and to injecting carbon dioxide gas in the container of splendid attire alcohol industry yellow water filtrate, sealing room temperature are placed 24h;
(4) lactic acid bacteria culturers is inoculated on MRS liquid nutrient medium, and be cultured to the logarithmic growth middle and later periods, it is for subsequent use as kind of daughter bacteria liquid when detection viable count is not less than 1 × 108 cfu/mL, wherein the making method of MRS liquid nutrient medium is: get beef protein powder 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium-acetate 5g, dibasic ammonium citrate 2g, tween 80 0.1mL, magnesium sulfate 0.58g, manganous sulfate 0. 28g, aquae destillata 1000mL mixing, at 121 DEG C of sterilizing 20min-30min, regulate pH6.3 ~ 6.5 with high-pressure sterilizing pot;
(5) according to 5%(v/v) ratio by step (4) gained kind daughter bacteria liquid be inoculated in the alcohol industry yellow water filtrate of step (3) gained, and cultivate 30h under the condition of 37.5 DEG C;
(6) in step (5), in the milk-acid bacteria of cultivation, add 2% cooked rice flour noodles according to weightmeasurement ratio, and leave standstill 10min under room temperature, then adopt large capacity whizzer, centrifugal in 3000r/min, obtain milk-acid bacteria solid mixture, for subsequent use;
(7) according to the weight of the milk-acid bacteria solid mixture of step (6) gained, in milk-acid bacteria solid mixture, add 2.5% ~ 3% sodium alginate according to weight ratio, 4.5% ~ 5% skim-milk, 5% glycerine and 8% ~ 10% cycloheptaamylose are as protective material, and mix, then spread out into the thickness of 0.3 ~ 0.5cm, be placed in vacuum freeze drier, in temperature-45 DEG C ~-50 DEG C, under the condition of vacuum tightness 10 ~ 20Pa, carry out lyophilize, time of drying 22 ~ 24h, after being dried, carry out vacuum packaging with laminated film, vacuum tightness is 0.085 ~ 0.095Mpa, can obtain lactobacillus completed product.
After measured, adopting number of viable in the method gained lactobacillus completed product in the present embodiment is (98.5 ± 9) × 10
7.
Embodiment 3: a kind of method of utilizing alcohol industry yellow water to produce milk-acid bacteria, what present method adopted is that corn is the yellow water of raw material production alcohol, concrete steps are as follows:
(1) the general absorbent gauze for alcohol industry yellow water (bilayer) of collecting is filtered, collect filtrate as milk-acid bacteria culture medium, for subsequent use;
(2) filtrate of step (1) gained is carried out to the mensuration of nutritive ingredient, its measurement result is shown in accompanying drawing 1, and carry out nutritional supplementation according to the nutritional need of measurement result and lactobacter growth breeding, concrete nutritive ingredient supplements kind and quantity is: often go up in the alcohol industry yellow water filtrate of stating and add urea 2.5g, glucose 10g, tween 80 0.5mL;
(3) the alcohol industry yellow water filtrate after nutritional supplementation in step (2) is carried out to sterilizing, and by pH regulator to 6.3 ~ 6.5 of the alcohol industry yellow water filtrate after sterilizing, for subsequent use, wherein, concrete sterilising method is: by pH regulator to 3.2 ~ 3.5 of the alcohol industry yellow water filtrate after supplementing the nutrients, and to injecting carbon dioxide gas in the container of splendid attire alcohol industry yellow water filtrate, sealing room temperature are placed 24h;
(4) lactic acid bacteria culturers is inoculated on MRS liquid nutrient medium, and be cultured to the logarithmic growth middle and later periods, it is for subsequent use as kind of daughter bacteria liquid when detection viable count is not less than 1 × 108 cfu/mL, wherein the making method of MRS liquid nutrient medium is: get beef protein powder 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium-acetate 5g, dibasic ammonium citrate 2g, tween 80 0.1mL, magnesium sulfate 0.58g, manganous sulfate 0. 28g, aquae destillata 1000mL mixing, at 121 DEG C of sterilizing 20min-30min, regulate pH6.3 ~ 6.5 with high-pressure sterilizing pot;
(5) according to 5%(v/v) ratio by step (4) gained kind daughter bacteria liquid be inoculated in the alcohol industry yellow water filtrate of step (3) gained, and cultivate 27h under the condition of 37.2 DEG C;
(6) in step (5), in the milk-acid bacteria of cultivation, add 2% cooked rice flour noodles according to weightmeasurement ratio, and leave standstill 7min under room temperature, then adopt large capacity whizzer, centrifugal in 3000r/min, obtain milk-acid bacteria solid mixture, for subsequent use;
(7) according to the weight of the milk-acid bacteria solid mixture of step (6) gained, in milk-acid bacteria solid mixture, add 2.5% ~ 3% sodium alginate according to weight ratio, 4.5% ~ 5% skim-milk, 5% glycerine and 8% ~ 10% cycloheptaamylose are as protective material, and mix, then spread out into the thickness of 0.3 ~ 0.5cm, be placed in vacuum freeze drier, in temperature-45 DEG C ~-50 DEG C, under the condition of vacuum tightness 10 ~ 20Pa, carry out lyophilize, time of drying 22 ~ 24h, after being dried, carry out vacuum packaging with laminated film, vacuum tightness is 0.085 ~ 0.095Mpa, can obtain lactobacillus completed product.
After measured, adopting number of viable in the method gained lactobacillus completed product in the present embodiment is (104.3 ± 7) × 10
7.
Below in conjunction with test example, the present invention is further illustrated.
Test example 1: inquire into and adopt method provided by the present invention to carry out milk-acid bacteria cultivation, the impact of different culture temperature on the quantity of viable bacteria in final lactobacillus completed product, and find out optimum culture temperature.
In the cultivation of microorganism, the impact of temperature is most important, directly has influence on the success or not of microorganism culturing, and whether final colony counts meets the requirements.According to method provided by the invention, adopt single factor design, culture temperature is set to respectively to the differing temps gradient of 36 DEG C, 36.5 DEG C, 36.8 DEG C, 37 DEG C, 37.2 DEG C, 37.5 DEG C, 38 DEG C and 40 DEG C, carries out the cultivation of milk-acid bacteria, test-results is shown in accompanying drawing 2.
By finding out in accompanying drawing 2, while carrying out the cultivation of milk-acid bacteria according to method of the present invention, when culture temperature is during at 37 DEG C to 37.5 DEG C, in milk-acid bacteria, viable count is higher, especially 37.2 DEG C time, viable count is the highest, and therefore, 37.2 DEG C is to adopt method of the present invention to cultivate the optimum culture temperature of milk-acid bacteria.
Test example 2: inquire into and adopt method provided by the present invention to carry out milk-acid bacteria cultivation, the impact of different incubation times on the quantity of viable bacteria in final lactobacillus completed product, and find out optimum incubation time.
The incubation time of milk-acid bacteria is unsuitable long, because overlong time, milk-acid bacteria there will be autolysis, causes bacterial number sharply to decline, and therefore, suitable incubation time is most important.According to method of the present invention, adopt single factor design, incubation time is set to respectively to 12h, 18h, 24h, 30h, 36h and 48h, to cultivate, and measure the quantity of viable bacteria in different incubation time gained milk-acid bacterias, it the results are shown in accompanying drawing 3.
From accompanying drawing 3, can find out, when incubation time is 24h ~ 30h, in milk-acid bacteria, number of viable is more, and in the time that incubation time is 30h, it is maximum that its viable count reaches, and therefore according to method of the present invention, optimum incubation time is 30h.
Claims (6)
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| CN104543402A (en) * | 2015-01-20 | 2015-04-29 | 河南科技大学 | Preparation method of fermentation-state iron feed additive |
| CN104543400A (en) * | 2015-01-20 | 2015-04-29 | 河南科技大学 | Preparation method of fermentation state zinc feed additive |
| CN104543398A (en) * | 2015-01-20 | 2015-04-29 | 河南科技大学 | Preparation method of fermentation state copper feed additive |
| CN104543400B (en) * | 2015-01-20 | 2018-04-20 | 河南科技大学 | A kind of preparation method for the state zinc feed additive that ferments |
| CN106497732A (en) * | 2016-12-08 | 2017-03-15 | 彭万洪 | A kind of method that yellow water safeguards pit mud |
| CN106591041A (en) * | 2016-12-08 | 2017-04-26 | 彭万洪 | Yellow water reutilization method |
| CN106665837A (en) * | 2016-12-16 | 2017-05-17 | 常州市鼎日环保科技有限公司 | Preparation method of high-activity ready-to-use yoghurt starter |
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| CN108865922A (en) * | 2018-05-02 | 2018-11-23 | 山东出入境检验检疫局检验检疫技术中心 | listeria monocytogenes standard sample and preparation method thereof |
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