CN101475923A - Method for high-density cultivation for lactobacillus by using bone collagen polypeptide powder - Google Patents
Method for high-density cultivation for lactobacillus by using bone collagen polypeptide powder Download PDFInfo
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- CN101475923A CN101475923A CNA2009100425477A CN200910042547A CN101475923A CN 101475923 A CN101475923 A CN 101475923A CN A2009100425477 A CNA2009100425477 A CN A2009100425477A CN 200910042547 A CN200910042547 A CN 200910042547A CN 101475923 A CN101475923 A CN 101475923A
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Abstract
The invention discloses a process for using bone collagen polypeptide powder to perform high-density culture to the lactic acid bacteria. The method takes the lactic acid bacteria as bacteria. The method includes: firstly suing the lactic acid bacteria basic medium to perform activating and expanding culture to the bacterial, and then inoculating to the fermentation medium for fermentation culture 12-18h, collecting the thalli and adding it in the protein protective agent for dissolving, and finally obtaining the completed product by vacuum freezing and drying. Bone collagen polypeptides powder is added in the fermentation medium. The inventive culture method has advantages of low cost, simple operation, etc., and can obtain lactobacillus completed product with high activity and high concentrations.
Description
Technical field
The present invention relates to a kind of method for culturing microbes, be specifically related to the high-density cultivation method of a kind of milk-acid bacteria.
Background technology
Milk-acid bacteria is can ferment lactose, product is based on the general designation of a big bacterioid of lactic acid, they mainly are distributed in the enteron aisle of people and animal, controlling the colony balance of human intestinal with bifidus bacillus, remove the toxic substance in the enteron aisle, resist pathogenic bacterium in the enteron aisle to its host's invasion and attack, common pathogenic bacterium (as dysentery bacterium, Corynebacterium diphtheriae, pathogenic colon bacillus, staphylococcus etc.) are had antagonistic action.In addition, milk-acid bacteria also have anti-treat constipation, the effect of diarrhea and gastrointestinal disorders, it produces a large amount of lactic acid, can promote intestines wall wriggling, help digest, material such as synthesise vitamins, amino acid in enteron aisle, simultaneously the absorption of human body be can help, but also children's brain and neural growth helped nutrient substances such as calcium, phosphorus, iron ions.Therefore, be that the beverage and the food formulation of main raw material all has very vast market at home and abroad with the milk-acid bacteria.
Although the market demand amount of lactobacillus product is very big, but milk-acid bacteria is a class entero-bacte, under situation about exsomatizing, the cultural method of milk-acid bacteria still is the technological difficulties that those skilled in the art faced, be that lactic acid bacterium number is not high in the unit nutrient solution of milk-acid bacteria, be difficult to reach 109CFU/mL.If the production lactobacillus product then needs a large amount of culture space and time.At present, both at home and abroad all in the high-density culture technology of inquiring into milk-acid bacteria, the method that is adopted has carries out that controlled atmosphere cultivates, and has to add somatomedins such as amino acid, also has to adopt the material that adds oligosaccharides or tea-polyphenol class to cultivate.Utilize above-mentioned technology to cultivate, have problems such as cost is big, operation difficulty, apply then more apparent being difficult at food enterprise.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, provides a kind of cost bone collagen polypeptide powder that utilizes low, simple to operate, that can obtain high reactivity, high density milk-acid bacteria milk-acid bacteria to be carried out the method for high-density culture.
For solving the problems of the technologies described above, the technical scheme that the present invention proposes is a kind of method of utilizing bone collagen polypeptide powder milk-acid bacteria to be carried out high-density culture, this method is to be bacterial classification with milk-acid bacteria (for example lactobacillus sake Lactobacillus Sake), at first adopt the milk-acid bacteria basic medium that bacterial classification is activated, enlarged culturing, inoculate (generally by 3% inoculum size) fermentation culture 12～18h (preferred 14h) in the fermention medium, collect thalline and join in the protein protective agent and dissolve, after vacuum freezing (vacuum tightness＜20Pa generally speaking, temperature-40 ℃～-50 ℃), the dry finished product that obtains; Be added with bone collagen polypeptide powder in the described fermention medium.
In the technique scheme, described milk-acid bacteria can select for use wherein a kind of lactobacillus sake as the indication bacterial classification, this lactobacillus sake can separate acquisition in fermented meat, utilize the growth of this bacterium to represent the growing state of milk-acid bacteria in cultural method of the present invention, the milk-acid bacteria that can certainly select other kinds for use is as the indication bacterial classification.
In the technique scheme, each component and the massfraction thereof of described fermention medium are: bone collagen polypeptide powder 0.5～1.5%, yeast extract paste 0.5%, glucose 0.5%, KH
2PO
40.3%, tween 80 0.5%, tomato juice 5%, sodium acetate 0.5%, the water of Trisodium Citrate 0.2% and surplus.
In the technique scheme, the pH value of described fermention medium is controlled at 6.4～6.8, the initial pH value that is fermention medium is controlled at 6.4～6.8, and adding alkali (for example NaOH of 2mol/L) by stream in culturing process is 6.4～6.8 as neutralizing agent with the pH value of controlling fermented liquid; Temperature during described fermentation culture is controlled at 35～40 ℃ (preferred 37 ℃).
In the technique scheme; the component of described protein protective agent and massfraction thereof are: the water of skimming milk 10%, glycerine 2%, trehalose 2% and surplus; described protein protective agent is mainly used in the thalline that dissolving is collected, and the thalline of described collection is 1: 4 with the mass ratio of the protein protective agent of dissolving thalline.
In the technique scheme, described milk-acid bacteria basic medium is formulated by the component of following mass fraction: 10 parts of beef protein powder, 10 parts of flesh of fish juice, 5 parts of Yeast diffusion juice powder, 20 parts of glucose, 5 parts of sodium-acetates, 2 parts of dibasic ammonium citrates, 0.1 part of tween 80,0.58 part of sal epsom, 0.28 part of manganous sulfate and 1000 parts of aquae destillatas, the pH value of described milk-acid bacteria basic medium is controlled at 6.2～6.4.
In the technique scheme, described bone collagen polypeptide powder specifically is to make by the following method: get Fresh Os Sus domestica and clean, first broken back boiling water boiling drains the bone piece and drying behind the grease of removal cooking liquor top layer; Again the bone piece is carried out coarse reduction, micronizing is made bone meal, adds water bone meal is prepared skeletonization mud liquid, high-temperature sterilization (15min sterilizes under 85 ℃ of temperature) postcooling (naturally cooling to below 50 ℃) also adds the Protamex compound protease and carries out enzymolysis, enzyme goes out; Get filtered liquid vacuum concentration and lyophilize after the filtration, obtain bone collagen polypeptide powder.In the preparation process of bone collagen polypeptide powder, the mass ratio of bone piece and water is 1: 1～1: 2 during the boiling water boiling, and the temperature during the boiling water boiling is controlled at 110 ℃～120 ℃, and pressure is controlled at 143kPa～198kPa, and the time is controlled at 30～60min.The addition of described Protamex compound protease is 2～2.5 ‰ of an enzymolysis substrate bone meal quality, and the pH value during described Protamex compound protease enzymolysis is 7.5, and temperature is controlled at 55 ℃, and enzymolysis time is 10～15h.Temperature when the bone piece is carried out drying is controlled at 80～85 ℃, and is dried to water content less than 5%; When the bone piece was carried out coarse crushing, micronizing, described bone piece was ground into the granularity of bone meal less than 160 orders; The mass concentration of described bone mud liquid is 10～15%.
Compared with prior art, the invention has the advantages that: the present invention adopts bone collagen polypeptide powder as additive, need not to add again under the situation of other materials, can effectively prolong the logarithmic phase of lactobacillus sake by cultural method of the present invention, (wherein the lactobacillus sake viable count reaches 2 * 10 to obtain the bacteria suspension of high reactivity, high density
9～9 * 10
9CFU/mL), the bacteria suspension that makes is carried out centrifugal collection and add composite protectant after can make the starter of dry powder type.If microbial numbers is controlled to be 10 in the dry powder leaven
10CFU/g, then 10mL fermention medium of the present invention can be prepared the 1g dry powder leaven.If adopt common culture method to cultivate milk-acid bacteria, it is 10 that the microbe population in the final fermented liquid generally has only
8CFU/mL, and prepare the dry powder leaven of milk-acid bacteria with common cultural method, then need to use a large amount of fermentation culture (approach cultural method nutrient solution consumption of the present invention 10 times), and need take bigger fermenting space, also must use jumbo fermentor tank in the production.As seen, cultural method of the present invention is by utilizing a spot of microbial fermentation nutrient solution, in the small-sized fermentation jar, can finish the high-density culture of milk-acid bacteria, on production cost, significantly reduced the use of nutritive ingredient, reduced the heat exhaustion in the fermenting process, simultaneously can obtain the higher lactobacillus product of concentration, be the milk-acid bacteria high-density cultivation method that a kind of cost is little, operation is easy, effective.
Embodiment
Embodiment 1:
A kind of method of utilizing bone collagen polypeptide powder milk-acid bacteria to be carried out high-density culture; this method is to be bacterial classification with lactobacillus sake (Lactobacillus Sake); at first adopt milk-acid bacteria basic medium (MRS substratum) that lactobacillus sake is activated; enlarged culturing; be inoculated in the fermention medium by 3% inoculum size again; cultivated 14 hours at 37 ℃ of temperature bottom fermentations; the NaOH solution control pH value that adds 2mol/L by stream in the fermentation culture process is 6.8; cultivate and finish the centrifugal collection thalline in back and join dissolving in the protein protective agent (thalline of collection is 1: 4 with the mass ratio of the protein protective agent of dissolving thalline), after vacuum freezing; the dry dry powder type lactobacillus starter that obtains.
The component and the massfraction of above-mentioned fermention medium are as follows: bone collagen polypeptide powder 0.5%, yeast extract paste 0.5%, glucose 0.5%, KH
2PO
40.3%, tween 80 0.5%, tomato juice 5%, sodium acetate 0.5%, the water of Trisodium Citrate 0.2% and surplus, regulating initial pH value is 6.8.
The prescription of the above-mentioned MRS substratum of using is as follows: beef protein powder 10g, flesh of fish juice 10g, Yeast diffusion juice powder 5g, glucose 20g, sodium-acetate 5g, dibasic ammonium citrate 2g, tween 80 0.1g, sal epsom 0.58g, manganous sulfate 0.28g, aquae destillata 1000g, the pH value is controlled at 6.2～6.4.
The component of the above-mentioned protein protective agent of using and massfraction thereof are: the water of skimming milk 10%, glycerine 2%, trehalose 2% and surplus.
The above-mentioned bone collagen polypeptide powder of using is to prepare by the following method: get Fresh Os Sus domestica and clean the preliminary fragmentation in back, bone piece after the fragmentation is put into steamer vessel, add water in the container with quality such as bone piece, boiling 40min under 115 ℃ of temperature, 143kPa pressure, remove the upper strata grease then, drain, chopped obtains the bone piece of 3～4cm, dry 7h under 85 ℃ of temperature, after the drying with skeletal grain through the pulverizer coarse reduction, carry out micronizing through supper micron mill again, the bone meal after the micronizing is crossed 160 mesh sieves; Bone meal after sieving is added bone mud liquid that water is mixed with 10% mass concentration packs in the container, 15min sterilizes in 85 ℃ of water-baths, be cooled to again below 40 ℃, regulate pH value to 7.5, the Protamex compound protease (Denmark Novozymes Company produce, the mark vigor is 1.5AU/g (Anson unit/gram)) that adds enzymolysis substrate bone meal quality 2.2 ‰ then is enzymolysis 12h under 55 ℃, the condition of pH value 7.5 in temperature, the enzyme 15min that goes out in 85 ℃ of water-baths then filters and collects enzymolysis solution; Use the rotary evaporation concentrating instrument that the enzymolysis solution of collecting is concentrated into 1/4 of original volume, then concentrated solution is placed-30 ℃ of refrigerators to freeze,, collect bone collagen polypeptide powder and be stored in-24 ℃ of refrigerators again through lyophilize 24h.
Lactobacillus sake viable bacteria number is 2 * 10 in the dry powder leaven that cultural method by present embodiment prepares
10CFU/g, the viable bacteria number in the fermented liquid is 2.1 * 10
9CFU/mL.
Embodiment 2:
A kind of method of utilizing bone collagen polypeptide powder milk-acid bacteria to be carried out high-density culture; this method is to be bacterial classification with the lactobacillus sake; at first adopt milk-acid bacteria basic medium (MRS substratum) that lactobacillus sake is activated; enlarged culturing; be inoculated in the fermention medium by 3% inoculum size again; cultivated 12 hours at 37 ℃ of temperature bottom fermentations; the NaOH solution control pH value that adds 2mol/L by stream in the fermentation culture process is 6.5; cultivate and finish the centrifugal collection thalline in back and join dissolving in the protein protective agent (thalline of collection is 1: 4 with the mass ratio of the protein protective agent of dissolving thalline), after vacuum freezing; the dry dry powder type lactobacillus starter that obtains.
Component and massfraction in the above-mentioned fermention medium are as follows: bone collagen polypeptide powder 1%, yeast extract paste 0.5%, glucose 0.5%, KH
2PO
40.3%, tween 80 0.5%, tomato juice 5%, sodium acetate 0.5%, the water of Trisodium Citrate 0.2% and surplus, regulating initial pH value is 6.8.The prescription of the above-mentioned MRS substratum of using, protein protective agent is identical with embodiment 1, and the preparation method of the above-mentioned bone collagen polypeptide powder of using is identical with embodiment 1.
Lactobacillus sake viable bacteria number is 9.0 * 10 in the dry powder leaven that cultural method by present embodiment prepares
10CFU/g, the viable bacteria number in the fermented liquid is 9.0 * 10
9CFU/mL.
Embodiment 3:
The massfraction of the bone collagen polypeptide powder that only fermention medium among the embodiment 1 is added in the present embodiment is adjusted into 1.5%, and remaining processing step and processing parameter are identical with embodiment 1.
Lactobacillus sake viable bacteria number is 8.8 * 10 in the dry powder leaven that cultural method by present embodiment prepares
10CFU/g, the viable bacteria number in the fermented liquid is 8.9 * 10
9CFU/mL.
Claims (9)
1, a kind of method of utilizing bone collagen polypeptide powder milk-acid bacteria to be carried out high-density culture, this method is to be bacterial classification with the milk-acid bacteria, at first adopt the milk-acid bacteria basic medium to bacterial classification activate, enlarged culturing, inoculate fermentation culture 12～18h in the fermention medium, collect thalline and join in the protein protective agent and dissolve, after vacuum freezing, the dry finished product that obtains; Be added with bone collagen polypeptide powder in the described fermention medium.
2, the bone collagen polypeptide powder that utilizes according to claim 1 carries out the method for high-density culture to milk-acid bacteria, and that it is characterized in that described milk-acid bacteria selects for use is lactobacillus sake (Lactobacillus Sake).
3, the method for utilizing bone collagen polypeptide powder milk-acid bacteria to be carried out high-density culture according to claim 1, each component and the massfraction thereof that it is characterized in that described fermention medium are: bone collagen polypeptide powder 0.5～1.5%, yeast extract paste 0.5%, glucose 0.5%, KH
2PO
40.3%, tween 80 0.5%, tomato juice 5%, sodium acetate 0.5%, the water of Trisodium Citrate 0.2% and surplus.
4, the bone collagen polypeptide powder that utilizes according to claim 1 carries out the method for high-density culture to milk-acid bacteria, it is characterized in that the pH value of described fermention medium is controlled at 6.4～6.8, and the temperature during fermentation culture is controlled at 35～40 ℃.
5, the bone collagen polypeptide powder that utilizes according to claim 1 carries out the method for high-density culture to milk-acid bacteria, it is characterized in that the component of described protein protective agent and massfraction thereof are: the water of skimming milk 10%, glycerine 2%, trehalose 2% and surplus; The thalline of described collection and the mass ratio of protein protective agent are 1: 4.
6, the method for utilizing bone collagen polypeptide powder milk-acid bacteria to be carried out high-density culture according to claim 1, it is characterized in that described milk-acid bacteria basic medium is formulated by the component of following mass fraction: 10 parts of beef protein powder, 10 parts of flesh of fish juice, 5 parts of Yeast diffusion juice powder, 20 parts of glucose, 5 parts of sodium-acetates, 2 parts of dibasic ammonium citrates, 0.1 part of tween 80,0.58 part of sal epsom, 0.28 part of manganous sulfate and 1000 parts of aquae destillatas, the pH value of described milk-acid bacteria basic medium is controlled at 6.2～6.4.
7, according to each described method of utilizing bone collagen polypeptide powder milk-acid bacteria to be carried out high-density culture in the claim 1～6, it is characterized in that described bone collagen polypeptide powder is to prepare by following steps: get Fresh Os Sus domestica and clean, first broken back boiling water boiling drains the bone piece and drying behind the grease of removal cooking liquor top layer; Again the bone piece is carried out coarse reduction, micronizing is made bone meal, adds water bone meal is prepared skeletonization mud liquid, the high-temperature sterilization postcooling also adds the Protamex compound protease and carries out enzymolysis, enzyme goes out; Get filtered liquid vacuum concentration and lyophilize after the filtration, obtain bone collagen polypeptide powder.
8, the method for utilizing bone collagen polypeptide powder milk-acid bacteria to be carried out high-density culture according to claim 7, it is characterized in that: the addition of described Protamex compound protease is 2～2.5 ‰ of an enzymolysis substrate bone meal quality, pH value during described Protamex compound protease enzymolysis is 7.5, temperature is controlled at 55 ℃, and enzymolysis time is 10～15 hours.
9, the method for utilizing bone collagen polypeptide powder milk-acid bacteria to be carried out high-density culture according to claim 7, it is characterized in that: during described boiling water boiling, the mass ratio of bone piece and water is 1: 1～1: 2, temperature during the boiling water boiling is controlled at 110 ℃～120 ℃, pressure during the boiling water boiling is controlled at 143kPa～198kPa, and the time of boiling water boiling is controlled at 30～60min.
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| CN2009100425477A CN101475923B (en) | 2009-01-19 | 2009-01-19 | Method for high-density cultivation for lactobacillus by using bone collagen polypeptide powder |
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| CN2009100425477A CN101475923B (en) | 2009-01-19 | 2009-01-19 | Method for high-density cultivation for lactobacillus by using bone collagen polypeptide powder |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191202A (en) * | 2011-04-08 | 2011-09-21 | 石家庄君乐宝乳业有限公司 | High-density culture method for lactic acid bacteria |
| CN109837222A (en) * | 2017-11-28 | 2019-06-04 | 广州中国科学院先进技术研究所 | A kind of cultural method of Bacillus acidi lactici culture medium and preparation method thereof and Bacillus acidi lactici |
| CN112450428A (en) * | 2020-10-23 | 2021-03-09 | 内蒙古蒙肽生物工程有限公司 | Bone mud fermentation liquor and preparation method thereof |
| CN113403353A (en) * | 2021-07-14 | 2021-09-17 | 四川大学 | Culture medium and method for producing undecyl prodigiosin |
-
2009
- 2009-01-19 CN CN2009100425477A patent/CN101475923B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191202A (en) * | 2011-04-08 | 2011-09-21 | 石家庄君乐宝乳业有限公司 | High-density culture method for lactic acid bacteria |
| CN102191202B (en) * | 2011-04-08 | 2013-05-22 | 石家庄君乐宝乳业有限公司 | High-density culture method for lactic acid bacteria |
| CN109837222A (en) * | 2017-11-28 | 2019-06-04 | 广州中国科学院先进技术研究所 | A kind of cultural method of Bacillus acidi lactici culture medium and preparation method thereof and Bacillus acidi lactici |
| CN112450428A (en) * | 2020-10-23 | 2021-03-09 | 内蒙古蒙肽生物工程有限公司 | Bone mud fermentation liquor and preparation method thereof |
| CN113403353A (en) * | 2021-07-14 | 2021-09-17 | 四川大学 | Culture medium and method for producing undecyl prodigiosin |
| CN113403353B (en) * | 2021-07-14 | 2022-09-30 | 四川大学 | Culture medium and method for producing undecyl prodigiosin |
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Application publication date: 20090708 Assignee: Hunan Hait Food Co., Ltd. Assignor: Hunan Agricultural University Contract record no.: 2012430000162 Denomination of invention: Method for high-density cultivation for lactobacillus by using bone collagen polypeptide powder Granted publication date: 20110420 License type: Exclusive License Record date: 20120803 |
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Application publication date: 20090708 Assignee: Hunan Hait Food Co., Ltd. Assignor: Hunan Agricultural University Contract record no.: 2012430000162 Denomination of invention: Method for high-density cultivation for lactobacillus by using bone collagen polypeptide powder Granted publication date: 20110420 License type: Exclusive License Record date: 20120803 |
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