CN104962493A - Hydrolysis rice residue protein bacillus subtilis HP 90 fermentation medium and cultivating method - Google Patents
Hydrolysis rice residue protein bacillus subtilis HP 90 fermentation medium and cultivating method Download PDFInfo
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Abstract
Provided is a hydrolysis rice residue protein bacillus subtilis HP 90 fermentation medium. The fermentation medium comprises 9.7 g/L of rice residues, 5.0 g/L of sodium chloride, 0.2 g/L of magnesium sulfate heptahydrate, 0.1 g/L of calcium chloride, 5.0 g/L of dipotassium phosphate and the balance water. The pH value of the fermentation medium is 7.35. A cultivating method comprises the steps that bacillus subtilis HP90 are inoculated into a seed medium, cultivating is carried out for 24 hours at the temperature of 30 DEG C and the speed of 180 rotates per minute, and a seed solution used for carrying out fermentation is prepared; the fermentation seed solution is inoculated into the fermentation medium according to the inoculating amount of 7%, and the cultivating is carried out for 60 hours under the temperature of 30 DEG C and the speed of 180 rotates per minute. The HP90 fermentation medium is an optimized medium, hydrolysis of rice residue protein can be efficiently catalyzed by protease used for fermentation producing, the fermentation condition is easy to achieve, the condition is warm, controlling is easy, and the industrial producing is facilitated.
Description
Technical field
The present invention relates to the substratum of microorganism, be particularly hydrolyzed the fermention medium of the subtilis HP90 of rice residue protein.
Background technology
Rice residue protein is byproduct residue remaining after the products such as the happy sugar of raw material production, monosodium glutamate, glucose, dextrin, starch with early rice, ageing rice etc., and its price is low, and output is large, almost often consumes 7kg rice and just have 1kg rice residue and generate.In rice residue, protein content is 40% ~ 70%, is 5 ~ 8 times of protein content in the whole grain of rice, and higher than protein content in soybean, is considered to the best in grain protein.At present, China mainly by these rice residue proteins through simple disintegrating process processing procedure dry powder, feed factory of selling at a low price makes feed, and it is worth and can obtains far away rationally and make full use of.If these albumen to be changed into polypeptide and the amino acid of high added value, this to improve its biological value, economic worth and China's paddy deep processing technology content all significant.
Rice residue protein changes into polypeptide and amino acid common acid or commercially available protein enzyme.Acid hydrolysis is rapid, thorough, but it carries out under extreme temperature and pH, to amino acid, protein breakdown seriously, destroys and makes hydrolyzed solution be black, be unfavorable for subsequent handling to monose and polysaccharide major part; Poisonous and carcinogenic propylene chlorohydrin class material can also be generated, contaminate environment with grease in raw material.And commercial protease can improve protein physics and chemistry function and organoleptics property, green safety, and be widely used in food, washing composition, medicine and leather industry etc.Meanwhile, also there is certain deficiency in commercial enzyme: commodity enzyme process, due to the specificity of enzyme, multiple protein enzyme keying action must be used in rice residue, and the action condition of different enzyme is different, causes it active affected by environment very large; Because in raw material, some albumen are wrapped up by starch etc., also need to add saccharifying enzyme etc. and discharge protein, make technical process complicated; Because of the cost of enzyme own and cost recovery higher, industrial applications is high in rice residue cost.Bacterium producing multi enzyme preparation directly applies to protein, can make full use of substrate because its enzyme system is abundant; Enlarged culturing can make seed liquor or the extensive use cost of freeze-dried vaccine powder is lower; Its enzyme comparatively commercial enzyme alive is stablized during the fermentation; For bacterium producing multi enzyme preparation replaces commercial enzyme hydrolysis rice residue protein to provide theoretical foundation.This method not only has the advantage of enzyme process concurrently, and can reduce production cost, reusable edible, simple and convenient.In addition, producing rice residue protein lytic enzyme bacterial strain, to directly apply to the hydrolysis of rice residue protein also very rare.For this reason, applicant works out " the strain hydrolysis protease-producing bacterium subtilis of rice residue and screening and using method " thereof, and has applied for Chinese patent, and application number is 201410474539.4, and this application part is announced on March 04th, 2015.
In current Chinese patent database, relate to the application part nearly up to a hundred of baccilus medium, such as, No. ZL2010105960593 " a kind of haemophilus parasuis 5 type high-density fermentation medium and application ", No. ZL201110099582X " a kind of subtilis submerged fermentation culture medium being applied to microbial fertilizer ", No. ZL2012102784847 " a kind of Tribactur emulsification fermentation culture medium ", No. ZL2013100415664 " a kind of promote the fermention medium that Bacillus licheniformis BL63516 grows and fermentation culture method " and No. ZL2013101899388 " a kind of fermentation of bacillus subtilis substratum " etc.Up to now, there is no the application part of the fermentation of bacillus subtilis substratum of hydrolysis rice residue protein.
Summary of the invention
The present invention aims to provide the fermention medium of the subtilis HP90 of hydrolysis rice residue protein, improves the degree of hydrolysis of subtilis HP90 to rice residue protein by optimizing fermention medium.
Another object of the present invention is to provide the cultural method optimizing fermention medium, makes aforesaid fermention medium be able to practical application.
The each component concentration of fermention medium of the subtilis HP90 of the hydrolysis rice residue protein that contriver provides is (g/L): rice residue 9.7, sodium-chlor 5.0, magnesium sulfate heptahydrate 0.2, calcium chloride 0.1, dipotassium hydrogen phosphate 5.0, and all the other are water; Medium pH is 7.35;
Described rice residue is purchased from Boxing County, Shandong Xiao Xu feed trade Co., Ltd.
The subtilis HP90 of said hydrolyzed rice residue protein is preserved in Wuhan China typical culture collection center on May 18th, 2014, and this centre address is: Wuhan, China Wuhan University, postcode: 430072; Be called for short subtilis HP90, deposit number is CCTCC NO.M2014201, Classification And Nomenclature: subtilis (Bacillus subtilis).This bacterial strain system is separated and obtains from Guizhou In China tradition fermented soya bean.
The cultural method of the HP90 fermention medium that contriver provides is that subtilis HP90 is inoculated in seed culture medium, in 30 DEG C, cultivate 24h under 180r/min, obtained fermentation seed liquor; Again by fermentation seed liquid according to 7% inoculum size be inoculated in above-mentioned fermention medium, 30 DEG C, cultivate 60h under 180r/min; According to said method, rice residue protein degree of hydrolysis can reach 28.23%.
Each component (g/L) of above-mentioned seed culture medium is: rice residue 15.0, sodium-chlor 5.0, magnesium sulfate heptahydrate 0.2, calcium chloride 0.1, dipotassium hydrogen phosphate 5.0, natural pH, and all the other are water, in 121 DEG C of sterilizing 20min.
Calculation formula=(bacterial strain is hydrolyzed the content of rice residue protein in the content/fermention medium of rice residue protein) × 100% of rice residue protein degree of hydrolysis
The subtilis HP90 fermention medium of hydrolysis rice residue protein of the present invention is a kind of Optimal Medium, proteolytic enzyme for fermentative production can the hydrolysis of efficient catalytic rice residue protein, and fermentation condition easily realizes, mild condition, be easy to control, be conducive to suitability for industrialized production.Substratum after adopting the present invention to optimize and condition, this subtilis HP90 can reach 28.23% to the degree of hydrolysis of rice residue protein, improves 42% compared with before optimization.The present invention is applicable to cultivate subtilis.
Accompanying drawing explanation
Fig. 1 is that fermented liquid pH is hydrolyzed the impact of rice residue protein degree of hydrolysis to bacterial strain HP90;
Fig. 2 is that different vaccination amount is hydrolyzed the impact of rice residue protein degree of hydrolysis to bacterial strain HP90;
Fig. 3 is hydrolyzed the impact of rice residue protein degree of hydrolysis to bacterial strain HP90 the different fermentations time;
Fig. 4 is that different rice residue concentration is hydrolyzed the impact of rice residue protein degree of hydrolysis to bacterial strain HP90.
Embodiment
The present invention's the following example further illustrates summary of the invention, but protection scope of the present invention is not limited to the following example.
Embodiment 1
Be below the substratum that arrives involved in the present invention and method
(1) seed culture medium (g/L): rice residue 15.0, sodium-chlor 5.0, magnesium sulfate heptahydrate 0.2, calcium chloride 0.1, dipotassium hydrogen phosphate 5.0, natural pH, all the other are water, in 121 DEG C of sterilizing 20min
(2) preparation of fermentation seed liquid: inclined-plane subtilis HP90 is inoculated in seed culture medium, 30 DEG C, 180r/min cultivates 24h, is fermentation seed liquid.
(3) fermention medium (g/L): same to seed culture medium.
(4) preparation of fermented liquid: by fermentation seed liquid by 6% inoculum size to be inoculated in fermentation production medium 30 DEG C, to cultivate 48h under 180r/min, then at 4 DEG C, under the condition of 10000r/min, centrifugal 10min obtains fermented supernatant fluid.
Embodiment 2
Single factor experiment designs
30 DEG C, under 180r/min condition, the condition that subtilis HP90 is hydrolyzed rice residue is investigated respectively with different hydrolysis pH, time, inoculum size and rice residue concentration, with the action condition scope of rice residue protein degree of hydrolysis for index determination bacterial strain hydrolysis rice residue, specific as follows:
(1) pH value is hydrolyzed on the impact of rice residue degree of hydrolysis
Hydrolysis time 48h, inoculum size 6%, rice residue concentration 1.5g/100mL, fermention medium pH is adjusted to 5,6,7,8,9 respectively,
(2) hydrolysis time is on the impact of rice residue degree of hydrolysis
PH 7, inoculum size 6%, rice residue concentration 1.5g/100mL, fermented incubation time gets 24h, 36h, 48h, 60h, 72h respectively.
(3) inoculum size is on the impact of rice residue degree of hydrolysis
PH 7, hydrolysis time 48h, rice residue concentration 1.5g/100mL, inoculum size is respectively 2%, 4%, 6%, 8%, 10%.
(4) rice residue protein content is on the impact of its degree of hydrolysis
PH is 7, and hydrolysis time is 48h, and inoculum size is 6% of fermented liquid, and rice residue protein content is respectively 0.5%, 1.0%, 1.5%, 2.0%, 2.5%.
Single factor experiment the results are shown in Figure 1 ~ Fig. 4.
Box-Benhnken test design
Box-Benhnken test design is carried out on the basis of above-mentioned experiment of single factor, optimizes the action condition that subtilis HP90 is hydrolyzed rice residue.Choosing hydrolysis pH value, hydrolysis time, inoculum size and rice residue concentration is research object, is responsively worth, is optimized by response surface analysis to fermentation condition with rice residue protein degree of hydrolysis.Experimental design is in table 1, and experimental result table 2, interpretation is in table 3.
Table 1 bacterial strain HP90 is hydrolyzed response surface analysis factor and the level of rice residue
Table 2 bacterial strain HP90 is hydrolyzed the Box-Behnken test-results of rice residue protein
The data of the rice residue protein degree of hydrolysis in Design-Expert software his-and-hers watches 2 are adopted to carry out variance analysis and multiple regression matching, obtain the significance (table 3) of each factor of influence, obtain subtilis HP90 and be hydrolyzed rice residue protein degree of hydrolysis (Y) to the pH (X of independent variable(s) fermented liquid
1), fermentation time (X
2), inoculum size (X
3) and concentration of substrate (X
4) the multinomial regression model equation of secondary be:
Y＝+25.70+0.33X
1+0.65X
2+0.23X
3-0.35X
4+0.82X
1X
2+0.44X
1X
3-0.13X
1X
4+0.39X
2X
3-0.29X
2X
4-0.14X
3X
4-2.26X
1 2+0.39X
2 2+0.55X
3 2-6.74X
4 2。
Utilize Design-Expert software to be worth most regression equation to solve, obtain the pH (X of model extreme point and fermented liquid
1) be 7.35, inoculum size (X
3) be 7%, fermentation time (X
2) be 60h, rice residue concentration (X
4) for 0.97g/100mL time, reaching maximum value to rice residue protein degree of hydrolysis, is 28.23%.
Above embodiment is only object lesson of the present invention, and obvious realization of the present invention is not subject to the restrictions described above.As long as adopt method of the present invention to conceive and the unsubstantiality that carries out of technical scheme improves, or design of the present invention and technical scheme directly applied to other occasions, all within protection scope of the present invention without improvement.
Claims (4)
1. be hydrolyzed the fermention medium of the subtilis HP90 of rice residue protein, it is characterized in that each component concentration of this fermention medium is counted with g/L: rice residue 9.7, sodium-chlor 5.0, magnesium sulfate heptahydrate 0.2, calcium chloride 0.1, dipotassium hydrogen phosphate 5.0, all the other are water; Medium pH is 7.35;
Described rice residue is purchased from Boxing County, Shandong Xiao Xu feed trade Co., Ltd.
2. fermention medium as claimed in claim 1, it is characterized in that the subtilis HP90 of described hydrolysis rice residue protein is preserved in Wuhan China typical culture collection center on May 18th, 2014, this centre address is: Wuhan, China Wuhan University, postcode: 430072; Be called for short subtilis HP90, deposit number is CCTCC NO. M 2014201, Classification And Nomenclature: subtilis (
bacillus subtilis).
3. the cultural method of HP90 fermention medium as claimed in claim 1, is characterized in that subtilis HP90 to be inoculated in seed culture medium, in 30 DEG C, cultivate 24h, obtained fermentation seed liquor under 180r/min; Again by fermentation seed liquid according to 7% inoculum size be inoculated in above-mentioned fermention medium, 30 DEG C, cultivate 60 h under 180r/min; According to said method, rice residue protein degree of hydrolysis can reach 28.23%.
4. method as claimed in claim 3, it is characterized in that each component of described seed culture medium in g/L is: rice residue 15.0, sodium-chlor 5.0, magnesium sulfate heptahydrate 0.2, calcium chloride 0.1, dipotassium hydrogen phosphate 5.0, nature pH, all the other are water, obtain in 121 DEG C of sterilizing 20 min.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102115774A (en) * | 2010-11-30 | 2011-07-06 | 华东理工大学 | Method for preparing plant polypeptide by enzyme process |
| CN104087638A (en) * | 2014-07-29 | 2014-10-08 | 湖南农业大学东方科技学院 | Method for preparing antioxidative peptide through fermentation of rice residue by use of bacillus subtilis |
| CN104357346A (en) * | 2014-09-18 | 2015-02-18 | 贵州大学 | Rice dreg's protease hydrolysis producing strain bacillus subtilis and screening and application methods thereof |
| CN104450554A (en) * | 2014-09-18 | 2015-03-25 | 贵州大学 | Protease-producing strain, namely, bacillus amyloliquefaciens capable of hydrolyzing rice residues as well as screening and applying method thereof |
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2015
- 2015-06-24 CN CN201510351529.2A patent/CN104962493A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102115774A (en) * | 2010-11-30 | 2011-07-06 | 华东理工大学 | Method for preparing plant polypeptide by enzyme process |
| CN104087638A (en) * | 2014-07-29 | 2014-10-08 | 湖南农业大学东方科技学院 | Method for preparing antioxidative peptide through fermentation of rice residue by use of bacillus subtilis |
| CN104357346A (en) * | 2014-09-18 | 2015-02-18 | 贵州大学 | Rice dreg's protease hydrolysis producing strain bacillus subtilis and screening and application methods thereof |
| CN104450554A (en) * | 2014-09-18 | 2015-03-25 | 贵州大学 | Protease-producing strain, namely, bacillus amyloliquefaciens capable of hydrolyzing rice residues as well as screening and applying method thereof |
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