CN100419071C - Bacillus of high proteinase yield and its induction mutation breeding method and uses - Google Patents
Bacillus of high proteinase yield and its induction mutation breeding method and uses Download PDFInfo
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- CN100419071C CN100419071C CNB2004100865578A CN200410086557A CN100419071C CN 100419071 C CN100419071 C CN 100419071C CN B2004100865578 A CNB2004100865578 A CN B2004100865578A CN 200410086557 A CN200410086557 A CN 200410086557A CN 100419071 C CN100419071 C CN 100419071C
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Abstract
The present invention relates to a bacillus with high proteinase yield. The classification name of the bacillus with high proteinase yield is a bacillus collected in the common microorganism common micro-organisms center of the China Committee for the Culture Collection of Microorganisms on June 28th, 2004, and the collection number is CGMCC No. 1180. Bacillus subtilis CAU208 with the collection number of CGMCC No. 1.769 is used as an original strain, the single-cell suspension of the original strain is orderly carried out with ultraviolet mutation, nitrosoguanidine mutation, and ultraviolet and nitrosoguanidine compound mutation in a circulating mode, and finally, a strain of bacillus with high proteinase yield is obtained. The bacillus with high proteinase yield can be used for preparing soybean biological active peptide feed additives, enzyme preparations, rich enzyme probiotics and the like through liquid fermentation. The prepared peptide feed additives have various purposes of enhancing the quality of poultry eggs, meeting the nutritional requirements of animals with immature digestive systems, replenishing the protein requirements of animals with anaphylactic diseases or being used as high-quality protein sources of young animals, such as anthony pigs, piglets and the like, and preventing protein feed, such as soybean meal(cakes) from generating anaphylactic disease of mucosae of intestinal tracts, edema and the like for the animals.
Description
Invention field
The invention belongs to biological technical field, but specifically relate to a kind of genus bacillus and mutagenic breeding method and purposes of high proteinase yield.
Background technology
In animal rearing and feed, be extensive use of promotes growth health-care agents such as microbiotic, chemical synthetic drug, hormone for many years, caused livestock and poultry and aquatic products product Chinese traditional medicine residual serious, the animal products quality descends, bacterial drug resistance strengthens, Resistant strain increases, and brings very big threat for human security and ecotope.Because antibiotic many side effects, particularly in recent years the human consumer to the concern of food-safety problem, antibiotic use is being forbidden or limited in countries in the world all one after another, and accelerate development, popularization new type of safe green feed additive efficiently, to reduce or alternative antibiotic etc additive.
Probiotics and soybean biological bioactive peptide become the research focus of novel green feed additive field because of its unique physicochemical property and biological function.Can see soybean peptides on the domestic market at present, mostly be imported product, cost an arm and a leg, mainly produce by enzyme process.Enzyme process is to use plant protease such as proteolytic enzyme such as papoid, bromeline or with animal proteases such as trypsinase, stomach en-s, soybean protein is degraded to little peptide, but the soybean peptides that this method is produced is easy to generate unacceptable fishy smell or bitter taste, and the production cost height is unsuitable for using in livestock industry; Microbe fermentation method is compared with enzyme process and production by biological enzyme and enzymolysis can be united two into one, and shortens operation, reduce cost, and the soybean peptides product has natural aromatising flavour, and mouthfeel is good.
In the process of industrial microorganism fermentative production, the main factor of decision production level and production cost height has three: produce bacterial classification, zymotechnique and back extraction process or after-processing technology, wherein, the most important thing is to produce the characteristic of bacterial classification.From the isolated bacterial classification of nature,, often can not satisfy industrial needs because throughput is low.Because under normal physiological conditions, microorganism relies on its metabolism regulation system, trends towards growth fast and breeding.But the needs of fermentation industry need microorganism can accumulate a large amount of meta-bolitess in contrast, as enzyme or other activeconstituents etc.For this reason, adopt various methodologies to break the eubolism of bacterial classification, make it to lose the adjusting control of self-conservative property, thereby accumulate our needed target meta-bolites (as proteolytic enzyme) in a large number.Reach this purpose, major measure is carried out strain improvement exactly, as carries out physics and chemomorphosis etc.
Summary of the invention
The defective that the objective of the invention is to overcome the cost height of prior art for preparing activated peptide forage additive and be not easy to promote the use of in the animal cultivation industry, but by the genus bacillus of physics with the complex mutation breeding one strain high proteinase yield of chemistry, make it can be low through the addition that fermentative production goes out in feed, definite functions, and have improve animal to nutrient substance (as protein, mineral substance, VITAMIN etc.) absorption rate, improve milk, meat, egg output and throughput, and improve milk, meat, the quality of egg and local flavor, stimulate the animal immune allelotaxis, improve disease resistance and reduce sickness rate, and can obviously improve aquaculture of aquatic animal water quality, improve aquatic animal to grow speed and survival rate, can be widely used in fowl, ox, sheep, pig, aquatic products and economic animal can be used as the activated peptide forage additive of advantages such as antibiotic substitute.
But another object of the present invention is to provide the mutagenic breeding method of a kind of genus bacillus of high proteinase yield.
But a further object of the present invention is the genus bacillus of this high proteinase yield and is used for the application that liquid fermenting prepares soybean biological activated peptide forage additive or zymin, rich enzyme probiotics etc.
Technical scheme of the present invention is as follows:
But the genus bacillus of high proteinase yield provided by the invention, it is characterized in that, but be somebody's turn to do the classification called after of the genus bacillus of high proteinase yield: bacillus sp, be preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on June 28th, 2004, its preserving number is CGMCC No.1180.
Cell is direct rod shape, and electron microscope is measured as 0.5-0.6 * 1.1-3.5 μ m, often with paired or catenation; Great majority present Gram-positive when the children cultivates age; The flagellum adnation can move.Gemma ovalize, column, circle or ovum circle, middle life or wilfully, 0.8 * 1.5-1.8 μ m can resist many poor environments to swim, a little less than spore surface is painted.Bacterium colony circle on the agar medium, surface colour is dark, can be wrinkling, can become cream color or brown; Great changes have taken place with medium component is different for the shape of bacterium colony; When the agar medium surface moisture, bacterium colony is easy to diffusion; Be difficult for diffusion at the lawn of growing on the agar in liquid, look dark, gauffer, complete film, clear degree are muddy or haze-free in nutrient solution.Aerobic or amphimicrobian has heat, pH and the various multifarious physiological properties of salt.Chemoheterotrophic bacteria has fermentation or respiratory metabolism type, usually the catalase positive.Growth temperature is up to 45-55 ℃, and minimum is 5-20 ℃.
But the mutagenic breeding method of the genus bacillus of high proteinase yield provided by the invention, its mutagenic and breeding step is as follows:
1) choosing from " China Committee for Culture Collection of Microorganisms common micro-organisms center ", preserving number is that the subtilis CAU208 of CGMCCNo1.769 is as starting strain;
2) mutagenic and breeding
(1) preparation starting strain CAU208 single-cell suspension liquid
Starting strain genus bacillus CAU208 is inoculated among the liquid nutrient medium A after the sterilization, 28～32 ℃ of shake-flask culture 36～48h, centrifugal, wash with stroke-physiological saline solution, place in the aseptic triangular flask that granulated glass sphere is housed, vibration makes it be dispersed into single cell suspension, filter with aseptic glass wool, obtain the unicellular bacteria suspension of starting strain CAU208;
Contained component of described liquid nutrient medium A and proportioning are: peptone 1.3～2.5g, and glucose 3～5g, molasses 3～5g, Zulkovsky starch 10～20g, sucrose 10～30g, extractum carnis 2～3g, yeast soak powder 3.5～5g, water 1000ml;
(2) ultraviolet mutagenesis
The viable count of regulating step (1) gained starting strain CAU208 bacteria suspension is 10
6～10
8Between individual/ml, liquid layer thickness 0.2～0.4cmm; Carry out ultraviolet mutagenesis, the used ultraviolet lamp power of ultraviolet mutagenesis 15～18W, irradiation time 2～5min, irradiation distance 25～30cm; Dilution immediately is coated on the primary dcreening operation isolation medium B after the irradiation, cultivate 30～36h for 27～32 ℃, select 50～70 single bacterium colonies, carrying out shake flask fermentation sieves again, with ninhydrin method determination of color degree of hydrolysis, select the highest bacterial strain of 2～4 strain hydrolysis abilities, make shake flask fermentation more respectively and sieve again, select the bacterial strain CAU208-N of the highest and good stability of a strain hydrolysis ability, and make bacteria suspension and be used for next step nitrosoguanidine mutagenesis;
Described contained component of primary dcreening operation isolation medium B and proportioning are: peptone 1～3g, yeast soak powder 2～4g, glucose 4～6g, skim-milk 5～8g, agar 10～13g, water 1000ml;
The step that described shake flask fermentation sieves again is:
The preparation fermented liquid: by weight ratio behind the uniform mixing, under 0.1～0.15mpa pressure, 120～122 ℃ of sterilizations are cooled to 30～37 ℃ again, make fermented liquid with nitrogenous source, carbon source and water; Wherein, the proportioning of the weight part sum of nitrogenous source and carbon source and the weight part of water is 3～6: 100, and nitrogenous source and carbon source weight part proportioning are 2～4: 1; Described nitrogenous source is bean cake powder, soybean isolate protein powder etc.; Described carbon source is Semen Maydis powder or Semen Maydis powder and molasses etc.;
With 50～70 single bacterium colony seed liquor capacity of being inoculated in that obtain is that 250ml is equipped with in the triangular flask of 100ml fermented liquid, and seed liquor and fermented liquid be 0.5～1.0: 10 ratio uniform mixing by volume, ferments on shaking table with the speed of 110～140rpm; Leavening temperature is 30 ℃～35 ℃, uses ninhydrin method determination of color degree of hydrolysis during fermentation ends;
(3) nitrosoguanidine mutagenesis
The viable count of step (2) gained CAU208-N bacteria suspension is transferred to 10
5～10
8Individual/ml, adding final concentration in the bacteria suspension is the nitrosoguanidine of 800～1000ug/ml, 30～35 ℃ of water bath processing 20～30min, intermittently concussion; Be applied to immediately after the mutagenesis on the primary dcreening operation isolation medium B, cultivate 30～36h for 27～32 ℃, select 50～70 single bacterium colonies, carry out shake flask fermentation and sieve again, obtain the highest strain bacterial strain CAU208-N1 of hydrolysis ability, and make bacteria suspension be used for next step the ultraviolet ray with the nitrosoguanidine complex mutation; The same step of the method that described shake flask fermentation sieves again (2);
(4) ultraviolet ray and nitrosoguanidine complex mutation
The viable count of step (3) gained CAU208-N1 bacteria suspension is transferred to 10
7～10
9Individual/ml, adding final concentration in the bacteria suspension is the nitrosoguanidine liquid of 900～1300ug/ml, 30～35 ℃ of water bath processing 20～30min, and the liquid layer thickness 0.2～0.4cm after nitrosoguanidine is handled is got in intermittently concussion; Carry out ultraviolet mutagenesis, used ultraviolet lamp power 15～18W, irradiation time 6～9min, distance 25～30cm; Be applied to immediately after the uv irradiating mutagenesis on the primary dcreening operation isolation medium B, cultivate 30～36h for 27～32 ℃, select 50～70 single bacterium colonies, carry out shake flask fermentation and sieve again, obtain the highest strain bacterial strain CAU208-N5 of hydrolysis ability; The multiple same step of screen method (2) of described shake flask fermentation;
(5) circulation repetition ultraviolet mutagenesis → nitrosoguanidine mutagenesis → ultraviolet ray and nitrosoguanidine complex mutation are 2 times, obtain superior strain CAU208-1 at last.
But the mutagenic breeding method of the genus bacillus of high proteinase yield provided by the invention comprises that also the superior strain CAU208-1 that will obtain is kept on the solid slant culture base C, 4 ℃ of preservations, and switching in per 3 months is once;
Described solid slant culture base consists of: peptone 1.3～2.5g, and glucose 3～5g, molasses 3～5g, Zulkovsky starch 10～20g, sucrose 10～30g, extractum carnis 2～3g, yeast soak powder 3.5～5g, agar 10～13g water 1000ml.
But the purposes of the genus bacillus of high proteinase yield of the present invention is characterized in that, prepares the soybean biological activated peptide forage additive or is used to prepare microbial forage additives such as probiotics, zymin but the genus bacillus of this high proteinase yield is used for liquid fermenting.
Advantage of the present invention:
1. combine with physics and chemical mutagen and handle, the heritability of the change microorganism of energy multi-angle, and circular treatment is repeatedly, guarantees the stability of dissociant;
2. the mutagenic compound convenient sources is cheap, and effect is obvious;
3. can obviously reduce fermentation costs with its fermentative preparation soybean active peptide fodder additives, make the application of soybean active peptide in livestock industry become possibility;
4. the superior strain of mutagenic and breeding can be used for preparing feeding probiotics;
5. the superior strain of mutagenic and breeding can be used for preparing fodder enzyme preparation;
6. the superior strain of mutagenic and breeding can be used for preparing rich enzyme prebiotics feed additive.
Embodiment
But the genus bacillus of embodiment 1, mutagenic and breeding high proteinase yield of the present invention
According to mutagenic breeding method provided by the invention, but the genus bacillus of mutagenic and breeding high proteinase yield, and its step is as follows:
1, choosing from " China Committee for Culture Collection of Microorganisms common micro-organisms center ", preserving number is that the subtilis CAU208 of CGMCCNo1.769 is as starting strain;
2, mutagenic and breeding:
(1) preparation starting strain CAU208 single-cell suspension liquid
Starting strain genus bacillus CAU208 is inoculated among the liquid nutrient medium A after the sterilization, 30 ℃ of shake-flask culture 36h, it is centrifugal to get the 1ml seed liquor, with stroke-physiological saline solution washing 3 times, place in the aseptic triangular flask that granulated glass sphere is housed, vibration 20min makes it be dispersed into single cell suspension, filter with aseptic glass wool, obtain the unicellular bacteria suspension of starting strain CAU208;
Contained component of described liquid nutrient medium A and proportioning are: peptone 1.3 g, and glucose 3g, molasses 3g, potato immersion liquid 50ml (wherein soluble-containing starch 10g), sucrose 10g, extractum carnis 2g, yeast soak powder 3.5g, water 1000ml;
(2) ultraviolet mutagenesis:
The viable count of regulating step (1) gained starting strain CAU208 bacteria suspension is 10
7Individual/ml, liquid layer thickness 0.2cm; Carry out ultraviolet mutagenesis, the used ultraviolet lamp power of ultraviolet mutagenesis 15W, irradiation time 2min, irradiation distance 25cm; Dilution immediately is coated on the primary dcreening operation isolation medium B after the irradiation, cultivate 30h for 27 ℃, select single bacterium colony of 50 hydrolysis circles big (thereby the protein that the proteolytic enzyme that this bacterium produces can decompose in the substratum produces the hydrolysis circle), carrying out shake flask fermentation sieves again, with ninhydrin method determination of color degree of hydrolysis, select the highest bacterial strain of 2 strain hydrolysis abilities, making shake flask fermentation more respectively sieves again, select the bacterial strain CAU208-N of the highest and good stability of a strain hydrolysis ability, and make bacteria suspension and be used for next step nitrosoguanidine mutagenesis;
Described contained component of primary dcreening operation isolation medium B and proportioning are: peptone 3g, yeast soak powder 4g, glucose 6g, skim-milk 5g, agar 13g, water 1000ml;
The step that described shake flask fermentation sieves again is:
(2-1) preparation fermented liquid: by weight ratio behind the uniform mixing, under 0.15mpa pressure, 120 ℃ of sterilizations are cooled to 37 ℃ again, make fermented liquid with nitrogenous source, carbon source and water; Wherein, the proportioning of the weight part sum of nitrogenous source and carbon source and the weight part of water is 3: 100, and nitrogenous source and carbon source weight part proportioning are 2: 1; Described nitrogenous source is for being crushed to 40 purpose soybean isolate protein powders; Described carbon source is Semen Maydis powder and molasses;
50 single bacterium colony seed liquor that (2-2) will obtain respectively with 0.5: 10 by volume ratio uniform mixing of fermented liquid; Speed with 110rpm is fermented on shaking table; Leavening temperature is 30 ℃, uses ninhydrin method determination of color degree of hydrolysis during fermentation ends.
(3) nitrosoguanidine mutagenesis
The viable count of CAU208-N bacteria suspension is transferred to 10
5Individual/ml, be added in the nitrosoguanidine that concentration is 1000ug/ml 30 ℃ of water bath processing 30min, intermittently concussion; Be applied to immediately after the mutagenesis on the primary dcreening operation isolation medium B, cultivate 36h for 32 ℃, select 70 single bacterium colonies, carrying out shake flask fermentation sieves again, the same step of the method that its shake flask fermentation sieves again (2), obtain the highest strain bacterial strain CAU208-N1 of hydrolysis ability, and make bacteria suspension be used for next step the ultraviolet ray with the nitrosoguanidine complex mutation;
(4) ultraviolet ray and nitrosoguanidine complex mutation
The viable count of CAU208-N1 bacteria suspension is transferred to 10
9Individual/ml is added to nitrosoguanidine liquid, and making the nitrosoguanidine final concentration is 1300ug/ml, 30 ℃ of water bath processing 20min, and the liquid layer thickness 0.4cm after nitrosoguanidine is handled is got in intermittently concussion; Carry out ultraviolet mutagenesis, the used ultraviolet lamp power of ultraviolet mutagenesis 15W, irradiation time 6min is apart from 25cm; Be applied to immediately after the mutagenesis on the primary dcreening operation isolation medium B, cultivate 30h for 27 ℃, select 70 single bacterium colonies, carry out shake flask fermentation and sieve again, its shake flask fermentation sieves same step (2) again, obtains the highest strain bacterial strain CAU208-N5 of hydrolysis ability;
(5) circulation repetition ultraviolet mutagenesis → nitrosoguanidine mutagenesis → ultraviolet ray and nitrosoguanidine complex mutation are 2 times, obtain superior strain CAU208-1 at last, be preserved on June 28th, 2004 at " China Committee for Culture Collection of Microorganisms common micro-organisms center ", its called after of classifying: bacillus sp, its preserving number are CGMCCNo.1180.
The superior strain CAU208-1 that obtains is deposited on the solid slant culture base C in 4 ℃, and switching in per 3 months once;
Described solid slant culture base C consists of: peptone 2.5g, and glucose 3g, molasses 5g, Zulkovsky starch 20g, sucrose 30g, extractum carnis 3g, yeast soak powder 3.5g, agar 10g, water 1000ml.
But the genus bacillus of embodiment 2, mutagenic and breeding high proteinase yield of the present invention
According to mutagenic breeding method provided by the invention, but the genus bacillus of mutagenic and breeding high proteinase yield, and its step is as follows:
1, choosing from " China Committee for Culture Collection of Microorganisms common micro-organisms center ", preserving number is that the subtilis CAU208 of CGMCCNo1.769 is as starting strain;
2, mutagenic and breeding:
(1) preparation starting strain CAU208 single-cell suspension liquid
Starting strain genus bacillus CAU208 is inoculated among the liquid nutrient medium A after the sterilization, 28 ℃ of shake-flask culture 48h, it is centrifugal to get the 1ml seed liquor, with stroke-physiological saline solution washing 3 times, place in the aseptic triangular flask that granulated glass sphere is housed, vibration 20min makes it be dispersed into single cell suspension, filter with aseptic glass wool, get the unicellular bacteria suspension of starting strain CAU208;
Contained component of described liquid nutrient medium A and proportioning are: peptone 2.5 g, and glucose 4g, molasses 4g, potato immersion liquid 50ml (wherein soluble-containing starch 20g), sucrose 20g, extractum carnis 3g, yeast soak powder 5g, water 1000ml;
(2) ultraviolet mutagenesis:
The viable count of regulating step (1) gained starting strain CAU208 bacteria suspension is 10
8Between individual/ml, liquid layer thickness 0.4cm; Carry out ultraviolet mutagenesis, the used ultraviolet lamp power of ultraviolet mutagenesis 18W, irradiation time 5min, irradiation distance 25cm; Dilution immediately is coated on the primary dcreening operation isolation medium B after the irradiation, cultivate 36h for 27 ℃, select 70 single bacterium colonies, carrying out shake flask fermentation sieves again, measure degree of hydrolysis, select the highest bacterial strain of 3 strain hydrolysis abilities, make shake flask fermentation more respectively and sieve again, select the bacterial strain CAU208-N of the highest and good stability of a strain hydrolysis ability, and make bacteria suspension and be used for next step nitrosoguanidine mutagenesis;
Described contained component of primary dcreening operation isolation medium B and proportioning are: peptone 1g, yeast soak powder 3g, glucose 4g, skim-milk 8g, agar 10g, water 1000ml;
The step that described shake flask fermentation sieves again is:
(2-1) preparation fermented liquid: by weight ratio behind the uniform mixing, under 0.1mpa pressure, 122 ℃ of sterilizations are cooled to 30 ℃ again, make fermented liquid with nitrogenous source, carbon source and water; Wherein, the proportioning of the weight part sum of nitrogenous source and carbon source and the weight part of water is 6: 100, and nitrogenous source and carbon source weight part proportioning are 4: 1; Described nitrogenous source is for being crushed to 40 purpose soybean isolate protein powders; Described carbon source is Semen Maydis powder and molasses;
70 single bacterium colony seed liquor that (2-2) will obtain and 1.0: 10 by volume ratio uniform mixing of fermented liquid carry out shake flask fermentation on shaking table;
(3) nitrosoguanidine mutagenesis
The viable count of CAU208-N bacteria suspension is transferred to 10
7Individual/ml, be added in the nitrosoguanidine that concentration is 800ug/ml 35 ℃ of water bath processing 20min, intermittently concussion; Be applied to immediately after the mutagenesis on the primary dcreening operation isolation medium B, cultivate 30h for 27 ℃, select 50 single bacterium colonies, carrying out shake flask fermentation sieves again, the same step of the method that its shake flask fermentation sieves again (2), obtain the highest strain bacterial strain CAU208-N1 of hydrolysis ability, and make bacteria suspension be used for next step the ultraviolet ray with the nitrosoguanidine complex mutation;
(4) ultraviolet ray and nitrosoguanidine complex mutation
The viable count of CAU208-N1 bacteria suspension is transferred to 10
7Individual/ml is added to nitrosoguanidine liquid, and making the nitrosoguanidine final concentration is 900ug/ml, 35 ℃ of water bath processing 30min, and the liquid layer thickness 0.2cm after nitrosoguanidine is handled is got in intermittently concussion; Carry out ultraviolet mutagenesis, the used ultraviolet lamp power of ultraviolet mutagenesis 18W, irradiation time 9min is apart from 30cm; Be applied to immediately after the mutagenesis on the primary dcreening operation isolation medium B, cultivate 36h for 32 ℃, select 70 single bacterium colonies, carry out shake flask fermentation and sieve again, its shake flask fermentation sieves same step (2) again, obtains the highest strain bacterial strain CAU208-N5 of hydrolysis ability;
(5) circulation repetition ultraviolet mutagenesis → nitrosoguanidine mutagenesis → ultraviolet ray and nitrosoguanidine complex mutation are 2 times, obtain superior strain CAU208-1 at last.
The superior strain CAU208-1 that obtains is deposited on the solid slant culture base C in 4 ℃, and switching in per 3 months once;
Described solid slant culture base C consists of: peptone 2, and glucose 4g, molasses 4g, Zulkovsky starch 15g, sucrose 20g, extractum carnis 2.5g, yeast soak powder 4g, agar 12g, water 1000ml.
But the genus bacillus of embodiment 3, mutagenic and breeding high proteinase yield of the present invention
According to mutagenic breeding method provided by the invention, but the genus bacillus of mutagenic and breeding high proteinase yield, and its step is as follows:
1, choosing from " China Committee for Culture Collection of Microorganisms common micro-organisms center ", preserving number is that the subtilis CAU208 of CGMCCNo1.769 is as starting strain;
2, mutagenic and breeding:
(1) preparation starting strain CAU208 single-cell suspension liquid
Starting strain genus bacillus CAU208 is inoculated among the liquid nutrient medium A after the sterilization, 30 ℃ of shake-flask culture 42h, it is centrifugal to get the 1ml seed liquor, with stroke-physiological saline solution washing 3 times, place in the aseptic triangular flask that granulated glass sphere is housed, vibration 20min makes it be dispersed into single cell suspension, filter with aseptic glass wool, obtain the unicellular bacteria suspension of starting strain CAU208;
Contained component of described liquid nutrient medium A and proportioning are: peptone 2g, and glucose 5g, molasses 5g, potato immersion liquid 50ml (wherein soluble-containing starch 15g), sucrose 30g, extractum carnis 2.5g, yeast soak powder 4g, water 1000ml;
(2) ultraviolet mutagenesis
The viable count of the starting strain CAU208 bacteria suspension of regulating step (1) gained is 10
6Individual/ml, liquid layer thickness 0.3cm; Carry out ultraviolet mutagenesis, the used ultraviolet lamp power of ultraviolet mutagenesis 17W, irradiation time 3.5min, irradiation distance 28cm; Dilution immediately is coated on the primary dcreening operation isolation medium B after the irradiation, cultivate 33h for 30 ℃, select 60 single bacterium colonies, carrying out shake flask fermentation sieves again, measure degree of hydrolysis, select the highest bacterial strain of 4 strain hydrolysis abilities, make shake flask fermentation more respectively and sieve again, select the bacterial strain CAU208-N of the highest and good stability of a strain hydrolysis ability, and make bacteria suspension and be used for next step nitrosoguanidine mutagenesis;
Described contained component of primary dcreening operation isolation medium B and proportioning are: peptone 2g, yeast soak powder 2g, glucose 5g, skim-milk 7g, agar 11g, water 1000ml;
The step that described shake flask fermentation sieves again is:
(2-1) preparation fermented liquid: by weight ratio behind the uniform mixing, under 0.12mpa pressure, 121 ℃ of sterilizations are cooled to 35 ℃ again, make fermented liquid with nitrogenous source, carbon source and water; Wherein, the proportioning of the weight part sum of nitrogenous source and carbon source and the weight part of water is 4: 100, and nitrogenous source and carbon source weight part proportioning are 3: 1; Described nitrogenous source is for being crushed to 40 purpose soybean isolate protein powders; Described carbon source is a Semen Maydis powder;
60 single bacterium colony seed liquor that (2-2) will obtain and 10: 0.5 by volume ratio uniform mixing of fermented liquid carry out shake flask fermentation on shaking table;
(3) nitrosoguanidine mutagenesis
The viable count of CAU208-N bacteria suspension is transferred to 10
8Individual/ml, be added in the nitrosoguanidine that concentration is 900ug/ml 33 ℃ of water bath processing 25min, intermittently concussion; Be applied to immediately after the mutagenesis on the primary dcreening operation isolation medium B, cultivate 33h for 30 ℃, select 65 single bacterium colonies, carrying out shake flask fermentation sieves again, the same step of the method that its shake flask fermentation sieves again (2), obtain the highest strain bacterial strain CAU208-N1 of hydrolysis ability, and make bacteria suspension be used for next step the ultraviolet ray with the nitrosoguanidine complex mutation;
(4) ultraviolet ray and nitrosoguanidine complex mutation
The viable count of CAU208-N1 bacteria suspension is transferred to 10
8Individual/ml is added to nitrosoguanidine liquid, and making the nitrosoguanidine final concentration is 1100ug/ml, 31 ℃ of water bath processing 25min, and the liquid layer thickness 0.3cm after nitrosoguanidine is handled is got in intermittently concussion; Carry out ultraviolet mutagenesis, the used ultraviolet lamp power of ultraviolet mutagenesis 17W, irradiation time 7min is apart from 28cm; Be applied to immediately after the mutagenesis on the primary dcreening operation isolation medium B, cultivate 34h for 29 ℃, select 55 single bacterium colonies, carry out shake flask fermentation and sieve again, its shake flask fermentation sieves same step (2) again, obtains the highest strain bacterial strain CAU208-N5 of hydrolysis ability;
(5) set by step (4) circulation repetition ultraviolet mutagenesis → nitrosoguanidine mutagenesis → ultraviolet ray and nitrosoguanidine complex mutation are 2 times, obtain superior strain CAU208-1 at last.
The superior strain CAU208-1 that obtains is deposited on the solid slant culture base C in 4 ℃, and switching in per 3 months once;
Described solid slant culture base C consists of: peptone 1.3g, and glucose 5g, molasses 3g, Zulkovsky starch 10g, sucrose 10g, extractum carnis 2g, yeast soak powder 5g, agar 13g, water 1000ml.
But the genus bacillus ability of being hydrolyzed to above-mentioned three embodiment gained high proteinase yields is tested, but the genus bacillus hydrolysis ability of embodiment 1 gained high proteinase yield the strongest be 25.78% (as shown in table 1).But the genus bacillus of embodiment 1 gained high proteinase yield is preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on June 28th, 2004, the classification called after: bacillus sp, its preserving number are CGMCCNo.1180.
The hydrolysis ability to the soybean protein fermentation of each step mutant strain and maternal plant relatively sees the following form 1 among the embodiment 1:
The comparison of table 1, starting strain and mutagenic fungi hydrolysis ability
Proteinic degree of hydrolysis is meant and utilizes method biological or chemistry that natural protein is carried out catalytic hydrolysis that the ratio that the peptide bond that is ruptured by hydrolysis accounts for total peptide bond in the protein molecule claims degree of hydrolysis (DH%).
Ninhydrin method determination of color degree of hydrolysis (Degree of Hydrolysis, method DH) is as follows:
1. the drafting of typical curve: at first prepare the standard glycine solution, its concentration is 20ug/ml; Then according to the listed application of sample amount order of table 2 application of sample.
Table 2, glycine, triketohydrindene hydrate and alcoholic acid application of sample amount
Press the listed application of sample amount of table 2, in tool plug test tube, add standard glycine solution, distilled water and triketohydrindene hydrate developer successively, in boiling water, heat 15min, in cold water, cool off 5min then; Add 40% ethanolic soln termination reaction again, place 15min; Return to zero in the mensuration A (absorbancy) of 570nm place value with the blank pipe.
2. in the Hydrolyzed protein liquid-NH
2Determination on content
Get a certain amount of protein hydrolyte centrifugal 15min under 4000rpm, get supernatant liquor 100 μ l in the 25ml volumetric flask, constant volume, that is dilute 250 times.Absorption 2ml diluent is in tool plug test tube and add 1ml triketohydrindene hydrate developer, heats 15min behind the mixing in boiling water; In cold water, cool off 5min then; The ethanolic soln termination reaction that adds 5ml 40% again, behind the placement 15min, to be measured.At last, utilize typical curve to calculate in the protein hydrolyte-NH
2Content (umol/ml).
3. degree of hydrolysis (DH%) calculates
In following formula:
The relative glycine concentration of protein hydrolyte: after being meant the absorbancy of the protein hydrolyte of measuring as stated above, the glycine concentration that converses by typical curve.
The protein concentration of fermentation substrate: the protein concn that is meant fermentation substrate (as soybean protein etc.).
75.07: the molecular weight that is glycine.
7.8mmol/g: be the peptide bond number (constant) after the soybean protein complete hydrolysis.
0.33mmol/g: be the peptide bond number (constant) in the fermentation substrate (raw material).
But the genus bacillus of high proteinase yield of the present invention can be used for liquid fermenting and prepares the soybean biological activated peptide forage additive.
Its detailed directions:
1) preparation seed liquor
But in the liquid nutrient medium after genus bacillus～Bacillus sp access sterilization of high proteinase yield of the present invention,, obtain seed liquor in 25～35 ℃ of cultivations 24～48 hours;
Described liquid nutrient medium is prepared in following ratio: peptone 1.3～2.5 g, glucose 3～5g, molasses 3～5g, potato immersion liquid 50～100ml, Zulkovsky starch 10～20g, sucrose 10～30g, extractum carnis 2～3g, yeast soak powder 3.5～5g, dregs of beans 1.5～2 g, somatomedin 1～2ml, water 1000ml; Somatomedin wherein is for containing MgSO
4.7H
2O 20.00～30.00mg/ml, CaCl
2.2H
2O 2.20～3.50mg/ml, ZnSO
4.7H
2O 0.81～1.10mg/ml, FeSO
4.7H
2O 0.44～0.60mg/ml, MnSO
4.H
2O 0.13～0.20mg/ml and CuSO
4.5H
2The mixture of O0.02～0.04mg/ml;
2) fermentative degradation prepares peptide feedstuff additive
Put into fermentor tank after fermentation substrate is crushed to 40～120 orders, add entry and carbon source successively, add entry weight and the proteinaceous weight part proportioning of fermentation substrate be 100: 3～20, add the nitrogen content of the carbon content of carbon source and fermentation substrate the weight part proportioning be 1: 2～30, with fermented liquid at 0.1～0.13MPa, sterilized 10～30 minutes for 115～125 ℃, be cooled to 37 ℃, the seed liquor that adds the above-mentioned steps preparation again, the volume ratio of seed liquor and fermented liquid is 0.3～1: 10, mix the back at 25～37 ℃, speed with 0.5～2.5vvm feeds sterile air, stirs fermentation 24～120 hours with 150～300rpm speed, obtains protein hydrolyte, in 140～145 ℃ of sterilizations 2～8 seconds, obtain liquid peptide feedstuff additive; Also this peptide feedstuff additive can be carried out centrifugal slagging-off, ultrafiltration, concentrate, drying etc., obtain the powdery peptide feedstuff additive.
Described fermentation substrate is one or more the mixture that is selected from soybean protein, the gluten;
Described soybean protein comprises soybean protein isolate, dregs of beans, soya-bean cake, soybean;
Described gluten comprises zein, Zein powder and wheat protein;
Described carbon source comprises Semen Maydis powder, brown sugar, edible white sugar, maize treacle, starch, cane molasses, beet sirup.
The peptide feedstuff additive of preparation serves many purposes:
1) this peptide feedstuff additive can be used for improving quality of poultry eggs, increases substantially yolk colourity, increases beneficiating ingredient in the yolk, improves production performances such as eggshell quality and laying rate.
2) this peptide feedstuff additive can absorb rapidly, and the nutritional need when satisfying the prematurity still of young animal Digestive tract can be used in the feed of young animal.
3) this peptide feedstuff additive has low-allergen, can be used to replenish to suffer from anaphylactic disease animal protein requirement or be used as the feed of young animals such as sucking pig, piglet, to prevent that dregs of beans (soya-bean cake) protein fodder of etc.ing is to diseases such as the intestinal mucosa allergy of its generation and oedema.
4) this peptide feedstuff additive can improve the utilization ratio of animal to mineral element, is ideal mineral substance sequestrant.
5) this peptide feedstuff additive has blood fat reducing and cholesterol, adjusting metabolism of fat, improves the livestock products quality, as the nutrition reallocation agent of animal.
6) this peptide feedstuff additive can be used as seasonings, improves the feed palatability.
But the activated peptide forage additive of the genus bacillus preparation of embodiment 4, use high proteinase yield of the present invention is used in Swine Production
Table 3, the result of use in Swine Production
| Processing/repetition | 29～50 age in days average daily gains (Kg) | 29～50 age in days material anharmonic ratioes |
| The blank group | 0.2707±0.01 | 2.23±0.09 |
| Microbiotic control group (duomycin 100mg/kg) | 0.3261±0.02 | 1.68±0.08 |
| 1 group of bioactive peptide (40mg/kg) | 0.3049±0.01 | 1.76±0.07 |
| Peptide 2 group (120mg/kg) | 0.3364±0.02 | 1.68±0.07 |
| 3 groups of bioactive peptides (200mg/kg) | 0.3171±0.01 | 1.84±0.17 |
As can be seen from Table 3, the average daily gain and the feed efficiency of having added 3 bioactive peptide groups of this activated peptide forage additive all are better than the blank group, can obviously improve pig production performance only, i.e. day weight gain and feed efficiency.
Wherein the average daily gain of peptide 2 group (effective constituent 120mg/kg feed) and feed efficiency significantly are better than blank group and microbiotic group, can substitute microbiotic fully.
But the application of activated peptide forage additive in layer breeding of the genus bacillus preparation of embodiment 5, use high proteinase yield of the present invention
Table 4, the application in layer breeding
Annotate: the digital right shoulder of going together indicates different lowercase persons (a, b, c are the method for expressing of test of significance on the statistics) and is significant difference (P＜0.05), and it is difference not remarkable (p＞0.05) that same letter person is arranged.
As can be seen from Table 4, along with the increase of the addition of activated peptide forage additive, laying rate obviously increases, and all has significant difference (p＜0.05) between three groups of A, B, C, wherein, adds bioactive peptide 140ppm and makes laying rate improve 4.6%.Aspect feed efficiency (material/egg ratio), add active Toplink and obviously reduce feedstuff-egg ratio, promptly obviously improved feed efficiency, and significant difference (p＜0.05), but the B group is organized difference not remarkable (p＞0.05) with C; In layer diets, add bioactive peptide 140ppm can obviously increase egg size reach 3.5 grams/piece, increasing degree reaches 5.4%, with control group significant difference (p＜0.05), also obviously can reduce death rate and rate of broken eggs, these all help to reduce aquaculture cost.In a word, this test shows, adds activated peptide forage additive and can obviously improve egg fowl laying rate in egg feedstuff, increases egg size, reduces feedstuff-egg ratio, rate of broken eggs, reduces death rate, improves efficiency of feed utilization, production performance and reduces production costs.
But the application of activated peptide forage additive in the egg duck is raised of the genus bacillus of embodiment 6, high proteinase yield of the present invention preparation
The egg duck in 55 ages in week is divided into 5 groups at random, and each organizes 100 ducks, is respectively
The A group, blank group, the egg duck basal diet of only feeding;
The B group is added the peptide feedstuff additive group, and except that the egg duck basal diet of feeding, feed per ton adds the peptide feedstuff additive of the raising quality of poultry eggs of 2kg embodiment 1;
The C group is added the peptide feedstuff additive group, and except that the egg duck basal diet of feeding, feed per ton adds the peptide feedstuff additive of the raising quality of poultry eggs of 1.92kg embodiment 55;
The D group is added the peptide feedstuff additive group, and except that the egg duck basal diet of feeding, feed per ton adds the peptide feedstuff additive of the raising quality of poultry eggs of 3.8kg embodiment 56;
The E group is added the peptide feedstuff additive group, and except that the egg duck basal diet of feeding, feed per ton adds the peptide feedstuff additive of the raising quality of poultry eggs of 2.6kg embodiment 57;
30 days trial periods, and press above-mentioned grouping revision test 4 times, 4 mean values of character such as laying rate of each group of record, egg size, yolk colourity, shell thickness, dense albumen height are listed in table 6.
Egg duck basal diet, prescription (weight percent) is a corn 59.0, dregs of beans 20.0, the dish dregs of rice 6.0, wheat bran 2.72, stone flour 8.46, hydrogen calcium 1.5, salt 0.32, vegetables oil 1.0,1% egg duck Preblendes 1.0.
Table 5, respectively organize the comparison of duck's egg quality
| Performance | The A group | The B group | The C group | The D group | The E group |
| Average egg weight (g) | 64.32 | 67.28 | 67.78 | 68.34 | 68.11 |
| Dense albumen height (mm) | 5.41 | 5.72 | 5.70 | 5.78 | 5.76 |
| Yolk colourity * | 6.55 | 10.97 | 11.28 | 11.48 | 11.45 |
| Eggshell strength (kg/mm 2) | 3.02 | 3.58 | 3.43 | 3.75 | 3.64 |
| Shell thickness (um) | 360.22 | 380.31 | 380.56 | 381.45 | 381.23 |
| Laying rate (%) | 80.5 | 85.3 | 85.6 | 86.9 | 85.7 |
| Feedstuff-egg ratio material * * | 2.57 | 2.30 | 2.24 | 2.11 | 2.13 |
* yolk colourity is Luo Shi colorimetric fan colourimetric number.
The * feedstuff-egg ratio is consumed forage volume (kg) by every product 1kg egg.
As can be seen from Table 5, the feed peptide feedstuff additive of raising quality of poultry eggs provided by the invention, every index of duck's egg is all significantly better than common mixed feed (A group), and this illustrates that this kind peptide class additive has played obvious facilitation in production performance that improves the egg duck and egg product matter.Particularly improve yolk colourity aspect, can improve nearly 5 colourities, illustrate that this peptide feedstuff additive has extraordinary hyperchromic function.This hyperchromic function mainly is that activated peptide forage additive has promoted being transported in yolk of the carotenoid in the feed, and carotenoid is orange, so the color of yolk has increased.Yolk color is an important indicator of quality of poultry eggs, in the food sensation, important effect is arranged also, the human consumer often links together flavous yolk and health, thereby the peptide feedstuff additive of the raising quality of poultry eggs provided by the invention of feeding makes duck's egg have more market outlook and value.
Claims (2)
1. the genus bacillus of a high proteinase yield, its called after genus bacillus (Bacillus sp) of classifying, its preserving number is CGMCC No.1180.
2. the described genus bacillus of claim 1 prepares application in soybean biological activated peptide forage additive, probiotics and the zymin at liquid fermenting.
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| CN102329787A (en) * | 2011-05-30 | 2012-01-25 | 濮阳泓天威药业有限公司 | Breeding method for improving producing resistant ability of apramycin strains |
| BR112014003950B1 (en) | 2011-08-24 | 2022-01-11 | Dupont Nutrition Biosciences Aps. | USE OF ISOLATED ENZYME-PRODUCING STRAPS OF BACILLUS AND AN ANIMAL FEEDING |
| CN102827788B (en) * | 2012-06-13 | 2014-07-09 | 江南大学 | Bacillus for degrading main allergen of soybean and application of bacillus |
| CN103355501A (en) * | 2013-08-01 | 2013-10-23 | 青岛和美饲料有限公司 | Feed with addition of bioactive peptides and of grow-finishing pigs and preparation method |
| CN105859839B (en) * | 2016-05-24 | 2019-08-30 | 湖北博大生物股份有限公司 | A kind of biologically active peptide and its preparation method and application promoting piglet growth |
| CN110079518A (en) * | 2019-05-23 | 2019-08-02 | 安徽吾悦农业科技有限公司 | The selection of High-Cellulase-Yielding bacterium |
| CN113801825A (en) * | 2021-10-21 | 2021-12-17 | 厦门海嘉成生物科技有限公司 | Bacillus subtilis for producing active peptide and application thereof |
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