CN101914513A - Method for fermenting and producing rennin by utilizing streptococcus lactis hydrolyzate as partial nitrogen source - Google Patents
Method for fermenting and producing rennin by utilizing streptococcus lactis hydrolyzate as partial nitrogen source Download PDFInfo
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Abstract
The invention discloses a method for fermenting and producing rennin by utilizing streptococcus lactis hydrolyzate as a partial nitrogen source, and relates to a method for producing rennin by using hydrolyzate. The invention solves the problem of high cost in the prior method for fermenting and producing rennin by microbes, and the problem of environmental pollution caused by direct discharge of streptococcus lactis fermentation waste liquor. The invention hydrolyzes streptococcus lactis by using animal proteases or hydrochloric acid. The production method of the rennin comprises the following steps: adding NaH2PO4, Na2HPO4, glucose, thermostable amylase, Polysorbates 80, corn flour, the streptococcus lactis hydrolyzate and soybean tissue proteins into a triangular flask, and culturing for 96 hours to obtain the rennin. The invention produces the rennin by utilizing the streptococcus lactis hydrolyzate as the nitrogen source instead of soybean tissue proteins, thereby reducing the pollutant discharge and lowering the production cost of the rennin. The invention can be widely used in the field of microbial fermentation.
Description
Technical field
The present invention relates to a kind of microbial fermentation and produce the method for rennin.
Background technology
Rennin belongs to aspartic protease, claims aspartate protease again, is to produce the indispensable preparation of cheese.The tradition source of rennin is the abomasum of phase lactation calf, the high ratio of animal rennet and curdled milk vigor and proteolytic activity becomes makes caseic first-selected enzyme, but growth of animal slowly and cost an arm and a leg, increase easily enterprise cost, and from animal, extract enzyme liquid program and technology is complicated.All contain the proteolytic enzyme that can make curdling solid in the plants such as pawpaw, Fructus Fici, pineapple, pumpkin, acacia, ginkgo, the rennin of plant origin is because of there being too high proteolysis vigor or own poisonous, and therefore a lot of plant rennets do not obtain the large-scale commercial applications application.
Microbial rennet wide material sources, but kind of microorganism fermenting and producing rennin surplus finding have 40 at present, if these microbial host actinomycetes, bacterium and fungi etc., because the control of the method for microbial fermentation easily, cost is low, and safety non-toxic, and is widely used.The nitrogenous source of the rennin of microbial fermentation production at present is mainly from textured soybean protein and Semen Maydis powder, and production cost is higher.
The refuse thalline that is produced in the nisin production directly discharging pollutes environment, make animal feeding-stuff containing somatic protein cost height, added value is low, the refuse thalline then contains abundant organic nitrogen source after hydrolysis, can be used as rennet-fermented raw material, reduce the production cost in the fermenting and producing rennin on the other hand.
Summary of the invention
The present invention produces the cost height of rennin in order to solve present microbial fermentation, the refuse thalline that is produced in the nisin production problem that directly discharging causes environmental pollution, makes animal feeding-stuff containing somatic protein cost height, added value is low, and a kind of method of utilizing streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin is provided.
The present invention utilizes the method for streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin, can realize according to the following steps: one, utilize enzymic hydrolysis or acid-hydrolyzed method hydrolysed lactic acid suis, obtain streptococcus lactis hydrolyzate; Two, the streptococcus lactis hydrolyzate preparation substratum that utilizes step 1 to obtain: with NaH
2PO
4, Na
2HPO
4, glucose, alpha-amylase, tween 80, Semen Maydis powder, streptococcus lactis hydrolyzate and textured soybean protein be dissolved in the water preparation substratum, wherein NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, streptococcus lactis hydrolyzate: 60～160g/L, textured soybean protein: 0～20g/L, with mass concentration is that 5～6% sodium hydroxide is transferred pH to 6.7～6.8, and in the 250mL triangular flask of packing into, 30min sterilizes under 0.14Mpa pressure; Three, inoculation: rice black root Mucor is inserted in the seed liquor substratum, rotating speed at shaking table is 260～280rpm then, temperature is to cultivate 20～24h under 37～38 ℃ of conditions, 1: 40 by volume the amount of seed liquor substratum after the inoculation culture is inoculated in the triangular flask in the step 2 again; Four, cultivate: triangular flask is placed down on the shaking table in 37～38 ℃ of conditions cultivates, the rotating speed of shaking table is 260～280rpm, cultivates 96h, promptly obtains rennin; Wherein total nitrogen content of streptococcus lactis hydrolyzate is 0.922% (quality) in the step 2, and the nitrogen content of textured soybean protein is 8% (quality); The Intake Quantity of substratum is 20% of a triangular flask volume in the step 2; The seed liquor substratum is prepared according to following concentration in the step 3: NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, textured soybean protein: 18g/L, they are dissolved in the water, and 250mL triangular flask substratum loading amount is 20%, is 5～6% sodium hydroxide accent pH to 6.7～6.8 with mass concentration, the 30min that sterilizes under 0.14Mpa pressure obtains finally that rice black root Mucor concentration is 2～6 * 10 in the seed liquor
6Cfu/mL.
The present invention utilizes streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin, reduced the direct environmental pollution caused by discharge of refuse thalline that is produced in the nisin production, as rennet-fermented raw material, reduced the cost of fermenting and producing rennin.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment is utilized the method for streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin, realize according to the following steps: one, utilize enzymic hydrolysis or acid-hydrolyzed method hydrolysed lactic acid suis, obtain streptococcus lactis hydrolyzate; Two, the streptococcus lactis hydrolyzate preparation substratum that utilizes step 1 to obtain: with NaH
2PO
4, Na
2HPO
4, glucose, alpha-amylase, tween 80, Semen Maydis powder, streptococcus lactis hydrolyzate and textured soybean protein be dissolved in the water preparation substratum, wherein NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, streptococcus lactis hydrolyzate: 60～160g/L, textured soybean protein: 0～20g/L, with mass concentration is that 5～6% sodium hydroxide is transferred pH to 6.7～6.8, and in the 250mL triangular flask of packing into, 30min sterilizes under 0.14Mpa pressure; Three, inoculation: rice black root Mucor is inserted in the seed liquor substratum, rotating speed at shaking table is 260～280rpm then, temperature is to cultivate 20～24h under 37～38 ℃ of conditions, again with the seed liquor substratum after the inoculation culture by volume the amount of 1:40 be inoculated in the triangular flask in the step 2; Four, cultivate: triangular flask is placed down on the shaking table in 37～38 ℃ of conditions cultivates, the rotating speed of shaking table is 260～280rpm, cultivates 96h, promptly obtains rennin; Wherein total nitrogen content of streptococcus lactis hydrolyzate is 0.922% (quality) in the step 2, and the nitrogen content of textured soybean protein is 8% (quality); The Intake Quantity of substratum is 20% of a triangular flask volume in the step 2; The seed liquor substratum is prepared according to following concentration in the step 3: NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, textured soybean protein: 18g/L, they are dissolved in the water, and 250mL triangular flask substratum loading amount is 20%, is 5～6% sodium hydroxide accent pH to 6.7～6.8 with mass concentration, the 30min that sterilizes under 0.14Mpa pressure obtains finally that rice black root Mucor concentration is 2～6 * 10 in the seed liquor
6Cfu/mL.
A rice black root Mucor (Rhizomucor miehei) is preserved in Chinese common micro-organisms culture presevation administrative center in the present embodiment, and culture presevation is numbered AS 3.4960.
Embodiment two: present embodiment and embodiment one are different is that the method for enzymic hydrolysis in the step 1 is undertaken by following operation: use the animal prolease hydrolysis streptococcus acidi lactici, the interpolation weight of animal protease is 0.45% of streptococcus acidi lactici thalline weight in wet base, be 6.75 at pH, temperature is hydrolysis 8h under 55 ℃ of water bath condition, and then be warming up to 85 ℃ of maintenance 10min, obtain streptococcus lactis hydrolyzate.Other are identical with embodiment one.
Embodiment three: what present embodiment and embodiment one were different is that acid-hydrolyzed method is undertaken by following operation in the step 1: adopt hydrochloric acid that the pH value of streptococcus acidi lactici is transferred to 1.0, hydrolysis 12h under 121 ℃ condition obtains streptococcus lactis hydrolyzate then.Other are identical with embodiment one.
Embodiment four: what present embodiment and embodiment one, two or three were different is that the streptococcus lactis hydrolyzate concentration that adds in the step 2 is 70～150g/L.Other are identical with embodiment one, two or three.
Embodiment five: what present embodiment and embodiment four were different is that the streptococcus lactis hydrolyzate concentration that adds in the step 2 is 75～140g/L.Other are identical with embodiment four.
Embodiment six: what present embodiment and embodiment five were different is that the streptococcus lactis hydrolyzate concentration that adds in the step 2 is 78g/L.Other are identical with embodiment five.
Embodiment seven: what present embodiment and embodiment six were different is that the textured soybean protein's concentration that adds in the step 2 is 3.6～9.0g/L.Other are identical with embodiment six.
Embodiment eight: what present embodiment and embodiment seven were different is that the textured soybean protein's concentration that adds in the step 2 is 5.4g/L.Other are identical with embodiment seven.
Embodiment nine: present embodiment is utilized the method for streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin, realize according to the following steps: one, use the animal prolease hydrolysis streptococcus acidi lactici, the interpolation weight of animal protease is 0.45% of streptococcus acidi lactici thalline weight in wet base, be 6.75 at pH, temperature is hydrolysis 8h under 55 ℃ of water bath condition, and then be warming up to 85 ℃ of maintenance 10min, obtain streptococcus lactis hydrolyzate; Two, the streptococcus lactis hydrolyzate preparation substratum that utilizes step 1 to obtain: with NaH
2PO
4, Na
2HPO
4, glucose, alpha-amylase, tween 80, Semen Maydis powder and streptococcus lactis hydrolyzate be dissolved in the water preparation substratum, wherein NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, streptococcus lactis hydrolyzate: 156g/L, with mass concentration is that 5～6% sodium hydroxide is transferred pH to 6.7～6.8, and 30min sterilizes under 0.14Mpa pressure; Three, inoculation: rice black root Mucor is inserted in the seed liquor substratum, rotating speed at shaking table is 260～280rpm then, temperature is to cultivate 20～24h under 37～38 ℃ of conditions, again with the seed liquor substratum after the inoculation culture by volume the amount of 1:40 be inoculated in the triangular flask in the step 2; Four, cultivate: triangular flask is placed down on the shaking table in 37～38 ℃ of conditions cultivates, the rotating speed of shaking table is 260rpm, cultivates 96h, promptly obtains rennin; Wherein total nitrogen content of streptococcus lactis hydrolyzate is 0.922% (quality) in the step 2; The Intake Quantity of substratum is 20% of a triangular flask volume in the step 2; The seed liquor substratum is prepared according to following concentration in the step 3: NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, textured soybean protein: 18g/L, they are dissolved in the water, and 250mL triangular flask substratum loading amount is 20%, is 5～6% sodium hydroxide accent pH to 6.7～6.8 with mass concentration, the 30min that sterilizes under 0.14Mpa pressure obtains finally that rice black root Mucor concentration is 2～6 * 10 in the seed liquor
6Cfu/mL.
The rennin that present embodiment is produced vigor after testing is 47.5IMCU/ml.
Embodiment ten: present embodiment is utilized the method for streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin, realize according to the following steps: one, use the animal prolease hydrolysis streptococcus acidi lactici, the interpolation weight of animal protease is 0.45% of streptococcus acidi lactici thalline weight in wet base, be 6.75 at pH, temperature is hydrolysis 8h under 55 ℃ of water bath condition, and then be warming up to 85 ℃ of maintenance 10min, obtain streptococcus lactis hydrolyzate; Two, the streptococcus lactis hydrolyzate preparation substratum that utilizes step 1 to obtain: with NaH
2PO
4, Na
2HPO
4, glucose, alpha-amylase, tween 80, Semen Maydis powder, streptococcus lactis hydrolyzate and textured soybean protein be dissolved in the water preparation substratum, wherein NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, streptococcus lactis hydrolyzate: 140g/L, textured soybean protein: 1.8g/L, with mass concentration is that 5～6% sodium hydroxide is transferred pH to 6.7～6.8, and 30min sterilizes under 0.14Mpa pressure; Three, inoculation: rice black root Mucor is inserted in the seed liquor substratum, rotating speed at shaking table is 260～280rpm then, temperature is to cultivate 20～24h under 37～38 ℃ of conditions, 1: 40 by volume the amount of seed liquor substratum after the inoculation culture is inoculated in the triangular flask in the step 2 again; Four, cultivate: triangular flask is placed down on the shaking table in 37～38 ℃ of conditions cultivates, the rotating speed of shaking table is 260rpm, cultivates 96h, promptly obtains rennin; Wherein total nitrogen content of streptococcus lactis hydrolyzate is 0.922% (quality) in the step 2, and the nitrogen content of textured soybean protein is 8% (quality); The Intake Quantity of substratum is 20% of a triangular flask volume in the step 2; The seed liquor substratum is prepared according to following concentration in the step 3: NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, textured soybean protein: 18g/L, they are dissolved in the water, and 250mL triangular flask substratum loading amount is 20%, is 5～6% sodium hydroxide accent pH to 6.7～6.8 with mass concentration, the 30min that sterilizes under 0.14Mpa pressure obtains finally that rice black root Mucor concentration is 2～6 * 10 in the seed liquor
6Cfu/mL.
The rennin that present embodiment is produced vigor after testing is 55.2IMCU/ml.
Embodiment 11: present embodiment is utilized the method for streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin, realize according to the following steps: one, use the animal prolease hydrolysis streptococcus acidi lactici, the interpolation weight of animal protease is 0.45% of streptococcus acidi lactici thalline weight in wet base, be 6.75 at pH, temperature is hydrolysis 8h under 55 ℃ of water bath condition, and then be warming up to 85 ℃ of maintenance 10min, obtain streptococcus lactis hydrolyzate; Two, the streptococcus lactis hydrolyzate preparation substratum that utilizes step 1 to obtain: with NaH
2PO
4, Na
2HPO
4, glucose, alpha-amylase, tween 80, Semen Maydis powder, streptococcus lactis hydrolyzate and textured soybean protein be dissolved in the water preparation substratum, wherein NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, streptococcus lactis hydrolyzate: 125g/L, textured soybean protein: 3.6g/L, with mass concentration is that 5～6% sodium hydroxide is transferred pH to 6.7～6.8, and 30min sterilizes under 0.14Mpa pressure; Three, inoculation: rice black root Mucor is inserted in the seed liquor substratum, rotating speed at shaking table is 260～280rpm then, temperature is to cultivate 20～24h under 37～38 ℃ of conditions, 1: 40 by volume the amount of seed liquor substratum after the inoculation culture is inoculated in the triangular flask in the step 2 again; Four, cultivate: triangular flask is placed down on the shaking table in 37～38 ℃ of conditions cultivates, the rotating speed of shaking table is 260rpm, cultivates 96h, promptly obtains rennin; Wherein total nitrogen content of streptococcus lactis hydrolyzate is 0.922% (quality) in the step 2, and the nitrogen content of textured soybean protein is 8% (quality); The Intake Quantity of substratum is 20% of a triangular flask volume in the step 2; The seed liquor substratum is prepared according to following concentration in the step 3: NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, textured soybean protein: 18g/L, they are dissolved in the water, and 250mL triangular flask substratum loading amount is 20%, is 5～6% sodium hydroxide accent pH to 6.7～6.8 with mass concentration, the 30min that sterilizes under 0.14Mpa pressure obtains finally that rice black root Mucor concentration is 2～6 * 10 in the seed liquor
6Cfu/mL.
The rennin that present embodiment is produced vigor after testing is 75.1IMCU/ml.
Embodiment 12: present embodiment is utilized the method for streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin, realize according to the following steps: one, use the animal prolease hydrolysis streptococcus acidi lactici, the interpolation weight of animal protease is 0.45% of streptococcus acidi lactici thalline weight in wet base, be 6.75 at pH, temperature is hydrolysis 8h under 55 ℃ of water bath condition, and then be warming up to 85 ℃ of maintenance 10min, obtain streptococcus lactis hydrolyzate; Two, the streptococcus lactis hydrolyzate preparation substratum that utilizes step 1 to obtain: with NaH
2PO
4, Na
2HPO
4, glucose, alpha-amylase, tween 80, Semen Maydis powder, streptococcus lactis hydrolyzate and textured soybean protein be dissolved in the water preparation substratum, wherein NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, streptococcus lactis hydrolyzate: 109g/L, textured soybean protein: 5.4g/L, with mass concentration is that 5～6% sodium hydroxide is transferred pH to 6.7～6.8, and 30min sterilizes under 0.14Mpa pressure; Three, inoculation: rice black root Mucor is inserted in the seed liquor substratum, rotating speed at shaking table is 260～280rpm then, temperature is to cultivate 20～24h under 37～38 ℃ of conditions, 1: 40 by volume the amount of seed liquor substratum after the inoculation culture is inoculated in the triangular flask in the step 2 again; Four, cultivate: triangular flask is placed down on the shaking table in 37～38 ℃ of conditions cultivates, the rotating speed of shaking table is 260rpm, cultivates 96h, promptly obtains rennin; Wherein total nitrogen content of streptococcus lactis hydrolyzate is 0.922% (quality) in the step 2, and the nitrogen content of textured soybean protein is 8% (quality); The Intake Quantity of substratum is 20% of a triangular flask volume in the step 2; The seed liquor substratum is prepared according to following concentration in the step 3: NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, textured soybean protein: 18g/L, they are dissolved in the water, and 250mL triangular flask substratum loading amount is 20%, is 5～6% sodium hydroxide accent pH to 6.7～6.8 with mass concentration, the 30min that sterilizes under 0.14Mpa pressure obtains finally that rice black root Mucor concentration is 2～6 * 10 in the seed liquor
6Cfu/mL.
The rennin that present embodiment is produced vigor after testing is 91.8IMCU/ml.
Embodiment 13: present embodiment is utilized the method for streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin, realize according to the following steps: one, use the animal prolease hydrolysis streptococcus acidi lactici, the interpolation weight of animal protease is 0.45% of streptococcus acidi lactici thalline weight in wet base, be 6.75 at pH, temperature is hydrolysis 8h under 55 ℃ of water bath condition, and then be warming up to 85 ℃ of maintenance 10min, obtain streptococcus lactis hydrolyzate; Two, the streptococcus lactis hydrolyzate preparation substratum that utilizes step 1 to obtain: with NaH
2PO
4, Na
2HPO
4, glucose, alpha-amylase, tween 80, Semen Maydis powder, streptococcus lactis hydrolyzate and textured soybean protein be dissolved in the water preparation substratum, wherein NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, streptococcus lactis hydrolyzate: 93.6g/L, textured soybean protein: 7.2g/L, with mass concentration is that 5～6% sodium hydroxide is transferred pH to 6.7～6.8, and 30min sterilizes under 0.14Mpa pressure; Three, inoculation: rice black root Mucor is inserted in the seed liquor substratum, rotating speed at shaking table is 260～280rpm then, temperature is to cultivate 20～24h under 37～38 ℃ of conditions, again with the seed liquor substratum after the inoculation culture by volume the amount of 1:40 be inoculated in the triangular flask in the step 2; Four, cultivate: triangular flask is placed down on the shaking table in 37～38 ℃ of conditions cultivates, the rotating speed of shaking table is 260rpm, cultivates 96h, promptly obtains rennin; Wherein total nitrogen content of streptococcus lactis hydrolyzate is 0.922% (quality) in the step 2, and the nitrogen content of textured soybean protein is 8% (quality); The Intake Quantity of substratum is 20% of a triangular flask volume in the step 2; The seed liquor substratum is prepared according to following concentration in the step 3: NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, textured soybean protein: 18g/L, they are dissolved in the water, and 250mL triangular flask substratum loading amount is 20%, is 5～6% sodium hydroxide accent pH to 6.7～6.8 with mass concentration, the 30min that sterilizes under 0.14Mpa pressure obtains finally that rice black root Mucor concentration is 2～6 * 10 in the seed liquor
6Cfu/mL.
The rennin that present embodiment is produced vigor after testing is 115.3IMCU/ml.
Embodiment 14: present embodiment is utilized the method for streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin, realize according to the following steps: one, use the animal prolease hydrolysis streptococcus acidi lactici, the interpolation weight of animal protease is 0.45% of streptococcus acidi lactici thalline weight in wet base, be 6.75 at pH, temperature is hydrolysis 8h under 55 ℃ of water bath condition, and then be warming up to 85 ℃ of maintenance 10min, obtain streptococcus lactis hydrolyzate; Two, the streptococcus lactis hydrolyzate preparation substratum that utilizes step 1 to obtain: with NaH
2PO
4, Na
2HPO
4, glucose, alpha-amylase, tween 80, Semen Maydis powder, streptococcus lactis hydrolyzate and textured soybean protein be dissolved in the water preparation substratum, wherein NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, streptococcus lactis hydrolyzate: 78g/L, textured soybean protein: 9g/L, with mass concentration is that 5～6% sodium hydroxide is transferred pH to 6.7～6.8, and 30min sterilizes under 0.14Mpa pressure; Three, inoculation: rice black root Mucor is inserted in the seed liquor substratum, rotating speed at shaking table is 260～280rpm then, temperature is to cultivate 20～24h under 37～38 ℃ of conditions, 1: 40 by volume the amount of seed liquor substratum after the inoculation culture is inoculated in the triangular flask in the step 2 again; Four, cultivate: triangular flask is placed down on the shaking table in 37～38 ℃ of conditions cultivates, the rotating speed of shaking table is 260rpm, cultivates 96h, promptly obtains rennin; Wherein total nitrogen content of streptococcus lactis hydrolyzate is 0.922% (quality) in the step 2, and the nitrogen content of textured soybean protein is 8% (quality); The Intake Quantity of substratum is 20% of a triangular flask volume in the step 2; The seed liquor substratum is prepared according to following concentration in the step 3: NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, textured soybean protein: 18g/L, they are dissolved in the water, and 250mL triangular flask substratum loading amount is 20%, is 5～6% sodium hydroxide accent pH to 6.7～6.8 with mass concentration, the 30min that sterilizes under 0.14Mpa pressure obtains finally that rice black root Mucor concentration is 2～6 * 10 in the seed liquor
6Cfu/mL.
The rennin that present embodiment is produced vigor after testing is 134.0IMCU/ml.
Embodiment 15: present embodiment is utilized the method for streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin, realize according to the following steps: one, adopt hydrochloric acid that the pH value of streptococcus acidi lactici is transferred to 1.0, hydrolysis 12h under 121 ℃ condition obtains streptococcus lactis hydrolyzate then; Two, the streptococcus lactis hydrolyzate preparation substratum that utilizes step 1 to obtain: with NaH
2PO
4, Na
2HPO
4, glucose, alpha-amylase, tween 80, Semen Maydis powder and streptococcus lactis hydrolyzate be dissolved in the water preparation substratum, wherein NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, streptococcus lactis hydrolyzate: 160g/L is dissolved in the water them, with mass concentration is that 5～6% sodium hydroxide is transferred pH to 6.7～6.8, and 30min sterilizes under 0.14Mpa pressure; Three, inoculation: rice black root Mucor is inserted in the seed liquor substratum, rotating speed at shaking table is 260～280rpm then, temperature is to cultivate 20～24h under 37～38 ℃ of conditions, 1: 40 by volume the amount of seed liquor substratum after the inoculation culture is inoculated in the triangular flask in the step 2 again; Four, cultivate: triangular flask is placed down on the shaking table in 37～38 ℃ of conditions cultivates, the rotating speed of shaking table is 260rpm, cultivates 96h, promptly obtains rennin; Wherein total nitrogen content of streptococcus lactis hydrolyzate is 0.922% (quality) in the step 2; The Intake Quantity of substratum is 20% of a triangular flask volume in the step 2; The seed liquor substratum is prepared according to following concentration in the step 3: NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, textured soybean protein: 18g/L, they are dissolved in the water, and 250mL triangular flask substratum loading amount is 20%, is 5～6% sodium hydroxide accent pH to 6.7～6.8 with mass concentration, the 30min that sterilizes under 0.14Mpa pressure obtains finally that rice black root Mucor concentration is 2～6 * 10 in the seed liquor
6Cfu/mL.
The rennin that present embodiment is produced vigor after testing is 84.0IMCU/ml.
Embodiment 16: present embodiment is utilized the method for streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin, realize according to the following steps: one, adopt hydrochloric acid that the pH value of streptococcus acidi lactici is transferred to 1.0, hydrolysis 12h under 121 ℃ condition obtains streptococcus lactis hydrolyzate then; Two, the streptococcus lactis hydrolyzate preparation substratum that utilizes step 1 to obtain: with NaH
2PO
4, Na
2HPO
4, glucose, alpha-amylase, tween 80, Semen Maydis powder, streptococcus lactis hydrolyzate and textured soybean protein be dissolved in the water preparation substratum, wherein NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, streptococcus lactis hydrolyzate: 80g/L, textured soybean protein: 9g/L, be that 5～6% sodium hydroxide is transferred pH to 6.7～6.8 with mass concentration then, 30min sterilizes under 0.14Mpa pressure; Three, inoculation: rice black root Mucor is inserted in the seed liquor substratum, rotating speed at shaking table is 260～280rpm then, temperature is to cultivate 20～24h under 37～38 ℃ of conditions, 1: 40 by volume the amount of seed liquor substratum after the inoculation culture is inoculated in the triangular flask in the step 2 again; Four, cultivate: triangular flask is placed down on the shaking table in 37～38 ℃ of conditions cultivates, the rotating speed of shaking table is 260rpm, cultivates 96h, promptly obtains rennin; Wherein total nitrogen content of streptococcus lactis hydrolyzate is 0.922% (quality) in the step 2, and the nitrogen content of textured soybean protein is 8% (quality); The Intake Quantity of substratum is 20% of a triangular flask volume in the step 2; The seed liquor substratum is prepared according to following concentration in the step 3: NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, textured soybean protein: 18g/L, they are dissolved in the water, and 250mL triangular flask substratum loading amount is 20%, is 5～6% sodium hydroxide accent pH to 6.7～6.8 with mass concentration, the 30min that sterilizes under 0.14Mpa pressure obtains finally that rice black root Mucor concentration is 2～6 * 10 in the seed liquor
6Cfu/mL.
The rennin that present embodiment is produced vigor after testing is 118.5IMCU/ml.
Claims (8)
1. utilize the method for streptococcus lactis hydrolyzate, it is characterized in that it realizes according to the following steps: one, utilize enzymic hydrolysis or acid-hydrolyzed method hydrolysed lactic acid suis, obtain streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin; Two, the streptococcus lactis hydrolyzate preparation substratum that utilizes step 1 to obtain: with NaH
2PO
4, Na
2HPO
4, glucose, alpha-amylase, tween 80, Semen Maydis powder, streptococcus lactis hydrolyzate and textured soybean protein be dissolved in the water preparation substratum, wherein NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, streptococcus lactis hydrolyzate: 60～160g/L, textured soybean protein: 0～20g/L, with mass concentration is that 5～6% sodium hydroxide is transferred pH to 6.7～6.8, and in the 250mL triangular flask of packing into, 30min sterilizes under 0.14Mpa pressure; Three, inoculation: rice black root Mucor is inserted in the seed liquor substratum, rotating speed at shaking table is 260～280rpm then, temperature is to cultivate 20～24h under 37～38 ℃ of conditions, again with the seed liquor after the inoculation culture by volume the amount of 1:40 be inoculated in the triangular flask in the step 2; Four, cultivate: triangular flask is placed down on the shaking table in 37～38 ℃ of conditions cultivates, the rotating speed of shaking table is 260～280rpm, cultivates 96h, promptly obtains rennin; Wherein total nitrogen content of streptococcus lactis hydrolyzate is 0.922% (quality) in the step 2, and the nitrogen content of textured soybean protein is 8% (quality); The Intake Quantity of substratum is 20% of a triangular flask volume in the step 2; The seed liquor substratum is prepared according to following concentration in the step 3: NaH
2PO
4: 3.25g/L, Na
2HPO
4: 3.11g/L, glucose: 2.14g/L, alpha-amylase: 20g/L, tween 80: 0.13g/L, Semen Maydis powder: 70g/L, textured soybean protein: 18g/L, they are dissolved in the water, and 250mL triangular flask substratum loading amount is 20%, is 5～6% sodium hydroxide accent pH to 6.7～6.8 with mass concentration, the 30min that sterilizes under 0.14Mpa pressure obtains finally that rice black root Mucor concentration is 2～6 * 10 in the seed liquor
6Cfu/mL.
2. the method for utilizing streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin according to claim 1, the method that it is characterized in that enzymic hydrolysis in the step 1 is undertaken by following operation: use the animal prolease hydrolysis streptococcus acidi lactici, the interpolation weight of animal protease is 0.45% of streptococcus acidi lactici thalline weight in wet base, be 6.75 at pH, temperature is hydrolysis 8h under 55 ℃ of water bath condition, and then be warming up to 85 ℃ of maintenance 10min, obtain streptococcus lactis hydrolyzate.
3. the method for utilizing streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin according to claim 1, it is characterized in that acid-hydrolyzed method is undertaken by following operation in the step 1: adopt hydrochloric acid that the pH value of streptococcus acidi lactici is transferred to 1.0, hydrolysis 12h under 121 ℃ condition obtains streptococcus lactis hydrolyzate then.
4. according to claim 1, the 2 or 3 described methods of utilizing streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin, it is characterized in that the streptococcus lactis hydrolyzate concentration that adds in the step 2 is 70～150g/L.
5. the method for utilizing streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin according to claim 4 is characterized in that the streptococcus lactis hydrolyzate concentration that adds in the step 2 is 75～140g/L.
6. the method for utilizing streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin according to claim 5 is characterized in that the streptococcus lactis hydrolyzate concentration that adds in the step 2 is 78g/L.
7. the method for utilizing streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin according to claim 6 is characterized in that the textured soybean protein's concentration that adds in the step 2 is 3.6～9.0g/L.
8. the method for utilizing streptococcus lactis hydrolyzate as the partial nitrogen source fermenting and producing rennin according to claim 7 is characterized in that the textured soybean protein's concentration that adds in the step 2 is 5.4g/L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045485A (en) * | 2011-10-14 | 2013-04-17 | 中国农业大学 | Rhizomucor miehei strain and application thereof to preparation of Beta-glucanase and chymosin |
| CN112961796A (en) * | 2021-01-29 | 2021-06-15 | 湖北华扬科技发展有限公司 | Method for producing clostridium butyricum by fermenting enterococcus faecium fermentation waste liquid |
-
2010
- 2010-09-15 CN CN2010102819531A patent/CN101914513A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045485A (en) * | 2011-10-14 | 2013-04-17 | 中国农业大学 | Rhizomucor miehei strain and application thereof to preparation of Beta-glucanase and chymosin |
| CN103045485B (en) * | 2011-10-14 | 2015-04-22 | 中国农业大学 | Rhizomucor miehei strain and application thereof in preparation of Beta-glucanase and chymosin |
| CN112961796A (en) * | 2021-01-29 | 2021-06-15 | 湖北华扬科技发展有限公司 | Method for producing clostridium butyricum by fermenting enterococcus faecium fermentation waste liquid |
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