CN102488176B - Application of bacillus subtilis aminopeptidase in preparation of yeast extract - Google Patents
Application of bacillus subtilis aminopeptidase in preparation of yeast extract Download PDFInfo
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Abstract
Application of bacillus subtilis aminopeptidase to preparation of yeast extract belongs to the technical field of enzyme preparations and food additives. The application includes: carrying processes during enzymolysis, blending bacillus subtilis Zj016 solid aminopeptidase and commercial neutral protease at a certain stage under certain conditions, proportionally adding the mixture into yeast extract, and shaking for enzymolysis for 24 hours, wherein peptide with molecular weight no less than 500Da in the yeast extract accounts for more than 80% of total peptide weight, total content of free amino acid in yeast extract is increased to more than 11mg/mL, main flavor amino acids are all increased, nutrition value of the yeast extract is further improved, freshness of the yeast extract is improved and can be equivalent to the effect of flavored enzyme, and the yeast extract has the potential of substituting Novozymes's enzymes applied to the yeast extract.
Description
Technical field
The application of a kind of food-grade bacillus subtilis solid ammonia peptase in the yeast extract preparation belongs to enzyme preparation, technical field of food additives.
Background technology
Enzyme preparation refers to the class material with enzyme characteristic that extracts from biology, be widely used in food, brewage, every field such as pharmacy, organic acid, starch sugar, feed, weaving, leather, washing agent and health products, and application more and more widely.Food and people's life is closely related, so enzyme is worth more and more importantly in Application in Food Industry, is curing, is obtaining very big embodiment in the hydrolysis of dairy produce, meat packing, novel nourishing food and protein at present.
Along with development of science and technology, the application of enzyme preparation more and more widely, the application of aminopeptidase is also in continuous expansion.Circumscribed complex enzyme in the existing trade wind flavor Protease F lavorzyme(of Novi belongs in the world); The LAP(of AB company liquid complex enzyme has only part aminopeptidase vigor); There is serial peptase ProteAX etc. in a Japan day wild company, and domestic aminopeptidase product still belongs to blank at present.
Yeast extract is the natural senior flavouring of a new generation, contains rich in protein, free amino acid, flavour nucleotide and heterocyclic compound, has the strong flavor performance that is, and meat fragrance is arranged, and is that collection nutrition, seasoning and health care are the good food dressing of one.Therefore yeast extract is described as third generation monosodium glutamate, has very high nutritive value.
This laboratory has filtered out the bacillus subtilis that aminopeptidase is produced in a strain voluntarily, and bacterial classification derives from traditional food, no security risks, extraction process maturation.This kind of enzyme has the potentiality with the composite application of multiple inscribe protease.Aminopeptidase and restriction endonuclease is composite, be applied in the yeast extract, can obtain higher degree of hydrolysis, the peptide of small-molecular weight obviously increases, and being the amino acid whose amount of flavor simultaneously also has raising, significantly increases delicate flavour, further improved the nutritive value of yeast extract, the effect of composite enzyme enzymolysis yeast is suitable with Novi's letter food flavor enzyme, can replace Novi's letter food flavor enzyme to be applied in the yeast extracting, reduces production cost.
Summary of the invention
The object of the invention provides the application of a kind of bacillus subtilis solid ammonia peptase in the yeast extract preparation, and small molecular weight titanium content significantly improves in the yeast extract that makes, and being the flavor amino acid content also has significantly raising, as the foodstuff flavouring additive.
Technical scheme of the present invention:
The zymotic fluid refrigerated centrifuge of bacillus subtilis Zj016, clarification, ultrafiltration concentrate, desalination, freeze drying, get food-grade solid ammonia peptase (Chinese patent 201010578579.1 is open, publication number CN102078012A, an open day 2011-6-1).The application of a kind of bacillus subtilis solid ammonia peptase in the yeast extract preparation, the yeast autolysis process is after optimizing, with aminopeptidase and neutral proteinase yeast is carried out composite enzymolysis, be foundation with free aminoacid content and glutamic acid content mainly, form the best preparation technology of yeast extract.Extraction process is:
The optimization of condition in the A yeast autolysis process
Investigated the yeast autolysis time by experiment of single factor, the addition of autolysis promoter salt, the concentration of sizing mixing is to the influence of yeast autolysis, be foundation with the amino nitrogen content, draw the optimum condition of yeast autolysis: the yeast autolysis time is 24～26h, the addition of autolysis promoter salt is system concentration 2%, and the concentration of sizing mixing is 8.5% for good.
The optimization of B enzymatic hydrolysis condition
(1) selection of composite enzyme: under the same conditions, behind the yeast autolysis 26h, add solid ammonia peptase (E/S=960U/g) and neutral proteinase (E/S=36U/g) respectively, the composite enzyme combination of solid ammonia peptase (E/S=960U/g) and alkali protease (E/S=36U/g), behind the enzymolysis 10h, 95 ℃ of enzyme 15min that go out, cooling back 5000r/min, 4 ℃ of centrifugal 10min, calculate amino nitrogen content, ammonia nitrogen yield and solid yield, best composite enzyme combination is the combination of solid ammonia peptase and neutral proteinase.
(2) optimization of enzymolysis time: yeast behind the self-dissolving 26h, adds solid ammonia peptase and neutral proteinase under the same conditions, and every 2h sampling and measuring amino nitrogen content, best enzymolysis time is 10h.
(3) optimization of composite enzyme enzyme concentration: yeast is under the same conditions behind the self-dissolving 26h, the composite enzyme that adds different enzyme amounts, behind the enzymolysis 10h, 95 ℃ of enzyme 15min that go out, cooling back 5000r/min, 4 ℃ of centrifugal 10min, measure free amino acid total amount and glutamic acid content in the supernatant, suitable enzyme concentration is E/S=20-30U/g for the neutral proteinase addition, solid ammonia peptase addition is E/S=700-800U/g, and best enzyme concentration is neutral proteinase (E/S=28U/g) and aminopeptidase (E/S=774U/g).
(4) optimization of Novi's trade wind flavor protease enzyme concentration: yeast is under the same conditions behind the self-dissolving 26h, the food flavor enzyme that adds E/S=500U/g, E/S=1000U/g, E/S=5000U/g, E/S=10000U/g, E/S=15000U/g, E/S=20000U/g respectively, behind the enzymolysis 10h, 95 ℃ of enzyme 15min that go out, cooling back 5000r/min, 4 ℃ of centrifugal 10min, measure free amino acid total amount and glutamic acid content in the supernatant, the best enzyme concentration of food flavor enzyme is E/S=1000U/g.
(5) the self-dissolving enzymolysis carries out simultaneously and successively carries out the selection of process: the composite enzyme of aminopeptidase and neutral proteinase initially adds in self-dissolving, or add behind the self-dissolving 26h, again behind the enzymolysis 10h, 95 ℃ of enzyme 15min that go out, cooling back 5000r/min, 4 ℃ of centrifugal 10min, measure free amino acid total amount and glutamic acid content in the supernatant, by comparing, both free aminoacid contents are suitable with glutamic acid content.So process of selecting the self-dissolving enzymolysis to carry out simultaneously.
(6) the self-dissolving enzymolysis carries out the selection of enzymolysis time simultaneously: in the scope of 4～30h, every the 4h sampling, measure the content of ammonia nitrogen in the supernatant, best enzymolysis time is 24h.
C under the self-dissolving enzymatic hydrolysis condition of the best, the comparison of separately self-dissolving, composite enzyme and Novi's trade wind flavor protease
(1) take by weighing 8.5g active dry yeast powder and be dissolved in 100mL distilled water, add 2g salt, add the food flavor enzyme of E/S=1000U/g respectively, or the composite enzyme of aminopeptidase (E/S=774U/g) and neutral proteinase (E/S=28U/g), and do not add protease.At 55 ℃, under the natural pH condition, put into shaking table, 150rpm vibration self-dissolving 24h.
(2) 95 ℃ of enzyme 15min that go out, cooling back 5000r/min, 4 ℃ of centrifugal 10min measure in the supernatant free aminoacid content and mainly are the amino acid whose content of flavor and the peptide molecular weight distribution.
After adding Novi's letter food flavor enzyme, the total content of free amino acid is 11.5494mg/mL in the supernatant behind the enzymolysis, mainly be flavor amino acid: the content of glutamic acid is 1.8412 mg/mL, the content of glycine is 0.4896mg/mL, the content of aspartic acid is 0.4515mg/mL, the content of alanine is 1.3726mg/mL, and molecular weight accounts for 84.67% of total peptide amount less than the peptide of 500Da in the supernatant.
After adding composite enzyme, the total content of free amino acid is 11.7325mg/mL in the supernatant behind the enzymolysis, mainly be flavor amino acid: the content of glutamic acid is 1.7204mg/mL, the content of glycine is 0.5254mg/mL, the content of aspartic acid is 0.5363mg/mL, the content of alanine is 1.4345mg/mL, and molecular weight accounts for 81.56% of total peptide amount less than the peptide of 500Da in the supernatant.
Separately after the self-dissolving (not enzyme-added), the total content of free amino acid is 5.1831mg/mL in the supernatant behind the enzymolysis, mainly be flavor amino acid: the content of glutamic acid is 0.8513mg/mL, the content of glycine is 0.2444mg/mL, the content of aspartic acid is 0.3181mg/mL, and the content of alanine is 1.0830mg/mL.
The enzyme of solid ammonia peptase is lived
The LNA method is measured.During mensuration, at first join the Tris-HCl buffer solution, get 6.075gTris and be dissolved in 900mL distilled water, transfer to pH8.5 with dense HCl, be settled to 1L then., get the 0.1g substrate and be dissolved in 1mL alcohol as substrate with L-leucine paranitroanilinum L-leu-pNA, use Tris-HCl buffer solution constant volume to 100mL then.Get 0.1g solid ammonia peptase powder with Tris-HCl buffer solution constant volume to 100mL as enzyme liquid to be measured.The Tris-HCl buffer solution and the 1mL substrate (L-leucine paranitroanilinum L-leu-pNA) that add 2mL, one adds 1mL enzyme liquid, and another adds 1mL Tris-HCl buffer solution in contrast, and behind 50 ℃ of water-bath 10min, the 405nm colorimetric determination of enzyme is lived.
Enzyme formula alive: enzyme work=A * 105.7 * 4 * n/10
Wherein: A is the absorbance of enzyme liquid to be measured; N is the extension rate of enzyme liquid to be measured.
The enzyme activity definition: 50 ℃, per minute decomposition L-leucine-paranitroanilinum produces the required enzyme amount of 1 micromolar paranitroanilinum and is enzyme unit alive.
Formol titration is measured amino nitrogen content.
The mensuration of supernatant solid content: the self-dissolving supernatant of getting constant weight after centrifugal is dried to constant weight, surveys its solid content.
Ammonia nitrogen yield=(the preceding yeast dry weight of supernatant amino nitrogen content/self-dissolving after the self-dissolving) * 100%
Solid yield=[yeast dry weight before (supernatant gross weight after the self-dissolving * supernatant solid content-additional substance dry weight)/self-dissolving ］ * 100%, in the formula: the wall breaking enzyme that additional substance refers to add, protease, autolysis promoter etc.
The mensuration of free amino acid in the enzymolysis liquid behind the enzymolysis yeast.Behind the composite enzyme enzymolysis of amino acid analysis liquid chromatograph (Ag1100) in the supernatant total content of free amino acid bring up to 11.7325mg/mL from 5.1831mg/mL, the content that mainly is the amino acid alanine of distinguishing the flavor of is brought up to 1.4345mg/mL from 1.0830mg/mL, the content of glycine is brought up to 0.5254mg/mL from 0.2444mg/mL, the content of aspartic acid is brought up to 0.5363mg/mL from 0.3181mg/mL, the content of glutamic acid is brought up to 1.7204mg/mL from 0.8513mg/mL, result such as Fig. 1.
Peptide molecular weight Determination of distribution in the enzymolysis yeast enzymolysis liquid.Waters 600 high performance liquid chromatographs (joining 2487 UV-detectors and Empower work station).Make behind the composite enzyme enzymolysis molecular weight in the enzymolysis liquid by oneself and account for 81.56% of total peptide amount less than the peptide of 500Da, as Fig. 4.Molecular weight accounts for 84.67% of total peptide amount less than the peptide of 500Da in Novi's trade wind flavor protease hydrolyzed liquid, as Fig. 5.
Beneficial effect of the present invention: testing used bacillus subtilis solid ammonia peptase is that this seminar develops voluntarily, behind solid ammonia peptase and the composite enzymolysis yeast of neutral proteinase, the content of free amino acid significantly increases in the enzymolysis gained supernatant, be the amino acid whose amount of flavor significantly raising is also arranged, delicate flavour increases, the peptide content of small-molecular weight increases behind the enzymolysis simultaneously, further improved the nutritive value of yeast extract, after optimizing, form the best preparation technology of yeast extract, relatively the back is found, the hydrolysis result of the hydrolysis result after neutral proteinase and aminopeptidase are composite and Novi's trade wind flavor protease is suitable, possesses the potentiality of using in high-end yeast extract preparation technology.
Description of drawings
Fig. 1 adds free amino acid collection of illustrative plates in the yeast enzymolysis extract of composite enzyme;
Fig. 2 adds free amino acid collection of illustrative plates in the yeast enzymolysis extract of Novi trade wind flavor protease;
Free amino acid collection of illustrative plates in the enzymolysis extract after the not enzyme-added self-dissolving of Fig. 3 yeast;
Fig. 4 adds peptide molecular weight distribution collection of illustrative plates in the yeast enzymolysis extract of composite enzyme;
Fig. 5 adds peptide molecular weight distribution collection of illustrative plates in the yeast enzymolysis extract of Novi trade wind flavor protease;
The composite adding process section of protease schematic diagram in the preparation of Fig. 6 yeast extract.
The specific embodiment
At 4 ℃, the centrifugal 10min of 6000rpm collects the 2.5L zymotic fluid with bacillus subtilis Zj016 zymotic fluid, adds (NH down at 4 ℃
4)
2SO
4500 g (stirring of limit edged), 4 ℃, the centrifugal 10min of 6000rpm collects clarified solution 2.5L, selects for use the milipore filter of 50kDa to be concentrated to 0.85L, flow velocity 30mL/min; Select 0.85L after the milipore filter desalination of 30kDa for use, flow velocity 40 mL/min obtain the solid ammonia peptase after concentrating and desalinating liquid cooling freeze-drying is dry.The enzyme work of the solid ammonia peptase that obtains is 7 * 10
5U/g.Take by weighing 8.5g active dry yeast powder and be dissolved in 100mL distilled water, add 2g salt, with neutral proteinase and the composite enzymolysis that carries out of solid ammonia peptase, wherein the neutral proteinase addition is E/S=28U/g, solid ammonia peptase addition is E/S=774U/g, at 55 ℃, under the natural pH condition, put into shaking table, 150rpm vibration self-dissolving 24h.95 ℃ of enzyme 15min that go out, cooling back 5000r/min, 4 ℃ of centrifugal 10min, be precipitated as the yeast thalline this moment, and supernatant is mixtures of polypeptides.Behind the enzymolysis in the supernatant molecular weight account for 81.56% of total peptide amount less than the peptide of 500Da, and the total content of free amino acid is brought up to 11.7325mg/mL from 5.1831mg/mL, mainly be flavor amino acid: the content of alanine is brought up to 1.4345mg/mL from 1.0830mg/mL, the content of glycine is brought up to 0.5254mg/mL from 0.2444mg/mL, the content of aspartic acid is brought up to 0.5363mg/mL from 0.3181mg/mL, and the content of glutamic acid is brought up to 1.7204mg/mL from 0.8513mg/mL.
At 4 ℃, the centrifugal 10min of 6000rpm collects the 2.5L zymotic fluid with bacillus subtilis Zj016 zymotic fluid, adds (NH down at 4 ℃
4)
2SO
4500 g (stirring of limit edged), 4 ℃, the centrifugal 10min of 6000rpm collects clarified solution 2.5L, selects for use the milipore filter of 50kDa to be concentrated to 0.85L, flow velocity 35mL/min; Select 0.85L after the milipore filter desalination of 30kDa for use, flow velocity 40 mL/min will obtain the solid ammonia peptase in the dry back of concentrating and desalinating liquid cooling freeze-drying.The enzyme work of the solid-state aminopeptidase that obtains is 7 * 10
5U/g.Take by weighing 8.5g active dry yeast powder and be dissolved in 100mL distilled water, add 2g salt, with neutral proteinase and the composite enzymolysis that carries out of solid ammonia peptase, wherein the neutral proteinase addition is E/S=20U/g, solid ammonia peptase addition is E/S=800U/g, at 55 ℃, under the natural pH value condition, put into shaking table, 150rpm vibration self-dissolving 24h.95 ℃ of enzyme 15min that go out, cooling back 5000r/min, 4 ℃ of centrifugal 10min, the total content of free amino acid reaches 11.0738mg/mL, mainly be flavor amino acid: the content of alanine reaches 1.4015mg/mL, the content of glycine reaches 0.5184mg/mL, and the content of aspartic acid reaches 0.5000mg/mL, and the content of glutamic acid reaches 1.7018mg/mL.
Claims (1)
1. the application of bacillus subtilis solid ammonia peptase in the yeast extract preparation is characterized in that:
(1) takes by weighing 8.5g active dry yeast powder and be dissolved in 100mL distilled water, add 2g salt, with neutral proteinase and the composite enzymolysis that carries out of solid ammonia peptase, wherein the neutral proteinase addition is E/S=20-30U/g, solid ammonia peptase addition is E/S=700-800U/g, at 55 ℃, under the natural pH condition, put into shaking table 150rpm vibration self-dissolving 24～26h;
(2) 95 ℃ of enzyme 15min that go out cool off back 4 ℃, the centrifugal 10min of 5000r/min, and be precipitated as the yeast thalline this moment, and supernatant is mixtures of polypeptides, i.e. yeast extract product; Behind the enzymolysis in the supernatant molecular weight account for more than 80% of total peptide amount less than the peptide of 500Da, and the total content of free amino acid is brought up to more than the 11mg/mL, the content that mainly is the amino acid alanine of distinguishing the flavor of is brought up to more than the 1.4mg/mL, the content of glycine is brought up to more than the 0.5mg/mL, the content of aspartic acid is brought up to more than the 0.5mg/mL, and the content of glutamic acid is brought up to more than the 1.7mg/mL.
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Non-Patent Citations (6)
| Title |
|---|
| 刘琦等.氨肽酶的特性及在虾风味调料制备中的应用研究.《江苏食品与发酵》.2006,(第2期), |
| 吕广林等.氨肽酶与中性蛋白酶协同水解大豆分离蛋白的研究.《食品与生物技术学报》.2011,第30卷(第1期), |
| 外加蛋白酶对酵母抽提物酶解效果的工艺研究;陈军等;《广西轻工业》;20111031(第10期);第4-5页 * |
| 氨肽酶与中性蛋白酶协同水解大豆分离蛋白的研究;吕广林等;《食品与生物技术学报》;20110131;第30卷(第1期);第101-105页 * |
| 氨肽酶的特性及在虾风味调料制备中的应用研究;刘琦等;《江苏食品与发酵》;20061231(第2期);第12-15页 * |
| 陈军等.外加蛋白酶对酵母抽提物酶解效果的工艺研究.《广西轻工业》.2011,(第10期), |
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