CN110742260A

CN110742260A - Corn-derived flavoring liquid and preparation method thereof

Info

Publication number: CN110742260A
Application number: CN201911162260.8A
Authority: CN
Inventors: 袁利; 栾小茜
Original assignee: Qingdao Kesu Biotechnology Co Ltd
Current assignee: Qingdao Kesu Biotechnology Co Ltd
Priority date: 2019-11-25
Filing date: 2019-11-25
Publication date: 2020-02-04

Abstract

The invention provides a preparation method of corn-derived flavoring liquid, which comprises the steps of carrying out enzymolysis on corn protein and solid corn syrup (or hydrolyzed corn starch sugar) by using compound aminopeptidase, taking the enzymolysis product as a substrate, and adding immobilized corynebacterium glutamicum and immobilized cells of ammonia-producing brevibacterium to carry out biotransformation. In the invention, aminopeptidase with stronger specificity is used for hydrolysis, and amino acid in the raw material is released to the maximum extent. And the immobilized cells are used for recycling components such as starch, sugar, peptide, inorganic nitrogen and the like in the raw materials to produce flavor amino acid and nucleotide. The final product obtained by the invention has high content of total nitrogen and amino acid nitrogen, has few impurities, and can be used for flavoring food.

Description

Corn-derived flavoring liquid and preparation method thereof

Technical Field

The invention belongs to the technical field of food preparation, and particularly relates to a preparation method of a corn-derived flavoring liquid.

Background

Amino acid seasoning liquid is usually used for preparing seasoning products, and generally, a large amount of flavor development amino acids (glutamic acid, glycine, alanine, proline, aspartic acid, serine and the like) are required, the higher the content of the flavor development amino acids is, the higher the quality of the seasoning liquid is, and the quality of the seasoning liquid is usually measured by amino acid nitrogen and total nitrogen. The corn protein has low price and high protein content, and is an ideal protein raw material. However, zein contains zein, gluten, globulin, protein and the like, has complex composition, rough mouthfeel and poor water solubility, and limits the application in the food industry. The corn protein can increase water solubility through hydrolysis, and can release various flavor amino acids and functional oligopeptides.

The corn protein hydrolysis method comprises an acid-base method, an enzyme method, a microbial method and the like, wherein the acid-base hydrolysis is thorough, but the hydrolysis is excessive, and chloropropanol and other substances harmful to human bodies are generated. The microbial method generally uses aspergillus and bacillus for fermentation, has rich enzyme systems, can produce a large amount of amino acids, but needs to consume carbon and nitrogen sources for the growth of thalli, has long growth period, is easy to pollute, and is not easy to separate products from the thalli. The enzymatic reaction condition is mild, environment-friendly and harmless, and an acid-base method is gradually replaced, but the enzymatic degradation efficiency is not as thorough as that of the acid-base method, protein can not be directly decomposed into amino acid, and a plurality of oligopeptides with the molecular weight below 1000Da are generated.

It has been reported that the distribution of amino acids in zein is not uniform, and the content of hydrophobic amino acids (such as leucine, alanine, isoleucine, valine, etc.) is high, so that the water solubility is poor. The traditional protease hydrolysis method or microbial fermentation method is used for conversion, the hydrolysis rate is only about 60%, the amino acid generation rate is low, and the index requirements of the seasoning liquid are difficult to meet.

Disclosure of Invention

The invention aims to provide a corn-derived flavoring liquid and a preparation method thereof, so as to solve the problems.

The invention firstly provides a corn protease hydrolysate, which is an enzymolysis solution obtained by carrying out enzymolysis on corn protein and corn syrup by using flavourzyme, then adding proline aminopeptidase and leucine aminopeptidase for enzymolysis, and finally adding lysine aminopeptidase for enzymolysis

Wherein the corn syrup may be replaced in whole or in part by hydrolyzed corn starch sugar;

the flavourzyme carries out enzymolysis, and the enzymolysis conditions are as follows: pH6.5-7.5, and temperature is 50 deg.C;

the proline aminopeptidase and the leucine aminopeptidase are subjected to enzymolysis, wherein the enzymolysis condition is pH6.5-7, and the temperature is 40 ℃;

the lysine aminopeptidase is used for enzymolysis, wherein the enzymolysis condition is pH 7.5 and the temperature is 40-50 ℃;

the invention also provides a corn protein fermentation liquor, which is prepared by fermenting the enzymolysis liquor by using corynebacterium glutamicum and brevibacterium ammoniagenes;

the corynebacterium glutamicum and the brevibacterium ammoniagenes are fermented under the fermentation condition of 30-37 ℃.

The corynebacterium glutamicum and the brevibacterium ammoniagenes adopt immobilized corynebacterium glutamicum and brevibacterium ammoniagenes.

The corn protein fermentation liquor prepared by the invention can be used for preparing foods such as seasoning liquid and the like.

The invention adopts the immobilized corynebacterium glutamicum and brevibacterium ammoniagenes to ferment in the prepared maize protein, and can further utilize and transform the maize protein; the method can produce flavor development substances such as glutamic acid, threonine, lysine, inosinic acid, guanylic acid and the like, does not introduce additional impurities, can be recycled, and has a simpler material separation mode by an immobilization mode.

Drawings

FIG. 1: a pH and temperature profile of proline aminopeptidase enzymolysis optimum;

FIG. 2: leucine aminopeptidase enzymolysis optimal pH and temperature graphs;

FIG. 3: the optimum pH and temperature diagram of the enzymolysis of lysine aminopeptidase;

FIG. 4: amino acid composition of hydrolyzed corn protein.

Detailed Description

The corn flour is also called corn flour, is a crude product obtained by drying and grinding corn, and mainly contains carbohydrate (starch) with the content of about 70 percent and protein of about 8 percent. Corn starch can be obtained by refining corn flour, corn protein is a by-product of the processing, the main components are alcohol soluble protein and globulin, the protein content is more than 65%, the starch content is 15%, and the cellulose content is 2%. Corn protein is used as a cheap protein source, has poor solubility, is difficult to utilize, has unbalanced amino acid composition, can only be used for feed generally, and even is discharged along with waste water, thereby not only wasting but also polluting the environment. To make high-value use of zein, it is first hydrolyzed.

At present, neutral protease, alkaline protease and flavor protease (flavor enzyme) are mainly used for corn protein hydrolysis, the protease can hydrolyze corn protein into polypeptide, the flavor enzyme can reduce the molecular weight of the polypeptide, and partial peptide is further degraded into amino acid to improve the taste and flavor of hydrolysate. The flavourzyme is extracted by fermenting aspergillus oryzae, is a mixed enzyme and has the characteristics of incision and excision enzyme. The flavor enzyme has poor specificity and random exo-site, and has certain limit on the generation rate of amino acid.

According to the invention, corn protein and solid corn syrup (or hydrolyzed corn starch sugar) are mixed, treated by flavourzyme, hydrolyzed by proline aminopeptidase, the Pro concentration is increased by 2.7 times, and after the mixture is treated by leucine aminopeptidase in a synergistic manner, Leu and Ile are 2.75 and 4 times before enzymolysis, and the content of other free hydrophobic amino acids in hydrolysate is also greatly increased. Further, the use of immobilized Corynebacterium glutamicum is one of industrially important glutamic acid-producing bacteria, and is also a main producing bacterium of threonine and lysine, and it is possible to secrete amino acids using various carbon sources such as glucose, fructose, sucrose, organic acids, and the like. Brevibacterium ammoniagenes is one of flavor nucleotide producing bacteria such as disodium inosinate and disodium guanylate, and corn protein can be further utilized and transformed by fermenting by using a method of co-immobilizing cells by using the two bacteria. Increasing the content of flavoring substances such as threonine, lysine, adenylic acid, guanylic acid, etc.

EXAMPLE 1 conditions of action of aminopeptidase

The enzyme activities of proline aminopeptidase, leucine aminopeptidase and lysine aminopeptidase are marked by taking L-proline-p-nitroaniline Pro-pNA, L-leucine-p-nitroaniline Leu-pNA and L-lysine-p-nitroaniline Lys-pNA as substrates (a substrate storage solution is prepared by Tris-HCl buffer solution with pH 7.5 and the concentration is 4.25 mM). The reaction system comprises 2mL of Tris-HCl pH 7.5 buffer solution, 1mL of substrate storage solution and 1mL of diluted enzyme solution, and is put into ice water immediately after water bath at 50 ℃ for 10min to stop the reaction and quickly measure the absorbance at the wavelength of 405 nm. The amount of enzyme required to decompose Xaa-pNA at 50 ℃ per minute to produce 1. mu.M p-nitroaniline was one enzyme activity unit (U).

The corn protein is composed of macromolecular protein and is rich in proline, leucine and the like, after the corn protein is treated by the flavor enzyme, the corn protein is internally cut into corn peptide, and after the corn peptide is continuously cut by the aminopeptidase, free amino acid is released. The amino group of the free amino acid can react with ninhydrin to produce a bluish-violet compound whose shade of color is proportional to the content of free amino acid. Taking a 20ml test tube, taking 1ml of a sample to be tested, adding l ml of distilled water, adding ninhydrin (0.6 g of recrystallized ninhydrin is weighed and put in a beaker, adding 15ml of n-propanol, 30ml of n-butanol, 60ml of ethylene glycol and 9ml of acetate buffer solution with pH4.54 for uniform mixing) 3.0ml and 0.1ml of 0.1% ascorbic acid for uniform mixing, and sealing. Heating in boiling water bath for 15min, shaking in cold water for cooling, dissolving in 60% ethanol to 20ml, shaking, and measuring absorbance A at 570 mn.

The flavourzyme-treated zein was further hydrolyzed at various pH values and at various temperatures by various aminopeptidases, added at 20U/ml, and the value of free amino acids was measured to determine the optimum reaction pH and temperature.

The pH value of the reaction suitable for hydrolyzing the corn protein by the proline aminopeptidase is 7, and the enzyme activity is seriously lost in an acidic environment. The catalytic activity is highest when the catalyst is reacted at 40-50 ℃, the activity is basically lost after the catalyst is reacted for 1h at 60 ℃, and the high temperature tolerance is poor (figure 1). The suitable reaction pH of the leucine aminopeptidase is 6.5, and the pH is 5.0-8.0, so that the leucine aminopeptidase has good stability. The catalytic activity is highest when the reaction is carried out at about 40 ℃, and the catalytic effect is good at low temperature (figure 2). A suitable reaction pH for lysine aminopeptidase is 7.5, and is a neutral enzyme. The catalyst has good catalytic activity when reacting at 40-50 ℃ and better tolerance to high temperature and low temperature (figure 3).

The action substrate of proline aminopeptidase, leucine aminopeptidase and lysine aminopeptidase was analyzed to determine the order of addition.

1mM of the polypeptide substrate was prepared with 50mM of PBS buffer pH 7.5, 0.1mL of the diluted enzyme solution (diluted with 50mM PBS buffer pH 7.5) and the polypeptide solution were added first, reacted at 50 ℃ for 10min, 2.5mL of acetic acid and 2.5mL of acidic ninhydrin solution were immediately added, mixed well, cooled on ice after 30min in a boiling water bath, and the absorbance at 480nm was measured. The blank was treated the same way with 0.1ml PBS buffer instead of the diluted enzyme solution. The amount of enzyme required to break down the polypeptide at 50 ℃ per minute to release 1. mu.M amino acid is defined as one unit of hydrolytic activity (U) of the oligopeptide by the aminopeptidase.

The oligopeptides were first dissolved in a small amount of DMSO solvent and then diluted to 1mM with 0.05M PBS buffer pH 7.5. Mixing the enzyme solution (diluted with 50mM PBS buffer solution with pH 7.5) and the polypeptide solution, reacting at 50 deg.C for 10min, immediately adding 2.5mL acetic acid and 2.5mL acidic ninhydrin solution, mixing, boiling in water bath for 30min, cooling on ice, and measuring absorbance at 480 nm. The blank was treated the same way with 0.1ml PBS buffer instead of the diluted enzyme solution. The amount of free amino acids released was determined by ninhydrin assay to determine the ability of the aminopeptidase to hydrolyze the di-tripeptide, with the highest ability being 100%.

Table 1: substrate selectivity profile for proline aminopeptidase

Substrate	Relative Activity (%)
		Pro-pNA (Standard control)	100.00
Pro-Phe	92.51
		Pro-Phe-Gly	104.50
Leu-Pro	11.05
		Pro-Leu	67.14
Pro-Leu-Ser	72.87
		Pro-Pro	57.30

Table 2: substrate specificity of leucine aminopeptidase

Substrate	Relative Activity (%)
		Leu-pNA (Standard control)	100.00
Pro-pNA	32.40
		Gly-Pro	0.41
Gly-pNA	12.74
		Glu-pNA	29.22
Ile-pNA	88.31

Table 3: substrate specificity of lysine aminopeptidase

Substrate	Relative Activity (%)
		Lys-pNA (Standard control)	100.00
Leu-pNA	81.14
		Arg-pNA	83.57
Met-pNA	79.66
		Val-pNA	74.75

From the results, it was found that proline aminopeptidase can specifically act on the proline residue at the N-terminal, and has the best hydrolysis effect on Pro-pNA and Pro-Phe-Gly, and the hydrolysis effect on tripeptide is superior to that of dipeptide. In addition, the proline aminopeptidase also has certain salt tolerance, salt ions with certain concentration have promotion effect on the activity of the proline aminopeptidase, and the proline aminopeptidase can tolerate the concentration of 25% of sodium chloride at most. At 8% salt concentration, the relative enzyme activity was 147% of the control. Leucine aminopeptidase has very strong hydrolytic activity on leucine at the N end of small peptide, has good hydrolytic effect on isoleucine, has poor hydrolysis on other amino acids, and belongs to specific enzyme. The enzyme is a metalloprotease, Ca2+ has activating effect on the aminopeptidase, and the activity is increased to 127%. Lysine aminopeptidase has the highest efficiency of decomposing lysine residues and high efficiency of cleaving hydrophobic amino acid residues, and its substrate is more extensive than proline aminopeptidase and leucine aminopeptidase.

From the above results, it was found that proline aminopeptidase and leucine aminopeptidase had high selectivity for the substrate and that lysine aminopeptidase had low selectivity. Zein can form a plurality of intermediate peptides after being hydrolyzed by endonucleases in flavor enzymes, and non-specific proteases generally can not cut an imino bond, so that polypeptides with proline at the N terminal can not be cut, and the number of proline carried at the tail end of the intermediate peptides is very large. Proline aminopeptidase specifically cleaves the N-terminal proline residue and breaks the hydrolysis barrier. Therefore, proline aminopeptidase is added firstly for reaction, and then leucine aminopeptidase is added to specifically cut the tail ends of leucine and isoleucine. Finally, lysine hydrolase is used to further release hydrophobic amino acid with rich content.

Example 2: hydrolysis of zein by flavourzyme and aminopeptidase

The free amino acids were determined by DABS-Cl derivatization and analyzed by HPLC. After the enzymolysis, the centrifuged supernatant was added with 7.5mM DABS-Cl at a ratio of 1:1, sealed and mixed well. The reaction mixture was incubated at 70 ℃ for 15min, and 3-fold reaction terminator (50% of 50mM disodium hydrogenphosphate solution pH7.0 and 50% ethanol) was added immediately after completion of the reaction in ice bath. The mixture was centrifuged to take the supernatant and subjected to HPLC detection. An RP-C18 chromatographic column (150X 4.6mm, inner diameter 5 μm) is adopted, the flow rate is 1mL/min, the sample injection amount is 20 μ L, and the detection is carried out by an ultraviolet detector at 436 nm.

The hydrolysis effect of the synergistic effect of the flavor protease + proline aminopeptidase, the flavor protease + proline aminopeptidase + leucine protease + lysine protease and the like on the corn protein is investigated,

preparing 20% corn protein concentrated slurry with 50mM PBS buffer solution with pH 7.5, adding flavourzyme, reacting for 3h at 50 ℃, boiling water bath for 15min, cooling to 40-50 ℃, and sampling as an initial control. Adding proper proline aminopeptidase in sequence for reaction for 2h, adding leucine aminopeptidase for reaction for 2h, and finally adding lysine aminopeptidase in water bath at 45 ℃ for overnight. Sampling in steps at 10000rpm, centrifuging for 5min, and measuring the molecular weight distribution and the flavor amino acid content of the polypeptide in the supernatant.

As shown in figure 4, after the zein is treated by the flavor enzyme and hydrolyzed by proline aminopeptidase, the Pro concentration is increased by 2.7 times, and after the zein is treated by the leucine aminopeptidase, Leu and Ile are 2.75 and 4 times before enzymolysis, and the content of other free hydrophobic amino acids in the hydrolysate is also greatly increased. After the three enzymes are used for synergistic hydrolysis, the hydrolysis rate of most of proteins is improved to different degrees.

Finally, adding flavor enzyme for reaction at 50 ℃ for 2h, inactivating and cooling to 40-50 ℃, then sequentially adding a proper amount of proline aminopeptidase, leucine aminopeptidase and lysine aminopeptidase, carrying out water bath at 45 ℃ overnight, and carrying out prehydrolysis on the substrate in a three-enzyme synergistic manner.

Through hydrolysis, amino acids in the corn protein are completely released, the residual oligopeptides can be used as a compound nitrogen source, starch and solid corn syrup (or hydrolyzed corn starch sugar) can be used as a carbon source, and a small amount of ions and vitamin components are released to become an excellent substrate for microbial fermentation and conversion.

EXAMPLE 3 cell immobilization of Corynebacterium glutamicum and Brevibacterium ammoniagenes and preparation of zein fermentation broth by conversion of zein hydrolysate

Seed medium (g/L): glucose 5, yeast extract 1, beef extract 1, tryptone 2, and pH 7.2;

fermentation medium (g/L): glucose 10, yeast extract 0.5, urea 0.5, potassium dihydrogen phosphate 0.1, dipotassium hydrogen phosphate 0.1, sodium chloride 0.1, magnesium sulfate 0.2 and pH 8.0.

The strain of Corynebacterium glutamicum (Corynebacterium glutamicum) and Brevibacterium ammoniagenes (Brevibacterium ammoniagenes) frozen at-70 ℃ is streaked on a slant culture medium, the strain is cultured for 24-36 hours at 37 ℃, activated fresh lawn is picked by using an inoculating loop and inoculated in a fermentation culture medium, the fresh lawn is cultured for 48 hours at 37 ℃ at 200r/min, the thallus is collected by centrifugation, and the thallus suspension with the thallus concentration of 30% is prepared by using physiological saline.

Preparing immobilized cells: preparing 2L of sodium alginate solution with a certain concentration, adding 100ml of thallus suspension, uniformly mixing, dropwise adding the mixed solution into 2% CaCl2 solution which is continuously stirred by using a self-prepared immobilization reactor to form smooth balls, and standing and hardening for 1h at room temperature of 4 ℃. Washing with distilled water several times to remove excessive ions to obtain granular immobilized cells with particle size of about 1mm, and storing in a sealed bag at 4 deg.C.

Mixing corn protein and solid corn syrup (or hydrolyzed corn starch sugar) at a ratio of 1:2, and adding water until the total solid content is 20-30%; adding 0.5-2% of enhanced compound aminopeptidase into the mixed solution, carrying out prehydrolysis according to the mode of the example 2, firstly adding flavor enzyme to react for 2 hours at 50 ℃, inactivating and cooling to 40-50 ℃, then sequentially adding a proper amount of proline aminopeptidase, leucine aminopeptidase and lysine aminopeptidase, and carrying out water bath at 45 ℃ overnight; after enzymolysis, keeping the temperature above 85 ℃ for 15min to inactivate enzyme.

Regulating the concentration of hydrolysate to make the sugar content in the hydrolysate about 14%, loading 200g of immobilized cell beads into a reaction column with the diameter of 2cm and the height of 20cm, introducing the hydrolysate treated by aminopeptidase from the bottom, controlling the conversion temperature at 35 ℃, and regulating the flow rate to repeatedly circulate until the residual sugar content is reduced to below 0.5%. Then, continuously adding the enzymolysis liquid to the inside for circular reaction.

The fermentation method effectively solves the problems that the pH value in the fermentation liquor is sharply reduced and the product inhibition is easily generated; without the need to add ammonia or introduce other ingredients.

Continuously concentrating the fermentation liquor, caramelizing at 115 deg.C for 40-60min, and filtering to remove impurities to obtain corn protein seasoning liquid.

The corn protein (20 percent, paste) is directly hydrolyzed overnight by using the hydrochloric acid with the concentration of 6mol/l, compared with the seasoning liquid obtained by the process provided by the invention:

table 4: comparison of the seasoning liquid of the invention with acid hydrolyzed zein

Item	The seasoning liquid of the invention	Acid hydrolyzed corn protein
			Loss on drying	29.9％	80％
Total nitrogen (dry weight ratio)	10.46g/100g	10.34g/100g
			Amino acid nitrogen (dry weight ratio)	5.43g/100g	5.03g/100g
3-chloro-1, 2-propanediol	Not detected (less than or equal to 0.01)	Not detected (less than or equal to 0.01)
			Ammonium salts	Not detected (less than or equal to 0.01)	0.61g/100g

The detection shows that no other substances are added in the production process of the seasoning liquid, the total nitrogen (in dry weight ratio) is about 10 percent, the amino acid nitrogen (in dry weight ratio) is 5 percent, the total amount of the amino acids is 29.55 percent, the amino acids are all derived from the corn protein and the amino acids generated by the immobilized cells through conversion, and the seasoning liquid does not contain 3-MCPD. Compared with acidolysis zein, the total nitrogen and the amino nitrogen are equivalent, an acidolysis method can be completely replaced, the content of ammonium salt is low and far exceeds the limit requirement of acid hydrolysis vegetable protein in the SB/T10338-2000 specification (the total nitrogen is more than or equal to 1.5 percent, the amino acid nitrogen is more than or equal to 1 percent, and the content of 3-chlorine-1, 2-propylene glycol is less than or equal to 1mg/kg)

The corn protein processing product prepared by the invention can be directly used as a flavoring liquid for food; or in combination with other ingredients commonly used in the food art for preparing flavored liquids.

Claims

1. The corn protein enzymolysis liquid is characterized in that corn protein and corn syrup are subjected to enzymolysis by using flavourzyme, proline aminopeptidase and leucine aminopeptidase are added for enzymolysis, and lysine aminopeptidase is added for enzymolysis; the obtained enzymolysis liquid is fermented by corynebacterium glutamicum and brevibacterium ammoniagenes to finally prepare a processed product.

2. The corn protein hydrolysate of claim 1, wherein the corn syrup is replaced in whole or in part with hydrolyzed corn starch sugar.

3. The corn protein hydrolysate of claim 1, wherein the flavourzyme performs enzymolysis under the following conditions: pH6.5-7.5, and temperature 50 deg.C.

4. The corn protein hydrolysate of claim 1, wherein the proline aminopeptidase + leucine aminopeptidase is subjected to enzymolysis under conditions of pH6.5-7 and at a temperature of 40 ℃.

5. The corn protein hydrolysate of claim 1, wherein the lysine aminopeptidase is subjected to enzymolysis under conditions of pH 7.5 and a temperature of 40-50 ℃.

6. A corn protein fermentation broth, wherein the corn protein fermentation broth is prepared by fermenting the corn protein enzymatic hydrolysate of any one of claims 1 to 5 with Corynebacterium glutamicum and Brevibacterium ammoniagenes.

7. The zein fermentation broth of claim 6, wherein the C.glutamicum and Brevibacterium ammoniagenes are in immobilized form during fermentation.

8. The corn protein fermentation broth according to claim 6 or 7, wherein the fermentation with Corynebacterium glutamicum and Brevibacterium ammoniagenes is carried out by adding corn protein enzymatic hydrolysate in running water using immobilized bacteria.

9. Use of the zein fermentation liquor of any of claims 6 to 8 in the preparation of a seasoning liquor.

10. A seasoning liquid for food, which is prepared by using the zein fermentation liquid as claimed in claim 6.