CN115039865A

CN115039865A - Method for preparing diacetyl natural milk flavor base material by enzymolysis

Info

Publication number: CN115039865A
Application number: CN202210685324.8A
Authority: CN
Inventors: 白卫东; 汪薇; 陈清; 王俊; 赵文红; 任文彬
Original assignee: Zhongkai University of Agriculture and Engineering
Current assignee: Zhongkai University of Agriculture and Engineering
Priority date: 2022-06-16
Filing date: 2022-06-16
Publication date: 2022-09-13

Abstract

The invention discloses a method for preparing diacetyl natural milk flavor base material by enzymolysis, which comprises the following steps: (1) weighing a substrate, an auxiliary substrate and water, uniformly mixing, heating for sterilization, adding a complex enzyme, performing enzymolysis for 8-12 hours at 40-45 ℃, and performing enzyme deactivation treatment to obtain an enzymolysis liquid; the substrate is non-dairy creamer; the auxiliary substrate is whey powder; the compound enzyme comprises Lipase AY and protease MSD, and the mass ratio of the Lipase AY to the protease MSD is (2-4): 1; (2) cooling the enzymolysis liquid, and subpackaging to obtain the diacetyl natural milk flavor base material. According to the method, specific base material selection is matched with a complex enzyme enzymolysis preparation process, under the condition of specific parameters, the diacetyl content of the obtained diacetyl type natural milk flavor base material is high, the milk flavor of the product is pure and has high stability, the fragrance-retaining time is long, the flavor is coordinated, and no pungent smell exists.

Description

Method for preparing diacetyl natural milk flavor base material by enzymolysis

Technical Field

The invention relates to a preparation method of a milk-flavor base material, in particular to a method for preparing a diacetyl natural milk-flavor base material by enzymolysis.

Background

The milk flavor essence is one of the most widely applied essences in the food industry, the flavor components of the common milk flavor essence comprise lactone substances, medium-short chain fatty acids, esters, thiazoles, ethyl vanillin, acetoin, diacetyl, delta-decalactone and other compounds, but the milk flavor essence has complex composition of flavor substances and extremely low content, so that how to increase the content of the flavor components becomes a key factor in the production and processing process of the milk flavor essence.

At present, the main processing methods of the milk flavor base materials are divided into three main types: chemical synthesis, microbial fermentation and enzymatic hydrolysis. However, these three methods have obvious advantages, but the defects are also not negligible. The chemical synthesis method has the advantages of simple process flow, low production cost and high production stability, but the obtained product has single and unnatural flavor, and has certain inharmonious flavor if the concentration is too high; although the microbial fermentation method and the enzymatic hydrolysis method can obtain products with more flavor levels, the production efficiency is low, meanwhile, the fragrance is difficult to control, the selection of the types of raw materials is more sophisticated, more production parameters are involved to control, and the harmony of the flavor of the products is extremely easy to cause if the enzymolysis or the fermentation is excessive.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for preparing diacetyl natural milk flavor base material by enzymolysis.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for preparing diacetyl natural milk flavor base material by enzymolysis comprises the following steps:

(1) weighing a substrate, an auxiliary substrate and water, uniformly mixing, heating for sterilization, adding a complex enzyme, performing enzymolysis for 8-12 hours at 40-45 ℃, and performing enzyme deactivation treatment to obtain an enzymolysis liquid; the substrate is non-dairy creamer; the auxiliary substrate is whey powder; the compound enzyme comprises Lipase AY and protease MSD, and the mass ratio of the Lipase AY to the protease MSD is (2-4): 1;

(2) cooling the enzymolysis liquid, and subpackaging to obtain the diacetyl natural milk flavor base material.

In the technical scheme of the invention, vegetable fat powder with various flavors and rich fat content (up to 80%) is taken as a raw material substrate (the component is also called as a creamer, and has more aromatic flavor and more stable property compared with milk powder), whey powder which is a dairy product processing byproduct rich in high protein is taken as an auxiliary substrate, the two substrates are compounded and are respectively hydrolyzed by complex enzyme of lipase and protease, and a natural milk flavor base material with various and durable aroma levels can be obtained.

Through repeated experimental research of the inventor, when the enzymolysis steps of the protein and the fat in the raw materials need to be carried out synchronously, the Lipase AY which enables the product to have strong and soft milk fragrance is selected, and the matching effect of the protease MSD which enables the enzymolysis product to have pure and strong milk fragrance is better than that of other common enzyme types. However, the enzyme ratio is not arbitrary and can obtain good effect, and if the ratio is not proper, the content of diacetyl in the product can be inhibited, the fragrance of the product can be reduced, and even an unpleasant odor can be generated.

The action time and the enzymolysis temperature of the enzyme have certain influence on the flavor quality of the formed milk-flavor base material, but when the lipase and the protease are subjected to enzymolysis synchronously, the selection of the two parameters is different from the selection of a single enzyme during enzymolysis, and the composite effect of the synchronous enzymolysis needs to be comprehensively considered. If the action time is too short or the enzymolysis temperature is too low, the enzymolysis degree is insufficient, and the flavor product diacetyl cannot be fully formed. However, if the enzymolysis time is too long, too much zymolyte can inhibit the subsequent reaction of enzyme, so that the final enzymolysis efficiency is lower; too high temperature easily causes enzyme inactivation or low enzymolysis efficiency, affects the flavor of the product, consumes too much time and wastes cost, so that the proper enzymolysis time and enzymolysis temperature need to be controlled.

Preferably, in the step (1), the mass ratio of Lipase AY to protease MSD is 2:1, the temperature of the compound enzyme during enzymolysis is 40 ℃, and the time is 8 h.

Through careful screening by the inventor, the diacetyl natural milk-flavor base material prepared under the conditions has the highest diacetyl content, and meanwhile, the milk-flavor is the most mellow and full-bodied, and is sweet and attractive, the flavor is synergistic and lasting, and the evaluation is the highest.

Preferably, the addition amount of the complex enzyme is 0.24-0.36% of the weight percentage of the substrate.

More preferably, the addition amount of the complex enzyme is 0.24-0.3% of the weight percentage of the substrate.

More preferably, the addition amount of the complex enzyme is 0.24 percent of the weight percentage of the substrate.

The compound enzyme contains lipase and protease, fat and protein in the substrate and the cosubstrate are respectively subjected to effective enzymolysis and converted into fragrance substances such as diacetyl and the like, and experiments show that when the ratio of the lipase to the protease in the compound enzyme is not changed, the total addition is too little, the yield or fragrance of the obtained product not only can not reach the ideal standard, but if the total addition is too much, the diacetyl content can be reduced on the contrary due to reverse inhibition of the enzyme product or further enzymolysis of the product, and meanwhile, the fragrance is reduced rapidly.

Preferably, the addition amount of the cosubstrate is 40-120% of the weight percentage of the strate.

More preferably, the addition amount of the cosubstrate is 40-80% of the weight percentage of the strate.

Preferably, the co-substrate is added in an amount of 80% by weight of the substrate.

The auxiliary substrate mainly provides enzymolysis raw material protein of protease MSD and participates in the generation of final product aroma components, and the inventor finds out through study that the addition amount of the auxiliary substrate is properly matched with the substrate, so that the content multiple of the aroma components in the product can be improved, and the aroma evaluation is higher.

Preferably, the pH of a mixture obtained by uniformly mixing the substrate, the cosubstrate and water in the step (1) is 6.5-7.5.

More preferably, the pH of the mixture obtained by uniformly mixing the substrate, the co-substrate and the water in the step (1) is 7.

The pH value of the enzymolysis raw material has a large influence on the enzymolysis activity of the compound enzyme, and the proper acid-base environment can ensure that the enzymolysis efficiency of the compound enzyme is higher and the content of the product diacetyl is higher.

The invention also provides the diacetyl natural milk flavor base material prepared by the method for preparing the diacetyl natural milk flavor base material by enzymolysis.

The diacetyl natural milk flavor base material prepared by the method has high diacetyl content ratio, can obtain ideal flavor by adding a small amount of the diacetyl natural milk flavor base material in practical application, has mellow, natural and sweet milk flavor without greasy feeling, harmonious, stable and lasting flavor, and has simple process for producing the product, low production cost and high commercial performance-price ratio.

The invention has the beneficial effects that: the invention provides a method for preparing diacetyl natural milk flavor base material by enzymolysis, which is characterized in that specific base material selection is adopted and a complex enzyme enzymolysis preparation process is matched, under the condition of specific parameters, the diacetyl content in the prepared diacetyl natural milk flavor base material is high, the milk flavor of the product is pure and has high stability, the fragrance-retaining time is long, the flavor is coordinated, and no pungent smell exists.

Drawings

FIG. 1 is a graph showing the results of tests for the investigation of the selection of the types of cosubstrate in effect example 1 of the present invention;

FIG. 2 is a graph showing the results of tests conducted on the selective addition of cosubstrate in effect example 2 of the present invention;

FIG. 3 is a test result diagram for exploring pH value selection of an enzymolysis raw material in effect example 3 of the present invention;

FIG. 4 is a diagram showing the results of tests on the selection of the ratio of lipase to protease in complex enzyme in effect example 4 of the present invention;

FIG. 5 is a diagram showing test results of the selection and investigation of the amount of complex enzyme added in effect example 5 of the present invention;

FIG. 6 is a graph showing the results of examination of the selection of lipase species in effect example 6 of the present invention;

FIG. 7 is a graph showing the results of the test on the enzymatic hydrolysis temperature selection in example 7 of the effect of the present invention;

FIG. 8 is a graph showing the results of the examination of the selection of enzymatic hydrolysis time in example 7 of the effect of the present invention.

Detailed Description

The main experimental equipment used in the present invention is shown in table 1.

TABLE 1 Main test Equipment

The criteria for diacetyl content determination and fragrance assessment in the present invention are as follows:

(mono) diacetyl content determination

20mL of sample is taken and added with trichloroacetic acid with the same volume as 8 percent to be mixed, then the mixture is centrifuged for 10min at 10000r/min at the temperature of 4 ℃ in a high-speed refrigerated centrifuge, the supernatant is taken and filtered by filter paper once, 10mL of clarified liquid is respectively taken and added into two test tubes, 0.5mL of 1 percent o-phenylenediamine is added into the first test tube, and the second test tube is not used as a blank control. After mixing, after reacting for 30min in the dark, 2mL of 4mol/L hydrochloric acid was added to the first tube, 2.5mL of 4mol/L hydrochloric acid was added to the second tube, after mixing, the absorbance was measured at 335 nm.

(II) evaluation of fragrance

15 specially trained personnel are selected to form a milk flavor base material fragrance sensory evaluation and identification group, the milk flavor intensity, the sweet flavor intensity, the fragrance harmony and the fragrance remaining time of the milk flavor base material are respectively graded, the grading result is the sum of 4 persons, and the total score is 10.

The detailed scoring criteria are shown in table 2.

TABLE 2

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

The raw materials used in the examples/comparative examples of the present invention were as follows:

whole milk powder, skim milk powder, new zealand constant natural ltd;

non-dairy creamer, whey powder, guangdong mingkang essence and flavor ltd;

lipase G50, MER, A12, 20000L, AY, R, TL, TG; protease MSD, shanghai representative of japan tianye enzyme products;

o-phenylenediamine, trichloroacetic acid, HCl, etc., commercially available in analytical purity.

Example 1

The method for preparing the diacetyl natural milk flavor base material by enzymolysis comprises the following steps:

(1) weighing 10g of substrate non-dairy creamer, 8g of auxiliary substrate whey powder and 32mL of water, uniformly mixing in a triangular flask (pH 7), heating in a constant-temperature water bath kettle at 75 ℃ for sterilizing for 20min, adding complex enzyme with 0.24% of substrate content, performing enzymolysis for 8h in a constant-temperature shaking table at 40 ℃, and performing enzyme deactivation treatment for 10min at 90 ℃ to obtain an enzymolysis liquid; the compound enzyme comprises Lipase AY and protease MSD, and the mass ratio of the Lipase AY to the protease MSD is 2: 1;

(2) cooling the enzymolysis liquid and packaging to obtain the diacetyl natural milk flavor base material.

Example 2

(1) weighing 10g of substrate non-dairy creamer, 4g of auxiliary substrate whey powder and 32mL of water, uniformly mixing in a triangular flask (pH 7), heating in a constant-temperature water bath kettle at 75 ℃, sterilizing for 20min, adding complex enzyme with 0.24% of substrate content, performing enzymolysis for 8h in a constant-temperature shaking table at 45 ℃, and performing enzyme deactivation treatment for 10min at 90 ℃ to obtain an enzymolysis solution; the compound enzyme comprises Lipase AY and protease MSD, and the mass ratio of the Lipase AY to the protease MSD is 4: 1;

Comparative example 1

The comparative example differs from example 2 only in that the whey powder is replaced by a whole milk powder.

Comparative example 2

The present comparative example differs from example 2 only in that the whey powder was replaced with skim milk powder.

Comparative example 3

This comparative example differs from example 2 only in that the preparation step does not add cosubstrates.

Example 3

This example differs from example 2 only in that the cosubstrate is added in an amount of 8 g.

Example 4

This example differs from example 2 only in that the cosubstrate is added in an amount of 12 g.

Comparative example 4

The comparative example differs from example 3 only in that the pH of the mixture obtained by uniformly mixing the substrate, the co-substrate and water in step (1) is 5.

Comparative example 5

The comparative example differs from example 3 only in that the pH of the mixture obtained by uniformly mixing the substrate, the co-substrate and water in step (1) is 6.

Comparative example 6

This comparative example differs from example 3 only in that the pH of the mixture obtained by uniformly mixing the substrate, the co-substrate and water in the step (1) is 8.

Comparative example 7

The comparative example differs from example 3 only in that the pH of the mixture obtained by uniformly mixing the substrate, the co-substrate and water in step (1) is 9.

Example 5

The difference between the embodiment and the embodiment 3 is only that the Lipase AY and the protease MSD have a mass ratio of 3: 1.

example 6

The difference between the embodiment and the embodiment 3 is only that the Lipase AY and the protease MSD have the mass ratio of 2: 1.

comparative example 8

The difference between the comparative example and the example 3 is only that the Lipase AY and the protease MSD have the mass ratio of 1: 1.

comparative example 9

The difference between the comparative example and the example 3 is only that the Lipase AY and the protease MSD have the mass ratio of 5: 1.

example 7

The difference between the embodiment and the embodiment 6 is only that the adding amount of the compound enzyme in the step (1) is 0.3 percent of the weight percentage of the substrate.

Example 8

The difference between the embodiment and the embodiment 6 is only that the adding amount of the complex enzyme in the step (1) is 0.36 percent of the weight percentage of the substrate.

Comparative example 10

The difference between the comparative example and the example 6 is only that the addition amount of the compound enzyme in the step (1) is 0.12 percent of the weight content of the substrate.

Comparative example 11

The difference between the comparative example and the example 6 is only that the addition amount of the compound enzyme in the step (1) is 0.18 percent of the weight content of the substrate.

Comparative example 12

This comparative example differs from example 6 only in that the Lipase AY was replaced with Lipase G50.

Comparative example 13

The comparative example differed from example 6 only in that the Lipase AY was replaced with the Lipase MER.

Comparative example 14

The comparative example differs from example 6 only in that the Lipase AY is replaced with Lipase 20000L.

Comparative example 15

The comparative example differed from example 6 only in that the Lipase AY was replaced with Lipase R.

Comparative example 16

The comparative example differed from example 6 only in that the Lipase AY was replaced with Lipase TL.

Comparative example 17

The comparative example differed from example 6 only in that the Lipase AY was replaced with Lipase TG.

Comparative example 18

This comparative example differs from example 6 only in that the Lipase AY was replaced with Lipase A12.

Example 9

The difference between this example and example 1 is only that the temperature during the enzymatic hydrolysis in step (1) is 45 ℃.

Comparative example 19

The comparative example differs from example 1 only in that the temperature at the time of the enzymatic hydrolysis in the step (1) is 35 ℃.

Comparative example 20

The comparative example differs from example 1 only in that the temperature at the time of enzymatic hydrolysis in the step (1) is 50 ℃.

Comparative example 21

The comparative example differs from example 1 only in that the temperature at the time of the enzymatic hydrolysis in the step (1) is 55 ℃.

Example 10

The difference between this embodiment and embodiment 1 is only that the time for enzymolysis in step (1) is 12 h.

Comparative example 22

The comparative example differs from example 1 only in that the time for enzymatic hydrolysis in step (1) is 0 h.

Comparative example 23

The comparative example differs from example 1 only in that the time for enzymatic hydrolysis in step (1) is 4 hours.

Comparative example 24

The comparative example differs from example 1 only in that the time for enzymatic hydrolysis in step (1) is 16 hours.

Effect example 1

In order to verify the selection preference of the types of the cosubstrates in the method, the products of example 2 and comparative examples 1-3 are subjected to performance tests, and the results are shown in FIG. 1.

As can be seen from FIG. 1, when other conditions are not changed, the type of the cosubstrate has a great influence on the diacetyl content and the fragrance score of the product, wherein the diacetyl content of the natural milk fragrance base prepared by using whey powder and skimmed milk powder as the cosubstrates reaches 10mg/mL, while the fragrance score of the product prepared by using the whey powder is higher, and in sum, the effect of preparing the diacetyl natural milk fragrance base by using the whey powder as the cosubstrate is the best.

Effect example 2

In order to verify the optimization of the addition amount of the cosubstrate in the method, the products of examples 2-4 and comparative example 3 are subjected to performance tests, and the results are shown in fig. 2.

As can be seen from FIG. 2, when other conditions are not changed, the addition amount of the cosubstrate is not in a linear relationship with the increase of the diacetyl content in the product, and when the cosubstrate is whey powder, the addition amount of the cosubstrate is 8g, the highest diacetyl yield and the fragrance score can be obtained, and further, the addition amount is increased continuously, so that the diacetyl content is reduced, and the fragrance score is less than 5 points.

Effect example 3

In order to verify the optimization of the pH value of the enzymolysis raw material in the method, the products of example 3 and comparative examples 4 to 7 are subjected to performance tests, and the results are shown in fig. 3.

It can be seen that when the pH of the enzymolysis raw material mixture is changed from 5 to 9, the diacetyl content and aroma evaluation of the product do not fluctuate much, and in general, when the pH is 7, the aroma score is the highest and can be more than 8.

Effect example 4

In order to verify the optimization of the ratio of lipase to protease in the complex enzyme in the method, the products of examples 3, 5-6 and 8-9 are subjected to performance test, and the result is shown in FIG. 4.

As can be seen from FIG. 4, when the quality of lipase and protease in the complex enzyme is the same, although the produced diacetyl content is higher, the fragrance score of the product is not high and is less than 7.5 points, which shows that the fragrance of the product is related to milk fragrance, sweet fragrance, harmony and durability, and is not in direct linear relation with the diacetyl concentration; along with the increase of the specific gravity of the lipase, the aroma score of the product is gradually increased, when the ratio of the two enzymes reaches 2:1, the aroma score of the product reaches more than 8 points, and the diacetyl content is also higher than 22 mg/L; as the specific gravity of the lipase was further increased, the aroma score of the product decreased instead, indicating that excessive lipolysis in the feedstock may cause the product to develop an unpleasant odor.

Effect example 5

After the enzyme mixture ratio of the compound enzyme in the preparation process of the product is determined, the optimization of the addition amount of the compound enzyme needs to be further studied, and the products of examples 6-8 and comparative examples 10-11 are subjected to performance tests, and the results are shown in FIG. 5.

As can be seen from FIG. 5, when the addition amount of the complex enzyme is 0.12% of the substrate content, both the substrate and the cosubstrate are not sufficiently enzymolyzed, the production amount of diacetyl is insufficient, and the fragrance of the product is insufficient; along with the increase of the addition amount, the diacetyl concentration in the product is increased, the fragrance score is also improved, and the fragrance score reaches a peak value when the addition amount reaches 0.24; with further increase in the amount of addition, the product's aroma score decreased significantly, the product began to develop an inconsistent flavor, and the product of excessive enzymatic hydrolysis also began to inhibit the activity of the enzyme.

Effect example 6

As the substrate is the non-dairy creamer rich in fat, the type of lipase has a decisive role in the quality of the milk-flavor base material obtained by enzymolysis of the raw materials, and the products of example 6 and comparative examples 12-18 are subjected to performance tests, and the results are shown in figure 6. It can be seen that the diacetyl content of the natural milk flavor base material obtained by using the Lipase AY in the same enzymolysis environment is the highest, and the fragrance score is the highest, while if some other enzymes are used for compound enzymolysis, the fragrance of the product is possibly insufficient, and even an incompatible pungent smell appears, as shown by Lipase TG, the fragrance score of the milk flavor base material product prepared by using the Lipase AY is only 4.5 points, which obviously cannot be used or sold.

Effect example 7

After the type of the complex enzyme and the enzymolysis environment are determined, in order to further confirm the optimization of the method in temperature and time selection during enzymolysis, the products of the embodiments 1, 9-10 and 19-24 are subjected to performance tests, and the results are shown in fig. 7 and 8, so that when the enzymolysis temperature is low, the diacetyl content produced by enzymolysis is high, and the obtained product has insufficient aroma; the content of diacetyl is slightly increased along with the temperature increase of 40-45 ℃, and the fragrance score is increased from the original 7 points to about 8 points; when the temperature is increased to 50 ℃ or even 60 ℃, the activity of the enzyme is inhibited, so that the content of the produced diacetyl is reduced, the fragrance score is reduced to below 6 points, and the harmonious smell and the durability of the product are obviously reduced.

On the other hand, along with the increase of the enzymolysis time, the content of diacetyl in the product is gradually increased, meanwhile, the quality of the fragrance is also improved, however, after the enzymolysis time is too long, partial byproducts can be generated, so that the odor of the product is reduced, unnatural smell occurs, and the score of the fragrance is only 4 minutes at least.

Through screening, the enzymolysis temperature of the compound enzyme is optimal at 40-45 ℃, and the time is optimal at 8-12 h.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing diacetyl natural milk flavor base material by enzymolysis is characterized by comprising the following steps:

2. The method for preparing diacetyl natural milk flavor base material by enzymolysis according to claim 1, wherein in the step (1), the mass ratio of Lipase AY to protease MSD is 2:1, the temperature of the compound enzyme during enzymolysis is 40 ℃, and the time is 8 h.

3. The method for preparing the diacetyl natural milk flavor base material through enzymolysis according to claim 1, wherein the addition amount of the complex enzyme is 0.24-0.36% of the weight percentage of the substrate.

4. The method for preparing the diacetyl natural milk flavor base material through enzymolysis according to claim 3, wherein the addition amount of the complex enzyme is 0.24-0.3% of the weight percentage of the substrate.

5. The method for preparing the diacetyl natural milk flavor base material by enzymolysis according to claim 1, wherein the addition amount of the cosubstrate is 40-120% of the weight percentage of the substrate.

6. The method for preparing the diacetyl natural milk flavor base material through enzymolysis according to claim 5, wherein the addition amount of the cosubstrate is 40-80% of the weight percentage of the substrate.

7. The method for preparing the diacetyl natural milk flavor base material by enzymolysis according to claim 1, wherein the pH of a mixture obtained by uniformly mixing the substrate, the cosubstrate and water in the step (1) is 6.5-7.5.

8. The method for preparing diacetyl natural milk flavor base according to claim 7, wherein the pH of the mixture obtained by uniformly mixing the substrate, the co-substrate and the water in step (1) is 7.

9. The diacetyl natural milk flavor base material is prepared by the method for preparing the diacetyl natural milk flavor base material through enzymolysis according to any one of claims 1 to 8.