CN117179065A

CN117179065A - Cell-free extract and enzyme system and application thereof in enzyme modified cheese

Info

Publication number: CN117179065A
Application number: CN202310934069.0A
Authority: CN
Inventors: 罗洁; 赵艳; 沈清武; 周辉; 罗杨; 樊科
Original assignee: Hunan Agricultural University
Current assignee: Hunan Agricultural University
Priority date: 2023-07-27
Filing date: 2023-07-27
Publication date: 2023-12-08

Abstract

The invention belongs to the field of foods, and particularly relates to a cell-free extract, an enzyme system and application thereof in enzyme modified cheese. The preparation method of the cell-free extract comprises the following steps: (1) Inoculating lactobacillus starter into skim milk culture medium for culturing to obtain culture; (2) Performing solid-liquid separation on the culture to obtain bacterial liquid and bacterial mud; (3) Resuspending the bacterial sludge in a buffer solution for cracking to obtain a cracking solution; (4) Obtaining a lysate from the lysate, and optionally mixing with the bacterial liquid to obtain the cell-free extract. The invention has the beneficial effects that: the invention ferments lactobacillus by using skim milk, prepares the fermentation product into cell-free extract, and adds the cell-free extract into enzyme modified cheese to obtain the enzyme modified cheese with the flavor similar to that of mature cheddar cheese, thus providing a foundation for research and development of domestic enzyme modified cheese.

Description

Cell-free extract and enzyme system and application thereof in enzyme modified cheese

Technical Field

The invention belongs to the field of foods, and particularly relates to a cell-free extract, an enzyme system and application thereof in enzyme modified cheese.

Background

Cheese may be defined as the coagulation of milk solids, wherein milk fat is surrounded by coagulated proteins (casein), typically in a solid or semi-solid state. World Health Organization (WHO) and united nations grain and agriculture organization (FAO) define the concept of cheese as a fresh or fermented product that is produced by using milk/goat milk, cream, skim milk/partially skim milk, etc. as raw materials, curding with chymosin or another means, and then draining the whey. Cheese and its fermentation products are an important component of the human diet and play an important role in human health.

The ripening of cheese refers to the process by which some of its flavor, texture and properties of itself develop, a series of consecutive and complex physiochemical changes, a process in which microorganisms act together with enzymes. The ripening period of cheese varies from 3 months to 36 months, and in industrial production, the cheese requires a certain storage space during ripening, and the temperature, humidity, etc. of the ripening environment need to be controlled, which increases the production cost. The longer the maturation period of cheese will result in longer production cycles and higher manufacturing costs, so how to shorten the maturation period of cheese is a common concern in the scientific and industry.

Preparation of enzyme modified cheeses using exogenous enzymes on cheeses can accelerate the maturation of the cheese to some extent, but studies have found that neither the Flavourage FR nor DCA50 (a mixture of proteases and peptidases) significantly promote the production of volatile flavour substances and in some cases can cause poor flavour or structural defects in the cheese. Fan Junhua and the like compare the flavors of the self-made enzyme-modified cheese powder and the natural mature cheese, and the result shows that the self-made enzyme-modified cheese has stronger flavor, obvious pungent sour taste, softer flavor of the natural cheese and balanced aroma.

Thus, the flavor of enzyme modified cheeses prepared using exogenous enzymes has not yet fully reached the level of natural mature cheeses. Thus, how to produce an enzyme modified cheese that approximates the flavor of a mature cheese is a current research focus.

Disclosure of Invention

The object of the present invention is to overcome the above drawbacks of the prior art by providing an enzyme modified cheese with a ripened cheddar cheese flavor, in order to achieve this object, a first aspect of the present invention provides a method for the preparation of a cell free extract, comprising:

(1) Inoculating lactobacillus starter into skim milk culture medium for culturing to obtain culture;

(2) Performing solid-liquid separation on the culture to obtain bacterial liquid and bacterial mud;

(3) Resuspending the bacterial sludge in a buffer solution for cracking to obtain a cracking solution;

(4) Obtaining a lysate from the lysate, and optionally mixing with the bacterial liquid to obtain the cell-free extract.

In a second aspect the invention provides a cell-free extract prepared by the method of the first aspect.

In a third aspect the invention provides an enzyme system comprising leucine transaminase, glutamate dehydrogenase, hydroxydehydrogenase, optionally keto acid decarboxylase, keto acid dehydrogenase, aldehyde dehydrogenase and alcohol dehydrogenase;

wherein the enzyme activity of leucine transaminase is 10-15 mu mol/g/h, the enzyme activity of glutamate dehydrogenase is 25-35 mu mol/g/h, the enzyme activity of hydroxyl dehydrogenase is 250-300 mu mol/g/h, the enzyme activity of keto acid decarboxylase is 0-0.001 mu mol/g/h, the enzyme activity of keto acid dehydrogenase is 55-65 mu mol/g/h, the enzyme activity of aldehyde dehydrogenase is 10-15 mu mol/g/h, and the enzyme activity of alcohol dehydrogenase is 90-110 mu mol/g/h.

In a fourth aspect the invention provides the use of the cell-free extract of the first aspect or the enzyme system of the second aspect for increasing the fermentation flavour and/or 3-methylbutanal content of an immature cheese and/or an enzymatically modified cheese.

In a fifth aspect, the invention provides a method of increasing cheese flavor and/or 3-methylbutyrate in an enzyme-modified cheese, the method comprising: the cell-free extract as described above or the enzyme system as described above is contacted with an enzyme modified cheese.

In a sixth aspect the invention provides a food product to which the cell-free extract of the first aspect or the enzyme system of the second aspect is added, for example a flavoured cheese produced by the method of the fifth aspect.

The invention has the beneficial effects that: the invention uses skim milk to ferment lactobacillus to simulate the matrix environment of the mature cheese, prepares the fermentation product into cell-free extract, and adds the cell-free extract into enzyme modified cheese to obtain the enzyme modified cheese with the flavor similar to that of the mature cheddar cheese, thus providing a foundation for the research and development of domestic enzyme modified cheese.

Drawings

FIG. 1 shows a protein fraction electrophoresis pattern of hydrolyzed cheese simulants using SDS-PAGC polyacrylamide gel electrophoresis.

FIG. 2 shows the effect of the addition of different neutral proteases on the pH4.6-WSN content of the enzyme-modified cheese.

FIG. 3 shows the effect of the amount of added protease of different flavors on the pH4.6-WSN content of the enzyme-modified cheese.

FIG. 4 shows the effect of different enzymatic hydrolysis times on the pH4.6-WSN content of the enzyme-modified cheese.

Figure 5 shows the effect of different cell-free extracts on flavor substances in enzyme modified cheeses.

FIG. 6 shows the enzyme content of different cell-free extracts.

Detailed Description

In a first aspect, the present invention provides a method for preparing a cell-free extract, the method comprising:

In the present invention, the term "skim milk" refers to a product obtained after fat removal from a milk source containing fat, typically having a fat content of less than 0.1% by weight.

The skim milk in the skim milk culture medium of the invention may be raw skim milk or skim milk powder. In some embodiments, the skim milk medium is formulated from skim milk powder and water.

It will be appreciated by those skilled in the art that the concentration of skim milk in the skim milk medium should be premised on the ability to ferment the lactic acid bacteria starter culture, and in some preferred embodiments the concentration of skim milk in the skim milk medium is 8 to 20% by weight on a dry weight basis, for example, may be 8, 10, 12, 14, 16, 18, 20% by weight, preferably 10 to 15% by weight.

In some embodiments, the skim milk medium is formulated from skim milk powder and water, the skim milk powder being present in an amount of 8 to 20% by weight, preferably 10 to 15% by weight.

In the present invention, the term "culture" means that the lactic acid bacteria starter can be fermented to obtain the target product, and the conditions of the culture are therefore not particularly limited as long as fermentation of the starter can be ensured. In some preferred embodiments, the temperature of the culture is 20-45 ℃ (e.g., may be 20, 25, 30, 35, 40, 45 ℃). In some embodiments, the culturing is for a time such that the concentration of bacterial cells in the culture is greater than 10 ⁸ cfu/mL。

In some embodiments, the lactic acid bacteria starter is selected from the group consisting of lactobacillus bulgaricus (Lactobacillus bulgaricus), lactobacillus lactis (Lactobacillus lactis), lactobacillus helveticus (Lactobacillus helveticus), lactobacillus delbrueckii subspecies lactis (Lactobacillus delbrueckii ss. Lactis), streptococcus salivarius thermophilus (streptococcus thermophilus), lactococcus lactis subspecies lactis (Lactococcus lactis subsp. Lactis), lactococcus lactis subsp. Lactis (Lactococcus lactis subsp. Cremoris), lactococcus lactis subspecies diacetyl milk biological variant (Lactococcus lactis subsp. Lactis biovar diacetylactis), leuconostoc lactis (Leuconostoc lactis), leuconostoc mesenteroides subsp. (Leuconostoc mesenteroides subsp. Cremoris), pediococcus pentosaceus (Pediococcus pentosaceus), and lactobacillus casei (lactobacillus) and mixtures thereof.

In some embodiments, the lactic acid bacteria starter is lactococcus lactis.

In a particularly preferred embodiment, the lactic acid bacteria starter is composed of lactococcus lactis subspecies lactis and lactococcus lactis subspecies milk fat. In some embodiments, the lactic acid bacteria starter is r704 starter, which is commercially available.

In the present invention, any technique known in the art can be used by those skilled in the art to perform solid-liquid separation of the culture. In some embodiments, the method is by centrifugation. In some embodiments, by filtration. In some embodiments, by a method of resting.

In a specific embodiment, the culture is centrifuged (5000-10000 r/min) at 2-10℃for 5-15min to obtain a first supernatant with extracellular enzyme, which contains a precipitate of intracellular enzyme, bacterial sludge.

In a more specific embodiment, the bacterial sludge is dissolved in a buffer, centrifuged for 5-15min at 5000-10000r/min, repeated twice, and the obtained supernatants are combined to obtain a second supernatant.

In one embodiment, the first supernatant and the second supernatant are combined to obtain a supernatant with extracellular enzyme.

In the present invention, the bacterial sludge may be resuspended in any buffer which is capable of facilitating the disruption of the bacterial cells, and in a specific embodiment, the buffer is a PBS buffer.

The method of lysing the cells may be ultrasonic or adding a lysing enzyme. In order not to introduce foreign substances, the present invention preferably employs an ultrasonic method. In a preferred embodiment, the ultrasound conditions are: the power is 50-70W, the working time is 2-4s, the power is stopped for 6-10s, and the total power is 8-12min. And (3) carrying out high-speed centrifugation on the crushed liquid obtained after ultrasonic treatment for 10000-15000r/min and 15-25min to obtain a lysate, and taking the lysate to pass through a 0.22 mu m filter membrane to obtain a lysate.

In some embodiments, the cell-free extract is a first supernatant; in some embodiments, the cell-free extract is the supernatant with extracellular enzymes; in some embodiments, the cell-free extract is the lysate; in some embodiments, the cell-free extract is a mixture of the extracellular enzyme-bearing supernatant and the lysate.

In a second aspect, the invention provides a cell-free extract prepared by the method described above.

In a preferred embodiment, the cell-free extract comprises leucine transaminase (LeuAT), glutamate Dehydrogenase (GDH), hydroxydehydrogenase (HADH), optionally ketoacid decarboxylase (KADC), ketoacid dehydrogenase (KADH), aldehyde dehydrogenase (AlcDH) and alcohol dehydrogenase (AlcDH).

Accordingly, in a third aspect, the present invention provides an enzyme system comprising leucine transaminase, glutamate dehydrogenase, hydroxydehydrogenase, optionally keto acid decarboxylase, keto acid dehydrogenase, aldehyde dehydrogenase and alcohol dehydrogenase;

In the present invention, the leucine aminotransferase may have an enzyme activity of 10, 11, 12, 13, 14, 15. Mu. Mol/g/h. In some embodiments, the leucine transaminase enzyme activity is determined by the method of PERALTAG H, WOLF I V, BERGAMINI C V, et a.evaluation of volatile compounds produced byLactobacillus paracaseiI in a hard-cooked cheese model using solid-phase microextraction, dairy Science & Technology,2014,94 (1): 73-81, which is incorporated herein by reference.

In the present invention, the glutamate dehydrogenase may have an enzyme activity of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35. Mu. Mol/g/h. In some embodiments, the glutamate dehydrogenase enzyme activity is determined by the method of reference TANOUS C, CHAMELLON E, LE BARSD, et al, glutamate Dehydrogenase Activity Can Be Transmitted Naturally to Lactococcus lactis Strains To Stimulate Amino Acid Conversion to Aroma Compounds, appl Environ Microbiol,2006,72 (2): 1402-1409.6, which is incorporated herein by reference.

In the present invention, the enzyme activity of the hydroxydehydrogenase may be 250, 260, 270, 280, 290, 300. Mu. Mol/g/h. In some embodiments, the enzymatic activity of the hydroxydehydrogenase is measured by the method of BRANDSMAJ B, FLORIS E, DIJKSTRAAR D, et al Nature diversity of aminotransferases and dehydrogenase activity in a large collection of Lactococcus lactis strain International Dairy Journal,2008,18 (12): 1103-1108, which is incorporated herein by reference.

In some embodiments of the invention, the enzymatic activity of a ketoacid decarboxylase is determined by the method of reference BADARO A, MORIMIITSU F, FERREIRAA, et al identification of fiber added to semolina by Near Infrared (NIR) spot technologies, FOOD CHEMISTRY,2019,289:195-203, which is incorporated herein by reference.

In the present invention, the enzyme activity of the ketoacid dehydrogenase may be 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65. Mu. Mol/g/h. In some embodiments, the enzymatic activity of the ketoacid dehydrogenase is determined by the method of reference DANNER D J, LEMMON S K, BESHARSE J C, et al purification and characterization of branched chain alpha-ketoacid dehydrogenase from bovine liver mitochondria journal of Biological Chemistry,1979,254 (12): 5522-5526, which is incorporated herein by reference.

In the present invention, the enzyme activity of the aldehyde dehydrogenase may be 10, 11, 12, 13, 14, 15. Mu. Mol/g/h. In some embodiments, the enzymatic activity of the aldehyde dehydrogenase is determined by a method of reference AFZAL M I, DELAUNAY S, PARIS C, et al identification of metabolic pathways involved in the biosynthesis of flavor compound 3-methylbutanal from leucine catabolism by Carnobacteriummaltaromaticum LMA 28.International Journal of Food Microbiology,2012,157 (3).

In the present invention, the enzyme activity of the alcohol dehydrogenase may be 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110. Mu. Mol/g/h. In some embodiments, the method of determining the enzymatic activity of an alcohol dehydrogenase comprises: the total volume of the reacted system was 250. Mu.L, and the system contained 100mM PBS buffer, 150mM 3-methylbutyraldehyde, 0.5mM NADH, and 10. Mu.L CFE. The sample was kept at 30℃for 30min, and the OD was measured with a microplate reader at a wavelength of 340nm. The final enzyme activity was evaluated using the oxidation rate of NADH. The unit of enzyme activity is expressed as NADH decrease μmol/min/g.

In a fourth aspect, the present invention provides the use of a cell-free extract as described above or an enzyme system as described above for increasing the fermentation flavor and/or 3-methylbutanal content of an immature cheese and/or an enzyme modified cheese.

In a fifth aspect, the present invention provides a method of increasing cheese flavor and/or 3-methylbutyrate in an enzyme-modified cheese, the method comprising: the cell-free extract as described above or the enzyme system as described above is contacted with an enzyme modified cheese.

In the present invention, the amount of the cell-free extract to be added may be varied within a wide range, and it is preferable that the amount of the cell-free extract or enzyme system to be added is 0.6 to 1g, for example, 0.6, 0.7, 0.8, 0.9, 1g, relative to 100g of the enzyme-modified cheese in order to obtain a desired cheese flavor or 3-methylbutyraldehyde content range.

In the present invention, the contact time is not particularly limited, and in a preferred embodiment, the contact time is 40 to 60 hours, for example, 40, 45, 50, 55, 60 hours.

In the present invention, the temperature of the contact is not particularly limited, and in a preferred embodiment, the temperature of the contact is room temperature, for example, may be 40 to 50 ℃, for example, may be 42, 44, 45, 46, 48, 50 ℃.

Although the addition of the cell-free extract or enzyme system of the present invention to enzyme modified cheese increases its cheese flavor and increases the 3-methylbutyrate content, the inventors of the present invention have found in research that the cell-free extract or enzyme system of the present invention is particularly useful for use in a combination enzyme modified cheese consisting of neutral protease and flavourzyme.

In some embodiments, more preferably, the method of modifying comprises: adding 0.3-0.5 wt% flavourzyme and 0.2-0.5 wt% neutral proteinase to the cheese base; then modifying for 30-45 hours at 45-60 ℃.

In some embodiments, the cheese foundation may be an immature natural cheese. In some embodiments, the cheese foundation is a self-made cheese.

In some embodiments, the cheese foundation comprises

The cheese foundation is obtained by mixing the above materials as above.

In a sixth aspect, the invention provides a food product, e.g. a flavoured cheese prepared by a method as described above, to which a cell-free extract as described above or an enzyme system as described above is added.

Examples

Skim milk powder was purchased from sapropel milk company, ltd, with a fat content of 0.1%;

r704 starter was purchased from hansen, kogaku.

Preparation example 1: preparation of cheese mimics

Under the water bath of 70-80 ℃, after 30 parts by weight of skim milk and 31 parts by weight of water are uniformly mixed, 20 parts by weight of butter, 19 parts by weight of casein and emulsifying salt (wherein, the emulsifying salt is 0.15 part by weight of sodium citrate and 0.2 part by weight of citric acid) are sequentially added, and the obtained uniform substances are uniformly stirred respectively, and are cheese mimics.

Preparation example 2: preparation of enzyme modified cheese

(1) Five different combinations of neutral protease, flavourzyme, aminopeptidase and leucine aminopeptidase were performed: (1) neutral proteases and aminopeptidases (designated NA); (2) neutral protease + leucine aminopeptidase (NL); (3) flavourzyme+aminopeptidase (FA); (4) flavourzyme + leucine aminopeptidase (FL); (5) neutral protease + flavourzyme (NF). Each enzyme was added at 0.5% to each group and the cheese analogue was hydrolyzed at 45 ℃. The hydrolyzed cheese analogue was placed in an oven at 80 ℃ for 20min to inactivate the enzymes. The next experiment was performed with each combination of pH4.6 soluble nitrogen at 52-55% incubation time (i.e., selected for 60h NA, 48h NL, 36h FA, 48h FL and 36h NF).

Determination of soluble nitrogen at pH 4.6:

0.75g of a sample is weighed, 25mL of pH4.6 acetic acid-sodium acetate buffer solution is added, the sample is sufficiently ground, 25mL of buffer solution is added for washing, centrifugation is carried out for 15min at the rotating speed of 4500r/min, the supernatant is poured into a digestion tube, automatic Kai-type nitrogen determination is carried out after digestion by a digestion furnace, and the result is expressed by mass percent (%) of total nitrogen.

(2) Determination of Free Amino Acids (FAA)

The sample was accurately weighed 0.2g (to the nearest 0.01 mg) and the soluble nitrogen at pH4.6 was obtained as described above. The supernatant was collected and processed, and then measured by a fully automatic amino acid analyzer. The results are shown in Table 1.

TABLE 1

The nut flavor of cheddar cheese is mainly caused by 3-methylbutyraldehyde, 2-methylbutyraldehyde and 2-methylpropionaldehyde in branched aldehydes, which are produced by the metabolism of leucine, isoleucine and valine, respectively. The threshold for 3-methylbutyrate is minimal so it has a greater impact on the presentation of nut flavor. As can be seen from Table 1, the total content of these 3 key free amino acids is, in order, NF.gtoreq.FL > FA > NA.gtoreq.NL.

(3) Determination of protein composition

The protein component of the hydrolyzed cheese simulant was identified by SDS-PAGC polyacrylamide gel electrophoresis. 0.1g of cheese was dissolved in 2mL of a solution (400 mgSDS+1mL mercaptoethanol+4 mg bromophenol blue+8 g sucrose+4 mL 0.05mol/L Tris-HCl buffer, 15mL distilled water), centrifuged, and the supernatant was collected, the protein concentration was measured by BCA protein concentration method, the mass concentration was adjusted to be uniform, diluted with 5 Xloading buffer, and boiled for 5min, and cooled for use. The concentration of the concentrated gel is 4%, the concentration of the separation gel is 12%, and the voltages of the concentrated gel and the separation gel are 70V and 110V respectively. The results are shown in FIG. 1.

The intensity ratio analysis of the gel Image by Image J software shows that the intensity ratio of casein decomposition products of the NA group is highest, and other groups are FA, NF, NL, FL (arranged from large to small), namely, the degradation degree of casein of each group is NA > FA > NF > NL > FL from large to small.

(4) Selection of enzyme combinations

The leucine weight ratio of the NF group is larger than that of the FL group, and the strength ratio of casein decomposition products of the NF group is higher than that of the FL group by combining with the result of SDS, and finally, the NF group is selected for subsequent single-factor orthogonal experiments.

(5) Selection of conditions

5.1 neutral protease addition amount

Neutral protease is added in an amount of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and flavourzyme is added in an amount of 0.5%, respectively, and the mixture is hydrolyzed at 45℃for 36 hours.

The neutral protease is an endoprotease, and the larger the amount of the enzyme added, the more peptides are produced and the greater the degree of hydrolysis of proteins is within a certain range of the amount of the enzyme added. As shown in fig. 2, an addition amount of 0.2% -0.5% was selected.

5.2 amount of flavourzyme to be added

The addition amounts of flavourzyme and neutral protease were 0.5%, and they were hydrolyzed at 35℃at 40℃at 45℃at 50℃at 55℃at 60℃for 36 hours, respectively.

Flavourzyme is a protease-aminopeptidase, which has the property of an exoenzyme and degrades peptides into amino acids. As shown in fig. 3, an addition amount of 0.3% -0.5% was selected.

5.3 culture temperature

As a result, as shown in FIG. 4, the culture was performed at 45-60 ℃.

Experimental group: the addition amount of the flavourzyme is 0.4%, the addition amount of the neutral protease is 0.4%, and the flavourzyme is hydrolyzed for 36 hours at 55 ℃.

Table 2 shows a comparison of the degree of hydrolysis of the cheese and commercial EMC at different maturation times, blank, experimental group.

TABLE 2

Table 3 shows the volatile flavors of the blank, experimental set, different maturation time cheeses and commercial EMC, together 42 volatile flavors were identified, including 7 alcohols, 11 esters, 4 ketones, 12 acids, 5 aldehydes and 3 other flavor compounds.

TABLE 3 Table 3

The addition of flavoured proteases and neutral proteases causes the formation of proteolytic and flavour substances in the cheese analogue, although the degree of proteolysis reaches the level of 12 months of ripened cheese at a certain level, the type and content of volatile flavour substances cannot fully reach that of ripened cheese, such as the absence of the alcohol substances 2-pentanol, 3-methylbutanol and hexanol, which impart respectively the enzymatic modified cheese with fruit, malt and floral flavour; the delta-nonanolactone with nut taste and the branched aldehyde 2-methyl butyraldehyde, 3-methyl butyraldehyde content are lower than those in the mature cheese.

Example 1

Skim milk powder and water are used for preparing a skim milk culture medium with the concentration of 11 weight percent, 0.1 percent of r704 starter is inoculated, and the culture is carried out at 37 ℃ until the total number of colonies is more than 8 times. Centrifuging the cultured starter and culture solution at 4deg.C (8000 r/min) for 10min, collecting supernatant which is liquid with extracellular enzyme, and collecting the lower precipitate which is bacterial mud with intracellular enzyme. The bacterial sludge was dissolved in phosphate buffer (PBS, 100mM,pH6.0), centrifuged at 8000r for 10min and repeated twice. And redissolving the centrifuged bacterial sludge in PBS buffer solution, and uniformly mixing to obtain bacterial suspension. Crushing thalli of the suspension by using an ultrasonic cell crusher, wherein the ultrasonic conditions are as follows: the power is 60W, the working time is 3s, the power is stopped for 8s, and the total time is 10min. Centrifuging the crushed liquid obtained after ultrasonic treatment at high speed for 12000r/min and 20min, collecting supernatant, filtering with 0.22 μm filter membrane to obtain lysate, and mixing with the supernatant to obtain cell-free extract.

The obtained cell-free extract was added in an amount of 0.6% by weight to the enzyme-modified cheese prepared according to the above experimental group, and cultured at room temperature for 48 hours to obtain a flavor cheese.

Comparative example 1

The preparation of flavor cheese was performed as in example 1, except that 16 wt% of the whole milk medium was inoculated with r704 starter, using whole milk powder and water.

Comparative example 2

Preparation of flavor cheese was performed as in example 1, except that 37% strength cheddar cheese culture broth was used to inoculate r704 starter.

Comparative example 3

The preparation of flavor cheese was performed as in example 1, except that the fermentation was performed without inoculating r704 starter.

Test case

1) Determination of flavour substances

3.0g of the sample was weighed accurately, placed in a 15mL headspace bottle, added with 2-methyl-3-heptanone (internal standard) at a concentration of 0.816. Mu.g/mL, and quickly capped and sealed. The headspace bottle was preheated in a 55 ℃ water bath for 20min and then the headspace was adsorbed for 40min. Analyzing at 250deg.C gas phase sample inlet for 3min, and sample injection analyzing.

Chromatographic conditions: the chromatographic column was DB-WAX (30.0mX250 um,0.25 um); heating to 40deg.C for 5min, heating to 120deg.C at 5deg.C/min, heating to 230deg.C at 10deg.C/min, and maintaining for 10min; the carrier gas is helium, the flow rate of the carrier gas is 1.0mL/min, and the carrier gas is not split.

Mass spectrometry conditions: an EI ion source; electron energy 70eV; the ion source temperature is 230 ℃; the temperature of the quadrupole rod is 150 ℃; the scanning quality range is 35-500 u.

As a result, as shown in FIG. 5, the amount of the volatile flavor substances in the three enzyme-modified cheeses to which CFE was added was increased, indicating that CFE promoted the production of the volatile flavor substances in the enzyme-modified cheeses as a whole. The amount of flavor substance in the skim milk culture fluid group increased the most relative to the amounts of flavor substance in the cheese group and the whole milk culture fluid group. The variety of alcohol volatile flavor substances is increased, and 3-methyl butanol and hexanol are increased. The total content of volatile flavour of esters increased significantly, with 0.12mg/kg increase in delta-nonanolactone having a nutty taste in the skim milk culture broth group, which may be the result of more esters being formed by alcohol and acid reactions over time. The increase in total content of ketone volatile flavor is primarily due to the increase in 3-hydroxy-2-butanone, which imparts a butter and creamy taste to the enzyme modified cheese. The increase of aldehyde volatile flavor substances is relatively visual, the content of 3-methyl butyraldehyde with nut taste in the enzyme modified cheese after 3 CFE culture is increased compared with that of an orthogonal group (without CFE), and the cheese culture solution group, the skim milk culture solution group and the full-fat milk culture solution group are obviously different, the content of the comparative example 3 group is 0.15+/-0.01 mg/kg, the content of the cheese culture solution group is 0.26+/-0.01 mg/kg, the content of the skim milk culture solution group is 0.40+/-0.02 mg/kg, and the content of the full-fat milk culture solution group is 0.16+/-0.00 mg/kg. 0.48.+ -. 0.02mg/kg was detected in the 12 month ripened cheese, and the 3-methylbutyrate of the cheese in the skim milk group was closer to the 12 month ripened level. In conclusion, the cell-free extract prepared from the skim milk culture solution effectively improves the volatile flavor substances of the enzyme modified cheese.

2) Determination of enzyme content

The results of measurement of leucine aminotransferase (LeuAT), glutamate Dehydrogenase (GDH), hydroxydehydrogenase (HADH), ketoacid decarboxylase (KADC), ketoacid dehydrogenase (KADH), aldehyde dehydrogenase (AldDH) and alcohol dehydrogenase (AlcDH) according to the method described in the present invention are shown in FIG. 6. It can be seen that the whole milk broth group converts alpha-ketoisocaproic acid to another downstream substance alpha-hydroxyisohexanoic acid by the action of transaminase and hydroxy acid dehydrogenase, resulting in a smaller increment of 3-methylbutyrate; the cheese culture broth group converts leucine into 3-methyl butyraldehyde by the action of transaminase and decarboxylase (although the cheese group has all enzyme activities, the activity of aldehyde dehydrogenase of the cheese group is low, and the generated product trimethyl butyraldehyde is less, so the cheese culture broth group is presumed not to have undergone the metabolic pathway of 3-methyl butyric acid); the skim milk culture broth group not only generates 3-methylbutanal by ammonia conversion and decarboxylation, but also converts 3-methylbutanoic acid into 3-methylbutanal by the action of ketoacid dehydrogenase and aldehyde dehydrogenase, producing more 3-methylbutanal.

Example 2

The inventors further prepared cell-free cultures in 10%, 12%, 13%, 14%, 15%, 18% and 20% skim milk medium, which was then added to enzyme modified cheeses.

The protein and fat content in the skim milk medium at the 10-15% series concentration was close to that in commercial medium MRS, with substantially consistent formation of enzyme modified cheese flavor (3-methylbutyrate).

At skim milk concentrations of 18% and 20%, the effect of formation of enzyme modified cheese flavor (3-methylbutyraldehyde) was reduced, but still superior to whole milk media.

Example 3

To verify the effect of the enzyme system, the enzyme system was formulated according to the respective enzyme components and the corresponding amounts determined in example 1 and then added to the enzyme-modified cheese.

The results show that optimizing the flavor of enzyme modified cheese by formulating the enzyme system also effectively forms the flavor material (3-methylbutanal), but the effect is somewhat less than that of cell-free extracts prepared with skim milk, and still has advantages over cell-free extracts prepared with whole milk and cheddar cheese cultures.

Comparative example 4

Skim milk medium at 10-15% series concentration, whole milk medium at 15-20% series concentration, mature 3 month cheddar cheese culture at 35-40% series concentration, protein and fat content approaches that in commercial medium MRS, therefore, to further demonstrate the effect of compared to commercial MRS, applicant inoculated r704 starter culture on MRS medium basis for cell-free culture preparation.

The results show that the formation of flavor substances (3-methylbutyraldehyde) in enzyme-modified cheeses still does not achieve the desired effect, i.e., an effect similar to skim milk culture medium, by adding the cell-free culture as described above to the enzyme-modified cheese in the same amount, it is seen that the fat content and protein content in the culture medium are not factors that primarily affect the formation of flavor substances.

Claims

1. A method of preparing a cell-free extract, the method comprising:

2. The method of claim 1, wherein the fat content in the skim milk is less than 0.1 wt%; and/or

The concentration of the skim milk in the skim milk culture medium is 8-20 wt%, and preferably, the skim milk culture medium is prepared from skim milk powder and water; and/or

The conditions of the culture include: the temperature of the culture is 30-55 ℃; the culture is carried out for a time period such that the concentration of the bacterial cells in the culture is more than 10 ⁸ cfu/mL; and/or

The lactobacillus starter is lactococcus lactis, streptococcus thermophilus and the like; preferably r704 starter.

3. A cell-free extract prepared by the method of claim 1 or 2;

preferably, the cell-free extract comprises leucine transaminase, glutamate dehydrogenase, hydroxydehydrogenase, optionally ketoacid decarboxylase, ketoacid dehydrogenase, aldehyde dehydrogenase and alcohol dehydrogenase.

4. An enzyme system comprising leucine transaminase, glutamate dehydrogenase, hydroxydehydrogenase, optionally ketoacid decarboxylase, ketoacid dehydrogenase, aldehyde dehydrogenase and alcohol dehydrogenase;

5. Use of the cell-free extract of claim 3 or the enzyme system of claim 4 for increasing the ripeness flavor and/or 3-methylbutanal content of an immature cheese and/or an enzyme modified cheese.

6. A method of increasing cheese flavor and/or 3-methylbutyrate in an enzyme-modified cheese, the method comprising: contacting the cell-free extract of claim 3 or the enzyme system of claim 4 with an enzyme modified cheese.

7. The method of claim 6, wherein the cell-free extract or enzyme system is added in an amount of 0.6-1g relative to 100g of enzyme modified cheese; and/or

The contact time is 40-60h, and the temperature is 40-50 ℃.

8. The method of claim 6 or 7, wherein the enzyme modified cheese is a modified cheese consisting of neutral protease and flavourzyme;

more preferably, the method of modifying comprises: adding 0.3-0.5 wt% flavourzyme and 0.2-0.5 wt% neutral proteinase to the cheese base; then modifying for 30-45 hours at 45-60 ℃.

9. The method of claim 8, wherein the cheese foundation comprises

10. A food product added with the cell-free extract of claim 3 or the enzyme system of claim 4, e.g. a flavor cheese prepared by the method of any of claims 6-9.