Cell-free extract and enzyme system and use thereof in enzymatically modified cheese

Abstract

The application belongs to the field of food, and particularly relates to a cell-free extract and an enzyme system and application thereof in enzyme-modified cheese. A preparation method of the cell-free extract comprises the following steps: (1) inoculating a lactic acid bacteria starter into a skim milk culture medium to culture, to obtain a culture; (2) performing solid-liquid separation on the culture to obtain a bacterial liquid and a bacterial slurry; (3) resuspending the bacterial slurry in a buffer to lyse, to obtain a lysate; and (4) obtaining a lysate supernatant from the lysate, and optionally mixing the lysate supernatant with the bacterial liquid, to obtain the cell-free extract. The application has the following beneficial effects: the lactic acid bacteria is fermented by using skim milk, and the fermentation product is prepared into a cell-free extract, which is added into enzyme-modified cheese, so that enzyme-modified cheese with a flavor close to mature cheddar cheese is obtained, and a basis is provided for research and development of domestic enzyme-modified cheese.

Description

Technical Field

[0001] This invention relates to the food industry, specifically to cell-free extracts and enzyme systems and their application in enzyme-modified cheese. Background Technology

[0002] Cheese can be defined as a coagulation of milk solids, in which milk fat is encapsulated by coagulated protein (casein), generally in a solid or semi-solid state. The World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) define cheese as: a fresh or fermented product made from cow's / sheep's milk, cream, skim / partially skim milk, etc., through the coagulation of rennet or other methods, followed by the removal of whey. As an important part of the human diet, cheese and its fermentation products play a vital role in human health.

[0003] Cheese maturation refers to the gradual formation of its flavor, texture, and other inherent characteristics. It is a continuous and complex process of physiological and chemical changes, involving the combined action of microorganisms and enzymes. The maturation period for cheese ranges from 3 to 36 months. In industrial production, cheese requires a certain amount of storage space during maturation, and the temperature and humidity of the maturation environment need to be controlled, which increases production costs. A longer maturation period results in a longer production cycle and higher production costs; therefore, shortening the maturation period is a key focus of both the scientific and industrial communities.

[0004] Using exogenous enzymes to prepare enzyme-modified cheese can accelerate cheese maturation to some extent. However, studies have found that Flavorage FR and DCA50 (a mixture of protease and peptidase) do not significantly promote the production of volatile flavor compounds, and in some cases, can even cause unpleasant flavors or structural defects in the cheese. Fan Junhua et al. compared the flavors of homemade enzyme-modified cheese powder and naturally matured cheese. The results showed that the homemade enzyme-modified cheese had a more intense flavor with a noticeable pungent sourness, while the natural cheese had a milder flavor and a more balanced aroma.

[0005] Therefore, the flavor of enzyme-modified cheese prepared using exogenous enzymes cannot yet fully match that of naturally ripened cheese. Thus, how to prepare enzyme-modified cheese with a flavor close to that of ripened cheese is currently a hot research topic. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned deficiencies in the prior art and provide an enzyme-modified cheese with the flavor of mature cheddar cheese. To achieve this purpose, a first aspect of this invention provides a method for preparing a cell-free extract, the method comprising:

[0007] (1) Inoculate the lactic acid bacteria starter into the skim milk culture medium and culture it to obtain the culture;

[0008] (2) The culture is subjected to solid-liquid separation to obtain bacterial solution and bacterial sludge;

[0009] (3) The bacterial sludge is resuspended in a buffer solution for lysis to obtain a lysate;

[0010] (4) The lysate obtained from the lysate is optionally mixed with the bacterial solution to obtain the cell-free extract.

[0011] A second aspect of the invention provides a cell-free extract prepared by the method of the first aspect.

[0012] A third aspect of the present invention provides an enzyme system comprising leucine transaminase, glutamate dehydrogenase, hydroxyl dehydrogenase, optionally keto acid decarboxylase, keto acid dehydrogenase, aldehyde dehydrogenase and alcohol dehydrogenase.

[0013] The enzyme activities of leucine transaminase, glutamate dehydrogenase, hydroxyl dehydrogenase, keto acid decarboxylase, keto acid dehydrogenase, aldehyde dehydrogenase, and alcohol dehydrogenase are 10-15 μmol / g / h, 0-0.001 μmol / g / h, 55-65 μmol / g / h, 10-15 μmol / g / h, and 90-110 μmol / g / h, respectively.

[0014] The fourth aspect of the invention provides the use of the cell-free extract of the first aspect or the enzyme system of the second aspect in improving the fermentation flavor and / or 3-methylbutyraldehyde content of immature cheese and / or enzyme-modified cheese.

[0015] A fifth aspect of the present invention provides a method for increasing cheese flavor and / or 3-methylbutyraldehyde in enzyme-modified cheese, the method comprising: contacting the cell-free extract as described above or the enzyme system as described above with the enzyme-modified cheese.

[0016] The sixth aspect of the present invention provides a food containing the cell-free extract of the first aspect or the enzyme system of the second aspect, for example, flavored cheese prepared by the method of the fifth aspect.

[0017] The beneficial effects of this invention are as follows: This invention uses skim milk to ferment lactic acid bacteria, simulating the substrate environment for cheese maturation, and prepares the fermentation product into a cell-free extract, which is then added to enzyme-modified cheese to obtain enzyme-modified cheese with a flavor close to that of mature cheddar cheese, thus providing a foundation for the research and development of domestically produced enzyme-modified cheese. Attached Figure Description

[0018] Figure 1The electrophoretic diagram of protein components of hydrolyzed cheese mimicry is shown using SDS-PAGC polyacrylamide gel electrophoresis.

[0019] Figure 2 The effect of different amounts of added neutral protease on the pH 4.6-WSN content of enzyme-modified cheese was shown.

[0020] Figure 3 The effect of the amount of different flavor proteases added on the pH 4.6-WSN content of enzyme-modified cheese was shown.

[0021] Figure 4 The effect of different enzymatic hydrolysis times on the pH 4.6-WSN content of enzyme-modified cheese was shown.

[0022] Figure 5 The effects of different cell-free extracts on flavor compounds in enzyme-modified cheese were shown.

[0023] Figure 6 The enzyme content in different cell-free extracts is shown. Detailed Implementation

[0024] In a first aspect, the present invention provides a method for preparing a cell-free extract, the method comprising:

[0025] (1) Inoculate the lactic acid bacteria starter into the skim milk culture medium and culture it to obtain the culture;

[0026] (2) The culture is subjected to solid-liquid separation to obtain bacterial solution and bacterial sludge;

[0027] (3) The bacterial sludge is resuspended in a buffer solution for lysis to obtain a lysate;

[0028] (4) The lysate obtained from the lysate is optionally mixed with the bacterial solution to obtain the cell-free extract.

[0029] In this invention, the term "skimmed milk" refers to a product after removing fat from a milk source containing fat. Typically, the fat content in the "skimmed milk" is less than 0.1% by weight.

[0030] In this invention, the skim milk in the skim milk culture medium can be skimmed raw milk or skimmed milk powder. In some embodiments, the skim milk culture medium is prepared from skim milk powder and water.

[0031] Those skilled in the art will understand that the concentration of skim milk in the skim milk culture medium should be sufficient to ferment the lactic acid bacteria starter culture. In some preferred embodiments, the concentration of skim milk in the skim milk culture medium, on a dry weight basis, is 8-20% by weight, for example, 8, 10, 12, 14, 16, 18, or 20% by weight, preferably 10-15% by weight.

[0032] In some embodiments, the skim milk culture medium is prepared from skim milk powder and water, wherein the content of the skim milk powder is 8-20% by weight, preferably 10-15% by weight.

[0033] In this invention, the term "cultivation" refers to the ability to ferment the lactic acid bacteria starter culture to obtain the target product. Therefore, the cultivation conditions are not particularly limited, as long as fermentation of the starter culture is guaranteed. In some preferred embodiments, the cultivation temperature is 20-45°C (e.g., 20, 25, 30, 35, 40, 45°C). In some embodiments, the cultivation time is such that the bacterial concentration in the culture is greater than 10⁻⁶. 8 cfu / mL.

[0034] In some embodiments, the lactic acid bacteria starter is selected from *Lactobacillus bulgaricus*, *Lactobacillus lactis*, *Lactobacillus shelveticus*, *Lactobacillus delbrueckii* subsp. lactis, *Streptococcus thermophilus*, *Lactococcus lactis subsp. lactis*, *Lactococcus lactis subsp. cremoris*, *Lactococcus lactis subsp. lactis biovardiacetylactis*, *Leuconostoc lactis*, and *Leuconostoc mesenteroides*. *Lactobacillus subsp. cremoris*, *Pediococcus pentosaceus*, and *Lactobacillus casei*, and mixtures thereof.

[0035] In some embodiments, the lactic acid bacteria starter is Lactococcus lactis.

[0036] In a particularly preferred embodiment, the lactic acid bacteria starter culture comprises *Lactococcus lactis* subsp. *lactolaccos* and *Lactococcus lactis* subsp. *fatty acid*. In some embodiments, the lactic acid bacteria starter culture is R704, which is commercially available.

[0037] In this invention, those skilled in the art can use any technique known in the art to perform solid-liquid separation of the culture. In some embodiments, centrifugation is used. In some embodiments, filtration is used. In some embodiments, settling is used.

[0038] In one specific embodiment, the culture is centrifuged at 2-10°C (5000-10000 r / min) for 5-15 min to obtain a first supernatant containing extracellular enzymes and a precipitate containing intracellular enzymes—bacterial sludge.

[0039] In a more specific embodiment, the bacterial sludge is dissolved in a buffer solution, centrifuged at 5000-10000 r / min for 5-15 min, and repeated twice. The resulting supernatants are then combined to obtain a second supernatant.

[0040] In one embodiment, the first supernatant and the second supernatant are combined to obtain a supernatant containing extracellular enzymes.

[0041] In this invention, the bacterial sludge can be resuspended in any buffer solution, as long as it is conducive to the disruption of the bacterial cells. In one specific embodiment, the buffer solution is PBS buffer.

[0042] The bacterial cells can be lysed using methods such as ultrasound or by adding lysin. To avoid introducing exogenous substances, ultrasound is preferred in this invention. In a preferred embodiment, the ultrasound conditions are: power 50-70W, working time 2-4s, pause 6-10s, total 8-12min. The resulting lysate is then centrifuged at high speed (10000-15000 r / min) for 15-25min to obtain the lysate. The lysate is then filtered through a 0.22μm filter membrane to obtain the supernatant lysate.

[0043] In some embodiments, the cell-free extract is a first supernatant; in some embodiments, the cell-free extract is the supernatant containing extracellular enzymes; in some embodiments, the cell-free extract is the lysate; in some embodiments, the cell-free extract is a mixture of the supernatant containing extracellular enzymes and the lysate.

[0044] Secondly, the present invention provides a cell-free extract prepared by the method described above.

[0045] In a preferred embodiment, the cell-free extract contains leucine transaminase (LeuAT), glutamate dehydrogenase (GDH), hydroxyl dehydrogenase (HADH), optionally keto acid decarboxylase (KADC), keto acid dehydrogenase (KADH), aldehyde dehydrogenase (AlcDH), and alcohol dehydrogenase (AlcDH).

[0046] Accordingly, in a third aspect, the present invention provides an enzyme system containing leucine transaminase, glutamate dehydrogenase, hydroxyl dehydrogenase, optionally keto acid decarboxylase, keto acid dehydrogenase, aldehyde dehydrogenase and alcohol dehydrogenase.

[0047] The enzyme activities of leucine transaminase, glutamate dehydrogenase, hydroxyl dehydrogenase, keto acid decarboxylase, keto acid dehydrogenase, aldehyde dehydrogenase, and alcohol dehydrogenase are 10-15 μmol / g / h, 0-0.001 μmol / g / h, 55-65 μmol / g / h, 10-15 μmol / g / h, and 90-110 μmol / g / h, respectively.

[0048] In this invention, the enzyme activity of the leucine transaminase can be 10, 11, 12, 13, 14, or 15 μmol / g / h. In some embodiments, the enzyme activity of the leucine transaminase is determined according to the method of PERALTAG H, WOLF IV, BERGAMINI CV, et al. Evaluation of volatile compounds produced by Lactobacillus paracaseiI90 in a hard-cooked cheese model using solid-phase microextraction. Dairy Science & Technology, 2014, 94(1):73-81, which is incorporated herein by reference.

[0049] In this invention, the enzyme activity of glutamate dehydrogenase can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 μmol / g / h. In some embodiments, the enzyme activity of the glutamate dehydrogenase is determined by referring to the method described in TANOUS C, CHAMBELLON 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.

[0050] In this invention, the enzyme activity of hydroxy dehydrogenase can be 250, 260, 270, 280, 290, or 300 μmol / g / h. In some embodiments, the enzyme activity of hydroxy dehydrogenase is determined by the method of BRANDSMAJ B, FLORIS E, DIJKSTRAAR D, et al. Natural diversity of aminotransferases and dehydrogenase activity in a large collection of Lactococcus lactis strains. International Dairy Journal, 2008, 18(12): 1103-1108, which is incorporated herein by reference.

[0051] In some embodiments of the present invention, the enzyme activity of keto acid decarboxylase was determined by reference to the method of BADARO A, MORIMITSU F, FERREIRAA, et al. Identification of fiber added to semolina by near infrared (NIR) spectral techniques. FOOD CHEMISTRY, 2019, 289:195-203, which is incorporated herein by reference.

[0052] In this invention, the enzyme activity of ketoacid dehydrogenase can be 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 μmol / g / h. In some embodiments, the enzyme activity of ketoacid dehydrogenase is determined by reference to the method of DANNER DJ, LEMMON SK, BESHARSE JC, 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.

[0053] In this invention, the enzyme activity of aldehyde dehydrogenase can be 10, 11, 12, 13, 14, or 15 μmol / g / h. In some embodiments, the enzyme activity of aldehyde dehydrogenase is determined by referring to the method described in AFZAL MI, DELAUNAY S, PARIS C, et al. Identification of metabolic pathways involved in the biosynthesis of flavor compound 3-methylbutanal from leucine catabolism by Carnobacterium maltaromaticum LMA28. International Journal of Food Microbiology, 2012, 157(3).

[0054] In this invention, the enzyme activity of alcohol dehydrogenase can be 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 μmol / g / h. In some embodiments, the method for determining the enzyme activity of alcohol dehydrogenase includes: a total reaction volume of 250 μL, containing 100 mM pH 6.0 PBS buffer, 150 mM 3-methylbutyraldehyde, 0.5 mM NADH, and 10 μL CFE. The reaction is incubated at 30°C for 30 min, and the OD value is measured using a microplate reader at a wavelength of 340 nm. The final enzyme activity is evaluated using the NADH oxidation rate. The unit of enzyme activity is expressed as the reduction of NADH in μmol / min / g.

[0055] Fourthly, the present invention provides the use of the cell-free extracts as described above or the enzyme systems as described above in improving the fermentation flavor and / or 3-methylbutyraldehyde content of immature cheese and / or enzyme-modified cheese.

[0056] Fifthly, the present invention provides a method for increasing cheese flavor and / or 3-methylbutyraldehyde in enzyme-modified cheese, the method comprising: contacting the cell-free extract as described above or the enzyme system as described above with the enzyme-modified cheese.

[0057] In this invention, the amount of the cell-free extract can be varied within a wide range. Preferably, in order to obtain an ideal cheese flavor or 3-methylbutyraldehyde content range, the amount of the cell-free extract or enzyme system added is 0.6-1g relative to 100g of enzyme-modified cheese, for example, it can be 0.6, 0.7, 0.8, 0.9, or 1g.

[0058] In this invention, the contact time is not particularly limited. In a preferred embodiment, the contact time is 40-60 hours, for example, 40, 45, 50, 55, or 60 hours.

[0059] In this invention, the contact temperature is not particularly limited. In a preferred embodiment, the contact temperature is room temperature, for example, 40-50°C, or for example, 42, 44, 45, 46, 48, or 50°C.

[0060] Although adding the cell-free extract or enzyme system of the present invention to enzyme-modified cheese can improve its cheese flavor and increase the content of 3-methylbutyraldehyde, the inventors of the present invention have found in their research that the cell-free extract or enzyme system of the present invention is particularly suitable for cheese modified by a combination of neutral proteases and flavor proteases.

[0061] In some embodiments, more preferably, the modification method comprises: adding 0.3-0.5% by weight of flavor protease and 0.2-0.5% by weight of neutral protease to a cheese base; and then modifying it at a temperature of 45-60°C for 30-45 hours.

[0062] In some embodiments, the cheese base may be unripened natural cheese. In some embodiments, the cheese base may be homemade cheese.

[0063] In some embodiments, the cheese base contains

[0064]

[0065] The cheese base is obtained by mixing the above materials as described above.

[0066] In a sixth aspect, the present invention provides food products containing the cell-free extracts or enzyme systems described above, such as flavored cheese prepared by the method described above.

[0067] Example

[0068] The skim milk powder was purchased from Saputo Dairy Co., Ltd., with a fat content of 0.1%.

[0069] R704 starter culture was purchased from Chr. Hansen.

[0070] Preparation Example 1: Preparation of Cheese Analog

[0071] In a water bath at 70-80℃, 30 parts by weight of skim milk and 31 parts by weight of water are mixed evenly, and then 20 parts by weight of butter, 19 parts by weight of casein and emulsifying salt (of which the emulsifying salt is 0.15 parts by weight of sodium citrate and 0.2 parts by weight of citric acid) are added in sequence and stirred evenly. The resulting homogeneous substance is the cheese imitator.

[0072] Preparation Example 2: Preparation of Enzyme-Modified Cheese

[0073] (1) Five different combinations of neutral protease, flavor protease, aminopeptidase, and leucine aminopeptidase were prepared: ① neutral protease and aminopeptidase (named NA); ② neutral protease + leucine aminopeptidase (NL); ③ flavor protease + aminopeptidase (FA); ④ flavor protease + leucine aminopeptidase (FL); ⑤ neutral protease + flavor protease (NF). Each enzyme was added at 0.5% in each group, and the cheese simulant was hydrolyzed at 45℃. The hydrolyzed cheese simulant was placed in an 80℃ oven for 20 min to inactivate the enzymes. The incubation times (pH 4.6, soluble nitrogen at 52-55% soluble nitrogen) for each combination were selected for the next stage of the experiment (i.e., NA for 60 h, NL for 48 h, FA for 36 h, FL for 48 h, and NF for 36 h).

[0074] Method for determining soluble nitrogen at pH 4.6:

[0075] Weigh 0.75 g of sample, add 25 mL of pH 4.6 acetate-sodium acetate buffer, grind the sample thoroughly, add another 25 mL of buffer for rinsing, centrifuge for 15 min at 4500 r / min, pour the supernatant into a digestion tube, digest with a digestion oven, and perform automatic Kjeldahl nitrogen determination. The result is expressed as the mass fraction (%) of total nitrogen.

[0076] (2) Determination of free amino acids (FAA)

[0077] Accurately weigh 0.2 g of sample (accurate to 0.01 mg) and obtain soluble nitrogen at pH 4.6 as described above. After processing the supernatant, determine the soluble nitrogen content using a fully automated amino acid analyzer. The results are shown in Table 1.

[0078] Table 1

[0079]

[0080] The nutty flavor of cheddar cheese is mainly due to 3-methylbutyraldehyde, 2-methylbutyraldehyde, and 2-methylpropionaldehyde, which are branched aldehydes produced by the metabolism of leucine, isoleucine, and valine, respectively. 3-methylbutyraldehyde has the lowest threshold, so it has a greater impact on the nutty flavor. According to Table 1, the total content of these three key free amino acids is in the following order: NF≥FL>FA>NA≥NL.

[0081] (3) Determination of protein composition

[0082] Protein components of hydrolyzed cheese mimics were identified using SDS-PAGC polyacrylamide gel electrophoresis. 0.1 g of cheese was dissolved in 2 mL of dissolving buffer (400 mg SDS + 1 mL mercaptoethanol + 4 mg bromophenol blue + 8 g sucrose + 4 mL 0.05 mol / L pH 8.0 Tris-HCl buffer, 15 mL distilled water), centrifuged, and the supernatant was collected. Protein concentration was determined using the BCA protein concentration method. The concentrations were adjusted to be consistent, diluted with 5× loading buffer, boiled for 5 min, and cooled before use. The stacking gel concentration was 4%, and the separating gel concentration was 12%. The voltages for the stacking and separating gels were 70 V and 110 V, respectively. Results are shown below. Figure 1 As shown.

[0083] Image J software was used to analyze the intensity ratio of gel images. The results showed that the NA group had the highest intensity ratio of casein degradation products, followed by FA, ​​NF, NL, and FL (in descending order). In other words, the degree of casein degradation in each group was NA > FA > NF > NL > FL.

[0084] (4) Selection of enzyme combination

[0085] The leucine content in the NF group was greater than that in the FL group. Combined with the results of SDS, the intensity of casein degradation products in the NF group was higher than that in the FL group. Finally, the NF group was selected for subsequent single-factor orthogonal experiments.

[0086] (5) Selection of conditions

[0087] 5.1 Amount of neutral protease added

[0088] The neutral protease was added at amounts of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%, respectively, and the flavor protease was added at an amount of 0.5%. The mixture was hydrolyzed at 45°C for 36 hours.

[0089] Neutral protease is an endopeptidase. Within a certain range of dosage, the higher the dosage, the more peptides are generated, and the greater the degree of protein hydrolysis. For example... Figure 2 As shown, select an addition amount of 0.2%-0.5%.

[0090] 5.2 Dosage of Flavor Protease Added

[0091] The amount of flavor protease and neutral protease added was 0.5%, and hydrolysis was carried out at 35℃, 40℃, 45℃, 50℃, 55℃ and 60℃ for 36 h, respectively.

[0092] Flavor proteases are proteases—aminopeptidases—that possess exonuclease properties, breaking down peptides into amino acids. For example... Figure 3 As shown, select an addition amount of 0.3%-0.5%.

[0093] 5.3 Incubation temperature

[0094] The amount of flavor protease and neutral protease added was 0.5%, and hydrolysis was carried out at 35℃, 40℃, 45℃, 50℃, 55℃ and 60℃ for 36 h, respectively.

[0095] The results are as follows Figure 4 As shown, culture should be carried out at 45-60℃.

[0096] Experimental group: The amount of flavor protease added was 0.4%, and the amount of neutral protease added was 0.4% for 36 hours at 55℃.

[0097] Table 2 shows a comparison of the degree of hydrolysis of blank, experimental group, cheese at different maturation times, and commercial EMC.

[0098] Table 2

[0099]

[0100] Table 3 shows the volatile flavor compounds of the blank, experimental group, cheeses at different maturation times, and commercial EMC. A total of 42 volatile flavor compounds were identified, including 7 alcohols, 11 esters, 4 ketones, 12 acids, 5 aldehydes, and 3 other flavor compounds.

[0101] Table 3

[0102]

[0103]

[0104] Adding flavor proteases and neutral proteases causes protein degradation and flavor compound formation in cheese mimics. Although the degree of protein hydrolysis reaches the level of 12-month-aged cheese by a certain standard, the types and contents of volatile flavor compounds cannot fully reach those of mature cheese. For example, it lacks alcohols such as 2-pentanol, 3-methylbutanol, and hexanol, which respectively impart fruity, malty, and floral flavors to enzyme-modified cheese. The contents of δ-nonanolactone, which has a nutty flavor, and branched aldehydes 2-methylbutanal and 3-methylbutanal are also lower than those in mature cheese.

[0105] Example 1

[0106] A skim milk culture medium with a concentration of 11% by weight was prepared using skim milk powder and water. 0.1% of R704 starter culture was inoculated and cultured at 37°C until the total bacterial count exceeded 8^8. The incubated starter culture and culture medium were centrifuged at 4°C (8000 rpm) for 10 min. The supernatant contained extracellular enzymes, and the precipitate contained intracellular enzymes. The precipitate was dissolved in phosphate-buffered saline (PBS, 100 mM, pH 6.0) and centrifuged at 8000 rpm for 10 min, repeated twice. The centrifuged precipitate was then redissolved in PBS buffer and mixed thoroughly to obtain a bacterial suspension. The bacterial cells in the suspension were disrupted using an ultrasonic cell disruptor. The ultrasonic conditions were: power 60 W, working time 3 s, pause 8 s, for a total of 10 min. The lysate obtained after sonication was centrifuged at 12000 r / min for 20 min. The supernatant was filtered through a 0.22 μm filter to obtain the lysate. The lysate was then mixed with the supernatant to obtain the cell-free extract.

[0107] The obtained cell-free extract was added at 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 flavored cheese.

[0108] Comparative Example 1

[0109] The flavored cheese was prepared according to the method of Example 1, except that a 16% by weight whole milk culture medium was prepared using whole milk powder and water and inoculated with R704 starter culture.

[0110] Comparative Example 2

[0111] The flavored cheese was prepared according to the method of Example 1, except that a 37% concentration of cheddar cheese culture that had been matured for 3 months was used to inoculate the R704 starter culture.

[0112] Comparative Example 3

[0113] The flavored cheese was prepared according to the method of Example 1, except that the R704 starter culture was not used for fermentation.

[0114] Test case

[0115] 1) Determination of flavor compounds

[0116] Accurately weigh 3.0 g of sample and place it in a 15 mL headspace vial. Add 0.816 μg / mL of 2-methyl-3-heptanone (internal standard) and quickly seal the vial. Preheat the headspace vial in a 55 °C water bath for 20 min, then perform headspace adsorption for 40 min. Desorb at a 250 °C gas chromatograph for 3 min, then inject for analysis.

[0117] Chromatographic conditions: The chromatographic column was DB-WAX (30.0m×250um, 0.25um); the temperature program was as follows: initial temperature 40℃, hold for 5 min, then increase to 120℃ at a rate of 5℃ / min, then increase to 230℃ at a rate of 10℃ / min, hold for 10 min; the carrier gas was helium, the carrier gas flow rate was 1.0 mL / min, and the flow was splitless.

[0118] Mass spectrometry conditions: EI ion source; electron energy 70 eV; ion source temperature 230 °C; quadrupole temperature 150 °C; scan mass range 35–500 u.

[0119] The results are as follows Figure 5 As shown, overall, the content of volatile flavor compounds in the three groups of enzyme-modified cheeses with added CFE increased, indicating that CFE can promote the formation of volatile flavor compounds in enzyme-modified cheese. Compared to the cheese group and the whole milk culture medium group, the skim milk culture medium group showed the largest increase in flavor compounds. The variety of alcohol volatile flavor compounds increased, with the addition of 3-methylbutanol and hexanol. The total content of ester volatile flavor compounds increased significantly, with the skim milk culture medium group showing an increase of 0.12 mg / kg in δ-nonanolide, which has a nutty flavor. This may be due to the increased ester formation from alcohol and acid reactions with longer culture time. The increase in the total content of ketone volatile flavor compounds was mainly due to the increase in 3-hydroxy-2-butanone, a compound that imparts a buttery and creamy flavor to the enzyme-modified cheese. The increase in volatile aldehyde flavor compounds was also relatively obvious. The content of 3-methylbutyraldehyde, a nutty flavor compound, was increased in all three CFE-cultured enzyme-modified cheeses compared to the orthogonal group (without CFE). Significant differences were observed between the cheese culture broth group, the skim milk culture broth group, and the whole milk culture broth group. The content was 0.15±0.01 mg / kg in the three comparative groups, 0.26±0.01 mg / kg in the cheese culture broth group, 0.40±0.02 mg / kg in the skim milk culture broth group, and 0.16±0.00 mg / kg in the whole milk culture broth group. 0.48±0.02 mg / kg was detected in 12-month-matured cheese, with the 3-methylbutyraldehyde content in the skim milk group being closer to the 12-month maturity level. In conclusion, the cell-free extract prepared from skim milk culture broth effectively improves the volatile flavor compounds in enzyme-modified cheese.

[0120] 2) Determination of enzyme content

[0121] Leucine transaminase (LeuAT), glutamate dehydrogenase (GDH), hydroxyl dehydrogenase (HADH), keto acid decarboxylase (KADC), keto acid dehydrogenase (KADH), aldehyde dehydrogenase (AldDH), and alcohol dehydrogenase (AlcDH) were measured according to the method described in this invention, and the results are as follows: Figure 6 As shown in the figure, the whole milk culture medium group converts α-ketoisocaproic acid to another downstream substance, α-hydroxyisocaproic acid, through the action of transaminases and hydroxy acid dehydrogenases, resulting in a smaller increase in 3-methylbutyraldehyde. The case culture medium group converts leucine to 3-methylbutyraldehyde through the action of transaminases and decarboxylases (although the case group has all the enzyme activities, the aldehyde dehydrogenase activity in the case group is lower, and the product trimethylbutyraldehyde is produced in less, so it is speculated that the case culture medium group does not have the metabolic pathway of 3-methylbutyric acid). The skim milk culture medium group not only produces 3-methylbutyraldehyde through transamination and decarboxylation, but also converts 3-methylbutyric acid to 3-methylbutyraldehyde through the action of keto acid dehydrogenases and aldehyde dehydrogenases, producing more 3-methylbutyraldehyde.

[0122] Example 2

[0123] The inventors further prepared cell-free cultures using skim milk culture media of 10%, 12%, 13%, 14%, 15%, 18%, and 20%, which were then added to enzyme-modified cheese.

[0124] The protein and fat contents in the 10-15% series of skim milk culture media are close to those in the commercial MRS culture medium, and are basically consistent with the formation of enzyme-modified cheese flavor substances (3-methylbutyraldehyde).

[0125] When the skim milk concentration was 18% and 20%, the effect on the formation of enzyme-modified cheese flavor compounds (3-methylbutyraldehyde) decreased, but it was still better than the whole milk medium.

[0126] Example 3

[0127] To verify the function of the enzyme system, the enzyme system was prepared according to the enzyme components and their corresponding contents as determined in Example 1, and then added to the enzyme-modified cheese.

[0128] The results showed that optimizing the flavor of enzyme-modified cheese by formulating an enzyme system could also effectively form flavor compounds (3-methylbutyraldehyde), but the effect was slightly inferior to that of cell-free extracts prepared from skim milk. However, it still had an advantage in effect compared to cell-free extracts prepared from whole milk and cheddar cheese cultures.

[0129] Comparative Example 4

[0130] The protein and fat content of the 10-15% series of skim milk medium, the 15-20% series of whole milk medium, and the 35-40% series of mature cheddar cheese culture medium at 3 months were close to those in the commercial MRS medium. Therefore, in order to further demonstrate the effect compared with the commercial MRS, the applicant prepared cell-free cultures by inoculating the MRS medium with r704 fermentation agent.

[0131] The results showed that adding the same amount of cell-free culture to enzyme-modified cheese did not achieve the desired effect in the formation of flavor compounds (3-methylbutyraldehyde), that is, the effect was similar to that of skim milk culture medium. This indicates that the fat and protein content in the culture medium is not the main factor affecting the formation of flavor compounds.

Claims

1. A method for increasing cheese flavor and / or 3-methylbutyraldehyde in enzyme-modified cheese, characterized in that, The method includes: contacting at least one of (1) and (2) below with enzyme-modified cheese; (1) Cell-free extract The method for preparing the cell-free extract includes: a. Inoculate the lactic acid bacteria starter into skim milk medium and culture it to obtain a culture; b. The culture is subjected to solid-liquid separation to obtain bacterial solution and bacterial sludge; c. Resuspend the bacterial sludge in a buffer solution for lysis to obtain a lysate; d. The lysate obtained from the lysate is then mixed with the bacterial culture to obtain the cell-free extract; (2) Enzyme system The enzyme system contains leucine transaminase, glutamate dehydrogenase, hydroxyl dehydrogenase, keto acid decarboxylase, keto acid dehydrogenase, aldehyde dehydrogenase, and alcohol dehydrogenase. Among them, the enzyme activity of leucine transaminase is 10-15 μmol / g / h, the enzyme activity of glutamate dehydrogenase is 25-35 μmol / g / h, the enzyme activity of hydroxy dehydrogenase is 250-300 μmol / g / h, the enzyme activity of keto acid decarboxylase is 0-0.001 μmol / g / h, the enzyme activity of keto acid dehydrogenase is 55-65 μmol / g / h, the enzyme activity of aldehyde dehydrogenase is 10-15 μmol / g / h, and the enzyme activity of alcohol dehydrogenase is 90-110 μmol / g / h. The enzyme-modified cheese is a cheese modified by a combination of neutral proteases and flavor proteases.

2. The method according to claim 1, wherein, The fat content of the skim milk is less than 0.1% by weight; and / or The concentration of skim milk in the skim milk culture medium is 8-20% by weight; and / or The cultivation conditions include: a cultivation temperature of 30-55℃; and a cultivation time such that the bacterial cell concentration in the culture is greater than 10. 8 cfu / mL; and / or The lactic acid bacteria starter contains Lactococcus lactis and Streptococcus thermophilus.

3. The method according to claim 2, wherein, The skim milk culture medium is prepared from skim milk powder and water.

4. The method according to claim 2, wherein, The lactic acid bacteria starter is R704 starter.

5. The method according to claim 1, wherein, The amount of the cell-free extract or enzyme system added relative to 100g of enzyme-modified cheese is 0.6-1g; and / or The contact time is 40-60 hours, and the temperature is 40-50℃.

6. The method according to claim 1, wherein, The modification method includes: adding 0.3-0.5% by weight of flavor protease and 0.2-0.5% by weight of neutral protease to a cheese base; and then modifying it at a temperature of 45-60°C for 30-45 hours.

7. The method according to claim 6, wherein, The cheese base is composed of the following ingredients: 25-35 parts by weight of skim milk powder; 15-25 parts by weight of casein; 15-25 parts by weight of anhydrous butter; Sodium citrate 0.1-0.2 parts by weight; Citric acid 0.1-0.2 parts by weight; Add water to bring the total to 100 parts by weight.

8. Flavored cheese prepared by the method according to any one of claims 1-7.

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