KR101814664B1 - The Gaussian with the manufacture and functional solution for curing this cheese ripened cheese manufacturing method using Chlorella vulgaris culture - Google Patents

Abstract

The present invention relates to a method for producing gouda cheese using a drench solution containing chlorella enzyme hydrolyzate and a method for preparing a drench solution for aged cheese using chlorella enzyme hydrolyzate and a method for producing functional gouda cheese will be.

Description

[0001] The present invention relates to a method for producing gouda cheese using a drench solution containing an enzyme hydrolyzate of chlorella,

The present invention relates to a method for producing gouda cheese using a drench solution containing chlorella enzyme hydrolyzate and a method for preparing a drench solution for aged cheese using chlorella enzyme hydrolyzate and a method for producing functional gouda cheese will be.

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Recently, more than 37% of new materials found in marine resources have been found to be microalgae, and interest in marine microalgae is increasing. Commercial use of marine microalgae has been increasing not only in medicines such as antibiotics, vitamins, anticancer drugs, and health functional foods, but also as a purification of contaminated soil and as a biological fertilizer. The marine microalgae can increase the productivity of the bioactive material depending on the culture method, and the availability of the microalgae can also be increased.

Among marine microalgae, chlorella and spiruna are widely used as health food. They are used for inhibiting accumulation and excretion of heavy metals in the body, biological degradation of environmental toxins, inhibition of arteriosclerosis and hepatic disorder, anticancer activity, It is known to contain ingredients that can inhibit aging by resurfacing, moisturizing effects of food, and harmful substances such as active oxygen species and free radicals. Chlorella is one of the microalgae that is used in various fields of academic and research and is highly utilized as a food additive.

Conventionally, a method for manufacturing Manchuria using sea mustard containing chlorella has been disclosed. However, there has been no description of a method for producing a drench solution for aged cheeses using chlorella having high physiological functions and a method for producing cheese using the same.

Japanese Patent Application Laid-Open No. 10-1604385

SUMMARY OF THE INVENTION The present invention has been made to overcome the above problems, and it is an object of the present invention to provide a drench solution for aged cheeses using chlorella having high physiological function and to provide a well-being type aged Gauda cheese manufacturing method using the same.

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A method for manufacturing gouda cheese using a drench solution containing chlorella enzyme hydrolyzate according to the present invention comprises: sterilizing crude oil; Cooling the sterilized crude oil; Culturing the cooled crude oil after adding a lactic acid bacteria starter and calcium chloride thereto; Adding rennet to the cultured crude oil and solidifying to produce a curd; Cutting and stirring the curd; Eliminating whey from the curd and adding and stirring the whey; Pressing the curd to produce cheese; Adding the cheeses to a drench solution to which chlorella enzyme hydrolyzate has been added to effect dying; And aging the bred cheese in an aging room.

In addition, in the present invention, the chlorella enzyme hydrolyzate is hydrolyzed by using at least one of Cellulase, Trypsin and Pepsin enzymes in chlorella, and the enzyme and chlorella are mixed at a weight ratio of 1: 100 Followed by hydrolysis for 12 hours.

In the present invention, the step of dying may be carried out by pouring the drench solution containing 0.5-1 parts by weight of the chlorella enzyme hydrolyzate into 100 parts by weight of cheese for 5-6 hours.

In the present invention, the chlorine-containing hydrolyzate is prepared by adjusting the Brix concentration of the chlorella enzyme hydrolyzate to 1 to 5% and the pH to 5.2, and adding salt to the salt to a salt concentration of 20 to 25% .

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The present invention as described above has an effect of producing functional dated Gauda cheese by preparing a dipping solution for aged cheese using chlorella with high physiological function.

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1 shows the appearance of Gauda cheese according to the present invention.
FIG. 2 is a microscope photograph of a 1,000-fold magnification of Chlorella vulgaris.
FIG. 3 is a photograph showing the basic culture of fresh C. vulgaris.
Figure 4 shows the effect of chlorella enzymatic hydrolysis culture on cell viability.
5 shows the effect of chlorella enzymatic hydrolysis culture on the production of LPS-induced NO.
FIG. 6 shows a standard curve according to Gallic acid concentration and a standard curve according to quercetin concentration.
Fig. 7 shows changes in viable counts of gouda cheese according to aging period.
8 is a graph showing changes in pH of gouda cheese according to aging.

The present invention relates to a method for producing gouda cheese using chlorella enzyme hydrolyzate, and is intended to prepare a drench solution for aged cheese using chlorella having high physiological function and to produce a well-being type aged cheese using the same.

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end. Materials and methods

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1. Securing Chlorella (Chlorella vulgaris) and mass culture

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1) Introduction and characteristics of species

It is a single cell belonging to the green algae and has a spherical shape. The cell wall is made of cellulosic and is characterized by a somewhat thick. Its size is very small with diameters of 2 ~ 5 ㎛ and it has no flagella. It is also the most commercially used species in the world because it enables large-scale mass cultivation. In the first place, health foods have been used mainly, but recently various studies on various pharmacological functions and functions have been revealed and they have been widely used.

This species is also effective for leukopenia, wounds, hypertension, diabetes, infant development, anemia, etc. The nutritional composition is protein, fat, chlorophyll, carotenoid, calcium, iron, magnesium, potassium, minerals, linolenic acid, linoleic acid, vitamins C and 16 kinds of vitamins are also included. Due to these various nutrients, pharmacological products have recently been used in foods ranging from drinks, vinegar, tea, tofu, liquor, noodles, candy and confections.

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2) Species Sale and Preservation

Chlorella vulgalis (Chlorella vulgalis) was distributed from Korea Microalgae Culture Center (KMCC) and stored in laboratory until cell density stabilized.

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3) Cultivation of microalgae

5 L of sterilized fresh water (3 L of culture) was prepared to cultivate stabilized microalgae at larger doses through species conservation. Here, the species that were preserved species were inoculated and cultured in a batch culture. Conway medium was prepared based on the composition table as shown in Table 1 to supply nutrients. The nutrient medium was supplied only once at the time of inoculation and no additional supply was made even after the incubation days had elapsed. The light condition was a 3-wavelength fluorescent lamp as an illumination light, and the light-dark cycle (light period) of 20L: 4D was set and then cultured in a laboratory at a temperature of 22 ± 1 ° C.

On the other hand, the total amount was harvested at the highest cultivating days and used as an experimental raw material source.

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Table 1 shows the Conway badge composition table. No. Component Contents g / Liter One NaNO ₃ 100 g 2 Na ₂ , (Na ₂ C ₃ H ₅ (OH) ₂ PO ₄ .5½H ₂ O) 17 g 3 _{FeSO 4 (NH 4) · 6H} 2 O 7 g 4 Na ₂ EDTA 3 g 5 H ₃ BO ₃ 2 g 6 Thiamine B ₁ 100 mg 7 Vitamine B ₁₂ 1 mg 8 Biotin 2 mg 9 B medium 10 mL 10 Filtered water 1 L

A badge.

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2. Enzymatic hydrolysis of chlorella culture

The enzymes were hydrolyzed with enzymes to enhance the functional material of chlorella, the enzymes and chlorella were mixed at a weight ratio of 1: 100, hydrolyzed for 12 hours, the enzyme was inactivated for 10 minutes, and stored at -70 ° C . The enzyme used here is one of cellulase, trypsin and pepsin.

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3. Functional verification of chlorella enzyme hydrolyzate

1) Cell culture

Raw 264.7 cells were purchased from the Korean Cell Line Research Foundation and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin and 100 μg / ml streptomycin at 37 ° C, Cells were cultured in 5% CO ₂ incubator and used for experiments.

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2) Cell viability measurement

Raw 264.7 cells were plated at 5 × 10 ⁴ cells / well in a 96-well plate and cultured for 16 hours. The chlorella enzyme hydrolyzate was treated at 100, 500 and 1,000 ppm for 1 hour. LPS was then treated and incubated in a 5% CO ₂ incubator at 37 ° C for 20 hours. Cells were treated with MTT at a concentration of 0.5 mg / mL and cultured for 4 hours. After removing the medium, the formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm using an ELISA microplate reader. Cell viability was expressed as a percentage of control cells.

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3) NO production measurement

Raw 264.7 cells were plated at 1 × 10 ⁵ cells / well in a 96-well plate and cultured for 16 hours. The chlorella enzyme hydrolyzate was treated at 100, 500 and 1,000 ppm for 1 hour. After that, the cells were treated with LPS and cultured in a 5% CO ₂ incubator at 37 ° C for 20 hours. After that, the amount of nitric oxide (NO) was measured by Griess reagent using cell culture medium. In other words, 100 μL of cell culture solution and 100 μL of Griess reagent (1% sulfanilamide in 5% phosphoric acid + 1% α-naphthylamide in H20) were mixed and reacted on 96-well plates for 5 minutes. Were measured. Absorbance was measured with sodium nitrite (NaNO2) to obtain a standard curve, and the concentration of NO was calculated.

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4. Production of dyestuff solution using chlorella enzyme hydrolyzate

The chlorella enzymatic hydrolyzate was added with salt to prepare a drench solution of Gouda cheese with 20 ~ 25% salinity.

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5. Gauda cheese manufacturing

The process for preparing gouda cheese using chlorella enzyme hydrolyzate is as follows, and it is prepared by adding chlorine enzyme hydrolyzate 0.5-1 parts by weight to 100 parts by weight of cheese, , And its appearance was as shown in Fig.

1) Sterilization: sterilization treatment at 63 ° C for 30 minutes

2) Cooling: cooling to 31 ℃ ~ 32 ℃

3) Starter, calcium chloride - incubate for 45 minutes with temperature maintained (after adding mesophilic and calcium chloride additives)

- Mesophilic bacteria: 10 U / 100 kg of crude oil (when using DVS strain)

- Calcium chloride: 0.02% of crude oil (20g / 100kg, 10 times diluted in warm water)

4) Addition of rennet: Add 19ml of rennet to 100kg of crude oil. 10 times dilution in distilled water

5) Solidification: Solidification at 32 ℃ for 25 ~ 30 minutes

6) Cutting: Cut 8 ~ 10mm in width and length

7) Stirring: 20 to 30 minutes

8) Primary whey exclusion: 35% (35ℓ / 100kg) of crude oil

9) Primary singer: 30% of crude oil is added with warm water so that the temperature of crude oil is 35 ℃ (30ℓ, temperature of heating water 60 ~ 65 ℃)

10) Stirring: 15 minutes

11) Secondary whey exclusion: 20% of crude oil (20ℓ)

12) Secondary singer: Add 10% of crude oil to hot water to make the temperature of crude oil up to 38 ℃ (10ℓ, temperature of warm water 70 ~ 80 ℃)

13) Stirring: Check the condition of the curd after 15 minutes

14) Pre-squeezed in whey: squeezed by the weight of curd for 30 minutes

15) Squeeze: Weighs about 5 times the weight of cheese

16) Clarified enzymes hydrolyzate to chlorine (5 to 6 hours per kilogram of chlorine)

17) Aging in the aging room

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6. Physiological activity of Gauda cheese with chlorella enzyme hydrolyzate

1) Total polyphenol content and total flavonoid content

The total polyphenol content was determined as follows. 1.9 mL of distilled water and 0.2 mL of Folin-Ciocalteau's phenol reagent were added to 0.1 mL of the sample solution and reacted at room temperature for 3 minutes. To this, 0.4 mL of saturated Na ₂ CO ₃ solution and 1.9 mL of distilled water were added and reacted at room temperature for 1 hour and absorbance was measured at 725 nm. The standard polyphenol content was determined by preparing a standard curve for each concentration using tannic acid. Total flavonoid content was determined by adding 0.6 mL of 1 N NaOH and 4 mL of diethylene glycol to 0.2 mL of the sample solution, reacting at 37 ° C for 1 hour, and then measuring the absorbance at 420 nm. The standard curve was prepared using naringin as a reference material and the total flavonoid content was determined.

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2) Measurement of DPPH radical scavenging ability

The DPPH radical scavenging ability of the sample solution was determined by adding 2 mL of 0.1 mM DPPH solution to 0.2 mL of the sample solution, reacting at room temperature for 30 minutes, and then measuring the absorbance at 517 nm. The absorbance ratio (%) between the sample solution- Respectively.

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7. Quality characteristics of Gouda cheese during ripening

1) General composition analysis of Gouda cheese according to immersion liquid

The general composition of gouda cheese according to the immersion liquid was analyzed by the following method. Moisture was obtained by atmospheric pressure drying method, crude fat was extracted by soxhlet method, crude protein was used by semimicro kjedahl method, and inquiry was by filtration method.

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2) pH change according to ripening period

pH was measured by using a pH meter.

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3) Changes in viable counts according to aging period

The number of viable cell counts was measured at 1 month intervals, diluted in sterilized saline by decidual dilution method, and cultured for 48 hours at 37 ° C using a BCP agar culture medium. colony forming unit / ml.

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I. result

1. Securing chlorella and mass culture

1) Microalga species: Chlorella vulgaris (freshwater fish)

5 L of sterilized fresh water (3 L of culture) was prepared to cultivate stabilized microalgae at larger doses through species conservation. Here, the species that were preserved were inoculated and cultured on a batch cuture principle. To supply nutrients, Conway medium was prepared based on the composition table and supplied. The nutrient medium was supplied only once at the time of inoculation and no additional supply was made even after the incubation days had elapsed. As a light condition, a 3-wavelength fluorescent lamp was set as an illumination light, and a light-dark cycle (light period) of 20L: 4D was set and then cultured in a laboratory having a temperature of 22 ± 1 ° C. .

On the other hand, the whole culture was harvested to the highest density culture days, and used as the raw material for the experiment. As shown in FIG. 3, the basic culture of C. vulgaris from fresh medium to medium cultivation was shown.

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2. Enzymatic hydrolysis of chlorella culture

The enzymes were hydrolyzed with enzymes to enhance the functional material of chlorella, and enzymes and chlorella were mixed at a weight ratio of 1: 100, hydrolyzed for 12 hours, and the enzyme was inactivated for 10 minutes and stored at -70 ° C . The enzyme used was trypsin and pepsin, and the polyphenol content of the hydrolyzate was as shown in Table 2 below.

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Table 2 shows changes in polyphenol content of chlorella enzyme hydrolyzate. division Polyphenol Chlorella culture 0.2% Chlorella enzyme hydrolyzate 0.3%

3. Functional verification of chlorella enzyme hydrolyzate

Inflammation is the reaction of the organism to recover to the state by removing the injury to the local body damage by the physical or chemical toxic stimulus or the microbial toxic substance, and the stimulation in the inflammatory reaction acts directly on the body part, It is also reported that it is transmitted indirectly through the endogenous chemical messenger to the blood vessels or cells of the bulb and damages it. It has been reported that various diseases such as arthritis, asthma, autoimmune disease and arteriosclerosis are caused by this, and that various diseases are caused by active oxygen species, and antioxidative activities In this study, we investigated the antioxidant activity of chlorella culture solution by enzymatic hydrolysis in vitro.

1) Effects of chlorella enzymatic hydrolysates on the production of NO induced by LPS and cell viability

The effect of the chlorella enzymatic hydrolysis culture medium on the cell viability is shown in Fig. 4. The effect of the chlorella enzymatic hydrolysis culture on the production of LPS-induced NO is shown in Fig. In order to measure the inhibitory effect of chlorella enzymatic hydrolysates on the production of NO induced by LPS in RAW264.7 cells, the concentration of chlorella enzymatic hydrolyzate was adjusted to 10, 50 and 250 ppm. Compared with the control group, LPS (10 μL / mL) increased about 95-fold and chlorella enzymatic hydrolysates significantly inhibited LPS-induced high NO production in a dose-dependent manner. The control group, chlorella culture, showed that the inhibitory effect of NO production was 30% lower than that of enzymatic hydrolysis. The cytotoxic effect did not cause cytotoxicity up to a concentration of 250 ppm.

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4. Production of dyestuff solution using chlorella enzyme hydrolyzate

The preparation of the drench solution using the chlorella enzyme hydrolysis culture solution is as follows. Adjust the Brix concentration of the chlorella enzyme hydrolysis culture to 1 ~ 5%, and then add salt to make a salt solution so that the salinity becomes 20 ~ 25%. At this time, the pH is adjusted to 5.2.

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5. Physiological activity of Gauda cheese with chlorella enzyme hydrolyzate

Total polyphenols, total flavonoids and DPPH scavenging activities of the control and experimental groups (Gauda cheese prepared by immersion in chlorine solution with chlorella enzyme hydrolyzate) were measured at 4 months after the aging. As shown in Table 3, the total polyphenol contents were 0.4 .mu.g / ml and 1.4 .mu.g / ml in the control and experimental groups, respectively, and the total flavonoids were 0.00088 .mu.g / ml and 0.01 .mu.g / ml. The DPPH radical scavenging activity was compared with the absorbance of the distilled water group without addition of the sample. As a result, the DPPH radical scavenging activity was not observed in the control and the DPPH radical scavenging activity was 10% in the experimental group.

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Table 3 shows the total polyphenol, total flavonoid, and DPPH radical scavenging activity of Gouda cheese prepared by immersing in chlorine liquid added with chlorella enzyme hydrolyzate. Total polyphenol ([mu] g / ml) Total flavonoid ([mu] g / ml) DPPH radical scavenging activity (%) Control 0.4 0.00088 - Experimental Section ¹⁾ 1.4 0.01 10

Experimental group ¹⁾ : Gouda cheese prepared by immersing in a drench solution containing chlorella enzyme hydrolyzate.

FIG. 6 shows a standard curve according to the total polyphenol content Gallic acid concentration and a quercetin concentration based on the total polabonoid content.

6. Quality characteristics of Gouda cheese during fermentation

1) General composition analysis of Gouda cheese according to immersion liquid

Table 4 shows the results of the general composition analysis of the gouda cheese prepared by immersing in chlorine solution with the chlorella enzyme hydrolysis culture solution. Moisture, protein, fat and ash contents were 43.2, 20.3, 31.3 and 3.4%, respectively, and there was no difference in physicochemical properties from control (gouda cheese prepared by general method). The contents of fat, protein and ash were increased with increasing ripening period, and moisture content decreased with increasing ripening period.

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Table 4 shows the general composition of gouda cheese prepared with chlorella enzyme hydrolyzate. division
Control Experimental Section ¹⁾ 0 weeks 3 months 0 weeks 3 months moisture 43.0 ± 0.8 39.5 ± 0.6 43.0 ± 1.3 39.8 ± 1.0 protein 20.3 ± 0.8 21.4 ± 0.5 20.3 ± 0.5 21.9 ± 0.6 Fat 31.3 ± 0.9 32.1 ± 1.2 31.3 ± 0.3 32.7 ± 1.0 Ash 3.4 ± 0.2 3.3 ± 0.4 3.4 ± 0.1 3.5 ± 0.2

Experimental group ¹⁾ : Gouda cheese prepared by immersing in a drench solution containing chlorella enzyme hydrolyzate.

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2) Measurement of viable cell count and pH change

As the aging time increased, the number of viable cells decreased in both the control and experimental groups (Gauda cheese prepared by immersing in the drench solution prepared using the chlorella enzyme hydrolysis culture) as shown in FIG. As shown in FIG. 8, the pH was slightly decreased in the experimental group from 5.30 to 4, and was 5.20 in the control group and 5.22 in the experimental group. The pH of the experimental group was varied according to the proteolytic degradation and ammonia formation 5.50 and 5.49, respectively, but there was no significant difference.

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<Examples>

Crude oil was sterilized at 63 占 폚 for 30 minutes and cooled to 32 占 폚.

To the cooled crude oil, 10 U of a lactic acid bacteria starter DVS (Direct-Vat-Set) strain and 20 g of calcium chloride were added, followed by incubation for 45 minutes at 32 ° C.

19 ml of lantern was added to 100 kg of the cultured crude oil, and the mixture was allowed to stand at 32 캜 for 25 minutes to solidify to prepare a curd.

The curd was cut horizontally and vertically using a cheese knife in a size of 8 mm and agitated while stirring for 20 minutes.

Thereafter, the primary whey drainage (35 wt% of the crude oil) was carried out, and the primary syrup (30 wt% of the crude oil) was warmed with warm water at 65 캜 and stirred at 35 캜 for 15 min.

Thereafter, secondary whey drainage (20 wt% of crude oil) was carried out, and secondary water (10 wt% of crude oil) was added with heating water at a temperature of 80 째 C, followed by stirring at 38 째 C for 15 min.

The curd was preliminarily pressed for 30 minutes, followed by compression bonding at 5 times the weight of the curd to produce cheese.

Next, 100 parts by weight of the crushed cheese was added to a drench solution to which 1 part by weight of chlorella enzyme hydrolyzate was added, followed by dying for 6 hours.

Here, the chlorella enzyme hydrolyzate was hydrolyzed using Trypsin and Pepsin enzymes, and the enzyme and chlorella were mixed at a weight ratio of 1: 100, hydrolyzed for 12 hours, and then the enzyme was inactivated for 10 minutes, And stored at 70 DEG C to prepare an enzyme hydrolyzate of chlorella culture broth.

Herein, the chlorine enzyme hydrolysis culture solution was adjusted to a Brix concentration of 1% and a pH of 5.2, and salt was added thereto so as to have a salinity of 20%.

The banned cheese was aged in the aging room for 3 months to prepare gouda cheese.

Claims (4)

Sterilizing the crude oil;
Cooling the sterilized crude oil;
Culturing the cooled crude oil after adding a lactic acid bacteria starter and calcium chloride thereto;
Adding rennet to the cultured crude oil and solidifying to produce a curd;
Cutting and stirring the curd;
Eliminating whey from the curd and adding and stirring the whey;
Pressing the curd to produce cheese;
Adding the cheeses to a drench solution to which chlorella enzyme hydrolyzate has been added to effect dying; And
Aging the bred cheese in a fermentation room;
Wherein the chlorella enzyme hydrolyzate is added to the chlorella enzyme hydrolyzate.
The method according to claim 1, wherein the chlorella enzyme hydrolyzate is hydrolyzed by using at least one of Cellulase, Trypsin and Pepsin enzymes in chlorella, wherein the enzyme and chlorella are mixed at a weight ratio of 1: 100 And the mixture is hydrolyzed for 12 hours. The method for producing gouda cheese using the drench solution to which the chlorella enzyme hydrolyzate is added.
[3] The method according to claim 2, wherein the dying step is carried out by adding the chlorella enzyme hydrolyzate to 0.5 to 1 part by weight of the chlorella enzyme hydrolyzate to 100 parts by weight of the cheese for 5 to 6 hours. A method for making Gauda cheese using a saline solution.
[Claim 4] The method according to claim 3, wherein the salt solution is prepared by adjusting the Brix concentration of the chlorella enzyme hydrolyzate to 1 to 5% and the pH to 5.2, and adding salt to the salt concentration of 20 to 25% Wherein the chlorella enzyme hydrolyzate is added to the chlorella enzyme hydrolyzate.

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