KR101486522B1 - Aspergillus oryzae improving the flavor of the food - Google Patents

Abstract

The present invention relates to a strain for enhancing the flavor of food, and more particularly to Aspergillus oryzae M1-4 (KCTC 12159BP). The present invention also relates to a method for enhancing the flavor of a food by using the strain as a fermenting bacteria. More specifically, the food may be at least one selected from the group consisting of miso, kochujang, chongkukjang, soy sauce, yogurt, kimchi, and cheese.
The choice of consumers is changing to be natural and health-oriented due to the problems of bird flu, BSE, pig cholera, livestock feed antibiotics, MSG, and GMO. At the same time, The industry is stagnating. In response to these trends, domestic seasoning companies are developing flavoring materials using natural extracts in accordance with consumers' preference, but they lack quality competitiveness such as depth of taste, richness and persistence. Accordingly, premium quality high quality natural and fermented flavoring materials with characteristics of no meat, no MSG, no HVP, and non-GMO will progressively replace existing flavoring materials.

Description

[0002] Aspergillus oryzae is known to improve flavor of the food,

The present invention relates to a strain for enhancing the flavor of food, and more particularly to Aspergillus oryzae M1-4 (KCTC 12159BP).

Another aspect of the present invention relates to a method for enhancing the flavor of a food by using the strain as a fermenting bacteria. More specifically, the food may be at least one selected from the group consisting of miso, kochujang, chongkukjang, soy sauce, yogurt, kimchi, and cheese.

Domestic flavoring materials have been manufactured by acid hydrolysis and enzymatic decomposition methods based on low cost and low quality concentrated aquatic products, processed products thereof, and byproducts generated in the food industry based on cost competitiveness and efficient production.

On the other hand, overseas flavoring materials are produced / sold mainly in Japan as shown in the table below. These products have been combined with acid decomposition technology, enzymatic decomposition technology, brewing fermentation technology, etc., and their use in the food industry in Japan is gradually increasing, and the inflow into the country is also increasing.

Japanese soy sauce products having a flavor component centered on amino acids can be seen as a kind of flavor materials, but they have characteristics that flavors peculiar to soy sauce are strong and colors are not suitable for flavor materials. In addition, there are no products that use only natural soybean as main ingredient.

In addition, high TN content materials such as Ajinomoto's "koji-aji" products are mostly made by decomposing wheat gluten into powder (or added with yeast extract etc.) And thus the price competitiveness is low.

In addition, due to westernization and high quality of "appetite", which is accompanied by an increase in income and living standard, the composition of flavoring materials that are well-suited to Korean cuisine as well as Western cuisine is increasing day by day. It seems that the market environment is changing with good flavoring materials that are well suited to Korean and Western cuisines.

Although the method of hydrolyzing a protein to an acid to obtain a high concentration of amino acid is advantageous in that the reaction time is very short, the utilization rate of the raw material is high and it is economical.

As a result, the decomposition method using an enzyme reaction has been attempted mainly in industry, but this also causes a problem that the production unit price is increased due to the use of expensive enzyme and the taste is not simple and abundant.

On the other hand, the conventional natural brewing method is a method in which a microorganism source is added to a raw protein to naturally mature it, and it is pointed out that it is difficult to obtain a highly concentrated fine substance because proteolysis is not efficient and the occurrence of an undesirable odor is difficult. It has a great advantage that it is suitable for use as a material which is weak in odor and is natural-oriented and economical in the production method. Therefore, the scientific and industrialization of traditional natural brewing methods has been transformed into a direction of technology development that can complement the shortcomings of traditional natural brewing methods and obtain high-quality, precision materials.

Sampyo Foods Co., Ltd. has 60 years of experience in brewing fermentation and enzyme decomposition technology as key technologies. In particular, brewing fermentation technology is domestically domesticated from microbiological fermentation based on vegetable raw materials to industrialization technology. Sam-Pyo Foods Co., Ltd. will be able to lead the development of advanced flavor materials manufacturing technology based on the acquired technology.

The background art relating to the present invention will be described below.

1. Jeong, SY; Chung, SJ; Shu, DS; Shu, BC; Kim, KO Developing a descriptive analysis procedure for evaluating the sensory characteristics of soy sauce. J. Food . Sci . 2004, 69, S319-S325

2. Baek, HH; Kim, HJ Solid phase microextraction-gas chromatography-olfactometry of soy sauce based on sample dilution analysis. Food Sci . Biotechnol . 2004, 13, 90-95

3. Lee, SM; Seo, BC; Kim, Y.-S. Volatile compounds in fermented and acid-hydrolyzed soy sauces. J. Food . Sci . 2006, 71, C146-C156

4. Cho, IH; Choi, H.-K .; Kim, Y.-S. Difference in the volatile composition of pine-mushrooms ( Tricholoma matsutake Sing.) According to their grades. J. Agric. Food Chem. 2006, 54, 4820-4825

5. Steinhaus, P .; Schieberle, P. Characterization of the key aroma compounds in soy sauce using approaches to molecular sensory science. J. Agric . Food Chem . 2007, 55, 6262-6269

6. Cho, IH; Lee, K.-W .; Kim, Y.-S .; Choi, H.-K. Determination of differences in the non-volatile metabolites of pine-mushrooms ( Tricholomamatsutake Sing.) According to ¹ HNMRandprincipalcomponentanalysis. J. Microbiol. Biotechnol. 2007, 17, 1682-1687

7. Cho, IH; Lee, SM; Kim, SY; Choi, HK; Kim, KO; Kim, YS. Differentiation of aroma characteristics of pine-mushrooms ( Tricholoma matsutake Sing.) of different grades using gaschromatography-olfactometry (GC-O) and sensoryanalysis. J. Agric. Food Chem. 2007, 55, 2323-2328

8. Cho, IH; Kim, Y.-S .; Choi, H.-K. Metabolomic discrimination of different grades of pine-mushroom ( Tricholoma matsutake Sing.) Using ¹ H NMR spectrometry and multivariate data analysis. J. Pharm. Biomed. Anal. 2007, 43, 900-904

9. Park, M. K .; Choi, H.-K .; Kwon, D.-Y .; Kim, Y.-S. Study of volatile organic acids in freeze-dried Cheonggukjang formed during fermentation using SPME and stable-isotope dilution assay (SIDA). Food Chem. 2007, 105, 1276-1280

10. Park, HK; Kim, JK Optimal manufacturing conditions for Korean soybean paste and soy sauce, using Aspergillus oryzae AJ 100 as a flavor improver. Food Sci . Biotechnol . 2008, 17, 208-211

11. Gao, SL; Zhao, HF; Zhao, MM; Cui, C .; Ren, JY Comparative study on volatile flavor compounds of traditional Chinese-type soy sauce prepared with soybean and defatted soy meal. Food Sci . Biotechnol . 2009, 18, 1447-1458

12. Ko, B.-K .; Ahn, H. -J .; van den Berg, F .; Lee, C.-H .; Hong, Y.-S. Metabolomic insight soy sauce through 1H NMR spectroscopy. J. Agric. Food Chem. 2009, 57, 6862-6870

13. Introduction, A .; Osako, K .; Okamoto, A .; Ohshima, T. Olfactometric characterization of aroma active compounds in fermented fish paste in comparison with fish sauce, fermented soy paste and sauce products. Food Res . Intl . 2010 (In Press) .1.

14. Seong-Hee Seo, Hwang In-Kyoung, Yoo, Hyeung-Seung, Odor component and sensory characteristics of conventional soy sauce. Korea Soybean Research Association. 1995, 12, 21-32

15. Park, O., Son, Kyunghee, and Park Hyun-kyung. Analysis of Taste Components of Korean Traditional Soy Sauce by Dipping Container. Korean Journal of Food Culture. 1996. 11, 229-233

16. Park, Hyung Kyung, Son Kyung Hee, Park Ok Jin. Analysis of Major Flavor Factors Related to Taste and Flavor of Korean Traditional Soy Sauce (1) - General Characteristics and Analysis of Sugars and Organic Acids - Korean Society of Food Culture. 1997, 12, 63-69

17. Park, Hyun-kyung, and Son Kyunghee. Analysis of the main flavor factors involved in the taste and flavor of Korean traditional soy sauce (2) - Analysis of nitrogen compounds, free amino acids and nucleic acid related substances. Korean Society of Food Culture. 1997, 12, 53-61

18. Park Hyun-kyung, Son Kyung-hee, Park Ok-jin. Analysis of Major Flavor Factors Related to Taste and Flavor of Korean Traditional Soy Sauce (3) - Analysis of Flavor Components - Korean Society of Food Culture. 1997, 12, 173-182

The object of the present invention is to provide a strain for improving the flavor of food, more specifically, Aspergillus oryzae M1-4 (KCTC 12159BP).

Further, the present invention aims to provide a method for enhancing the flavor of at least one food selected from the group consisting of miso, kochujang, chongkukjang, soy sauce, yogurt, kimchi, and cheese by using the strain as a fermenter.

Hereinafter, the aiming point of the present invention will be described in detail.

1. Need to develop natural flavor enhancing material

Our ancestors have used natural seasonings such as soy sauce, miso, kochujang, and vinegar to taste food. Double soy sauce is a flavoring material based on soybean and has the characteristic that it is composed mainly of free amino acids and peptides. Despite these advantages, however, total nitrogen (TN) is 0.5 ~ 0.8%, which is not rich in flavor, and has a distinctive strong flavor (smoother, unpleasant odor) It is not suitable for Western cuisine and its utilization is low. The seasoning market is based on the second-generation seasoning ('mixed seasoning': a mixture of MSG and other additives) in 'MSG (monosodium glutamate)' called first-generation cooking liquor. In order to meet the consumer trend of "well- ('Natural ingredients seasonings'), but they are getting out of touch with consumers because they are not rich in sensual taste. (Domestic seasoning market has been steadily declining in both 2002 and 2007, and it has been growing by -7% in 2007.) Therefore, the trend of "well-being" It has a gentle flavor, and the composition of the flavoring material that is well suited to Korean cuisine as well as Western cuisine is increasing day by day.

2. Necessity of development of flavoring materials using natural vegetable soybeans

Animal raw materials have been used as raw materials for flavoring materials until now. However, due to the problems of mad cow disease, avian influenza and antibiotics, interest in plant raw materials has been increasing, and in particular, soybean has advantages in terms of functional materials Unlike other grains, it is very high in protein (30-45%) and fat (16-22%). Since the conventional flavoring materials have been developed in order to improve only the taste itself, defatted soybeans in which the fat component has been removed have been mainly used rather than using the soybean itself in order to maximize the amino acid content, which is a qualitative substance, Of the fat component was not noticed. However, recently, research has been conducted to dissolve and remove the fat-soluble odor components generated during the fermentation process of soybean oil, and to prevent the oxidation of fermented product by preventing oxygen from being oxidized. It is known that glycerol induces a mild taste, and it is important to develop a technology that can obtain high quality substances (amino acids, peptides) while using soybean itself in order to develop flavor enhancing flavor materials.

3. Necessity of replacing imported materials through development of natural flavoring material

Domestic flavoring materials have been manufactured by methods such as acid hydrolysis and enzymatic decomposition based on low cost and low quality concentrated aquatic products, processed products thereof, and byproducts generated in the food industry, based on cost competitiveness and efficient production. However, in recent years, there has been a growing demand for health-oriented flavoring materials that are natural-oriented and harmless as well as improving income and living standards. Therefore, existing flavoring materials are not satisfying consumers' desires. Unlike in the domestic market, Japanese and European companies are developing and selling high value added natural flavoring materials using traditional fermented products such as wheat and soybean and enzymatic hydrolysates. It is a high value added material that gives depth, richness and sustainability. It is used not only in the overseas food industry but also in the domestic market. In Korea, high-quality natural flavoring materials have not been developed in Japan, and Japanese flavors are being imported by European and Japanese manufacturers. Therefore, in Korea, it is urgent to develop new natural and fermented flavoring materials that combine traditional food industry and biotechnology.

The technical objects to be achieved by the present invention are not limited to the above-mentioned technical problems, and other technical subjects which are not mentioned can be clearly understood by those skilled in the art from the description of the present invention .

The present invention provides a strain that promotes the flavor of food. More specifically, Aspergillus oryzae M1-4 (KCTC 12159BP) is provided.

The aspergillus oryzae may be characterized by high activity for protease, amylase, and glutaminase.

The aspergillus oryzae may be characterized as having no aflatoxin-producing ability known as a toxic substance.

The aspergillus oryzae may be characterized in that the color of the spores is not rich because it does not affect the color of the flavoring material.

The present invention also provides a method for enhancing the flavor of a food by using the strain as a fermenting microorganism.

The food may be at least one selected from the group consisting of miso, kochujang, chongkukjang, soy sauce, yogurt, kimchi, and cheese.

Hereinafter, the technical content of the present invention will be further described. Domestic flavoring materials have been manufactured by methods such as acid hydrolysis and enzymatic decomposition based on low-cost, low-quality concentrated aquatic products and their processed products and by-products generated in the food industry based on cost competitiveness and efficient production. However, in recent years, there has been an increasing demand for health-oriented flavoring materials that are natural-oriented and harmless as well as improving income and living standards, and the existing flavoring materials are not satisfying consumer's desires. Unlike in the domestic market, Japanese and European companies develop and sell high value-added natural flavoring materials using traditional fermented products such as wheat and soybean and enzymatic hydrolysates, , High-value added materials that give a sense of richness and persistence, are used not only in the overseas food industry but also in the domestic market. In Korea, high-quality natural flavoring materials have not been developed in Japan. As a result, the taste of seasoning foods is reinforced by importing materials developed by Japanese and European companies. Therefore, in Korea, it is necessary to participate in the development of new natural and fermented flavoring materials that combine traditional food industry and biotechnology.

In addition to increasing income and living standards, health-oriented and natural-oriented flavor ingredients are increasing day by day. However, the present flavor ingredients are not only a few natural oriented products in terms of raw material and manufacturing method, Quality is also weak. Therefore, this R & D product has developed natural and fermented flavoring materials through the improvement of the traditional brewing method using the natural vegetable raw material, soybean, rather than animal, in terms of raw material, and it is suitable for the trend of well-being, It has a rich and gentle flavor and is characterized by good flavor in both Korean and Western dishes. In accordance with the trend of "well-being", the natural and fermented flavors that have a deep flavor, abundant flavor and soft flavor are becoming bigger and bigger. The use of natural vegetable soybeans and improved traditional brewing technology .

It is important to minimize the mail-rod response in order to achieve bright colors that can be used in a variety of dishes and foods. Therefore, it is important to reduce the duration of the protein degradation by shortening the duration of the protein degradation. For this, a strain with high protease activity should be used. Protease and amylase activity were improved by 52% and 55%, respectively. One yeast strain with good aroma, high alcohol production ability, no acid membrane production ability, one kind of lactic acid bacteria with high lactic acid production ability, good flavor, inhibiting acid production and lacking histidine decarboxylase activity was finally selected and used .

Protein degradation / fermentation conditions were optimized through the setting of raw material costs, salinity content and aging period to achieve TN = 1.5 or more, AN = 0.9 or more and Color (OD ₅₀₀ ) = 3 or less.

Based on the above-mentioned conditions, we aimed to prevent quality deterioration due to contaminated microorganisms and to maximize production efficiency by shortening the time for each manufacturing process by mass-production, and by stabilizing the quality through application of homogenization and de- We have constructed / optimized the system for safety.

The natural and fermented flavor produced through the mass production system achieved not only the physicochemical target level but also the general consumers' sensory evaluation results showed that the soybean flavor was strong, the chemical flavor was weak, the color was bright and the customer satisfaction was high (3.8 Point), and customer satisfaction (3.2 ~ 3.4 points for similar products) was evaluated positively.

Based on the above-mentioned first natural and fermented flavor manufacturing technology, in the second year, we will develop a technology to manufacture a second natural and fermented flavor (TN 2.0% or more, AN 1.2% or more) It is thought that it will be able to develop application products suitable for eastern and western dishes using fermented flavor.

The choice of consumers is changing to be natural and health-oriented due to the problems of bird flu, BSE, pig cholera, livestock feed antibiotics, MSG, and GMO. At the same time, The industry is stagnating. In response to these trends, domestic seasoning companies are developing flavoring materials using natural extracts in accordance with consumers' preference, but they lack quality competitiveness such as depth of taste, richness and persistence. Accordingly, premium quality high quality natural and fermented flavoring materials with characteristics of no meat, no MSG, no HVP, and non-GMO will progressively replace existing flavoring materials.

Figure 1 relates to a protease plate assay.
Figure 2 relates to the Amylase Plate assay.
Fig. 3 relates to changes in the number of spores and the total number of bacteria in the aspartate culture according to the variation of the water content.
Figure 4 relates to a decarboxylating agar (left: decarboxylase formation; right: Decarboxylase non-production).
Fig. 5 relates to the characteristic intensity of flavor due to fermentation of yeast / lactic acid bacteria.
Figure 6 relates to the change in TN content due to yeast / lactic acid fermentation.
Figure 7 relates to the pH change due to yeast / lactic acid fermentation.
FIG. 8 relates to a flow of the manufacturing process of the flavoring material.
Fig. 9 relates to the results of the sensory characteristics (characteristic strength) analysis of the scale-up prototype.
Fig. 10 relates to the results of the sensory characteristics (characteristic intensity) analysis of the scale-up prototype.
11 is a photograph of a prototype of the present invention.

The present invention provides a strain that promotes the flavor of food. More particularly, to Aspergillus oryzae M1-4 (KCTC 12159BP). The aspergillus oryzae may be characterized by high activity against protease, amylase and glutaminase, and may be characterized by having no aflatoxin-producing ability known as a toxic substance. In order not to affect the color of the flavoring material And the color of the spores is not rich.

The present invention also relates to a method for enhancing the flavor of a food by using the strain as a fermenting microorganism. The food may be at least one selected from the group consisting of miso, kochujang, chongkukjang, soy sauce, yogurt, kimchi, and cheese.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Section 1 Overview of Product Development

The present invention aims to develop high quality natural and fermented flavoring materials and applied products by combining microorganism fermentation technology with soybean which is a vegetable natural raw material.

Section 2 Research Contents and Results

[1] Development of new flavor enhancing fermentation taste material using soybean fermentation technology

1. Materials and Experimental Methods

end. Culture and selection

Fungal strains belonging to Sampo Foods Research Institute are subcultured on Potato dextrose agar (PDA) medium and cultured at 30 ℃ for 5 days. A lab-scale empire is conducted by first selecting strains that are fast growing mycelium. Imperial conditions were carried out under the empire culture conditions of Sampyo Foods Co., Ltd.

I. Lab-scale Empire

The bran or soybean was mixed with distilled water so that the water content was 48%, and the mixture was transferred to an Erlenmeyer flask. The autoclave was performed at 121 ° C for 20 minutes.

All. protease plate assay

Lab-scale emulsion was obtained and the obtained broth was suspended in physiological saline to extract the enzyme solution. The extracted enzyme solution was assayed for clear zone production on potato dextrose agar (PDA) medium containing 2% casein.

la. Amylase plate assay

The enzyme solution was extracted from Lab-scale broth and the clear zone production was measured on potato dextrose agar (PDA) medium containing 1% starch.

hemp. Enzyme determination of germs

The activity of Protease, Amylase, Glutaminas, and Pectinase was measured according to each enzymatic method.

bar. Establish culture condition of Aspergillus

The selected strains are firstly cultured in potato dextrose agar (PDA) medium and then transferred to a different wheat bran at different water contents and incubated at 30 ° C for 5 days. After incubation for 5 days, the germs were suspended in physiological saline, and then sprouted on a PDA medium to measure the amount of spores produced.

four. Screening and selection of organic acid-producing microorganisms

Bacteria from Sampyo Food Technology Research Institute were plated on MRS medium and cultured in aerobic jar at 30 ℃ for 3 days. Single colony was cultured in MRS medium (5%, 10%, 15%) according to salinity concentration to select salt tolerant microorganisms.

Ah. Selection of non-producing strains of histidine decarboxylase

Decarboxylating agar medium was inoculated with Lactobacillus and incubated at 30 ℃ for 3 days. When the color of the medium changed to red, Histidine decarboxylase activity was determined.

Table 1 below describes decarboxylating agar medium.

Constituent content (%) Tryptone 0.125 Yeast extract 0.125 (NH _{4) 2} SO ₄ 0.75 NaCl 0.5 Glucose 0.1 MgSO ₄ 7H ₂ O 0.02 MnSO ₄ 7H ₂ O 0.005 FeSO ₄ 7H ₂ O 0.004 Tween 80 0.05 Cresol red 0.02 agar 3 L-histidine hydrochloride monohydrate 2

 character. Organic acid analysis

Standard curves of each standard sample (lactic acid, citric acid, malic acid, succinic acid) were prepared using HPLC and quantitative analysis of organic acids was performed by comparing with the samples (culture medium of lactic acid bacteria, .

car. Screening and screening of high alcohol-producing yeast

Yeast strains belonging to the Sungpyo Food Technology Research Institute were subcultured on Yeast modified agar (YM) medium and cultured at 30 ° C for 72 hours. Ethanol and glucose contents in the culture medium were analyzed at 24, 48, and 72 hours, respectively, and the strains in which glucose was converted to ethanol were firstly selected. The selected strains were inoculated with 1% each in 8% and 16% salinity, respectively, and cultured at 30 ℃ for 1 month.

Car. Formalin nitrogen

5 ml of the sample is weighed into a 100 ml beaker and distilled water is added to the flask. After thoroughly stirring, the flask is placed in a 250 ml m-flask. The beaker is thoroughly washed with distilled water and placed in an m-flask. Ml Erlenmeyer flask, and 25 ml was taken with a hole pipet. Then, 20 ml of formaldehyde titrated to pH 8.4 with 0.1 N NaOH was added, titrated until the pH was 8.4, and the amount consumed was recorded and calculated.

A: the number of 0.1N NaOH solution consumed for neutralizing the sample solution

B: the number of 0.1N NaOH solution consumed in the background test

f: concentration coefficient of 0.1N NaOH

S: Sample weight (g)

Get on. saline

5 ml of the sample was put in a 250 ml m-flask and massed up. Then 5 ml of 2% K ₂ CrO ₄ was added thereto, and the amount consumed at the time of changing from yellow to red while titrating with 0.02 N AgNO ₃ was calculated .

wave. T-N

50 ml of the sample (diluted when the nitrogen content is 0.1 mg or more) is placed in a syringe bottle, 10 ml of an alkali potassium persulfate solution is added thereto, the stopper is closed and shaken, and the mixture is heated in a high pressure steam sterilizer. And the mixture was heated and decomposed for 30 minutes. The supernatant of the pretreated sample was filtered through a glass fiber filter (GF / C), 25 ml of the filtrate was accurately taken, and 5 ml of hydrochloric acid was added thereto to adjust the pH to 2 to 3. A part of this solution was transferred to a 10 mm absorption cell of the bed length and used as a sample solution. 50 ml of water was taken and tested according to the pretreatment test method of the sample, and used as a base test solution. The absorbance of the test solution was measured at 220 nm using the basal test solution as a reference, and the amount of nitrogen was determined from the calibration curve prepared beforehand, and the total nitrogen concentration (mg / l) in the sample was calculated by the following equation.

a: Amount of nitrogen (mg) obtained from the calibration curve

V: Amount of sample used in the pretreatment (ml)

Ha. Amino acid composition analysis

The amino acid composition was analyzed using the AccQ-Tag method. That is, 50 ml of a solution obtained by sonication with 0.5 g of distilled water was added to 350 ml of a borate buffer and 100 ml of AccQ-Tag derivative reagent, followed by derivatization at 55 ° C for 10 minutes . Each 10 ml of each of the derivatized samples was placed in an automatic sample injector of HPLC and analyzed under the same conditions as in Table 1. Amino acid analysis column (AccI-Tag C ₁₈ , Millipore Co. Milford, MA, USA) was used to analyze the amino acid by injecting 40 ml of the standard material and 10 ml of the sample solution. ) And the mobile phase was eluted with a solution of 0.14 M sodium acetate and 10% triethylamine in 1% phosphoric acid at pH 5.02 (eluent A), water and acetonitrile 4: 6 (eluent B) was eluted with a linear gradient.

Table 2 below describes the HPLC analysis condition for amino acids.

 Item  Experimental conditions  Instrument Waters 1525 Binary Pump, Waters 717 plus Autosampler,
Waters 474 Fluorescence detector  Column AccQ-Tag column C ₁₈  Column size  3.9 x 150 mm  Solvent A (Eluent A)  0.14 M sodium acetate, 10% triethylamine in water, pH 5.02  Solvent B (Eluent B)  Acetonitrile: water = 6: 4  Gradient  AccQ-Teg method  Flow rate  1.0 mL / min  Run time  45 min  Wavelength  ex. = 250 nm, em. = 395 nm

roughness. Microbial toxin Aflatoxin content measurement

The enzyme-linked immu- noosorbent assay (ELISA) was used to determine Aflatoxin in the fermentation process.

you. Biogenic amine measurement for identification of fermented food safety

The biosynthetic amine (Histamine, Tyramine) was analyzed by the HPLC method proposed by Korea Food & Drug Administration.

more. Nitrogen utilization rate

Nitrogen utilization was calculated as the factor showing how much of the nitrogen component derived from the raw material was eluted into the flavoring material as follows.

* TN (%) of soybean = 5.5

It is. Soy sauce paste manufacture

The heat - treated soybean (or defatted soybean) and wheat flour were mixed and fermented for 3 days at 30 ° C, mixed with an appropriate amount of salt water, and aged for several days. At this time, the concentration of salt water and the amount of added salt were adjusted according to the final salinity of the soy sauce.

what. Sensory test

- panel selection and training

Among the general consumers, 20 people with excellent taste were selected and composed of sensory panels. These are sweet, 5% sucrose solution, sour, 0.08% lactic acid solution, salty, 1% sodium chloride solution, glutamate-like, umami, 0.3% MSG solution, bitter , 0.05% caffein solution) were trained as reference materials.

- Sample preparation and presentation

For the evaluation of the appearance / fragrance, samples were placed in a 50 ml beaker with 10 ml of each sample and closed under the incandescent lamp. The flavor evaluation was carried out by placing 5 ml of the sample in a plastic container, Respectively.

- Evaluation contents and procedures

The tests were conducted at 11:00 am and 5:00 pm. For the sensory testees participating in the experiment, drinkers were advised to avoid drinks, food and mouthwashes other than water 1 hour before the test, and to avoid use of fragrant cosmetics. The 15 - point item scale was used for the character strength evaluation and the sample was provided at random, and the evaluation was divided into training and experiment. In addition, up to three samples were presented to one person to prevent slowing of taste. The 5 - point scale was used for the evaluation of preference.

- definition of flavor

Alcohol: Volatile incense from Ethyl alcohol

Chemical Chemical (Chemical): artificial flavor felt in soy sauce

Soybean: The fragrant aroma of boiled soybeans

Meju (Meju): A unique flavor in Meju

Roasted soybean: Fragrant incense from roasted beans

Roasted sesame oil: sesame oil

Burnt: Smell of bells or trees

Sweet: Sweet potatoes such as molasses and syrup

Sour: Acetic acid is a mixture of

Briny: Warm fragrance in seawater, water kelp

Kumkumham: The old-fashioned incense of old soy sauce

Caramel flavor: caramel flavor

Tofu: Dust, a mysterious cleanse from the basement

Fermented (Fermented): I have a unique flavor from the party that took effect by the East

Laver: I have a unique smell in Kim

Salty: The taste associated with sodium ion, such as NaCl, felt in the tongue.

Sweet: The taste associated with a single substance such as sugar, artificial sweetener felt in the tongue

Sour: The taste of the substance associated with the tongue

MSG flavor, richness (MSG): The unique taste of MSG in the tongue

Bitter: bitter taste of caffeine, quinine, etc., felt from the back of the tongue

Bing: A stimulating sensation in front of the tongue

Pungent sensation: a sense of touching sensation in the sense of smell

Alcohol: Volatile flavor from Ethyl alcohol

Chemical Chemical: The artificial chemical flavor felt in soy sauce

Meju (Meju): Meju is a unique flavor of Meju

Roasted soybean: Flavored flavor from roasted beans

Boiled soybean: Boiled soybean flavor

Roasted sesame oil: The sweet flavor from sesame oil

Metallic: Metallic flavors from metals such as iron and copper

Kim, Nam-mi (Laver): I have a unique flavor

Burnt: burnt flavor from tan or charcoal

Tuck-in / tingling: Feeling tanned or tanned by tannins

Overall Taste Strength: Strong and weak in taste

Mouthfull: Complexity of taste

Concentration: thick, body sensation

Burr. Consumer Research

Prototype's customer satisfaction survey was conducted by Home Usage Test (HUT). The sample was prepared from 72 samples of 28-year-old and 45-year-old Sampei panel, and 200ml of sample was provided and freely used for heating and non- Respectively.

2. Detailed performance results

end. Separation and Biological Characterization of Flavor Enhanced Fine Fermentation Microorganisms

(1) Isolation of high protease activity

Soybeans contain about 40% protein, which is very high in protein compared to other common grains. Soybean protein must be effectively decomposed into amino acids, which are essential amino acids, to make flavorings. In order to effectively decompose the soybean protein, it is necessary to isolate / apply a high-quality microorganism having high protease activity, and among the various microorganisms producing the protease, it is possible to take a highly active protease when solid phase fermentation It is known.

In the solid state fermented product used in the manufacture of conventional flavorings such as brewed soy sauce, the protease activity is about 600 to 800 unit / g, and it takes about 5 to 6 months to decompose the enzyme into the amino acid, . However, if it is decomposed for such a long period of time, the color becomes black due to the mailard reaction. Such dark color changes the color of the food and the food itself when it is used for cooking and food, so it is not suitable as a flavoring material because the width of the application area is narrow.

For this reason, selection and application of a good strain with high protease activity is important in that it can shorten the time for proteolytic cleavage, improve the color of the flavor material, and improve the content of the amino acid (amino acid). Amylase is also known to be an important enzyme because it contains the raw material itself and can decompose carbohydrates into saccharides to enhance sweetness. Glutaminase is an important enzyme in that glutamine can be converted into glutamic acid to enhance its richness. Therefore, in order to prevent aflatoxin production, which has a high activity of protease, amylase and glutaminase and toxic substance and does not affect the color of the flavoring material,

(A) 1st screening - Plate assay

As shown in Table 3, each of the germs was suspended in physiological saline, and the obtained enzyme solution was reacted on a plate containing skim milk to measure the size of the clear zone (FIG. 1) M1-4 and M2-1 strains were found to have higher protease activity than the control.

The following Table 3 relates to the protease plate assay by germ.

Strain number Clear zone size (mm) 1st 2nd 3rd average (mm) Control 5.3 5.3 5.1 5.2 M1-1 3.3 3.3 3.3 3.3 M1 -2 6.5 6.4 6.5 6.5 M1-3 4.5 4.6 4.6 4.6 M1 -4 6.0 5.8 6.0 6.0 M2 -One 5.8 5.6 5.7 5.7 M2-2 2.9 3.1 2.9 3.0 M3-1 3.0 3.2 3.1 3.1 M3-2 1.8 1.9 2.1 1.9 M4-1 3.5 3.7 3.5 3.6 M5-1 5.4 5.3 5.1 5.3

As shown in Table 4, each of the germs was suspended in physiological saline after solid-phase culture, and the obtained enzyme solution was reacted on a plate containing starch to measure the size of the clear zone (FIG. 2) to obtain M1-4, M3-1, 2 strains were found to have higher amylase activity than the control (primary pathogen).

The following Table 4 relates to the amylase plate assay by germ.

Strain number Clear zone size (mm) 1st 2nd 3rd average (mm) Control 5.3 5.5 5.7 5.5 M1-1 5.0 5.2 5.1 5.1 M1-2 4.4 4.3 4.5 4.4 M1-3 4.2 4.1 4.2 4.2 M1 -4 7.0 7.0 7.2 7.1 M2-1 4.0 3.8 3.9 3.9 M2-2 3.5 3.4 3.5 3.5 M3 -One 7.1 7.5 7.3 7.3 M3 -2 5.5 5.4 5.5 5.5 M4-1 3.7 3.1 3.5 3.4 M5-1 4.5 4.8 4.2 4.5

(B) 2nd screening - Enzyme assay & aflatoxin analysis

Plate assays were used to culture M1-2, M1-4, M2-1, and M1-4, M3-1, and M3-2, excellent in protease activity, at 30 ° C. for 40 hours, The activity of various enzymes and aflatoxin were analyzed and the M1-4 strain, which had high activity and had no aflatoxin-producing ability and no dark color, was finally selected.

Glutaminase activities of the M1-4 strain were comparable to those of the control (primary pathogens), but protease activity and amylase activity were 52% and 55% higher than those of the control (primary pathogens), respectively, and were not aflatoxin-producing and dark (yellow) This strain was finally selected.

 The following Table 5 relates to the enzyme titer and Aflatoxin of the solid fermentation product of Aspergillus.

Bacterium
No. Protease
(U / g) Amylase
(U / g) Glutaminase
(U / g) pH Aflatoxin Remarks Control 616 ± 25 220.2 ± 10.5 1.81 + - 0.12 6.7.1 ± 0.28 voice blue M1-2 916 ± 25 120.2 ± 10.5 1.78 + - 0.12 7.10 ± 0.28 voice Dark blue M1 -4 934 ± 28 342.1 + - 24.1 1.84 + 0.11 6.70 ± 0.21 voice yellow M2-1 685 ± 11 170.1 + - 13.2 1.81 + - 0.11 6.73 ± 0.18 voice yellow M3-1 498 ± 21 247.4 ± 3.8 0.83 ± 0.09 6.74 ± 0.21 voice White M3-2 611 ± 31 96.3 ± 6.4 0.79 ± 0.08 6.65 + 0.17 voice yellow

(2) Establishment of culturing conditions of isolated Escherichia coli

Generally, when cultivating industrial germs, water is added to raw materials such as grains, and then heat treatment is performed to soften the raw materials. At the same time, microorganisms adhered to the raw material are removed, and then pure germs are inoculated and cultured. In this case, the water content of the raw material itself is low (generally 10% or more in the case of grain), and water is added so that heat transfer can be smoothly performed and the growth of germs can be promoted.

Most microorganisms including aspergillus are preferred to add a large amount of water in order to smooth the growth (higher water activity) as the moisture content is higher. However, unlike liquid culture, in the case of solid culture, If the water content in the heat-treated raw material is excessive, there arises a problem that microorganisms of bacteria grow more explosively than the germ. Therefore, it is important to establish the water content which can smoothly cultivate the germs and minimize the growth of the germs by taking advantage of the fact that the growth of the germs is smooth at lower water activity than that of the germs.

By controlling the amount of water added to the wheat bran, the water content of the germ culture can be changed to 35, 40, 45, 50, 55, 60%, and the maximum number of spores can be obtained. The conditions that could be considered were investigated.

As the moisture content increased from 35%, the number of spores increased gradually, reaching the peak at 50% of water content, and the number of spores tended to decrease with increasing water content. Total bacterial counts were maintained at less than 10 cfu / g up to 50% moisture content, and rapidly increased at more than 55% of water content. The optimum conditions for obtaining the maximum number of bacteria and minimizing total bacterial count were 50% .

(2.0 × 10 ⁹ cfu / g) and total bacterial counts (7 cfu / g) at 50% moisture content were higher than that of seeds (1.6 × 10 ⁹ cfu / g) (About 10 cfu / g) (Fig. 3).

(3) Separation and Characterization of High Alcohol Producing Yeast

(A) 1st screening - Alcohol production

Yeast is known to produce various alcohol components including ethanol. Such alcohol components are known to have an effect of inhibiting the growth of common bacteria while imparting flavor. Ethanol and glucose contents were measured in yeast modified agar (YM) medium containing 8% NaCl for 24, 48 and 72 hours. The ethanol and glucose contents were measured and the yeast Y2-1, Y2-3, Y3-3 , Y4-1 and Y8-3 were first selected.

Table 6 below shows the amount of ethanol produced and the amount of glucose consumed according to the incubation time of each yeast strain.

Strain name 24 hr  48 hr 72 hr Ethanol
(%) Glucose
(%) Ethanol
(%) Glucose
(%) Ethanol
(%) Glucose
(%) Y2 -One 2.25 14.4 6.46 5.50 6.95 4.19 Y2 -3 3.36 12.0 7.35 3.80 7.80 2.68 Y3 -3 3.38 11.8 5.97 4.20 6.57 3.39 Y4 -One 4.01 11.0 7.56 3.80 8.06 2.44 Y6-2 1.55 15.8 1.70 15.0 2.88 12.53 Y8-1 1.51 15.6 2.01 14.8 2.95 13.00 Y8 -3 2.58 13.6 6.40 4.50 6.95 4.13

(B) 2nd screening - Induction of acid and ethanol production

The first selected yeast was inoculated with 1% NaCl in 8% and 16% soy sauce and fermented at 30 ℃ for one month. As shown in Table 7, Y2-1, Y2-3 and Y4-1 showed the highest ethanol production ability.

Table 7 below shows the ethanol production ability and the production of acid film in the soy sauce by yeast strains.

Strain name Soy sauce (NaCl 8%) Soy Sauce (NaCl 16%) Ethanol (%) Occurrence Ethanol (%) Occurrence Y2 -One 1.28 X 0.61 X Y2 -3 1.40 X 0.88 X Y3-3 1.17 O 0.19 O Y4 -One 1.42 X 0.80 X Y8-3 1.20 O 0.17 O

(C) 3rd Screening - Sensory Characteristics

The three yeasts selected (Y2-1, Y2-3, Y4-1) were inoculated 1% each in soy sauce with NaCl content of 16% and fermented at 30 ℃ for one month. Respectively. As shown in Table 8, the flavor characteristics of each yeast strain were confirmed by sensory evaluation. In the analysis of character strength, Y4-1 strain, which had a strong tendency and weak chemical and fragrance, and fragrant fragrance in the descriptive analysis, was finally selected.

The following Table 8 relates to the sensory characteristics of soy sauce pasta by yeast strains.

division Y2-1 Y2-3 Y4 -One Significance level characteristic

Strength ^* Alcohol flavor 7.2 7.7 7.6 0.75 Chemical incense 5.1b 6.8a 5.2b 0.05 SOUND 6.8 6.5 6.2 0.62 Heron 6.0b 5.2c 6.8a 0.03 Creation 4.8b 6.1a 4.7b 0.04 Kumkumham 5.5 5.7 5.8 0.85 Salty taste 8.8 8.5 8.7 0.99 sweetness 7.7 7.2 7.3 0.92 Sour taste 5.6 6.1 5.6 0.71 Richness 5.6 5.7 5.1 0.81 Soy flavor 7.5 7.2 7.1 0.80 bitter 5.7 5.6 5.4 0.91 Arin degree 5.7 5.4 5.8 0.72 Seed analysis Sweet scent It is strong in the fragrance and medicine. Fragrant incense -

^* 15 points Scale: Strength (1 point is very weak, 8 points is appropriate, 15 points is very strong)

Target audience: 10 food research institutes

a, b, c: significant difference (p <0.05)

(4) Separation and Characterization of Organic Acid-Producing Microorganisms

(A) 1st screening - Histidine decarboxylase production ability and salt tolerance

Biogenic amines found in fermented foods are known to act directly or indirectly as neurotransmitters in the body and affect cardiovascular system such as blood pressure control and blood flow. In the United States and Europe, biogenic amine content is regulated in foods. Efforts to reduce biogenic amine in fermented foods are underway in all parts of the world. Biogenic amines can be divided into aliphatic compounds (putrescine, cadaverine, agmatine, spermine, spermidine), aromatic compounds (tyramine, 2-phenylethylamine) and heterocyclic compounds (histamine and tryptamine). Of these, histamine and tyramine are widely known biogenic amines, and the KFDA provides a guideline of 500 ppm or less based on histamine.

The decarboxylase produced by lactic acid bacteria is an enzyme that converts amino acids to biogenic amines. The lower the decarboxylase activity, the lower the biogenic amine content in fermented foods. In order to produce safe fermented food, lactic acid bacteria which can grow in deciduous state while decarboxyase activity was low were selected. Histidine decarboxylase activity was considered to be Histidine decarboxylase producing ability when red color was shown on a decarboxylating agar medium and Histidine decarboxylase producing ability when it was yellow (see FIG. 4).

As shown in Table 9, L1-1, L3-2, L4-1, L5-1, L5-2, L6-1, L6-2, and L7-2, which are capable of growing in a high salt state, but not producing histidine decarboxylase, L9-2 and L9-3 strains were first selected.

The following Table 9 relates to the ability to produce histidine decarboxylase and salt tolerance.

Strain number Viability Histidine decarboxylase production ability MRS
(5% NaCl) MRS
(10% NaCl) MRS
(15% NaCl) L1 -One + + + - L1-2 + + + + L1-3 + + + + L1-4 + + - - L2-1 + - - + L2-2 + + + + L2-3 + + - - L3-1 + + + + L3 -2 + + + - L4 -One + + + - L5 -One + + + - L5 -2 + + + - L6 -One + + + - L6 -2 + + + - L6-3 + + - - L7-1 + + + + L7 -2 + + + - L9 -2 + + + - L9 -3 + + + -

(B) 2nd screening - Organic acid production ability & inhibition of acid generation

Soy sauce was prepared by adding salt water to fermented fungi mainly used for soybean, and it has a high salt content and a large amount of solids, so that it is similar to the intermediate product of the flavoring material. Therefore, it is considered that the selection of strains having excellent organic acid production ability in soy sauce is more suitable for selecting strains having high organic acid production ability than measuring the production of organic acids according to the growing time on the general medium. The growth rate of organic acid was determined by the growth time of the organic acid.

Excessive production of acetic acid is known to exhibit negative signs due to strong acidity. Lactic acid, on the other hand, has positive effects on the growth of polluted microorganisms, while imparting moderate acidity.

In this experiment, the primary selected lactic acid bacteria were inoculated with 1% NaCl in 16% soy sauce and fermented at 30 ℃ for one month. The contents of lactic acid, acetic, biogenic amine (histamine, tyramine) , L1-2, L3-2, L5-1 and L5-2 strains, which have high lactic acid content, low acetic acid content and inhibited acid deposition, were selected.

The following Table 10 relates to the quality change in soy sauce over time by lactic acid bacteria.

Strain number pH Organic acid (ppm) Biogenic amine (ppm) Occurrence
Inhibition Lactic acid Acetic acid Histamine Tyramine L1 -One 5.4 9,061 3,126 12.7 4.5 O L3 -2 4.9 8,045 2,254 20.6 8.9 O L4-1 5.5 154 412 14.8 10.3 X L5 -One 5.4 7,012 2,458 16.8 9.9 O L5 -2 4.8 11,875 6,120 8.5
7.9
O L6-1 5.5 624 421 14.6
11.6
X L6-2 5.5 978 741 7.8
10.9
X

(C) 3rd screening - Sensory characteristics

Four kinds of lactic acid bacteria selected (L1-2, L3-2, L5-1, L5-2) were inoculated 1% in soy sauce with 16% NaCl content and fermented at 30 ℃ for one month. Sensory evaluation was performed. As shown in Table 11, the flavor characteristics of each lactic acid bacterium were confirmed, and L1-2 strain having the strongest soymilk / soybean flavor and the least amount of chemical flavor and Kumkhambum was finally selected.

The following Table 11 relates to the sensory characteristics (characteristic intensity analysis ^* ) of soy sauce pasta by lactic acid bacteria.

division L1 -2 L3-2 L5-1 L5-2 Significance level Alcohol flavor 5.3 5.6 5.5 5.2 0.92 Chemical incense 5.1c 6.8a 5.1c 6.0b 0.05 SOUND 7.8a 7.5a 5.2c 6.6b 0.02 Heron 6.5 6.2 6.8 6.8 0.67 Creation 4.8 5.1 4.5 4.7 0.83 Kumkumham 5.7c 6.8a 5.3c 6.1b 0.05 Salty taste 8.4 8.2 8.5 8.7 0.78 sweetness 7.4 7.6 7.6 7.2 0.58 Sour taste 5.8 6 5.6 5.7 0.51 Richness 5.6 5.7 6.1 6.2 0.21 Soy flavor 8.1a 8.2a 7.2b 7.6ab 0.04 bitter 5.5 5.5 5.4 5.6 0.91 Arin degree 6.4 6.5 6.3 6.5 0.92

^* 15 points Scale: Strength (1 point is very weak, 8 points is appropriate, 15 points is very strong),

Target audience: 10 food research institutes

a, b, c: significant difference (p <0.05)

I. First taste (clean taste) Technology development and optimal fermentation condition establishment

(1) Protein degradation condition setting and optimization

As shown in Table 12, full factorial experiments were performed with the treatment conditions for each factor, as shown in Table 12, where the use ratio of soybeans and salt water, the salt content, and the ripening period, which can affect the protein content of the flavoring material,

Table 12 below relates to experimental design-3 factor complete factorial experiments.

factor Number of levels Treatment condition Percentage of use of soybeans and salt water 3 1: 1.4, 1: 1.3, 1: 1.2 Salinity Content (%) 2 21%, 16% Aging period 3 1 month, 2 months, 3 months

The results of the complete factorial test are shown in Table 13. Flavoring physicochemical target level of TN = 1.5 or later, AN = 0.9 or higher, Color (OD ₅₀₀₎ = condition can be satisfied more than 3.0 of the material raw material = 1: 1.3, salinity = 16%, when aged 3 months And raw material ratio = 1: 1.2, salt concentration = 16%, and two cases when ripening for three months. However, in the first condition (raw material ratio = 1: 1.3, salt concentration = 16%, aged 3 months), the OD ₅₀₀ = 2.1 of the flavoring material was bright and the nitrogen utilization rate was as high as 82.7% (Raw material ratio = 1: 1.3, salt concentration = 16%, 3 months) was optimal because the OD _{500 was} 2.7, and the nitrogen utilization rate was as low as 79.9% Condition.

Table 13 below shows the results of the analysis of general compositional changes according to the raw material cost, salinity concentration and aging period.

Raw material cost
(Soybean: salt water) salt
content
(% w / v) ferment
term
(month) General component Nitrogen utilization rate
(%) TN
(%) AN
(%) pH Color
(OD ₅₀₀ ) 1: 1.4 21 One 1.22 ± 0.05 0.59 + 0.11 5.34 ± 0.01 1.2 ± 0.02 69.17 2 1.28 + 0.04 0.73 ± 0.08 5.30 ± 0.01 1.5 ± 0.02 72.91 3 1.32 ± 0.03 0.79 ± 0.05 5.27 ± 0.00 1.6 + 0.04 74.84 16 One 1.29 + 0.06 0.71 ± 0.09 5.31 ± 0.00 1.3 ± 0.02 71.63 2 1.38 + 0.03 0.78 + 0.07 5.23 ± 0.01 1.5 ± 0.03 77.48 3 1.46 + 0.03 0.82 + 0.07 5.21 ± 0.01 1.8 ± 0.04 82.89 1: 1.3 21 One 1.26 + 0.06 0.62 ± 0.08 5.35 ± 0.00 1.5 ± 0.03 69.06 2 1.31 + 0.04 0.73 + 0.11 5.30 ± 0.00 1.8 ± 0.02 72.14 3 1.35 + 0.03 0.79 ± 0.05 5.26 ± 0.01 2.0 ± 0.04 73.29 16 One 1.33 + 0.04 0.78 ± 0.03 5.35 ± 0.01 1.6 ± 0.02 71.51 2 1.43 + 0.03 0.85 + 0.04 5.22 ± 0.01 1.9 ± 0.04 77.33 3 1.52 + 0.02 0.92 + 0.03 5.19 ± 0.01 2.1 ± 0.03 82.67 1: 1.2 21 One 1.29 ± 0.05 0.65 + 0.07 5.37 ± 0.01 1.9 ± 0.03 66.42 2 1.36 + 0.06 0.77 ± 0.06 5.33 ± 0.01 2.1 ± 0.03 70.36 3 1.39 ± 0.05 0.83 0.03 5.29 ± 0.02 2.4 ± 0.03 71.92 16 One 1.37 + 0.04 0.80 + 0.08 5.34 ± 0.01 2.1 ± 0.03 68.99 2 1.47 + 0.03 0.88 ± 0.05 5.23 ± 0.02 2.4 ± 0.03 74.89 3 1.55 + 0.06 0.94 + 0.07 5.20 ± 0.01 2.7 ± 0.04 79.91

(2) Setting and optimizing fermentation conditions of yeast / lactic acid bacteria

In order to establish the fermentation condition by the excellent microorganisms (yeast, lactic acid bacteria) separated in the manufacturing of the flavoring material, when the yeast and the lactic acid bacteria were simultaneously fermented after confirming the physicochemical and sensory characteristics of the yeast and lactic acid bacteria simultaneously or separately, It was confirmed that the flavoring material most suitable for sensory superiority and bright color can be manufactured.

(A) Sensory evaluation

[Characteristic Strength Investigation]

As a result of confirming the sensory characteristics of the yeast and lactic acid bacteria after simultaneous or separate fermentation of the yeast and lactic acid bacteria in order to set the fermentation conditions by the superior microorganisms (yeast, lactic acid bacteria) In the case of the flavoring material (Experiment 1), fermented soybean / soybean flavor showed weak characteristics. In the fermented yeast fermentation material (Experiment 2), alcohol and drug flavor were strong. The flavor material (Experiment 3) which had fermented lactic acid bacteria showed strong characteristics of soybean / soybean flavor, monomorphic flavor, and kumquam bean with low alcohol and chemical flavor. The fermented yeast fermentation broth (Experiment 2) and fermented lactic acid fermentation broth (Experiment 3) showed strong fermentation of soybean / soybean flavor and soybean, ), Respectively.

The fermentation conditions (simultaneous fermentation with lactic acid bacteria and yeast) of the experimental group in which the soybean flavor and soybean flavor were strong and the chemical aroma was relatively weak were found through the above-described sensory characteristics (FIG. 5).

The following Table 14 relates to the sensory properties (characteristic intensity ^* ) of the yeast / lactic acid fermentation experiment.

division No additives leaven Lactobacillus Yeast + lactic acid bacteria Significance level Alcohol flavor 6.3b 6.9a 5.5c 6.2ab 0 Chemical incense 6.0b 6.8a 5.1c 5.8bc 0 SOUND 5.8b 5.5b 7.2a 7.6a 0 Heron 6.5ab 5.9b 6.9a 6.8a 0.02 Creation 5.2 4.8 4.5 5 0.23 Kumkumham 5.7b 5.9b 7.3a 6.6ab 0 Salty taste 8.5 8.1 8 8.7 0.28 sweetness 6.4 6.7 6.5 6.1 0.5 Sour taste 5.1 6 5.6 5.7 0.1 Richness 6.6 6.9 7.2 7.2 0.21 Soy flavor 6.3c 7.1b 8.0a 7.5 ab 0 bitter 6.5 6.4 6.2 6.5 0.91 Arin degree 6.9 6.5 6.6 6.9 0.6 zest 6.9 7.5 7.3 7.2 0.58

^* 15 points Scale: Strength (1 point is very weak, 8 points is appropriate, 15 points is very strong)

Target audience: 20 generic consumer sensibility panels

a, b, c: significant difference (p <0.05)

(B) Physicochemical analysis

As shown in FIG. 6, there was no difference in the TN content between the fermentation conditions, but the pH (FIG. 7), the organic acid (Table 15), and the organic acid There was a difference in the analysis of amino acids (Table 16).

TN of the fermented taste material was confirmed to be the same regardless of the fermentation conditions of yeast / lactic acid bacteria, and it was confirmed that all experiments were at the level of 1.6, which is the target value of 1.5 or more.

As shown in FIG. 7, pH values of all the experimental groups were similar until the first month of fermentation, but the pH of the experimental groups (Experiments 3 and 4) in which the lactic acid bacteria were fermented was lower than that of the experimental groups without lactic acid fermentation. The reason why the pH of the experimental group subjected to the lactic acid fermentation was lower was considered to be due to the increase of lactic acid and acetic acid when referring to the organic acid analysis data of Table 15 (FIGS. 6 and 7).

The results of analyzing organic acids are shown in Table 15. Citric acid was fermented with lactic acid bacteria at concentrations of 11,220 ~ 11,311 ppm in experimental groups (Experiments 1 and 2), which were not fermented with lactic acid bacteria, while those of experimental groups (Experiments 3 and 4) with lactic acid fermentation were 7,676 ~ 7,751 ppm The citric acid content was reduced by about 30%. Lactic acid was found to be 9,701 ~ 9,787 ppm in the experimental groups (Experiments 3 and 4) in which lactic acid bacteria were fermented, whereas lactic acid in the fermented flavorings was not detected in the experimental groups (Experiment Groups 1 and 2) And it was confirmed that it was produced by fermentation of lactic acid bacteria. Acetic acid was found to be 2,457 ~ 2,803 ppm in lactic acid fermentation (Experiments 3 and 4) and 824 ~ 835 ppm in lactic acid fermentation (Experiments 1 and 2) ~ 0.2%, respectively.

Table 15 below shows the content of organic acids in the yeast / lactic acid fermentation experiment period.

division Fermentation condition Organic acid (ppm) leaven
Fermentation Lactobacillus
Fermentation Citric acid Succinic acid Malic acid Lactic acid Acetic acid Experiment 1 X X 11,220 5,479 4,838 Non-detection 824 Experiment 2 O X 11,311 5,476 4,203 Non-detection 835 Experiment 3 X O 7,751 5,321 13,747 9,701 2,803 Experiment 4 O O 7,676 4,657 5,877 9,787  2,457

The free amino acid composition of all experimental groups was almost the same. However, in the case of Arginine, lactic acid bacteria fermentation (Experiment 3, Experiment 4) was slightly lower than that of lactic acid fermentation (Experiment 1, Experiment 2), and lactic acid bacteria Tetragenococcus halophillus Was known to have metabolic ability to arginine.

The following Table 16 relates to the free amino acid composition of the yeast / lactobacillus fermentation experimental period.

division Free amino acid content (%) Remarks Experiment 1
(No additives) Experiment 2
(Yeast fermentation) Experiment 3
(Fermentation of lactic acid bacteria) Experiment 4
(Yeast & lactobacillus) ASP 0.95 0.93 0.86 0.90 THR 0.41 0.41 0.40 0.41 SER 0.56 0.55 0.54 0.54 GLU 1.41 1.30 1.26 1.26 PRO 0.34 0.33 0.33 0.34 GLY 0.33 0.32 0.31 0.31 ALA 0.44 0.43 0.47 0.45 CYS 0.07 0.06 0.07 0.08 VAL 0.57 0.56 0.55 0.56 MET 0.15 0.15 0.15 0.15 ILE 0.44 0.48 0.47 0.48 LEU 0.74 0.72 0.72 0.74 TYR 0.06 0.06 0.06 0.06 PHE 0.42 0.41 0.40 0.41 HIS 0.28 0.27 0.27 0.28 LYS 0.61 0.60 0.61 0.62 ARG 0.32 0.34 0.06 0.06 Decrease tendency in fermentation of lactic acid bacteria Sum(%) 8.09 7.93 7.52 7.65

Table 17 below shows the total amino acid composition of the yeast / lactobacillus fermentation experimental period.

division Total amino acid content (%) Remarks Experiment 1
(No additives) Experiment 2
(Yeast fermentation) Experiment 3
(Fermentation of lactic acid bacteria) Experiment 4
(Yeast & lactobacillus) ASP 1.33 1.31 1.24 1.25 THR 0.44 0.44 0.43 0.43 SER 0.59 0.59 0.58 0.58 GLU 2.04 2.01 1.97 1.94 PRO 0.48 0.48 0.47 0.48 GLY 0.44 0.44 0.43 0.43 ALA 0.47 0.46 0.50 0.47 CYS 0.06 0.07 0.08 0.09 VAL 0.57 0.57 0.55 0.55 MET 0.13 0.13 0.13 0.13 ILE 0.50 0.49 0.49 0.48 LEU 0.74 0.73 0.73 0.72 TYR 0.08 0.08 0.07 0.07 PHE 0.43 0.43 0.43 0.41 HIS 0.34 0.34 0.32 0.32 LYS 0.66 0.65 0.65 0.63 ARG 0.34 0.36 0.08 0.08 Decrease tendency in fermentation of lactic acid bacteria Sum(%) 9.66 9.57 9.16 9.07

Regardless of yeast / lactobacillus fermentation, it was confirmed that only trace amounts of biogenic amines were detected below 40ppm as shown in Table 18, ensuring safety. Biogenic amines are known to be produced mainly by lactic acid bacteria. Lactic acid bacteria used in this fermented taste material can be confirmed to have no biogenic amine production ability by screening strains which do not have decarboxylase activity.

The following Table 18 relates to the analysis of the biogenic amines by the yeast / lactobacillus fermentation experiment sections.

division Presence of microorganism addition Biogenic amines (ppm) leaven Lactobacillus Histamine Tyramine Experiment 1 X X 2.51 1.34 Experiment 2 O X 2.4 1.65 Experiment 3 X O 3.72 2.66 Experiment 4 O O 3.26 4.94

(3) Product suitability investigation through sensory evaluation of fermented taste material

Sensory evaluation of the fermented flavor materials prepared by the previously set proteolytic conditions and yeast / lactic acid fermentation conditions was carried out and the purchasing intention of the consumers was confirmed. As shown in Table 19, the satisfaction score of the 72 consumers was very high, 3.9, and the ratio of the good and the good was 17% and 62%, respectively. 79% of the respondents answered positively and only 7 Only% of the respondents gave negative evaluations and showed very high overall satisfaction.

In addition, it was found that the overall satisfaction was high (3.8 points) regardless of the type of dish. The results were as follows. (Table 20) Showing no difference. As a result, it was confirmed that this fermented flavor material is suitable for both heating and non-heating cooking.

As a result of examining the purchasing intention of the consumers (Table 21), the intention to purchase was as high as 3.7 as the satisfaction level, and 65% of the evaluators were very positive.

Table 19 below shows the results of the customer satisfaction survey of fermented flavoring materials.

Number of evaluators Overall satisfaction Percentage of responses (%) Not good usually good Very good n = 72 3.9 7 13 62 17

Table 20 below shows the results of the satisfaction and the characteristic strength test according to the type of the dishes to which the fermented taste material was applied (n = 72).

Cooking form Overall
satisfaction Percentage of responses (%) Likelihood Affirmation good
Not usually good Very good Exterior flavor Salty taste Richness sweetness Flood
A taste heating
Cooking 3.8 3 26 62 9 3.7 3.8 3.5 3.8 3.5 3.8 Non-heat cooking 3.8 9 25 45 22 3.8 3.8 3.7 3.8 3.6 3.6 Significance level 0.83 - - - - 0.28 0.70 0.19 0.87 0.92 0.64

Table 21 below shows the results of a consumer purchase surveys of fermented flavoring materials.

Number of evaluators Purchase physician Percentage of responses (%) Do not buy Perhaps
Non-sale Half Perhaps
purchase Always buy n = 72 3.7 One 12 22 48 17

(4) Physicochemical analysis of fermented taste material

The results of physicochemical analysis of the fermented flavoring material are shown in Table 22. It was 1.7% higher than the target level of 1.5% of TN, and 1.03% higher than the target level of 0.9%. In addition, it was confirmed that all of the values below 3.0 which _is the target level of Color (OD ₅₀₀₎ were satisfied.

Table 22 below shows the results of physicochemical analysis of the flavoring material.

Item Analyzer Remarks TN (%) 1.7 Target level = 1.5% or more AN (%) 1.03 Target level = 0.9% or more pH 5.0 Color (OD ₅₀₀ ) 2.05 Target level = 3.0 or less Reducing sugar (%) 0.27 Total (%) 1.13

All. Scale-up and standardize the first taste (clean taste)

(1) Construction and optimization of mass production system

(A) Manufacturing process and problems in mass production

In order to produce the flavoring material on the plant scale, the fermentation (solid fermentation) of the mold was carried out by adding 30,000 kg of soybean as shown in FIG. 8, and the salt, yeast culture and lactobacillus cultures prepared beforehand were added thereto, Proteolysis / microbial fermentation was performed for 3 months, and the mixture was compressed / filtered to finally produce a primary flavoring material.

Since the plant scale is about 100 ~ 300 times larger than the Lab scale, it is important to predict and solve problems that may occur during mass production because it is difficult to repeat the scale.

As a problem that can occur in mass production, the processing time of each process may become longer due to its large size. Therefore, it is important to shorten each processing time as much as possible. When 30 tons of soybeans are used, it is necessary to transfer the raw material in the shortest time, transfer it after transferring it to the next process, and it is possible to prevent quality deterioration and reduction of production efficiency due to microbial contamination.

In the plant scale, homogenization and dephosphorization are very important. Therefore, it is necessary to devise and apply a method that can effectively homogenize each process and a method of removing the foreign matter, so as to improve the stability and safety of the quality.

In order to construct a mass production system, the process of producing the flavor material is divided into soybean pretreatment, salt water production, protein decomposition / microbial fermentation, and compression / filtration process, and a method for increasing the processing speed, homogenizing, And applied (FIG. 8).

(B) Soybean pretreatment

When soybean is soaked, it takes much time to absorb moisture, and productivity is reduced because it is required to work in a batch type. In order to shorten the time of water absorption and to work in a continuous type, the soybean is sprayed evenly through the spray nozzle after the pressing, so that the soybean can absorb the water in a short time. In order to shorten the heat treatment time, short time heating was performed at a high temperature. The heat - treated soybeans were inoculated and mixed evenly with the self - cultured soybean germ, and fermented for 42 hours in a solid fermentation chamber. In order to prevent moisture evaporation of the fermented product, the humidity of the fermentation room was maintained at over 90% and the fermented product was partially crushed and mixed to eliminate the accumulation of mold mycelium and inhomogeneity of the upper and lower layers during the fermentation process.

Table 23 below compares LAB and Plant conditions during soybean pretreatment.

division Lab scale condition Plant scale condition Remarks Soybean processing none Stamp Improved water absorption rate through soybean scrap Singer method Batch type
Immersion Continuous
watering Increase throughput per hour Heat treatment condition Batch type
120 DEG C
30 to 40 min Continuous
135 ° C (2 kgf / cm ² ), 5 to 10 min Increase throughput per hour

(C) Proteolysis / microbial fermentation

The fermented product (soybean), salt water, yeast culture solution and lactobacillus culture solution prepared as described above were mixed and transferred to a fermentation tank having a capacity of 100 KL. After that, the temperature was maintained for 3 months to decompose soybean protein and fermentation of salt tolerant microorganism Respectively.

The fungus fermentation product is aggregated due to the mycelium of the fungus, which causes the brine to delay the penetration time into the fungus fermentation product. In order to allow the brine to penetrate into the fermentation product quickly, a crusher was applied to loosen the fused fermented product. Also, since the specific gravity of the fungus fermented product is low, the product floats on the upper part of the brine, Air was blown from the bottom of the tank to induce homogeneous mixing of the fermented fungi and salt water.

Table 24 below compares LAB and Plant conditions during proteolytic / microbial fermentation.

division Lab scale condition Plant scale condition Remarks Mold fermentation product
Pretreatment Shred by hand Application of crusher Prompt rapid penetration of salt water into fungal fermentation products. Homogenization Proceed by hand Apply aeration device Through homogenization
Quality stabilization

(D) Compression and filtration

In order to effectively separate the fermented fermented material from the fermented fermented medium, the intermediate product that has undergone proteolytic degradation and microbial fermentation process is firstly subjected to a pressure filtration process to increase the yield and processing speed of the fermented fermentation material. As a result, the microfiltering process has secured clear and refined fermentation flavor materials and enhanced safety through the removal of foreign substances.

Table 25 below compares LAB and Plant conditions during compaction / filtration.

division Lab scale condition Plant scale condition Remarks Crimping / Filtration - Step 1: Natural filtration
Filter paper NO.4


- Step 2: None - Step 1: Pressurized filtration
Filter press application

- Step 2:
Micro Filteration
(pore size = 0.1 mu m) Pressure filtration improves yield and throughput

Two-step process increases throughput per hour

Increased safety through application of Micro Filteration

(2) Scale-up prototype analysis and security

(A) Physicochemical analysis results

Table 27 shows the results of the general analysis of the primary flavoring materials prepared on the plant scale. TN showed 1.63%, which is about 0.13% higher than the target standard of 1.5%, and AN achieved 1.05%, 0.15% higher than the target standard of 0.9%. In the case of Color, the target standard OD ₅₀₀ = 1.46, which is much lower than 3.0 or less. As a whole, the primary flavor material (primary soybean fermentation flavor material) is high in TN and AN, and is rich in flavor and very bright in color and can be applied to various foods and dishes .

Table 26 below shows the results of the physico-chemical analysis of the scale-up prototype.

division Analyzer Remarks TN (%) 1.63 Achieve a goal of 1.5 or higher AN (%) 1.05 Achieved target of 0.9 or higher pH 5.41 Color (OD ₅₀₀ ) 1.46 Achieved goal below 3.0 PH I 6.7 PH II 8.2

(B) Amino acid composition

Free amino acids were analyzed for free amino acid composition and total amino acid composition as factors directly related to the sensory characteristics of the flavoring material. Among the free amino acids, glutamic acid was a typical flavor ingredient, which was 1.17%, which is 0.17% higher than the target standard of 1.0%.

Total free amino acids and total amino acids were 6.80% and 8.40%, respectively, indicating that most of the protein components were degraded into free amino acids, indicating that proteolysis was effective.

Table 27 below shows the amino acid analysis results of the scale-up prototype.

division Amino acid content (%) Free amino acid Total amino acid ASP 0.02 0.40 THR 0.38 0.40 SER 0.51 0.56 GLU 1.17 1.86 PRO 0.31 0.48 GLY 0.31 0.43 ALA 0.98 0.99 CYS 0.16 0.19 VAL 0.50 0.50 MET 0.15 0.13 ILE 0.46 0.47 LEU 0.70 0.70 TYR 0.06 0.06 PHE 0.39 0.42 HIS 0.05 0.09 LYS 0.59 0.63 ARG 0.06 0.09 Sum(%) 6.80 8.40

(3) Blind test (comparison with similar products)

A blind test was conducted to compare the first taste material prepared on the plant scale with four similar products.

(A) Characteristic strength analysis results

The analysis of the characteristics of the samples was carried out by a trained panel of experts on a scale of 15 points. The results were as shown in Figures 9-10 (* significant difference between products was p <0.05) There was a significant difference in meju, soybean, chemical, alcohol, concentration, salty, sour, soybean, and metal flavor. Especially, the primary flavoring material shows a characteristic that it has a strong flavor and strong flavor, strong acidity, chemical flavor, and low alcohol content compared to other similar products. (P < 0.05), compared to the products (C, D, and C) (FIGS. 9 and 10).

(B) Consumer survey

As shown in Table 31 below, the overall satisfaction level and the preference preference by item were found to be 3.8, 4.0, 3.8, and 3.6, respectively, in color, flavor, and savory taste compared to other samples Point scale), and overall satisfaction was also significantly better.

Table 28 below shows the results of a survey of consumer preference scores of scale-up prototypes and similar products.

Sample name Overall
satisfaction Symbol color Taste harmony Savory taste Salty taste sweetness Richness First taste 3.8a 4.0a 3.8a 3.6a 3.1 3.2 3.4 A product 3.3b 3.4ab 3.3b 3.1b 3.1 3.3 3.2 B products 3.4b 3.2b 3.4b 3.4ab 3.0 3.3 3.2 C products 3.2b 3.4ab 3.2b 3.1b 2.9 3.1 3.1 D Products 3.4b 3.1b 3.3b 3.3b 3.3 3.5 3.2 Significance level 0.05 0.00 0.05 0.04 0.56 0.55 0.52

Target audience: 38 consumers

a, b, c: significant difference (p <0.05)

Although the present invention has been described in connection with the specific embodiments of the present invention, it is to be understood that the present invention is not limited thereto. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. In addition, the materials of each component described herein can be readily selected and substituted for various materials known to those skilled in the art. Those skilled in the art will also appreciate that some of the components described herein can be omitted without degrading performance or adding components to improve performance. In addition, those skilled in the art may change the order of the method steps described herein depending on the process environment or equipment. Therefore, the scope of the present invention should be determined by the appended claims and equivalents thereof, not by the embodiments described.

Korea Biotechnology Research Institute KCTC12159 20120328

Claims (4)

delete delete Aspergillus oryzae M1, which has a clear zone size (mm) of 5.8 to 6.0 mm in the protease plate assay, a clear zone size (mm) in the amylase plate assay of 7.0 to 7.2 mm, and no Aflatoxin- -4 (KCTC 12159BP) and a yeast that does not have lactic acid bacteria and acid membrane-forming ability which do not have histidine decarboxylase activity. [4] The method of claim 3, wherein the food is at least one selected from the group consisting of miso, kochujang, chongkukjang, soy sauce, yogurt, kimchi and cheese.

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