KR20160097422A - Salt-tolerant Yeast Isolated from Soybean Paste and Manufacturing Method of Yeast Salt - Google Patents

Abstract

The present invention relates to a yeast extract using salt-tolerant yeast, Zygosaccharomyces mellis TK-01 (Accession number KCCM-11605), isolated from a fermented soybean paste, and a method for manufacturing fermented yeast salt using the same. More specifically, the present invention relates to a method for manufacturing fermented yeast salt having reduced sodium by preparing a yeast extract containing a high content of KCl using a strain isolated from a highly salty traditional fermented soybean paste, performing enzymatic decomposition, performing a high-temperature reaction, and adding a predetermined ratio of salt. The fermented yeast salt for salt substitute, which has a reduced content of sodium, of the present invention can reduce the use amount of sodium.

Description

[0001] The present invention relates to a salt-tolerant yeast extract,

The present invention relates to a yeast extract using a salt tolerant yeast Zygosaccharomyces mellis TK-01 (Deposit No. KCCM11605P) isolated from soybean, which is a traditional food having a high salt concentration, and a process for producing fermented yeast salt using the yeast extract. More specifically, the present invention relates to a method for preparing a fermented yeast salt in which a yeast extract is prepared by using a strain isolated from doenjang, enzymatically decomposed, and subjected to a high temperature reaction and a certain amount of salt to reduce sodium.

Sodium salt, which is a component of salty salt, is an essential element for maintaining human equilibrium, but there is no concern about deficiency because the amount required is very small. However, when excess sodium is consumed by the excessive intake of salt, the blood draws the surrounding water in order to adjust the concentration. As a result, the whole blood volume increases and the blood pressure rises. When the state becomes chronic, hypertension occurs, Hypertension can cause cardiac overload by causing heart overload, and can also affect the blood vessels and increase the risk of stroke.

Salt is a multipurpose food additive, added directly or indirectly in food processing, and sodium ion intake is also increasing due to the recent increase in salt intake due to the rapid development and spread of processed foods. Concerns about oversupply of sodium have led to efforts by WHO and other food regulatory agencies to reduce sodium. According to the National Health and Nutrition Survey released in 2012, the average sodium intake of Koreans is 4583 mg, which is twice as high as the WHO recommended maximum intake (2000 mg). As a result, the government of the Republic of Korea has launched a sodium reduction program to reduce sodium, and aims to reduce it by 20% to 3900 mg by 2017.

According to the 2010 National Health and Nutrition Examination Survey, the average potassium intake in Korean adults was about 2800 mg, which is lower than the recommended intake. Potassium is known to increase the amount of sodium excreted in vitro, and Dietary Approaches to Stop Hypertension (DASH) recommends that potassium is enriched and sodium is reduced. However, current intake of sodium in Koreans is more than twice as high as the recommended intake, but potassium intake is about 80% of the recommended amount. Potassium intake should be increased considering sodium and potassium intake.

Studies on the development and commercialization of substitute salts for reducing sodium have been progressing steadily, and salt substitutes such as inorganic salts having salty taste including potassium chloride (KCl) have been mainly used. In particular, potassium chloride has been sold worldwide as a substitute for salt since 1982, and more than 1,000 of the world's advanced food companies are using potassium chloride as a substitute for sodium chloride. However, inorganic salts such as potassium chloride have a unique bitter taste and are already very limited in use.

The reason why it is difficult to reduce the salt in various efforts to reduce the salt is that it is difficult to find a substitute to reproduce the salty taste of the salt in the same way, and when the salt is reduced, the function to strengthen the unique taste of the salt, It breaks. In other words, in order to reduce the salt intake, the salty taste of the salt as well as the taste enhancement function should be supplemented by using an alternative ingredient to enrich the overall food taste.

Yeast is a safe microorganism in the GRAS (generally recognized as safe) category and is used in a variety of foods, including baking, brewing and other foods. Yeast extracts produced from yeast are also widely used as natural flavor materials (Nagodawithana T. Food Technol. 1992) because they are relatively inexpensive compared to other materials having the same strength.

Yeast extract is a natural seasoning material that has been attracting attention as a substitute for acid-degraded hydrolyzed vegetable protein (HVP) and monosodium glutamate (MSG) according to consumer's health trend. In addition, the yeast extract contains amino acids, nucleic acids and peptides, and has complexity and rich taste. Therefore, it already has a function that can not be seen in other seasonings such as masking odor.

Generally, yeast extract is produced mainly by baker's yeast, brewer's yeast, and some Candida's yeast. In the case of brewer's yeast, which is a by-product of beer production, it is necessary to remove the unique bitter taste derived from the hop, and these yeasts are relatively resistant to salt and inhibit growth in the presence of high salt concentration . Salt-tolerant microorganisms include Zygosaccharomyces rouxii and some Candida Only a few strains, including the genus, have been known and little research has been done using them.

Doenjang is a traditional fermented food that produces unique flavor and aroma in the process of decomposing soybean protein into peptides and amino acids by enzymes produced by fermentation of microorganisms such as fungi, bacteria and yeast at high salt concentration. And has been used as a vegetable seasoning for flavoring food. Especially, it is known that the yeast involved in the fermentation of soybean paste is involved in the production of deep flavor of soybean paste in the salt - tolerant environment, and thus the quality of soybean paste is influenced.

In order to reduce the intake of sodium, the present invention can replace sodium salt added to food or food to reduce the sodium content while minimizing the loss of taste due to the decrease in salt, thereby maintaining the original taste and flavor Which is a high concentration inorganic salt, and to develop the technology of fermented yeast salt using this.

In order to achieve the above object, the present invention provides a salt tolerant Zygosaccharomyces mellis strain TK-01 isolated from miso TK-01, Accession No .: KCCM-11605).

The present invention also provides a method for producing fermented yeast salt using Zygosaccharomyces mellis TK-01.

Hereinafter, the present invention will be described in detail.

The present invention provides a yeast of the genus Zygosaccharomyces sp. Used in the present invention. Specifically, the present invention relates to a method for producing a microorganism, which comprises culturing 6 commercially available yeast strains (Saf-Instant Red, Saf-Instant Gold, Bakels Instant Active Drye Yeast, Vega Instant Yeast, Mauripan Instant Dry Yeast, Two species (Saccharomyce scerevisiae KCTC7951, Zygosaccharomyces rouxii KCCM50054) and Zygosaccharomyces spp. Isolated from three kinds of yeast isolated from commercial soybean miso, preferably Zygosaccharomyces melis TK- 01 (Zygosaccharomyces mellis TK-01).

Further, the step of evaluating the culture characteristics of the selected strain

1) It is cultured by inoculating 1 ~ 10% strain with medium containing 0 ~ 4M of KCl due to the influence of KCl (potassium chloride) concentration.

2) Carbon source and its concentration can be selected by adding carbon source (corn syrup, HFCS, dextrin, glucose, sucrose, maltose, fructose)

3) Due to the influence of the nitrogen source, the source of nitrogen and its concentration can be selected by adding ammonium sulfate (soypeptone, eHVP).

4) Depending on the initial pH, the pH of the culture medium is adjusted to 3.0 ~ 7.0, and the initial pH suitable for culturing can be selected by inoculating the strain.

5) Due to the effect of the inoculation amount, it is possible to select the initial inoculation amount suitable for culturing according to 1 to 10% of the inoculum amount of the strain.

The yeast extract using the strain and the fermented yeast salt using the same can be prepared by the following steps.

1) Pre-incubation step

The yeast extracts were prepared by the following steps: yeast extract 0.1-0.9%, malt extract 0.1-0.5%, amiga plus 1-2%, KCl 1-10%, glucose 1-7%, ammonium sulfate 0.1-0.8% , potassium phosphate monobasic 0.05 to 0.1%, Zygosaccharomyces mellis TK-01 1 to 10%, and purified water 50 to 80% Time can be cultured.

2) In the present culture step

The cells are cultured at a temperature of 20 to 30 ° C. for 6 to 72 hours under the condition of air of 0.1 to 1 vvm and glucose for 6 to 12 hours periodically.

3) Enzymatic decomposition step

Enzymatic digestion with 5 proteases, 1 nuclease, 1 lytic enzyme, 1 deaminase, etc. in the cultured medium is completed. When the temperature of the culture reaches 30 to 50 ° C, the enzymes can be decomposed by combining the enzymes.

The amount of the enzyme to be added is 0.01 to 5% based on the solid content of the culture medium.

Enzymatic degradation of the culture solution is carried out at 30 to 50 ° C for 1 to 48 hours while stirring at 90 to 350 rpm.

4) Deactivation and filtration phase

In order to inactivate the two proteases used in step 3), it is inactivated at 80 to 95 ° C for 20 to 60 minutes.

In addition, the undegraded cells are removed by centrifugation or diatomaceous filtration at 5,000 to 8,000 g for 20 to 60 minutes.

5) Mixing step

In step 4, a certain amount of purified salt is added to the filtered supernatant solids, and the mixture is mixed at 60 to 80 ° C for 20 to 60 minutes and concentrated to 20 to 30 Bricks.

6) Drying step

The mixed enzyme decomposition solution may be dried and pulverized. There is no limitation on the drying method, and general drying methods such as vacuum drying, hot air drying, spray drying and freeze drying are used.

The present invention provides a salt substitute yeast extract containing a high concentration of inorganic salt and a fermented yeast salt utilizing the salt-resistant yeast Zygosaccharomyces mellis TK-01 isolated from soybean paste, a traditional salt having high salt concentration. The fermented yeast salt prepared by the above method is a natural fermented yeast salt which has no bitter taste and no imitation and has a reduced sodium content, and can be widely used for food or food to reduce the intake of sodium chloride.

1 is a graph showing the amount of Z. mellis TK-01 cells according to the concentration of potassium chloride (KCl).
FIG. 2 is a graph showing the amount of the cells of Z. mellis TK-01 according to the carbon source.
3 is a graph showing the amount of Z. mellis TK-01 cells according to the nitrogen source.
Figure 4 is a graph showing the effect of glucose concentration on Z. mellis Lt; RTI ID = 0.0 > TK-01. ≪ / RTI >
FIG. 5 is a graph showing the effect of growth of Z. mellis TK-01 on the nitrogen source concentration.
FIG. 6 is a graph showing the influence of Z. mellis TK-01 growth on initial pH.
Figure 7 shows the effect of growth of Z. meills TK-01 on the amount of aeration. (a) 0.1 vvm, (b) 1.0 vvm
8 is a graph showing the growth of yeast extract for fermentation yeast salt production according to the presence or absence of KCl.
Drawing 9. Process for the production of fermented yeast salt.

Hereinafter, the present invention will be described in more detail by way of examples.

Example 1: Preparation of Zygosaccharomyces mellis TK-01 (Zygosaccharomyces mellis TK-01) culture

[1-1] Influence of KCl concentration

In order to evaluate the salt tolerance of the Zygosaccharomyces mellis TK-01 strain used in the present invention, YMAD (yeast extract 7 g / L, malt extract 3 g / L, amiga puls 15 g / L, dextrose 45 g / L, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 M was added to the YMAD broth and YMAD broth at a concentration of 1% in a 1% 1% in each medium. The medium containing 4.5 M and 5.0 M KCl was not tested because KCl was precipitated after sterilization. After incubation at 30 ° C and 180 rpm for 10, 24, and 36 hours, the absorbance was measured at OD 600. After 36 hours of incubation, the culture was filtered through a 0.45 μm GN-6 membrane filter, washed twice with distilled water, After drying, the weight was measured.

As a result, as shown in FIG. 1, when the OD value and the DW (dry weight) of the Zygosaccharomyces mellis TK-01 were compared with each other, the OD value and DW (dry weight) And the value decreased. However, since it can survive even at a high salt concentration, it can be used in the present invention.

[1-2] Influence of carbon source type

The change of the amount of yeast cells according to the type of carbon source was investigated by changing the type of carbon source in the culture medium of Zygosaccharomyces mellis TK-01 strain of the present invention. The cryopreserved strain was treated with a pH 5.5 solution consisting of 3 g / L of yeast extract, 3 g / L of malt extract, 223.62 g / L of KCl, 1 g / L of potassium phosphate monobasic, 10 g / L of amiga plus, 5 g / L of ammonium sulfate and 20 g / After 50% solution of corn syrup, HFCS, maltodextrin (MD), glucose (Glc.), Sucrose, maltose (Mal.) And fructose (Fru. 20 g / L of glucose and 10 g / L of glucose and maltose or fructose were added, respectively, so that the concentration of total sugar was 20 g / L. The culture was inoculated with 1% each of different kinds of sugars and cultivated at 30 ° C and 180 rpm for 48 hours. The culture was filtered with a whatman filter GF / C (1.2 ㎛ grass microfiber filter) to remove the culture medium, Washed twice and weighed.

As a result, Zygosaccharomyces mellis TK-01 showed the highest production of yeast cream when glucose and fructose were used together, and glucose production was slightly higher when glucose and fructose were added. In addition, sucrose was not available at all and no yeast cream was produced, and maltose showed a low yeast cream yield of about 1/10 of glucose (Fig. 2). Therefore, in order to increase the yield of yeast cream, glucose or fructose may be used as the carbon source of the medium.

[1-3] Effect of type of nitrogen source

The changes of the amount of yeast cells according to the type of nitrogen source were examined by changing the type of nitrogen source in the culture medium of Zygosaccharomyces mellis TK-01 strain of the present invention. L of ammonium malate was added to the medium consisting of 3 g / L of yeast extract, 3 g / L of malt extract, 223.62 g / L of KCl, 1 g / L of potassium phosphate monobasic, 10 g / L of amiga plus, 5 g / (NH4) 2SO4) and 10 g / L of soypeptone and eHVP (amiga plus) were used as organic substances. Organic and inorganic nitrogen sources 5 g / L + eHVP 10 g / L and ammonium sulfate 5 g / L + soypeptone 10 g / L) were adjusted to pH 5.5 with 1M KOH and sterilized. The carbon source was sterilized separately with 50% aqueous glucose solution, added at 20 g / L during inoculation, and incubated at 30 ° C and 180 rpm for 48 hours inoculated with 1% each of different nitrogen sources. The yeast culture was filtered with whatman filter GF / C (1.2 ㎛ grass microfiber filter) to remove the culture medium, and the cells were recovered, washed twice with distilled water and weighed.

As a result, as shown in Figure 3, Zygosaccharomyces mellis TK-01 showed maximum yeast cream yield when soypeptone was used as a nitrogen source, but soypeptone is an unauthorized nitrogen source for food. When ammonium sulfate and inorganic sulfate were added, the bacteria did not grow much better than the organic nitrogen source. When ammonium sulfate and organic matter were added together, . In the case of eHVP, a sweetener used as an organic raw material, the production amount of yeast cream of about 85% of soypeptone was shown, and it can be used as an organic material in the present invention.

[1-4] Effect of Glucose Concentration

L of glucose, 3 g / L of malt extract, 223.62 g / L of KCl, 1 g / L of potassium phosphate monobasic, 10 g / L of amiga plus, 10 g / L of potassium phosphate monobasic to investigate the effect of glucose concentration on the production of Zygosaccharomyces mellis TK- , ammonium sulfate 5 g / L and glucose concentrations of 5, 10, 20, 30, 40 and 50 g / L, respectively. The culture was adjusted to pH 5.5 with 1M KOH and inoculated with 1% And cultured at 30 DEG C at 180 rpm.

As a result, as shown in FIG. 4, the absorbance was increased with increasing glucose concentration. As a result, similar absorbances were observed at 30 g / L, 40 g / L, and 50 g / L, indicating that the strain used in the present invention is suitable for culturing at a glucose concentration of 30 to 50 g /.

[1-5] Effect of organic matter concentration

Soypeptone, which is an organic nitrogen source, is an effective nitrogen source for increasing the amount of yeast cells. However, the relationship between the amount of organic matter and the amount of biomass was investigated by varying the concentration of amiga plus which can be used as a food additive. The cryopreserved strains were cultured in the medium containing yeast extract 7 g / L, malt extract 3 g / L, KCl 223.62 g / L, potassium phosphate monobasic 1 g / L, ammonium sulfate 5 g / L and amiga plus 5, 10 and 15 g / L was adjusted to pH 5.5 with 1M KOH, sterilized, and 1% of the pre-cultured strain was inoculated and cultured at 30 ° C at 180 rpm.

As a result, as shown in FIG. 5, the amount of amiga plus was increased rapidly in a concentration-dependent manner up to 40 hours after the incubation, but the increase in the amount of the bacterial cell was not observed after 40 hours.

[1-6] Influence of initial medium pH

To investigate the effect of pH on the Zygosaccharomyces mellis TK-01 strain of the present invention, yeast extract 7 g / L, malt extract 3 g / L, KCl 223.62 g / L, potassium phosphate monobasic 1 g / L and ammonium sulfate 5 g / The medium was adjusted to pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 using 1 M KOH and 1 M HCl in the medium and sterilized. The carbon source was glucose 50% And sterilized separately and added at 30 g / L each time of inoculation. The culture medium was inoculated with 1% of the preculture strain and cultured at 30 ° C at 180 rpm.

As a result, as shown in FIG. 6, it was confirmed that the initial pH of the culture broth greatly affected the growth of the strain. Growth up to pH 3.5 6.5 was possible, but optimum pH was 5.0.

<Example 2> Preparation of yeast extract containing high concentration of inorganic salt by self-extinguishing and enzymatic decomposition

[2-1] Culture of yeast for yeast extract preparation

For the production of yeast extract, Zygosaccharomyces mellis TK-01 strain was cultured for 3 days using a 50 L fermenter and all the cells containing the cells were used for yeast extract preparation. The culture of the strain was cultured at 30 ° C at 120 rpm in the medium composition (Table 1). The culture broth was inoculated with 1% of Zygosaccharomyces cerevisiae TK-01 cultured in the same medium as in Table 1. To confirm the effect on the aeration, 0.1 or 1.0 vvm of air was supplied and cultured.

Zygosaccharomyces mellis TK-01 culture medium composition Media composition One Yeast extract 2 Malt extract 3 Amiga plus 4 KCl 5 Glucose 6 Ammonium sulfate 7 Potassium phosphate monobasic 8 Antifoam 9 Inoculum 10 Water

As a result, as shown in FIG. 7, 8.439 g / L of the cells were recovered when 0.1 vvm of air was supplied to Zygosaccharomyces mellis TK-01, and 17.042 g / L of cells were recovered when 1 vvm of air was supplied . Therefore, it was confirmed that the supply of air was necessary to cultivate Zygosaccharomyces mellis TK-01. The cultures were cultured in aeration at 1 vvm for the yeast extract preparation.

[2-2] Selection of Enzymes for the Production of Yeast Extracts

It is known that the self-digestion method has a relatively low degradation rate of cell components, resulting in a low yield of yeast extract. Therefore, the present invention intends to increase the yield by using enzymes at the same time as self-extinguishing. The yeast culture medium cultured in Example 2-1 was adjusted to pH 6.0 with 1M KOH, and each commercially available enzyme (Table 2) was treated. In addition, TN (total nitrogen) and AN (amino nitrogen) were analyzed and compared in order to determine the decomposition degree of the yeast extract under the conditions of Table 3.

Enzyme type used Enzyme Source Optimum temperature ℃ pH Protease A Aspergillus oryzae 50 7 ProteAX Aspergillus oryzae 55 5-6 Protamex Bacillus subtillis 55 5-7 Flavourzyme Aspergillus oryzae 50 7 Amylase R Rhizypus oryzae 40 5-6 Nuclease Penicillium citrinum 70 6 Lytic enzyme Bacillus subtillis 50 7 Deaminase Aspergillus melleus 50 5-6

Evaluation of Enzyme Decomposition Solution of Yeast Extracts by Enzyme Type No. Enzyme process
Way Amino nitrogen
(AN) Total nitrogen
(TN) One Self-extinguishing


Step by step
process 1.27 1.93 2 Self-digestion + lytic enzyme 1.31 2.02 3 lytic enzyme + protease A 1.38 2.08 4 Self-digestion + lytic Enzyme + protease A 1.27 2.02 5 lytic enzyme + amylase R 1.27 1.97 6 Self-digestion + lytic enzyme + amylase R 1.28 2.13 7 lytic enzyme + protease A + amylase R 1.59 2.64 8 Self-digestion + lytic enzyme + protease A + amylase R 1.45 2.22 9 Self-extinguishing


The same time
process 1.20 2.02 10 Self-extinguishing + proteax + amylase R + glutaminase 1.28 2.02 11 Self-extinguishing + protamex + flavourzyme + glutaminase 1.43 2.04 12 Self-extinguishing + proteax + amylase R + nuclease + deaminase 1.32 2.10 13 Self-extinguishing + proteax + amylase R 1.29 2.05

As a result, as shown in Table 3, the addition of lytic enzyme did not have a significant effect on the increase of self-digestibility, and the enzyme was treated at the same time as the stepwise treatment, and the TN value and AN value It does not have much effect, but it differs by the combination of enzymes. Among them, protease A, amylase R, protamex and flavorzyme enzymes show good tendency and it is better to treat enzymes at the same time as self-digestion rather than stepwise treatment of enzymes for efficient processing.

Example 3: Preparation of fermented yeast salt using salt tolerant yeast

[3-1] Culture of resistant yeast for fermented yeast salt production

Yeast extract was cultured under the conditions of Table 2 and Table 4 for the preparation of fermented yeast salt using the strain Zygosaccharomyces mellis TK-01 of the present invention.

Yeast extract for fermentation yeast salt production Culture conditions Medium Table 1 Initial pH 5.0 Growth Temperature 30 ℃ Growth Time 48hr Agitation speed 120 rpm Aeration 1.0 v / v / m

 As a result, as shown in FIG. 8, 80.5 g / L of cells were cultured without KCl, but 38.0 g / L was cultured with 3M KCl. In the present invention, since yeast culture medium was used as a food additive, the culture medium containing the cells was used for yeast salt production without collecting the cells and removing the impurities.

 [3-2] Preparation of fermented yeast salt

In the present invention, the culture broth using the strain Zygosaccharomyces mellis TK-01 was adjusted to pH 6.0 with 1M KOH, and flavomezyme (Novo, Denmark) as exopeptidase and protamex (Novo, Denmark) as endopeptidase 0.01 to 5% (w / w), respectively, and reacted at 30 to 50 ° C for 24 hours with stirring. After completion of the reaction, the yeast extract solution was heated at 80 ° C. for 30 minutes to terminate the reaction. Cells and insoluble component-free yeast extracts were obtained through Celite bed filtration. The solid content of the produced yeast extract was confirmed and the ratio of the yeast extract and sodium chloride was 2: 4: 6 to 8 to prepare a final fermented yeast salt (FIG. 9).

Korea Microorganism Conservation Center (overseas) KCCM11605P 20141120

Claims (5)

1) Yeast extract 0.1-0.9%, malt extract 0.1-0.5%, amiga plus 1-2%, KCl 1 (2%), and yeast extract were used as the pre-culture stage using the resistant yeast Zygosaccharomyces mellis TK-01 (Accession No. KCCM-11605) , Potassium phosphate monobasic 0.05 ~ 0.1%, Zygosaccharomyces mellis TK-01 1 ~ 10%, and purified water 50 ~ 80% were added and incubated at 20 ~ 30 ℃ Culturing for 6 to 72 hours; and 2) culturing the cells at a culture temperature of 20 to 30 ° C for 6 to 12 hours, adding air of 0.1 to 1 vvm and a carbon source periodically for 6 to 12 hours to the culture conditions of step 1) Culturing for 72 hours; and 3) adding 0.01 ~ 5% of the protease, nuclease, lytic enzyme, and diaminase to the culture medium in which the present cultivation step has been completed as the enzymatic degradation step, stirring the mixture at 90~350 rpm, 50 ° C for 1 to 48 hours; and 4) inactivating the enzyme used in step 3) as the enzyme inactivation and filtration step Followed by inactivation at 80 to 95 ° C for 20 to 60 minutes, followed by centrifugation at 5,000 to 8,000 g for 20 to 60 minutes, or removal of undissociated cells using diatomaceous filtration A method for producing yeast extract using a salt tolerant yeast isolated from soybean paste The yeast extract according to claim 1, wherein the yeast and the enzyme are mixed and cultured simultaneously 2. The method according to claim 1, wherein the enzyme is protease A, amylase R, protamex and flavourzyme, and the enzyme is simultaneously treated with autolysis A process for producing fermented yeast salt using a yeast extract comprising mixing yeast extract prepared by the method of any one of claims 1 to 3 with sodium chloride in a ratio of 2-4: 6-8 The fermented yeast salt prepared in the fourth method

Images