KR20140092061A - The fermentation method for mixed grains using novel lactic acid bacteria isolated from grains and its production methods - Google Patents

Abstract

The present invention relates to a fermentation method of mixed grains using a novel lactic acid bacteria separated from grains and a mass production method thereof, and more specifically, to a method for producing fermentation mixed grains in which the titer of protease and amylase is high by fermenting mixed grains with Lactobacillus plantarum, Streptococcus thermophiles, and Enterococcus fecalis. According to the present invention, the disadvantages of general mixed grains, that the digestion is difficult and the texture is rough, can be supplemented by a lactic acid bacteria fermentation process, and fermentation mixed grains with excellent functions that the ingestion and digestion are facilitated by protease and amylase can be produced. Moreover, mixed grains, which were avoided by the above problems in spite of being useful to health, can be easily ingested, so the fermentation method of mixed grains is expected to contribute to the health improvement of modern people.

Description

Description: TECHNICAL FIELD The present invention relates to a fermentation method of mixed ginseng using fresh lactic acid bacteria isolated from grains and a mass production method therefor,

The present invention relates to a method of fermenting a mixed tune using a novel complex lactic acid bacteria isolated from grains and a mass production method using the fermentation method, and more particularly, to a fermentation method using Lactobacillus plantarum, Streptococcus thermophilus, And Enterococcus faecalis. The present invention also relates to a method for producing a fermented mixed tuna having a high protease and amylase titer by fermenting a mixed tuna with mixed lactic acid bacteria.

The most important source of energy for people is cereals, which are best for people because they contain nutrients evenly in whole grains that have not been removed. This is because most important nutrients, antioxidants and minerals are contained in various parts of the grain's shell and shell. Therefore, the rice is more nutritious than brown rice. Even in the case of other grains, it is useful for health because nutrients are uniformly contained in the grains with the outer skin and eyes attached. These facts are well known, but they are avoiding the ingestion of whole grains with their husks and shells intact. The reason for this is that grains attached to the outer skin are hard to eat, tasteless and difficult to digest.

Due to the habit of avoiding whole grains, modern diseases have been increasing in recent years, such as diabetes, obesity and hypertension. People suffering from these diseases often try to eat grains, but their digestive absorption is so low that the effect is not great. There are many reports that ingestion of grains is beneficial to health. It is necessary to eat the grains efficiently to prevent various adult diseases and to increase digestion efficiency.

In addition, the number of lactic acid bacteria in the intestines has been drastically reduced due to irregular eating habits of modern people, ingestion of various kinds of instant foods, and drinking of various chemical drugs. The intake of lactic acid bacteria is very important for this purpose. Most fermented foods are made from liquid fermentation. It is an important task to ferment cereals, which are always consumed as stocks, in a solid state to exhibit fermentation effects.

The present invention provides a fermented product of lactic acid bacterium and a method for producing the fermented product of lactic acid bacterium by using lactic acid bacteria which grow well in mixed grains containing brown rice and white rice as main ingredients, thereby promoting the consumption of cereals, We provide this excellent fermented product of lactic acid bacteria to contribute to improvement of health of consumers both at home and abroad.

Patent application publication number 10-1192673 A fermentation method and a mass production method of mixed tuna using a mixed fermenting microorganism

Accordingly, it is a main object of the present invention to provide a fermented mixed tuna having a high nutritional value, easy to ingest, and excellent in functionality by fermenting a mixed tuna with mixed lactic acid bacteria that grow well in a mixed tuna.

Another object of the present invention is to provide a fermented mixed musical piece produced by the above-described method for producing fermented mixed musical compositions using the above-mentioned complex lactic acid bacteria.

The present invention relates to a method for producing a complex lactic acid bacterium by mixing one or more cultures obtained by culturing Lactobacillus plantarum, Streptococcus thermophilus and Enterococcus faecalis, Culturing the complex lactic acid bacterium in a liquid medium, culturing the culture medium of each strain cultivated individually in an amount of 1 to 3% (v / v) in a liquid medium containing 0.1 to 5% by weight molasses, Cultivating the fermented mixed ginseng at 30 to 40 DEG C for 1 to 3 days, adding the fermented mixed culture to 100 parts by weight of the mixed culture, and culturing the mixture at 30 to 40 DEG C for 1 to 3 days. And a manufacturing method thereof.

The cereals used in the fermented mixed tuna production method are brown rice, black rice, barley brown rice, wheat rice, millet, sorghum, yulmu, white rice and black rice, and the mixing ratio is 30 to 45 parts by weight each of brown rice, 30 to 45 parts by weight, 10 to 25 parts by weight of Korean millet, sorghum and yulmu are mixed to prepare a mixed tune. The mixed tune may be used as it is in the form of a grain, or may be used by milling into a powder, and the particle size of the powder is irrelevant.

When the mixed tune having the above-described structure is used, a fermented mixed tune having excellent nutritional content and functionality can be produced at the time of fermentation due to the correlation of ingredients provided to the strain of the complex.

In the method for producing the fermented mixed tuna of the present invention, the individual seed culture of the Lactobacillus plantarum, Streptococcus thermophilus and Enterococcus faecalis is carried out in an MRS liquid medium or glucose glucose, yeast extract and sodium acetate, preferably at 30 to 38 DEG C for 24 to 48 hours.

The components of the MRS liquid medium were 20.0 g of Pancreatic digest of gelatin, 16.0 g of beef extract, 40 g of dextrose, 4.0 g of Dipotassium phosphate, 2.0 g of Polysolate 80, 10.0 g of sodium acetate, , And 0.4 g of magnesium sulfate.

The individual species cultivation is for increasing the activity of each strain and controlling the number of cultivars. If the seed culture is carried out in accordance with the above-mentioned conditions, a culture medium in a state suitable for the present cultivation can be obtained.

In the fermented mixed tuna producing method of the present invention, the strain concentration of the individual cultivated culture of each of the lactic acid bacterium is 1 × 10 ⁸ to 2 × 10 ⁹ cfu / ml.

When the above-mentioned culture broth is added to the present culture, the present culture broth in a state suitable for producing a fermented mixed tune can be produced.

When the present culture broth is added to the mixed broth and mixed, it is preferable that the broth is sprayed with mist, and the broth can be mixed using a mixer or the like.

After the fermentation is completed, the fermented product can be dried and pulverized.

According to another aspect of the present invention, there is provided a fermented mixed musical piece produced by a method of fermenting a mixed tune using the complex lactic acid bacterium.

According to the present invention, there is provided a method for preparing a mixed fermented lactic acid bacteria, which is easy to ingest and extinguish and which is excellent in functionality, which is difficult to digest and complements the disadvantages of ordinary grains due to rough texture.

In modern humans, ingestion of lactic acid bacteria is very important because irregular eating habits and ingestion of various kinds of instant foods greatly shorten the number of lactic acid bacteria in the intestines. In order to do so, it is necessary to ferment lactic acid bacteria not only in liquid fermentation but also in a solid state of cereals to prepare foods containing lactic acid bacteria and ingest them.

The present invention provides a fermented product of lactic acid bacteria and a method for producing the fermented product of a lactic acid bacterium by using a mixed lactic acid bacterium as a main ingredient of brown rice and white rice, thereby promoting the consumption of cereals and thereby solving the economic problems of farmers, We will contribute to the health improvement of domestic and foreign consumers by providing fermented products of lactic acid bacteria.

In addition, although it is beneficial for health, it is expected that it is possible to easily eat the grains avoided due to the above-mentioned problems and to improve the health of the modern people.

1 is a process diagram showing a fermentation process of lactic acid bacteria mixed with mixed grains.

The present invention relates to a method for producing a fermented mixed piece by fermenting mixed grains mixed with brown rice, black rice, barley brown rice, Korean millet, sorghum, yulmu, white rice, and black rice using a new lactic acid microorganism as a new microorganism.

The complex lactic acid bacterium is produced by mixing one or more cultures obtained by culturing the new microorganisms Lactobacillus plantarum, Streptococcus thermophilus and Enterococcus faecalis.

Lactobacillus plantarum, a new microorganism among multiple lactic acid bacteria, is a lactic acid bacterium isolated from brown rice, inhibits the growth of harmful pathogens such as Salmonella, and has acid resistance, bile acid resistance and heat resistance. Another new microorganism, Streptococcus thermophilus, ferment glycated grains to lactic acid and lower the neutral pH to 4.0 to 4.5. When cereals are fermented by Streptococcus thermophilus, the quality characteristics of the fermented grains, such as soft taste and flavor, are excellent as well as the nutritional and physiological values of the fermented grains. Another new microorganism, Enterococcus faecalis, promotes the growth of beneficial bacteria in the intestine, inhibits the growth of harmful microorganisms and keeps the intestinal microflora in a balanced manner, thus acting as a grain fermentation strain that has a beneficial effect on the host. do.

The novel microorganisms constituting the complex lactic acid bacteria of the present invention were respectively identified as Lactobacillus plantarum, Streptococcus thermophilus and Enterococcus picalis, which have 99% homology based on the 16S rDNA nucleotide sequence. Analysis of sugar availability, proteolytic enzyme activity, acid resistance, bile salt acidity and lyophilization yield were compared to confirm that the strain was a new strain. Lactobacillus plantarum, Streptococcus thermophilus, and Enterococcus picalis, which are novel lactic acid bacteria of the present invention, have different characteristics such as protein resolution, acid resistance, bile acid resistance and yield during freeze drying, which are different from known lactic acid bacteria.

In order to ferment the mixed grains of the present invention, individual cultivating the complex lactic acid bacterium in a liquid medium, and culturing each strain cultivated individually. The present cultivation is carried out by adding 1 to 3% (v / v) of the culture medium of the strain, which is a new microorganism individually cultured to a liquid medium containing 0.1 to 5% by weight of molasses, at 1 to 2 Cultured for one day.

1 to 10 parts by weight of the present culture cultured for fermenting mixed grains is added to 100 parts by weight of a mixed tang, mixed and cultured at 30 to 40 ° C for 1 to 3 days.

The grains used in the fermented mixed grain production method consist of brown rice, black rice, barley brown rice, wheat rice, millet, sorghum, yulmu, white rice and black rice, and the mixing ratio is 30 to 45 parts by weight each of brown rice, 30 to 45 parts by weight, 10 to 25 parts by weight of Korean millet, sorghum and yulmu are mixed to prepare a mixed tune. The mixing tumbles are milled and mixed with the powder so that the particle size of the powder is irrelevant.

When the mixed tune having the above-described structure is used, a fermented mixed tune having excellent nutritional content and functionality can be produced at the time of fermentation due to the correlation of ingredients provided to the strain of the complex.

(Example 1)

Separate Species Culture of Each Strain MRS solid medium (Lactobacillus plantarum, Streptococcus thermophilus, Enterococcus faecalis), which is commercially produced and sold by taking an appropriate amount of separated lactic acid bacteria Lactobacillus plantarum, Streptococcus thermophilus and Enterococcus faecalis Beef Extract 10.0g, Yeast Extract 5.0g, Dextrose 20.0g, Sorbitan Monooleate 1.0g, Ammonium Citrate 2.0g, Sodium Acetate 5.0g, MnSO ₄ .H ₂ O 0.05g, Na ₂ HPO ₄ 2.0 g, Agar 15 g; 55 g / L) and cultured for about one day at 35, then a single colony was taken and inoculated into 300 ml of MRS liquid medium of the same composition except for agar. It was cultured at 35 ° C for 2 days and used as a seed. The numbers of bacteria were 9.2 × 10 ⁸ cfu / ml for Lactobacillus plantarum, 9.4 × 10 ⁸ cfu / ml for Streptococcus thermophilus, 1.1 × 10 ^{9 for} Enterococcus faecalis, cfu / ml.

(Example 2)

    Mixed Lactobacillus culture

The mass culture of complex microorganisms was prepared by using the culture medium of each strain prepared in Example 1 as a seed. In order to cultivate the complex lactic acid bacteria in a large amount, 60 g of edible molasses was sufficiently dissolved in 10 liters of tap water, and 200 ml of the seed culture of Example 1 was added to each well. Water was then added to bring the final volume to 20 liters. The microbial mixture was incubated at 35 DEG C for 2 days. The culture pH of the cultured complex lactic acid bacteria was 4.3. The number of the cultures was 6.8 x 10 ⁸ cfu / ml for Lactobacillus plantarum, 4.5 x 10 ⁸ cfu / ml for Streptococcus thermophilus, and 6.1 x 10 ⁸ cfu / ml for Enterococcus faecalis. × 10 ⁸ cfu / ml.

 (Example 3)

  Enzyme activity investigation

 3-1 a-amylase

 ① Reagent

  ㉮ 1% soluble starch solution: 1 g of soluble starch (best quality product) is dissolved in water and gelatinized to 100 mL.

  Mc Mcllvaine buffer (pH 6.0 or 7.0): 0.1 N citric acid solution is added to a constant amount of 0.1 N sodium phosphate monobasic solution to make pH 6.0 and 7.0, respectively.

  ㉰ 0.1% calcium chloride solution: 1 g of calcium chloride (pure product) is dissolved in water to make 1 L.

  Iodine solution: Dissolve 0.2 g of iodine and 2 g of potassium iodide in water to make 100 mL. Add 1 mL of 1 N hydrochloric acid to 1 mL and make 100 mL with water.

 ② Device

  Spectrophotometer

 ③ Preparation of Test Solution

   5.0 g of sample is precisely weighed and dissolved in water or buffer to make 100 mL, and then filtered to give the sample solution. Two 20 mL test tubes shall be prepared for each test and blank test. Add 5 mL of 1% soluble starch solution, 13 mL of McBain buffer (pH 6.0 or 7.0) and 1 mL of 0.1% calcium chloride solution in a test tube, warm to 37 ° C, add 1 mL of the sample solution, and allow to stand at 37 ° C for 30 minutes . Separately, heat the sample at 100 ° C for 30 minutes to remove 1 mL of the sample solution. Test solution is prepared by adding 10 mL of iodine TS to 0.2 mL of test solution and blank test solution.

 ④ Test operation

Absorbance is measured at 660 nm at 1 cm wavelength in the liquid layer using water as a reference solution. At this time, the absorbance of the test solution should be less than 0.030 of the absorbance of the blank test. If the degree of color development is too great to be measured, dilute the test solution and apply dilution.

3-2 Protease

 ① Reagent

  ㉮ 0.6% casein solution: After drying the casein (the best product), 0.6 g of the product is dissolved by heating in 20 mL of 0.1 N sodium hydroxide solution. After cooling, the solution is adjusted to pH 7.0 by adding 0.1 M phosphoric acid and 20 mL of pH 7.0 buffer mL.

  ㉯ 0.4 M trichloroacetic acid solution: 65.4 g of trichloroacetic acid (pure product) is dissolved in water to make 1 L.

  ㉰ 0.4 M sodium carbonate solution: 42.5 g of sodium carbonate (pure product) is dissolved in water to make 1 L.

  ㉱ Porin solution: It is made according to the food additive revolution and is made into the undiluted solution.

  ㉲ Buffer: 0.1 M phosphate buffer (pH 6.0 or 8.0) or 0.1 M acetate buffer (pH 6.0 or 8.0)

 ② Device

Spectrophotometer

 ③ Preparation of Test Solution

Approximately 5.0 g of the sample is precisely weighed and dissolved in water or buffer to make 100 mL, and then filtered to give the sample solution. Add 1 mL of 0.6% casein in a test tube, warm in a 37 ° C warm bath, add 1 mL of the test solution, and shake well. It is added to an incubation bath at 37 ° C for exactly 10 minutes, then 2 ml of 0.4 M trichloroacetic acid solution is added, and the mixture is left at 37 ° C for 25 minutes and then filtered. Take exactly 1 mL of the filtrate in a test tube, add 5 mL of 0.4 M sodium carbonate solution, and 1 mL of purine solution (3-fold diluted solution), and shake well. Allow to stand for 20 minutes at 37 ° C and use the developed solution as the test solution. Separately, add 1 mL of the sample solution, place it in a test tube, incubate at 37 ° C for 10 minutes, add 2 mL of 0.4 M trichloroacetic acid solution, mix 1 mL of 0.6% casein solution and incubate at 37 ° C for 25 minutes. The same procedure is followed to prepare a blank test solution.

 ④ Test operation

Using water as a reference, absorbance is measured at 1 cm in the liquid layer and 660 nm in wavelength. At this time, the absorbance of the test solution should be greater than 0.030 of the absorbance of the blank test. If the degree of color development is too great to be measured, dilute the test solution and apply dilution.

(Example 4)

      Mass production and fermentation of lactic acid bacteria and enzymatic ability

In order to ferment the mixed tastes, various grains were constituted at the same weight ratio as in Table 1. Each grain was put into a mixer in proportions and mixed. Based on the weight of the mixed grains, 10% of the combined microbial cultures were mixed for 20 minutes while spraying with mist spray. The mixture was thoroughly mixed in an aluminum pouch at a rate of 1 kg, sealed, and then fermented in a culture room at 35 ° C for 2 days. After the fermentation, it was dried so that the moisture content was within 10% in the dryer at 50 ° C.

And protease and amylase activity were as follows (see Table 2).

grain Test section 1 Test Area 2 Experiment 3 Brown rice 20 25 30 Black rice 10 10 10 Barley brown rice 10 10 10 various phases 20 30 30 Black 10 10 5 Domestic 10 5 5 Sorghum 10 5 5 Yulmu 10 5 5

Analysis item Test section 1 Test Area 2 Experiment 3 Number of lactic acid bacteria (cfu / g) 4.5 × 10 ⁵ 6.3 × 10 ⁵ 7.1 x 10 ⁵ Protease (AU / g) 128 256 256 Amylase (AU / g) 256 512 512

In Example 4, the fermented mixed rice was brown rice, black rice, barley brown rice, Korean millet, sorghum, yulmu, white rice and black rice. The mixing ratio of the cereals used in Example 4 was 30 to 45 parts by weight each of brown rice, black rice and barley brown rice, 30 to 45 parts by weight of white rice, 30 to 45 parts by weight of black rice, 10 to 25 parts by weight of Korean millet, To prepare a mixed tune. The mixed tune may be used as it is in the form of a grain, or may be used by milling into a powder, and the particle size of the powder is irrelevant.

As a result, the number of lactic acid bacteria and the enzyme activity were increased as the mixing ratio of each test group was increased. In Table 1 and Table 2, protease and amylase reverse activity showed the highest enzyme activity in Experiment 2 and Experiment 3, and Experiment 3 was the most abundant in the number of lactic acid bacteria. The activity of lactic acid bacteria was higher in the mixed fermented rice with high content of brown rice and black rice.

Claims (7)

Lactobacillus plantarum CQE 131 (KCTC-), which has acid resistance, bile acid resistance, and heat resistance and has the function of inhibiting the growth of harmful pathogens, Streptococcus thermophilus CQE 135 (KCTC-), which promotes fermentation and lowers the pH of cereals,
To promote the growth of beneficial bacteria in the intestinal yen Tero Lactococcus blood faecalis (Enterococcus faecalis) CQE (KCTC-) 137 ℃
One or more cultures obtained by culturing Lactobacillus plantarum, Streptococcus thermophilus and Enterococcus faecalis are cultured to produce a complex lactic acid bacterium, Culturing individual seeds in a liquid medium; And
Culturing the individual seed cultured medium in a liquid medium; And
And 1 to 10 parts by weight of the culture medium cultured in the main culturing step is added to 100 parts by weight of the mixed cereal, and the mixture is cultured at 30 to 40 DEG C for 1 to 3 days.
The method of claim 4, wherein
Wherein the mixed grains are composed of brown rice, black rice, barley brown rice, common wheat, sorghum, yulmu, white rice and black rice.
The method according to claim 4 or 5,
Wherein the mixing ratio of the mixed grains is 30 to 45 parts by weight each of brown rice, black rice, barley brown rice, 30 to 45 parts by weight of white rice, 30 to 45 parts by weight of each of black rice, 10 to 25 parts by weight of common rice, Method of making fermented mixed grains.
5. The method of claim 4,
The components of the liquid medium were 20.0 g of Pancreatic digest of gelatin, 16.0 g of beef extract, 40 g of dextrose, 4.0 g of Dipotassium phosphate, 2.0 g of Polysolate 80, 10.0 g of sodium acetate, 4.0 g of ammonium citrate 4.0 g, and 0.4 g of magnesium sulfate.

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