WO2020004852A1 - Lactic acid bacteria-fermented rice product having increased lysine amount, and preparation method therefor - Google Patents

Lactic acid bacteria-fermented rice product having increased lysine amount, and preparation method therefor Download PDF

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WO2020004852A1
WO2020004852A1 PCT/KR2019/007290 KR2019007290W WO2020004852A1 WO 2020004852 A1 WO2020004852 A1 WO 2020004852A1 KR 2019007290 W KR2019007290 W KR 2019007290W WO 2020004852 A1 WO2020004852 A1 WO 2020004852A1
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rice
lactic acid
acid bacteria
fermentation
lactobacillus plantarum
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PCT/KR2019/007290
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French (fr)
Korean (ko)
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WO2020004852A9 (en
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최혜선
한상익
김선림
최인덕
박혜영
이석기
박지영
오세관
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대한민국(농촌진흥청장)
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Publication of WO2020004852A1 publication Critical patent/WO2020004852A1/en
Publication of WO2020004852A9 publication Critical patent/WO2020004852A9/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/20—Malt products
    • A23L7/25—Fermentation of cereal malt or of cereal by malting
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/02—Acid
    • A23V2250/06—Amino acid
    • A23V2250/063—Lysine
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2300/00—Processes
    • A23V2300/14—Extraction
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2300/00—Processes
    • A23V2300/26—Homogenisation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/169—Plantarum

Definitions

  • the present invention relates to a method for producing a rice lactic acid bacteria fermentation with increased lysine content, specifically, a method for producing a rice lactic acid bacteria fermentation comprising the step of fermenting a rice extract using a Lactobacillus plantarum strain, by the method
  • the present invention relates to a rice lactic acid bacteria fermented product with increased lysine content and a food composition containing the rice lactic acid bacteria fermented product.
  • Common yogurt is a kind of fermented milk, and is prepared by inoculating and fermenting lactic acid bacteria in milk. Fermented milk consumption is estimated at 573 thousand tonnes in 2013 from 52,000 thousand tonnes in 2013, as it is known that lactic acid bacteria contained in yogurt can make various vitamin B in the intestine and suppress intestinal toxins. The consumption is growing rapidly, with an increase of about 26% over three years.
  • yogurt when the yogurt is manufactured using rice, there is a problem that the rice and milk are not fully luxury and the rice and the milk feel heterogeneous.
  • the number of strict vegetarians who do not eat meat, milk, or eggs is increasing, and it is necessary to develop yogurt, which is composed only of grains and increased in taste and functionality.
  • lysine essential amino acid lysine
  • tissues such as cartilage and ligaments, and necessary for the production of hormones, antibodies, enzymes, and the like, but as a deficient amino acid of rice, its content is very little or almost no.
  • the present inventors did not include dairy products but only cereals as main ingredients, and as a result of diligent efforts to produce rice lactic acid bacteria fermented products with increased lysine content, rice germ can be used to ferment rice without dairy products. In addition, it was confirmed that the lysine content is increased through fermentation using Lactobacillus plantarum strain, to complete the present invention.
  • One object of the present invention is to provide a method for producing a fermented rice lactic acid bacteria increased lysine, comprising the step of fermenting the rice extract using Lactobacillus plantarum ( Lactobacillus plantarum ).
  • Another object of the present invention is to provide a method for increasing the content of lysine in rice lactic acid bacteria fermentation, comprising the step of fermenting rice extract using Lactobacillus plantarum in the production of fermented rice lactic acid bacteria.
  • Yet another object of the present invention is to provide a fermented rice lactic acid bacterium with an increased lysine content prepared by the above method.
  • Another object of the present invention is to provide a food composition comprising the rice lactic acid bacteria fermentation.
  • Rice lactobacillus fermentation according to the present invention is increased in the lysine content of the deficient amino acid of rice, it is excellent in quality characteristics compared to the lactobacillus fermentation product prepared without using the Lactobacillus plantarum strain, lysine content is increased and only grains It can be actively used in the production of yogurt containing and the development of food containing the same.
  • FIG. 1 is a view showing a change in lysine content when producing a lactic acid bacteria fermented by the manufacturing method according to the present invention.
  • Figure 2 is a view showing an amino acid analysis chromatogram when producing the lactic acid bacteria fermentation by the production method according to the present invention.
  • FIG. 3 is a view showing a change in lysine content according to the fermentation strain when producing the lactic acid bacteria fermented by the production method according to the present invention.
  • Figure 4 is a view showing the results confirming the gene involved in lysine synthesis of lysine biosynthesis pathway and Lactobacillus plantarum.
  • one embodiment of the present invention provides a method for producing a fermented rice lactic acid bacteria fermentation with increased lysine content, comprising the step of fermenting the rice extract using Lactobacillus plantarum ( Lactobacillus plantarum ) .
  • the term "rice” refers to white rice which has been refined by milling rice.
  • the rice may be completely removed from the embryo and rice bran layer through the milling.
  • the rice varieties are not particularly limited to this, but may be Samgwang, Hana powder, Haiami, Cheonggeum, A La Carte, Dodam, Goami No. 2, Goami No. 4, Hong Jinju, Red Pearl, Choeun Black Rice, Suwon 542, Milyang 317 It may be, specifically, Samgwang, a la carte, Goami No. 4, Haiami, powdered powder, Suwon 542, Milyang 371, Zoeun black rice, and more specifically, Zoeun black rice.
  • the lysine content increased 1.5 to 3 times in other varieties.
  • the lysine content was increased by about 6 times or more compared to before fermentation, which is an increase of about 18 times compared to the positive control group. It could be seen (Fig. 1 and Fig. 2).
  • lactic acid bacterium fermentation product refers to a substance fermented using lactic acid bacteria (lactic acid bacteria), and may be used in combination with "yoghurt” in the present invention.
  • the yogurt may be one containing no dairy.
  • the dairy product as a whole made up of food made by processing the milk of livestock, milk, butter, cheese, condensed milk, milk powder and the like belong to this, in the present invention is characterized in the production of yogurt without dairy products containing milk.
  • the yogurt prepared using Lactobacillus plantarum as a fermentation strain without using dairy products, the lysine content is significantly increased compared to yogurt prepared using other species of lactic acid bacteria. Confirmed. That is, when the fermentation is performed by Lactobacillus plantarum, the present invention solves the conventional problem of lacking the lysine content of rice lactic acid bacteria fermentation, unlike fermentation by other species of lactic acid bacteria.
  • the method for producing a fermented rice lactic acid bacteria fermented lysine content is characterized in that using Lactobacillus plantarum as a fermentation strain. That is, lysine was a deficient amino acid of rice in the past, but in the present invention, by using Lactobacillus plantarum as a fermentation strain, the lysine content is markedly increased.
  • Lactobacillus plantarum may be inoculated into the mixture at 0.05 to 0.15% (v / v), specifically 0.07 to 0.13% (v / v), most specifically 0.1% (v / v), but fermented. It is not limited to this as long as it can be performed.
  • the Lactobacillus plantarum may be the Lactobacillus plantarum JSA22, which is described in Korean Patent Publication No. 10-1634270, which is the prior patent of the inventors, that is, the Lactobacillus plantarum having the accession number KACC91973P.
  • the Lactobacillus plantarum JSA22 strain was converted to an international deposit under the Budapest Treaty by the National Agricultural Research and Development Institute (KACC) National Agricultural Research and Development Institute (KACC) on June 12, 2019, and has been granted the International Deposit No. KACC81097BP.
  • the strain is a lactic acid bacterium isolated and selected from traditional Jangjang, characterized by excellent antimicrobial activity, digestibility and adhesion.
  • Lactobacillus fermentation products of significantly increased lysine content can be produced through the production method of the present invention using Lactobacillus plantarum as a fermentation strain.
  • the dairy product is not used in the production of the fermented rice lactic acid bacteria produced in the present invention. That is, in the past, dairy products were used to carry out fermentation by lactic acid bacteria, but in the present invention, rice embryos are used instead of dairy products.
  • rice extract means a product extracted from rice.
  • the rice extract means rice itself, or an extract obtained by extracting and processing rice, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a crude or purified product of the extract, or a mixture thereof Extracts of all formulations that can be formed using the extract itself and the extract, such as mixtures.
  • the method of extracting the rice is not particularly limited and may be extracted according to methods commonly used in the art.
  • the rice extract comprises the steps of (a) mixing rice and water to prepare a rice paste, and mixing rice germ and water to prepare rice germ oil; And (b) may be prepared by mixing the rice paste and rice germ oil to prepare a mixture.
  • rice germ means an embryo of rice.
  • the rice embryo may be separated from the rice through milling.
  • the step (a) includes a step of obtaining rice paste by mixing rice and water, wherein the mixing ratio of rice and water maximizes the quality and sensory properties of yogurt, and does not inhibit fermentation by lactic acid bacteria.
  • the "rice paste” refers to a mixture of rice and water, it may mean that the mixture is ground.
  • the rice paste may be prepared by mixing 5 to 35% (w / w) of rice and 65 to 95% (w / w) of water, more specifically 15 to 35% (w / w) It can be prepared by mixing rice and 65 to 85% (w / w) of water, most specifically 20 to 30% (w / w) of rice and 70 to 80% (w / w) of water It may be prepared by, but is not limited thereto.
  • a mixing ratio outside the above range there is a problem that the quality and palatability of the yogurt is lowered.
  • the step (a), comprising the step of mixing the rice embryo and water to obtain rice germ oil, the mixing ratio of the rice embryo and water to maximize the quality and sensory properties of yogurt, inhibit the fermentation by lactic acid bacteria It is appropriate not to do it.
  • the "rice germ oil” may refer to a mixture of rice germ and water, and may mean that the mixture is ground or the mixture is ground and filtered.
  • the rice germ oil may be prepared by mixing 5 to 15% (w / w) rice germ and 85 to 95% (w / w) water, more specifically 8 to 12% (w / w) w) can be prepared by mixing rice germ and 88 to 92% (w / w) of water, but is not limited thereto.
  • a mixing ratio outside the above range, there is a problem that the quality and palatability of yogurt is lowered.
  • Step (b) is a step of obtaining a mixture by mixing the rice paste and rice germ oil obtained in the step (a), the mixing ratio of the rice paste, rice germ oil maximizes the quality characteristics and sensory characteristics of yogurt, It is appropriate to not inhibit the fermentation by lactic acid bacteria.
  • the mixture may be prepared by mixing the rice paste, rice germ oil in the same volume ratio.
  • the mixture is prepared at a mixing ratio other than the above range, there is a problem in that the quality and palatability of the yogurt are reduced.
  • the mixture in order to enhance the fermentation efficiency, may be fermented by including 2 to 6% (w / v) of sugar, specifically 3% (w / v) of sugar to ferment It may be, but is not limited thereto.
  • sugar is added at a mixing ratio other than the above range, there is a problem that the quality and palatability of the yogurt are lowered.
  • the preparation method of the present invention may further comprise the step of sterilizing and homogenizing the mixture after step (b).
  • the step is to sterilize the mixture of rice paste and rice germ oil obtained in the step (b), to prevent the desired fermentation is not progressed by other bacteria.
  • the sterilization can be carried out at 90 to 110 ° C, more specifically at 95 to 105 ° C, most specifically at 100 ° C, and also for 50 to 70 minutes, specifically 55 to 65 minutes, most specifically 60 minutes It may be carried out, but is not limited to so long as sterilization can be performed.
  • the sterilization may be performed by a method known in the art, and specifically, may be performed by a method such as dry heat sterilization and autoclaving, but is not limited thereto.
  • the step is to homogenize the mixture of the rice paste and rice germ oil obtained in the step (b), to enhance the efficiency of the fermentation. Homogenization results in an increase in the surface area of the particles, a uniform viscosity, a smooth texture and a stable tissue.
  • the homogenization can be carried out at 20 to 40 ° C., more specifically at 25 to 35 ° C., most specifically at 30 ° C., and also for 35 to 45 hours, specifically 37 to 43 hours, most specifically 40 hours. It may be performed, but is not limited thereto.
  • the homogenization may be performed by a method known in the art, and specifically, may be performed using a high speed homogenization, a high pressure homogenizer, a colloid mill, an ultrasonic wave, or the like, but is not limited thereto.
  • Another aspect of the present invention provides a method for increasing lysine content in rice lactic acid bacteria fermentation, comprising the step of fermenting rice extract using Lactobacillus plantarum in the production of rice lactic acid bacteria fermentation.
  • Another aspect of the present invention provides a rice lactic acid bacteria fermentation with an increased lysine content prepared by the above production method.
  • Lactobacillus fermentation product with increased lysine content of the present invention is characterized by using the Lactobacillus plantarum strain as a fermentation strain, the lysine content is significantly increased compared to the lactic acid bacteria fermentation products prepared using other species of lactic acid bacteria .
  • the lactic acid bacteria fermentation may be prepared by a method for preparing yogurt using Lactobacillus plantarum JSA22 used in one embodiment of the present invention as a fermentation strain, Lactobacillus plantarum strain increases the lysine content It is not limited to this as long as it has an effect to make it.
  • Another aspect of the invention provides a food composition comprising the lactic acid bacteria fermentation.
  • lactic acid bacteria fermentation product (yogurt)
  • yogurt lactic acid bacteria fermentation product
  • the term "food” includes all foods in a common sense such as snacks, confectionery, bread, pizza, ramen, other noodles, rice, instant rice, ice cream, dairy products, and health foods. As long as fermentation may be included, it is not limited to this.
  • yogurt containing rice and rice germ were prepared.
  • the selected rice was pulverized with a test mill, and then rice flour passed through a 50 mesh sieve was used.
  • the rice flour and water were mixed at a weight ratio of 1: 9 to 3: 7, and ground to prepare rice paste.
  • rice germ and water were mixed at a weight ratio of 1:10, and then pulverized and coagulated with a cheese cloth to prepare rice germ oil.
  • the mixture of rice paste and rice germ oil was mixed at the same volume ratio to prepare a mixture of rice paste and rice germ oil, and 3% (w / v) of sugar was added to the mixture, followed by sterilization (luxury, autoclaving, 100). C, 60 minutes). After cooling to room temperature and homogenizing, the lactic acid bacteria were inoculated at a constant concentration (0.1% (v / v), OD (600 nm)), fermented at 30 ° C. for 40 hours, and rice containing rice germ Yogurt was prepared.
  • the lactic acid bacteria was Lactobacillus plantarum JSA22, and the control strain was Lactobacillus rhamnosus GG. Lactobacillus strains were used in two subcultures on MRS broth.
  • the rice embryo of the present invention is a fermentation aid, that is, a protein source of lactic acid bacteria, used for the purpose of increasing the fermentation efficiency of yogurt, yogurt containing milk used as a fermentation aid in the art (Korea Yagurt, Mechinnikov) Was used as a positive control.
  • Example 2-1 Rice Manufactured Using Various Rice Varieties Lactic acid bacteria fermentation Determination of Amino Acid Content
  • Free amino acid was extracted by sonication by adding 800 uL of 80% ethanol to 0.1 g of the lyophilized sample, and 500 uL of 80% ethanol was extracted two more times.
  • the extract was centrifuged (13,500 rpm, 10 minutes), the supernatant was taken, and the supernatant extracted three times was combined and concentrated under reduced pressure with CentriVap DNA Concentrator (LABCONCO CORPORATION, KANSAS CITY, USA).
  • the extracted solvent was dissolved in 1 mL of 3% TCA in a tube completely removed, and shaken at 200 rpm for 1 hour sonication.
  • the extract was centrifuged (13,500 rpm, 20 minutes) and the supernatant was filtered through a 0.45 ⁇ m syringe filter.
  • an Amino Acid Analyzer L-8900, HITACHI, Japan
  • the ion exchange column # 2622PF Column, 4D3846
  • column temperature 38 to 70 °C
  • reactor heater temperature was maintained at 135 °C
  • the injection volume was analyzed 20.0 uL, flow rate 0.35 mL / min
  • the free amino acid standard was mixed with Type AN-II and Type B (Wako, Japan) at 1: 1 (v / v), diluted 20-fold, and used for analysis.
  • Example 2-2 Lactobacillus Rhamnosus Rice using strain With lactic acid bacteria fermentation compare
  • Example 2-1 it was confirmed that the rice lactobacillus fermentation product using the Lactobacillus plantarum strain of the present invention has an excellent lysine increasing effect, and compared with the rice lactic acid bacterium fermentation product using the Lactobacillus rhamnosus strain. It was.
  • Samkwang and coarse black rice were used as rice varieties, and Lactobacillus plantarum JSA22 strain and Lactobacillus rhamnosus GG strain were used as fermentation strains.
  • the content of lysine and aspartic acid before fermentation (0 hour) and the content of lysine and aspartic acid (40 hour) after 40 hours of fermentation were measured, respectively.
  • the fermented with other species of lactic acid bacteria compared to the case of producing a lactic acid bacteria fermented product, significantly increased the lysine content when prepared by fermentation with Lactobacillus plantarum, especially Choeun black rice as a rice variety When it was confirmed that the increase efficiency of the lysine content is excellent.
  • Lysine synthesis is synthesized using aspartic acid as a precursor, in order to identify the gene involved in the synthesis, DNA extraction and PCR of the Lactobacillus plantarum strain was performed.
  • Genomic DNA extraction of Lactobacillus plantarum JSA22 strain was performed using a Qiagenprep spin miniprep kit (Qiagen, Valencia, CA, USA). It was centrifuged for 10 minutes at 13,500 rpm. After centrifugation, the supernatant was carefully removed, suspended by addition of 180 uL of 20 mg / mL lysozyme, and reacted in a 37 ° C. constant temperature water bath for 30 minutes. 20 uL proteinase K and 200 uL AL buffer were added to react at 56 ° C. for 30 minutes. After the reaction, 200 uL ethanol was added and turbid. The reaction solution was transferred to a mini spin column and centrifuged. AW buffer was added to the spin column and all samples passed through the column were discarded. DNA was extracted by adding 50 uL AE buffer and centrifuging for 1 minute at 8,000 rpm. DNA concentration was measured using a spectrophotometer.
  • PCR was performed using a thermal cycler (T100 Thermal Cycler, Bio-Rad, Calif., USA), PCR reaction solution consisting of amfiEco PCR premix kit (GenDEPOT, Calif., USA), 2 ⁇ l forward and reverse primers, 25 ⁇ l total DNA sample.
  • PCR reaction conditions were denatured at 94 ° C for 2 minutes, then denatured at 94 ° C for 2 minutes, annealing at 46-60 ° C for 2 minutes depending on the Tm value of the primer, and 1 minute / kb elongation at 72 ° C.
  • the reaction was carried out in 30 cycles, followed by a final elongation reaction at 72 ° C. for 10 minutes.
  • the amplified product was identified on a 0.8% agarose gel.
  • yogurt prepared using rice has a higher lysine content than milk-based fermented milk, and in addition, Lactobacillus plantarum significantly increases lysine content when Lactobacillus plantarum is used as a fermentation strain. It was confirmed that the fermentation of the strain can produce a fermented rice lactic acid bacteria increased lysine content.

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Abstract

The present invention relates to a method for preparing a lactic acid bacteria-fermented rice product having an increased lysine amount and, more specifically, to: a method for preparing a lactic acid bacteria-fermented rice product, comprising a step of fermenting a rice extract by using a Lactobacillus plantarum strain; a lactic acid bacteria-fermented rice product having an increased lysine amount, prepared by the preparation method; and a food composition containing the lactic acid bacteria-fermented rice product. A lactic acid bacteria-fermented rice product according to the present invention has an increased amount of lysine, which is an amino acid deficient in rice, so as to have quality superior to those of lactic acid bacteria fermented products that are prepared without using a Lactobacillus plantarum strain, thereby being actively usable in the preparation of yogurt containing only cereals and in the development of food comprising same.

Description

라이신 함량이 증가된 쌀 유산균발효물 및 이의 제조방법Rice Lactic Acid Bacteria Fermented with Increased Lysine Content
본 발명은 라이신 함량이 증가된 쌀 유산균발효물의 제조방법에 관한 것으로, 구체적으로 쌀 추출물을 락토바실러스 플란타럼 균주를 이용하여 발효시키는 단계를 포함하는 쌀 유산균발효물의 제조방법, 상기 제조방법에 의해 제조된 라이신 함량이 증가된 쌀 유산균발효물 및 상기 쌀 유산균발효물을 포함하는 식품조성물에 관한 것이다.The present invention relates to a method for producing a rice lactic acid bacteria fermentation with increased lysine content, specifically, a method for producing a rice lactic acid bacteria fermentation comprising the step of fermenting a rice extract using a Lactobacillus plantarum strain, by the method The present invention relates to a rice lactic acid bacteria fermented product with increased lysine content and a food composition containing the rice lactic acid bacteria fermented product.
일반적인 요구르트는 발효유의 일종이며, 우유류에 젖산균을 접종 및 발효시켜 제조된다. 요구르트에 포함된 젖산균이 장내에서 각종 비타민B를 만들고, 장내에서 독소를 생성하는 유해균을 억압하여 장 건강에 도움이 될 수 있다는 것이 알려짐에 따라, 발효유 소비량은 2010년 502천 톤에서 2013년 573천 톤으로, 3년 동안 약 26%가 증가하는 등 그 소비량이 급성장하고 있다.Common yogurt is a kind of fermented milk, and is prepared by inoculating and fermenting lactic acid bacteria in milk. Fermented milk consumption is estimated at 573 thousand tonnes in 2013 from 52,000 thousand tonnes in 2013, as it is known that lactic acid bacteria contained in yogurt can make various vitamin B in the intestine and suppress intestinal toxins. The consumption is growing rapidly, with an increase of about 26% over three years.
이에 따라, 우유를 이용한 요구르트의 제조에 관한 연구는 무수히 진행되어 왔으며 그 제조기술 역시 잘 정립되어 있다. 현재까지 국내에는 많은 요구르트 관련 특허가 공개 또는 등록되었고, 대표적인 공개 특허는 아래와 같다. 대한민국 등록특허공보 제10-1754233호는 요구르트의 커드 형성을 단단히 하고 체지방 개선 효과가 있는 요구르트 제조용 조성물에 관한 것이고, 대한민국 공개특허공보 제10-2016-0082305호는 점도가 향상된 요구르트의 제조방법에 관한 것이다. 그러나, 곡류를 이용한 요구르트의 제조에 관한 연구는 매우 제한적으로 수행되어, 유산균 발효식품 시장에서는 우유를 주원료로 하는 요구르트가 대부분을 차지하는 실정이다.Accordingly, the research on the production of yogurt using milk has been innumerable and the production technology is well established. To date, many yogurt related patents have been published or registered in Korea, and representative public patents are as follows. Republic of Korea Patent Publication No. 10-1754233 relates to a composition for preparing yogurt that has a curd formation of yogurt firmly and has a body fat improvement effect, Republic of Korea Patent Publication No. 10-2016-0082305 relates to a method for producing a yogurt with improved viscosity will be. However, research on the production of yogurt using cereals has been very limited, and in the market of lactic acid bacteria fermented foods, yogurt mainly containing milk occupies most of the situation.
이와 관련하여, 쌀을 사용하여 요구르트를 제조하는 경우 쌀과 우유가 완전히 호화되지 못하여 쌀과 우유가 이질감이 느껴지는 등의 문제점이 있었다. 아울러, 최근 채식에 대한 관심이 높아지면서 고기는 물론 우유, 달걀도 먹지 않는 엄격한 채식주의자의 수가 증가하고 있는바, 곡류로만 구성되어 있으며 그 맛뿐만 아니라 기능성까지 증대된 요구르트의 개발이 필요한 실정이었다.In this regard, when the yogurt is manufactured using rice, there is a problem that the rice and milk are not fully luxury and the rice and the milk feel heterogeneous. In addition, as the interest in vegetarian foods has increased recently, the number of strict vegetarians who do not eat meat, milk, or eggs is increasing, and it is necessary to develop yogurt, which is composed only of grains and increased in taste and functionality.
또한, 필수 아미노산인 라이신(lysine)은 연골과 인대 등의 조직형성에 필요하며, 호르몬, 항체, 효소 등의 생성에 반드시 필요하지만, 쌀의 결핍 아미노산으로서, 그 함량이 매우 적거나 거의 없다.In addition, the essential amino acid lysine (lysine) is required for the formation of tissues such as cartilage and ligaments, and necessary for the production of hormones, antibodies, enzymes, and the like, but as a deficient amino acid of rice, its content is very little or almost no.
본 발명자들은 유제품을 포함하지 않고 주재료로서 오로지 곡류만 포함하며, 라이신 함량이 증가된 쌀 유산균발효물을 제조하고자 예의 노력 연구한 결과, 쌀배아를 사용하는 경우 유제품 없이도 쌀의 발효를 수행할 수 있을 뿐만 아니라, 락토바실러스 플란타럼 균주를 이용한 발효를 통해 라이신 함량이 증가됨을 확인하여, 본 발명을 완성하였다.The present inventors did not include dairy products but only cereals as main ingredients, and as a result of diligent efforts to produce rice lactic acid bacteria fermented products with increased lysine content, rice germ can be used to ferment rice without dairy products. In addition, it was confirmed that the lysine content is increased through fermentation using Lactobacillus plantarum strain, to complete the present invention.
본 발명의 하나의 목적은 쌀 추출물을 락토바실러스 플란타럼(Lactobacillus plantarum)을 이용하여 발효시키는 단계를 포함하는, 라이신 함량이 증가된 쌀 유산균발효물의 제조방법을 제공하는 것이다.One object of the present invention is to provide a method for producing a fermented rice lactic acid bacteria increased lysine, comprising the step of fermenting the rice extract using Lactobacillus plantarum ( Lactobacillus plantarum ).
본 발명의 다른 하나의 목적은 쌀 유산균발효물 제조에 있어, 쌀 추출물을 락토바실러스 플란타럼을 이용하여 발효시키는 단계를 포함하는, 쌀 유산균 발효물 내 라이신 함량 증가 방법을 제공하는 것이다.Another object of the present invention is to provide a method for increasing the content of lysine in rice lactic acid bacteria fermentation, comprising the step of fermenting rice extract using Lactobacillus plantarum in the production of fermented rice lactic acid bacteria.
본 발명의 또 다른 하나의 목적은 상기 제조방법에 의해 제조되는 라이신 함량이 증가된 쌀 유산균발효물을 제공하는 것이다.Yet another object of the present invention is to provide a fermented rice lactic acid bacterium with an increased lysine content prepared by the above method.
본 발명의 또 다른 하나의 목적은 상기 쌀 유산균발효물을 포함하는 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition comprising the rice lactic acid bacteria fermentation.
본 발명에 따른 쌀 유산균발효물은 쌀의 결핍 아미노산인 라이신 함량이 증가되어, 락토바실러스 플란타럼 균주를 이용하지 않고 제조한 유산균발효물에 비하여 품질특성이 우수하므로, 라이신 함량이 증가되고 곡류만을 포함하는 요구르트의 제조 및 이를 포함하는 식품의 개발에 적극 이용될 수 있다.Rice lactobacillus fermentation according to the present invention is increased in the lysine content of the deficient amino acid of rice, it is excellent in quality characteristics compared to the lactobacillus fermentation product prepared without using the Lactobacillus plantarum strain, lysine content is increased and only grains It can be actively used in the production of yogurt containing and the development of food containing the same.
도 1은 본 발명에 따른 제조방법에 의해 유산균 발효물을 제조하는 경우, 라이신 함량의 변화를 보여주는 도이다.1 is a view showing a change in lysine content when producing a lactic acid bacteria fermented by the manufacturing method according to the present invention.
도 2는 본 발명에 따른 제조방법에 의해 유산균 발효물을 제조하는 경우, 아미노산 분석 크로마토그램을 보여주는 도이다.Figure 2 is a view showing an amino acid analysis chromatogram when producing the lactic acid bacteria fermentation by the production method according to the present invention.
도 3은 본 발명에 따른 제조방법에 의해 유산균 발효물을 제조하는 경우, 발효 균주에 따른 라이신 함량 변화를 보여주는 도이다.3 is a view showing a change in lysine content according to the fermentation strain when producing the lactic acid bacteria fermented by the production method according to the present invention.
도 4는 라이신 생합성 경로 및 락토바실러스 플란타럼의 라이신 합성에 관여하는 유전자를 확인한 결과를 보여주는 도이다.Figure 4 is a view showing the results confirming the gene involved in lysine synthesis of lysine biosynthesis pathway and Lactobacillus plantarum.
상기 목적을 달성하기 위하여, 본 발명의 하나의 양태는 쌀 추출물을 락토바실러스 플란타럼(Lactobacillus plantarum)을 이용하여 발효시키는 단계를 포함하는, 라이신 함량이 증가된 쌀 유산균발효물의 제조방법을 제공한다.In order to achieve the above object, one embodiment of the present invention provides a method for producing a fermented rice lactic acid bacteria fermentation with increased lysine content, comprising the step of fermenting the rice extract using Lactobacillus plantarum ( Lactobacillus plantarum ) .
본 발명의 용어, "쌀"은 벼를 도정하여 정제한 백미를 의미한다. 본 발명의 목적상, 상기 쌀은 도정을 통하여 배아와 쌀겨층을 완전히 제거한 것일 수 있다. 상기 쌀의 품종은 특별히 이에 제한되지 않으나, 삼광, 한가루, 하이아미, 청품, 일품, 도담, 고아미2호, 고아미4호, 홍진주, 적진주, 조은흑미, 수원542, 밀양317일 수 있으며, 구체적으로는 삼광, 일품, 고아미4호, 하이아미, 한가루, 수원542, 밀양371, 조은흑미일 수 있고, 보다 구체적으로는 조은흑미일 수 있다.As used herein, the term "rice" refers to white rice which has been refined by milling rice. For the purposes of the present invention, the rice may be completely removed from the embryo and rice bran layer through the milling. The rice varieties are not particularly limited to this, but may be Samgwang, Hana powder, Haiami, Cheonggeum, A La Carte, Dodam, Goami No. 2, Goami No. 4, Hong Jinju, Red Pearl, Choeun Black Rice, Suwon 542, Milyang 317 It may be, specifically, Samgwang, a la carte, Goami No. 4, Haiami, powdered powder, Suwon 542, Milyang 371, Zoeun black rice, and more specifically, Zoeun black rice.
본 발명의 구체적인 일 실시예에서는, 락토바실러스 플란타럼을 발효균주로 사용하여 제조된 쌀 유산균발효물의 라이신 함량을 측정한 결과, 다른 품종의 경우에는 라이신 함량이 1.5배 내지 3배 정도 증가하였으나, 조은흑미 품종의 경우에는 발효 전에 비하여 약 6배 이상 라이신 함량이 증가한 것을 확인하였으며, 이는 양성 대조군에 비하여 약 18배의 증가된 수치임을 확인함으로써, 조은흑미 품종을 사용할 경우에는 라이신 함량 증가가 보다 현저함을 알 수 있었다(도 1 및 도 2).In a specific embodiment of the present invention, as a result of measuring the lysine content of rice lactic acid bacteria fermentation produced using Lactobacillus plantarum as a fermentation strain, the lysine content increased 1.5 to 3 times in other varieties, In the case of black rice varieties, it was confirmed that the lysine content was increased by about 6 times or more compared to before fermentation, which is an increase of about 18 times compared to the positive control group. It could be seen (Fig. 1 and Fig. 2).
본 발명의 용어, "유산균발효물"은 유산균(젖산균)을 이용하여 발효된 물질을 총칭하여 이르는 것으로서, 본 발명에서 "요구르트"와 혼용되어 사용될 수 있다. 또한, 본 발명의 목적상, 상기 요구르트는 유제품을 포함하지 않는 것일 수 있다. 상기 유제품은 가축의 젖을 가공하여 만든 식품을 통틀어 이르는 것으로서, 우유, 버터, 치즈, 연유, 분유 등이 이에 속하며, 본 발명에서는 우유를 포함하는 유제품 없이 요구르트를 제조한 데에 특징이 있다.As used herein, the term "lactic acid bacterium fermentation product" refers to a substance fermented using lactic acid bacteria (lactic acid bacteria), and may be used in combination with "yoghurt" in the present invention. In addition, for the purposes of the present invention, the yogurt may be one containing no dairy. The dairy product as a whole made up of food made by processing the milk of livestock, milk, butter, cheese, condensed milk, milk powder and the like belong to this, in the present invention is characterized in the production of yogurt without dairy products containing milk.
본 발명에서는, 유제품을 사용하지 않고, 쌀을 사용하였으며, 발효 균주로서 락토바실러스 플란타럼을 이용하여 제조한 요구르트가, 다른 종의 유산균을 이용하여 제조한 요구르트에 비하여 라이신 함량이 현저하게 증가됨을 확인하였다. 즉, 본 발명은 락토바실러스 플란타럼에 의해 발효를 수행하는 경우, 다른 종의 유산균에 의한 발효와 달리, 쌀 유산균발효물의 라이신 함량이 결핍되었던 종래의 문제점을 해결한 것이다.In the present invention, the yogurt prepared using Lactobacillus plantarum as a fermentation strain, without using dairy products, the lysine content is significantly increased compared to yogurt prepared using other species of lactic acid bacteria. Confirmed. That is, when the fermentation is performed by Lactobacillus plantarum, the present invention solves the conventional problem of lacking the lysine content of rice lactic acid bacteria fermentation, unlike fermentation by other species of lactic acid bacteria.
본 발명의 목적상, 상기 라이신 함량이 증가된 쌀 유산균발효물의 제조방법은 발효 균주로서 락토바실러스 플란타럼을 사용하는 것을 특징으로 한다. 즉, 종래에 라이신은 쌀의 결핍 아미노산이었으나, 본 발명에서 락토바실러스 플란타럼을 발효균주로 사용함으로써, 라이신 함량을 현저히 증가시킴을 특징으로 한다.For the purpose of the present invention, the method for producing a fermented rice lactic acid bacteria fermented lysine content is characterized in that using Lactobacillus plantarum as a fermentation strain. That is, lysine was a deficient amino acid of rice in the past, but in the present invention, by using Lactobacillus plantarum as a fermentation strain, the lysine content is markedly increased.
또한, 상기 락토바실러스 플란타럼은 0.05 내지 0.15%(v/v), 구체적으로 0.07 내지 0.13%(v/v), 가장 구체적으로 0.1%(v/v)로 혼합물에 접종될 수 있으나, 발효를 수행할 수 있는 한 이에 제한되지 않는다. In addition, the Lactobacillus plantarum may be inoculated into the mixture at 0.05 to 0.15% (v / v), specifically 0.07 to 0.13% (v / v), most specifically 0.1% (v / v), but fermented. It is not limited to this as long as it can be performed.
본 발명에서, 상기 락토바실러스 플란타럼은 본 발명자의 선행특허인 한국 등록특허공보 제10-1634270호에 기재된 락토바실러스 플란타럼 JSA22, 즉 기탁번호 KACC91973P를 갖는 락토바실러스 플란타럼일 수 있다. 상기 락토바실러스 플란타럼 JSA22 균주는 2019년 6월 12일자로 농촌진흥청 국립농업과학원 미생물은행(KACC)에서 국내기탁을 부다페스트 조약 하의 국제기탁으로 전환하여, 국제기탁번호 KACC81097BP를 부여받은 균주이다. 상기 균주는 전통 장류에서 분리 및 선발된 유산균으로서, 항균활성, 인공소화액 생존율 및 부착성이 우수한 것이 특징이다. In the present invention, the Lactobacillus plantarum may be the Lactobacillus plantarum JSA22, which is described in Korean Patent Publication No. 10-1634270, which is the prior patent of the inventors, that is, the Lactobacillus plantarum having the accession number KACC91973P. The Lactobacillus plantarum JSA22 strain was converted to an international deposit under the Budapest Treaty by the National Agricultural Research and Development Institute (KACC) National Agricultural Research and Development Institute (KACC) on June 12, 2019, and has been granted the International Deposit No. KACC81097BP. The strain is a lactic acid bacterium isolated and selected from traditional Jangjang, characterized by excellent antimicrobial activity, digestibility and adhesion.
본 발명의 구체적인 일 실시예에서는, 다양한 쌀 품종에 대하여 락토바실러스 플란타럼을 발효균주로 사용하여 제조된 쌀 유산균발효물의 아미노산 함량을 측정한 결과, 사용된 모든 쌀 품종에서 발효 전과 발효 후의 총 아미노산의 함량에는 변화가 미미하였으나, 라이신 함량에 있어서는 발효 전 대비 발효 후의 라이신 함량이 최소 1.5배 이상으로 현저하게 증가한 것을 확인하였다. 아울러, 이는 양성 대조군에 비해서는 최소 4배 이상 높은 수치로서, 락토바실러스 플란타럼을 발효균주로 사용하여 제조된 요구르트에서 라이신의 함량이 현저하게 증가하였음을 확인할 수 있었다. 상기 결과를 통해, 우유 기반의 발효유에 비하여, 쌀을 포함하는 요구르트는 락토바실러스 플란타럼을 이용하여 제조함으로써 라이신 함량을 현저히 증가시킬 수 있음을 확인하였다(표 1). In a specific embodiment of the present invention, as a result of measuring the amino acid content of rice lactic acid bacteria fermentation produced using Lactobacillus plantarum as a fermentation strain for various rice varieties, the total amino acid before and after fermentation in all of the rice varieties used Although the content was insignificant, it was confirmed that the content of lysine increased significantly at least 1.5 times after fermentation compared to before fermentation. In addition, it was confirmed that the content of lysine was significantly increased in yogurt prepared using Lactobacillus plantarum as a fermentation strain at least four times higher than the positive control. Through the above results, it was confirmed that compared with milk-based fermented milk, yogurt containing rice can significantly increase the lysine content by using Lactobacillus plantarum (Table 1).
특히, 락토바실러스 플란타럼 JSA22 균주를 발효균주로 사용하여 제조한 요구르트가 다른 종의 유산균인 락토바실러스 람노서스 GG 균주에 비하여, 라이신 함량을 현저히 증가시킴을 확인하였다(도 3).In particular, it was confirmed that yoghurt prepared using Lactobacillus plantarum JSA22 strain as a fermentation strain significantly increased the lysine content compared to other strains of Lactobacillus rhamnosus GG strains (Fig. 3).
즉, 이는 락토바실러스 플란타럼을 발효 균주로 사용하여 유산균발효물을 제조하는 본 발명의 제조방법을 통해 라이신 함량의 현저히 증가된 유산균발효물을 제조할 수 있음을 시사하는 것이다.In other words, this suggests that Lactobacillus fermentation products of significantly increased lysine content can be produced through the production method of the present invention using Lactobacillus plantarum as a fermentation strain.
또한, 본 발명에서 제조된 쌀 유산균발효물의 제조시에 유제품을 사용하지 않는 것을 특징으로 한다. 즉, 종래에는 유산균에 의한 발효를 수행하기 위하여 유제품을 사용하였으나, 본 발명에서는 유제품을 사용하지 않고, 유제품 대신에 쌀배아를 사용하는 것을 특징으로 한다. In addition, it is characterized in that the dairy product is not used in the production of the fermented rice lactic acid bacteria produced in the present invention. That is, in the past, dairy products were used to carry out fermentation by lactic acid bacteria, but in the present invention, rice embryos are used instead of dairy products.
본 발명에서 “쌀 추출물”은 쌀로부터 추출한 산물을 의미한다. 구체적으로, 본 발명에서 쌀 추출물이란 쌀 자체를 의미하거나, 쌀을 추출 처리하여 얻어지는 추출액, 상기 추출액의 희석액이나 농축액, 상기 추출액을 건조하여 얻어지는 건조물, 상기 추출액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함한다.In the present invention, "rice extract" means a product extracted from rice. Specifically, in the present invention, the rice extract means rice itself, or an extract obtained by extracting and processing rice, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a crude or purified product of the extract, or a mixture thereof Extracts of all formulations that can be formed using the extract itself and the extract, such as mixtures.
상기 쌀을 추출하는 방법은 특별히 제한되지 않으며, 당해 기술분야에서 통상적으로 사용하는 방법에 따라 추출할 수 있다. 일례로, 본 발명에서 쌀 추출물은 (a) 쌀 및 물을 혼합하여 쌀페이스트를 제조하고, 쌀배아 및 물을 혼합하여 쌀배아유를 제조하는 단계; 및 (b) 상기 쌀페이스트 및 쌀배아유를 혼합하여 혼합물을 제조하는 단계에 의해 제조된 것일 수 있다.The method of extracting the rice is not particularly limited and may be extracted according to methods commonly used in the art. For example, in the present invention, the rice extract comprises the steps of (a) mixing rice and water to prepare a rice paste, and mixing rice germ and water to prepare rice germ oil; And (b) may be prepared by mixing the rice paste and rice germ oil to prepare a mixture.
본 발명의 용어, "쌀배아"는 쌀의 배아를 의미한다. 본 발명의 목적상, 상기 쌀배아는 도정을 통하여 쌀로부터 분리된 것일 수 있다.As used herein, the term “rice germ” means an embryo of rice. For the purposes of the present invention, the rice embryo may be separated from the rice through milling.
상기 단계 (a)는, 쌀 및 물을 혼합하여 쌀페이스트를 얻는 단계를 포함하며, 상기 쌀 및 물의 혼합비는 요구르트의 품질특성 및 관능특성을 최대화시키고, 유산균에 의한 발효를 저해하지 않는 정도가 적당하다. 이때, 상기 "쌀페이스트"는 쌀 및 물의 혼합물을 의미하며, 상기 혼합물이 분쇄된 것을 의미할 수 있다.The step (a) includes a step of obtaining rice paste by mixing rice and water, wherein the mixing ratio of rice and water maximizes the quality and sensory properties of yogurt, and does not inhibit fermentation by lactic acid bacteria. Do. At this time, the "rice paste" refers to a mixture of rice and water, it may mean that the mixture is ground.
구체적으로, 상기 쌀페이스트는 5 내지 35%(w/w)의 쌀 및 65 내지 95%(w/w)의 물을 혼합하여 제조될 수 있고, 더욱 구체적으로 15 내지 35%(w/w)의 쌀 및 65 내지 85%(w/w)의 물을 혼합하여 제조될 수 있고, 가장 구체적으로 20 내지 30%(w/w)의 쌀 및 70 내지 80%(w/w)의 물을 혼합하여 제조될 수 있으나, 이에 제한되지 않는다. 상기 범위 이외의 혼합비로 쌀페이스트를 제조하는 경우, 요구르트의 품질 및 기호도가 저하되는 문제점이 있다.Specifically, the rice paste may be prepared by mixing 5 to 35% (w / w) of rice and 65 to 95% (w / w) of water, more specifically 15 to 35% (w / w) It can be prepared by mixing rice and 65 to 85% (w / w) of water, most specifically 20 to 30% (w / w) of rice and 70 to 80% (w / w) of water It may be prepared by, but is not limited thereto. When preparing the rice paste in a mixing ratio outside the above range, there is a problem that the quality and palatability of the yogurt is lowered.
또한, 상기 단계 (a)는, 쌀배아 및 물을 혼합하여 쌀배아유를 얻는 단계를 포함하며, 상기 쌀배아 및 물의 혼합비는 요구르트의 품질특성 및 관능특성을 최대화시키고, 유산균에 의한 발효를 저해하지 않는 정도가 적당하다. 이때, 상기 "쌀배아유"는 쌀배아 및 물의 혼합물을 의미하며, 상기 혼합물이 분쇄된 것 또는 상기 혼합물이 분쇄되어 여과된 것을 의미할 수 있다.In addition, the step (a), comprising the step of mixing the rice embryo and water to obtain rice germ oil, the mixing ratio of the rice embryo and water to maximize the quality and sensory properties of yogurt, inhibit the fermentation by lactic acid bacteria It is appropriate not to do it. In this case, the "rice germ oil" may refer to a mixture of rice germ and water, and may mean that the mixture is ground or the mixture is ground and filtered.
구체적으로, 상기 쌀배아유는 5 내지 15%(w/w)의 쌀배아 및 85 내지 95%(w/w)의 물을 혼합하여 제조될 수 있고, 더욱 구체적으로 8 내지 12%(w/w)의 쌀배아 및 88 내지 92%(w/w)의 물을 혼합하여 제조될 수 있으나, 이에 제한되지 않는다. 상기 범위 이외의 혼합비로 쌀배아유를 제조하는 경우, 요구르트의 품질 및 기호도가 저하되는 문제점이 있다.Specifically, the rice germ oil may be prepared by mixing 5 to 15% (w / w) rice germ and 85 to 95% (w / w) water, more specifically 8 to 12% (w / w) w) can be prepared by mixing rice germ and 88 to 92% (w / w) of water, but is not limited thereto. When manufacturing rice germ oil in a mixing ratio outside the above range, there is a problem that the quality and palatability of yogurt is lowered.
상기 단계 (b)는, 상기 (a) 단계에서 수득한 쌀페이스트 및 쌀배아유를 혼합하여 혼합물을 얻는 단계로서, 상기 쌀페이스트, 쌀배아유의 혼합비는 요구르트의 품질특성 및 관능특성을 최대화시키고, 유산균에 의한 발효를 저해하지 않는 정도가 적당하다. Step (b) is a step of obtaining a mixture by mixing the rice paste and rice germ oil obtained in the step (a), the mixing ratio of the rice paste, rice germ oil maximizes the quality characteristics and sensory characteristics of yogurt, It is appropriate to not inhibit the fermentation by lactic acid bacteria.
구체적으로, 상기 혼합물은 상기 쌀페이스트, 쌀배아유를 동량의 부피비로 혼합하여 제조될 수 있다. 상기 범위 이외의 혼합비로 혼합물을 제조하는 경우, 요구르트의 품질 및 기호도가 저하되는 문제점이 있다.Specifically, the mixture may be prepared by mixing the rice paste, rice germ oil in the same volume ratio. When the mixture is prepared at a mixing ratio other than the above range, there is a problem in that the quality and palatability of the yogurt are reduced.
본 발명의 목적상, 발효 효율을 증진시키기 위하여, 상기 혼합물에 2 내지 6%(w/v)의 설탕을 포함시켜 발효할 수 있고, 구체적으로 3%(w/v)의 설탕을 포함시켜 발효할 수 있으나, 이에 제한되지 않는다. 상기 범위 이외의 혼합비로 설탕을 첨가하는 경우, 요구르트의 품질 및 기호도가 저하되는 문제점이 있다.For the purposes of the present invention, in order to enhance the fermentation efficiency, the mixture may be fermented by including 2 to 6% (w / v) of sugar, specifically 3% (w / v) of sugar to ferment It may be, but is not limited thereto. When sugar is added at a mixing ratio other than the above range, there is a problem that the quality and palatability of the yogurt are lowered.
본 발명의 제조방법은, 상기 단계 (b) 이후에 상기 혼합물을 멸균하고 균질화하는 단계를 추가로 포함할 수 있다.The preparation method of the present invention may further comprise the step of sterilizing and homogenizing the mixture after step (b).
구체적으로, 상기 단계는 상기 (b) 단계에서 수득한 쌀페이스트 및 쌀배아유의 혼합물을 멸균하는 단계로서, 다른 균에 의해 목적하는 발효가 진행되지 않는 것을 막기 위한 것이다.Specifically, the step is to sterilize the mixture of rice paste and rice germ oil obtained in the step (b), to prevent the desired fermentation is not progressed by other bacteria.
더욱 구체적으로, 상기 멸균은 90 내지 110℃, 더욱 구체적으로 95 내지 105℃, 가장 구체적으로 100℃에서 수행될 수 있고, 또한 50 내지 70분, 구체적으로 55 내지 65분, 가장 구체적으로 60분 동안 수행될 수 있으나, 멸균을 수행할 수 있는 한 이에 제한되지 않는다. 상기 멸균은 당업계에 알려진 방법으로 수행할 수 있으며, 구체적으로 건열살균, 고압살균 등의 방법으로 수행할 수 있으나, 이에 제한되지 않는다.More specifically, the sterilization can be carried out at 90 to 110 ° C, more specifically at 95 to 105 ° C, most specifically at 100 ° C, and also for 50 to 70 minutes, specifically 55 to 65 minutes, most specifically 60 minutes It may be carried out, but is not limited to so long as sterilization can be performed. The sterilization may be performed by a method known in the art, and specifically, may be performed by a method such as dry heat sterilization and autoclaving, but is not limited thereto.
또한, 상기 단계는 상기 (b) 단계에서 수득한 쌀페이스트 및 쌀배아유의 혼합물을 균질화하는 단계로서, 발효의 효율을 증진시키기 위한 것이다. 균질화를 통해 입자의 표면적이 증가되고 점도가 균일하게 되며 질감이 부드럽게 되고 조직이 안정하게 된다.In addition, the step is to homogenize the mixture of the rice paste and rice germ oil obtained in the step (b), to enhance the efficiency of the fermentation. Homogenization results in an increase in the surface area of the particles, a uniform viscosity, a smooth texture and a stable tissue.
더욱 구체적으로, 상기 균질화는 20 내지 40℃, 더욱 구체적으로 25 내지 35℃, 가장 구체적으로 30℃에서 수행될 수 있고, 또한 35 내지 45시간, 구체적으로 37 내지 43시간, 가장 구체적으로 40시간 동안 수행될 수 있으나, 이에 제한되지 않는다. 상기 균질화는 당업계에 알려진 방법으로 수행할 수 있으며, 구체적으로 고속균질화, 고압균질기, 콜로이드밀, 초음파기 등을 이용하여 수행할 수 있으나, 이에 제한되지 않는다.More specifically, the homogenization can be carried out at 20 to 40 ° C., more specifically at 25 to 35 ° C., most specifically at 30 ° C., and also for 35 to 45 hours, specifically 37 to 43 hours, most specifically 40 hours. It may be performed, but is not limited thereto. The homogenization may be performed by a method known in the art, and specifically, may be performed using a high speed homogenization, a high pressure homogenizer, a colloid mill, an ultrasonic wave, or the like, but is not limited thereto.
본 발명의 다른 하나의 양태는 쌀 유산균발효물 제조에 있어, 쌀 추출물을 락토바실러스 플란타럼을 이용하여 발효시키는 단계를 포함하는, 쌀 유산균 발효물 내 라이신 함량 증가 방법을 제공한다. Another aspect of the present invention provides a method for increasing lysine content in rice lactic acid bacteria fermentation, comprising the step of fermenting rice extract using Lactobacillus plantarum in the production of rice lactic acid bacteria fermentation.
이때, 상기 용어 “쌀”, “유산균발효물(요구르트)”, “락토바실러스 플란타럼”, “쌀 추출물” 등에 대한 설명은 전술한 바와 같다.At this time, the terms "rice", "lactic acid bacteria fermented product (yogurt)", "Lactobacillus plantarum", "rice extract" and the like are described above.
본 발명의 또 다른 하나의 양태는 상기 제조방법에 의해 제조되는, 라이신 함량이 증가된 쌀 유산균발효물을 제공한다.Another aspect of the present invention provides a rice lactic acid bacteria fermentation with an increased lysine content prepared by the above production method.
이때, 상기 용어 "쌀", "유산균발효물(요구르트)" 등에 대한 설명은 전술한 바와 같다.At this time, the terms "rice", "lactic acid bacteria fermentation (yogurt)" and the like are described above.
본 발명의 라이신 함량이 증가된 유산균발효물은, 락토바실러스 플란타럼 균주를 발효 균주로 사용하는 것을 특징으로 하며, 유산균의 다른 종을 사용하여 제조한 유산균발효물에 비하여 라이신 함량이 현저히 증가된다.Lactobacillus fermentation product with increased lysine content of the present invention is characterized by using the Lactobacillus plantarum strain as a fermentation strain, the lysine content is significantly increased compared to the lactic acid bacteria fermentation products prepared using other species of lactic acid bacteria .
구체적으로, 상기 유산균발효물은 본 발명의 일 실시예에 사용된 락토바실러스 플란타럼 JSA22를 발효균주로 사용하는 요구르트의 제조방법으로 제조된 것일 수 있으나, 락토바실러스 플란타럼 균주가 라이신 함량을 증가시키는 효과를 갖는 한 이에 제한되지 않는다.Specifically, the lactic acid bacteria fermentation may be prepared by a method for preparing yogurt using Lactobacillus plantarum JSA22 used in one embodiment of the present invention as a fermentation strain, Lactobacillus plantarum strain increases the lysine content It is not limited to this as long as it has an effect to make it.
본 발명의 또 다른 하나의 양태는 상기 유산균발효물을 포함하는 식품 조성물을 제공한다.Another aspect of the invention provides a food composition comprising the lactic acid bacteria fermentation.
이때, 상기 용어 "유산균발효물(요구르트)"에 대한 설명은 전술한 바와 같다.In this case, the term "lactic acid bacteria fermentation product (yogurt)" is as described above.
본 발명의 용어, "식품"은 스낵류, 과자류, 빵, 피자, 라면, 기타 면류, 밥류, 즉석밥류, 아이스크림류를 포함한 낙농제품, 건강식품 등 통상적인 의미에서의 식품을 모두 포함하며, 상기 유산균발효물이 포함될 수 있는 한, 이에 제한되지 않는다.As used herein, the term "food" includes all foods in a common sense such as snacks, confectionery, bread, pizza, ramen, other noodles, rice, instant rice, ice cream, dairy products, and health foods. As long as fermentation may be included, it is not limited to this.
이하 실시예를 통하여 본 발명을 더욱 상세하게 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것으로 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only intended to illustrate the invention and are not to be construed as limiting the scope of the invention by these examples.
실시예 1: 쌀 유산균발효물의 제조Example 1: Preparation of Rice Lactic Acid Bacteria Fermentation
다음과 같은 방법으로, 쌀 및 쌀배아를 포함하는 요구르트를 제조하였다.In the following manner, yogurt containing rice and rice germ were prepared.
먼저, 정선한 쌀을 시험용 분쇄기로 분쇄한 후, 50 메쉬(mesh) 체를 사용하여 통과한 쌀가루를 사용하였다. 상기 쌀가루와 물을 1:9 내지 3:7의 중량비로 혼합하고, 이를 분쇄하여 쌀페이스트를 제조하였다.First, the selected rice was pulverized with a test mill, and then rice flour passed through a 50 mesh sieve was used. The rice flour and water were mixed at a weight ratio of 1: 9 to 3: 7, and ground to prepare rice paste.
또한, 쌀배아와 물을 1:10의 중량비로 혼합하고, 이를 분쇄한 후 cheese cloth로 조여과하여 쌀배아유를 제조하였다.In addition, rice germ and water were mixed at a weight ratio of 1:10, and then pulverized and coagulated with a cheese cloth to prepare rice germ oil.
이후, 상기 쌀페이스트 및 쌀배아유를 동량의 부피비로 혼합하여 쌀페이스트 및 쌀배아유의 혼합물을 제조하였고, 상기 혼합물에 3%(w/v)의 설탕을 가한 후, 멸균(호화, autoclaving, 100℃, 60분)하였다. 이를 상온에서 방냉하고, 균질화 과정을 거친 후, 유산균을 일정농도(0.1%(v/v), OD(600nm))로 접종하였고, 30℃에서 40시간 동안 발효시켜 쌀 및 쌀배아가 포함된 쌀요구르트를 제조하였다. Thereafter, the mixture of rice paste and rice germ oil was mixed at the same volume ratio to prepare a mixture of rice paste and rice germ oil, and 3% (w / v) of sugar was added to the mixture, followed by sterilization (luxury, autoclaving, 100). C, 60 minutes). After cooling to room temperature and homogenizing, the lactic acid bacteria were inoculated at a constant concentration (0.1% (v / v), OD (600 nm)), fermented at 30 ° C. for 40 hours, and rice containing rice germ Yogurt was prepared.
상기 유산균는 락토바실러스 플란타럼 JSA22이며, 대조 균주는 락토바실러스 람노서스 GG를 사용하였다. 유산균주는 MRS broth에 2회 계대배양하여 사용하였다.The lactic acid bacteria was Lactobacillus plantarum JSA22, and the control strain was Lactobacillus rhamnosus GG. Lactobacillus strains were used in two subcultures on MRS broth.
한편, 본 발명의 쌀배아는 발효보조제, 즉 유산균의 단백질원으로써, 요구르트의 발효 효율을 증가시키기 위한 목적으로 사용한바, 당업계에서 발효보조제로 사용되는 우유를 포함하는 요구르트(한국야구르트, 메치니코프)를 양성 대조군으로 사용하였다.On the other hand, the rice embryo of the present invention is a fermentation aid, that is, a protein source of lactic acid bacteria, used for the purpose of increasing the fermentation efficiency of yogurt, yogurt containing milk used as a fermentation aid in the art (Korea Yagurt, Mechinnikov) Was used as a positive control.
실시예Example 2-1: 다양한 쌀 품종을 이용하여 제조한 쌀  2-1: Rice Manufactured Using Various Rice Varieties 유산균발효물의Lactic acid bacteria fermentation 아미노산 함량의 측정 Determination of Amino Acid Content
상기 실시예 1에서 제조한, 락토바실러스 플란타럼 균주를 이용하여 제조된 요구르트의 라이신 함량을 확인하기 위하여, 유리아미노산의 함량을 측정하였다.In order to confirm the lysine content of the yogurt prepared using the Lactobacillus plantarum strain prepared in Example 1, the content of free amino acid was measured.
이때, 쌀 품종으로는 삼광, 일품, 고아미4호, 하이아미, 한가루, 수원542, 밀양317, 조은흑미를 이용하였다.At this time, the rice varieties Samgwang, a gem, Goami No. 4, Haiami, powdered, Suwon 542, Milyang 317, was used as black rice.
유리아미노산은 동결건조된 시료 0.1 g에 80 % 에탄올 800 uL을 가하여 음파처리(sonication)로 추출, 80 % 에탄올 500 uL을 추가 2회 반복하여 추출하였다. 추출액은 원심분리(13,500 rpm, 10분)하여 상등액을 취하고 3회 추출한 상층액을 모두 합하여 CentriVap DNA Concentrator(LABCONCO CORPORATION, KANSAS CITY, USA)로 감압농축하였다. 추출 용매를 완전히 제거한 튜브에 3% TCA 1mL로 용해하여 1시간 음파처리, 1시간 200rpm으로 진탕(shaking)시켰다. 이 추출액을 원심분리(13,500 rpm, 20분)하여 상등액을 0.45 ㎛의 실린지 필터(syringe filter)로 여과하였다. Amino Acid Analyzer (L-8900, HITACHI, Japan)에 주입하여, 이온 교환 컬럼(#2622PF Column, 4D3846), 컬럼 온도는 38 내지 70 ℃, reactor heater 온도는 135 ℃를 유지하며 분석하였고, 주입 부피는 20.0 uL, 유속 0.35 mL/분, 이동상은 PF1, PF2, PF3, PF4, PF-RG, R3, 닌히드린 용액, 닌히드린 완충액(Wako, Japan)을 사용하였으며, Detector UV 570 nm, 440 nm에서 검출하였다. 유리아미노산 표준물질은 Type AN-II와 Type B(Wako, Japan)를 1:1(v/v)로 혼합하여, 20배 희석한 후 분석에 사용하였다. Free amino acid was extracted by sonication by adding 800 uL of 80% ethanol to 0.1 g of the lyophilized sample, and 500 uL of 80% ethanol was extracted two more times. The extract was centrifuged (13,500 rpm, 10 minutes), the supernatant was taken, and the supernatant extracted three times was combined and concentrated under reduced pressure with CentriVap DNA Concentrator (LABCONCO CORPORATION, KANSAS CITY, USA). The extracted solvent was dissolved in 1 mL of 3% TCA in a tube completely removed, and shaken at 200 rpm for 1 hour sonication. The extract was centrifuged (13,500 rpm, 20 minutes) and the supernatant was filtered through a 0.45 μm syringe filter. Injected into an Amino Acid Analyzer (L-8900, HITACHI, Japan), the ion exchange column (# 2622PF Column, 4D3846), column temperature of 38 to 70 ℃, reactor heater temperature was maintained at 135 ℃, the injection volume was analyzed 20.0 uL, flow rate 0.35 mL / min, mobile phase used PF1, PF2, PF3, PF4, PF-RG, R3, ninhydrin solution, ninhydrin buffer (Wako, Japan) and detected at Detector UV 570 nm, 440 nm It was. The free amino acid standard was mixed with Type AN-II and Type B (Wako, Japan) at 1: 1 (v / v), diluted 20-fold, and used for analysis.
발효 전 유리아미노산의 함량(0 hour) 및 40시간 발효 후 유리아미노산의 함량(40 hour)을 각각 측정하였고, 양성 대조군인 우유기반 발효유(한국야구르트, 메치니코프)의 유리아미노산의 함량을 측정하였다.The content of free amino acid before fermentation (0 hour) and the content of free amino acid (40 hour) after 40 hours of fermentation were measured, respectively, and the content of free amino acid in milk-based fermented milk (Korean yogurt, Mechinnikov), which was a positive control, was measured.
Figure PCTKR2019007290-appb-T000001
Figure PCTKR2019007290-appb-T000001
그 결과, 상기 표 1에서 볼 수 있는 바와 같이, 사용된 모든 쌀 품종에서 발효 전과 발효 후의 총 아미노산의 함량에는 변화가 미미하였으나, 라이신 함량에 있어서는 발효 전 대비 발효 후의 라이신 함량이 최소 1.5배 이상으로 현저하게 증가한 것을 확인하였다. 아울러, 이는 양성 대조군에 비해서는 최소 4배 이상 높은 수치로서, 상기 방법에 의해 제조된 요구르트에서 라이신의 함량이 현저하게 증가하였음을 확인할 수 있었다. 상기 결과를 통해, 우유 기반의 발효유에 비하여, 쌀을 포함하는 요구르트는 락토바실러스 플란타럼을 이용하여 제조함으로써 라이신 함량이 현저히 증가시킬 수 있음을 확인하였다.As a result, as shown in Table 1 , the total amino acid content before and after fermentation was insignificant in all of the rice varieties used, but the lysine content after fermentation was at least 1.5 times higher than before fermentation. It was confirmed that the increase significantly. In addition, this is at least four times higher than the positive control, it was confirmed that the content of lysine significantly increased in the yogurt prepared by the above method. Through the above results, it was confirmed that compared to milk-based fermented milk, yogurt containing rice can be significantly increased lysine content by manufacturing using Lactobacillus plantarum.
특히, 도 1 및 도 2에서 볼 수 있는 바와 같이, 다른 품종의 경우에는 라이신 함량이 1.5배 내지 3배 정도 증가하였으나, 조은흑미 품종의 경우에는 발효 전에 비하여 약 6배 이상 라이신 함량이 증가한 것을 확인하였으며, 이는 양성 대조군에 비하여 약 18배의 증가된 수치임을 확인함으로써, 조은흑미 품종을 사용할 경우에는 라이신 함량 증가가 보다 현저함을 알 수 있었다.In particular, as can be seen in Figures 1 and 2, in the case of other varieties, the lysine content increased 1.5 to 3 times, but in the case of the crude black rice varieties, the lysine content was increased by about 6 times or more compared to before fermentation. By confirming that this is an increase of about 18 times compared to the positive control, it can be seen that the increase in lysine content is more significant when using the crude black rice varieties.
실시예Example 2-2:  2-2: 락토바실러스Lactobacillus 람노서스Rhamnosus 균주를 이용한 쌀  Rice using strain 유산균발효물과의With lactic acid bacteria fermentation 비교 compare
상기 실시예 2-1에서 본 발명의 락토바실러스 플란타럼 균주를 이용한 쌀 유산균발효물이 우수한 라이신 증가 효과가 있음을 확인하였는바, 락토바실러스 람노서스 균주를 이용한 쌀 유산균발효물과의 비교를 수행하였다.In Example 2-1, it was confirmed that the rice lactobacillus fermentation product using the Lactobacillus plantarum strain of the present invention has an excellent lysine increasing effect, and compared with the rice lactic acid bacterium fermentation product using the Lactobacillus rhamnosus strain. It was.
구체적으로는, 쌀 품종으로는 삼광 및 조은흑미를 사용하였으며, 발효 균주로서 락토바실러스 플란타럼 JSA22 균주 및 락토바실러스 람노서스 GG 균주를 이용하였다. 발효 전 라이신 및 아스파르트산의 함량(0 hour) 및 40시간 발효 후 라이신 및 아스파르트산의 함량(40 hour)을 각각 측정하였다.Specifically, Samkwang and coarse black rice were used as rice varieties, and Lactobacillus plantarum JSA22 strain and Lactobacillus rhamnosus GG strain were used as fermentation strains. The content of lysine and aspartic acid before fermentation (0 hour) and the content of lysine and aspartic acid (40 hour) after 40 hours of fermentation were measured, respectively.
그 결과, 도 3에서 확인할 수 있는 바와 같이, 락토바실러스 플란타럼 JSA22로 제조된 쌀발효물의 경우에는 발효 전(조은흑미: 5.26mg%, 삼광: 4.27mg%)에 비하여 발효 후(조은흑미: 49.52mg%, 삼광: 39.08mg%)에 약 10배 정도 라이신 함량이 증가하였고, 반면에 락토바실러스 람노서스 GG(LGG)로 제조된 쌀발효물의 경우에는 발효 전(조은흑미: 5.53 mg%, 삼광:3.96mg%)에 비하여 발효 후(조은흑미: 4.81mg%, 삼광 3.59mg%)에 라이신 함량의 변화가 미미하였다. As a result, as can be seen in Figure 3, in the case of the rice fermented product prepared with Lactobacillus plantarum JSA22, after fermentation (roughly black rice: 5.26mg%, Samkwang: 4.27mg%) compared to before fermentation (Choeun black rice: The amount of lysine increased about 49.52mg% and 39.08mg%), whereas rice fermented with Lactobacillus rhamnosus GG (LGG) was added before fermentation (crude black rice: 5.53 mg%, Samkwang). The change in lysine content was insignificant after fermentation (Choeun black rice: 4.81mg%, Samgwang 3.59mg%) compared to: 3.96mg%).
상기 결과를 통해, 다른 종의 유산균으로 발효하여 쌀 유산균발효물을 제조한 경우에 비하여, 락토바실러스 플란타럼으로 발효하여 제조한 경우에 라이신 함량을 현저히 증가시키며, 특히 조은흑미를 쌀 품종으로 선택하는 경우 라이신 함량의 증가 효율이 우수함을 확인하였다.Through the above results, the fermented with other species of lactic acid bacteria, compared to the case of producing a lactic acid bacteria fermented product, significantly increased the lysine content when prepared by fermentation with Lactobacillus plantarum, especially Choeun black rice as a rice variety When it was confirmed that the increase efficiency of the lysine content is excellent.
실시예 3: 락토바실러스 플란타럼의 라이신 합성유전자 확인Example 3 Identification of Lysine Synthesis of Lactobacillus Plantarum
라이신 합성은 아스파르트산을 전구체로 하여 합성이 되는데, 합성에 관여하는 유전자를 확인하기 위해, 락토바실러스 플란타럼 균주의 DNA 추출 및 PCR을 수행하였다.Lysine synthesis is synthesized using aspartic acid as a precursor, in order to identify the gene involved in the synthesis, DNA extraction and PCR of the Lactobacillus plantarum strain was performed.
락토바실러스 플란타럼 JSA22 균주의 게놈 DNA 추출은 Qiagenprep spin miniprep kit (Qiagen, Valencia, CA, USA)을 이용하였다. 13,500 rpm에서 10분간 원심분리하였다. 원심분리 후 상층액을 조심스럽게 제거하고, 180 uL의 20 mg/mL 라이소자임을 첨가하여 현탁한 후, 37 ℃ 항온수조에서 30분 동안 반응시켰다. 20 uL 프로테인나아제 K 및 200 uL AL 완충액을 첨가하여 56 ℃에서 30분 동안 반응시켰다. 반응 후 200 uL 에탄올을 첨가하여 혼탁하였다. 반응액을 미니 스핀 컬럼(mini spin column)에 옮기고 원심분리하였다. 스핀 컬럼에 AW 완충액을 첨가하였고 컬럼을 통과한 시료는 모두 폐기하였다. 50 uL AE 완충액을 첨가하여 8,000 rpm에서 1분간 원심분리하여 DNA를 추출하였다. DNA 농도는 분광광도계를 이용하여 측정하였다. Genomic DNA extraction of Lactobacillus plantarum JSA22 strain was performed using a Qiagenprep spin miniprep kit (Qiagen, Valencia, CA, USA). It was centrifuged for 10 minutes at 13,500 rpm. After centrifugation, the supernatant was carefully removed, suspended by addition of 180 uL of 20 mg / mL lysozyme, and reacted in a 37 ° C. constant temperature water bath for 30 minutes. 20 uL proteinase K and 200 uL AL buffer were added to react at 56 ° C. for 30 minutes. After the reaction, 200 uL ethanol was added and turbid. The reaction solution was transferred to a mini spin column and centrifuged. AW buffer was added to the spin column and all samples passed through the column were discarded. DNA was extracted by adding 50 uL AE buffer and centrifuging for 1 minute at 8,000 rpm. DNA concentration was measured using a spectrophotometer.
PCR은 thermal cycler (T100 Thermal Cycler, Bio-Rad, CA, USA)을 사용하여 amfiEco PCR premix kit (GenDEPOT, CA, USA), 2 μl의 정방향 및 역방향 프라이머, DNA 시료 총 25 μl로 구성된 PCR 반응액으로 실시되었다. PCR 반응 조건은 94℃에서 2분간 변성 후, 94℃에서 2분 변성(denaturation), 프라이머의 Tm값에 따라 46∼60℃에서 2분 어닐링(annealing), 72℃에서 1분/kb 신장(elongation) 반응을 30회 주기로 수행한 후, 72℃ 10분간 최종 신장 반응을 수행하였다. 증폭된 산물은 0.8% 아가로스 겔에서 확인하였다.PCR was performed using a thermal cycler (T100 Thermal Cycler, Bio-Rad, Calif., USA), PCR reaction solution consisting of amfiEco PCR premix kit (GenDEPOT, Calif., USA), 2 μl forward and reverse primers, 25 μl total DNA sample. Was carried out. PCR reaction conditions were denatured at 94 ° C for 2 minutes, then denatured at 94 ° C for 2 minutes, annealing at 46-60 ° C for 2 minutes depending on the Tm value of the primer, and 1 minute / kb elongation at 72 ° C. The reaction was carried out in 30 cycles, followed by a final elongation reaction at 72 ° C. for 10 minutes. The amplified product was identified on a 0.8% agarose gel.
<라이신 합성 유전자 검출 프라이머><Lysine synthetic gene detection primer>
번호number 유전자gene 프라이머primer 서열번호SEQ ID NO:
1-11-1 ThrA2-5ThrA2-5 (5-ATTGATGACTCTGAACCGTC-3) (5-ATTGATGACTCTGAACCGTC-3) 1One
ThrA2-6ThrA2-6 (5-TGCTGACGAACGAGTATCCG-3) (5-TGCTGACGAACGAGTATCCG-3) 22
1-21-2 ThrA1-1ThrA1-1 (5-TTCAATAAGGACTACTTCTTG-3) (5-TTCAATAAGGACTACTTCTTG-3) 33
ThrA1-2 ThrA1-2 (5-ATCTAGGTCAACAAAAAAGC-3) (5-ATCTAGGTCAACAAAAAAGC-3) 44
1-31-3 ThrA2-1ThrA2-1 (5- AATTAAATTCTCGTGAGTGG-3) (5-AATTAAATTCTCGTGAGTGG-3) 55
ThrA2-2ThrA2-2 (5-AACACGGCCATAAAAAATG-3) (5-AACACGGCCATAAAAAATG-3) 66
2-12-1 Asd2-11Asd2-11 (5-TTGAGTGGCGTATTCAACGG-3)(5-TTGAGTGGCGTATTCAACGG-3) 77
Asd2-12Asd2-12 (5-AGGACAAGGATAAAGGACAC-3)(5-AGGACAAGGATAAAGGACAC-3) 88
2-22-2 Asd1-1Asd1-1 (5-CATAGGAAGAAAAAAGTGCG-3)(5-CATAGGAAGAAAAAAGTGCG-3) 99
Asd1-2Asd1-2 (5-AGCCTCCTCAATAATTCTCC-3)(5-AGCCTCCTCAATAATTCTCC-3) 1010
2-32-3 Asd2-1Asd2-1 (5-ATCTGCGTGTGCAAAACAG-3)(5-ATCTGCGTGTGCAAAACAG-3) 1111
Asd2-2Asd2-2 (5-GGATCAGCTTTTCATTAC-3)(5-GGATCAGCTTTTCATTAC-3) 1212
2-42-4 asd-F asd-F 5-GCGCACATGTGGGTCAAGAAAATGGTCTTC-35-GCGCACATGTGGGTCAAGAAAATGGTCTTC-3 1313
asd-R asd-R 5-ATGGTACCTGCCCCCGAATTTTTGAAC-35-ATGGTACCTGCCCCCGAATTTTTGAAC-3 1414
3-13-1 Hom1-1 Hom1-1 (5-TTAGCAAACGTCGCTGATG-3)(5-TTAGCAAACGTCGCTGATG-3) 1515
Hom1-2 Hom1-2 (5-TGATATCTACGTTCTCGTG-3)(5-TGATATCTACGTTCTCGTG-3) 1616
3-23-2 Hom2-1 Hom2-1 (5-GGGCCAATTAAATTAGTCTG-3)(5-GGGCCAATTAAATTAGTCTG-3) 1717
Hom2-2Hom2-2 (5-ACACTTGTAGCGTTAGTTGC-3) (5-ACACTTGTAGCGTTAGTTGC-3) 1818
4-14-1 DapA1-3 DapA1-3 (5-GGTTAGCCGTGATTGTTG-3)(5-GGTTAGCCGTGATTGTTG-3) 1919
DapA1-4 DapA1-4 (5-AACTGAAGTCCGGAATCAC-3)(5-AACTGAAGTCCGGAATCAC-3) 2020
4-1’4-1 ’ DapA1-1DapA1-1 (5-GGTTAGCCGTGATTGTTG-3)(5-GGTTAGCCGTGATTGTTG-3) 2121
DapA1-2DapA1-2 (5-AACTGAAGTCCGGAATCAC-3)(5-AACTGAAGTCCGGAATCAC-3) 2222
4-24-2 DapA2-1DapA2-1 (5-ATGGCATCACACAACTGTC-3)(5-ATGGCATCACACAACTGTC-3) 2323
DapA2-2DapA2-2 (5-CTGATCCCTGATGATGAAAC-3)(5-CTGATCCCTGATGATGAAAC-3) 2424
4-34-3 dapA-F dapA-F 5-GCGCACATGTGGTTTCATTTGGTCGAATATC-35-GCGCACATGTGGTTTCATTTGGTCGAATATC-3 2525
dapA-R dapA-R 5-ATGGTACCGGCAGTAAAAACTCCATTGAT-35-ATGGTACCGGCAGTAAAAACTCCATTGAT-3 2626
5-15-1 DapB-1 DapB-1 (5-AATAGTGTTCGACCGAGCG-3) (5-AATAGTGTTCGACCGAGCG-3) 2727
DapB-2 DapB-2 (5-AATATCGACGTCATGTAGGGG-3)(5-AATATCGACGTCATGTAGGGG-3) 2828
10-110-1 lysA-F lysA-F 5-GCGCACATGTGTATTTTCATGGCACAACA-35-GCGCACATGTGTATTTTCATGGCACAACA-3 2929
lysA-R lysA-R 5-ATGGTACCGCAGCTTAGTATCTTACTCT-35-ATGGTACCGCAGCTTAGTATCTTACTCT-3 3030
그 결과, 도 4에서 볼 수 있는 바와 같이, 락토바실러스 플란타럼 균주의 DNA에는 번호 1(ThrA), 2(Asd), 3(Hom), 4(DapA), 5(DapB)에 해당하는 유전자, 즉 라이신 합성에 관여하는 유전자를 가짐을 확인하였다.As a result, as shown in Figure 4 , the DNA corresponding to the number 1 (ThrA), 2 (Asd), 3 (Hom), 4 (DapA), 5 (DapB) in the DNA of the Lactobacillus plantarum strain That is, it was confirmed that it has a gene involved in lysine synthesis.
이상의 내용을 종합하면, 쌀을 사용하여 제조한 요구르트는 우유 기반의 발효유에 비하여 라이신 함량이 높은데, 그에 더하여 락토바실러스 플란타럼을 발효 균주로 이용할 경우 라이신 함량을 현저히 증가시키므로, 락토바실러스 플란타럼 균주의 발효를 통해 라이신 함량이 증가된 쌀 유산균발효물을 제조할 수 있음을 확인하였다.In summary, yogurt prepared using rice has a higher lysine content than milk-based fermented milk, and in addition, Lactobacillus plantarum significantly increases lysine content when Lactobacillus plantarum is used as a fermentation strain. It was confirmed that the fermentation of the strain can produce a fermented rice lactic acid bacteria increased lysine content.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.
Figure PCTKR2019007290-appb-I000001
Figure PCTKR2019007290-appb-I000001

Claims (14)

  1. 쌀 추출물을, 락토바실러스 플란타럼(Lactobacillus plantarum)을 이용하여 발효시키는 단계를 포함하는, 라이신 함량이 증가된 쌀 유산균발효물의 제조방법.A method for producing a rice lactic acid bacteria fermentation with increased lysine, comprising the step of fermenting the rice extract, Lactobacillus plantarum ( Lactobacillus plantarum ).
  2. 제1항에 있어서, 상기 쌀 추출물은,The method of claim 1, wherein the rice extract,
    (a) 쌀 및 물을 혼합하여 쌀페이스트를 제조하고, 쌀배아 및 물을 혼합하여 쌀배아유를 제조하는 단계; 및(a) mixing rice and water to prepare a rice paste, and mixing rice germ and water to prepare rice germ oil; And
    (b) 상기 쌀페이스트 및 쌀배아유를 혼합하여 혼합물을 제조하는 단계에 의해 제조된 것인, 라이신 함량이 증가된 쌀 유산균발효물의 제조방법.(b) prepared by mixing the rice paste and rice germ oil to prepare a mixture, lysine content of the increased production of lactic acid bacteria fermentation.
  3. 제2항에 있어서, 상기 단계 (a)의 쌀페이스트는 5 내지 35%(w/w)의 쌀 및 65 내지 95%(w/w)의 물을 혼합하여 제조된 것인, 라이신 함량이 증가된 쌀 유산균발효물의 제조방법.The lysine content of claim 2, wherein the rice paste of step (a) is prepared by mixing 5 to 35% (w / w) of rice and 65 to 95% (w / w) of water. Method of fermented rice lactic acid bacteria fermentation.
  4. 제2항에 있어서, 상기 단계 (a)의 쌀배아유는 5 내지 15%(w/w)의 쌀배아 및 85 내지 95%(w/w)의 물을 혼합하여 제조된 것인, 라이신 함량이 증가된 쌀 유산균발효물의 제조방법.The lysine content of claim 2, wherein the rice germ oil of step (a) is prepared by mixing 5 to 15% (w / w) rice germ and 85 to 95% (w / w) water. Method for producing this increased rice lactic acid bacteria fermentation.
  5. 제2항에 있어서, 상기 단계 (b)는 상기 쌀페이스트 및 쌀배아유를 동량의 부피비로 혼합한 것인, 라이신 함량이 증가된 쌀 유산균발효물의 제조방법.The method of claim 2, wherein step (b) is a mixture of the rice paste and rice germ oil in the same volume ratio, lysine content of the increased production of lactic acid bacteria fermentation.
  6. 제2항에 있어서, 상기 단계 (b)의 혼합물은 2 내지 6(w/v)의 설탕을 포함하는 것인, 라이신 함량이 증가된 쌀 유산균발효물의 제조방법The method according to claim 2, wherein the mixture of step (b) comprises 2 to 6 (w / v) sugar, wherein the lysine content is increased.
  7. 제2항에 있어서, 상기 제조방법은 상기 (b) 단계 이후에 상기 혼합물을 멸균하고 균질화하는 단계를 추가로 포함하는 것인, 라이신 함량이 증가된 쌀 유산균발효물의 제조방법.The method of claim 2, wherein the method further comprises the step of sterilizing and homogenizing the mixture after the step (b).
  8. 제2항에 있어서, 상기 락토바실러스 플란타럼은 0.05 내지 0.15%(v/v)로 접종되는 것인, 라이신 함량이 증가된 쌀 유산균발효물의 제조방법.The method of claim 2, wherein the Lactobacillus plantarum is inoculated at 0.05 to 0.15% (v / v).
  9. 제2항에 있어서, 상기 단계에 사용된 쌀은 조은흑미인 것인, 라이신 함량이 증가된 쌀 유산균발효물의 제조방법. The method of claim 2, wherein the rice used in the step is coarse black rice, lysine content increased rice lactic acid bacteria production method.
  10. 제1항에 있어서, 상기 제조방법은 유제품을 사용하지 않는 것을 특징으로 하는 것인, 라이신 함량이 증가된 쌀 유산균발효물의 제조방법.The method of claim 1, wherein the production method is characterized in that it does not use dairy products.
  11. 제1항에 있어서, 상기 락토바실러스 플란타럼은 기탁번호 KACC91973P 및 KACC81097BP로 기탁된 락토바실러스 플란타럼 JSA22인 것인, 라이신 함량이 증가된 쌀 유산균발효물의 제조방법.The method according to claim 1, wherein the Lactobacillus plantarum is Lactobacillus plantarum JSA22 deposited under the accession numbers KACC91973P and KACC81097BP, lysine content of the increased production of lactic acid bacteria fermentation.
  12. 쌀 유산균발효물 제조에 있어,In the production of fermented rice lactic acid bacteria,
    쌀 추출물을, 락토바실러스 플란타럼을 이용하여 발효시키는 단계를 포함하는, 쌀 유산균 발효물 내 라이신 함량 증가 방법.A method of increasing the lysine content in rice lactic acid bacteria fermentation, comprising the step of fermenting rice extract using Lactobacillus plantarum.
  13. 제1항 내지 제11항 중 어느 한 항의 제조방법에 의해 제조된, 라이신 함량이 증가된 쌀 유산균발효물.A rice lactic acid bacterium fermentation with an increased lysine content produced by the method of any one of claims 1 to 11.
  14. 제13항의 쌀 유산균 발효물을 포함하는 식품조성물.A food composition comprising the fermented rice lactic acid bacteria of claim 13.
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