KR102223309B1 - Lactobacillus plantarum MB-0601 strain having gluten degradation activity from salted seafood and uses thereof - Google Patents

Abstract

The present invention relates to a Lactobacillus plantarum MB-0601 strain having gluten-degrading ability isolated from salted fish and its use, and to noodles, udon, pizza and bread using the Lactobacillus plantarum MB-0601 strain according to the present invention. It is possible to manufacture popular gluten-free processed foods, so it can be very useful to people who have not been able to eat grain flour processed foods due to Celiac disease or gluten allergy caused by gluten intolerance.

Description

Lactobacillus plantarum MB-0601 strain having gluten degradation activity from salted seafood and uses thereof {Lactobacillus plantarum MB-0601 strain having gluten degradation activity from salted seafood and uses thereof}

The present invention relates to a Lactobacillus plantarum MB-0601 strain having a gluten-degrading ability isolated from salted fish and its use.

Flour is one of the most important raw materials in Korea's food industry, and its main uses are noodle, confectionery, and bakery. The production of flour in 2001 was 1.779,000 tons, of which 37.7% for noodles and 21.9% for confectionery and bakery were used. The flour produced in Korea is strong flour, gravity flour, and powerful flour, and there are mixed flour and whole wheat flour produced since 1986, and mixed flour belongs to gravity flour for making noodles. Whole wheat flour is ground whole wheat and is used for special baking and confectionery purposes, and strong flour is mainly made from American hard spring wheat (Dark Northern Spring (DNS)), and Canada's CWRS (Canada Western Red Spring) has also been used since 1991. It began to be used for baking.

Gravity flour is produced by mixing and milling white wheat (Western White) and hard red winter wheat (Hard Red Winter) in the same ratio when American wheat is used as a raw material, and is mainly used for making noodles and for household multipurpose flour. Recently, Australian wheat is sometimes used as flour for making noodles, and Australian wheat is largely composed of Australian Standard White, Australian White, and Australian Hard wheat. As the preference of ASW, the main species of wheat in Australia, has greatly increased due to the diversification of products and the high quality of quality, the amount of imported wheat is also increasing year by year. In the case of wheat flour, over 100 years of systematic research and accumulation and industrialization, hundreds of products optimized for each food are being produced.In Western society, where wheat is the staple food, most wheat processed foods such as bread, sweets, noodles, fried food, and pizza It has been developed as such, and in processing mainly carbohydrates, wheat processed foods are almost all. In recent years, as the food culture gradually became westernized, rice is the staple food, but the annual consumption of rice per capita has decreased by 23% from 992 kg in 1998 to 774 kg in 2009, and the consumption of flour food instead of rice is gradually increasing. It is an increasing trend. However, grains such as barley and wheat contain gluten, which is an insoluble protein, so it is difficult to digest by itself and may cause gluten intolerance.

Gluten is a by-product generated during the production of wheat starch. It is a protein insoluble in water. It is mainly composed of gliadin and glutenin. Gliadin has a molecular weight of 30,000 to 80,000 Da, and glutenin is millions of Da. It has a high molecular weight. In particular, gluten's gliadin is soluble in alcohol, but glutenin is insoluble, but glutenin plays an important role in baking. That is, when wheat flour is mixed with water, gliadin and glutenin combine to form gluten. Among gluten proteins, gliadin has viscosity and elongation, and glutenin has elasticity. The proportions of these two proteins differ depending on the type of wheat flour, and this is a unique property that only wheat flour has. For example, 65% of gliadin and 35% of glutenin are found in wheat for strong flour used to make bread. %, and the soft flour wheat used to make cakes contains 75% of gliadin and 25% of glutenin. Its role is related to the volume of the bread, and its role is related to the skeletal formation of the dough and the gas generated during fermentation. You will have the function you have. Gluten by itself is difficult to digest, and when gluten intolerance occurs, skin, nervous system, immune system, stamina, joints, teeth, and even behavior and mood are adversely affected.It is commonly known as a gluten allergy, but the actual name is'gluten-sensitive bowel disease. 'to be.

Intolerance is a reaction that our body rejects and cannot absorb nutrients when nutrients come into the body. Representative examples are gluten intolerance, which is accompanied by digestive disorders such as diarrhea, abdominal pain, constipation, and abdominal distension. Gluten-sensitive bowel disease is a symptom of lack or lack of an enzyme that digests gluten. When gluten is consumed, the intestine is stimulated and the protrusions of the intestinal membrane become atrophy, making it impossible to absorb nutrients. These gluten side effects are caused by genetic factors. As an outbreak, it is a genetic disease that occurs only in people who do not have gluten digestive enzymes by nature. Once diagnosed with gluten-sensitive bowel disease, it indicates discomfort that must be controlled through diet for life.

As is commonly known, celiac disease, which is a gluten resistance, is an allergic disease that occurs in the small intestine, and is a disease that manifests as a symptom of a susceptibility to gluten that inhibits the absorption of nutrients in the intestine. There are cases. Among Americans, at least one in 133 people suffer from celiac disease, but it is estimated that more than three times the population has a sensitivity to gluten. Blacks and Asians are also on the rise.

On the other hand, Korean Patent Publication No. 2017-0068894 discloses'Bicella confussa CJE-0201 strain having gluten-degrading ability isolated from salted fish and its use', and Korean Patent Publication No. 2015-0083899 discloses'Into Sauerdough. Microencapsulated bacterial group for gluten decomposition of and a method for producing the sourdough' are disclosed, but as in the present invention,'Lactobacillus plantarum MB-0601 strain having gluten-degrading ability isolated from salted fish and its use Nothing has been revealed about'.

The present invention was derived from the above requirements, and the present inventors searched for lactic acid bacteria capable of decomposing gluten causing celiac disease from domestic fermented foods, and analyzed gluten decomposition ability by selecting lactic acid bacteria having gluten decomposition ability among them. I did. As a result, it was confirmed that when the gluten-containing grains (wheat, rye, barley, oats, etc.) were fermented using the Lactobacillus plantarum MB-0601 strain selected in the present invention, it was possible to decompose gluten present in the grain flour. And, it was confirmed that the Lactobacillus plantarum MB-0601 strain produced a gluten-degrading enzyme having a molecular weight of about 67,000 Da. Therefore, it is possible to manufacture popular gluten-free processed foods such as noodles, udon, pizza, and bread by using the gluten-decomposed grain powder prepared by fermentation as described above, and this invention is expected to be very useful in the related food industry. Was completed.

In order to achieve the object of the present invention, the present invention provides a Lactobacillus plantarum (Lactobacillus plantarum) MB-0601 strain isolated from salted fish and having a gluten resolution of KCTC 13532BP as an accession number.

In addition, the present invention provides a microbial preparation for decomposing gluten comprising the strain or a culture solution thereof as an active ingredient.

In addition, the present invention provides a method for producing a gluten-free fermented grain flour comprising the step of decomposing gluten present in the grain flour by fermenting the strain or its culture broth after treating the grain flour. .

In addition, the present invention provides a gluten-free (gluten-free) grain flour fermented product prepared by the above method.

In addition, the present invention provides a gluten-free processed food prepared from the gluten-free fermented grain flour.

In the case of gluten-reduced cotton products fermented using the Lactobacillus plantarum MB-0601 strain (KCTC 13532BP) according to the present invention, gastrointestinal digestion is improved by promoting digestive enzymes and secretion of gastrin related to digestive enzymes through animal experiments. The efficacy was confirmed. Therefore, the Lactobacillus plantarum MB-0601 strain isolated in the present invention has excellent gluten-degrading ability, and therefore, by decomposing wheat starch or gluten present in grains through grain fermentation using the strain, gluten-containing grains are consumed. It can improve gluten-sensitive bowel disease, also known as allergy.

Therefore, by using the Lactobacillus plantarum MB-0601 strain according to the present invention, popular gluten-free processed foods such as noodles, udon, pizza, and bread can be manufactured, resulting in celiac disease caused by gluten intolerance. Alternatively, it may be very useful for those who have not been able to consume processed grain flour foods due to gluten allergy or the like.

1 shows a photograph of the culture of the Lactobacillus plantarum MB-0601 strain isolated in the present invention.
Figure 2 shows the 16s rDNA nucleotide sequence (SEQ ID NO: 1) for genetic identification of the Lactobacillus plantarum MB-0601 strain isolated in the present invention.
Figure 3 shows the results of screening the protease activity of the dialysate obtained through ammonium sulfate precipitation from the culture supernatant of the Lactobacillus plantarum MB-0601 strain isolated in the present invention.
4 is a result of purification of a dialysate having protease activity selected from the culture supernatant of the Lactobacillus plantarum MB-0601 strain isolated in the present invention through ammonium sulfate precipitation using a DEAE column chromatography method.
5 is a result of confirming the peak of gluten decomposition activity through primary (A) and secondary (B) purification by performing affinity column chromatography on the active fraction obtained using the DEAE column chromatography method.
6 is a result of measuring the molecular weight of the gluten-degrading enzyme produced by the Lactobacillus plantarum MB-0601 strain isolated in the present invention.
Figure 7 shows the feces of mice induced digestive disorders caused by dextran sulfate sodium.
8 shows the results of enhancing secretion of amylase, a digestive enzyme, when ingesting fermented noodles using the Lactobacillus plantarum MB-0601 strain isolated in the present invention.
9 shows the results of enhancing the secretion of gastrin, a digestive hormone, when ingesting fermented noodles using the Lactobacillus plantarum MB-0601 strain isolated in the present invention.

In order to achieve the object of the present invention, the present invention provides a Lactobacillus plantarum (Lactobacillus plantarum) MB-0601 strain isolated from salted fish and having a gluten resolution of KCTC 13532BP as an accession number.

Accordingly, the present inventors searched for lactic acid bacteria capable of decomposing gluten from salted fermented food, a domestic fermented food, and analyzed gluten decomposition ability by selecting lactic acid bacteria having excellent gluten decomposition ability among them. The present invention was completed by depositing the finally secured lactic acid bacteria with the Korea Research Institute of Bioscience and Biotechnology Gene Bank (KCTC, Korean Collection for Type Cultures) on May 28, 2018, and receiving the accession number KCTC 13532BP.

In addition, the Lactobacillus planta MB-0601 strain of the present invention can produce a gluten-degrading enzyme having a molecular weight of about 67,000 Da, but is not limited thereto.

In the present invention, Celiac disease caused by gluten intolerance is an enterophatic, chronic, immune-mediated type caused by the intake of gluten, a "storage" protein naturally contained in some grains. It is an inflammatory disease. This food intolerance is the intake of typical protein fractions of wheat gluten, spelled (a type of wheat flour), kamut and spella (a type of wheat flour), barley, rye, and rye (a hybrid of wheat and rye) and their derivatives. It affects people who are genetically predisposed to have an immune system that responds in an abnormal way. Some individuals are intolerant to oat protein.

In the present invention, the term "gluten" technically refers to a simple prolaminic (proline-rich) protein called "gliadin" and a glutenic called "glutenin" which is a protein of the above-mentioned grains. A combination of (glutelinic) (glutamine-rich) proteins. With regard to celiac disease, "gluten" is used to collectively refer to proteins commonly included in cereals, including various protein fractions that form gluten, of which gliadin is considered to be the most harmful. The possibility of preventing or treating the development of celiac disease has not existed so far, so eating a strictly gluten-free diet throughout life is the only treatment that can guarantee perfect health for people with celiac disease.

The present invention also provides a microbial preparation for decomposing gluten, comprising the strain of the present invention or a culture solution thereof as an active ingredient. The strain is preferably Lactobacillus plantarum , more preferably Lactobacillus plantarum MB-0601 strain (KCTC 13532BP).

In addition, the present invention provides a method for producing a microbial preparation for decomposing gluten, including the step of culturing the strain.

The strain is preferably Lactobacillus plantarum MB-0601 strain (KCTC 13532BP). The method of culturing the strain of the present invention may use a method commonly used in the art.

In addition, the lactic acid bacteria strain and lactic acid bacteria culture solution provided in the present invention, granular, solid, etc. can be formulated and supplied according to the type and purpose of use of the target grain.

In addition, the present invention provides a method for producing a gluten-free fermented grain flour comprising the step of decomposing gluten present in the grain flour by fermenting the strain or its culture broth after treating the grain flour. .

In the method according to an embodiment of the present invention, the grain may be one or more selected from the group consisting of wheat, rye, barley, oats, spelt, and kamut, but is not limited thereto.

In addition, the present invention provides a gluten-free (gluten-free) grain flour fermented product prepared by the above method. The "gluten-free grain flour fermentation" is interpreted as including a grain flour fermentation product from which gluten is mostly removed in addition to a grain flour fermentation product from which gluten is completely removed.

In addition, the present invention provides a gluten-free processed food prepared from the gluten-free fermented grain flour. The gluten-free processed food may be noodles, udon, confectionery, ramen, pizza, or bread made of fermented gluten-free grain flour, but is not limited thereto.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example 1. Securing gluten lactic acid bacteria

To separate the lactic acid bacteria having gluten-degrading ability from various salted salted salted fish, chop various salted fish aseptically, take 1 g of it, suspend it in 9 ml of sterile physiological saline (0.85% NaCl), and dilute stepwise to contain 1% gluten. Each was plated on MRS solid medium. After incubation at 30° C. for 48 hours, only microorganisms forming a clear zone generated by decomposing gluten around the colonies that appeared were isolated. Subsequently, the microorganisms purely isolated from the salted fish were spread on MRS solid medium and cultured at 30° C., and the colonies formed at this time were suspended in a 20% glycerol solution and stored in a -80° C. freezer to search for gluten-decomposing lactic acid bacteria.

Example 2. Genetic Identification of Lactobacillus Isolated from Salted Fish

For the genetic identification of the isolated strain, the strain was inoculated in MRS liquid medium, followed by subculture at 37° C. for 18 hours, and 2 ml of the culture solution was centrifuged at 4,000 rpm for 10 minutes. The cell precipitate obtained by centrifugation was washed 3 times with 0.85% NaCl. After washing, 0.5 ml of lysozyme (10 mg/ml) was added, followed by treatment at 37°C for 1 hour. After adding 20 μl of protease K (Protease K) and 25 μl of 10% sodium dodecyl sulfate (SDS), treated in a water bath at 60° C. for 30 minutes and then RNA degrading enzyme 1 µl of RNase was added, followed by treatment at 37°C for 1 hour. The same amount of phenol (Phenol)-chloroform (Isoamyl alcohol) was added and suspended in a ratio of 25:24:1, followed by centrifugation at 4℃ for 5 minutes at 14,000 rpm to take only the supernatant. Next, this process was repeated twice. Half the amount of the supernatant was added with 3M ammonium acetate (ammonium acetate, pH 4.8) and twice the amount of 100% alcohol, and the mixture was allowed to stand at -20°C for 1 hour. Centrifugation was performed at 3,000 rpm for 5 minutes at 4° C. to check the cell precipitate, and after completely removing the supernatant, 1 ml of 70% ethanol was added and centrifuged again at 3,000 rpm for 5 minutes at 4° C. After removing the supernatant, it was dried and suspended in TE buffer or distilled water to separate DNA. To amplify the 16s rRNA of the microorganism, the forward primer (27f, 5'-AGA GTT TGA TCM TGG CTC AG -3': SEQ ID NO: 2) and the reverse primer (1492r, 5'-GGT TAC CTT TGT TAC GAC TT -3' : SEQ ID NO: 3) was used. To a PCR premix (Bioneer, Cat No. K-2012), 17 µl of distilled water, 1 µl of forward primer, 1 µl of reverse primer, and 1 µl of DNA were added and mixed, followed by PCR. PCR conditions were treated at 94° C. for 5 minutes, then repeated 30 times at 94° C. for 1 minute, 62° C. for 40 seconds, and 72° C. for 40 seconds, and the reaction was terminated at 72° C. for 7 minutes. The PCR reaction product was confirmed by electrophoresis with 0.8% agarose gel. The final nucleotide sequence (SEQ ID NO: 1, Fig. 2) was analyzed and compared with the database to identify lactic acid bacteria, and this is shown in Fig. 2.

As a result, as shown in FIGS. 1 and 2, the strain isolated by the present invention was identified as Lactobacillus plantarum , and the lactic acid bacteria was named Lactobacillus plantarum MB-0601, and the Korea Research Institute of Bioscience and Biotechnology It was deposited with the Center for Biological Resources as of May 28, 2018 (Accession No. KCTC 13532BP).

Example 3. Lactobacillus Planta Room Gluten resolution of MB-0601 strain

Gluten-fermented lactic acid bacteria Lactobacillus plantarum MB-0601 strain was applied to the process for the production of noodles to evaluate the gluten decomposition ability in flour. In the experimental method, 500 g of water was added to 1 kg of wheat flour, and at the same time, gluten-fermented lactic acid bacteria (prototype) were added and kneaded. At this time, the group to which 1.5 g (0.15%) of gluten-fermented lactic acid bacteria was added to the dough was experimental group 1, the group to which 0.6 g (0.06%) was added was experimental group 2, and the group to which 0.3 g (0.03%) was added was experimental group 3 Recorded as. After kneading the flour, the gluten decomposition power was evaluated through a aging process at 30°C. The method for evaluating gluten content was analyzed using the method of separating food protein (separation of gluten from wheat flour) of the National Institute of Food Science and Technology of the Rural Development Administration. Gluten resolution according to the addition of lactic acid bacteria was shown in Table 1 below.

Evaluation of Gluten Degradation Rate of Udon Noodles Applying Gluten Fermented Lactobacillus Amount of lactic acid bacteria added Dry gluten (g) Decomposition rate (%) Average(%) Control 1.85 0.00 0 Experimental group 1 0.49 73.51 67.027 0.73 60.54 Experimental group 2 0.69 62.70 69.730 0.43 76.76 Experimental group 3 0.75 59.46 58.108 0.8 56.76

Example 4. Securing the gluten-degrading enzyme secreted by the Lactobacillus plantarum MB-0601 strain

1) ammonium sulfate precipitation

1% Lactobacillus plantarum MB-0601 strain was inoculated in 1 L of MRS medium adjusted to pH 7.0 and cultured for 24 hours. The culture solution was placed in a 400 mL centrifuge tube, and the supernatant obtained by centrifugation at 6,000 rpm for 20 minutes to remove the cells was used in the experiment. For the separation and purification of gluten-degrading enzyme, ammonium sulfate was first added in a saturated concentration of 0-20%, 20-40%, 40-60%, 60-80%, and 80-100% at each concentration. The precipitated protein was dissolved in 20 mM Tris-HCl (pH 7.0) buffer containing 10 _{mM CaCl 2 and dialyzed at 4° C. for 12 hours with the same buffer.} Each 100 µl aliquot of the dialysate was used as a coenzyme, and the activity was measured in MRS solid medium containing 1.5% skim milk using the agar diffusion method. As a result, as disclosed in FIG. 3, since the protease activity was measured only in the 40-60% saturation section of ammonium sulfate, the above section was selected for the purification of the proteolytic enzyme. Proteins recovered from ammonium sulfate (40-60%) were dialyzed with 20 mM Tris-HCl (pH 7.0) buffer containing _{20 mM CaCl 2 as an initial buffer for ion exchange chromatography.}

2) DEAE column chromatography

The coenzyme obtained through the ammonium sulfate precipitation was subjected to a purification experiment using a DEAE-sepharose column chromatography method. At this time, NaCl gave a concentration gradient of 0-1M. 100 µl of each fraction was aliquoted, and the activity was measured in MRS solid medium containing 1.5% skim milk using the agar diffusion method, and the enzyme activity and protein content of 1% casein were analyzed. As disclosed in FIG. 4, it was found that the amount of the eluted enzyme protein gradually increased as the NaCl content in the column increased. The enzyme active portion was eluted in the 49-55 fractions, and the enzyme activity was the highest in the 52 fractions with the highest protein content. Only 49-55 fractions were collected, the enzyme solution was dialyzed three times in distilled water at intervals of 2 hours, and then concentrated with an Amicon concentrator (Amicon) using an Amicon membrane (M.W. 1,000). The concentrated sample was dissolved in a 20 mM Tri-HCl buffer solution and used as a sample for the next experiment.

3) Affinity column chromatography

The active fractions obtained from the DEAE-sepharose column chromatography experiment were collected and an affinity column chromatography experiment was performed. Each fraction was aliquoted by 100 µl, and the activity was measured in MRS solid medium containing 1.5% skim milk using the agar diffusion method, and the enzyme activity and protein content for 1% casein were also analyzed. As a result, as disclosed in FIG. 5A, a peak having gluten-decomposing activity was confirmed in fractions 9-16. 9-16 fractions obtained due to different types of protein peaks were collected and a secondary affinity column chromatography experiment was performed to collect the active fraction of a single peak (FIG. 5B). The specific activity of the enzyme finally purified was 49.4 unit/mg, the yield was 4.9%, and the degree of purification was 17.4 times.

4) Measurement of gluten-degrading enzyme activity

Enzyme activity was measured using the tyrosine quantification method (Folin, 1927, J. Biol. Chem., 73, 627-650) to determine the degree of hydrolysis of the substrate casein. 100 µl of the sample solution was pre-incubated for 5 minutes in a shaking incubator at 40°C, then 900µl of 1% substrate solution was added and hydrolyzed for 60 minutes at the same temperature, and 1,000µl of 10% TCA solution was added to stop the enzyme reaction. . Proteins that were not hydrolyzed were allowed to stand at room temperature for 10 minutes and then centrifuged at 10,000 rpm for 10 minutes to remove them. 200 µl of the supernatant and _{800 µl of 0.55M Na 2} CO ₃ were added, mixed, and left for 15 minutes. 200 µl of 1.0N Folin Ciocalteu's phenol solution was added thereto, mixed, and allowed to stand for 30 minutes, and the absorbance was measured at 660 nm. As for the enzyme activity unit, 1 μM of tyrosine produced per minute by 1.0 mL of the sample solution was defined as 1 unit, and the units were calculated from a standard curve prepared using tyrosine.

Enzyme activity by step of purification of gluten-degrading enzyme Purification steps Total activity (Unit) Total protein (mg) Specific activity ⁽¹⁾
(U/mg) Yield ⁽²⁾
(%) Purification fold Supernatant 38,456 13,564 2.9 100 One Ammonium sulfate 16,877 1,856 9.1 43.9 3.2 DEAE-sepharose 6,587 448 14.7 17.1 5.2 1st Affinity
chromatography 2,566 66 38.8 6.7 13.7 2nd Affinity
chromatography 1,878 38 49.4 4.9 17.4

1) Specific activity = total activity/total protein.

2) Yield = (total activity)/(total activity of supernatant) × 100.

5) Measurement of molecular weight of gluten-degrading enzyme

The molecular weight of the final purified gluten-degrading enzyme was measured according to Laemmli's method (1970, Nature, 227, 680-685), and the molecular weight was calculated as the ratio of the eluted volume of the protease to the void volume (Ve/Vo). Standard curves for molecular weight measurement were prepared with standard proteins cytochrome C (MW 12,400 Da), carbonic anhydrase (MW 29,000 Da), albumin (MW 66,000 Da), alcohol dehydrogenase (MW 150,000 Da), and apoferritin (MW 443,000 Da). . A standard curve was created through the ratio of the elution volume of each standard protein. At this time, the standard curve was created with an exponential equation, and the standard curve was y = 1E+07e ^-2.932x , and the reliability was R² = 0.9968. The molecular weight was calculated by introducing the elution volume of the purified gluten-degrading enzyme into the exponential equation of the standard curve thus prepared, and the calculation formula for measuring the molecular weight of the gluten-degrading enzyme produced by the Lactobacillus plantarum MB-0601 strain is as follows.

One.

2.

3.

Vt = total bed volume, total volume in column

Vo = void volume, space volume between gel particles in DEAE column chromatography

Vi = inner volume, volume of gel particles containing moisture

Ve = elution volume, volume until the gluten-degrading enzyme is eluted

Vt = Vo + Vi

K = partition coefficient, volume of protein moved through the inside of gel particles/volume of stationary phase

Kav = average distribution coefficient

Vm = volume of mobile phase = Vo

Vs = volume of stationary phase = Vi

Vr = volume of gluten-degrading enzyme = Ve

Therefore, as a result of measuring the molecular weight of the gluten-degrading enzyme produced by the Lactobacillus plantarum MB-0601 strain through the above formula, the ratio of the elution volume of the gluten-degrading enzyme (Ve/Vo) was 1.8, and the molecular weight was confirmed to be about 67,000 Da. Became (Fig. 6).

Example 5. Analysis of Digestive Efficacy of Gluten Fermented Noodle Products Using Animal Models

Digestive efficacy in animals was confirmed for cotton products fermented with Lactobacillus plantarum MB-0601 strain. A total of 60 4-week-old C57BL/6 male mice were randomly grouped, and each test group was divided in a manner as shown in Table 3 below to perform an efficacy test.

Animal experiment design group UT Control Gluten-free Low concentration Medium concentration High concentration % kcal from fat 18 18 18 18 18 % kcal from wheat - 60 60 60 60 Gluten cotton (%, w/w) - 30 27 18 0 Gluten free cotton
(%.w/w) - 0 3 12 30 Number of animals (mouse) 12 12 12 12 12 gender cock cock cock cock cock Feeding period (weeks) 8 8 8 8 8

1) General feed (UT; untreated)

2) General feed + general flour 30%(w/w) (CTL; control, negative control)

3) General feed + 27% of regular flour + 3% of gluten-free flour (low concentration)

4) Regular feed + 18% regular wheat flour + 12% gluten-free wheat flour (medium concentration)

5) General feed + gluten-free flour 30% (high concentration)

After the end of the acclimatization period for one week, 5% Dextran Sulfate Sodium (DSS) in sterile distilled water was fed negatively to all groups except for the UT group for one week. DSS is 2-5% of the negative water, which induces digestive disorders, which can be seen through body weight and bowel movements (FIG. 7).

Among gluten-related digestive enzymes in vivo, amylase is an enzyme that hydrolyzes the glycosidic bonds of polysaccharides, and can help digestion when its activity increases. As a result of measuring pancreatic amylase by decomposing pancreatic tissue, the negative control group was 0±0.0005 mU/ml. Gluten-free wheat flour low concentration fed group 0.004±0.001 mU/mL, medium concentration fed group 0.005±0.002 mU/mL, high concentration fed group 0.008±0.002 mU/mL, respectively, significantly increased by 40, 50 and 80%. . This increased the activity of pancreatic glycolytic enzyme by gluten-free flour (all p <0.001) (Fig. 8).

In addition, among the digestive enzymes and related hormones in the blood, gastrin is a hormone secreted from G cells in the antrum and promotes the secretion of gastric juice and aids gastrointestinal movement. Blood gastrin was measured using a commercial diagnostic kit. As a result of the measurement, the negative control group showed 37.33±9.06 pg/ml, and the gluten-free wheat flour high concentration was 59.64±18.48 pg/ml, indicating a 60% increase in the gluten-free wheat flour high concentration compared to the negative control group ( p <0.01) . It was confirmed that feeding gluten-free flour could help improve gastrointestinal digestion by promoting gastrin secretion (FIG. 9).

Korea Research Institute of Bioscience and Biotechnology KCTC13532BP 20180528

<110> Korea Research Institute of Bioscience and Biotechnology Microbiome Co., Ltd. <120> Lactobacillus plantarum MB-0601 strain having gluten degradation activity from salted seafood and uses thereof <130> PN19215 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 1017 <212> DNA <213> Lactobacillus plantarum MB-0601 <400> 1 tgcagtcgaa cgaactctgg tattgattgg tgcttgcatc atgatttaca tttgagtgag 60 tggcgaactg gtgagtaaca cgtgggaaac ctgcccagaa gcgggggata acacctggaa 120 acagatgcta ataccgcata acaacttgga ccgcatggtc cgagcttgaa agatggcttc 180 ggctatcact tttggatggt cccgcggcgt attagctaga tggtggggta acggctcacc 240 atggcaatga tacgtagccg acctgagagg gtaatcggcc acattgggac tgagacacgg 300 cccaaactcc tacgggaggc agcagtaggg aatcttccac aatggacgaa agtctgatgg 360 agcaacgccg cgtgagtgaa gaagggtttc ggctcgtaaa actctgttgt taaagaagaa 420 catatctgag agtaactgtt caggtattga cggtatttaa ccagaaagcc acggctaact 480 acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggattt attgggcgta 540 aagcgagcgc aggcggtttt ttaagtctga tgtgaaagcc ttcggctcaa ccgaagaagt 600 gcatcggaaa ctgggaaact tgagtgcaga agaggacagt ggaactccat gtgtagcggt 660 gaaatgcgta gatatatgga agaacaccag tggcgaaggc ggctgtctgg tctgtaactg 720 acgctgaggc tcgaaagtat gggtagcaaa caggattaga taccctggta gtccataccg 780 taaacgatga atgctaagtg ttggagggtt tccgcccttc agtgctgcag ctaacgcatt 840 aagcattccg cctggggagt acggccgcaa ggctgaaact caaaggaatt gacgggggcc 900 cgcacaagcg gtggagcatg tggtttaatt cgaagctacg cgaagaacct taccaggtct 960 tgacatacta tgcaaatcta agagattaga cgttcccttc ggggacatgg atacagg 1017 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agagtttgat cmtggctcag 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggttaccttt gttacgactt 20

Claims (6)

Separated from salted fish, Lactobacillus plantarum MB-0601 strain having an accession number of KCTC 13532BP, gluten and casein resolution. A microbial preparation for decomposing gluten and casein comprising the strain of claim 1 or a culture solution thereof as an active ingredient. A method for producing a gluten-free fermented grain flour comprising the step of decomposing gluten present in the grain flour by fermenting the strain of claim 1 or a culture broth thereof in grain flour. The gluten-free grain flour fermentation according to claim 3, wherein the grain is at least one selected from the group consisting of wheat, rye, barley, oats, spelt and kamut. Method of making water. delete delete

Images