KR101575867B1 - Method for producing a grain composition having high free calcium level - Google Patents

Abstract

The present invention relates to a grain composition having an improved free calcium content and a composition for preventing osteoporosis using the same, and more particularly, to a grain composition having an increased free calcium content according to the present invention, which comprises cereal and Lactobacillus case Wikim010 strain It can not only increase the content of free calcium in grains by decomposing the picanoic acid contained in the grain, but also the osteoblast proliferation effect is superior. Therefore, the grain composition of the present invention having an increased free calcium content can be used as a composition for prevention of osteoporosis.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing a grain composition having improved free calcium content,

The present invention relates to a cereal composition having improved free calcium content and a composition for preventing osteoporosis using the same. More particularly, the present invention relates to a method for preventing osteoporosis using a lactobacillus casei strain capable of degrading picanoic acid contained in cereals, And a composition having an osteoporosis-preventing effect using the improved grain composition.

Phytic acid (myo-inositol 1,2,3,4,5,6-hexakisphosphate) is an important storage material of causalinositol (inositol) among the seeds of grains and leguminous plants. The action of phytic acid acts as an anti-nutritional factor that interferes with the digestion and absorption of minerals by strongly binding to useful minerals such as calcium in the food. In particular, phytic acid is known to be an indirect cause of osteoporosis by suppressing the absorption of calcium in digestive tract intestinal tract.

On the other hand, calcium is not only a component of skeletal tissue growth, bone and teeth, but also activates various enzymes in the body, regulates nerve excitement, muscle contraction and relaxation, regular heartbeat and blood coagulation, Is an important minerals. However, due to the increase in the living standard of modern people, intake of animal protein, lack of exercise, excessive drinking, smoking, high fat diet, caffeine and stress, calcium absorption rate is further decreased and the need for calcium intake is further increased. However, not only is calcium insoluble in water, but it is also difficult to add to food due to its unique bitter taste and its absorption rate is also very low, so almost all of it is excreted outside the body.

Therefore, although chemical treatment has been attempted as a method for decomposing phytic acid, it has not been practically used since it destroys coexisting nutrients. After phytase was proved to be most effective as a means to decompose phytate without nutritional adverse effects on food or feedstuffs, research to search for microorganisms producing phytase has been conducted since the 1960s, The activity of the enzyme has been studied extensively from abroad. Especially, when making bread by making Sourdough, a symbiotic culture of lactic acid bacteria and yeast, which is used to bake bread with whole wheat flour, lactic acid bacteria culture lowers the pH of dough, Lactobacillus paracasei subsp. Paracasei isolated from follicular feces has excellent phytic acid degrading ability, and it has been reported that separation of fructose from wheat flour The strains were added to the finishing feed and the feed efficiency improvement effect was obtained. In rats fed with low phosphorus diet, saccharomyces cerevisiae yeast culture was supplemented with 4-6% yeast preparation ( Saccharomyces cerevisiae yeast culture) (Phytate-P) of citric acid was increased. In addition, the P accumulation rate was improved by the addition of lactic acid bacteria to the feed containing low phosphorus content (0.25%) in the laying hens, which is the result of the contained phytase and intestinal pH drop. However, studies on reduction of phytic acid in animal feeds have been mainly focused on the degradation of phytic acid in food, and most studies on the decomposition of phytic acid in food have concentrated on bread products using flour as a material.

Therefore, the inventors of the present invention have been conducting research to develop a grain composition having an increased content of free calcium by decomposing phytic acid in grains, and isolating and identifying Lactobacillus case Wikim010 strain capable of decomposing phytic acid in grains, The present inventors have completed the present invention by confirming that the content of free calcium in the fermented composition increases the osteoporosis-preventing effect when it is edible.

Accordingly, a technical problem to be solved by the present invention is to provide a grain composition with increased free calcium content in the grain.

Another object of the present invention is to provide a method for producing a cereal composition in which the content of free calcium is increased.

Another object of the present invention is to provide a composition for preventing osteoporosis comprising a cereal composition having an increased content of free calcium.

In order to accomplish the above object, the present invention provides a grain composition with increased free calcium content, which comprises a grain and a strain of Lactobacillus sac Wikim010.

According to another aspect of the present invention, there is provided a grain composition with an increased free calcium content, which comprises the following steps.

(S1): 45 to 55% by weight of brown rice, 5 to 10% by weight of black rice, 5 to 10% by weight of glutinous rice, 5 to 10% by weight of waxy rice, 5 to 10% by weight of waxy rice, 5 to 10% 10 to 10% by weight of soybeans, 5 to 10% by weight of soybeans, 5 to 10% by weight of seabort and 5 to 10% by weight of red beans;

(S2) Inoculating the strain Lactobacillus sac Wikim010 in an amount of 1 to 10% by weight based on the total weight of the mixed grains; And

(S3) reacting the mixed grains inoculated with the strain at 25 to 35 DEG C for 20 to 30 hours.

According to another aspect of the present invention, there is provided a composition for preventing osteoporosis comprising a cereal composition having an increased free calcium content.

The grain composition with an increased free calcium content according to the present invention not only can increase the content of free calcium in the grain by decomposing the carbonic acid contained in the grain but also the osteoblast proliferation effect is superior. Therefore, the grain composition of the present invention having an increased free calcium content can be used as a composition for prevention of osteoporosis.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments of the invention and, together with the description of the invention, It should not be construed as limited.
Fig. 1 is a graph showing changes in the content of phytic acid after culturing inoculated with Lactobacillus casei Wikim010 in brown rice.
FIG. 2 is a graph showing changes in the content of phytic acid after culturing inoculated with a strain of Lactobacillus casei Wikim010.
FIG. 3 is a graph showing changes in the content of phytic acid after culturing inoculated with Lactobacillus casei Wikim010.
4 is a graph showing the results of analysis of calcium content in brown rice according to the treatment of Lactobacillus case Wikim010 strain.
5 is a graph showing cell proliferation results of a human-like osteoblast cell line SaOS-2 according to the treatment of Lactobacillus casei Wikim010 strain.

The grain composition with increased free calcium content of the present invention is characterized in that it comprises cereal and Lactobacillus sac Wikim010 strain.

Preferably, the grain in the present invention is brown rice or a mixed grain consisting of a mixture of at least one grain selected from the group consisting of black rice, glutinous rice, charred rice, barley rice, burdock rice, sorghum, soybean, have.

Preferably, in the present invention, in the case of mixed grains mixed with brown rice and other grains as grain, 45 to 55% by weight of brown rice; And 45 to 55 wt% of at least one grain selected from the group consisting of black rice, glutinous rice, charred rice, barley rice, barley rice, sorghum, soybean, seaweed and red beans.

More preferably, in the present invention, the mixed grains contain 45 to 55% by weight, 5 to 10% by weight of black rice, 5 to 10% by weight of glutinous rice, 5 to 10% by weight of charred rice, 5 to 10% by weight of barley rice, 10 to 10% by weight, 5 to 10% by weight, 5 to 10% by weight of soybeans, 5 to 10% by weight and 5 to 10% by weight of red beans.

The grains used in the present invention are grains that can be easily obtained in the market, and there is no particular limitation thereto.

The Lactobacillus case Wikim010 strain used in the present invention is a strain capable of reducing the content of free calcium in grains and having 99% homology with Lactobacillus casei strain. Barruto Bacillus can be identified as Saccharomyces cerevisiae. In the present invention, the strain was designated as " Lactobacillus sakei Wikim010 "and entrusted to the Korean Culture Center of Microorganisms as deposit No. KFCC 11571P on Dec. 3, 2013 .

The grain composition with an increased free calcium content of the present invention is characterized in that the strain Lactobacillus sac Wikim010 is contained in an amount of 1 to 10% by weight based on the total weight of the grain.

The grain composition with increased free calcium content of the present invention

(S1): 45 to 55% by weight of brown rice, 5 to 10% by weight of black rice, 5 to 10% by weight of glutinous rice, 5 to 10% by weight of waxy rice, 5 to 10% by weight of waxy rice, 5 to 10% 10 to 10% by weight of soybeans, 5 to 10% by weight of soybeans, 5 to 10% by weight of seabort and 5 to 10% by weight of red beans;

(S2) Inoculating the strain Lactobacillus sac Wikim010 in an amount of 1 to 10% by weight based on the total weight of the mixed grains; And

(S3) reacting the mixed grains inoculated with the strain at 25 to 35 DEG C for 20 to 30 hours.

The grain composition with an increased free calcium content according to the present invention not only can increase the content of free calcium in the grain by decomposing the carbonic acid contained in the grain but also the osteoblast proliferation effect is superior.

Therefore, the grain composition of the present invention having an increased free calcium content can be used as a composition for prevention of osteoporosis.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example 1 Isolation and Identification of Lactobacillus saccharum Strain Having Pyric Acid Resolution Ability

<1-1> Isolation of lactic acid bacteria from kimchi

The kimchi samples were diluted with physiological saline at a concentration that allows the bacteria to be separated and cultured on MRS solid medium at 30 ℃ for 48 hours. Among the 150 colonies grown in the medium, the colonies with different color and shape were separated purely.

Lactic acid bacteria were cultured using MRS liquid medium (Difco, USA), and added to 25% (v / v) glycerol in the medium at the beginning of the growing season. , Cultured at 30 ° C for 24 hours, cultured in MRS liquid medium, and cultured in MRS liquid medium at 30 ° C for 24 hours. The activity of phytase was assayed by modification of the method of Quan and Shimizu. Intracellular phytase activity was obtained by harvesting the cells by centrifugation, washing the cells 2-3 times with 0.85% NaCl solution, The phytase activity of the lactic acid bacteria was measured in an acetate buffer (pH 3.5), and a strain having excellent phytase activity was selected.

The intracellular phytase activity was expressed as the sum of the intracellular enzyme activities based on the dose of the enzyme activity of the cells contained in the unit culture solution. The activity of 2mM sodium phytate used as a substrate was expressed as the sum of intracellular enzyme activities based on extracellular activity and dose. 0.9 ml of a 2 mM sodium phytate solution used as a substrate was sufficiently preheated at 37 ° C, 0.1 ml of crude enzyme solution or cell suspension was added and reacted for 10 minutes, followed by the addition of 1 ml of 10% TCA to terminate the enzyme activity. To the reaction mixture was added 1.5 ml of a color developing reagent (a solution of ammonium molybdate in an amount of 1.5% in 5.5% H2SO4 and a solution of 2.7% ferrous sulfate in distilled water at a ratio of 4: 1) Solution) was added, and the amount of free phosphoric acid was measured at 700 nm after coloring for 10 minutes, which was expressed as the activity of phytase. Enzyme activity was defined as the amount of enzyme that converts 1 몰 mol of PO ₄ ^-3 in phytic acid to 1 unit in 1 min.

<1-2> Identification of selected lactic acid bacteria

a. Analysis using API system

The strain was identified as an API system (La Balme-les-Grottes, France). First, the sterilized platinum was taken in each of the bacterial colonies, and then suspended in sterilized distilled water. The mycelial liquid was suspended in sterilized distilled water at a concentration of MccFaland Standard Solution No. 2 supplied by API 50CH kit (BioMerieux, France). The supernatant was added to the liquid medium of the API 50CH kit and homogenized. Then, 50 tubes of the API 50CH kit were overlaid with tubes of neral oil and cultured at 30 DEG C for 48 hours. The fermentation patterns of 49 carbohydrates were analyzed by the API system, and the results are shown in Table 1 below. The results were input to the ATB identification computer system and identified.

As shown in Table 1, it was found that the lactic acid bacteria selected in <1-1> were strains belonging to the genus of Lactobacillus having a carbohydrate fermentation pattern.

b. 16S rRNA sequencing

The nucleotide sequences of 16S rRNA of the selected lactic acid bacteria were analyzed according to a conventional method known in the art. As a result, the 16S rRNA base sequence of the lactic acid bacterium was 99% identical to the 16S rRNA base sequence of Lactobacillus sacae. Therefore, the present inventors named the identified lactic acid bacteria as " Lactobacillus sakei Wikim010 &quot;, and referred to the Korean Culture Center of Microorganisms as deposit No. KFCC 11571P on Dec. 3, Respectively.

<Example 2> Determination of the activity of reducing the content of phytic acid in Lactobacillus case Wikim010 strain in cereals

200 g of brown rice, barley and salted rice were reacted with Lactobacillus case Wikim010 and incubated in an incubator at 30 ° C for 24 hours. Then, 5 g of the solution was added to 30 ml of distilled water, and the mixture was ground for 1 minute and diluted 10-fold. Add 1 mL of Wade reagent (dissolved in 0.03% FeCl ₃ H ₂ O and 0.3% sulfosalicylic acid in distilled water) to 3 mL of the diluted sample 10 times with distilled water, mix for 10 seconds with a vortex mixer, add 0.45 μm syringe filter ) And the absorbance of the filtrate was measured at 500 nm. Phytic acid content was calculated using Na-phytate standard curve. Lactobacillus casei Wikim010 was treated with each of the brown rice, barley and seaweed, which were substrates of the actual food, based on the reaction pattern on the standard substrate, and the reaction pattern was observed.

The results obtained by inoculating Lactobacillus casei Wikim010 strain into brown rice, barley and salty rice and culturing for 24 hours are shown in FIGS. 1 to 3, respectively.

As shown in Figs. 1 to 3, the Lactobacillus case Wikim010 strain was found to significantly reduce the content of phytic acid in brown rice, barley and seabream.

<Example 3> Measurement of calcium content in cereals according to Lactobacillus case Wikim010 strain

Analysis of calcium content of brown rice by Lactobacillus casei Wikim010 strain was commissioned by Jeonnam Province Food Industry Research Center.

The results are shown in FIG. 4. As shown in FIG. 4, the calcium content of brown rice was 6.3682 mg / 100 g, and the calcium content of brown rice treated with Lactobacillus case Wikim010 was 10.2470 mg / 100 g.

From the above results, it can be concluded that the lactobacillus case Wikim010 strain of the present invention secretes phytase and converts the calcium associated with phytic acid contained in brown rice into a usable form.

<Example 4>

Confirmation of osteoblast proliferation effect of brown rice by Lactobacillus case Wikim010 strain

The SaOS-2 cells used in this experiment are human osteoblast cells, which are cultured in KCLB and supplemented with 500 μL of RPMI 1640 (Gibco, USA) liquid medium containing 10% FBS (Gibco) and 1% streptomycin (Gibco) And subcultured for 48-60 hours. Cell culture was performed in a 5% CO ₂ incubator at 37 ° C.

To investigate the effect of brown rice extract on osteoblast proliferation, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphynyltetrazolium bromide) assay was performed. Brown rice extract was prepared by treating Lactobacillus case Wikim010 strain with 200 g of brown rice, removing the air from the zipper bag, flattening it, sealing it and incubating at 30 ℃ for 24 hours. Brown rice and Lactobacillus saccharum were treated with 100 ml of sterilized water, 270 ml of 70% ethanol, and stirred at room temperature for 24 hours, 48 hours, and 72 hours Followed by 1, 2, and 3 times of filtration. The brown rice 70% ethanol filtrate was concentrated at 65 ° C using a concentrator, and the concentrate was recovered with distilled water. The concentrate was lyophilized to obtain a powder, which was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 mg / ml and used in the experiment.

SaOS-2 cells were inoculated in 96-well plates at 2 × 10 ⁴ cells per well. After incubation for 24 hours, the cells were treated with the brown rice ethanol extract at 0, 50, 125, 250 and 500 μg / ml And cultured for 24 hours.

MTT was added at a concentration of 5 mg / ml and incubated for 4 hours in the same conditions. After removing the medium, insoluble formazan crystals were dissolved in DMSO and absorbance was measured at 550 nm.

As a control group, brown rice ethanol extract without Lactobacillus casei Wikim010 was used. As a positive comparison group, NaF (sodium fluoride, Sigma aldrich) and CaCl ₂ (Calcium chloride, Sigma aldrich) Respectively.

The results are shown in Fig. As shown in FIG. 5, in the cell proliferation experiment of SaOS-2, a human osteoblast cell line, NaF and CaCl ₂ treatment groups, which are known to increase cell proliferation and activity of osteoblasts, were treated at a concentration of 10 ^-4 mg / The cell proliferation effect of Lactobacillus casei Wikim010 was significantly higher than that of the control group at the concentration of 500 ug / ml for 24 hours. On the other hand, it was confirmed that there was no cell proliferation effect by ethanol extract of general brown rice (500 ug / ml).

From these results, it can be seen that the brown rice extract treated with the Lactobacillus case Wikim010 strain has an osteoblast activity increasing effect comparable to that of the reference material having the effect of increasing the osteoblast activity.

<Experimental Example 5>

Sensory evaluation of the grain composition of the present invention was performed.

Composition 1 using 100% by weight of brown rice as a cereal; 5 wt% of soybean, 10 wt% of seaweed, 5 wt% of black rice, 5 wt% of black rice, 5 wt% of glutinous rice, 5 wt% of charred rice, 5 wt% of barley rice, 5 wt% Composition 2 containing 50% by weight of brown rice, 20% by weight of charcoal rice and 30% by weight of seaweed was used. In the case of large grains, soybeans, frosts, and red beans, grit was prepared. Gritted grains were placed in a grinder and filtered through a shoe to produce a mixture of 5 (4 mm) to 10 (2 mm) mash Size.

20 g (10 ⁸ cfu / ml) of Lactobacillus casei Wikim010 strain was added to 200 g of each of the obtained grains, followed by incubation in a 30 ° C incubator for 24 hours.

The thus-obtained culture was added to an electric rice cooker (KUKU Model: CKU Homis, CR-0352FR) for 50 minutes, and 10 minutes of steam for 10 minutes. And then used as a sample.

The sensory test was performed on 15 persons in 20 ~ 30 age group. The containers of each sample were marked with three-digit numbers selected from the random number table, and the order of presentation was random. In order to reduce the confusion with the previous sample and the slowing of sensation according to the characteristics of the sample before evaluating the sample by providing purified water (20 ° C and 1 ° C) and spitting cup so that the mouth can be caught between the sample and the sample, I rinse the mouth sufficiently. The strength and likelihood were evaluated by the 7 point item scale (1 = very low, 4 = moderate, 7 = very high). Statistical significance was tested using the SPSS program using ANOVA ANOVA and Duncan's multi-range test.

As a control, a composition having the same composition as that of Composition 2 was used, except that Lactobacillus case Wikim010 strain was not added. All values are mean ± SD. The mean ± SD indicated by the different subscripts in the column means that there is a significant difference ( p <0.05) in the Duncan's multiple range test.

As shown in Table 2, the compositions 1, 2 and 3 according to the present invention were evaluated in terms of appearance, taste, flaver and texture, and in particular, In the case of the adjusted composition 2, the best evaluation was recorded. On the other hand, the control group without Lactobacillus casei Wikim010 showed a significantly lower evaluation result.

The grain composition with an increased free calcium content according to the present invention not only can increase the content of free calcium in the grain by decomposing the carbonic acid contained in the grain but also the osteoblast proliferation effect is superior. Therefore, the grain composition of the present invention having an increased free calcium content can be used as a composition for prevention of osteoporosis.

Korea Microorganism Conservation Center (Domestic) KFCC11571P 20131203

Claims (8)

delete delete delete delete delete delete CLAIMS What is claimed is: 1. A method of making a grain composition with increased free calcium content comprising the steps of:
(S1): 45 to 55% by weight of brown rice, 5 to 10% by weight of black rice, 5 to 10% by weight of glutinous rice, 5 to 10% by weight of waxy rice, 5 to 10% by weight of waxy rice, 5 to 10% 10 to 10% by weight of soybeans, 5 to 10% by weight of soybeans, 5 to 10% by weight of seabort and 5 to 10% by weight of red beans;
(S2) Inoculating Lactobacillus casei Wikim010 strain (Korea Microorganism Conservation Center Accession No .: KFCC11571P) in an amount of 1 to 10% by weight based on the total weight of the mixed grains; And
(S3) reacting the mixed grains inoculated with the strain at 25 to 35 DEG C for 20 to 30 hours.
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