KR102685690B1 - A process for the preparation of sikhye containing lactobacillus and the sikhye containing lactobacillus prepared therefrom - Google Patents

Abstract

The present invention relates to a method for producing lactic acid sikhye and the lactic acid sikhye produced thereby, comprising: (S1) cooking godubap; (S2) preparing malt extract; (S3) mixing the malt extract prepared in step (S2) with the godubap cooked in step (S1) and saccharifying it at 50 to 70°C for 5 to 7 hours; (S4) Removing 15 to 25% (v/v) of godubap from the malt saccharification liquid saccharified in step (S3), washing it with cold water, heating at 95 to 100°C for 50 to 70 minutes, and reintroducing it. ; (S5) adding a mixture of lactic acid bacteria metabolites, fructooligosaccharides and sugar to the mixture obtained in step (S4) and then heating at 95 to 100° C. for 50 to 70 minutes; (S6) sterilizing and filtering the mixture obtained in step (S5) to obtain lactic acid bacteria sikhye; and (S7) cooling the lactic acid bacteria sikhye obtained in step (S6) at 60 to 80°C, transferring it to a filling machine, filling it, and packaging it. Lactobacillus sikhye is not only effective in relieving fatigue and improving digestive function due to the various effects of lactic acid bacteria, but also has organ strengthening, anti-obesity, and anti-inflammatory effects. Additionally, preference can be further increased by using it with lactic acid bacteria. Therefore, the lactic acid bacteria sikhye of the present invention can be used as a fatigue recovery, anti-inflammatory and diet drink, and is expected to be very useful as a well-being health drink without the worry of obesity for studying students and office workers.

Description

Method for manufacturing lactic acid bacteria sikhye and lactic acid sikhye prepared accordingly {A process for the preparation of sikhye containing lactobacillus and the sikhye containing lactobacillus prepared therefrom}

The present invention relates to a method for producing lactic acid sikhye and the lactic acid sikhye produced accordingly. More specifically, by adding lactic acid bacteria metabolites during the production of sikhye, it improves digestive function, helps in recovery from fatigue, and has anti-obesity and anti-inflammatory effects. In addition, it relates to a method of manufacturing lactic acid bacteria sikhye, which can be used as a diet and health drink because it has an effect of strengthening organ function, and the lactic acid bacteria sikhye manufactured accordingly.

Lactic acid bacteria are safe microorganisms that are easily found in the intestines of humans and animals and in fermented foods (Orrhge, K. et al., 2000, Bifidobacteria and lactobacilli in human health, Drugs Exptl. Clin. Res ., 26, 95-111) It attaches to the intestinal epithelial cells and becomes parasitic, improving the properties of the intestinal flora, stabilizing the intestinal flora, reducing the production of decay products by suppressing the settlement of harmful bacteria, preventing diseases, activating immunity, and anti-cancer. It is known to have many benefits to the human body, such as lowering cholesterol. The reason lactic acid bacteria have a growth-inhibiting effect on various putrefactive and pathogenic microorganisms is due to several metabolic characteristics. This is because lactic acid bacteria produce antibacterial active factors such as organic acid, hydrogen peroxide, reuterin, diacetyl, acetaldehyde, and bacteriocin as metabolites. (Fuller, K., 1989, Probiotics in man and animals, J. Appl. Bacteriol., 66, 365-378). However, most of the conventional lactic acid bacteria products are fermented dairy products made with milk as the main ingredient, and cannot be considered suitable foods for Koreans who cannot digest milk's lactose well. In addition, lactic acid bacteria are heterotrophic microorganisms that basically require various nutrients such as sugar, various amino acids, and vitamins. Therefore, if lactic acid bacteria fermentation broth is made using only plants, the lactic acid bacteria containing a large amount of lactic acid bacteria like yogurt due to lack of nutrients. It is difficult to make a fermentation broth and it is difficult to homogenize the product.

Meanwhile, sikhye is one of Korea's representative umcheongryu and is a traditional dessert mainly used at feasts and ancestral rites. In Korea, the first record of sikhye can be found in the Joseon Dynasty's document ≪Sumunsaseol≫, where it is referred to as gamju. Later, in the late 1800s, the method of making amju using malt and the method of growing malt were introduced in ≪Siuijeonseo≫, and in ≪Gyugonyoram≫, the method of making sikhye using malt was recorded.

Sikhye uses rice and malt as its main ingredients. At this time, the malt produces a large amount of amylase, a starch-decomposing enzyme, and the starch in the rice is saccharified by amylase to produce dextrin, maltose, glucose, etc. It is broken down into monosaccharides and disaccharides. The sugars produced in this way provide Sikhye's unique sweetness and flavor, and it is known that an increase in the amount of malt used increases the smell, flavor, and reducing sugar content of Sikhye, thereby increasing its sweetness.

Research on malt, the raw material of sikhye, includes the length of malt leaf buds and buds with strong saccharification power, the length of barley, malt soaking time, comparative study of sikhye with different types of rice and cooking method, changes in sikhye composition during the saccharification process, and malt Research has been conducted on the raw material, barley, and germination conditions. In addition, research was reported on the quality of sikhye according to the rice variety, which is the main ingredient, and the quality of sikhye using rice barley.

Research was also conducted on functional sikhye to improve the quality of sikhye, including the quality characteristics of sikhye according to the addition of puffed rice powder, sikhye with powdered green tea, sikhye with mulberries, sikhye with different sweet pumpkin additives, and astragalus extract. Research has been reported on the production of sikhye with various functionalities, including Sikhye with added sikhye, sikhye with porridge extract, colored sikhye, sikhye with bellflower root powder, sikhye with corn silk extract, and ginseng sikhye.

In addition, as related patent documents, Republic of Korea Patent No. 10-1916843 reported sikhye using bitter melon, and Republic of Korea Patent No. 10-1702812 disclosed sikhye using black garlic.

However, sikhye containing lactic acid bacteria metabolites and its manufacturing method have not yet been disclosed.

Accordingly, the present inventors discovered that adding lactic acid bacteria metabolites with various effects to sikhye can not only improve fatigue recovery, improve digestive function, anti-obesity and anti-inflammatory effects, but also improve intestinal function and improve taste and aroma. The invention was completed.

Therefore, the technical problem to be solved by the present invention is to provide a method for producing lactic acid sikhye that can not only relieve fatigue, improve digestive function, and have anti-obesity and anti-inflammatory effects, but also improve intestinal function and improve taste and aroma.

In addition, another technical problem to be solved by the present invention is to provide lactic acid bacteria sikhye prepared according to the above production method.

In order to solve the above technical problems, the present invention provides a method for producing lactic acid bacteria Sikhye, comprising the following steps:

(S1) Step of making godubap;

(S2) preparing malt extract;

(S3) mixing the malt extract prepared in step (S2) with the godubap cooked in step (S1) and saccharifying it at 50 to 70°C for 5 to 7 hours;

(S4) Removing 15 to 25% (v/v) of godubap from the malt saccharification liquid saccharified in step (S3), washing it with cold water, heating at 95 to 100°C for 50 to 70 minutes, and reintroducing it. ;

(S5) adding a mixture of lactic acid bacteria metabolites, fructooligosaccharides and sugar to the mixture obtained in step (S4) and then concentrating by heating at 95 to 100° C. for 50 to 70 minutes;

(S6) sterilizing and filtering the mixture obtained in step (S5) to obtain lactic acid bacteria sikhye; and

(S7) Cooling the lactic acid bacteria sikhye obtained in step (S6) at 60 to 80°C, transferring it to a filling machine, filling it, and packaging it.

Preferably, the lactic acid bacteria metabolite is one or more lactic acid bacteria selected from the group consisting of Lactobacillus plantarum ( L.plantarum ), Lactobacillus casei ( L.casei ), and Bifidobacterium infantis ( B. infantis ). It can be prepared by inoculating a medium and culturing at 20 to 40°C for 12 to 48 hours and freeze-drying the resulting fermentation broth.

Preferably, the mixture of fructooligosaccharide and sugar in step (S5) is characterized in that fructooligosaccharide and sugar are mixed at a weight ratio of 2:8.

Preferably, grapefruit seed extract is additionally added in step (S5).

Preferably, in step (S6), sterilization is performed at 95 to 100° C. for 20 to 30 minutes.

In order to solve the above-mentioned other technical problems, the present invention provides lactic acid bacteria sikhye prepared according to the above production method.

As such, lactic acid sikhye containing lactic acid bacteria metabolites prepared according to the present invention not only has the effect of relieving fatigue and improving digestive function by adding various effects of lactic acid bacteria, but also has organ strengthening, anti-obesity, and anti-inflammatory effects. Additionally, preference can be further increased by using it with lactic acid bacteria. Therefore, the lactic acid bacteria sikhye of the present invention can be used as a fatigue recovery, anti-inflammatory and diet drink, and is expected to be very useful as a well-being health drink without the worry of obesity for studying students and office workers.

Hereinafter, the present invention will be described in more detail.

The present invention provides a method for producing lactic acid bacteria Sikhye, comprising the following steps:

(S1) Step of making godubap;

(S2) preparing malt extract;

(S3) adding godubap cooked in step (S1) to the malt extract prepared in step (S2) and saccharifying it at 50 to 70°C for 5 to 7 hours;

(S4) Removing 15 to 25% (v/v) of godubap from the malt saccharification liquid saccharified in step (S3), washing it with cold water, heating at 95 to 100°C for 50 to 70 minutes, and reintroducing it. ;

(S5) adding a mixture of lactic acid bacteria metabolites, fructooligosaccharides and sugar to the mixture obtained in step (S4) and then concentrating by heating at 95 to 100° C. for 50 to 70 minutes;

(S6) sterilizing and filtering the mixture obtained in step (S5) to obtain lactic acid bacteria sikhye; and

(S7) Cooling the lactic acid bacteria sikhye obtained in step (S6) at 60 to 80°C, transferring it to a filling machine, filling it, and packaging it.

The malt added to the lactic acid sikhye of the present invention is made by first pouring 1L of distilled water into 600g of malt powder, immersing it in water at 30°C for 1 hour, adding a total of 5L of distilled water, extracting it 5 times, and then separating the malt extract and foil using a 100 mesh sieve. After separation, the malt extract can be prepared by precipitating it in a 4℃ refrigerator for 10 hours to remove the precipitate, then taking the supernatant and using it to saccharify Sikhye.

According to one embodiment of the present invention, in the method for producing lactic acid sikhye of the present invention, step (S3) is performed by adding 80 to 100 parts by weight of godubap based on 100 parts by weight of the extracted malt and fermenting at 50 to 70 ° C. for 5 to 7 hours. It is characterized by saccharification, and it is desirable to set the saccharification end point when 10 or more rice grains float.

According to one embodiment of the present invention, in the method for producing lactic acid sikhye of the present invention, step (S4) is performed by removing 15 to 25% (v/v) of godubap from the saccharified malt and saccharification liquid and washing with cold water. It is characterized by heating at 95 to 100°C for 50 to 70 minutes and then reintroducing it. At this time, in order to produce clean sikhye without being dry, the remaining 75 to 85% (v/v) of godubap, which is removed from the saccharification liquid, is discarded without being used again. This means that if more godubap (75 to 85% (v/v) godubap) is added than necessary, the carbohydrates contained in the rice are dissolved by the malt, which increases the turbidity and viscosity of the sikhye, increasing the dull taste and impairing the clean taste. Because there is.

According to one embodiment of the present invention, step (S5) in the method for producing lactic acid sikhye of the present invention is characterized by adding a mixture of lactic acid bacteria metabolites, fructooligosaccharides, and sugar. At this time, the lactic acid bacteria metabolite is added in an amount of 0.02 to 0.03 parts by weight based on 100 parts by weight of malt, and the mixture of fructooligosaccharide and sugar is added in an amount of 110 to 130 parts by weight based on 100 parts by weight of malt. .

The fructooligosaccharide and sugar are preferably mixed at a weight ratio of 2:8, and the fructooligosaccharide is added as food for lactic acid bacteria.

In the present invention, lactic acid bacteria metabolites are substances produced by lactic acid bacteria when they eat food, and representative examples include dead cells and bacteriocins. Unlike lactic acid bacteria, these lactic acid bacteria metabolites are not in a living form, so they are not affected by stomach acid and bile acid, so they can stably reach the intestines and directly kill harmful bacteria, helping to quickly stabilize the intestines.

Lactic acid bacteria metabolites have their own efficacy, and in the case of dead cells, they help alleviate atopic dermatitis. According to a study published in Biological and Pharmaceutical Bulletin in 2008, when dead cells were injected into experimental mice with atopic dermatitis, the production of IgE (immunoglobulin E), which causes allergies such as atopic dermatitis, was suppressed. appear.

The lactic acid bacteria used in the present invention are preferably one or more lactic acid bacteria selected from the group consisting of Lactobacillus plantarum ( L.plantarum ), Lactobacillus casei ( L.casei ), and Bifidobacterium infantis ( B. infantis ) Inoculate mixed lactic acid bacteria mixed with Lactobacillus plantarum ( L.plantarum ), Lactobacillus casei ( L.casei ), and Bifidobacterium infantis ( B. infantis ) in a ratio of 1:1:1. It is characterized by

Lactobacillus Plantarum, one of the lactic acid bacteria, is a rod-shaped bacillus of the genus Lactobacillus and is Gram-positive. The size is 0.7 to 1.0 x 3.0 to 8.0 μ, the optimal growth temperature is 29 to 33 ℃, and it is slightly aerobic. Lactobacillus plantarum contains arabinose, glucose, fructose, galactose, maltose, sucrose, dextran, and raffinose. and trehalose are fermented to produce lactic acid. Lactobacillus plantarum is mainly isolated from milk, cheese, butter, kefir, fermented sausages, fermented potatoes, grains, acidic bread dough, pickled vegetables such as pickles and kimchi, and tomatoes. It is one of the most widely distributed lactic acid bacteria in nature. In particular, lactic acid bacteria play an important role in the fermentation of Korean kimchi, and their salt tolerance is about 5.5%.

Additionally, Lactobacillus casei is an anaerobic microorganism of the Lactobacillus genus found in the human intestines or mouth and was isolated from milk and cheese. Lactobacillus casei, a probiotic, is a rod-shaped bacillus that is Gram-positive, non-motile, and resistant to acid and heat. It also helps the growth of Lactobacillus acidophilus and produces amylase, an enzyme that decomposes carbohydrates.

This bacterium is not killed by digestive juices in the human body, but travels to the small intestine and is very beneficial to the human body, normalizing the flora in the small intestine, reducing milk intolerance, and helping the intestines and digestion. Milk coagulates and becomes viscous in 3-5 days due to lactic acid produced by Lactobacillus casei, which uses casein, which is a very important bacteria in cheese ripening. Recently, Lactobacillus casei has been reported to inhibit the growth of Helicobacter pylori, which causes gastric ulcers and gastritis, and to help balance the microbial flora in the large intestine.

Bifidobacterium infantis ( B. infantis ) helps digest food and improves irritable bowel syndrome. It is known to be highly effective in strengthening immunity and suppressing harmful bacteria inside the intestines.

Liquid food medium for cultivating the lactic acid bacteria (liquid consisting of 2.0 to 4.0% by weight of glucose, 0.5 to 2.0% by weight of peptone, 0.2 to 1.0% by weight of yeast extract, 2.0 to 4.0% by weight of inorganic salts, and 89.0 to 95.3% by weight of purified water) Lactic acid bacteria metabolites can be prepared by inoculating 1x10 ⁸ cfu/mL or more of lactic acid bacteria into a culture medium and culturing at 20 to 40°C for 12 to 48 hours and freeze-drying the resulting fermentation broth.

According to one embodiment of the present invention, the lactic acid bacteria metabolite is a group consisting of Lactobacillus plantarum ( L.plantarum ), Lactobacillus casei ( L.casei ), and Bifidobacterium infantis ( B. infantis ) It can be prepared by inoculating a liquid medium with one or more lactic acid bacteria selected from and culturing at 20 to 40°C for 12 to 48 hours and freeze-drying the obtained fermentation broth.

According to one embodiment of the present invention, grapefruit seed extract is additionally added in step (S5). At this time, the grapefruit seed extract is preferably added in an amount of 0.1 to 0.3 parts by weight based on 100 parts by weight of malt.

By additionally adding the grapefruit seed extract, the storability of lactic acid sikhye can be increased.

According to one embodiment of the present invention, sterilization in step (S6) of the method for producing lactic acid sikhye of the present invention is performed at 95 to 100 ° C. for 20 to 30 minutes.

According to one embodiment of the present invention, in the method for producing lactic acid sikhye of the present invention, in step (S7), the lactic acid sikhye is cooled at 60 to 80° C. and then transferred to a filling machine and filled. It is characterized by packaging.

Meanwhile, the present invention provides lactic acid bacteria sikhye prepared according to the above production method.

As such, lactic acid sikhye containing lactic acid bacteria metabolites prepared according to the present invention not only has the effect of relieving fatigue and improving digestive function by adding various effects of lactic acid bacteria, but also has organ strengthening, anti-obesity, and anti-inflammatory effects. Additionally, preference can be further increased by using it with lactic acid bacteria. Therefore, the lactic acid bacteria sikhye of the present invention can be used as a fatigue recovery, anti-inflammatory and diet drink, and is expected to be very useful as a well-being health drink without worrying about obesity for studying students and office workers.

Hereinafter, the present invention will be described in detail using drawings and examples to aid understanding. However, the embodiments according to the present invention may 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 present invention are provided to more completely explain the present invention to those skilled in the art.

<Preparation Example 1> Production of lactic acid bacteria metabolites

The culture conditions for lactic acid bacteria are cultured in a liquid medium at a culture temperature of 20 to 40°C for 12 to 48 hours until the number of lactic acid bacteria reaches 1x10 ⁸ cfu/mL or more and pH 4.8 or less to prepare a lactic acid bacteria culture solution and then freeze-dry to metabolize lactic acid bacteria. The product was obtained.

Specifically, the food liquid medium for culturing the lactic acid bacteria contains 2.0 to 4.0% by weight of glucose, 0.5 to 2.0% by weight of peptone, 0.2 to 1.0% by weight of yeast extract, 2.0 to 4.0% by weight of inorganic salts, and 89.0 to 95.3% by weight of purified water. A liquid medium consisting of was prepared by sterilizing at 121°C for 30 minutes.

In the component composition constituting the liquid medium according to the present invention, the inorganic salts are the total weight of the Mg, Mn, and Co components commonly used in the medium composition.

The cultured lactic acid bacteria culture medium was centrifuged at 7,000 to 10,000 rpm to separate the lactic acid bacteria cells and the culture supernatant. The culture filtrate separated above was sterilized again (100°C, 30 minutes) and cooled to 90°C to prepare a lactic acid bacteria culture filtrate, and then concentrated under reduced pressure according to a conventional method to prepare a culture concentrate more than twice as concentrated.

The lactic acid bacteria culture concentrate of the present invention prepared by the above method mainly contains gamma aminobutyric acid (GABA), amino acids, peptides, vitamins, organic acids, and bacteriocins as metabolites produced during the fermentation process of lactic acid bacteria.

The obtained lactic acid bacteria culture concentrate was freeze-dried to prepare lactic acid bacteria metabolites.

<Example 1> Production of lactic acid sikhye

(1) Production of malt extract

First, 1 L of distilled water was poured into 600 g of malt powder, soaked in water at 30°C for 1 hour, then a total of 5 L of distilled water was added and extracted five times, and then the malt extract and the meal were separated using a 100 mesh sieve. The malt extract was allowed to settle for 1 hour at room temperature to remove the precipitate, and then the supernatant was taken and used for saccharification of sikhye.

(2) Manufacturing of godubap

After washing 500 g of glutinous rice or spicy rice 5 times, 2.0 liters of water was added and dehydrated at room temperature for 10 minutes, then 500 mL of water was added and godubap was prepared in an electric rice cooker (CR3031N, Cuckoo Korea). Glutinous rice or spicy rice composed of amylopectin, an α1.6 linkage of glucose, with a high content of marginal dextrin, an activator of bifidobacteria, was used as an ingredient for sikhye.

(3) Production of lactic acid sikhye

Godubap was added in an amount of 20 parts by weight to 100 parts by weight of the extracted malt and saccharified in an electric rice cooker (CR3031N, Cuckoo Korea) at 70°C (electric rice cooker temperature) for 5 to 7 hours, until 10 or more grains of rice floated. The time was set as the end point of saccharification, and 15 to 25% (v/v) of godubap was taken out from the saccharified malt saccharification liquid, washed with cold water, heated at 95 to 100°C for 50 to 70 minutes, and then reintroduced. The remaining 75 to 85% (v/v) of the godubap was discarded without being reused.

Next, the lactic acid bacteria metabolite obtained in Preparation Example 1 was added in an amount of 0.01 to 0.03 parts by weight based on 100 parts by weight of malt saccharification liquid, and the mixture of fructooligosaccharide and sugar mixed at a weight ratio of 2:8 was added to 100 parts by weight of malt. It was added in an amount of 110 to 130 parts by weight and concentrated by heating at 95 to 100°C for 50 to 70 minutes.

Afterwards, the obtained lactic acid bacteria sikhye was sterilized at 95 to 100°C for 20 to 30 minutes, cooled at 60 to 80°C, and then transferred to a filling machine and filled to package the lactic acid bacteria sikhye.

<Example 2>

The above example except that lactic acid bacteria metabolites were added in an amount of 0.01 to 0.03 parts by weight based on 100 parts by weight of malt saccharified liquid, and grapefruit seed extract was added in an amount of 0.1 to 0.3 parts by weight based on 100 parts by weight of malt saccharified liquid. Lactobacillus sikhye was prepared in a similar manner to 1.

<Comparative Example 1>

Regular sikhye without added lactic acid bacteria metabolites was prepared.

<Test Example 1> Sugar content measurement

The sweetness of the lactic acid sikhye prepared in Example 1 was measured with a sugar meter (digital refractometer, pocket PAL-1, Atago co., Japan) and expressed as ˚Brix. All samples were collected in an amount of 1 g, and the average value was repeated three times. For comparison, regular sikhye of Comparative Example 1 without lactic acid bacteria metabolites was used. The results are shown in Table 1 below.

Sample Sugar content (˚Brix) Lactobacillus Sikhye of Example 1 12 Lactic acid bacteria Sikhye of Example 2 12 General Sikhye of Comparative Example 1 12

As shown in Table 1, the ˚Brix of the lactic acid bacteria sikhye prepared in Examples 1 and 2 and the regular sikhye prepared in Comparative Example 1 was 12˚Brix, indicating that the addition of lactic acid bacteria did not significantly change the sweetness. .

<Test Example 2> pH measurement

The pH of lactic acid sikhye was measured with a pH/ISE Meter (pH/Temp Meter pH-200L). All samples were collected in 30 ml each and the average value was repeated three times. The results are shown in Table 2 below.

Sample pH Lactobacillus Sikhye of Example 1 5.65 Lactobacillus Sikhye of Example 2 5.63 General Sikhye of Comparative Example 1 5.71

As shown in Table 2, the pH of the lactic acid bacteria sikhye of the present invention was 5.65 and 5.63, which was found to have a pH similar to that of general sikhye.

<Test Example 3> Measurement of NO inhibition induced by LPS in macrophage cell line (RAW264.7) of lactic acid sikhye

When an inflammatory response occurs in macrophages due to external stimuli such as LPS, NO is secreted, various substances such as inflammatory cytokines are produced, and various pathological reactions that regulate the inflammatory response occur. The anti-inflammatory efficacy was investigated by treating RAW264.7 cells in which an inflammatory response was induced with LPS with the extract of Kodeulbae.

NO ₂ (Nitrite), a stabilized NO oxide, was measured using the Griess reaction. First, the mouse macrophage cell line (RAW264.7) was dispensed into a 96- ^well plate at ₅ LPS (lipopolysaccharide) was treated at a concentration of 1 μg/ml and CO₂ cultured for 1 hour. Then, the prepared Kodeulpae extract was treated at different concentrations and CO₂ cultured for 24 hours. Then, place each culture supernatant in a 96-well plate and add an equal amount of Griess reagent (0.1% N-1-naphtyl-ethylendiamine in H ₂ O : 1% sulfanilamide in 5% H ₃ PO ₄ = 1 : 1). After reacting for 10 minutes, the absorbance was measured at 550 nm with a microplate reader. The concentration of NO ₂ (Nitrite) was calculated by comparing it with a standard curve obtained by diluting NaNO ₂ (sodium nitrite) two-fold from 100 μM to 0.7 μM. LPS was used as a positive control.

The results are shown in Table 3 below.

Concentration(μg/ml) LPS Comparative Example 1 Example 1 Example 2 7.27±0.5 7.17±0.1 0.18±0.3 0.15±0.5

As shown in the results, it was confirmed that the lactic acid bacteria sikhye of Examples 1 and 2 of the present invention suppressed NO.

<Test Example 4> Measurement of the effect on inflammatory cytokines induced by LPS in the macrophage cell line (RAW 264.7) of lactic acid sikhye

RAW 264.7 cells, a macrophage cell line, were purchased from the Korea Cell Line Bank (KTCC) and cultured at 37°C using RPMI 1640 medium containing 10% FBS (fetalbovine serum), 1% antibiotics, and 2-mercaptoethanol (2-ME) at 4% CO. Cultured in an incubator controlled at CO ₂ .

RAW264.7 macrophages were dispensed into a 24-well plate at 5 x 10 ⁵ cells/1ml per well and incubated with CO₂ for 24 hours. Afterwards, the wells were treated with LPS (lipopolysaccharide) at a concentration of 1 μg/ml and lactic acid sikhye at various concentrations and incubated with CO₂ for 24 hours. Then, the cell culture supernatant was collected. The cytokines IL-6, TNF-α, GM-CSF, and IL-1β contained in the supernatant were measured using enzyme-linked immunosorbent assay (ELISA). That is, the primary antibody (capture antibody) was diluted in the coating solution (0.1 M sodium carbonate, pH 9.5) and dispensed at 100 μl/well into the plate-bottom micro-well, incubated overnight at 4°C, and then washed with a washing solution (0.05% Washed with Tween 20/PBS). The washed microwells were blocked with PBS containing 10% FBS, and the culture supernatant collected in the experiment was diluted at an appropriate ratio and dispensed into each well to react at room temperature. Next, after reacting with 100 μl/well of biotinylated secondary antibody at room temperature for a certain period of time, 100 μl/well of avidin-peroxidase (enzyme reagent) was added. Finally, a substrate (3,3′,5,5′-Tetramethylbenzidine Liquid Substrate, H ₂ O ₂ ) was added to develop color, and then measured using a microplate reader. The measured concentrations of IL-6, TNF-α, GM-CSF, and IL-1β were converted using a standard curve.

The results are shown in Table 4 below.

Cytokines LPS
(1 μg/ml) Comparative Example 1 Example 1 Example 2 IL-1β 526.31±3.1 521.16±13.4 86.23±2.5 84.11±5.1 GM-CSF 1711.51±21.2 1723.01±14.1 163.52±2.6 152.15±5.1 IL-6 16.82±1.5 18.32±0.4 1.20±0.4 1.21±1.1 TNF-α 7.35±0.5 8.47±0.2 3.23±1.5 3.21±1.3

As shown in the results, the effect of lactic acid sikhye on inflammatory cytokines induced by LPS in RAW 264.7 cells was confirmed, and the pro-inflammatory cytokines of IL-6, IL-1β, TNF-α, and GM-CSF were confirmed. It was confirmed that all cytokines were suppressed statistically significantly. These results confirm that lactic acid sikhye participates in anti-inflammatory function by effectively suppressing inflammatory mediator cytokines in RAW 264.7 cells.

<Test Example 5> Confirmation of improvement in fatigue recovery effect

The lactic acid bacteria sikhye of Examples 1 and 2 and the regular sikhye of Comparative Example 1 were administered to 20 men and women in their 10s to 40s, 10 each, 110 ml three times a day, 2 hours after lunch, for 30 days. After taking it, the degree of improvement in digestive function, lethargy, and fatigue is shown in Table 5 using the 5-point scoring system (5 points: very excellent, 4 points: excellent, 3 points: average, 2 points: bad, 1 point: very poor). It was expressed as . To this end, the effects of improving digestive function, feeling of lethargy, and overall fatigue were confirmed.

Digestive function
improvement effect helplessness
improvement effect fatigue
improvement effect Example 1 4.9 4.9 4.9 Example 2 4.9 4.9 4.9 Comparative Example 1 2.5 2.4 2.3

As shown in Table 5 above, the lactic acid bacteria sikhye of the present invention was very effective in improving digestive function, had the effect of reducing lethargy, and improved overall fatigue. In particular, when taken after lunch, it was evaluated that digestive function was improved and fatigue was improved so that one could concentrate more on studying or working in the afternoon.

<Test Example 6> Weight and blood cholesterol reduction experiment

Using ICR mice as experimental animals, a test control group and a normal group made from the lactic acid bacteria sikhye prepared in Examples 1 and 2 and the regular sikhye of Comparative Example 1 were fed and raised and compared.

In this experiment, the basic diet was AIN-76, the protein source was casein (Japan), the carbohydrate source was corn starch (CheilJedang), and the fat source was corn oil (target), and the normal group and test control group were compared. proceeded. The lactic acid sikhye used in this experiment was administered orally in 1 ml at a certain time every day, and food and drinking water were freely consumed. The test animals were raised separately in polycarbonate cages, maintaining constant conditions of constant temperature (20 ± 2°C), constant humidity (50 ± 5%), and a 12-hour photoperiod. In animals, cholesterol circulates through the bloodstream and is synthesized in several organs. If the cholesterol level in the bloodstream is high, it is a major cause of arteriosclerosis. Therefore, the analysis items are (1) weight change and dietary intake, and (2) measurement of blood cholesterol and neutral lipids. It proceeded with this.

After completing breeding, the experimental animals were fasted for 12 hours, anesthetized by inhaling ether, and then blood was collected from the abdominal inferior vena cava. After treatment with heparin, plasma was separated by centrifugation (15 mins, 3,000 RPM) and lipid content was measured. did.

The internal organs (liver, epididymis, kidney) of each experimental animal were removed immediately after blood collection, rinsed several times with PBS (Phosphate Buffered saline) solution, surface moisture was removed, and compared and quantified. Plasma neutral lipids were measured at 550 nm using a colorimetric neutral fat measurement reagent (Asan kit) using the enzymatic method of McGowan et al. (1983), and plasma total cholesterol was measured using the enzymatic method of Allain et al. (1974). A reagent for measuring total cholesterol (Asan kit) was used to develop color, and the absorbance was measured at 500 nm for quantification by comparison with the cholesterol standard curve. Organ lipids were cut into small pieces according to the method of Folch et al. (1957) and then quantified. ml, Chloroform: Methyl alcohol was extracted in a 2:1 solution, and neutral lipids and cholesterol were quantified using the same method as in plasma. Each item was statistically processed and taken as an average value.

(1) Weight change

The weight change and dietary intake of the experimental animals are shown in Tables 6 and 7, respectively.

Referring to Table 6 below, in the case of experimental animals orally fed the lactic acid bacteria sikhye prepared in Examples 1 and 2, the body weight was similar or decreased compared to before the experiment, while the experimental animals orally fed the regular sikhye prepared in Comparative Example 1 And in the case of the normal group, it was confirmed that body weight increased compared to before the experiment.

Referring to Table 7 below, it was confirmed that the dietary intake of the experimental animals and the normal group orally fed the lactic acid bacteria sikhye prepared in Examples 1 and 2 and the regular sikhye of Comparative Example 1 were similar.

Before experiment (g) After experiment (g) Example 1 27.51 26.05 Example 2 27.52 25.96 Comparative Example 1 27.75 28.37

Dietary intake (g) Example 1 4.0 Example 2 4.0 Comparative Example 1 4.0

Through this, despite the fact that the dietary intake of all the experimental animals was similar, the body weight of the experimental animals that consumed the lactic acid bacteria sikhye of Examples 1 and 2 did not increase, but was similar to or decreased compared to before the experiment, showing that it can be used as a diet drink. Confirmed.

(2) Blood cholesterol and neutral lipids

The plasma neutral lipids, plasma cholesterol, epididymal neutral lipids, renal tissue neutral lipids, and liver tissue cholesterol of the experimental animals are shown in Tables 8 and 9 below.

Referring to Table 8 below, the test control group that consumed the lactic acid sikhye of Examples 1 and 2 had a significant decrease in plasma neutral lipids compared to before the experiment, while the test control group that consumed the regular sikhye of Comparative Example 1 showed no significant change. .

Before experiment (mg/dL) After experiment (mg/dL) Example 1 121 117 Example 2 121 115 Comparative Example 1 120 121

Referring to Table 9 below, it can be seen that the test control group that consumed the lactic acid bacteria Sikhye of Examples 1 and 2 had a significant decrease in plasma cholesterol compared to before the experiment, while Comparative Example 1 showed no significant change.

Before experiment (mg/dL) After experiment (mg/dL) Example 1 156 121 Example 2 157 120 Comparative Example 1 157 157

Through the above results, it was confirmed that the lactic acid bacteria Sikhye prepared in Examples 1 and 2 according to the present invention reduced the amount of neutral lipids and improved fat and cholesterol.

<Test Example 7> Effect of improving intestinal function and constipation

In order to determine the effect of lactic acid sikhye prepared in Examples 1 and 2 on improving intestinal function and constipation, the following method was performed.

To measure the amount of defecation, Sprague Dawley rats (Daehan Biolink, Eumseong) with an average male weight of about 220-240g (Daehan Biolink, Eumseong) were used as animal models, 8 to 10 rats per group, and were acclimatized in a metabolic cage for 3 days. After adaptation, feed containing 1mg of loperamide (Sigma, USA) per 3g of feed was supplied to induce constipation from the 4th day. The control group (loperamide treatment group) was supplied only with drinking water, and the lactic acid bacteria sikhye of Examples 1 and 2 were dissolved in drinking water at a concentration of 3.2 mg/ml and administered until the end of the experiment. Feces were collected daily and weighed.

As a result of testing the composition's effect on improving constipation, it was confirmed that it had an effect on improving constipation by increasing the amount of defecation during the constipation induction period compared to the control group.

Increase in variance compared to control group (%) Number of days since sample administration D+1 D+1 D+1 D+1 D+1 D+1 D+1 Lactobacillus Sikhye of Example 1 42 46 34 28 35 24 35 Lactic acid bacteria Sikhye of Example 2 45 46 33 29 38 28 36 General Sikhye of Comparative Example 1 4 5 6 7 6 6 8

<Test Example 8> Sensory test

In order to evaluate the sensory properties of the lactic acid sikhye of the present invention, a sensory test was conducted comparing the lactic acid sikhye prepared in Examples 1 and 2 and the regular sikhye of Comparative Example 1.

Sensory tests for aroma, taste, color, texture, and overall preference were repeated three times with 30 sensory testers, and the results shown in Table 11 below were obtained.

The evaluation scale was a 9-point scoring system (9 points: very good, 7 points: good, 5 points: average, 3 points: a little bad, 1 point: very bad). The sensory test results were verified for significant differences between each sample using ANOVA and Duncan's multiple range test at the 5% level. The results are shown in Table 11 (average values) below.

division Example 1 Example 2 Comparative Example 1 incense 9.0 9.0 4.3 taste 9.0 9.0 4.1 color 9.0 9.0 4.0 Texture 9.0 9.0 4.2 overall preference 9.0 9.0 4.1

As a result of the experiment, the lactic acid bacteria sikhye of Examples 1 and 2 according to the present invention had very high aroma, taste, color and texture, and the overall preference was 9, while the general preference of the general sikhye prepared in Comparative Example 1 was lower than that of the lactic acid bacteria sikhye of the present invention. Compared to this, it was very low at 4.1. From this, it was confirmed that lactic acid bacteria metabolites are highly fused with sikhye and provide a synergistic effect on sensory characteristics.

<Test Example 9> Storage evaluation

The lactic acid bacteria sikhye prepared in Examples 1 and 2 and the regular sikhye of Comparative Example 1 were each stored at 1°C for 12 months and then evaluated for changes in color, taste and aroma in the same manner as Test Example 8. It is shown in Table 12 below.

storage duration color taste incense Example 1 6 months later 8.7 8.5 8.5 12 months later 6.6 6.3 6.7 Example 2 6 months later 8.9 8.9 8.9 12 months later 7.7 7.4 7.8 Comparative Example 1 6 months later 1.7 1.6 1.7 12 months later 0.6 0.4 0.5

As shown in Table 12, the lactic acid bacteria sikhye of Examples 1 and 2 had almost no unpleasant odor even after 12 months, and the taste and color were almost maintained the same, while the general sikhye of Comparative Example 1 had a low pungency rating after storage. received.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

(S1) Step of making godubap;
(S2) preparing malt extract;
(S3) mixing the malt extract prepared in step (S2) with the godubap cooked in step (S1) and saccharifying it at 50 to 70°C for 5 to 7 hours;
(S4) Removing 15 to 25% (v/v) of godubap from the malt saccharification liquid saccharified in step (S3), washing it with cold water, heating at 95 to 100°C for 50 to 70 minutes, and reintroducing it. ;
(S5) adding a mixture of lactic acid bacteria metabolites, fructooligosaccharides, and sugar to the mixture obtained in step (S4) and then concentrating by heating at 95 to 100° C. for 50 to 70 minutes;
(S6) sterilizing and filtering the mixture obtained in step (S5) at 95 to 100° C. for 20 to 30 minutes to obtain lactic acid bacteria sikhye; and
(S7) cooling the lactic acid bacteria sikhye obtained in step (S6) at 60 to 80° C. and transferring it to a filling machine to fill and package it,
The lactic acid bacteria metabolites are obtained by inoculating one or more lactic acid bacteria selected from the group consisting of Lactobacillus plantarum ( L.plantarum ), Lactobacillus casei ( L.casei ), and Bifidobacterium infantis ( B. infantis ) into a liquid medium. It is manufactured by freeze-drying the fermentation broth obtained by culturing at 20 to 40°C for 12 to 48 hours,
The liquid medium contains 2.0 to 4.0% by weight of glucose, 0.5 to 2.0% by weight of peptone, 0.2 to 1.0% by weight of yeast extract, 2.0 to 4.0% by weight of inorganic salts, and 89.0 to 95.3% by weight of purified water,
In step (S5), grapefruit seed extract is additionally added,
The lactic acid bacteria metabolites are added in an amount of 0.02 to 0.03 parts by weight based on 100 parts by weight of malt, and the mixture of fructooligosaccharide and sugar is added in an amount of 110 to 130 parts by weight based on 100 parts by weight of malt,
In the step (S5), fructooligosaccharide and sugar are mixed at a weight ratio of 2:8.
delete delete delete delete Lactic acid bacteria sikhye manufactured according to the manufacturing method of paragraph 1.