KR20190114585A - Starter containing of lactic acid bacteria with superior effect for alcohol degradation activity and cheese containing alcohol metabolism using the same - Google Patents

Abstract

The present invention relates to a starter comprising lactic acid bacteria, and a fermented food manufactured using the starter and a method for manufacturing the same. The starter comprising the lactic acid bacteria and a fig extract of the present invention has high alcohol dehydrogenase activity to have excellent alcohol decomposability, and high acetaldehyde dehydrogenase activity to have excellent hangover reliving activity, and is excellent in acid resistance and bile resistance. Therefore, the starter can be used as a dairy product, health functional food, or food additive for preventing alcoholic liver disease or relieving hangover.

Description

Starter containing of lactic acid bacteria with superior effect for alcohol degradation activity and cheese containing alcohol metabolism using the same}

The present invention relates to a starter comprising a lactic acid bacterium and a fermented food prepared using the starter and a method for producing the same.

Food containing lactobacillus probiotics is a health food that transcends the range of palates, and it is useful to introduce useful live bacteria in the human intestines to reduce the growth of intestinal pathogens and contribute to health, as well as healing indigestion of diarrhea in children Effects have also been reported and are widely ingested. In addition, probiotics have been reported to suppress the growth of harmful bacteria that produce ammonia, amines, etc. to suppress the production of toxic substances, thereby reducing the amount of ammonia and alpha amino nitrogen in the blood, thereby improving symptoms of liver disease.

Among these useful bacteria have been reported to protect the liver from alcohol and to improve liver specific function (Raibaud, P: The 3rd International Synposium on Lactic Acid Bacteria and Human Health (1983), P116-126), probiotics lactobacillus ( Useful useful lactic acid bacteria such as probiotics lactobacillus and Bifidobacterium actively promote alcohol metabolism to rapidly decompose alcohol and to metabolize and detoxify acetaldehyde harmful to human body Has been reported (Nosava T et al: Alcohol and Alcoholism 35 (2000), P561-568). Studies on alcohol degradation and dairy product application of Lactobacillus family plantarum and fermentum strains, cheese production and alcohol degradation activity through strain screening, etc. There is not much progress by application.

Cheese is not only important food nutritionally important as a representative food, but also fermented foods that go well with various liquors such as wine or beer. Compared to the widespread use of cheese, studies on cheese in functional aspects such as alcohol degradation have not been diverse.

Figs (Ficus carica L.) are reported to contain high calcium, dietary fiber, ficin, high cholesterol and polyphenols, and are effective in treating heart disease and obesity. Known. Ficin, a proteolytic enzyme, is widely used as a curdase additive for cheese making as a substitute for meat and animal enzymes. Figs are also rich in Aspartic acid and Glutamic acid, which are involved in the promotion of ADH, and are rich in minerals such as Ca.

The present inventors screened excellent strains that can be utilized in probiotics by checking the ethanol resistance and acid, bile resistance, and alcohol degrading activity of lactic acid bacteria, and grown together with lactic acid bacteria and fig extracts having alcohol degrading activity and then elevated alcohol degrading activity. It was confirmed that, by using a novel starter was confirmed that the cheese can be produced excellent alcohol resolution was completed the present invention.

Accordingly, it is an object of the present invention to provide a starter and a method of manufacturing the same that can be used to produce foods having excellent alcohol decomposition and hangover relief effects when used in preparing various fermented foods.

Another object of the present invention is to provide a fermented food prepared by the starter and greatly enhanced alcohol resolution and a method for producing the same.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention is Lactobacillus kitasatonis ( Lactobacillus kitasatonis ), Lactobacillus amylophillus ( Lactobacillus amylophillus ) and at least one selected from the group consisting of Leuconostoc mesenteroides sub. Provided are starters comprising lactic acid bacteria and fig extracts.

The present invention also provides a medium containing a fig extract Lactobacillus Kita Sato varnish (Lactobacillus kitasatonis), Lactobacillus amyl to fill loose (Lactobacillus amylophillus) and flow Pocono stock mesen steroid selected from the group consisting of (Leuconostoc mesenteroides sub.) 1 It provides a starter manufacturing method comprising the step of inoculating more than one species of lactic acid bacteria.

The present invention also provides a fermented food fermented with the starter.

Fermented food of the present invention is characterized in that the dairy product containing cheese, butter and fermented milk.

The present invention also provides a method for producing a fermented food comprising the step of adding the starter.

The present invention also provides a Lactobacillus Kita Sato varnish (Lactobacillus kitasatonis), Lactobacillus amyl to fill loose (Lactobacillus amylophillus) and flow Pocono stock mesen steroid (Leuconostoc mesenteroides sub.) One or more kinds of lactic acid bacteria and fig extract selected from the group consisting of Provide health functional foods that include.

The starter comprising the lactic acid bacteria and fig extract of the present invention has high alcohol dehydrogenase activity, and thus has high alcohol decomposability, and high acetaldehyde dehydrogenase activity, which is excellent in hangover elimination activity, and excellent in acid resistance and bile resistance. Therefore, it can be used as a dairy product, dietary supplement or food additive to prevent alcoholic liver disease or to relieve hangover.

1 is a schematic view showing a cheese manufacturing method according to an embodiment of the present invention.
Figure 2 shows the results confirming the ethanol resistance of 10 strains, including Lactobacillus amylophillus ( Lactobacillus amylophillus ).
Figure 3 is a result confirming the acid resistance and bile resistance of 10 strains including Lactobacillus amylophillus ( Lactobacillus amylophillus) .
Figure 4 is a result confirming the alcohol dehydrogenase (adh) activity of 10 strains including Lactobacillus amylophillus ( Lactobacillus amylophillus) .
Figure 5 shows the results of acetaldehyde dehydrogenase (aldh) activity of 10 strains, including Lactobacillus amylophillus ( Lactobacillus amylophillus) .
With Lactobacillus amyl when Figure 6 is the addition of a fig extract, field Ruth (Lactobacillus amylophillus), Lactobacillus Kita Sato varnish (Lactobacillus kitasatonis) and flow Pocono stock mesen steroid alcohol dehydrogenase (adh) increase in (Leuconostoc mesenteroid) The result of confirming the activity.
Figure 7 is a Lactobacillus-amyl the addition of a fig extract, field Ruth (Lactobacillus amylophillus), Lactobacillus Kita Sato varnish (Lactobacillus kitasatonis) and flow Pocono stock mesen steroid (Leuconostoc mesenteroid) The acetaldehyde dehydrogenase (aldh increase in ) It is the result of confirming the activity.
8 is a real view of the cheese produced using the fig extract and Lactobacillus amylophillus ( Lactobacillus amylophillus ).
9 is an alcohol dehydrogenase of cheese prepared using fig extract and Lactobacillus amylophillus , Lactobacillus kitasatonis or Leuconostoc mesenteroid Lactobacillus amylophillus . (adh) It is the result of confirming activity.
Figure 10 shows the acetaldehyde dehydrogenase ( Lactobacillus amylophillus ) , Lactobacillus kitasatonis ( Lactobacillus kitasatonis ) or leuconostoc mesenteroid ( leuconostoc mesenteroid ) of cheese prepared using aldh) activity was confirmed.

Hereinafter, the present invention will be described in detail.

The inventors have found that Lactobacillus kitasatonis , Lactobacillus amylophillus , and Leuconostoc mesenteroides sub. Strains are resistant to alcohol, acid, bile, and alcohol dehydrogenase. It was confirmed that the antase and acetaldehyde dehydrogenase activity is excellent, and when the strain was cultured with the addition of the fig extract, it was confirmed that the alcohol-degrading activity was excellent, thus leading to the present invention.

According to one embodiment of the invention, the invention is Lactobacillus kitasatonis ( Lactobacillus kitasatonis ), Lactobacillus amylophillus ( Lactobacillus amylophillus ) and Leuconostoc mesenteroides sub. It contains more than a species of lactic acid bacteria and fig extract, to provide a starter for dairy raw material fermentation.

As used herein, the term "lactic acid bacteria" refers to a generic term for bacteria that ferment sugars to obtain energy and produce large amounts of lactic acid. This is characterized in that it develops well in an environment with low oxygen to produce lactic acid from various sugars.

In the present invention, "starter" refers to a seed of lactic acid bacteria used for producing lactic acid fermentation of raw material oil and an energy component requiring fermentation in the manufacture of fermented foods such as cheese, butter and fermented milk.

In the present invention, "fermentation" is to produce lactic acid by lactic acid bacteria, two types of lactic acid fermentation, normal lactic acid fermentation and abnormal lactic acid fermentation to further produce alcohol. In addition, as a concept corresponding to corruption, it means that yeast or bacteria decompose carbohydrates in the absence of oxygen to obtain lactic acid and energy. Specifically, the present invention is not limited thereto. The process of obtaining fermented food may be included.

In the present invention, the "fermented food" refers to a generic name of a substance made by using an action of synthesizing new components by decomposing organic substances in raw oil by the microbial action of lactic acid bacteria, but is not limited thereto, for example, cheese, butter , Yogurt, yogurt, cream and the like.

Lactobacillus kitasatonis , Lactobacillus amylophillus , and Leuconostoc mesenteroides sub. Of the present invention have a growth reduction rate of 0.5 to 6% at 5% ethanol concentration. It is a strain selected by the present inventors as a strain having excellent alcohol resistance, excellent acid resistance and bile resistance, which can be used as a probiotic, and excellent in alcohol dehydrogenase activity and acetaldehyde dehydrogenase activity.

The lactic acid bacteria of the present invention is excellent in alcohol degrading activity alone, but may further include a fig extract to further increase the alcohol degrading activity of the lactic acid bacteria. Preferably, the fig extract may be included in an amount of 0.1 to 15% by weight based on the total weight of the starter. When the fig extract is included in an amount of less than 1% by weight, the synergistic effect by the fig extract is insignificant, and when the fig extract exceeds 15% by weight, the sour taste of the fermented food may become strong, and the appearance of the starter may be deteriorated.

The starter of the present invention is most preferably used to produce fermented foods such as cheese, yoghurt, fermented milk by fermenting milk, but, if necessary, dairy products (yogurt, cheese, fermented milk) prepared using the starter of the present invention. The fermentation can be changed to suit the taste.

In an embodiment of the present invention, the Lactobacillus kitasatonis has a deposit number of KCTC 3155, the Lactobacillus amylophillus has a deposit number of KCTC 3161, and the leukonostock mesene The steroid ( Leuconostoc mesenteroides sub. ) Used a deposit number of KCTC 3718, but is not limited thereto.

According to another embodiment of the present invention, Lactobacillus kitasatonis , Lactobacillus amylophillus , and Leuconostoc mesenteroides sub. It provides a method for producing a starter comprising the step of inoculating one or more lactic acid bacteria selected from the group.

In the starter manufacturing method of the present invention, the present invention can further increase the alcohol decomposition effect by including the fig extract in an appropriate blending ratio with lactic acid bacteria. Preferably the fig extract may be added in an amount of 1 to 15% by weight based on the total weight of the medium.

According to another embodiment of the present invention, the present invention provides a fermented food prepared using the starter.

The fermented food in the present invention is a product fermented by adding the starter according to the present invention in milk, for example, cheese, butter and yogurt, etc., if the food can be obtained by fermentation by adding lactic acid bacteria to milk It is not limited.

In addition, in the present invention, fermented foods having an alcohol resolution and a hangover removal effect can be prepared by adding fermentation secondary to the dairy products (cheese, butter, yogurt, fermented milk, etc.) as necessary, and fermenting secondary.

The fermented food in the present invention may be a food obtained by fermentation after adding the starter according to the present invention in dairy products. Herein, the fermentation temperature and time are not particularly limited and may be performed under process conditions generally used in the art according to the type of food desired.

According to another embodiment of the present invention, the present invention provides a method for producing a fermented food comprising the step of adding a starter according to the present invention to milk or a dairy product prepared using the same.

By using the starter according to the present invention it is possible to produce a fermented food excellent in alcohol resolution and hangover effect.

In the fermented food manufacturing method of the present invention, it may further comprise the step of fermentation after the addition step. Herein, the temperature and time of the fermentation process are not particularly limited and may be performed under process conditions generally used in the art according to the type of food desired.

According to another embodiment of the invention the invention is Lactobacillus Kita Sato varnish (Lactobacillus kitasatonis), Lactobacillus amyl to fill loose (Lactobacillus amylophillus) and flow Pocono stock mesen steroid selected from the group consisting of (Leuconostoc mesenteroides sub.) 1 It provides a health functional food comprising more than a species of lactic acid bacteria and fig extracts.

In the present invention, the "health functional food" is meant to bring a specific effect on health when ingested, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a raw material.

When formulating the health functional food in the form of powder, granules, tablets or capsules, it may further include appropriate carriers, excipients and diluents commonly used in food preparation. The carriers, excipients and diluents may include lactose, dexter Rose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy Benzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. . In addition to the above excipients, lubricants such as magnesium styrate and talc are also used.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples are intended to illustrate the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited thereby.

Preparation Example 1 Extraction of Fig Coenzyme

The figs used for the test were obtained from the Agricultural Cooperatives as a Masui cultivar, and the extract of fig coenzyme was chopped and the homogenizer was added by adding 200% (w / v) 0.1M sodium phosphate buffer (pH 7.0), 5 mM cysteine, and 2 mM EDTA Homogenized using (K555, Kitchenaid, Benton Harbor, NY, USA) and filtered twice with cheese cloth. The filtrate was centrifuged at 5,500 rpm for 20 minutes and the supernatant was taken. The supernatant was stored at minus 75 ℃ was used as the basic coenzyme solution.

Example 1. Selection of lactic acid bacteria excellent in ethanol resistance

1-1. Preparation of Microorganisms

The experimental microorganisms used in this study are test strains and are shown in Table 1 below. This Lactobacillus strain was purchased from KCTC, Korea, and each strain was cultivated and identified for 24 hours at each optimal culture temperature in MRS medium (Difco Lab., Spark, MD, USA). Prepared and used.

Sample No. KCTC No. Strain name culture
Temperature badge anaerobe One 3109 Lactobacillus casei 37 MRS Tong 2 3115 Lactococcus lactis 37 3 3139 Lactobacillus gasseri 37 4 3140 Lactobacillus acidophilus 37 5 3155 Lactobacillus kitasatonis 37 6 3161 Lactobacillus amylophillus 30 7 3237 Lactobacullus rhamnosus 37 8 3498 Lactobacillus brevis 30 9 3718 Leuconostoc mesenteroides subs . 37 10 3528 Leuconostoc lactis 30

1-2. Confirmation of Ethanol Resistance of 10 Lactic Acid Bacteria

After adding ethanol sterilized with a membrane filter to 5% and 10%, respectively, in the pre-sterilized MRS liquid medium, 10 lactic acid strains of Table 1 activated in the MRS liquid medium were adjusted to OD 1 level 1 % Was added and allowed to stand for 48 hours at each optimum incubation temperature. Thereafter, each strain was diluted 10 ^-6 times and smeared 0.1 ml in MRS solid medium to measure the number of viable cells after incubation for 48 hours at each optimum culture temperature.

As a result, as shown in FIG. 2, in the case of 5% ethanol concentration, all 10 strains showed a decrease in growth rate of up to 20% or less compared to ethanol-free, and thus it was confirmed that the ethanol concentration was excellent in 10%. In the case of L. kitasatonis, L. amylophillus, L. brevis, L. mesenteroides subs. All of the other strains except for four strains showed a significant reduction of more than 90%, indicating that the resistance to ethanol was greatly reduced. Among 10 strains, L. amylophillus strain showed 0.52% growth rate at 5% ethanol concentration, showing little decrease in growth rate by ethanol, and only 47% growth rate at 10% ethanol concentration. It was confirmed that the ethanol resistance is excellent. L. kitasatonis , L. brevis , L. mesenteroides subs . The strains also showed low reduction rates of 5%, 16%, and 6% at 5% ethanol concentrations, respectively, and growth rates of 51%, 45%, and 53% growth at 10% ethanol concentrations. there was.

Example 2 Screening of Lactic Acid Bacteria with Excellent Acid and Bile Resistance

2-1. Acid resistance confirmation experiment

10 kinds of lactic acid strains activated in MRS liquid medium were OD 1 level, centrifuged at 3,000 rpm for 20 minutes using a centrifuge (Combi514R, Hanil, Korea), and the supernatant was discarded. 1 N HCl adjusted to pH 2.5 was added 1% pepsin to the MRS liquid medium to prepare a gastric juice. Incubate the recovered bacteria with the same amount of supernatant as the supernatant, incubate for 2 hours at each optimal incubation temperature, dilute 10 ^-6 times, and smear 0.1 ml into MRS solid medium for 48 hours at each optimal incubation temperature. The viable cell count was then measured. The control group used a medium without adjusting the pH.

2-2. Bile Resistance Test

Artificial bile solution was prepared by adding 1% oxgall solution prepared in 10% MRS liquid medium. The cultured bacteria were incubated for 2 hours at the optimal culture temperature by diluting artificial bile solution with the supernatant, and diluted 10 ^-6 times, and smeared with 0.1 ml in MRS solid medium, and cultured for 48 hours at the optimum culture temperature. Was measured. As a control, a medium without 10% oxgall was used. After culturing the strain in the MRS liquid medium containing artificial gastric juice and artificial bile as shown in Figure 3 and measuring the number of viable cells, and cultured the strain without adding anything to the control group measured the number of viable cells and analyzed the results.

2-3. Acid and Bile Resistance Test Results

As shown in Figure 3, L. gasseri, L. kitasatonis, L. amylophillus, L. mesenteroides subs . The growth rate of the strain was significantly higher. L. gasseri showed about 11%, 12%, L. amylophillus , about 17%, 9%, and L. mesenteroides subs. About 6%, 8%, respectively. In particular, L. kitasatonis strains showed about 46% and 14% of acid and bile growth rate, indicating that acid and bile resistance were better than other strains. L. casei and L. rhamnosus showed low growth rates of 0.3-0.4% and 0.2-0.7%, respectively. Park et al. (143) reported that the growth rates of L. casei and L. rhamnosus strains from foods were high at 77% and 82%, respectively. However, L. casei and L. rhamnosus strains showed a level of 10 ⁷ CFU / mL, but the other strains were resistant to growth above 10 ⁸ CFU / mL.

Example 3 Screening of Lactic Acid Bacteria with Excellent Alcohol Metabolic Activity

3-1. Preparation of crude extract of strain

The cells of 10 strains of lactic acid strains activated in MRS liquid medium were prepared by O.D. At 1 level, the cells were disrupted for 5 minutes using an ultrasonic cell crusher (VC505, Sonics & Materials, INC., Newtown, CT, USA), and then at 12,000 rpm using an ultra-high speed centrifuge (Smart R17, Hanil, Korea). After centrifugation for 20 minutes, the supernatant was taken and used as crude extract of the strain. ADH and ALDH activity tests of crude extracts of each strain were performed by referring to and modifying Nosova's method. The crude extracts of each strain were measured for protein content using the bradford method, and the activity of ADH and ALDH enzymes was calculated using the standard curve obtained by measuring the NADH concentrations of the crude extracts of each strain. Calculated as the amount of NADH produced per hour.

3-2. Determination of ADH Activity in Crude Extracts of Strains

ADH activity of the crude extracts of each strain was 1 mL in total, and the final concentration was 0.1 M Glycine buffer (pH 9.6), 2.5 mM NAD ⁺ , 25 mM ethanol. The extract was added and reacted for 5 minutes in a 25 ° C. constant temperature water bath. After the reaction was completed, the amount of NADH was measured at 25 ° C. and 340 nm using a UV / VIS Spectrometer (Sunrise ^™ , TECAN, M * j * nnedorf, Switzerlan).

3-3. Determination of ALDH Activity of Crude Extracts of Strains

100 ml of each strain was prepared by preparing the reaction solution so that the final concentration of ALDH activity was 60 mM Sodium pyrophosphate buffer (pH 8.8), 1 mM NAD ⁺ , 0.1 mM Acetaldehyde and 0.1 mM 4-methylpyrazole. The crude extract of was added and reacted for 5 minutes in a 25 ℃ constant temperature water bath. After the reaction was completed, the amount of NADH was measured at 25 ° C. and 340 nm using a spectrophotometer.

To examine the specificity of the strain for selection as a probiotic for 10 strains, alcohol metabolism activity was determined by generating NADH in vitro using the activities of ADH and ALDH enzymes produced by each strain. Measurements are shown in FIGS. 4 and 5.

3-4. Alcohol metabolism activity measurement result

As a result, all 10 strains were found to have enzyme activity against ADH and ALDH, and confirmed a significant difference by strain. In particular, L. kitasatonis and L. amylophillus showed 30% higher activity of ADH and ALDH enzymes than other strains. L. kitasatonis strain showed high ADH and ALDH activities of about 660 nmole / min / mg protein and 579 nmole / min / mg protein, and L. amylophillus strain showed about 660 nmole / min / mg protein and 607 nmole / min / mg The protein showed the best ADH and ALDH activity compared to other strains. L. mesenteroides subs. The strains showed activity of about 591 nmole / min / mg protein and 454 nmole / min / mg protein, respectively . The strains of L. brevis were about 522 nmole / min / mg protein and 558 nmole / min / mg protein, respectively. Significantly high ADH and ALDH activity. The remaining strains also showed enzymatic activity of over 400 nmole / min / mg protein.

Example 4. Confirmation of synergistic effect according to the addition of fig extract

In order to investigate the synergistic effect between strains and figs, lactobacillus strain and fig enzyme extracted from figs, which were finally selected in the basic experiment, were inoculated into MRS liquid medium by concentrations (0, 1, 5, 10%). After culturing for 24 hours at the optimum incubation temperature, the enzyme activity, cheese production, antioxidant research, and component analysis were performed after adding figs.

4-1. Confirmation of Growth Status of Strain According to Fig Concentration

Crude crude extract was inoculated in MRS liquid medium by concentration (0, 1, 5, 10%) to three strains (OD 1 level) determined to be stable as probiotics through Examples 1 to 3 for 24 hours. After incubation, the growth state of each strain by fig concentration was measured by absorbance, and the results are shown in <Table 2>.

% L. kitasatonis L. amylophillus L. mesenteroides subs. 0 2.030 ± 0.13 ^bc 2.166 ± 0.25 ^abc 1.946 ± 0.16 ^c One 2.086 ± 0.11 ^abc 2.113 ± 0.18 ^abc 2.063 ± 0.03 ^bc 5 2.138 ± 0.16 ^abc 2.170 ± 0.14 ^abc 2.198 ± 0.04 ^abc 10 2.239 ± 0.07 ^ab 2.337 ± 0.04 ^a 2.131 ± 0.07 ^abc

All three strains showed no significant difference in growth according to the crude crude extract concentration. L. kitasatonis , L. amylophillus , L. mesenteroides subs. 3 strains absorbance Despite appear to roughly around 1.946 ~ 2.337 fig crude this absorbance value was increased slightly depending on the concentration of the extract, in particular L. In the case of strains amylophillus the absorbance at 10% fig crude extract solution was added a little higher than other strains to 2.337 The results were shown.

4-2. Confirmation of Alcohol Metabolism Activity by the Concentration of Fig Extract

The various enzymes produced by lactic acid bacteria exist in the cytoplasm and sometimes go outside through the cell wall. Therefore, alcohol metabolic activity was measured by dividing into intracellular and external strains. Intracellular activity experiments showed O.D. Washed three times with sterile 0.1 M potassium phosphate buffer (pH 7.4) at 1 level and crushed cells for 5 minutes using an ultrasonic cell crusher (VC505, Sonics & materials, INC., Newtown, CT, USA) After centrifugation at 12,000 rpm for 20 minutes using a centrifuge (Smart R17, Hanil, Korea), the supernatant was taken and used as crude extract of the strain. In the extracellular activity experiment, the culture medium was incubated for 24 hours at the optimum culture temperature for each selected strain, centrifuged at 12,000 rpm for 20 minutes, and the supernatant was taken as a crude extract of the strain. In addition, 10% of the melon and pineapple crude extracts were added and cultured as a control group to carry out an extracellular activity experiment. Alcohol metabolism activity measurement of each strain according to the concentration of the crude crude extract was carried out by separating the cells into cells, the results are shown in FIG.

As shown in Figure 6, ADH enzyme activity results can be confirmed the significant difference according to the crude crude extract concentration, strain. In addition, the enzyme activity in the extracellular was confirmed to be higher than the intracellular. L. amylophillus strain showed the best activity, ADH activity was increased with increasing the fig concentration. In particular, the addition of 10% crude crude extract in the extracellular showed activity of about 1,054.98 μmole / min / mg protein, and the addition of crude crude extract in the extracellular showed about 270% higher activity than the 390.18 μmole / min / mg protein. Addition of 10% crude extract showed about 350% higher activity than that of intracellular 300.99 μmole / min / mg protein. L. kitasatonis , L. mesenteroides subs. The strains also showed an increase in enzyme activity of up to 170% and 190% in extracellular with increasing crude extract concentration. L. mesenteroides subs. The extracellular and intracellular strains showed the highest activity at the crude extract concentration of 5%, and were similar or lower at 10%.

As shown in the results of FIG. 7, the results of ALDH enzyme activity also showed a similar tendency to ADH, and the significant differences according to the crude extract concentrations and the strains were confirmed. The ALDH enzyme activity of L. amylophillus was the highest in both extracellular and intracellular strains. Addition of 10% fig crude extract from the extracellular resulted in approximately 280% increase in activity with approximately 1,209 μmole / min / mg protein. L. kitasatonis , L. mesenteroides subs. The strain also exhibited high activity of about 170% and 190% compared to no addition. As with ADH, low ALDH activity was observed except for L. amylophillus strain, which was about 10% of extracellular solution.

Example 5 Preparation and Quality Analysis of Cottage Cheese

5-1. Preparation of Cottage Cheese

Cottage cheese was manufactured in the order and method shown in FIG. Lactic acid bacteria added to the cheese was used after inoculation and incubation of three selected strains and crude enzyme extracted from figs (0, 1, 5, 10%) by concentration (0, 1, 5, 10%). Raw milk was stabilized after heating commercially available milk (Busan milk) to 30 ~ 32 ℃. Stabilized milk was added 5% of the selected strain and 0.2% of the crude crude extract and fermented for 7-8 hours until the pH 4.6 ~ 4.8. The curd milk was cut evenly with 10 mm cheese knives, stabilized for about 20 minutes, and then heat-treated while stirring at a constant strength and speed while raising the temperature from 30 ° C. to 55 ° C. for 1 hour and 30 minutes at 30 minute intervals. After the heat treatment was completed, the whey was separated using a cheese cloth, and the cheese was cooled to 33-35 ° C. and 5 ° C. over 1 and 2 times using purified water. After removing water for 10 to 12 hours, half of the finished cheese sample was sealed in a vacuum pack, stored in a 4-8 ° C. refrigerator, and used for analysis. Some of the remaining cheese samples were freeze-dried using a freeze dryer (Freeze dryer FD8508, IlshinBioBase, Korea), sealed in a vacuum pack, stored in a -20 ° C freezer, and used for analysis. The form of the cheese prepared using the control cheese and the fig coenzyme solution and Lactobacillus amylophillus is shown in FIG. 8.

5-2. Measurement of Alcohol Metabolism Activity in Cheese

Alcohol metabolism activity of cheese was studied in the same way as strain screening study. Commercial cottage cheese (USA) was purchased and used as a control, and pretreatment of the cheese for use in the experiment was performed by referring to and modifying the method of Maytiya Konkit, Kuchroo. The fresh cheese sample was adjusted to the level of 100 mg / mL in distilled water, homogenized at 200 rpm using a homogenizer (Ultra Turrax T18, IKA, Germany) for 30 minutes, and the cheese components were eluted for 2 minutes using an ultrasonic cell crusher. After centrifugation at 12,000 rpm for 30 minutes using an ultrafast centrifuge, the supernatant between the fat layer and the sediment layer was taken and used as crude extract of cheese.

As a result, as shown in Figures 9 and 10, in the commercial control cheese used as a control enzyme activity of ADH and ALDH 32.84 μmole / min / mg protein, 23.85 μmole / min / mg protein, respectively, the present invention The cheese produced using the lactic acid bacteria showed the activity of about 5 to 10% of ADH and ALDH activity, it was confirmed that the alcohol metabolism activity of the cheese prepared using the lactic acid bacteria used in the present invention. As a result of ADH enzyme activity of cheese, it was confirmed the significant difference according to the crude crude extract concentration and strain. L. amylophillus strains tended to increase with the concentration of the crude crude extract, and the activity of about 1121.41 μmole / min / mg protein with 10% of the crude crude extract was approximately 445.62 μmole / min when the crude crude extract was not added. It was confirmed that 252% higher activity compared to / mg protein. However, the activity of L. kitasatonis strain increased until 5% crude crude extract was added, but decreased at 10% concentration. L. mesenteriodes subs. In the case of the strain, the activity decreased as the crude crude extract concentration increased, and the activity decreased by about 50% compared to the added group. The ALDH enzyme activity of the cheese showed a similar tendency to the ADH activity. In particular, L. amylophillus strains showed excellent ALDH activity. When the crude extract of 10% was added, the activity was about 516.2 μmole / min / mg protein. L. kitasatonis , L. mesenteriodes subs. The strains also showed 175% and 195% increased activity with 368.68 μmole / min / mg protein and 379.89 μmole / min / mg protein, respectively.

5-3. Ingredient Analysis of Cheese

Sample extract pretreatment for the analysis of the composition of the cheese was carried out as follows with reference to Shaiban et al. 1 g of a freeze-dried cheese sample was added to 50 ml of 95% methanol and 1% HCl, and homogenized at 50 ° C. at 200 rpm for 30 minutes. The mixed solution was filtered using a cheese filter cloth after cooling, and the remaining material was washed with the above solvent and used as a sample.

<Measurement of total polyphenol content>

Total polyphenol content was measured by applying the Folin-Denis method. Distilled water was mixed with the sample extract to dissolve at 1000 μg / mL, 500 μL of 0.1 N Folin-Ciocalteau reagent was added and reacted at room temperature for 5 minutes, and 500 μL of a 1 M Na 2 CO 3 solution was mixed. After reacting for 1 hour, absorbance at 765 nm was measured. The total polyphenol content of each sample was expressed as the dose of Gallic acid (mg GAE / 100g). As a result of the content measurement, the analysis results as shown in Table 3 were obtained.

% L. kitasatonis L. amylophillus L. mesenteroides subs. 0 805 ± 55 ^e 1038 ± 14 ^cd 1170 ± 42 ^b One 1052 ± 7 ^c 1130 ± 25 ^b 1161 ± 22 ^b 5 989 ± 7 ^cd 1050 ± 15 ^c 1184 ± 96 ^b 10 977 ± 4 ^d 1135 ± 4 ^b 1309 ± 11 ^a

As shown in Table 3, about 805-1,309 mg / 100 g of polyphenols were produced, and all three strains were found to increase significantly according to the concentration of the crude crude extract, and 10% of the crude crude extract was added. In the case of gourd cheese, about 977 ~ 1,309mg / 100g of polyphenol was produced, and it was confirmed that it increased significantly by about 10 ~ 20% compared to the non-added ball. Of these, L. mesenteroides subs. The highest polyphenol content of the strain was 1309 mg / 100 g. L. amylophillus strain was the second highest with 1,135 mg / 100 g. L. kitasatonis strain showed the highest content of 1,052 mg / 100g in 1% of fig extract, but it decreased after the concentration of crude extract increased.

5-4. Determination of Antioxidant Effect of Cheese-Measurement of ABTS Radical Scavenging Activity

ABTS radical scavenging ability was measured by applying Re's method. After mixing 2.6 mM potassium persulphate in 7.4 mM ABTS solution for 24 hours in the dark at room temperature, positive ion (ABTS · +) was formed and the absorbance value was 0.8 ± 0.02 at 734 nm (PBS). ), pH 7.4. 150 μL of the diluted ABTS · + solution and 5 μL of the sample extract (1000 μg / mL) were mixed, reacted at room temperature for 10 minutes, and the absorbance was measured at 734 nm. ABTS radical scavenging ability was expressed as a percentage of the control group using distilled water as a control (Table 4).

<Equation 1>

% L. kitasatonis L. amylophillus L. mesenteroides subs. 0 20.92 ± 0.70% ^cd 20.83 ± 0.55% ^cd 21.44 ± 0.42% ^bc One 19.92 ± 0.27% ^d 21.23 ± 0.93% ^bc 23.18 ± 0.37% ^a 5 21.20 ± 0.50% ^bc 22.75 ± 0.18% ^a 22.26 ± 0.63% ^ab 10 22.84 ± 0.85% ^a 22.81 ± 0.80% ^a 23.24 ± 0.45% ^a

As shown in Table 4, all of the cheeses produced using the three strains were able to confirm that the ABTS radical scavenging ability is 20% or more, and there was no significant difference in ABTS radical scavenging ability according to each strain. However, it was confirmed that the scavenging ability increased significantly with the addition of the crude crude extract, and when the crude crude extract was added 10%, the scavenging ability was 22.81 ~ 23.24%, which was increased by about 10% compared to the non-added extract. there was.

5-5. Antioxidant Effect of Cheese-Measurement of DPPH Radical Scavenging Activity

Electron donating ability (EDA) was measured by applying the method of Blois. 900 μL of 100 μM DPPH solution dissolved in ethanol and 100 μL of sample extract dissolved at 1000 μg / mL were mixed. This mixed sample was reacted for 30 minutes in the dark at room temperature, and the absorbance was measured at 517 nm. Electron donating ability was expressed as a percentage of the control group using distilled water as a control. As a result, the analysis results as shown in Table 5 were obtained.

<Equation 2>

% L. kitasatonis L. amylophillus L. mesenteroides subs. 0 14.8 ± 0.7% ^f 13.3 ± 0.5% ^g 17.9 ± 0.4% ^e One 26.6 ± 0.2% ^d 15.0 ± 0.8% ^f 15.7 ± 0.3% ^f 5 38.3 ± 0.5% ^b 34.9 ± 0.1% ^c 39.4 ± 0.6% ^a 10 40.4 ± 0.8% ^a 39.8 ± 0.9% ^a 37.6 ± 0.4% ^b

As shown in Table 5 can be confirmed the significant DPPH scavenging ability by the amount of fig extract crude extract by strain. In particular, L. kitasatonis strain exhibited the highest scavenging activity of 40.4% when 10% of fig extracts were added, and increased activity by more than about 270% compared to 14.8% scavenging activity. The L. amylophillus strain showed 39.8% scavenging activity in 10% of the crude extract, but showed a 3-fold increase in activity. L. mesenteroides subs. The strain showed the highest activity at 39.4% in the 5% addition of fig crude extract, and then decreased slightly at 10%.

5-6. Statistical analysis

All experiments were repeated three times and averaged. Statistical analysis was analyzed by analysis of variance of the SPSS program (Statistical package for the social sciences, version 22.0, SPSS Inc., Chicago, I L. , USA), and the mean value of the samples was tested by Duncan's multiple range test (p). <0.05) was performed.

In conclusion, in the present invention, the lactic acid bacteria and the fig extract having the alcohol-degrading activity were grown together, the cheese was prepared, and the alcohol-degrading activity and the antioxidant activity of the cheese were confirmed. These functional cheeses are expected to have high applicability due to hangover and antioxidant effects in the food market.

Claims (12)

Lactobacillus Kita Sato varnish (Lactobacillus kitasatonis), Lactobacillus amyl with loose fill (Lactobacillus amylophillus) and flow Pocono stock mesen steroid (Leuconostoc mesenteroides sub.) Starter containing lactic acid bacteria and at least one extract selected from the group consisting of fig. The starter of claim 1, wherein the fig extract is included in an amount of 1 to 15 wt% based on the total weight of the starter. The starter according to claim 1, wherein the lactic acid bacterium has excellent growth resistance at 0.5% to 6% at 5% ethanol concentration. The starter according to claim 1, wherein the starter is characterized by increased alcohol dehydrogenase and aldehyde dehydrogenase enzyme activities. The method according to claim 1, wherein the Lactobacillus kitasatonis ( Lactobacillus kitasatonis ) has a deposit number of KCTC 3155, the Lactobacillus amylophillus ( Lactobacillus amylophillus ) has a deposit number of KCTC 3161, the Lactobacillus kitasatonis Leuconostoc mesenteroides sub. ) Is a starter with an accession number of KCTC 3718. Nice to the addition of fig extract medium Lactobacillus Kita Sato (Lactobacillus kitasatonis), Lactobacillus amyl to fill loose (Lactobacillus amylophillus) and flow Pocono stock mesen steroid (Leuconostoc mesenteroides sub.) Inoculated with one or more lactic acid bacteria selected from the group consisting of Including a step of culturing, the starter manufacturing method. The method of claim 1, wherein the fig extract is added in an amount of 1 to 15% by weight based on the total weight of the medium. Fermented foods fermented with the starter of any one of claims 1 to 5. The fermented food product of claim 8, wherein the fermented food product is a dairy product including cheese, butter and fermented milk. Claim 1 to claim 5, comprising the step of adding a starter, a method for producing a fermented food. The method of claim 10, wherein the fermented food is dairy product including cheese, butter and fermented milk. Lactobacillus Kita Sato Nice (Lactobacillus kitasatonis), Lactobacillus amyl by Phil Ruth (Lactobacillus amylophillus) and Chapter Kono Stock mesen steroid supplements that contain lactic acid bacteria and fig extracts at least one selected from the group consisting of (Leuconostoc mesenteroides sub.) .

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