KR101159237B1 - Novel leuconostoc citreum enhancing anti-oxidant activity by garlic fermentation - Google Patents

Abstract

PURPOSE: A novel Leuconostoc citreum strain is provided to improve antioxidation and to increase sulfur compounds. CONSTITUTION: A composition for improving antioxidative activity of garlic contains Leuconostoc citreum strain KACC91593P as an active ingredient. A composition for enhancing sulfur compounds of garlic contains the strain as an active ingredient. A method for enhancing antioxidative activity comprises a step of fermenting garlic using the strain. A method for enhancing the sulfur compounds of garlic comprises a step of fermenting garlic with the strain.

Description

Novel Leuconostoc citreum enhancing anti-oxidant activity by garlic fermentation

The present invention relates to a novel Leuconostoc citreum strain and its application to enhance antioxidant activity through garlic fermentation.

Garlic in general ( Allium sativum L.) is an annual plant belonging to the genus Allium of the family Liliaceae, and is grown not only in Korea but also in various regions such as India, China, the United States, and Southern Europe (Kim et al. 2009, Korean J Culinary Res 15). : 59-66). Garlic is widely used as a spice because it contains a sulfur-containing volatile substance that exhibits a unique aroma, and it is known that garlic's sulfur-containing substances are known to have physiological activities such as antibacterial, anticancer, antithrombotic, and antioxidant properties. It is widely used as a functional material of (Song et al. 2009, Korean J Food Preserv 16: 929-937). As the usefulness of garlic is widely known, its cultivation volume has increased, and accordingly, the amount of garlic discarded due to lack of merchandise in processing has been increased, and researches on the treatment of garlic by-products are being conducted (Kim et al. 2009, Food Eng Prog 13: 352-359).

Fermentation is applied as one of the methods to improve the utilization efficiency of the plant, and it is reported that the fermentation process can increase the useful components of the plant and improve its efficacy (de Castro et al. 1998, International Journal of Food Microbiology). 39: 205-211; Sato et al. 2006, Plant Foods for Human Nutrition 61: 157-160). However, garlic has a wide range of antimicrobial activities against various microorganisms, which places considerable restrictions on the strains that can ferment garlic (Kim et al. 2009b, Food Eng Prog 13: 352-359). However, garlic has an antimicrobial activity through heat treatment, which increases the possibility of fermentation, and increases the amount of available carbohydrates, which is sufficient as a substrate for microorganisms (de Castro et al. 1998, International Journal of Food Microbiology 39: 205). -211). In fact, the water content of garlic is 62 ~ 68%, lower than other fruits and vegetables, and contains various minerals and vitamins such as calcium, phosphorus, niacin, thiamin, riboflavin, vitamin A, and vitamin C (Kim et al. 2009, Korean). J Culinary Res 15: 59-66). The crude protein content of garlic grown in Korea is about 8-10%, and potassium (559 mg%) and phosphate (469 mg%) are included as the main minerals. Garlic contains about 1172 mg% of sugar, 74% of which is composed of sucrose, and 10% and 17% of glucose and fructose, respectively (Kim et al. 2009, Korean J Culinary Res 15:59). -66). Other carbohydrates consist mainly of fructan, a polymer of fructose (Hwang et al. 2010, J Agri Life Sci 44: 61-67). Garlic is rich in essential amino acids, proteins such as cysteine, histidine, lysine, and carbohydrates, and also contains essential fatty acids (Kim et al. 2010, Food Eng Prog 14: 65-74). The rich nutrients of garlic are believed to be able to play a role as a substrate of microorganisms themselves. Therefore, in the present invention, garlic was performed to isolate useful strains that can be fermented through a selective medium consisting of a main carbon source and a nitrogen source.

The present invention solves the above problems and the object of the present invention as devised by the necessity of the above is to provide a useful strain that can ferment garlic through a selective medium consisting of a major carbon source and nitrogen source.

In order to achieve the above object, the present invention provides a novel Leuconostoc citreum strain KACC91593P using garlic as the only carbon and nitrogen source to improve the antioxidant activity in garlic fermentation.

In another aspect, the present invention provides a composition for improving the antioxidant activity of garlic comprising the strain of the present invention as an active ingredient.

The present invention also provides a composition for increasing the sulfur compound of garlic comprising the strain of the present invention as an active ingredient.

The present invention also provides a composition for increasing the sulfur compound of garlic comprising the strain of the present invention as an active ingredient.

In another aspect, the present invention provides a method for increasing the antioxidant activity of garlic, including the step of fermenting garlic using the strain of the present invention.

In another aspect, the present invention provides a method for increasing the sulfur compound of garlic comprising the step of fermenting garlic using the strain of the present invention.

Garlic fermentation products of the present invention can be used as food material and feed material.

When the garlic fermentation product of the present invention is applied to food or feed, the garlic fermentation composition of the present invention may be appropriately formulated into a food or feed material.

In principle, the raw material of the food is vegetables, but may be used in other meat, etc.

Examples of the feed material include grains, crude algae, vegetable oils, animal feed materials, other feed materials, and refined products. For example, grains such as myro, wheat, barley, oats, rye, brown rice, buckwheat, crude, Sorghum, blood, corn, soybean, and the like.

Examples of the crude steel include rice bran, defatted rice bran, wheat bran, horse meal, wheat, embryo, barley bran, screening, pellets, corn bran, and corn embryo.

Examples of vegetable oils include soybean meal, soybean meal, flaxseed meal, cottonseed meal, groundnut meal meal, safflower meal, palm oil palm, palm oil, fomarak, sunflower oil, rapeseed meal, cowpea oil and mustard oil.

As animal feed materials, for example, fish meal (North American wheat, imported wheat, whole wheat, coastal wheat), fish bulrush, meat meal, fish bone meal, blood meal, decomposing hair, bone meal, animal by-products, feather mill, silkworm, skim milk powder, Casein, dried whey, and the like.

Other feedstocks include plant foliage (alfalfa, haycube, alfalfa leaf powder, pseudo acacia powder, etc.), corn processing industrial by-products (corn gluten, wheat, corn gluten feed, corn staples, etc.), starch processed products, sugar, fermentation. Industrial products (yeast, beer foil, malt sprouts, alcohol foil, walnuts, etc.), agricultural production by-products (citrus fruits, tofu, coffee, cocoa, etc.), others (cassava, bean, guam, seaweed, Krill, spirulina, chlorella, minerals, and the like); and the like. Proteins include protein (casein, albumin, etc.), amino acids, sugars (starch, cellulose, sucrose, glucose, etc.), minerals, vitamins, and the like.

Hereinafter, the present invention will be described.

Screening of Garlic Fermented Microorganisms

In order to search for useful strains to ferment garlic, useful strains were screened from Korean traditional fermented foods (kimchi, soybean paste, pickled garlic) and soil. As a screening method, garlic was screened using a medium having a sole carbon source and a nitrogen source (K ₂ HPO ₄ , 0.1 g / l; MgSO ₄ , 0.01 g / l, 30% (w / v) garlic juice). As a result, a total of 104 strains were secured, and the screened strains grew well in MRS and LB medium. After culturing for 24 hours at 30 ℃ in the liquid medium for screening to evaluate the growth of the selected strains, the total viable cell count was measured as shown in the table of FIG.

Investigation of antimicrobial activity

In order to evaluate the physiological activity of the selected strains inoculated with 1% of each selected strain in the liquid medium containing garlic, and incubated for 24 hours at 30 ℃ using the supernatant Staphylococcus aureus , Salmonella gallinarum, Listeria monocytogens , Escherichia As a result of evaluating the antimicrobial activity of pathogens such as coli , the antimicrobial activity of the selected strains did not appear.

Investigation of antioxidant activity

The results of evaluating the antioxidant activity of the culture supernatant obtained after culturing each of the selected strains are as shown in the table of FIG.

Using relative activity index Selection  evaluation

As a result of evaluating the relative activity of the selected strains using the standard normal distribution, the best activity was shown in the SK2556 strain.

Selection of strains  Classification

16S rRNA gene sequences of the selected strains were used to investigate the molecular location between the strains. As a result, the selected strains were largely divided into five categories, most of which belonged to Leuconostoc and Weisella. The final selected SK2556 strain was Leuconostoc citreum (FIG. 3). The isolated strain was named Leuconostoc citreum SK2556, and on November 23, 2010, it was deposited with Leuconostoc citreum SK2556, Accessory Preservation Number (Accession Number): KACC91593, at the National Institute of Agricultural Science, Seodun-dong, Suwon-si, Gyeonggi-do, Korea.

Result of analysis of sulfur compound

Among the selected strains, the fermentation of garlic was carried out using the strain SK2556, which has an excellent relative activity index, and then investigated whether the sulfur compound was increased before and after fermentation. As a result, as shown in Figure 4, it was confirmed that the sulfur compounds investigated in all the selected strains were increased compared to the control group. The peak of the chromatogram is as shown in FIG. 5.

As can be seen through the present invention, the strain of the present invention has the effect of improving the antioxidant activity in garlic fermentation, sulfur compounds are increased compared to the control.

1 is a table showing the cell growth and culture pH of the isolated strain.
Figure 2 is a table showing the free radical scavenging activity of the isolated strain.
Figure 3 shows the 16S rRNA gene sequence of the selected SK2556 strain of the present invention.
Figure 4 shows the chromatogram of sulfur compounds in garlic fermented using the SK2556 strain of the present invention. a, b, c and d represent chromatograms of fermentations 0, 8, 16 and 24 hours, respectively.
5 shows a chromatogram of standard sulfur compounds used

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example  1: Composition and Preparation of Garlic Medium

Garlic was used as a major carbon and nitrogen source in the medium to search for useful microorganisms that can use garlic as a carbon and nitrogen source. Garlic used in the present invention was used in the general market marketed garlic and after homogenization using a grinder (Philips HR2860, Netherlands). 10% garlic medium was used as a medium for detecting and separating strains, and 30% garlic medium was used in the enrichment process of the selected strains. The detailed composition is as follows. 10% garlic medium is garlic, 100 g / L; potassium phosphate, 0.1 g / L; magnesium sulfate, 0.01 g / L; sodium acetate, 1 g / L; sodium chloride, 1 g / L; agar, 15 g / L, 30% garlic medium, 300 g / L garlic; potassium phosphate, 0.1 g / L; magnesium sulfate, 0.01 g / L; sodium acetate, 1 g / L; Sodium chloride, 1 g / L.

Example  2: Separation  Pretreatment

Soil, kimchi, soybean paste, and garlic pickling were used as the separation sources for the useful strains using garlic. Soil was collected and used near Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul, and the pretreatment process was performed to remove nutrients from soil samples in order to reduce microbial selection errors due to nutrients derived from the soil. 10 g of soil sample was suspended in 90 mL of sterile 0.8% NaCl solution, stirred for 1 hour in an ice bath, and centrifuged at 1,000 rpm for 5 minutes to remove soil and other particles. The centrifuged supernatant was again centrifuged at 10,000 rpm for 5 minutes to separate the microbial cells in the soil. The obtained cell-containing material was washed with the same amount of 0.8% NaCl, and then the cell-containing material was recovered by the same centrifugation method. . Washing process using 0.8% NaCl was carried out three times, and finally the cell-containing material was suspended in the same amount of 0.8% NaCl, DMSO was added to a final concentration of 10% and stored at -70 ℃ until the experiment. Kimchi, doenjang and garlic pickles were prepared using traditional methods. Each sample was homogenized using a grinder (Philips HR2860, Netherlands), and the washing process for removing nutrients from the sample was the same as the pretreatment of the soil sample.

Example  3: Selection of strain

After completion of the pretreatment, each of the isolate samples was diluted in sterile 0.8% NaCl solution and then plated in 10% garlic plate medium and incubated for 24 hours in a 30 ℃ thermostat. The number of colonies formed on the plate medium was about 500 to 1,000 plate medium for strain isolation, and morphologically different colonies were randomly selected. A total of 104 colonies were selected and separated into pure strains through three passages.

Example  4: culture conditions

Isolated strains were enriched in LB (Difco, USA) or MRS (Difco, USA) and used as inoculum for 30% garlic medium. Strains enriched in complex medium were centrifuged at 10,000 rpm for 5 minutes, washed twice with sterile 0.8% NaCl solution, suspended in the same amount of sterile 0.8% NaCl solution and used as inoculum. In a 100 mL Erlenmeyer flask containing 30 mL of 30% garlic medium and sterilized at 121 ° C. for 15 minutes, the prepared inoculum was inoculated at a rate of 1% (v / v), at a speed of 150 rpm in a 30 ° C. shaker. Incubated for 24 hours.

Example  5: Viable count  Measure

The viable cell number of the culture was measured to evaluate the growth of the isolates in 30% garlic medium. After incubation, 1 mL of the culture was taken and serially diluted in 9 ml of 0.85% sterile NaCl solution, and then 0.1 ml of the dilution of each step was aseptically plated in LB or MRS plate medium and incubated for 20 to 24 hours in a 30 ° C incubator. The number of colonies formed after incubation was measured. The effective colony count was set to 30 to 300, and the density of viable cells was calculated.

Example  6: Antioxidant activity measurement

In order to examine the effect of fermentation of the selected strains on the antioxidant activity of garlic medium, the antioxidant activity of the culture supernatant was evaluated using DPPH (α, α'-diphenyl-β-picrylhydrazyl, Sigma) solution. After the culture was completed, the culture solution was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was used. The reaction solution for antioxidant activity was mixed with the culture supernatant, sterile distilled water, 99.9% ethanol, and 4x10 ^-4 M DPPH solution at a ratio of 0.1: 0.4: 1.0: 1.0, respectively, and reacted at room temperature for 15 minutes, followed by spectrophotometer (SHIMADZU, Japan) was used to measure the absorbance of the reaction solution at 525 nm. The control group was reacted with sterile distilled water instead of the culture supernatant and then measured for absorbance.

Example  7: Selection of strains

The selection of excellent strains involved in garlic fermentation was assessed using relative activity index to evaluate both growth and antioxidant activity. Relative performance evaluation was performed using the standard normal distribution.

Example  8: Selection  Sympathy

Identification of the strains selected through relative activity index was performed using the 16S ribosomal RNA gene sequence, and the 16S rRNA gene sequence was composed of two primers, 27F (forward primer; 5GTT TGA TCC TGG CTC AG-3) and 1492R ( After amplification by PCR using a reverse primer; 5TAC CTT GTT ACG ACT T-3), the ABI 3730XL capillary DNA analyzer was analyzed. All analysis of the nucleotide sequence was performed by Solgent (Korea, Daejun). The analyzed sequencing was performed for similarity analysis in Genbank using BLAST of NCBI, and the selected strains were identified and named using the strain with the highest similarity.

Example  9: Analysis of sulfur compounds

Among the selected strains, the sulfur compounds present in the culture medium were measured by HPLC using SK2556 strain having the highest relative activity index and SK2554 strain having the second highest activity index after fermenting garlic. The measurement method of sulfur compound was performed according to the method of Block et al. (1992, J. Agric. Food Chem. 40: 2418-2430).

Agricultural Biotechnology Research Institute KACC91593P 20101123

Claims (6)

delete delete delete delete delete Leuconostoc citreum ( Leuconostoc citreum ) SK 2556 Method of increasing the sulfur compound of garlic comprising the step of fermenting garlic using KACC91593P strain.

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