KR101757223B1 - Method for preparing fermented citrus composition with improved bioactivity - Google Patents

Abstract

The present invention relates to a method for producing a citrus fermented product having improved physiological activity, and more particularly, to a method for producing a citrus fermented product having enhanced physiological activity, comprising the steps of: Lactobacillus sakei KACC92032P, Pediococcus acidilactici KACC92031P and Leuconostoc mesenteroides KCTC3505 to produce a citrus fermented product having an acetylcholine esterase inhibitory activity, an angiotensin converting enzyme inhibitory activity or an antimicrobial activity, a fermented product produced by the method, And a food composition comprising the fermented product.
According to the present invention, citrus and lactic acid bacteria can be used to produce a citrus fermented product having an acetylcholine esterase inhibitory activity, angiotensin converting enzyme inhibiting activity or antimicrobial activity. In addition, based on the excellent physiological activity of the citrus fermented product, it is possible to prevent or ameliorate Alzheimer's disease and hypertension, and produce a functional food having an excellent antibacterial effect.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for preparing a fermented citrus fruit having improved physiological activity,

The present invention relates to a method for producing a citrus fermented product having improved physiological activity, and more particularly, to a method for producing a citrus fermented product having enhanced physiological activity, comprising the steps of: Lactobacillus sakei KACC92032P, Pediococcus acidilactici KACC92031P and Leuconostoc mesenteroides KCTC3505 to produce a citrus fermented product having an acetylcholine esterase inhibitory activity, an angiotensin converting enzyme inhibitory activity or an antimicrobial activity, a fermented product produced by the method, And a food composition comprising the fermented product.

Modern people are increasingly interested in health due to the increasing number of chronic diseases due to Westernized diet and nutritional imbalance. In addition, there is an increasing effort to prevent through aging and disease prevention as well as medical approaches such as food nutritional approach. Among the various materials used as food, there are many things that contain ingredients that are beneficial to the human body. In particular, there are a wide variety of physiologically active substances in plant materials, and their physiological activities, separation and purification methods, and the like have been studied and utilized for treatment of various diseases.

Citrus is an edible plant of the Rutaceae Citrus which is one of the world's favorite fruits grown in more than 100 countries on six continents. It is reported that citrus fruits contain various physiologically active substances such as carotenoids, flavonoids, cellulose, pectin, limonoids and vitamins and exhibit various physiological functions such as antioxidant, anti-inflammation and anti-cancer. Citrus fruits are mainly consumed in raw and dried forms, and some have been used for juice processing. In recent years, various citrus processed products such as chocolate, crunch, and malt liquor have been spread.

Lactic acid bacteria are microorganisms that produce lactic acid and various metabolites by fermenting saccharides. Recently, researches as probiotics for prevention of health promotion and diseases have been extensively carried out and various fermented foods, feed additives and the like It is widely used. In addition, lactic acid bacteria taken into the human body through fermented foods are introduced into the intestines to support the growth of beneficial bacteria and inhibit harmful bacteria to maintain the balance of useful microorganisms in the intestines. And antioxidant, anticancer, immune activation, and blood cholesterol lowering effects are known to help prevent disease.

The present inventors tried to develop a more beneficial and excellent food by using citrus fruits, and in particular, to develop foods that can prevent or improve disease. Therefore, it is expected that the interaction of the enzymes contained in the citrus fruits with the enzymes possessed by the lactic acid bacteria will produce new physiologically active substances or increase the concentration of useful physiologically active substances inherent in citrus, and ferment the citrus fruits with various lactic acid bacteria Study.

As a result, the citrus fruits were fermented with one kind of lactic acid bacteria selected from the group consisting of Lactobacillus sakei KACC92032P, Pediococcus acidilactici KACC92031P and Leuconostoc mesenteroides KCTC3505 , It was confirmed that it is possible to produce a fermented product having excellent inhibitory activity on acetylcholine esterase inhibition, angiotensin converting enzyme inhibitory activity or antimicrobial activity. Based on this effect, it is possible to prevent or ameliorate Alzheimer's disease and hypertension, Functional food can be produced, and the present invention has been completed.

Albertazzi P, Steel SA, Clifford E, Bottazzi M. (2002) Attitude towards post-menopausal women. Climacteric 5: 374-382. Fang, YZ, Yang, S. and Wu, G. (2002) Free radical antioxidant and nutrition. Nutrition 18: 872-879. Choi SY, Ko HC, Ko SY, Hwang JH, Park JG, Kang SH, Han SH, Yun SH, Kim SJ. (2007) Correlation between flaconoid content and NO production inhibitory activity of peel extracts from various citrus fruits. Biol. Pharm. Bull. 30: 772-778. Kim YD. (2009) Study on the composition of flavonoids and biological activities from Jeju citrus fruits. Jeju National University of Korea. 6-57. Ahn MS, Kim HJ, Seo MS. (2007) A study on the anti-oxidative and antimicrobial activities of the citrus unshiu peel extracts. Korean J. Food Culture 22: 454-461. Cheung HS, Cushman DW. (1971) Spectrometric assay and properties of angiotensin-converting enzyme of rabbit lung. Biochem. Pharmacol 20: 1637-1648. Cotter PD, C. Hill, R. P. Ross. (2005) Bacteriocins: developing innate immunity for food. Nat. Rev. Microbiol. 3: 777-788.

Accordingly, a main object of the present invention is to provide a method for producing a foodstuff effective for prevention or improvement of disease by using citrus as a material.

Another object of the present invention is to provide a functional food prepared by the above production method.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising Lactobacillus sakei KACC92032P, Pediococcus acidilactici KACC92031P and Leuconostoc mesenteroides KCTC3505 selected from the group consisting of Lactobacillus sakei KACC92032P, Pediococcus acidilactici KACC92031P and Leuconostoc mesenteroides KCTC3505. (AChE) inhibiting activity, an angiotensin converting enzyme (ACE) inhibiting activity, or an antimicrobial activity, which comprises fermenting citrus fruits with one kind of lactic acid bacteria .

In the method for preparing a citrus fermented product according to the present invention, it is preferable that the fermentation is performed at 20 to 40 DEG C or more per day.

According to the present invention, Lactobacillus < RTI ID = 0.0 > sakei ) When KACC92032P is used, it is preferable that fermentation is carried out at 28 to 32 DEG C for 7 days or more in order to increase acetylcholine esterase inhibitory activity, angiotensin converting enzyme inhibitory activity or antimicrobial activity. More preferably 7 to 10 days.

According to the present invention, when Pediococcus acidilactici KACC92031P is used as a lactic acid bacterium, fermentation for more than 7 days at 28 to 32 ° C in order to enhance acetylcholine esterase inhibitory activity, angiotensin converting enzyme inhibitory activity or antimicrobial activity . More preferably 7 to 10 days.

According to the present invention, when Leuconostoc mesenteroides KCTC3505 is used as lactic acid bacteria, fermentation is preferably performed at 28 to 32 ° C for 3 to 7 days to enhance angiotensin converting enzyme inhibitory activity or antimicrobial activity.

According to another aspect of the present invention, And citrus pulverized product were inoculated with one kind of lactic acid bacteria selected from the group consisting of Lactobacillus sakei KACC92032P, Pediococcus acidilactici KACC92031P and Leuconostoc mesenteroides KCTC3505 And fermenting the mixture at 20 to 40 째 C, comprising the steps of: preparing a citrus fermented product having an inhibitory activity against angiotensin converting enzyme or an antimicrobial activity.

According to another aspect of the present invention, there is provided a citrus fermented product having the acetylcholine esterase inhibiting activity, angiotensin converting enzyme inhibiting activity or antimicrobial activity produced by the above production method.

According to another aspect of the present invention, the present invention provides a food composition comprising the citrus fermented product.

The food composition of the present invention may be a functional food for preventing or improving Alzheimer's disease, a functional food for preventing or improving hypertension, or a functional food for antibacterial activity.

In the present invention, the acetylcholine esterase inhibitory activity means an activity of inhibiting the action of acetylcholine esterase to decompose acetylcholine (ACh) into acetate and choline.

In the present invention, the angiotensin converting enzyme inhibitory activity refers to an activity of inhibiting the action of angiotensin converting enzyme to convert angiotensin I to angiotensin II.

In the present invention, the antimicrobial activity refers to an activity of inhibiting or delaying the growth of bacteria or killing bacteria.

The present invention provides a food containing the citrus fermented product itself or a composition comprising a pharmaceutically acceptable carrier according to the present invention as a dietary material, wherein the food is selected from the group consisting of meat products, fish meat products, tofu, Such as noodles such as soy sauce, soy sauce, soy sauce, soy sauce, soybean paste, kochujang, mixed potatoes, sauces such as sauces, confectionaries, fermented milk and cheese, pickles such as kimchi and pickles, fruits, vegetables, soy milk, fermented beverages It can be used in food. It will be apparent to those skilled in the art that the specific cooking methods and production methods are not described herein. The pharmaceutically acceptable carrier may also be a pharmaceutically acceptable carrier.

According to the present invention, citrus and lactic acid bacteria can be used to produce a citrus fermented product having an acetylcholine esterase inhibitory activity, angiotensin converting enzyme inhibiting activity or antimicrobial activity. In addition, based on the excellent physiological activity of the citrus fermented product, it is possible to prevent or ameliorate Alzheimer's disease and hypertension, and produce a functional food having an excellent antibacterial effect.

FIG. 1 is a graph showing the results of analysis of acetylcholinesterase (AChE) inhibitory activity of a citrus fermented product (× 10 dilution) fermented with seven kinds of lactic acid bacteria. a ~ g, w ~ z: Different alphabets in the same group bar or the same color bar mean that there is a significant difference (P <0.05). The inhibitory activity (%) was expressed as the mean ± standard deviation of the results of three replicates. UC: citrus sample not fermented, CWS: citrus sample fermented without inoculation, EL ~ No. 2: ELA: Enterococcus lactis , EF: Enterococcus faecalis , LM: Leuconostoc mesenteroides (KCTC3505), LA: Lactobacillus acidophilus , LP: Lactobacillus plantarum , No. 1: Pediococcus sp . (KACC92031P), No. 2: Lactobacillus sp (KACC92032P) .
FIG. 2 is a graph showing the results of assay of an angiotensin converting enzyme (ACE) inhibitory activity of a citrus fermented product (× 20 diluted) fermented with seven kinds of lactic acid bacteria, respectively. ag, vz: different alphabets in the same group bar or same color bar mean that there is a significant difference (P <0.05). The inhibitory activity (%) was expressed as the mean ± standard deviation of the results of three replicates. UC: citrus sample not fermented, CWS: citrus sample fermented without inoculation, EL ~ No. 2: A sample of fermented citrus fruit fermented by inoculation with each strain, EL: Enterococcus lactis , EF: Enterococcus faecalis , LM: Leuconostoc mesenteroides (KCTC3505), LA: Lactobacillus acidophilus , LP: Lactobacillus plantarum , No. 1: Pediococcus sp . (KACC92031P), No. 2: Lactobacillus sp (KACC92032P) .
Fig. 3 is a graph showing the results of Pediococcus sp . Lactic acid bacteria isolated from kimchi among the lactic acid bacteria used in the examples of the present invention . (SEQ ID NO: 2) (KACC92031P), which is a phylogenetic tree.
Fig. 4 is a graph showing the effect of Lactobacillus sp . (SEQ ID NO: 2) (KACC92032P), which is a phylogenetic tree.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example  1. Fermentation of lactic acid bacteria of citrus fruit

1-1. material

Tangerine was purchased in July 2014 from Citrus unshiu (Citrus unshiu), which is commercially available from Jeju Island. Acetylthiocholin esterase, acetylthiocholine iodide, rabbit lung acetone powder, hypouril histidine leucicne (HHL) and eserine were purchased from Sigma-Aldrich (St. Louis, Mo., USA). Sodium carbonate anhydrous and sodium bicarbonate were purchased from Daejung Chemical Co. (Siheung, Korea). Folin-Ciocalteu reagent was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

1-2. The strains used and culture conditions

For the citrus fermentation, five strains of Lactobacillus acidophilus KCTC 3140 (LA), Lactobacillus plantarum KCTC 3104 (LP), Enterococcus lactis KCTC 21015 (EL), Enterococcus faecalis KCTC 2011 (EF) and Leuconostoc mesenteroides KCTC 3505 (LM) were purchased and used. In addition, lactic acid bacteria Pediococcus sp . (No. 1) (KACC92031P) and Lactobacillus sp . (No. 2) (KACC92032P) was also utilized. All cultures of all strains were activated by shaking culture in MRS liquid medium (Difco, Detroit, MI, USA) at 30 ° C, 100 rpm for 2 days and inoculated into citrus fruit purée.

1-3. Manufacture of citrus fermented products

The citrus fruits were cleanly washed in tap water and then washed again with distilled water. To prevent contamination during the fermentation process, the washed citrus surface was irradiated with UV for 15 minutes and sterilized. The citrus fruits were evenly ground with a blender and placed in a pre-sterilized flask to prepare 270 ml of citrus puree.

Lactobacillus acidophilus KCTC 3140 (LA), Lactobacillus plantarum KCTC 3104 (LP), Enterococcus lactis KCTC 21015 (EL), Enterococcus faecalis KCTC 2011 (EF), Leuconostoc mesenteroides KCTC 3505 (LM), Pediococcus sp. (No. 1), Lactobacillus sp. (No. 2) before the culture was then inoculated in addition to the citrus puree prepared by each 30㎖ the pre-culture in a MRS liquid medium, each of the strains of 7 species. As a control group, sterilized distilled water was used instead of the strain pre-culture solution. Each strain was inoculated on citrus puree and fermented at 30 ° C, 100 rpm, shaking incubator for 3, 5, 7, and 10 days, and then the fermented product was recovered.

The recovered fermented product was centrifuged (4 ° C, 10 min, 5,000 × g) and the supernatant was passed through Whatman No.1 filter paper (Advantec, Tokyo, Japan).

Example 2. Analysis of physiological activities of citrus fermented products

2-1. Experimental Method

2-1-1. Acetylcholinesterase (AChE) Incompatibility Analysis

AChE inhibition was measured by using Prashanth 's method using acetylcholine iodide as a substrate. To 90 μl of the sample was added 160 μl of AChE (2 U / ml) and 30 μl of DTNB solution (3.96 mg of DTNB and 1.5 mg of sodium bicarbonate in 10 ml of phosphate buffer (pH 7.7)), For 5 minutes. 30 μl of acetylcholine iodide solution (10.85 mg of acetylthiocholine iodide dissolved in 5 ml of phosphate buffer (pH 7.7)) was added to the reaction mixture, followed by reaction at 25 ° C for 30 minutes. / TW / BC / 6Filter, Tecan Austria GmbH, Gradig, Austria), and the AChE inhibition was determined using the following equation.

AChE inhibition (%) = 1 - (absorbance of sample added / absorbance of untreated)

2-1-2. Angiotensin converting enzyme (ACE) inhibitory assay

ACE inhibition was measured by the method of Cushman and Cheung. 1 g of rabbit lung acetone powder was added to 20 ml of 50 mM sodium borate buffer (pH 8.3) at 4 ° C for 24 hours, and centrifuged at 4 ° C for 5 minutes to obtain ACE enzyme solution. 50 AC of the diluted sample was added with 50 AC of ACE enzyme solution, and the mixture was allowed to stand at 37 캜 for 10 minutes. Then, 100 25 of 25 mM HHL was added and reacted at 37 캜 for 60 minutes. 250 μl of 1M HCI was added and the mixture was stirred for 30 seconds and centrifuged (4 ° C, 10 minutes, 5,000 × g) to obtain 200 μl of a supernatant. The supernatant was completely dried at 80 ° C for 30 minutes, and after adding 1 ml of distilled water, the absorbance was measured at 228 nm. In the control group, distilled water was used instead of the sample, and captopril was used as a positive control. The ACE inhibition was calculated using the following equation.

ACE inhibition (%) = 1 - (absorbance of sample added / untreated)

2-1-3. Antimicrobial activity analysis

The fermented citrus fruits fermented with each lactic acid bacterium were collected during fermentation on days 3, 5, 7, and 10, centrifuged, and filtered to obtain a supernatant. On the 0.5% yeast extract agar plate, 10 ml of 0.8% NB soft agar containing 1% of the indicator culture medium and 0.1 M phosphate buffer (pH 7.0) was layered to prepare a test plate. The antimicrobial activity was measured by spotting 50 ㎕ of citrus fermented extract extract on a test plate, drying the supernatant, and culturing for 12 hours or more in a constant temperature incubator at 37 캜.

2-1-4. Statistical processing

All experiments were repeated 3 times and the results were processed with SPSS software (Ver. 12.0K, SPSS Inc., Chicago, IL, USA). The mean and standard deviation of each measurement group were calculated and the significance was tested by Duncan's multiple range test. The difference of all treatment values was compared with 95% confidence level (P <0.05).

2-2. Experiment result

2-2-1. Results of Acetylcholinesterase (AChE)

Acetylcholine (ACh), one of neurotransmitters in the body, is synthesized by acetyl CoA and choline under the action of choline acetyltransferase (ChAT) enzyme, which is decomposed into acetate and choline under the action of AChE enzyme. Therefore, the activation of ChAT and AChE enzymes, which affect ACh, is important for smooth neurotransmission in the body. In patients with Alzheimer disease (AD), which accounts for 50% of dementia, ChAT tends to decrease and AChE is inhibited in patients with AD to increase the concentration of ACh in the body.

The results of assaying acetylcholinesterase (AChE) inhibitory activity of a 10-fold diluted citrus fermented extract are shown in FIG. The positive control group, eserine, showed 87% inhibition activity at 100 μg / ml concentration. 1 ( Pediococcus sp. ) And No. 2 ( Lactobacillus sp. ) Fermented extract showed higher inhibitory activity as fermentation progressed than other extracts. It has been reported that donepezil, tacrine, rivastigmine, galactamine, etc., as AChE inhibitors, are allowed in the FDA and that coumarine phenol substances inhibit AChE in a manner similar to galactamine. Therefore, the results of DPPH and ABTS radical scavenging activities are different from those of DPPH and ABTS radical scavenging activity, which means that there are differences in the substances that affect antioxidant activity and AChE inhibitory activity.

2-2-2. Angiotensin converting enzyme (ACE) inhibitory assay results

Renin in the body decomposes angiotensinogen into angiotensin Ⅰ and inactive angiotensin Ⅰ becomes angiotensin Ⅱ with angiotensin-converting enzyme (ACE). This angiotensin Ⅱ is involved in the onset of hypertension by contracting the arterial blood vessels, raising blood pressure, and promoting the secretion of aldosterone from the adrenal gland, thereby increasing renal sodium and water reabsorption.

The ACE inhibitory activity of 20-fold diluted citrus fermented extract samples is shown in FIG. The ACE inhibitory effect was 4.62 ± 2.71 ~ 86.22 ± 1.57%. The inhibitory effect of captopril was 91% at 2 μg / ㎖. LA ( L. acidophilus ) fermented extract decreased from 38.17% to 14.19% as the fermentation period became longer. 1 ( Pediococcus sp. ), No. 2 ( Lactobacillus sp. ) Fermented extracts increased from 42.90% to 85.97% and from 41.94% to 83.88%, respectively. According to Lee et al.'S results of antioxidative and ACE inhibition studies on the fermentation of lactic acid bacteria in leek slurries, leucine fermentation showed 50.4 ~ 67% ACE inhibition depending on the type of lactic acid bacteria. Although the kinds of lactic acid bacteria used are different, And it was confirmed that it showed an inferiority. In addition, ACE inhibition is mainly caused by proteins or peptides. It is likely that the products produced from fermented citrus extracts using lactic acid bacteria are composed of proteins and peptides, and as a result, ACE inhibition is excellent.

2-2-3. Antimicrobial activity analysis result

The pathogenic microorganisms L. delbrueckii , B. subtilis , E. coli , S. aureus , E. faecium , X. citri , B. cereus , S. typhimurium , M. luteus , P. acidilactici , B. sphaericus , B. megaterium The antibacterial properties were examined and the results are shown in Tables 1 to 3. In the control group, no antimicrobial activity was observed for all the strains used in the experiment, and LP ( L. plantarum ), LM ( L. mesenteroides ), and No. 1 ( Pediococcus sp. ), No. 2 ( Lactobacillus sp. ) Fermented extract. E. coli , S. aureus , E. faecium , X. citri , and B. subtilis extracts of fermented LP ( L. plantarum ) showed higher antimicrobial activity with increasing fermentation period . cereus, S. typhimurium, M.luteus, P. acidilactici, B. sphaericus, B. in megaterium LM (L. mesenteroides) fermented extract of E. coli, X. citri, B. cereus , S. typhimurium, P. acidilactici , B. sphaericus , and B. megaterium . 1 ( Pediococcus sp. ) Fermented extracts were identified as L. delbrueckii , M. luteus , N. 2 ( Lactobacillus sp. ) Fermented extract showed antimicrobial activity against pathogenic microorganisms except L. delbrueckii . Especially No. 2 ( Lactobacillus sp. ) Fermented extract showed strong antimicrobial activity. No. 2 ( Lactobacillus sp. ), The antimicrobial activity of citrus fruit is considered to produce antimicrobial substance as a metabolite. Cho et al. Reported that the natural fermented Omija fermentation broth had no antimicrobial activity, but the fermentation showed an antimicrobial activity against the E. coli strain.

Strain Sample ^{1 )} Fermentation period (days) 0 3 5 7 10


L. delbrueckii CWS - ²⁾ - - - - EL - - - - - EF - - - - - LM - - - - - LA - - - - - LP - - - - - No. One - - - - - No. 2 - - - - -


B. subtilis CWS - - - - - EL - - - - - EF - - - - - LM - - - - - LA - - - - - LP - - - - ++ No. One - - + + ++ No. 2 - - + +++ +++


E. coli CWS - - - - - EL - - - - - EF - - - - - LM - - + + + LA - - - - - LP - - - - ++ No. One - - + ++ ++ No. 2 - - + ++ +++


S. aureus CWS - - - - - EL - - - - - EF - - - - - LM - - - - - LA - - - - - LP - - - - + No. One - - + + ++ No. 2 - - + ++ ++

1) CWS: citrus sample fermented without inoculation of strains, EL ~ No. 2: A sample of fermented citrus fruit fermented by inoculation with each strain, EL: Enterococcus lactis , EF: Enterococcus faecalis , LM: Leuconostoc mesenteroides , LA: Lactobacillus acidophilus , LP: Lactobacillus plantarum , No. 1: Pediococcus sp. , No. 2: Lactobacillus sp.

2) -: Antibacterial activity not detected, Antimicrobial activity: +, <1.0 mm; ++, 1.0 to 1.5 mm; +++, > 1.5mm.

Strain Sample ^{1 )} Fermentation period (days) 0 3 5 7 10


E. faecium CWS - ²⁾ - - - - EL - - - - - EF - - - - - LM - - - - - LA - - - - - LP - - - - + No. One - - + + + No. 2 - - - + +


X. citri CWS - - - - - EL - - - - - EF - - - - - LM - + + ++ + LA - - - - - LP - - - - ++ No. One - - + ++ ++ No. 2 - - + +++ +++


B. cereus CWS - - - - - EL - - - - - EF - - - - - LM - - - + + LA - - - - - LP - - - - + No. One - - + + + No. 2 - - + +++ +++


S. typhimurium CWS - - - - - EL - - - - - EF - - - - - LM - + + + + LA - - - - - LP - - - + ++ No. One - - ++ ++ ++ No. 2 - - + +++ +++

1) CWS: citrus sample fermented without inoculation of strains, EL ~ No. EL: Enterococcus lactis , EF: Enterococcus faecalis , LM: Leuconostoc mesenteroides , LA: Lactobacillus acidophilus , LP: Lactobacillus plantarum . 1: Pediococcus sp. , No. 2: Lactobacillus sp.

2) -: Antibacterial activity not detected, Antimicrobial activity: +, <1.0 mm; ++, 1.0 to 1.5 mm; +++, > 1.5mm.

Strain Sample ^{1 )} Fermentation period (days) 0 3 5 7 10


M. luteus CWS - ²⁾ - - - - EL - - - - - EF - - - - - LM - - - - - LA - - - - - LP - - - - + No. One - - - - - No. 2 - - - ++ ++


P. acidilactici CWS - - - - - EL - - - - - EF - - - - - LM - - + + + LA - - - - - LP - - - - ++ No. One - - + + + No. 2 - - + ++ +++


B. sphaericus CWS - - - - - EL - - - - - EF - - - - - LM - - + + + LA - - - - - LP - - - - + No. One - - + + + No. 2 - - + ++ ++


B. megaterium CWS - - - - - EL - - - - - EF - - - - - LM - - - + + LA - - - - - LP - - - - + No. One - - + + + No. 2 - - + +++ +++

1) CWS: citrus sample fermented without inoculation of strains, EL ~ No. EL: Enterococcus lactis , EF: Enterococcus faecalis , LM: Leuconostoc mesenteroides , LA: Lactobacillus acidophilus , LP: Lactobacillus plantarum . 1: Pediococcus sp. , No. 2: Lactobacillus sp.

2) -: Antibacterial activity not detected, Antimicrobial activity: +, <1.0 mm; ++, 1.0 to 1.5 mm; +++, > 1.5mm.

Example 3 Identification of lactic acid bacteria

As a result of functional analysis, highly active strains (No. 1 and No. 2) were identified by 16S rRNA gene analysis. The strain was sent to Solent (Daejeon) in the form of Colony for genetic analysis. PCR was performed using the universal primer 27F primer (5'-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R primer (5'-GGTTACCTTGTTACGACTT-3') used for 16S rRNA sequencing. The nucleotide sequence was analyzed using a 3730XL Capillary RNA Sequencer from Applied Biosystems, and a blast search of NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/) was performed using the analyzed nucleotide sequence Were identified and identified.

Result No. One strain of 16S rRNA was identified as Pediococcus lt ; / RTI &gt; 2 strains of 16S rRNA were found in Lactobacillus and 99% or more homology with sakei . These strains were deposited with the National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology, and were assigned accession numbers of KACC92031P and KACC92032P, respectively.

L. sakei is a lactic acid bacterium that produces alcohol or lactic acid bacteria from sugars and produces a bacteriocin called sakacin. Bacteriocin is an antimicrobial substance composed of peptides and proteins. It is a competitive inhibitor against other microorganisms that grow in the same environment and is isolated from various microorganisms. Especially, lactic acid bacteria plays an important role in kimchi fermentation and is known to produce various antimicrobial substances. Bacteriocin has a very broad antibacterial range and is effective in suppressing or killing the growth of spoilage bacteria, food poisoning bacteria, infectious disease bacteria and spore forming bacteria. Unlike antibiotics, bacteriocin is made up of proteins and is easily degraded by protein hydrolytic enzymes in the digestive tract when it enters the body, making it non-toxic and non-persistent to the human body. Antibiotics are secondary metabolites, whereas bacteriocins are biosynthesized directly from their genes. Sakacin, produced by L. sakei, has been reported as A, G, K, P and Q type, of which A and P type are well known. Sakacin produced by L. sakei identified in this Example is not a bacteriocin with a narrow inhibition range but a broad inhibition range such as nisin, a bacteriocin produced by Streptococcus lactis .

Agricultural Biotechnology Research Institute KACC92031 20150210 Agricultural Biotechnology Research Institute KACC92032 20150210

Claims (8)

Lactobacillus sakei KACC92032P, Pediococcus acidilactici KACC92031P and Leuconostoc mesenteroides KCTC3505. It is characterized by fermenting citrus fruits with one kind of lactic acid bacteria selected from the group consisting of Lactobacillus sakei KACC92032P, Pediococcus acidilactici KACC92031P and Leuconostoc mesenteroides KCTC3505. , An angiotensin converting enzyme inhibiting activity, or an antimicrobial activity. The method according to claim 1,
Wherein the fermentation is performed at 20 to 40 占 폚 for one day or more. Crushing citrus fruits; And
The citrus pulverized product was inoculated with one kind of lactic acid bacteria selected from the group consisting of Lactobacillus sakei KACC92032P, Pediococcus acidilactici KACC92031P and Leuconostoc mesenteroides KCTC3505 And fermenting at 20 to 40 캜, wherein the fermented product has an inhibitory activity against acetylcholine esterase, an angiotensin converting enzyme inhibitory activity or an antimicrobial activity. A citrus fermented product produced by the production method of any one of claims 1 to 3 and having an acetylcholine esterase inhibitory activity, an angiotensin converting enzyme inhibitory activity or an antibacterial activity. A food composition comprising the citrus fermented product of claim 4. 6. The method of claim 5,
Wherein the food composition comprises a citrus fermented product fermented with one kind of lactic acid bacterium selected from the group consisting of Lactobacillus sakei KACC92032P and Pediococcus acidilactici KACC92031P and the prevention or prevention of Alzheimer's disease Wherein the food composition is a functional food for improvement. 6. The method of claim 5,
Wherein the food composition is a functional food for preventing or improving hypertension. 6. The method of claim 5,
Wherein the food composition is a functional food for antibacterial activity.

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