KR101481793B1 - The method of Cottage cheese preparation containing ornithine using the strain of Pediococcus pentosaceus AYO-1 having ornithine production capacity - Google Patents

Abstract

The present invention relates to a method for producing cottage cheese comprising Pediococcus pentosaceus A -1 (KCCM11355P), a strain of Pediococcus pentosaceus AYO-1, which is capable of producing ornithine, and a method for producing anti-obesity, ammonia detoxification, The present invention relates to a cottage cheese comprising a novel Pediococcus pentosaceus AYO-1 strain (KCCM11355P), which has an effect on the improvement of the texture and the like.
a) pasteurizing the crude oil at 65 to 70 ° C for 4 to 6 seconds;
b) a cooling step of cooling the crude oil after the sterilization step to 30 to 36 ° C;
c) Inoculation step of inoculating functional crude lactic acid bacterium and industrial starter at 1.0 to 2.0% (v / v) into the crude oil after the cooling step;
d) culturing the mixture after the inoculation step at 30-35 ° C;
e) adding 0.001-0.02% (v / v) rennet to the mixture after the culturing step;
f) a step of allowing the mixture to stand for 50 to 70 minutes;
g) a cutting step of cutting the curd after completion of the setting step;
h) heating step wherein the cut curd is heated to 55 to 60 DEG C with stirring for 30 to 50 minutes;
i) a whey removing step of washing the curd after the heating step twice and removing the whole whey;
j) an evacuation step of evacuating the remaining whey from the curd after the whey removal step for 2 to 3 hours;
k) an addition step of adding 0.5-1.0 parts by weight of salt to 100 parts by weight of the curd having been subjected to the discharging step;
l) a storage step in which the cheese having been subjected to the adding step is packed and stored at -1 to 5 DEG C to prepare a cottage cheese.

Description

[0001] Description [0002] The present invention relates to a method for preparing cotyledon cheese, which comprises using Pediococcus pentosaceus AYO-1 strain having ornithine production ability, and a method for producing cottage cheese using the strain AYO-1 having ornithine production capacity of Pediococcus pentosaceus,

The present invention relates to a method for producing cottage cheese comprising Pediococcus pentosaceus A -1 (KCCM11355P), a strain of Pediococcus pentosaceus AYO-1, which is capable of producing ornithine, and a method for producing anti-obesity, ammonia detoxification, A novel Pediococcus pentosaceus AYO-1 strain ( Pediococcus pentosaceus AYO-1) (KCCM11355P).

It is also called popcorn cheese because it is similar to Dutch cheese, port cheese and popcorn. There are two types of soft cheese that are not aged, most of which are manufactured in many countries. There are two manufacturing methods. One is to form acid curd by lactic acid fermentation and the other is to add curd by addition of starch. Starters include Lactococcus lactis subsp., Leuconostoc mesenteroides subsp. .

L-ornithine is an amino acid normally contained in food, though it is not an amino acid constituting a protein. In particular, the content of L-ornithine in freshwater bivalve clams and mussels is relatively high. The content of free ornithine in 100 g of mulberry extract is 159.9 mg, and it is known that the free L-ornithine content is increased when the mulberry is treated at low temperature. L-ornithine is widely used in the United States as a food material that secretes growth hormone, increases muscle synthesis, promotes basic metabolism, and prevents obesity. Recently, it has been found that it has new functionalities such as a skin cosmetic effect such as improvement of wrinkles. Also, L-ornithine is in the form of L-ornithine-alpha ketoglutarylate (OKG), which contains L-ornithine and alpha-ketoglutaryl acid in a ratio of 2: Is widely used as an enhancing material, and in Europe, L-ornithine-L-aspartate is used as a medicine for improving hepatic disorder.

In Japan, it can be used as a food material in the form of L-ornithine hydrochloride. In the United States, it can be used as a dietary supplement, but in Korea, it is not yet listed as a food additive in the KFDA. Therefore, it is expected that when fermented food is fermented using ornithine high productivity strain derived from fermented food, fermented food having enhanced nutritional function, ornithine bacterium flavor and flavor, such as ornithine, A need was raised.

Accordingly, the present invention provides a method for producing ornithine cottage cheese by separating, identifying and characterizing lactic acid bacteria having ornithine-producing ability from starters and naturally fermented dairy products for the production of cottage cheese, and a cheese product prepared using the same .

Korean Registration No. 10-1190149 "Vegetable cheese and its preparation method" Korean Registration No. 10-0763037 "Novel Lactobacillus lambosus strains and uses thereof" Korean Utility Model No. 10-2011-0023136 "Cheese Containing Surimi & Korea Publication No. 10-2011-0139861 "Mozzarella cheese containing Streptococcus Massonikus LC 743 strain and its production method"

It is an object of the present invention to provide a strain of Pediococcus pentosaceus AYO-1 which is capable of producing ornithine.

Another object of the present invention is to provide a method for producing cottage cheese containing ornithine by using the Pediococcus pentosaceus strain AYO-1 having the ability to produce ornithine and a process for producing ornithine- I would like to offer cottage cheese.

Another object of the present invention is to provide a health functional food comprising Pseudococcus pentosaceus AYO-1 strain capable of producing ornithine.

In order to solve the above problems,

Pediococcus pentosaceus AYO-1 (KCCM11355P), which produces ornithine, was used as a strain of Pediococcus pentosaceus AYO-1

A culture solution obtained by culturing Pediococcus pentosaceus AYO-1 (KCCM11355P), Pediococcus pentosaceus producing ornithine, was used.

According to an aspect of the present invention,

a) pasteurizing the crude oil at 65 to 70 ° C for 4 to 6 seconds;

b) a cooling step of cooling the crude oil after the sterilization step to 30 to 36 ° C;

c) Inoculation step of inoculating functional crude lactic acid bacteria and starter at 1.0 to 2.0% (v / v) into the crude oil after the cooling step;

d) culturing the mixture after the inoculation step at 30-35 ° C;

e) adding 0.001-0.02% (v / v) rennet to the mixture after the culturing step;

f) a step of allowing the mixture to stand for 50 to 70 minutes;

g) a cutting step of cutting the curd after completion of the setting step;

h) heating step wherein the cut curd is heated to 55 to 60 DEG C with stirring for 30 to 50 minutes;

i) a whey removing step of washing the curd after the heating step twice and removing the whole whey;

j) an evacuation step of evacuating the remaining whey for 2-3 hours at the curd after the whey removal step;

k) an addition step of adding 0.5-1.0 parts by weight of salt to 100 parts by weight of the curd having been subjected to the discharging step;

l) a storage step of packing the cheese having passed through the adding step and storing the cheese at a temperature of -1 to 5 캜, thereby completing the present invention.

The present invention provides a strain of Pediococcus pentosaceus AYO-1 having ornithine production ability.

The present invention provides a method for producing cottage cheese including Pediococcus pentosaceus AYO-1 strain, and a method for producing cottage cheese such as anti-obesity, ammonia detoxification, fatigue recovery, skin texture improvement, and Pediococcus pentosaceus AYO- 1 < / RTI >

1 is a TLC showing the possibility of ornithine production of a candidate strain producing ornithine from a naturally fermented dairy product containing a yogurt industrial starter.
Fig. 2 shows the effect of Pediococcus pentosaceus ( PEG ) on the MRS medium (pH 6.5, 30 ° C) supplemented with 1, 3, 5% arginine AYO-1 and strains were cultured for 48 hours, respectively, and analyzed for ornithine content of each strain.
Fig. 3 shows the results of the fermentation of cottage cheese prepared by using Pottococcus pentosaceus AYO-1 (KCCM 11355P) at 58 ° C, and the content of ornithine according to the refrigerated storage period of 0, 3, .

The present invention shows Pediococcus pentosaceus AYO-1 (KCCM11355P), which is capable of producing ornithine.

The present invention relates to a method for producing cottage cheese, which comprises adding 1.0 to 2.0% (v / v) of Pediococcus pentosaceus AYO-1 (KCCM11355P) having ornithine-producing ability Pediococcus pentosaceus AYO- ≪ / RTI > to produce a cottage cheese containing ornithine.

The present invention relates to a method for producing cottage cheese, comprising the step of inoculating Pediococcus pentosaceus AYO-1 (KCCM11355P) having ornithine-producing ability Pediococcus pentosaceus AYO-1 (KCCM11355P) And adding 1.0 to 2.0% (v / v) of a culture solution obtained by culturing for 6 hours, to prepare a cottage cheese containing ornithine.

In the above, cottage cheese represents cottage cheese based on crude oil.

In the method for producing cottage cheese comprising the culture liquid obtained by culturing the Pediococcus pentosaceus AYO-1 (KCCM11355P) of the present invention in a culture medium, Detailed selection of cottage cheese ingredients will be omitted.

The above-mentioned cottage cheese containing ornithine according to the present invention prepared by the process for producing cottage cheese is stored at a temperature of -1 to 5 占 폚.

The present invention relates to a Pediococcus pentosaceus AYO-1 strain capable of producing ornithine ( Pediococcus pentosaceus AYO-1) (KCCM11355P) is 1.0 to 2.0% (v / v).

The present invention relates to a Pediococcus pentosaceus AYO-1 strain capable of producing ornithine ( Pediococcus pentosaceus (V / v) obtained by inoculating AIK-1 (KCCM11355P) into skim milk medium and culturing at 30 DEG C for 48 hours.

Hereinafter, the content of the present invention will be described in detail through Experimental Examples, Examples and Test Examples. However, these are for the purpose of illustrating the present invention in detail, and the scope of the present invention is not limited thereto.

<Experimental Example>

The present invention is described in more detail below for the isolation and identification of Pediococcus pentosaceus AYO-1 (KCCM11355P), which is capable of ornithine production, of Pediococcus pentosaceus AYO-1.

[Materials and Methods]

(1) From natural fermented dairy products containing starters for the production of yogurt, the genus Pediococcus Isolation of candidate strains

In order to improve the functionality of cottage cheese, Pediococcus Lactic acid bacteria having ornithine - producing ability were isolated from naturally fermented dairy products containing yogurt starters as a study on the ability of the genus strains to produce bioactive substances. RGOSA and SHARPE MRS agar medium (Difco, Detroit, MI, USA) was used to dilute to 10 ^-1 10 ^-8 by the number of peptones using naturally fermented dairy products containing yogurt starters, ). MRS medium was supplemented with 0.02% Bromocresol purple (BCP) (Sigma Chemical Co., St. Louis, USA) and cultured at 37 ° C for 48 hours to obtain colonies. The colonies were dark yellow without forming a circle, and Pediococcus Respectively. The possibility of ornithine production was then investigated using TLC. TLC was performed using silica gel F ₂₅₄ and standard ornithine (Merck, Damstadt, Germany) and mobile phase (Butanol: Acetic acid: Dichoromethanol: water = 5: 3: 3: 3).

(2) Pediococcus with ornithine- producing ability genus Identification of the strain

Genomic DNA was isolated from candidate strains by DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany). Purification of the DNA followed the manufacturer's instructions. For cloning of 16S rDNA, Primer was subjected to PCR amplification using 5'-AGAGTTTGATCMTGGCTCAG-3 '(Forward) and 5'-ACGGGCGGGTGTGTRC-3' (Reverse). PCR amplification was carried out at 95 ° C for 30 sec. Denaturation at 55 ° C using 55 μl of 100 ng template DNA, 0.5 μl of primer DNA, 0.2 mM dNTPs, 10 × Ex Taq buffer and Taq polymerase (Takara Bio Inc., Shiga, Japan) 35 cycles of annealing at 30 ° C for 30 seconds and annealing at 72 ° C for 1 minute and 30 seconds were performed with a Biometra thermocycler (Tampa, Fla., USA). PCR products were confirmed by 1% agarose gel electrophoresis. The purified PCR products were purified using Purification Kit (Bioneer, Korea) and ligated to pGEM T-easy vector (Promega, Madison, WI, USA) using T4 ligase. 16S rDNA sequencing The nucleotide sequence information of AYO-1 was obtained. Using the obtained sequence information of 16S rDNA, NCBI (www.ncbi.nlm.nih.gov) was used for the homology of DNA, and the homology with the nucleotide sequence of the published strain was analyzed by Clustal X program.

(3) starters for the manufacture of cottage cheese and natural fermented dairy products Pediococcus genus Characteristics of strains

The API 50 CHL kit (BioMerieux sa, Marcy-L'Etoile, France) was used according to the manufacturer's instructions to investigate the sugar fermentation pattern and biochemical characteristics of the isolated strains. (Lee, JS, KC Lee, JS Ahn, TI Mheen, YR Byun and YH Park. 2002. Pediococcus. Koreensis sp. nov., isolated from kimchi. Int'l J. Syst . Evol. Microbial . 52, 1257-1261.). Inoculated with the strain of 1% arginine is added to the MRS medium (Difco, Detroit, MI, USA, pH 6.5) to measure the ornithine producing ability of the selected strains, and were cultured at 30 ℃ 30 ~ 48 times, in 5,000 × g After centrifugation for 20 minutes, the cells and the culture broth were separated and analyzed for ornithine, respectively.

(4) Manufacture of cottage cheese

The cottage cheese was prepared by pasteurizing the raw milk and inoculating 1.0% (v / v) of Renat and Pediococcus pentosaceus AYO-1 (KCCM11355P) and industrial starter Flora danica See Table 1).

[Table 1]

(5) Analysis of ornithine

To analyze ornithine, 800 μl of a solvent (methanol: chloroform: water = 12: 5: 3 (v / v)) was mixed with 200 μl of the cell solution and the culture solution. The supernatant, which is an aqueous solution layer, was firstly recovered by centrifugation (13,000 × g, 4 ° C., 15 minutes). To the organic solvent layer, 600 μl of a mixture of chloroform and water (1: 2 (v / v) And the supernatant was recovered secondarily. The supernatant was recovered by lyophilization. The supernatant was dissolved in ultrapure water and passed through a 0.45 μm PVDF membrane to be used as a sample for HPLC analysis. The samples were derivatized with 6-aminoqualio-N-hydroxysuccinimidyl carbonate (AQC) for analytical HPLC (Waters, Milford, Mass., USA) and analyzed with a 3.9x150 mm AccQ ㆍ Tag ^™ (Nova-Pak ^™ C18, Waters) (Seok, JH, KB Park, YH Kim, MO Bae, MK Lee and SH Oh, 2008, Production and Characterization of Kimchi with Enhanced Levels of -aminobutyric acid, Food Sci. Biotechnol 17, 940-946. The ornithine content was calculated using a standard curve based on the results of HPLC analysis of standard ornithine.

[result]

(1) Separation of ornithine-producing Pediococcus candidate strains from starter and natural fermented dairy products for the manufacture of cottage cheese

In order to isolate lactic acid bacteria producing ornithine from starters for the production of cottage cheese and natural fermented dairy products, the colonies grown in the MRS medium supplemented with bromophenol blue were selected as candidate strains which did not form a ring but were dark yellow. The possibility of ornithine production of candidate strains was investigated using TLC. Four candidate strains having excellent ornithine-producing ability were selected using TLC (see Fig. 1). The morphological and biochemical characteristics of AYO-1 among the selected strains were investigated. The isolated strain was Gram-positive and produced carbon dioxide under anaerobic conditions. The isolated lactic acid strains produced acids by decomposing ribose, glucose, proctose and mannose, and did not decompose arabinose, sorbose and sorbitol (see Table 2).

[Table 2]

In Table 2, + means positive and negative means negative.

(2) Identification of the strain by 16S rDNA sequencing

The 16S rDNA sequence was analyzed to identify the selected strains. The starting strain Pediococcus sp. The 16S rDNA sequence of AYO-1 was 99% homologous to the 16S rDNA sequence of Pediococcus pentosaceus strain KDLLL3-4 (Access No. KC454283.1), respectively (see Pediococcus pentosaceus AYO-1 base sequence). These results are summarized and identified by Sharpe et al. (Sharpe, ME, TF Fryer, and DG Smith, 1996. Identification of lactic acid bacteria, pp. 65-79, In BM Gibbs and FA Skinner (ed. This microorganism was identified as Pediococcus pentosaceus strain and named Pediococcus pentosaceus AYO-1 by reference to Methods for Microbiologists: part A. Academic Press, Inc., New York, USA. The isolated strain Pediococcus pentosaceus AYO-1 was deposited with KCCM 11355P on Jan. 29, 2013 at the Korean Center for Microbiological Conservation.

(3) Ornithine production ability of Pediococcus pentosaceus strain AYO-1

In order to investigate the ornithine production ability of the selected strain Pediococcus pentosaceus A YO-1, strains were cultured in MRS medium supplemented with arginine-free and arginine at pH 6.5 and 30 ° C for 48 hours . The degree of ornithine production of the strain was analyzed qualitatively and quantitatively using TLC and HPLC. The strain produced ornithine, and as a result, the ornithine production increased with increasing arginine concentration and the ornithine concentration decreased at 5%. Pediococus pentosaceus The ornithine-producing ability of AYO-1 strain is shown in Table 3.

[Table 3]

&Lt; Example 1 >

Pasteurized raw milk was mixed with 1.0 to 2.0% (v / v) of Pediococcus pentosaceus AYO-1 (KCCM11355P) and industrial starter Flora danica. The cottage cheese was prepared through the above cheese process.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings. It should be understood, however, that there is no intention to limit the scope of the present invention, which is intended to be illustrative of the present invention, and that various equivalents may be substituted for the same. Also, the terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor shall appropriately define the concept of the term to describe its invention in the best way The present invention should be construed in accordance with the meaning and concept consistent with the technical idea of the present invention.

&Lt; Test Example 1 >: Consumer preference and sensory evaluation

The color and sensory properties of five students of Woosuk University were examined by using 5 kinds of color, flavor and taste of Pediococcus pentosaceus AYO-1 and Flora danica, which are capable of producing ornithine. The results are shown in Table 4 below.

[Table 4] Sensory properties of cheese prepared using functional strains

As shown in the table above, the cottage cheese with Pediococcus pentosaceus AYO-1 strain showed similar color change over time, but the taste was superior over time, and the aroma gradually increased over time .

&Lt; Test Example 2 >

<Test Example 2-1>: Measurement of Ornithine Generation Potential of Strain

When the ornithine standard is used as a standard, candidate samples 1 to 4, 8, and 12 produce spots on the same line as the ornithine reference material.

<Test Example 2-2>: Analysis of ornithine content of the strain cultured in MRS medium

The content of arginine was 179.35 / 100ml, 319.43mg / 100ml, and 296.01mg / 100ml in arginine 1, 3, and 5%, respectively. The content of arginine increased with increasing arginine content And 5%, respectively, and it was found to be slightly lower.

<Test Example 2-3>: Content of ornithine in refrigerated storage after fermentation of cottage cheese

When the concentration ratio of functional lactic acid bacteria and commercial starter was 1: 1 and 1: 2, the ornithine concentration was higher than that of the cheese of the other conditions at the initial stage of production and 3 days of refrigerated storage. On the sixth day, Respectively. However, the concentration of ornithine at the 6th day of refrigerated storage was relatively low when the concentration ratio of the active lactic acid bacteria to the commercial starter was 1: 2.

[Table 5] Sequence list comparison

The Pediococcus pentosaceus AYO-1 strain having the ability to produce ornithine of the present invention ( Pediococcus pentosaceus AYO-1) (KCCM11355P), which can provide cottage cheese with excellent characteristics containing ornithine when producing cottage cheese, thereby contributing to health promotion of cottage cheese consumers.

Also, as a manufacturer of cottage cheese, Pediococcus pentosaceus AYO-1 strain ( Pediococcus pentosaceus AYO-1) (KCCM11355P), it is expected that it will provide excellent nutritional value to consumers in the field of cottage cheese, and it is expected to contribute greatly to the dairy food industry.

Korea Microorganism Conservation Center (overseas) KCCM11355P 20130129

Claims (6)

delete delete a) pasteurizing the crude oil at 65 to 70 ° C for 4 to 6 seconds;
b) a cooling step of cooling the crude oil after the sterilization step to 30 to 36 ° C;
c) Inoculation step of inoculating functional crude lactic acid bacterium and industrial starter at 1.0 to 2.0% (v / v) into the crude oil after the cooling step;
d) culturing the mixture after the inoculation step at 30-35 ° C;
e) adding 0.001-0.02% (v / v) rennet to the mixture after the culturing step;
f) a step of allowing the mixture to stand for 50 to 70 minutes;
g) a cutting step of cutting the curd after completion of the setting step;
h) heating step wherein the cut curd is heated to 55 to 60 DEG C with stirring for 30 to 50 minutes;
i) a whey removing step of washing the curd after the heating step twice and removing the whole whey;
j) a draining step for draining the remaining whey for 2-3 hours at the curd after the whey removal step;
k) an addition step of adding 0.5 to 1.0% (w / v) of salt to 100 parts by weight of the curd subjected to the above step;
l) a storing step of packing the cheese having passed through the adding step and storing at -1 to 5 &lt; 0 &gt; C,

Wherein the functional lactic acid bacterium is Pediococcus pentosaceus AYO-1 (KCCM11355P) having the ability to produce ornithine and the industrial starter is Flora danica in step c) Gt;
delete In step c) of claim 3,
A culture solution obtained by inoculating Pediococcus pentosaceus AYO-1 (KCCM11355P), a Pediococcus pentosaceus AYO-1 having ornithine production ability, into skim milk medium and culturing at 42 ° C for 4 to 6 hours, % &lt; / RTI &gt; (v / v).
delete

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