KR20220142145A - Lactobacillus harbinensis VF for fermented foods production - Google Patents

Abstract

The present invention provides a novel Lactobacillus harbinensis VF capable of lactic acid fermentation and acetic acid fermentation, and providing a unique flavor by harmonizing sweetness and sourness during fermentation. The present invention is Lactobacillus habinensis VF (accession number: KACC 92345P) for fermented foods with lactic acid bacteria producing lactic acid and acetic acid.

Description

Lactobacillus harbinensis VF for fermented foods production

The present invention relates to lactic acid bacteria for manufacturing fermented food, and more particularly, lactic acid fermentation and vinegar fermentation using various plant materials, and Lactobacillus hobius for manufacturing a novel fermented food that provides a unique flavor by harmonizing sweet and sour taste It's about Nensis V.F.

Fermentation using microorganisms has been performed as a method of increasing the preference for food by using various ingredients and maintaining or increasing specific ingredients along with natural organic acids. In particular, as for the traditional fermentation technology, various fermented beverages have been developed through lactic acid fermentation and acetic acid fermentation. In the millennial generation, interest in environmental pollution, climate change, and animal welfare due to factory livestock is growing, and due to the expansion of vegetarianism and veganism, various attempts have been made to substitute plant-based fermented foods using animal ingredients. In addition, the meat and dairy substitute industry continues to expand after 2020, and the demand for plant-based alternative milk and dairy products using it to replace dairy products is increasing.

Rice is the main food source cultivated in Southeast Asia including Korea. Rice production is on a downward trend from 4.33 million tons in 2015 to 3.63 million tons in 2020. In Korea, in terms of demand and supply of rice, an average of 280,000 tons of rice is overproduced annually (the average annual production of rice is 4.17 million tons, which is a state of oversupply that greatly exceeds the proper demand of 3.7 million tons). Due to the oversupply of rice compared to the consumption of rice, the rate of processing using rice is increased, and the development of processed rice foods is actively progressing in response to changes in dietary habits that are becoming more convenient and westernized.

Research on rice reports on general ingredients of rice, amylose and resistant starch content, dietary fiber and antioxidants, and research and development of various types of special rice such as high amylose high amylose or colored rice with antioxidant activity. Because black rice is rich in flavonoid and anthocyanin content and has antioxidant activity similar to that of α-tocopherol, it is reported to have various physiological activities such as anti-aging, antioxidant and anti-thrombolysis compared to white or brown rice. It is reported that red yeast rice produced by culturing filamentous fungi of the genus Monascus on white rice produces Monacolin K, which is low in toxicity and has a strong cholesterol-lowering action, effectively reducing blood cholesterol in the body.

In particular, as gluten, a wheat protein, can cause celiac disease and wheat allergy, the development of alternative products using rice as 'gluten-free' is actively underway. As processed foods using rice, rice cakes and noodles account for more than half of all rice-using products, and they are produced in a form suitable for various consumption patterns such as rice crackers, rice flour, soy sauce, snacks, and beverages. However, the results of research on developing food with rice alone are very limited.

Although rice is saccharified or fermented rice in the form of yogurt with lactic acid bacteria added, it is used as a meal replacement and health drink, but it is mostly limited to cases developed as lactic acid bacteria fermented beverages based on milk products by adding milk. In some cases, development of rice fermented beverages using yeast has been progressed, but there is a problem accompanying alcoholic fermentation.

At a time when the demand for vegan food is increasing significantly, research and development on vegan-type lactic acid bacteria fermented beverages that do not contain animal milk while facilitating consumption of rice, a vegetable raw material, are urgently required due to their excellent sensory properties. .

The present invention has been proposed to solve the problems of the prior art described above, and the purpose is to enable lactic acid and acetic acid fermentation, and a novel Lactobacillus habinensis V that provides a unique flavor by harmonizing sweet and sour taste during fermentation It is to provide F.

The problems to be solved of the present invention are not limited to those mentioned above, and other problems to be solved that are not mentioned will be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description.

The technical problem of the present invention as described above is achieved by the following means.

(1) Lactobacillus habinensis VF (KACC 92345P) for fermented food with lactic acid bacteria producing lactic acid and acetic acid

(2) The fermented food according to the above (1), Lactobacillus habinensis VF (KACC 92345P), characterized in that the vegetable raw material including rice.

As described above, the present invention provides a novel Lactobacillus habinensis VF that can be fermented with lactic acid and acetic acid, and provides a unique flavor by harmonizing sweet and sour taste during fermentation.

1 is a manufacturing process diagram of the lactic acid bacteria fermented beverage of the present invention.
Figure 2 is the result of liquefaction and saccharification enzyme treatment of non-gelatinized rice (60 ℃, 30 minutes).
Figure 3 is a Neighbor-Joining-based phylogenetic tree of 16S rRNA of the strain of the present invention.
4 is a gel permeation chromatography result of AMG-treated rice hydrolyzate.
5 is a result of lactic acid fermentation in GY medium supplemented with 2.5% glucose and 1% yeast extract using L. plantarum and L. harbinensis VF mixed microorganisms.
6 is an acid resistance test result performed on the lactic acid bacteria strain of the present invention.
7 is a result of a biliary resistance test performed on the lactic acid bacteria strain of the present invention.
8 is an antibiotic resistance susceptibility test result performed on the lactic acid bacteria strain of the present invention.
9 is a surface hydrophobicity test results performed on the lactic acid bacteria strain of the present invention.
10 is a result of aggregation experiments performed on the lactic acid bacteria strain of the present invention.
11 is a result of analysis of aroma components according to the culture of lactic acid bacteria strains for each medium_PCA [(a) Analysis of aroma components according to the culture of lactic acid bacteria single strain in MRS medium_PCA, (b) according to the culture of lactic acid bacteria single strain in GY medium Fragrance component analysis_PCA, (c) Fragrance component analysis according to culturing of lactic acid bacteria single strain in NW medium_PCA, (d) Fragrance component analysis according to culture of lactic acid bacteria single strain in Black medium].
12 is a result of analysis of the aroma component according to the culture of the lactic acid bacteria strain for each medium_Bar graph [(a) Analysis of the aroma component according to the culture of the lactic acid bacteria single strain in the MRS medium_Bar graph, (b) the culture of the lactic acid bacteria single strain in the GY medium Analysis of aroma components according to _Bar graph, (c) Analysis of aroma components according to the culture of a single lactic acid bacteria in NW medium_Bar graph, (d) Analysis of aroma components according to the cultivation of a single strain of lactic acid bacteria in black medium_Bar graph].
13 is an analysis result of aroma components of LP + LH complex culture medium in NW (left) and Black (right) medium_PCA.
14 is a fragrance component analysis_Radar plot of the LP + LH complex culture medium.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described below in detail in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the technical field to which the present invention pertains It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.

In describing the embodiments of the present invention, if it is determined that a detailed description of a well-known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. In addition, the terms to be described later are terms defined in consideration of functions in an embodiment of the present invention, which may vary according to intentions or customs of users and operators. Therefore, the definition should be made based on the content throughout this specification.

Hereinafter, an embodiment of the present invention will be described with reference to the accompanying drawings.

The lactic acid bacterium L. harbinensis of the present invention was isolated from mulberry vinegar soaked in a farmhouse in Gimje, named L. harbinensis VF, and deposited with the Agricultural Genetic Resources Center Microbial Bank on March 30, 2021 with deposit number KACC 92345P.

The lactic acid bacterium L. harbinensis VF of the present invention is taxonomically considered to belong to L. harbinensis , but it shows a slight difference in phenotype from other strains of the same species reported previously, and simultaneously performs lactic acid fermentation and acetic acid fermentation using rice as a substrate. In particular, it has a difference in that it provides a unique flavor that is different from other lactic acid strains.

The lactic acid bacteria of the present invention may be used alone in the production of fermented food, as well as other publicly disclosed strains, for example, may be used together with lactic acid bacteria such as L. plantarum .

Hereinafter, the manufacturing process of the rice fermented beverage using the lactic acid bacteria of the present invention will be described.

As shown in FIG. 1, the method for producing a fermented beverage using lactic acid bacteria according to the present invention exemplifies a lactic acid bacteria fermented beverage using rice, but in addition to rice, various vegetable raw materials such as vegetables, fruits, etc. can be used. .

The manufacturing method of the lactic acid bacteria fermented beverage using the rice includes the steps of (1) preparation of raw material rice, (2) gelatinization of raw material rice, (3) liquefaction/saccharification of gelatinized rice, and (4) fermentation of lactic acid bacteria.

The raw material rice in step (1) is not particularly limited, and for example, spicy rice, glutinous rice, colored rice (black rice, red rice rice, etc.) may be mentioned, and preferably non-glutinous rice, black rice, red rice rice, or mixed rice thereof. can In particular, in the case of mixed rice, it is preferable in that various colors can be produced by adjusting the ingredients and composition ratio, and various flavors can be provided.

The gelatinization process of raw rice in step (2) is preferably carried out for at least 10 minutes using a 100°C bath, preferably for 15 minutes.

In the liquefaction/saccharification process of the gelatinized rice in step (3), it is also possible to perform the liquefaction process and the saccharification process sequentially, but it is preferable to perform the liquefaction process and the saccharification process at the same time to simplify the process.

Preferably, in the present invention, in the liquefaction / saccharification process, the gelatinized rice is 2-5% (w/v), preferably 3% (w/v). Although this will be described in the experimental example of the present invention to be described later, it is to match 2.5% (w/v) glucose, which is an optimal medium composition, as a carbon source for the lactic acid bacteria of the present invention, which is desirable not only for the growth of lactic acid bacteria, but also for the sensory characteristics of the final product. .

When liquefaction and saccharification are sequentially performed, the liquefaction process is preferably 2-5% (w/v) rice with liquefaction enzyme (e.g., Fungamyl) at 55-65 ℃ 0.005-0.075% (v/v) ), preferably 0.025% (v/v) is added and the reaction is carried out within 30 minutes, preferably for 30 minutes.

Subsequently, the saccharification process is preferably performed by adding 0.01 to 0.25% (v/v), preferably 0.02% (v/v) of a saccharification enzyme (eg, glucoamylase (AMG)) at 55 to 65° C. for 60 minutes. within, preferably for 60 minutes.

For efficient pretreatment in the present invention, it is preferable to simplify the process, and for this purpose, it is also possible to perform the liquefaction/saccharification process as a single process. For the liquefaction/saccharification process, a single enzyme (Fungamyl or AMG) may be used, or two or more complex enzymes (eg, a mixed enzyme of Funamyl and AMG) may be used, but in terms of process efficiency, preferably AMG Use a single enzyme.

The liquefaction/saccharification process using only AMG is preferably within 60 minutes by adding 0.01 to 0.25% (v/v), preferably 0.25% (v/v) at 55 to 65°C, preferably 60°C, preferably react for 30 minutes.

In the step (4), the lactic acid bacteria may be selected from known lactic acid bacteria, or purely isolated lactic acid bacteria may be selected alone or by mixing with a test strain. As the known lactic acid bacteria in the present invention, preferably, a representative plant lactic acid bacteria, Lactobacillus plantarum ( L. plantarum ) is used.

As a result of the present invention, both L. plantarum and L. harbinensis VF require 2.5% (w/v) glucose as a carbon source and 1% (w/v) yeast extract as a nitrogen source as optimal medium conditions.

When the two types of lactic acid bacteria are mixed and used, the initial composition ratio of L. plantarum and L. harbinensis VF is preferably 10 ^{5:10 6} ~ 10 ^{6:10 5} CFU/mL. The fermentation temperature is preferably 30-37°C, preferably 37°C, and the pH is preferably adjusted to 6-7, preferably pH 6.

Hereinafter, the contents of the present invention will be described in more detail with reference to examples, but these examples are only presented to help the understanding of the present invention, and the scope of the present invention should not be construed as being limited thereto.

[Experimental Example 1] Establishment and optimization of conditions for hydrolysis (liquefaction → saccharification) of gelatinized rice starch

In the pre-treatment process, the gelatinization process is necessary for enzymatic decomposition, and it was carried out for 15 minutes in a 100° hot water treatment method. In this experiment, the two-step process of liquefaction/saccharification was simplified, and it was considered that the optimum reaction temperature of the liquefaction enzyme and the saccharification enzyme was the same 60℃. As a result of confirming the optimal reaction conditions using liquefaction and saccharification enzymes with single enzyme and complex enzyme treatment, 0.25% (v/v) AMG as a single enzyme was treated within 30 minutes of reaction time and liquefaction and saccharification enzymes were combined. One result was the same (FIG. 2). Accordingly, in order to efficiently proceed with the pretreatment enzyme treatment process, it was decided to use only the glycosylation enzyme as a single enzyme.

The results of confirming the hydrolyzate of rice treated with AMG, a saccharification enzyme, by using GPC chromatography are shown in FIG. 3 . After gelatinizing 3% non-glutinous rice, 0.25% of AMG enzyme was treated to analyze the molecular weight of the hydrolyzate for each reaction time, and as a result, it can be confirmed that all high molecular substances other than the decomposition product, glucose, disappear after 30 minutes.

[Experimental Example 2] Lactobacillus selection and optimization of lactic acid bacteria culture conditions

1. Lactobacillus selection

As the test strain, L. plantarum KCTC 3108, a representative plant lactic acid strain, was purchased, and L. harbinensis VF (KACC 92345P) was used after pure separation by the present inventors through the following process.

Samples were collected from the traditional vinegar fermentation process using mulberry at Jaram Farm (Gimje, Jeollabuk-do), and GY plate medium containing 2% (v/v) ethanol (Glucose 5%, Yeast extract 1%, CaCO ₃ 3%, Acid-producing microorganisms were isolated using pH 6). In the GY plate medium, acetic acid-producing bacteria and lactic acid bacteria producing organic acids including lactic acid could be separated. At this time, the microorganisms growing on the BCP-containing MRS plate medium were determined as lactic acid bacteria among the various strains separated at this time, and pure separation was performed. After culturing the pure isolated lactic acid bacteria, they were extracted using a genomic DNA extraction kit (iNtRON Biotechnology, Korea) and used as a template for 16S rRNA gene amplification of the selected lactic acid bacteria. To amplify the 16S rRNA of lactic acid bacteria, common primers 27F (5'-AGA GTT TGA TCC TGG CTC AG-3', forward), 1492R (5'-GGC TAC CTT GTT ACG ACT T-3', reverse) were used. Thus, PCR was performed using Solg™ EF-Taq DNA Polymerase (Solgent Co., Ltd. Deajeon, Korea). Polymerase Chain Reaction (PCR) conditions were repeated 30 times by thermal denaturation at 95°C for 1 minute, primer binding at 45°C for 1 minute, and DNA extension at 72°C for 1 minute and 30 seconds. After confirming the band through electrophoresis to analyze the amplified DNA, the amplified product was purified using QIAquick Gel Extraction kit (QIAGEN GmbH, Hilden, Germany). 16S rRNA base sequence was determined using BigDye® Terminator v3.1 Cycle Sequencing Kits and ABI Prism 3730XL DNA Analyzer (Applied Biosystems, Foster city, CA, USA).

The strain isolated in the present invention, Lactobacillus spp, is a Blast N result, L. harbinensis (NBRC 100982 = SBT 10908) at 99% similarity, L. perolens (L 532) at 98% similarity, and L. shenzhenensis (LY) at 98% similarity. -73) (FIG. 4), and was determined as L. harbinensis in identification using 16S rRNA nucleotide sequence (SEQ ID NO: 1). The phenotype of the isolated strain of the present invention was compared with the existing test strains L. perolens and L. harbinensis , and it was found that there were differences as shown in Tables 1 and 2 below.

strain information


Substrate isolated strain L. perolens L. harbinensis DSM 12745 DSM 12744 KF 312679 KACC12409 K.V9.3.1Np FQ003
AB 196123 DSM 16991 SBT
10904 (SBT 10908) Glycerol - + - - w - - + + Erythritol - - - - w - - - - D-Arabinose - + - w + - w + + L-Arabinose + + + + + + + + + D-Ribose - - - - w - - - - D-Xylose + - - + - - + - + L-Xylose - - - - - - - - - D-Lyxose (Aldopentose) - - - - - - - - - D-Adonitol (Rbitol) - - - - - - - - - D-Arabitol - - - - - - - - - L-Arabitol - - - - - - - - - Xylitol - - - - - - - - - Methyl-β-D-Xylopyranoside - - - - - - - - + D-Galactose + + + + + + + + + D-Glucose + + + + - + + + + D-Fructose + + + + + + + + + D-Mannose + + + + + + + + + L-Sorbose (Ketose) - - - - - - - - - L-Rhamnose - - - - - - - - - D-Tagatose (Sweetner) w + + w w - w + + D-Fucose (Fucoidan) - - - - - - - - - L-Fucose (Fucoidan) - + - w - - w - + Dulcitol (Galactol) - - - - - - - - - Inositol - - - - - - - - - D-Mannitol + - - - - - - - - D-Sorbitol w + w - - - - - - Amygdalin + + + + + + + + + Arbutin + + + + + + + + + Esculin (Aesculin) + + + + + + + + + Salicin + + + + + + + + + α-Methyl-D-Mannopyranoside - + - - + - - - - α-Methyl-D-Glucopyranoside + + + + + - + + + N-Acetyl-Glucosamine + + + + + + + + + Gluconate w + + + w + + w w 2-Keto-Gluconate - + - - w - - w w 5-Keto-Gluconate - w - - - - - + +

strain information


Substrate isolated strain L. perolens L. harbinensis DSM 12745 DSM 12744 KF 312679 KACC12409 K.V9.3.1Np FQ003
AB 196123 DSM 16991 SBT
10904 (SBT 10908) Cellobiose + + + + + + + + + Maltose + + + + + + + + + D-Lactose + + + - w + - + + Melibiose + + + + w + + + + Sucrose + + + + + + + + + Gentiobiose w + + + + + + + + Trehalose + + + + + + + + + D-Turanose + + + w w - w + + Melezitose (Melicitose) + + + + w - + + + Raffinose + + + + + + + + + Starch - + - w w - w - + Inulin w + - - + - - - - Glycogen - - - - - - - - - growth temp
in MRS 5° - - - - 10° - + (-) + - - 15° (+) + + + 20° + + + 45° - - + 42 - - - 50° - - - Specific growth rate (h ^-1 ) 30° 0.20 37° 0.15 Growth pH
in MRS pH 3.0 + + 3.5 + pH 4.0 + + + + pH 5.0 + + + + pH 6.0 + + + + pH 7.0 + + + + pH 8.0 + + + + Salt (NaCl)　tolerance 3% + + + 6.5% - - - 7% - + Characteristics Shape Rod Rod Rod Gram + + +

(+): grow after 21 days, (-): No growth at 12° w: weak growth

2. Optimization of lactic acid bacteria culture conditions

(1) Optimization of medium composition for two types of selected lactic acid bacteria

In order to proceed with the fermentation of lactic acid bacteria after rice pretreatment, the composition of GY medium (Glucose-Yeast extract medium) was established by optimizing the carbon source (glucose) and nitrogen source (yeast extract).

go. Effect of carbon source (glucose) on the growth of each lactic acid bacteria and fermentation of lactic acid bacteria

Using batch culture, the growth and fermentation characteristics of lactic acid bacteria were investigated according to the 1-20% initial glucose concentration in a state where 1% (w/v) nitrogen source (YE) was fixed.

L. plantarum : When the concentration of the initial carbon source (glucose) was 5% (w/v) or more, the specific growth rate and the tendency to decrease the production of lactic acid were confirmed. In a state where the nitrogen source (YE) was fixed at 1%, the initial carbon source was 2.5% (w/v) glucose at the optimal specific growth rate (0.18±0.01h ^-1 ) and lactic acid (4.8 g/L) and acetic acid (0.14 g/L) L) was produced.

L. harbinensis VF: When the concentration of the initial carbon source (glucose) is 5% (w/v) or more, the specific growth rate and the production of lactic acid tend to decrease, but L. plantarum The negative effect is lower than the fermentation of lactic acid bacteria used. In a state where the nitrogen source (YE) was fixed at 1%, the initial carbon source was 2.5% (w/v) glucose at the optimal specific growth rate (0.18±0.01h ^-1 ) and lactic acid (7.8 g/L) and acetic acid (0.88 g/L) L) was produced.

me. Effect of nitrogen source (YE) on the growth of each lactic acid bacteria and fermentation of lactic acid bacteria

Growth and lactic acid bacteria fermentation characteristics were investigated according to the concentration of yeast extract corresponding to 0.05-2.5% (w/v) in a state where 2.5% (w/v) carbon source (glucose) was fixed using batch culture.

L. plantarum : As the concentration of yeast extract (YE), the initial nitrogen source, increased, the production of lactic acid and acetic acid continued to increase. Although it was used as a nitrogen source, it can also serve as a carbon source, so it is judged as a result of the continuous growth of L. plantrum . In the presence of 2.5% (w/v) carbon source (glucose), the initial nitrogen source concentration was determined with 1% (w/v) yeast extract to obtain the optimal specific growth rate (0.17±0.01h ^-1 ).

L. harbinensis VF: As the concentration of yeast extract (YE), the initial nitrogen source, increased, the production of lactic acid and acetic acid continued to increase. Although it was used as a nitrogen source, it can also serve as a carbon source, so it is judged as a result of the continuous growth of L. harbinensis VF. In the presence of 2.5% (w/v) carbon source (glucose), the initial nitrogen source concentration was determined with 1% (w/v) yeast extract to obtain the optimal specific growth rate (0.16±0.01h ^-1 ), and lactic acid bacteria It was observed that the unique yogurt flavor was strongly generated according to the fermentation.

All. Culture characteristics of two types of lactic acid bacteria using 2.5% glucose and 1% yeast extract selected as the optimal GY medium

As shown in Figure 5, the growth curves of the two types of lactic acid bacteria were the same in the GY medium, but the pH was confirmed to be lowered faster in L. harbinensis VF than in L. plantarum . This is due to the fact that the production of lactic acid and acetic acid was higher in L. harbinensis VF than in L. plantarum at the same incubation time. Accordingly, it was decided to use a seed strain mixed with two types of lactic acid bacteria to maximize the unique fermentation flavor.

[Experimental Example 3] Mixed culture of two types of lactic acid bacteria

1. Characteristics of fermentation of lactic acid bacteria using individual strains of two types of lactic acid bacteria

As a result of culturing a single lactic acid bacteria culture at 37° using a GY medium composed of 2.5% Glucose and 1% yeast extract, the number of viable cells under the same culture conditions as shown in Table 3 below was 2 in L. plantarum compared to L. harbinensis VF. In culture using L. harbinensis VF, the production of lactic acid and acetic acid was higher than that of L. plantarum by 2 times and 7 to 10 times, respectively. It was confirmed that the unique lactic acid bacteria fermentation flavor was produced during the cultivation of L. harbinensis VF.

* LP: L. plantarum , LH: Lactobacillus harbinensi s VF, YE: Yeast extract, △pH: Difference in pH

2. Characteristics of fermentation of lactic acid bacteria using a mixed strain of two types of lactic acid bacteria

Table 4 below shows the results of investigating the fermentation characteristics of mixed lactic acid bacteria according to the inoculation ratio (A~C) of the two types of lactic acid bacteria using GY medium composed of 2.5% Glucose and 1% yeast extract.

A = LP ( L. plantarum ) : LH ( L. harbinensis VF) = 10 ⁵ : 10 ⁶ (5:6)

B = LP ( L. plantarum ) : LH ( L. harbinensis VF) = 10 ⁶ : 10 ⁶ (6:6)

C = LP ( L. plantarum ) : LH ( L. harbinensis VF) = 10 ⁶ : 10 ⁵ (6:5)

As a result of the experiment, the specific growth rate was lower than the case of L. plantarum single and higher than the case of L. harbinensis VF single with a mixture of two types of lactic acid bacteria, indicating that it was in the middle. Although the OD showed a tendency to grow continuously, the number of viable cells reached the maximum CFU within about 15 hours, and the number of viable cells decreased as the pH of the medium was lowered to 3.5 or less.

As a result of lactic acid fermentation with two types of mixed lactic acid bacteria, the production of lactic acid obtained similar results to that of single lactic acid bacteria inoculation, whereas the production of acetic acid was lower than the case of using a single lactic acid bacteria (LH) according to the use of L and plantarum . The production of lactic acid and acetic acid gradually increased as the culture continued.

As such, the inoculation ratio of LP and LH for mixed lactic acid bacteria fermentation was determined as type A (LP:LH = 10 ⁵ : 10 ⁶ ) from the characteristics of fermentation of lactic acid bacteria according to the three inoculation amounts.

[Experimental Example 4] Preparation of lactic acid bacteria fermented beverage

Three types of rice (spicy rice, black rice, and red rice) were used for the fermentation of lactic acid bacteria. For the production of lactic acid bacteria fermented beverages using these rice, a GTY medium to which the optimized conditions obtained in the previous experiment were applied was prepared. That is, the concentration of rice was determined to be 3% (w/v) to correspond to 2.5% Glucose and applied, and 1.0% yeast extract was applied after rice pretreatment, and two types of individual lactic acid bacteria and mixed lactic acid bacteria (LP: LH = 10 ⁵ : 10 ⁶ ) was applied to perform lactic acid fermentation, and the results of each experiment are shown in Table 5.

(1) Specific growth rate

As shown in Table 3 for the results of fermentation of lactic acid bacteria, the measurement results of the specific growth rate of lactic acid bacteria by rice variety, in the case of L. plantarum single lactobacillus fermentation, when rice pretreated rather than GY medium is used, the specific growth rate of non-glutinous rice (Shin Dongjin) and black rice is was increased by 1.87 times in , and increased by 1.53 times in honggukmi. In the case of L. harbinensis VF single lactobacillus fermentation, the specific growth rate increased by 1.31 times in non-glutinous rice (Shin Dongjin), 1.16 times in black rice, and 1.09 times in red yeast rice when pretreated rice was used rather than GY medium. In the case of lactic acid fermentation using mixed lactic acid bacteria (LP: LH = 10 ⁵ : 10 ⁶ ), when rice pretreated rather than GY medium is used, the specific growth rate is 2.07 times in non-glutinous rice (Shin Dongjin), 1.88 times in black rice, and 1.62 times in red rice rice. doubled. When rice was pretreated compared to GY medium, the specific growth rate of lactic acid bacteria was increased by various minerals and other components such as protein.

(2) production of lactic acid

In the case of L. plantarum single lactobacillus fermentation, the production of lactic acid increased by 1.37 times in non-glutinous rice (Shindongjin), 2.34 times in black rice, and 1.18 times in red yeast rice when pretreated rice was used rather than GY medium. In the case of L. harbinensis VF single lactobacillus fermentation, lactic acid production increased by 1.05 times in non-glutinous rice (Shin Dongjin), 1.19 times in black rice, and 1.05 times in red yeast rice when pretreated rice was used rather than GY medium. In the case of lactic acid fermentation using mixed lactic acid bacteria (LP: LH = 10 ⁵ : 10 ⁶ ), lactic acid production was 0.93 times in non-glutinous rice (Shindongjin), 1.54 times in black rice, and 0.73 times in red yeast rice when using pretreated rice rather than GY medium. doubled. When rice was pretreated compared to GY medium, it was judged that the production of lactic acid by lactic acid bacteria was mainly effective for L. plantarum by various minerals and other ingredients such as proteins.

(3) production of acetic acid

In the case of L. plantarum single lactobacillus fermentation, it was confirmed that when pretreated rice was used rather than GY medium, the production of acetic acid increased by 0.46 times in non-glutinous rice (Shindongjin), 2.01 times in black rice, and decreased in red rice rice. In the case of L. harbinensis VF single lactobacillus fermentation, the production of acetic acid increased 0.74 times in non-glutinous rice (Shindongjin), 1.36 times in black rice, and 0.52 times in red yeast rice when pretreated rice was used rather than GY medium. In the case of lactic acid fermentation using mixed lactic acid bacteria (LP: LH = 10 ⁵ : 10 ⁶ ), when rice pretreated rather than GY medium was used, the production of acetic acid increased by 0.18 times in non-glutinous rice (Shindongjin) and 0.61 times in black rice, but decreased in red rice. . When rice was pretreated compared to GY medium, acetic acid production was found to increase only in black rice, and it was confirmed that it decreased when using complex lactic acid bacteria.

(4) consumption of carbon sources

In the case of L. plantarum single lactobacillus fermentation, about 3.5 g/L was consumed in GY medium and non-glutinous rice (Shindongjin), but 6.2 g/L in black rice and 7.41 g/L in red rice rice were used, so the use increased in colored rice than in white rice. . In the case of L. harbinensis VF single lactobacillus fermentation, 6.25 g/L in GY medium, 5.25 g/L in non-glutinous rice (Shin Dongjin), 5.57 g/L in black rice, and 4.33 g/L in red yeast rice were consumed, and it was used in colored rice rather than white rice. this decreased. In case of fermentation of lactic acid bacteria using mixed lactic acid bacteria (LP: LH = 10 ⁵ : 10 ⁶ ), 5.99 g/L in GY medium, 3.65 g/L in non-glutinous rice (Shindongjin), 6.53 g/L in black rice, and 2.94 g in red yeast rice. /L was used.

* ND: Not determined, LP+LH: Mixed culture with L. plantarum and L. harbinensis VF

[Experimental Example 5] Sensory evaluation

By adding milk instead of rice component, L. plantarum single lactic acid bacteria fermented beverage was used as a control, and sensory evaluation was performed on the fermented beverage prepared by Experimental Example 4 of the present invention using the rice component. A five-point scale method was used, and taste, aroma, color, and overall preference were used as the score criteria, with 5 being “good” and 1 being “bad”, and the results are shown in Table 6 below.

As shown in Table 6 above, in all items, the product according to Experimental Example 4 of the present invention showed good sensory properties compared to the control obtained by fermenting with milk, especially in the case of taste and flavor when a mixed strain was used. It appears to be particularly excellent, which is a result of simultaneous lactic acid and acetic acid fermentation by mixing the two strains, and is evaluated to provide a completely different flavor from the previous lactic acid fermented beverages.

[Experimental Example 7] Probiotic properties of lactic acid bacteria

(1) acid resistance (Fig. 6)

L. plantarum , a standard strain, was confirmed to have viability at pH 3, but at pH 2.5 or lower, viability decreased with increasing time. After 3 hours at pH 2.0, the survival rate was less than 20%, and the selected lactic acid bacteria L. The viability of harbinensis VF was confirmed at pH 2.5, but the viability decreased to 80% after 3 hours as time increased at pH 2.0 . was found to be excellent.

(2) bile resistance (Fig. 7)

As for the standard strain L. plantarum , the survival rate over time in the MRS medium containing the Bile salt was 88.6% after 6 hours when 0.3% Bile salt was contained, whereas 60 when 0.5% Bile salt was contained. % or less, and the survival rate over time of L. harbinensis VF, the selected lactic acid bacterium, was 75.7% after 6 hours when 0.3% bile salt was contained in MRS medium containing bile salt, whereas 0.5% bile salt In the case of containing salt, it was reduced to 71.8%. In general, the bile resistance of lactic acid bacteria tends to decrease as the bile salt increases, and it was confirmed that the selected lactic acid bacteria, L. harbinensis VF, had better bile resistance than the standard strain L. plantarum .

(3) antibiotic resistance and susceptibility

Disc diffusion method was used to confirm antibiotic resistance. Experiments were performed at two concentrations (10 ⁹ and 10 ⁶ CFU/mL) to confirm susceptibility to antibiotics, and the results are shown in FIG. 8 .

L. plantarum , a standard strain, is resistant to AK (Amikacin), CIP (Ciprofloxacin), K (Kanamycin), S (Streptomycin), and VA (Vancomycin), and L. harbinensis VF, the selected lactic acid bacteria, is SXT (Sulfamethoxazole). + Trimethoprim), it is sensitive to most antibiotics, and it was confirmed to be particularly sensitive to VA (Vancomycin), which is resistant to most lactic acid bacteria.

(4) surface hydrophobicity

Surface hydrophobicity, which is one type of surface characteristics of the selected lactic acid bacteria, was confirmed using three types of hydrocarbons and chloroform (FIG. 9). The surface hydrophobicity of L. plantarum , a standard strain, was 40% in octane and xylene and 60% in chloroform at 30 minutes of reaction time, but gradually decreased as the reaction time elapsed to 20% in the case of n-octane. decreased, and the surface hydrophobicity of L. harbinensis VF, the selected lactic acid bacterium, increased from 7% at 30 minutes in reaction time in three hydrocarbons and chloroform to 10-20% as the reaction time increased.

(5) Aggregation characteristics

The auto-aggregation ability of the selected lactic acid bacteria and the co-aggregation ability with 5 kinds of harmful microorganisms were confirmed using the PBS solution (FIG. 10). [ B. cereus (KCTC 1013), E. coli O157:H7 (ATCC 43895), S. typhimurium (KCTC 2514), S. aureus (KCTC 1621), L. monocytogenes (KCTC 3596)]

In L. plantarum , a standard strain, auto-aggregation increased with time, reaching the level of 20% after 6.5 hours, and co-aggregation of B cereus and S. aureus among harmful microorganisms was promoted, and selected lactic acid bacteria In L. harbinensis , auto-aggregation increased with time, reaching 17.5% after 6.5 hours, and among harmful microorganisms, co-aggregation with B cereus, S. aureus and L. monocytogenes tended to be promoted.

(6) antibacterial activity (Table 7)

The growth inhibition of 5 types of harmful microorganisms was confirmed using the culture medium of the two selected lactic acid bacteria in MRS and GY medium.

The inhibitory effect of harmful microorganisms of the lactic acid bacteria culture was confirmed only in the culture medium itself, and there was no inhibitory effect in the case of the culture medium adjusted to neutral pH (7.0*? The difference was confirmed.

In the case of L. plantarum , when cultured using MRS medium, there was no growth inhibitory effect on S. aureus , but a strong growth inhibitory effect on L. monocytogenes . It was found that the inhibitory effect of harmful microorganisms of the culture medium using the MRS medium was higher than that of the GY medium. In the case of L. harbinensis VF, results similar to those of L. plantarum were observed, and the growth inhibitory effect on S. typhimurium and L. monocytogenes was improved when cultured using GY medium.

LAB badge culture
hour harmful microorganisms B. cereus
(KCTC1013) E. coli O157 (ATCC43895) S. typhimurium (KCTC2514) S. aureus
(KCTC1621) L. monocytogenes (KCTC3596) 12h 24h 12h 24h 12h 24h 12h 24h 12h 24h LP MRS 24h - - + + ++ + - - +++ +++ 48h ++ ++ - - ++ + - - ++ ++ GY 24h + - + - + - - - - + 48h + - - - + - + - - + LH MRS 24h + + + + ++ + - - ++ ++ 48h ++ ++ ++ ++ ++ + - - ++ ++ GY 24h + - - + + - - - - + 48h + - + + ++ + +++ + - +

[Experimental Example 8] Analysis of fragrance components through electronic nose analysis of lactic acid bacteria culture medium

1. Sample and analysis method

1-1 2 strains

1) L. plantarum single strain: LP

2) L. harbinensis VF single strain: LH

3) L. plantarum + L. harbinensis VF complex strain: LP+LH

1-2 culture medium: 4 types

1) MRS broth: MRS

2) Glucose (2.5%) + YE (1%): GY medium

3) Saccharified non-waxy rice (3%) + YE (1%): NW medium

4) Saccharified black rice (3%)+ YE (1%): Black medium

1-3 Sample processing

1) Sample pretreatment: none

2) Sample volume: 2 mL

3) Other specifics: Conditions for culturing lactic acid bacteria

- Inoculation concentration: L. plantarum (10 ⁵ CFU/ml), L. harbinensis VF (10 ⁶ CFU/ml)

- Incubation time: 40 h, culture pH: 5.8

1-4. Analytical instrument conditions

1) Analysis device: Electronic nose (Heracles II, Alpha MOS, Toulouse, France)

2) Detectors: 2 flame ionization detectors (FID)

3) Column: MTX-5 / MTX-1701

4) Temperature: 80℃ (1℃/s) → 80~250℃ (3℃/s)

5) Injection volume: 5,000 μL

6) Data processing: AlphaSoft version 14.2

2. Results and Discussion

(1) Results of culturing 2 types of lactic acid bacteria in 4 types of culture media and comparing and analyzing the fragrance components using an electronic nose

● In the odor map analyzed based on PCA, the relative difference according to the composition of volatile compounds was confirmed: PC1 (x-axis) was 92.395 to 99.88%, and PC2 (y-axis) was 0.075 to 6.753%, all with a cumulative share of 99%. is shown, and the differentiation is determined by the x-axis (PC1).

- As a result of culturing LP ( L. plantarum ) as a single strain using 4 types of culture medium, control air (Control, Air), the medium itself (MRS, GY, NW, Black), and LP culture medium (MRS_LP, GY_LP, NW_LP, Black_LP) are identically identified on the x-axis (PC1), so it is determined that there is no particular fragrance component according to the LP culture (FIG. 11).

- However, as a result of culturing LH ( L. harbinensis VF) as a single strain using the same four culture media, the LH culture medium (MRS_LH, GY_LH, NW_LH, Black_LH) was clearly differentiated on the x-axis (PC1) and cultured. It is judged to have a unique fragrance component according to (FIG. 11).

● Fragrance components identified in the process of culturing two types of lactic acid bacteria as a single strain were confirmed in the bar graph according to the analysis column (MTX-5 & MTX-1701) (FIG. 12).

- In the LH monoculture medium using MRS, NW, and Black medium, the component of the peak that appears higher in the vicinity of RT 23-1A than in the medium or LP medium by the MTX-5 analysis column is Butane-2,3-dione or Butane-2 It was confirmed as -one, and this is mainly due to the flavor of butter, etc. [Fig. 12(a), (c), (d)].

- The component of the peak appearing high at RT 23.07-1A by the MTX-5 analysis column in the LH monomicrobial culture medium using the GY medium was confirmed to be formic acid or 1,1-dichloroethane, which is mainly due to the acidic flavor (Fig. 12). (b)).

- In the LH monoculture medium using MRS, NW, and Black medium, the component of the peak that was higher at RT 27.61-2A than in the medium or LP medium was confirmed by the MTX-1701 analysis column as Butan-2-one, which is mainly butter, etc. of (Fig. 12(a),(c),(d)).

- The component of the peak appearing at RT 27.67-2A by the MTX-1701 analysis column in the LH monoculture medium using GY medium was confirmed to be ethyl acetate, which is mainly due to the flavor of acidic and butter (Fig. 12(b)).

- Unlike the case of LP culture, it was confirmed that the aroma component of LH ( L. harbinensis VF) single strain culture was buttery, and the butterly flavor produced by LH culture in GY medium was weaker than that of MRS, NW, and Black medium. is considered to be generated.

(2) The result of comparing the characteristics of the LP+LH complex culture medium in NW and Black medium using electronic nose

1) In the odor map analyzed based on PCA, it can be seen that the volatile components differ depending on the culture medium: PC1 (x-axis) and PC2 (y-axis) show a ratio of 99:1, so the differentiation is on the x-axis ( PC1) (Fig. 13).

2) The main peaks in the culture medium of the LP+LH complex species are RT 23.10-1A and 27.72-2A, which are the same aroma pattern as the samples cultured by single treatment, but are judged to show a different intensity (FIG. 14).

The above description is merely illustrative of the technical spirit of the present invention, and various modifications and variations will be possible without departing from the essential characteristics of the present invention by those skilled in the art to which the present invention pertains. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain, and the scope of the technical spirit of the present invention is not limited by these embodiments. The protection scope of the present invention should be construed by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.

Agricultural Biotechnology Research Institute KACC92345 20210330

<110> Wonkwang University Center for Industry-Academy Cooperation <120> Lactobacillus harbinensis VF for fermented foods production <130> SP1236 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1444 <212> DNA <213> Lactobacillus sp. <400> 1 tgcagtcgaa cgaggtttgg tcagtttgcg gtggtgcttg catcaccaat taccgatcaa 60 accgagtggc ggacgggtga gtaacacgtg ggtaacctgc ccttcagcag gggataacat 120 ttggaaacag atgctaatac cgtataacca cggagaccgc atggtctccg ggtaaaagat 180 ggcgcaagct atcactgaag gatggacccg cggcgtatta gccagttggt ggggtaacgg 240 cctaccaaag cgatgatacg tagccgacct gagagggtaa tcggccacat tgggactgag 300 acacggccca aactcctacg ggaggcagca gtagggaatc ttccacaatg gacgcaagtc 360 tgatggagca acgccgcgtg agtgaagaag gctttcgggt cgtaaaactc tgttattgaa 420 gaagaacgtg tgtgacagta actggtcatg cagtgacggt attcaatcag aaagtcacgg 480 ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggatttattg 540 ggcgtaaagc gagtgcaggc ggtcttttaa gtctgatgtg aaagccttcg gcttaaccga 600 agaagggcat cggaaactgg gagacttgag tgcagaagag gagagtggaa ctccatgtgt 660 agcggtgaaa tgcgtagata tatggaagaa caccagtggc gaaggcggct ctctggtctg 720 taactgacgc tgaggctcga aagcgtgggt agcaaacagg attagatacc ctggtagtcc 780 acgccgtaaa cgatgaatac taagtgttgg agggtttccg cccttcagtg ctgcagctaa 840 cgcattaagt attccgcctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg 900 ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 960 aggtcttgac atcttctgcc aggctgagag atcagctgtt cccttcgggg acagaatgac 1020 aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080 gcgcaaccct tatgatcagt tgccagcatt cagttgggca ctctggtcag actgccggtg 1140 acaaaccgga ggaaggcggg gatgacgtca aatcatcatg ccccttatga cctgggctac 1200 acacgtgcta caatgggtgg tacaacgagc agcgagaccg cgaggtcaag cgaatctcta 1260 aaaaccatcc tcagttcgga ttgcaggctg caactcgcct gcatgaagct ggaatcgcta 1320 gtaatcgcgg atcagcacgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380 cacaccatga gagtttgtaa cacccaaagc cggtgagaca accgcaagga gtcagccgtc 1440 taag 1444

Claims (2)

Lactobacillus habinensis VF (KACC 92345P) for fermented lactic acid bacteria producing lactic acid and acetic acid [Claim 2] The Lactobacillus habinensis VF (KACC 92345P) according to claim 1, wherein the fermented food is made from grains or fruits and vegetables, which are food raw materials.

Images