KR20170036450A - A composition for inhibiting the growth of bacteria comprising lysozyme and organic acids - Google Patents

A composition for inhibiting the growth of bacteria comprising lysozyme and organic acids Download PDF

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KR20170036450A
KR20170036450A KR1020150135618A KR20150135618A KR20170036450A KR 20170036450 A KR20170036450 A KR 20170036450A KR 1020150135618 A KR1020150135618 A KR 1020150135618A KR 20150135618 A KR20150135618 A KR 20150135618A KR 20170036450 A KR20170036450 A KR 20170036450A
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lysozyme
gum
acid
organic acid
food preservative
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김미숙
오명근
이영승
정윤화
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단국대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/10—Preserving against microbes

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  • Food Science & Technology (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
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  • Molecular Biology (AREA)
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  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

The present invention relates to a composition for suppressing bacterial proliferation containing lysozyme and organic acid. By using both lysozyme and organic acid, the composition exhibits outstanding antibacterial functions.

Description

[0001] The present invention relates to a composition for inhibiting the growth of bacteria comprising lysozyme and organic acids,

The present invention relates to a fungal growth inhibiting composition comprising lysozyme and an organic acid, and more particularly to a fungal growth inhibiting composition having a stronger antimicrobial activity by using a mixture of lysozyme and an organic acid.

Listeria monocytogenes is a gram-positive anaerobic anaerobic bacillus, the flagella are predominant and the optimum temperature is 30 to 37 ° C. However, it can grow at refrigeration and freezing temperatures, and can survive for a relatively long time under dry conditions, resulting in infectious food poisoning.

The Listeria monocytogenes causes bacterial meningitis, and is also a bacterium with a mortality rate of 22%. It is also one of the three major causative agents of neonatal meningitis and is the most common bacterial meningitis in patients with immunosuppression due to lymphoma, organ transplantation, and steroids. In addition, it can cause other brain meningitis, brain abscess, endocarditis, and local infection. In recent studies, it has been reported that acute gastroenteritis can be induced in a group and cause acute diarrhea in normal people.

However, in healthy people, symptoms do not appear or symptoms of mild food poisoning are manifested. However, if a susceptible pregnant woman and a newborn infant with weak immunity, an elderly person, diabetes, epilepsy, chronic kidney disease and iron- The probability of disease occurrence increases. In addition, listeric meningitis is associated with a mortality rate of about 70%, sepsis 50%, and pregnant women or newborns 80%.

The Listeria monocytogenes has recently been attracted by large-scale food poisoning incidents in the United States and the increased risk of food poisoning caused by the bacteria as the consumption of simple cooked food to eat without cooking increases.

Lysozyme has been recognized for its excellent antibacterial and safety properties and is widely used as a natural antimicrobial agent and food preservative in foods, agricultural, livestock and pharmaceuticals.

The lysozyme is colorless, tasteless, and odorless. However, the lysozyme has been widely applied to food, but its antibacterial spectrum is narrow and its price competitiveness is low. Thus, studies using lysozyme have been conducted by mixing with EDTA and nisin.

However, the mixture of lysozyme and EDTA lowers the pH of the food, and nisin raises the cost.

Therefore, it is necessary to study the new antimicrobial substance against food harmful microorganisms such as food poisoning bacteria including Listeria monocytogenes which is harmless to human body and excellent in price competitiveness.

It is an object of the present invention to provide a composition comprising lysozyme and an organic acid capable of inhibiting the growth of bacteria.

It is another object of the present invention to provide a food preservative containing the composition to inhibit the growth of bacteria in food.

In order to achieve the above object,

The present invention provides a fungal growth inhibiting composition comprising lysozyme and an organic acid.

In addition, the present invention provides a food preservative containing the microbial growth inhibiting composition.

The fungal growth inhibiting composition of the present invention can have a higher antibacterial effect by using lysozyme and organic acid together.

Further, the bactericidal growth inhibiting composition of the present invention is water-soluble, stable, and can be used as a food preservative.

1 shows a checkerboard method.
2 is a flow chart illustrating a time-kill assay method.
FIG. 3 is a graph showing the antimicrobial synergy effect of lysozyme and acetic acid (AA) on Listeria monocytogenes KCTC 3710, as measured by the time-kill assay method.
FIG. 4 is a graph showing the antimicrobial synergy effect of lysozyme and citric acid (CA) on Listeria monocytogenes KCTC 3710, as measured by the time-kill assay method.
FIG. 5 is a graph showing the antimicrobial synergy effect of lysozyme and lactic acid (LA) on Listeria monocytogenes KCTC 3710, as measured by the time-kill assay method.
FIG. 6 is a graph showing the antimicrobial synergy effect of lysozyme and malic acid (MA) on Listeria monocytogenes KCTC 3710, as measured by the time-kill assay method.
FIG. 7 is a graph showing the antimicrobial synergy effect of lysozyme and succinic acid (SA) on Listeria monocytogenes KCTC 3710, measured by the time-kill assay method.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a fungal growth inhibiting composition comprising lysozyme and an organic acid.

Lysozyme is a substance with excellent antibacterial activity and is used for natural antimicrobial agents and food preservatives. However, since the lysozyme is dependent on the import, there is a problem that the cost is high.

In order to solve the above problems, EDTA or nisin has been used together with lysozyme. However, when EDTA is used together, the pH is low and can not be used in food preservatives and the like, and when the nisin is used together, the cost increases and the economical cost problem can not be solved.

Therefore, in the present invention, organic acids are used together with lysozyme to solve the above problems.

The use of the organic acid together can reduce the amount of lysozyme used, which can reduce the economic cost, and the antimicrobial activity of the lysozyme can be increased, so that the antifungal composition having higher antimicrobial activity can be provided.

The use of organic acids in combination with lysozyme also improves the antimicrobial activity.

Preferably, the organic acid is selected from the group consisting of malic acid (MA), lactic acid (LA), citric acid, citric acid, CA), succinic acid (SA), and acetic acid (AA).

The lysozyme and the organic acid are mixed at a weight ratio of 10: 1 to 80: 1, preferably 20: 1 to 40: 1.

When the lysozyme and the organic acid are mixed at a weight ratio of 10: 1 to 80: 1, excellent antibacterial activity can be exhibited.

In addition, the microbial growth inhibiting composition of the present invention does not particularly limit the kind of the microorganism, but preferably inhibits the proliferation of Listeria monocytogenes.

The Listeria monocytogenes is a bacterium that causes food poisoning, and is capable of proliferating at refrigeration and freezing temperatures, further increasing the risk of food poisoning.

The fungus growth inhibiting composition comprising lysozyme and organic acid of the present invention is excellent in antibacterial activity against the Listeria monocytogenes and can prevent food poisoning.

In addition, the antifungal composition of the present invention has a pH of 5 to 7, and preferably a pH of 6 to 7. If the pH is low, it is difficult to grow the microorganism and may cause a change in the food. However, since the pH of the mycelial growth inhibiting composition of the present invention is in the above range, the growth of bacteria can be suppressed without affecting the food.

The present invention also relates to a food preservative comprising the microbial growth inhibiting composition comprising the lysozyme and the organic acid of the present invention.

The antifungal composition of the present invention is water-soluble, stable, and can be used as a food preservative because it does not change food when added to food, thereby preventing diseases caused by bacteria.

More specifically, the antifungal composition of the present invention is excellent in antimicrobial activity against L. monocytogenes, so that the proliferation of the bacteria can be inhibited and food poisoning and the like can be prevented.

In addition, a thickener and a stabilizer can be added to the food preservative as a food additive, and examples thereof include methylcellulose, ethylcellulose, carboxymethylcellulose sodium, carboxymethylcellulose calcium, carboxymethylstarch sodium, propylene glycol alginate, But are not limited to, sodium, sodium caseinate, sodium alginate, polydextrose, modified starch, hydroxypropylmethylcellulose, guar gum, locust bean gum, xanthan gum, gum arabic, alginic acid, gelatin, gellan gum, carrageenan, , Tamarind gum, tragacanth gum, pectin, glucomannan, ghatti gum, microfibrous cellulose, silicate seed gum, arabinogalactan, chitin, persellaran, dhamma gum, dextrin, cyclodextrin and cyclodextrin syrup And at least one selected from the group consisting of The food preservative can be prepared in powder form by including these materials.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are intended to further illustrate the present invention, and the scope of the present invention is not limited by the following examples.

Comparative Example  One. Lysozyme  Minimum inhibitory concentration ( MIC ) Measure

Lysozyme (Lyz) was dissolved in a 0.1 M sodium phosphate buffer solution (pH 6.0) to prepare a 0.014 M lysozyme solution.

100 μL of the lysozyme solution was added to a sterile 96-well plate (SPL life science, Pocheon, Korea) containing 100 μL of BHI broth (Oxoid Ltd., Basingstoke, England), and L. monocytogenes KCTC 3710 7 log CFU / mL and incubated at 37 DEG C for 24 hours.

The above-mentioned L. monocytogenes KCTC 3710 was purchased from the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Bioscience and Biotechnology.

The 96-well plate immediately after the inoculation of the strain and incubated for 24 hours was measured for absorbance at 600 nm using an enzyme-linked immunosorbent assay (ELISA) reader (SpectraMax M2e, Molecular Devices, CA, USA) Minimal inhibitory concentrations (MIC) of lysozyme were set, and the results are shown in Table 1 below.

Comparative Example  2. Minimum inhibitory concentration of organic acids ( MIC ) Measure

Lactic acid (LA), citric acid (CA), succinic acid (SA) and acetic acid (AA) were used as organic acid (OA) .

The organic acid was dissolved in distilled water to prepare 0.33 M acetic acid solution, 0.1 M citric acid solution, 0.15 M lactic acid solution, 0.22 M malic acid solution and 0.17 M succinic acid solution.

Minimal inhibitory concentrations (MIC) of organic acids were measured in the same manner as in Comparative Example 1 except that an organic acid solution was used in place of the lysozyme solution of Comparative Example 1, and the results are shown in Table 1 below .

(Unit: ppm) division Lyz AA CA LA MA SA MIC 50,000 1,250 2,500 2,500 2,500 2,500

Example  1. Antimicrobial activity measurement by Checkerboard method

A sterile 96-well plate (SPL life science, Pocheon, Korea) containing 100 μL of BHI broth (Oxoid Ltd., Basingstoke, England) was prepared.

On the horizontal axis, the minimum inhibitory concentration of organic acids in Table 1 was increased from 1/32 to 2 times, and then 50 μL was added up to 8 times. On the vertical axis, the minimum inhibitory concentration of lysozyme was increased from 1/32 to 2 times 50 [mu] L was added up to 4 times so that the total volume became 200 [mu] L (Fig. 1).

L. monocytogenes KCTC 3710 was inoculated to a medium containing the lysozyme and an organic acid at 7 log CFU / mL and incubated at 37 ° C. for 24 hours. Minimal inhibitory concentrations (MIC) , And the fractional inhibitory concentration index (FICI) was calculated by the following Equation 1, and the results are shown in Table 3 below.

[Equation 1]

Figure pat00001

In addition, the FICI value

If FICI ≤ 0.5, synergy,

If 0.5 <FICI ≤ 1, additive,

If 1 <FICI ≤ 2, then indifference,

2 < FICI.

Organic acid (OA) Minimum inhibitory concentration (MIC) Lyzia Organic acid (OA) Lyz + OA Acetic acid (AA) 50 0.125 1.25 + 0.0078 0.625 + 0.0625 Citric acid (CA) 50 0.25 1.25 + 0.0156 0.625 + 0.25 Lactic acid (LA) 50 0.25 2.5 + 0.0078 1.25 + 0.0625 Malasan (MA) 50 0.25 1.25 + 0.0078 0.625 + 0.25 Succinic acid (SA) 50 0.25 2.5 + 0.0156 0.625 + 0.125

From the results shown in the above Table 2, it was confirmed that the minimum inhibitory concentration of lysozyme was at least 40-fold reduced in a composition in which malic acid (MA), citric acid (CA) and acetic acid (AA) were mixed with lysozyme.

In addition, it was confirmed that the minimum inhibitory concentration of lysozyme was at least 20 times lower in a composition in which lactic acid (LA) and succinic acid (SA) were mixed with lysozyme, respectively.

In addition, the minimum inhibitory concentration of lysozyme decreased as the concentration of organic acid increased.

Therefore, it can be seen that the microbial growth inhibiting composition comprising lysozyme and organic acid of the present invention exhibits higher antimicrobial activity by including both substances together.

division Lyz + AA Lyz + CA Lyz + LA Lyz + MA Lyz + SA FICI S S S S S

It can be seen from the results in Table 3 that lysozyme (Lyz) has a synergistic effect with organic acids such as acetic acid (AA), citric acid (CA), lactic acid (LA), malic acid (MA) and succinic acid (SA).

That is, the antifungal composition containing lysozyme and organic acid of the present invention has higher antibacterial activity than lysozyme alone.

Example  2. Measurement of antimicrobial activity by time kill curve method

Lysosyme (Lyz) was used 0.25 times and 0.5 times the minimum inhibitory concentration and malic acid (MA), lactic acid (LA), citric acid (CA) as organic acid ), Succinic acid (SA) and acetic acid (AA) were used, and the two materials were mixed using 0.25 times and 0.5 times of the minimum inhibitory concentration.

The L. monocytogenes KCTC 3710 in the log phase 7 log CFU / inoculated into BHI broth to a mL, and were cultured at 37 ℃ for 24 hours, 0 to 6, and 0.85% sterile collected the bacteria for 24 hours physiological saline 10 After dilution, the cells were spread on BHI agar.

After culturing the bacterium on the BHI agar for 24 hours at 37 ° C, the number of bacteria was measured to obtain an average value (FIG. 2).

When the number of bacteria is decreased by 2 log CFU / mL or more, synergy (S) decreases by 2 log CFU / mL or less, or by 2 log CFU / mL or more when the lysozyme is used alone. ).

The measurement results are shown in Table 4 below.

division Lyz + AA Lyz + CA Lyz + LA Lyz + MA Lyz + SA 0.25MIC Lyz + 0.25MIC OA I I I I I 0.25MIC Lyz + 0.5MIC OA I I I I I 0.5MIC Lyz + 0.25MIC OA I I I I S 0.5MIC Lyz + 0.5MIC OA I I I S S

In the results of Table 4, the antimicrobial activity of acetic acid (AA), citric acid (CA) and lactic acid (LA) was increased when lysozyme or organic acid was used alone with lysozyme (Figs. 3 to 5) (MA) and succinic acid (SA), when used together with lysozyme, significantly increased the antimicrobial activity compared to lysozyme or organic acid alone (FIGS. 6 and 7).

Therefore, it has been found that the microbial growth inhibiting composition comprising lysozyme and organic acid of the present invention has better antibacterial activity than when lysozyme or organic acid is used alone.

The pH of the fungal growth inhibiting composition containing lysozyme and organic acid was measured, and the results are shown in Table 5 below.

division Lyz + AA Lyz + CA Lyz + LA Lyz + MA Lyz + SA 0.25MIC Lyz + 0.25MIC OA 6.38 ± 0.01 6.30 ± 0.01 6.33 ± 0.01 6.31 ± 0.01 6.31 ± 0.00 0.25MIC Lyz + 0.5MIC OA 6.28 ± 0.01 6.18 ± 0.01 6.19 ± 0.01 6.15 ± 0.01 6.14 ± 0.01 0.5MIC Lyz + 0.25MIC OA 6.31 ± 0.00 6.20 ± 0.01 6.26 ± 0.01 6.22 ± 0.00 6.21 ± 0.00 0.5MIC Lyz + 0.5MIC OA 6.21 + 0.03 6.08 ± 0.01 6.08 ± 0.00 6.05 ± 0.01 6.04 ± 0.01

In the results of Table 5, the pH of the antifungal composition containing lysozyme and organic acid was slightly acidic or neutral to about 6.

This means that at a pH that does not affect the food, the antifungal composition of the present invention can inhibit the growth of bacteria in the food without affecting the food.

That is, the microbial growth inhibiting composition comprising lysozyme and organic acid of the present invention means that it can be used as a food preservative.

Claims (7)

A fungal growth inhibiting composition comprising lysozyme and an organic acid. The antifungal composition according to claim 1, wherein the organic acid comprises at least one member selected from the group consisting of malic acid, lactic acid, citric acid, succinic acid, and acetic acid. The antifungal composition according to claim 1, wherein the lysozyme and the organic acid are mixed at a weight ratio of 10: 1 to 80: 1. 2. The antifungal composition according to claim 1, wherein the bacterium comprises Listeria monocytogenes. The antifungal composition according to claim 1, wherein the antifungal composition is a pH of 5 to 7. A food preservative comprising the mycelial growth inhibiting composition of claim 1. [7] The food preservative according to claim 6, wherein the food preservative further comprises at least one selected from the group consisting of methylcellulose, ethylcellulose, sodium carboxymethylcellulose, carboxymethylcellulose calcium, carboxymethylstarch sodium, propylene glycol alginate, sodium polyacrylate, sodium caseinate, sodium alginate, polydextrose, Starch, hydroxypropylmethylcellulose, guar gum, locust bean gum, xanthan gum, gum arabic, alginic acid, gelatin, gellan gum, carrageenan, karaya gum, curdlan, chitosan, tara gum, tamarind gum, tragacanth, At least one member selected from the group consisting of glutamate, glutamate, glutamate, glutamate, gluconamine, gutiogam, microfibrous cellulose, silicate gum, arabinogalactan, chitin, persaleran, dhamma gum, dextrin, cyclodextrin, and cyclodextrin syrup Wherein the food preservative is a food preservative.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005000133A (en) * 2003-06-16 2005-01-06 Pharmafoods Kenkyusho:Kk Egg white peptide-blended storage duration improver for food
JP3928064B2 (en) * 2001-03-08 2007-06-13 エーザイ・アール・アンド・ディー・マネジメント株式会社 Food additive composition

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3928064B2 (en) * 2001-03-08 2007-06-13 エーザイ・アール・アンド・ディー・マネジメント株式会社 Food additive composition
JP2005000133A (en) * 2003-06-16 2005-01-06 Pharmafoods Kenkyusho:Kk Egg white peptide-blended storage duration improver for food

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
일본 특허공보 특허 제 3928064호(2007.06.13.) 1부. *

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