KR101847130B1 - A composition, and method for separation of microorganism in food - Google Patents

Abstract

The present invention relates to a composition for separating microorganisms in food and a method for separating microorganisms using the same. More specifically, the composition for separating microorganisms in food comprises: an ionic surfactant; a nonionic surfactant; and pyruvate. According to an aspect of the present invention, the composition for separating microorganisms is configured to effectively isolate and concentrate microorganisms in a food sample, such that a detection limit can be reached within a short time.

Description

TECHNICAL FIELD The present invention relates to a composition for separating microorganisms in foods, and a method for separating microorganisms using the same.

The present invention relates to a method for rapidly detecting microorganisms in food, and a composition for separating microorganisms in food and a method for separating microorganisms using the same.

Food poisoning is a disease accompanied by symptoms such as fever, nausea, vomiting, diarrhea and abdominal pain. Most of them are bacterial food poisoning. Bacterial food poisoning is classified as infectious or toxic according to the onset type.

Infection type of food poisoning is to develop and proliferate within Chapter microorganisms by ingestion of food contaminated with pathogenic microorganisms, Salmonella (Salmonella spp), Vibrio para Molly Tee hee Caicos (Vibrio paraheamolyticus), E. coli O157 (Escherichia coli O157: H7 ) are the main causative bacteria.

Poisonous food poisoning can occur by ingesting toxins produced by pathogenic microorganisms that multiply in food. In this case, the pathogens can already be caused by the presence of death, even if the toxins within foods, Staphylococcus aureus (Staphylococcus aureus), Clostridium botyul rineom (Clostridiumbotulinum) is the major causative agent.

In food poisoning, the rate of incidence is increasing due to changes in lifestyle patterns and global warming, such as the expansion of group meals or the increase in eating out, and the scale is also becoming larger and larger, so it is important to detect causative organisms early.

Nevertheless, pathogenic microorganisms including foodborne pathogens are usually detected by classical culture methods, and this method can not guarantee rapid detection results because it takes a long period of incubation and analysis period.

In order to overcome the above problem, the enzyme polymerization reaction (PCR) is applied. However, the electrophoresis method has to be performed in order to determine whether it is positive.

Immunosensor methods using liposome-based chemiluminescent immunosensors, real-time polymerase chain reaction (PCR), quartz crystal microbalance (QCM), and surface plasmon resonance (SPR) have been developed to detect food poisoning bacteria. are 10 ³ cfu / ml due to the low practical low sensitivity over (Yacoub-George, E. et al ., Analytica Chimica Acta 457, 3-12, 2002, Ho, JA, et al., Analytical Biochemistry, 339, 2004, Wu, VCH et al., Biosensors and Bioelectronics 22, 2967-2975, 2007, Fu, Z. et al., International Journal of Food Microbiology 99, 47-57, 2005, Yang, L. et al., Analytical Chemistry, 76, 1107-1113, 2004, Waswa, J., et al., LWT, 40, 187-192, 2007).

Therefore, various techniques are being developed to shorten the time required because the microorganisms present in the food have a very low concentration and therefore require a culture to reach the minimum microorganism concentration required by the detection technique.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and an object of the present invention is to provide a method for rapidly detecting microorganisms without expensive equipment.

According to one aspect of the present invention, there is provided an ionic surfactant composition comprising: an ionic surfactant; Nonionic surfactants; And pyruvate. The present invention also provides a composition for separating microorganisms in foods.

In one embodiment, the ionic surfactant is selected from the group consisting of sodium dodecyl sulfate (SDS), sodium bis (2-ethylhexyl) sulfosuccinate (AOT) , Sodium dodecanoate, sodium hexyl sulfate, sodium octyl sulfate, and sodium decyl sulfate. The amount of the salt may be selected from the group consisting of sodium dodecanoate, sodium hexyl sulfate, sodium octyl sulfate, and sodium decyl sulfate.

In one embodiment, the content of the ionic surfactant may be 0.03 to 0.5% by weight based on the total weight of the composition.

In one embodiment, the nonionic surfactant is selected from the group consisting of t-octylphenoxypolyethoxyethanol (Triton X-100), octylphenoxypolyethoxyethanol (IGEPAL), and Tween 80 , polyoxyethylene sorbitan monooleate).

In one embodiment, the content of the nonionic surfactant may be 1.0 to 5.0% by weight based on the total weight of the composition.

In one embodiment, the composition may further comprise a normal saline or buffer solution.

According to another aspect of the present invention, there is provided a method for separating a microorganism in a food, comprising the step of mixing the composition for separating microorganisms and a food sample.

In one embodiment, the method for separating microorganisms comprises: filtering the microorganisms in the mixed sample with a filter; And separating the filtered microorganisms into the filter.

The composition for separating microorganisms according to one aspect of the present invention can effectively reach the detection limit within a short time by efficiently isolating and concentrating the microorganisms in the food sample.

In addition, since the conventional preculture medium contains various components acting as inhibitors in the rapid detection method such as PCR, a separate inhibitory substance is unnecessary, so that it can be usefully used in the rapid detection method.

In particular, when the composition for separating microorganisms is used, the microorganisms can be rapidly concentrated without expensive equipment and the detection limit can be reached, so that the presence of microorganisms can be detected immediately on site.

It should be understood that the effects of the present invention are not limited to the above effects and include all effects that can be deduced from the detailed description of the present invention or the configuration of the invention described in the claims.

FIG. 1 is a diagram illustrating a method for rapidly detecting microorganisms using a microorganism separation method according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly explain the present invention, parts not related to the description are omitted, and like parts are denoted by similar reference numerals throughout the specification.

When an element is referred to as " comprising ", it means that it can include other elements, not excluding other elements unless specifically stated otherwise.

Unless otherwise defined, can be performed by molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing and routine techniques commonly used in the art of recombinant DNA within the skill of those skilled in the art. These techniques are known to those skilled in the art and are described in many standardized textbooks and references.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Various scientific dictionaries, including the terms contained herein, are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing of the present application, some methods and materials have been described. It is not intended that the invention be limited to the particular methodology, protocols, and reagents, as they may be used in various ways in accordance with the context in which those skilled in the art use them.

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

An aspect of the present invention relates to an ionic surfactant; Nonionic surfactants; And pyruvate. The present invention also provides a composition for separating microorganisms in foods.

The above-mentioned " composition for separating microorganisms " means a liquid composition for eluting a specific object, specifically, a microorganism bound to or proliferating in food from the food. By mixing the composition for isolating microorganisms with the food, a microorganism capable of proliferating the food can be eluted from the food.

The food may be a solid, liquid (e.g., solution, dispersion, emulsion, suspension) or semi-solid edible composition. The food may be selected from the group consisting of meat, poultry, turbulence, fish, seafood, vegetables, fruits, cooked foods (e.g. soups, sauces, pastes), cereal products (e.g. cereal, cereals, breads), canned foods, , Cheese, yogurt, sour cream), fat, oil, desserts, condiments, spices, pasta, beverages, water, animal feeds, but the type is not particularly limited as long as it can be ingested as edible.

The microorganism may be a microorganism capable of proliferating in food and causing food poisoning upon ingestion, for example, Escherichia coli O157 ( E. coli O157: H7 ), Staphylococcus aureus , Bacillus cereus , Vibrio parahaemolyticus , Listeria monocytogenes , Yersinia entericolitica), Clostridium botyul rineom (Clostridiumbotulinum), Clostridium flops presence (Clostridium perfringens), Salmonella sp (Salmonella spp.), or a break Gela sp (Shigella spp.) may be a, this limitation is that no.

The composition for separating microorganisms not only does not damage the microorganisms in the food but effectively separates them, but does not contain an inhibitory factor against the growth of microorganisms, and thus can be usefully used for the detection of microorganisms present in foods. In particular, since the composition for separating microorganisms contains an oxidizing component for recovering the separated microorganisms, the microorganism can be easily detected.

The composition for separating microorganisms may include an ionic surfactant and a nonionic surfactant. Since the surfactant alleviates the interface boundary, the microorganisms adsorbed on the food can be easily released.

The ionic surfactant can effectively separate microorganisms adsorbed or bound to food due to excellent detergency.

The ionic surfactant may be selected from the group consisting of sodium dodecyl sulfate (SDS), sodium bis (2-ethylhexyl) sulfosuccinate (AOT), sodium dodecanoate Sodium dodecanoate, sodium hexyl sulfate, sodium octyl sulfate, and sodium decyl sulfate, but preferably sodium dodecyl sulfate (SDS ).

The content of the ionic surfactant may be 0.03 to 0.5% by weight based on the total weight of the composition. If the content of the ionic surfactant is less than 0.03% by weight, the isolation efficiency of the microorganism may be deteriorated. If the content of the ionic surfactant is more than 0.5% by weight, damage to the membrane protein of the microorganism may be caused, .

On the other hand, the nonionic surfactant is excellent in emulsifying power so that the food ingredient can be uniformly dispersed in the composition, and the microorganism isolation efficiency can be increased without damaging the microorganisms due to hypoallergenic properties. Therefore, even when a small amount of the ionic surfactant is added, the nonionic surfactant can minimize the damage to the microorganism and improve the dissolution efficiency of the microorganism.

The nonionic surfactant may be selected from the group consisting of t-octylphenoxypolyethoxyethanol (Triton X-100), octylphenoxypolyethoxyethanol (IGEPAL) and Tween 80 (polyoxyethylene sorbitan monooleate) At least one of the groups may be selected, but preferably it may be twenty-eight.

The content of the nonionic surfactant may be 1.0 to 5.0 wt% based on the total weight of the composition. The content of the nonionic surfactant may be 1.0 to 5.0 wt% based on the total weight of the composition. If the content of the ionic surfactant is less than 1.0 wt%, the isolation efficiency of the microorganism may be deteriorated. If the content of the ionic surfactant is more than 5.0 wt%, the microorganism recovery and growth rate may be lowered.

In addition, the composition for separating microorganisms may comprise pyruvate. The pyruvic acid promotes the metabolism of microorganisms damaged by external force or stimulation of the surfactant, thereby helping the recovery of the cells and significantly improving the ability of the microorganisms to replicate. The composition for separating microorganisms may further include various additives capable of promoting the growth of microorganisms such as peptone and growth factors together with the pyruvic acid.

The composition may further comprise a normal saline or buffer solution.

The physiological saline or buffer solution contains salts at a predetermined concentration, so that cells can be stabilized without causing a change in osmotic pressure on the cells, and the composition can be maintained at a proper concentration.

The buffer solution may be a buffer solution commonly used in animal cell treatment in the art and may be, for example, a phosphate buffered saline (PBS), a Hank's balanced salt solution (HBSS), or a Tris buffered saline However, the present invention is not limited thereto.

Referring to FIG. 1, another aspect of the present invention provides a method for separating microorganisms, comprising the steps of: mixing a composition for separating microorganisms and a food sample; Filtering the microorganisms in the mixed sample with a filter; And separating the filtered microorganisms from the filter. The present invention also provides a method for separating microorganisms in a food.

The food sample is mixed with the composition for separating microorganisms and then filtered by a filter to separate and concentrate the microorganisms in the food sample. The food sample may be a subject separated from food in order to evaluate whether it is contaminated by microorganisms. The type of the food sample is not particularly limited, but may refer to a food suspected of contamination by a microorganism that may cause disease such as food poisoning.

The food sample may be homogenized in the composition for separating microorganisms and cultured to promote the growth of microorganisms, and may be recovered by the filter because it is suspended in a liquid composition.

Since the composition for isolating microorganisms contains nutritional ingredients such as pyruvic acid which can be used as an energy source, the microorganisms contained in the food sample can reach the maximum population within a short time due to active metabolism.

Although the composition for separating microorganisms may include common microorganisms as a commonly available energy source, if the content of the energy source is too small, the microorganisms may not be effectively proliferated, and in a case where the amount is too low, the cost efficiency may be lowered .

The food sample can be homogenized in the composition for separating microorganisms and uniformly dispersed in fine particles. The food sample may be pulverized into fine particles and introduced into the microbial separation composition and may be uniformly distributed in the composition by various homogenization apparatuses such as a homogenizer, a stomacher, and an ultrasonic disperser .

The composition for separating microorganisms and the food sample may be mixed at a weight ratio of 10 to 15: 1 to 2. If the proportion of the composition for separating microorganisms is too small, the food sample may not be uniformly dispersed in the composition for separating microorganisms, so that the filtration efficiency may be lowered and the growth of microorganisms may be slowed. And the process efficiency may be lowered.

The food sample mixed with the composition for separating microorganisms may be filtered by a filter. The filter is capable of effectively recovering the microorganisms contained in the food sample by filtering the uniformly dispersed solid microparticles.

The type of the filter is not particularly limited as long as it can filter a predetermined component having a fine pore size, but it may preferably be a cellulose-based filter. The cellulose-based filter may be, but not limited to, cellulose nitrate, cellulose acetate, or a mixed cellulose ester filter.

The cellulose-based filter has excellent filtering efficiency because the polar component of the fiber interacts with the microorganism electrostatically and the fiber layer is formed into a plurality of layers. Particularly, the cellulose-based filter has high crystallinity due to a fibrous three-dimensional network structure and is excellent in mechanical strength, adsorptivity, water retention, suspension stability and binding property, and thus is suitable for filtration of the microorganisms.

The filtered filter may be put into a composition for extracting a filter and then stomached or irradiated with ultrasound to separate the microorganisms bound to the filter.

The stomaching is usually performed by a stomacher, and the microbes adsorbed to the filter can be removed by vibrating the filtration filter at a predetermined frequency and amplitude after injecting the filtration filter into the composition for extracting a filter.

The ultrasonic wave may be irradiated by an ultrasonic generator, and the frequency of the ultrasonic wave may be 15KHz to 25KHz and the amplitude may be 5 to 50μm. The ultrasonic waves can effectively vibrate the filtered filter to efficiently separate the adsorbed solid components within the above range.

The microbial separation composition and the filter extract composition may be used in a weight ratio of 20 to 30: 1 to 3.

The density and the growth rate of the microorganism can be remarkably increased in a short time by injecting the test sample into the large amount of composition for separating microorganisms, filtering the microorganisms, and then separating the microorganisms into a relatively small amount of the composition for filter extraction. For example, a food sample mixed with 250 mL of the composition for separating microorganisms may be filtered and then resuspended in 2.5 mL of the filter-extracting composition to increase the microbial concentration by about 100 times or more.

At this time, the concentrated microorganism can be further cultured to proliferate at a high concentration. The propagation method is not particularly limited and may be carried out by putting the separated microorganism into a separate liquid medium and culturing it by a conventionally known method

On the other hand, the enriched and proliferated microorganisms can be detected by various methods well known in the art. Since the microorganisms can reach the detection limit within a short time by the above-mentioned concentration method, the culture time required for a long time is shortened to enable rapid detection of microorganisms.

It will be apparent to those skilled in the art that various changes and modifications may be made thereto without departing from the spirit and scope of the present invention as set forth in the appended claims. It is encompassed in the technical idea of.

The present invention will be further described with reference to the following examples, but it should be apparent that the present invention is not limited by the following examples.

Preparation Example: Culturing and Separation of Strain

Salmonella spp . Was anaerobically incubated at 37 ° C in a chamber (Whitley A35 anaerobic chamber; Don Whitley Scientific Ltd., Shipley, UK) using a brain heart infusion broth (Oxoid, Hampshire, England) Time. Then, a single colony showing green / yellow fluorescence in a long-wave ultraviolet light was applied to a selective agar (BD BBL, Sparks, USA) and used for the experiment.

In order to evaluate the separation efficiency of the composition for separating microorganisms, compositions for separating microorganisms having different compositions were prepared as shown in Table 1 below.

division Composition ratio Example 1 (Saline) + SDS (0.05%) + Tween 80 (1.5%) + pyruvic acid (0.1%) Example 2 (Saline) + SDS (0.01%) + Tween 80 (1.5%) + pyruvic acid (0.1%) Example 3 (Saline) + SDS (0.03%) + Tween 80 (1.5%) + pyruvic acid (0.1%) Example 4 (Saline) + SDS (0.1%) + Tween 80 (1.5%) + pyruvic acid (0.1%) Example 5 (Saline) + SDS (0.5%) + Tween 80 (1.5%) + pyruvic acid (0.1%) Example 6 (Saline) + SDS (0.7%) + Tween 80 (1.5%) + pyruvic acid (0.1%) Example 7 (Saline) + SDS (0.05%) + Tween 80 (0.1%) + pyruvic acid (0.1%) Example 8 (Saline) + SDS (0.05%) + Tween 80 (0.5%) + pyruvic acid (0.1%) Example 9 (Saline) + SDS (0.05%) + Tween 80 (1.0%) + pyruvic acid (0.1%) Example 10 (Saline) + SDS (0.05%) + Tween 80 (3.0%) + pyruvic acid (0.1%) Example 11 (Saline) + SDS (0.05%) + Tween 80 (5.0%) + pyruvic acid (0.1%) Example 12 (Saline) + SDS (0.05%) + Tween 80 (7.0%) + pyruvic acid (0.1%) Comparative Example 1 Saline Comparative Example 2 SDS (0.05%) Comparative Example 3 Tween 80 (1.5%) Comparative Example 4 Pyruvic acid (0.1%) Comparative Example 5 Buffered peptone water (BPW)

Experimental Example 1: Microbial separation efficiency test

100 g of the Salmonella spp . Strain was inoculated into lettuce and dried on a clean bench for 30 minutes to fix on the surface of lettuce.

As shown in Table 1, the dried lettuce was added to 225 mL of the composition for the separation of microorganisms having different compositions and homogenized. The homogenized mixed composition was filtered through filter paper of 45 [mu] m.

The filter paper was collected and incubated on TSA plates for 6 hours to allow the injured cells to resuscitate. The filter paper was transferred to XLD, a selective medium of Salmonella strain, and cultured. Salmonella bacteria were counted by observing the typical shape of the colonies formed on the filter paper, and the isolation rate was evaluated by comparing the number of inoculated bacteria and the number of bacteria recovered from the filter paper.

division Separation rate (%) Example 1 99.4 Comparative Example 1 76.2 Comparative Example 2 86.4 Comparative Example 3 82.3 Comparative Example 4 51.5 Comparative Example 5 73.4

As a result of the evaluation, the composition for separating microorganisms of Example 1 was measured by completely isolating salmonella strain inoculated on the surface of lettuce.

On the other hand, the composition of Comparative Example 4, which does not contain a surfactant, shows a marked decrease in the separation efficiency, which indicates that the isolation efficiency for the strains bound to the surface of the food is low due to the absence of the surfactant.

Furthermore, the composition comprising only the buffer (Comparative Example 1), the composition comprising only the surfactant (Comparative Examples 2 and 3), and the separation rate of BPW (Comparative Example 5) used as the preculture medium in the separation of food poisoning bacteria are also remarkably low , Which indicates that the recovery rate is decreased due to the damage of the filtered strains or the separation efficiency for Salmonella strains is low.

Experimental Example 2: Comparison of separation efficiency according to ionic surfactant concentration

The results are shown in Table 3, and the results are shown in Table 3. The results are shown in Table 3.

As shown in Table 3, when the concentration of SDS added as an ionic surfactant was 0.03% (w / w) or more, the separation rate was remarkably increased, and the separation rate was maximally measured at a concentration of 0.05% (w / w).

On the other hand, when the concentration of SDS exceeded 0.5% (w / w), the separation rate gradually decreased. This suggests that as the concentration of SDS increases, the cells become damaged and the recovery rate decreases.

Furtherance Separation rate (%) Example 1 99.4 Example 2 89.3 Example 3 95.4 Example 4 98.4 Example 5 96.1 Example 6 91.3

Experimental Example 3: Comparison of separation efficiency according to concentration of nonionic surfactant

The results are shown in Table 4, and the results are shown in Table 4. The results are shown in Table 4. [Table 4] < tb > < TABLE >

As shown in Table 4, when the concentration of Tween 80 added as a nonionic surfactant was 1.0% (w / w) or higher, the separation rate was markedly increased, and the separation rate was maximized at a concentration of 1.5% (w / w).

On the other hand, when the concentration of Tween 80 exceeded 5.0% (w / w), the separation rate gradually decreased. That is, as the concentration of Tween 80 gradually increases, it can be evaluated that the recovery of damaged cells is negatively affected or the separation of microorganisms by ionic surfactant is inhibited.

Furtherance Separation rate (%) Example 1 99.4 Example 7 90.1 Example 8 95.4 Example 9 97.3 Example 10 98.4 Example 11 96.9 Example 12 93.1

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (8)

Ionic surfactants; Nonionic surfactants; And pyruvate;
Wherein the content of the ionic surfactant is 0.03 to 0.5% by weight based on the total weight of the composition, and the content of the nonionic surfactant is 1.0 to 5.0% by weight based on the total weight of the composition. The method according to claim 1,
The ionic surfactant may be selected from the group consisting of sodium dodecyl sulfate (SDS), sodium bis (2-ethylhexyl) sulfosuccinate (AOT), sodium dodecanoate Wherein the composition is at least one selected from the group consisting of sodium dodecanoate, sodium hexyl sulfate, sodium octyl sulfate and sodium decyl sulfate. delete The method according to claim 1,
The nonionic surfactant may be selected from the group consisting of t-octylphenoxypolyethoxyethanol (Triton X-100), octylphenoxypolyethoxyethanol (IGEPAL) and Tween 80 (polyoxyethylene sorbitan monooleate) 0.0 > a < / RTI > delete The method according to claim 1,
Wherein the composition further comprises a normal saline or buffer solution. A method for separating a microorganism in a food, which comprises mixing the composition of any one of claims 1, 2, 4, and 6, and a food sample. 8. The method of claim 7,
Filtering the microorganisms in the mixed sample with a filter; And
And separating the filtered microorganisms into the filter.

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