JP4532165B2 - Method for separating and / or concentrating microorganisms and apparatus for use in those methods - Google Patents

Description

  The present invention relates to a method for separating and / or concentrating microorganisms, and an apparatus for use in those methods.

Chitosan is β-1,4-poly-D-glucosamine obtained by deacetylating chitin, which is abundant in shellfish such as shrimp and crab, and insects. Chitosan is known to have various physiological activities such as a cholesterol absorption inhibitory action and a blood glucose rise inhibitory action in addition to an antibacterial action. Moreover, since chitosan can adsorb | suck a microbial cell, it is known that it can utilize also as an adsorbent of microorganisms (refer patent document 1). However, as described in Patent Document 1 (paragraph [0012]), chitosan exhibits antibacterial properties that suppress the growth of microorganisms, so that the microorganisms remain alive when chitosan is used as an adsorbent. It is not possible to get separated in the state.
JP 2000-14380 A

  Then, this invention makes it a subject to provide the method which can isolate | separate and concentrate microorganisms alive.

  The inventors of the present invention have been diligently researching to find a method capable of separating microorganisms in a living state. In an aqueous medium in the presence of a compound having an SH group such as cysteine, a specific amino group is present. As a result, it was found that even when adsorbed on the chitosan coated with a microorganism, the microorganisms are maintained in a living state. Furthermore, in an aqueous medium in the presence of a compound having an SH group such as cysteine, the adsorption rate of this specific amino group-coated chitosan to microorganisms is different. Further, when a releasing agent is added to this aqueous medium, The detachment rate of microorganisms from this specific amino group-covered chitosan was also different, and it was found that the microorganisms were detached from chitosan in a living cell form, and the present invention was completed.

That is, the present invention
(1) A method for isolating a specific microorganism from a sample containing one or more kinds of microorganisms in the form of viable cells and / or concentrating the sample in the form of viable cells. And an amino group-coated chitosan in a water-based medium in the presence of an SH group-containing compound, the adsorption rate and the desorption rate of the specific microorganism with the amino group-coated chitosan A method for separating and / or concentrating microorganisms utilizing the difference from the other microorganisms.

  (2) Microbe-adsorbed chitosan produced by adsorbing the specific microorganisms to the amino group-coated chitosan by the first contact step, and the specific microorganisms can be desorbed from the microorganism-adsorbed chitosan into the aqueous medium. The separation method according to the above (1), further comprising a second contact step of bringing the releasing agent into contact.

  (3) The separation method according to (2), further comprising a separation step of separating the aqueous medium containing the desorbed specific microorganism.

  (4) A kit for separating a specific microorganism from a sample containing two or more types of microorganisms from other microorganisms in a living cell form, the kit adsorbing the specific microorganism contained in a container A kit comprising: an amino group-coated chitosan capable of desorbing the specific microorganism from the amino group-coated chitosan adsorbed by the specific microorganism.

I will provide a.

  According to the method for separating and / or concentrating microorganisms of the present invention (hereinafter, also referred to as “the method of separating and concentrating the present invention”), the microorganisms are separated and / or concentrated without losing the activity of the microorganisms. can do. Despite the antibacterial activity of chitosan, the amino group-coated chitosan (hereinafter also simply referred to as “coated chitosan”) is obtained by coating the amino group of chitosan and allowing SH group-containing compounds to exist in the medium. It was completely unexpected that microorganisms were not killed even if they were adsorbed and adsorbed while they were alive.

  Further, in the separation / concentration method of the present invention, the cells can be recovered in a live state, and therefore, by using a component known to cause a specific reaction with a certain bacterium, the bacterium can be easily obtained. Can be identified. For example, an antibody that specifically binds to a certain bacterium can be used, or a dye that specifically reacts to a certain bacterium can be used.

  Moreover, according to the separation / concentration method of the present invention, the detachment of the living cells adsorbed on the coated chitosan can be performed by an extremely simple operation of adding a detaching agent to the reaction medium. Since the detachment agent is specific to the type of microorganism, the microorganism can be selectively separated by selecting an appropriate type of detachment agent. Moreover, the concentration of the releasing agent can be carried out at a low concentration that does not impair the activity of the bacterial cells. Conventionally, cells once adsorbed to chitosan are not detached unless the salt concentration is high, and the cells are killed at such a high salt concentration. On the other hand, in the separation / concentration method of the present invention, the cells can be detached from the coated chitosan even when the salt concentration is low so that the cells can survive. This was completely unexpected.

  Furthermore, in particular, the concentration method of the present invention can be used for PCR pretreatment. In particular, when coated chitosan immobilized on a substrate is used, the microorganisms are concentrated alive, so that the microorganisms can be removed by adding the microorganisms immobilized on the substrate to the selective medium. It can be used for efficient culture. In addition, the microorganisms immobilized on the substrate can be frozen (added with a cryoprotectant if necessary) or dried to be used as a cell storage carrier. Furthermore, specific bacterial cells can be immobilized to efficiently produce enzymes and antibiotics.

  Since the coated chitosan used in the separation / concentration method of the present invention can be easily produced using chitin such as crustaceans that have been discarded in the past, the separation / concentration method of the present invention and The manufacturing cost of the apparatus can be kept low.

  The apparatus for carrying out the method of the present invention includes a kit in which a container containing amino group-coated chitosan and a releasing agent are set. The container used in the apparatus of the present invention may be as small as 1 to 2 mL, for example, and can reduce labor and cost required for transportation. Moreover, such an apparatus requires only a very simple operation of adding a sample and a desorbing agent to the container, and is easy to handle.

  On the other hand, as a container, a plate-like base material such as glass is used, and by fixing various types of amino group-coated chitosan on the base material, it is also used as a chip capable of detecting various types of microorganisms. be able to.

  In the method for separating and concentrating microorganisms of the present invention, a coated chitosan that can be used, an aqueous medium, an SH group-containing compound, a releasing agent and other components that can be contained therein, an immobilizing substrate, and a method of the present invention The microorganisms and the like that can be applied will be described in detail.

(1) Coated chitosan Coated chitosan used in the method of the present invention is coated with an amino group of chitosan and has the following characteristics.

  In this specification, the substance to be adsorbed with microorganisms is described as being coated with the amino group of chitosan, but it is obtained by coating the amino group of chitosan that can be used in the separation / concentration method of the present invention. The “amino group-coated chitosan” obtained can be treated as equivalent to the one obtained by coating the amino group of chitin, ie, “amino group-coated chitin”. Chitosan is an amino group obtained by deacetylating a predetermined amount of the acetyl group at the 2-position carbon of D-glucose, which is a repeating unit constituting chitin (usually a degree of deacetylation exceeding 0.5). This is because chitosan and chitin have the same skeleton. In addition, the reason demonstrated with the amino group covering chitosan in this specification is because reaction which coat | covers the amino group of chitosan can be easily performed compared with reaction which coat | covers the amino group of chitin.

  (i) Molecular weight: The molecular weight of chitosan is not particularly limited as long as the cells can be maintained alive in the presence of the SH group-containing compound described below. Chitosan is obtained by deacetylation by heating chitin in the presence of a concentrated alkaline solution, and is obtained as a mixture having different molecular weights depending on the heating conditions. In the separation / concentration method of the present invention, for example, those having a molecular weight over a wide range of about 10,000 to 1,000,000 can be used.

  (Ii) Deacetylation degree: The degree of deacetylation of chitosan (number of acetyl groups after deacetylation / number of acetyl groups of raw chitin) is determined by the presence of the SH group-containing compound described later. There is no particular limitation as long as it can be maintained as it is, for example, from a large value of about 0.99 to about 0.1 (usually 0.5 or less is called chitin), which has a deacetylation degree over a wide range be able to.

  (Iii): The covering ratio of the amino group of the coated chitosan is not particularly limited as long as the cells can be kept alive in the presence of the SH group-containing compound described later. For example, a wide range of about 40 to 100% The one with a covering rate over a wide range can be used.

  The amino group may be coated by coating at least one hydrogen atom of the primary amine with another group.

  Chitosan amino group coating methods include, but are not limited to, Schiff basification (using formaldehyde, acrolein, etc.) and alkylation (using alkyl aldehyde, etc.) with aldehydes. For example, acylation using a carboxylic acid anhydride or carboxymethylation using glyoxylic acid can be used as a method for coating an amino group. These amino group coating reactions themselves are known to those skilled in the art.

  The present inventors change the adsorption rate of a certain microorganism with the coated chitosan and the desorption rate from the coated chitosan by changing at least one of the above-described characteristics of the coated chitosan, and also the type of the microorganism. It was found that the adsorption rate with the coated chitosan and the desorption rate from the coated chitosan differed depending on the case. In the separation / concentration method of the present invention, this knowledge can be used to specifically separate microorganisms.

(2) Aqueous medium The separation / concentration method of the present invention can be carried out in an aqueous medium. The aqueous medium is typically an aqueous solution.

  The aqueous medium contains an adsorption aid such as an SH group-containing compound described below as an essential component at least while the cells and the coated chitosan are adsorbed. On the other hand, when detaching the cells from the coated chitosan, a desorbing agent described later is an essential component, and a surfactant can be used in combination with the desorbing agent. Moreover, the aqueous medium can contain the below-mentioned arbitrary components as needed throughout both the adsorption and desorption of the bacterial cells.

(2-1) SH group-containing compound, etc. (at least essential components during adsorption)
Examples of the SH group-containing compound include water-soluble organic compounds having both an amino group and a carboxyl group such as cysteine and glutathione.

  In addition, even if it does not contain an SH group, such a compound can be separated / concentrated according to the present invention as long as it can maintain a living cell by a compound containing a sulfur atom and other compounds. In the method, it can be used as an adsorption aid in the same manner as the SH group-containing compound.

  The concentration of the SH group-containing compound in the aqueous medium is not particularly limited as long as the microbial cells can be kept alive even if they are adsorbed to the coated chitosan in the aqueous medium, and range, for example, from about 1 mM to about 100 mM. Can be a concentration.

  The present inventors have found that the presence of the SH group compound described above enables efficient adsorption to the coated chitosan while the microorganisms remain alive.

(2-2) Desorbent (essential component for desorption)
Various releasing agents can be used depending on the type of microorganisms to be separated and concentrated, as long as the cells adsorbed on the coated chitosan can be detached in a living state. it can. It should be noted that it is preferable that the releasing agent is not present when the cells are adsorbed on the coated chitosan from the viewpoint of increasing the adsorption rate between the coated chitosan and the cells.

  Examples of the releasing agent are listed below. The concentration of these releasing agents is used below the concentration at which each microbial cell is killed depending on the type. Moreover, even if only 1 type is used for a releasing agent, it is also possible to use multiple types together.

-Sodium salts (eg sodium chloride, sodium carbonate, sodium sulfate, SP (sodium polyphosphate))
-Ammonium salts (eg ammonium sulfate)
・ Amino acids (eg, arginine, histidine)
Buffering agent (eg Tris)
-Heterocyclic compounds (eg, imidazole)
Organic acids (eg citric acid, malic acid)
The present inventors have obtained the knowledge that the above releasing agent has some specificity for the type of microorganism depending on the type and concentration. In the separation / concentration method of the present invention, separation / concentration can be performed specifically using such knowledge.

  Further, by using a surfactant in combination with the above releasing agent, the releasing action of the releasing agent can be enhanced.

  The surfactant to be used is one that maintains the living state of microorganisms, and is nonionic (for example, Tween 20, Tween 80 (both products) depending on the detachment status of microorganisms from the coated chitosan. Name)), a neutral surfactant (for example, CHAPS (3-[(3-cholamidopropyl) dimethyl-ammonio] propanesulfonic acid) can be used.

(2-3) Arbitrary components (common during adsorption and desorption)
The aqueous medium can contain the following optional components in order to keep the cells alive. The concentration of each arbitrary component can be appropriately set by those skilled in the art according to the purpose and the like.

Buffering agent (having a buffering pH range of 4.0 to 8.0 and satisfying the condition that the microorganism can be kept alive, for example, 3- (N-morpholino) propanesulfonic acid -Preservatives (eg sodium azide)
(2-4) pH
The pH of the aqueous medium varies depending on the optimum pH of the microorganism, but can usually be in the range of 4.0 to 8.0.

(3) Substrate The coated chitosan can also exist in a free state in an aqueous medium.

  In addition, the coated chitosan can be bonded to an appropriate immobilization substrate through a crosslinking agent as necessary.

  The immobilization substrate may be either a substrate that can flow, such as silica gel, or a substrate that does not, such as a glass tube. The material of the immobilization substrate is not particularly limited as long as it can immobilize the coated chitosan, such as silica gel (for example, silica for a column), glass tube, polystyrene, and the like.

  Any method may be used for immobilizing the coated chitosan on the substrate as long as it does not adversely affect the adsorbing action of the coated chitosan on microorganisms. For example, the amino group of the coated chitosan may be directly immobilized on the substrate, or may be immobilized on the substrate via an appropriate crosslinking agent. As the crosslinking agent, polycarboxylic acid, hexamethylene diisocyanate, or the like can be used. Moreover, what has functional groups, such as an amino group, a vinyl group, an epoxy group, can be used for a base material. The base material which has these functional groups can be created by a known method from a commercial item or a commercial item. Moreover, those skilled in the art can appropriately set reaction conditions for immobilizing the coated chitosan on the substrate based on known reactions.

  In particular, when silica for a column is used as an immobilization substrate, silica having an amino group as a functional group is used as an immobilization substrate, and glutaraldehyde is used as a cross-linking agent to coat the amino group of chitosan, This is convenient because the coated chitosan can be immobilized on the immobilization substrate. In this case, the coated chitosan immobilized on silica can be used in a suspended state in a dispersion medium, or can be used after being packed in a column or the like.

  When the coated chitosan having a predetermined degree of deacetylation is immobilized on the substrate, the degree of deacetylation is reduced by the amount that contributes to the immobilization. On the other hand, when a coated chitosan having a predetermined amino coverage is immobilized on a base material, the amino coverage is increased by the amount contributed to the immobilization. Therefore, when immobilizing the coated chitosan on the substrate, the degree of deacetylation and amino coverage of the coated chitosan before immobilization are determined in consideration of this decrease in deacetylation and increase in amino coverage. Remember what to do.

(4) Microorganisms As microorganisms that can be used in the separation / concentration method of the present invention, fungi (yeast), bacteria (which may be either gram positive or negative), such as Escherichia , Bacillus , Micrococcus , Staphylococcus Saccharomyces , Schizosaccharomyces , Staphylococcus , Vibrio , Aeromonas ) and the like. However, the microorganisms to which the separation / concentration method of the present invention can be applied are not limited to these.

  Next, the separation / concentration method of the present invention will be described.

  In the separation / concentration method of the present invention, the coated chitosan and the microorganism are contacted in an aqueous medium in the presence of an SH group-containing compound. The separation / concentration method of the present invention can separate and concentrate microorganisms as they are in living cells by utilizing the fact that the adsorption rate of the microorganisms to be contacted with the coated chitosan varies depending on the type of microorganisms. Is. Therefore, in the separation / concentration method of the present invention, other steps can be performed before and / or after the contact as long as the effect is exhibited.

  In the separation / concentration method of the present invention, contact conditions between the coated chitosan and the microorganism {for example, contact time, ambient environment (atmosphere, temperature, humidity, pH, etc.)} can be appropriately selected by those skilled in the art. It is. Usually, it can be performed at a contact time of 5 to 60 minutes, normal temperature, normal pressure, viable pH of the target microorganism, and osmotic pressure.

  In the separation / concentration method of the present invention, the adsorption rate with the coated chitosan differs depending on the microorganism, so that the coated chitosan adsorbed with the microorganism is separated from the microorganism remaining in the aqueous medium without being adsorbed on the coated chitosan. Thus, the plurality of microorganisms can be separated from each other.

  Hereinafter, separation and / or recovery procedures (hereinafter referred to as “separation / recovery procedures”) that can be carried out after contacting the coated chitosan with two or more types of microorganisms in the presence of an SH group-containing compound in an aqueous medium. However, the separation / concentration method of the present invention is not limited to these embodiments. Further, the following separation / recovery procedures can be performed alone or in combination of two or more types of separation / recovery procedures. Furthermore, one type of separation / collection procedure can be performed in two or more steps. At this time, each separation / recovery procedure may be performed continuously, or other steps may be interposed.

  The first aspect of the separation / recovery procedure is to separate / recover the microorganisms adsorbed on the coated chitosan and the microorganisms remaining in the aqueous medium. For example, using gravity (stationary, centrifugation, etc.), chromatography It can be performed using a chromatography (column chromatography or the like). These procedures can be selected as appropriate depending on whether or not the coated chitosan is immobilized on a carrier, the time spent for recovery, the number of types of microorganisms to be separated, and the like.

  When separating microorganisms by this method, separation with higher accuracy is possible when the difference in the adsorption rate of each microorganism is larger. In general, separation is possible if there is a difference in adsorption rate of at least about 20%.

Here, the adsorption rate (%) is represented by the optical density at 600 nm obtained by the following formula:
{(Optical density of aqueous medium after contact with coated chitosan) /
(Optical density of aqueous medium before contact with coated chitosan)} × 100
The measurement wavelength of the optical density was 600 nm because the absorption wavelength of the cell suspension is usually 600 nm, but the results obtained at 660 nm and 750 nm are the same.

  Another aspect is to separate a plurality of microorganisms adsorbed on the coated chitosan. At this time, an appropriate detachment agent is selected, and after detaching the microorganism from the coated chitosan, the detached microorganism is separated and recovered. The separation / recovery procedure at that time can use the above-mentioned ones.

  The use conditions of the release agent (type of release agent used, concentration, time until release is completed after addition of release agent, addition method of release agent, etc.) are determined by the adsorption rate between the coated chitosan and the microorganism { Specifically, it can be appropriately selected according to the selected releasing agent, the coated chitosan to be used, the type of microorganism to be separated, the desired separation accuracy, the separation / recovery procedure, etc. In general, the concentration of the desorbing agent is about 1 to 500 mM. Desorption starts immediately after the addition of the desorbing agent and can be desorbed in about 1 to 20 minutes. In addition, the releasing agent can be added directly to the aqueous medium, or it can be added to the aqueous medium as an aqueous solution, or can be added as an eluent when using chromatography.

  When separating and recovering microorganisms according to this aspect, separation can be performed with high accuracy if the difference in the rate of each microorganism is large. In general, separation is possible if there is a difference in the desorption rate of at least about 20%.

Here, the desorption rate (%) is represented by the optical density at 600 nm obtained by the following formula:
[(Optical density of aqueous medium after using release agent) /
{(Optical density of aqueous medium before contact with coated chitosan) − (Optical density of aqueous medium before using release agent)}] × 100
A typical separation / recovery procedure in this embodiment is a procedure in which a specific releasing agent is sequentially used for each of a plurality of types of adsorbed microorganisms. Specifically, when the first microorganism and the second microorganism are adsorbed to the coated chitosan, the first releasing agent (which can release the first microorganism but the second microorganism) The microorganisms cannot be detached) to release the first microorganisms into the aqueous medium. After removal of the aqueous medium, a second detachment agent (which can desorb the second microorganism) is used to release the second microorganism into the aqueous medium.

  Furthermore, as another aspect, it is also possible to fractionate the microorganisms adsorbed on the coated chitosan without using a desorbing agent after contacting the microorganism with the coated chitosan. In this embodiment, the coated chitosan used is at least one of properties such as molecular weight, degree of deacetylation, amino group coverage, whether the coated chitosan is immobilized on the substrate, and the type of substrate used. If they are different, those having different adsorption rates with microorganisms are used as a mixture.

  Although the separation / recovery method of the present invention has been described on the premise that at least one of these microorganisms is separated from other microorganisms for two or more types of microorganisms, one of the principles of the separation / recovery method of the present invention, That is, if the fact that the adsorption rate and desorption rate of microorganisms with the coated chitosan differ depending on the type of microorganisms, it can also be used as the following modification method. The present invention includes such a modification method.

  The modification includes a method for detecting whether or not a specific microorganism is present in a sample. Specifically, in the detection method, when a specific microorganism is present in the sample, the amino group-coated chitosan capable of adsorbing the specific microorganism and the sample are placed in the presence of the SH group-containing compound. A first contacting step for contacting in an aqueous medium; a coated chitosan adsorbed by the specific microorganism; and a desorbing agent capable of desorbing the specific microorganism from the amino group-coated chitosan into the aqueous medium. If the microorganism is present in the aqueous medium, it can be detected that the specific microorganism is present in the sample. On the other hand, when a specific microorganism is not present in the sample, the amino group-coated chitosan capable of adsorbing the specific microorganism and the sample are contacted in an aqueous medium in the presence of the SH group-containing compound. A second contacting step in which a coated chitosan to which the specific microorganism is not adsorbed and a releasing agent capable of desorbing the specific microorganism from the amino group-coated chitosan in an aqueous medium are contacted with each other. If the microorganism is not present in the aqueous medium, it can be detected that the specific microorganism is not present in the sample.

  According to this method, it is possible to determine whether or not the specific microorganism is present in the sample without using a reagent or the like for identifying the specific microorganism.

  In the separation / concentration method of the present invention, the following cases are included when microorganisms can be concentrated. When microorganisms are present in a certain liquid sample, and the coated chitosan is immobilized on the substrate and the microorganisms are adsorbed on the immobilized coated chitosan, the concentration of the microorganism immobilized on the substrate is It can be higher than the microbial concentration in the original sample. Further, after another liquid sample microorganism is adsorbed with the coated chitosan, the supernatant is removed, and then the releasing agent to be added is added in the form of a small amount of aqueous solution, whereby the microorganism is detached from the supernatant. In this case, the microorganism concentration in the supernatant can be higher than the microorganism concentration in the original sample.

  An apparatus (hereinafter also referred to as “apparatus of the present invention”) that can be used in the method of the present invention described above (including the above-described modification) will be described.

  The device of the present invention may be any device as long as it can provide a place where the coated chitosan can be brought into contact with the microorganism in the presence of an SH group-containing compound in an aqueous medium. In consideration of collecting and / or concentrating microorganisms, the following is particularly preferable.

Test tubes, columns, and perforated plates that contain coated chitosan In these devices, the coated chitosan may be fixed to the substrate or may not be fixed.

-Chip with immobilized coated chitosan When used as a chip, the coated chitosan is immobilized on a substrate such as a glass plate.

  The apparatus can be in the form of a kit that also includes a desorbing agent, if desired. This kit can further include a medium for culturing the separated microorganisms, a reagent for identifying the separated microorganisms, and the like. The type, amount, etc., of specific substances provided for the addition of these releasing agents and the like can be appropriately selected according to the target microorganism.

  Examples of the present invention will be described, but the present invention is not limited to these examples.

  In particular, microorganisms to which the separation / concentration method of the present invention can be applied are not limited to those used in the following examples. Even if the separation / concentration method of the present invention is other than the microorganisms used in the following examples, the microorganisms can be adsorbed with chitosan coated in an aqueous medium in the presence of an SH group-containing compound. It can be applied to any microorganisms if a preliminary test is conducted on whether or not it binds, and what kind of detachment agent can be used for detachment.

Example 1
1. As test strain, Escherichia coli XL1-blue, Bacillus subtilis IAM12118, Micrococcus Luteus IAM1056, Staphylococcus aureus IAM12544, Saccharomyces ludwigii IAM12243, Saccharomyces cerevisiae IAM4512, Schizosaccharomyces pombe IAM4863, Lactobacillus casei NRIC1046, Staphylococcus epiderimidis JCM2411, Vibrio parahaemolyticus IFO12711, Aeromonas hydrophyla IFO12978 Streptococcus mutans GS5, Streptococcus sobrinus 6715, Streptococcus salivarius ATCC9222 were used. These strains are all commercially available by other procedures. S. mutans , S. sobrinus and S. salivarius are conserved strains of the Department of Bacteriology, Nihon University Matsudo Dental School.

2. Preparation of Bacterial Solution Each of the above strains was cultured, and after collection, washed with a 10 mM MOPS (3- (N-morpholino) propanesulfonic acid) solution (pH 7.0) three times. Each strain was suspended in 10 mM MOPS, an L-cysteine solution (cysteine was dissolved in 10 mM MOPS) was added thereto, and the final concentration of L-cysteine was adjusted to 50 mM. The suspension of each strain was adjusted with a 50 mM L-cysteine + 10 mM MOPS solution so that the absorbance value at 600 nm of the optical density (OD) was 0.7.

3. Immobilization of chitosan Add 190μl of 0.1M acetic acid solution (pH5.0, 0.25mM sodium azide) to 50mg of silica NH-DM1020 (Fuji Silysia) in a microtube (2mL capacity) and deaerate for 10 minutes, then chitosan solution Of 1.5 mL was added. 0.3 mL of 25% glutaraldehyde was added and stirred rapidly at room temperature (3 hours). Thereafter, it was washed 5 times with 10 mM MOPS.

  The chitosan solution was dissolved in a 0.1 M acetic acid solution (pH 5.0, 0.25 mM sodium azide added) so that the chitosan concentration was 0.05%. The molecular weight of chitosan used here was 150,000 in weight average molecular weight, and the degree of deacetylation was 0.99.

4). Adsorption of microbial cells to chitosan 1.8 mL of the above prepared bacterial solution was added to chitosan-immobilized silica, and the mixture was stirred at room temperature for 1 hour. Thereafter, the mixture was allowed to stand to precipitate chitosan-immobilized silica, and the supernatant was removed. The absorbance value of this supernatant at OD600 nm was measured. To the precipitate excluding the supernatant, 1.8 mL of variously prepared eluates were added and stirred at room temperature for 15 minutes. Then, the mixture was allowed to stand to precipitate chitosan-immobilized silica, and the absorbance value at OD 600 nm of the eluate was measured. The proportion of bacteria separated by the addition of various eluates was calculated by the following formula.

Desorption rate (%) = absorbance value of OD600nm of supernatant after addition of eluate ÷ (0.7 (OD600 of first bacterial solution)-supernatant after adsorption) x 100
The above 14 types of strains were mixed with chitosan-immobilized silica, and the detachment rates of the bacteria when eluted with various eluates are summarized in Tables 1 and 2.

  From the results shown in Table 1 and Table 2, it was found that the type of eluate that can be detached differs depending on the bacterial species.

From the results in Table 1, the following was also found. Sacch. Cerevisiae , Sacch. Ludwigi and Schizo. Pombe all belong to yeasts.For example, when 1M sodium chloride is used as a desorbing agent, S. cerevisiae detaches 75% or more. Thus, S. ludwigi and S. pombe desorb less than 25%. From this result, at least under the conditions of Example 1, the ease of detachment of yeast varies greatly depending on the species. From this, it seems that the yeast can be separated at the species level. A similar phenomenon, even in the oral bacteria was observed (Table 2 S. mutans, see the results of S. sobrinus and S. salivarius.).

Among the strains shown in Table 1, E. coli is a gram-negative bacterium, and the other strains are gram-positive bacteria. Under the conditions of Example 1, it seems that Gram-negative bacteria can be more selectively separated when a mixture of 0.1M citric acid and 1% CHAPS is used as the releasing agent. Moreover, it seems that Gram-positive bacteria can be more selectively separated by using a mixed solution of 0.1 M arginine (Arg) and 1% CHAPS or a mixed solution of Tris buffer and 1% CHAPS.

  Moreover, under the conditions of Example 1, when the surfactant was contained in the desorbed liquid, the recovery rate increased compared to the case where it was not contained.

(Example 2)
Separation from different cell mixture
A cell mixture of E. coli XL1-blue and B. subtilis IAM12118 was prepared in the same manner as in Example 1. 1.8 mL of this mixed solution was added to chitosan-immobilized silica and stirred at room temperature for 1 hour. Thereafter, the mixture was allowed to stand to precipitate chitosan-immobilized silica, and the supernatant was removed. The absorbance value of this supernatant at 0.D.600 nm was measured.

  Add 1.8 mL of mixed aqueous solution (hereinafter referred to as “50 mM L-cysteine + 10 mM MOPS solution”) adjusted so that L-cysteine and MOPS are 50 mM and 10 mM, respectively, to the residue (precipitate) and wash the bacteria. After that, the supernatant was removed, and the absorbance value (OD600 nm) of this supernatant was measured as one cycle. This cycle was repeated three times, and the absorbance value (OD600 nm) of each supernatant was recorded ( Corresponding to fractions Nos. 2 to 4 in FIG. 1-1). Then add 1.8 mL of 0.1 M citric acid + 1% Tween 20 (trade name) solution, remove the supernatant after removing the bacteria, and measure the absorbance (OD600nm) of this supernatant in one cycle. This was repeated three times, and the absorbance value (OD600 nm) of each supernatant was recorded (corresponding to fractions Nos. 5 to 7 in FIG. 1-1). Thereafter, 1.8 mL of 50 nM L-cysteine + 10 mM MOPS solution was added, the bacteria were washed, the supernatant was removed, and the absorbance value (OD600 nm) of this supernatant was measured (fraction Nos. 8 to 9 in FIG. 1-1). Corresponding). Thereafter, 1.8 mL of 0.1 M Arg + 1% Tween solution was added to detach the bacteria, the supernatant was removed, and the absorbance value (OD600 nm) of this supernatant was measured as one cycle, and this cycle was repeated three times. The absorbance value (OD600 nm) of each supernatant was recorded repeatedly (corresponding to fractions Nos. 10 to 12 in FIG. 1-1).

The same operation as described above was performed using an E. coli XL1-blue single solution and a B. subtilis IAM12118 single solution instead of the E. coli XL1-blue and B. subtilis IAM12118 cell mixture.

The results are shown in FIGS. 1-1 and 1-2. FIG. 1-1 shows the absorbance value (vertical axis) of each fraction (horizontal axis). The black square is the cell mixture (Mix) of E. coli XL1-blue and B. subtilis IAM12118, the black circle is the E. coli XL1-blue single solution (E. coli), the black triangle is the B. subtilis The results with IAM12118 alone solution ( B. subtilis ) are shown. FIG. 1-2 is a photomicrograph (magnification 1,000) of each fraction No. 5 (Solution A) and fraction No. 10 (Solution B) obtained with E. coli , B. subtilis and Mix.

From this result, it can be seen that separation was possible in a living state by changing the detachment solution from the mixture of E. coli XL1-blue and B. subtilis IAM12118.

(Comparative Example 1)
The same experiment as in Example 2 was conducted except that L-cysteine was not contained, but it was confirmed that the bacteria were not detached at the concentration of the releasing agent used in this Example. Further, when the concentration of the releasing agent was increased (24% with NaCl), the bacteria were not detached.

(Example 3)
Instead of a mixture of E. coli XL1-blue and B. subtilis IAM12118, using a mixture of S. cerevisiae IAM4512 and B. subtilis IAM12118, also using a 1M NaCl and 0.1Arg + 1% Tween 20 as releasing liquid Except that, the same experiment as in Example 2 was performed. As a result, these bacteria could be isolated in a living state (see FIGS. 2-1 and 2-2).

Example 4
Instead of a mixture of E. coli XL1-blue and B. subtilis IAM12118, a mixture of E. coli XL1-blue and S. cerevisiae IAM4512 is used, and 0.01M NaCl and 0.1M citric acid + 1% Tween as the release agent. The same experiment as in Example 2 was performed except that 20. As a result, these bacteria could be isolated in a live state (see FIGS. 3-1 and 3-3).

(Example 5)
The same experiment as in Example 1 was performed using methionine and glutathione instead of L-cysteine as the SH group-containing compound, and S. mutans GS5 as the bacterial cell. Since only 10 mM MOPS was used as a control not containing an SH group-containing compound, the leaving solutions were 50 mM methionine + 10 mM MOPS and 50 mM glutathione + 10 mM MOPS.

The results are shown in Table 3.

  From the results shown in Table 3, it can be seen that even when various solutions (excluding SH groups) are used for the immobilized coated chitosan, the cells are adsorbed. However, it was found that the use of SH group-containing glutathione has a higher adsorption rate and is more suitable for separation and concentration.

(Measurement Example 1)
In the same manner as in Example 1, the cells ( E. coli XL1-blue, B. subtilis IAM12118, M. Luteus IAM1056, S. aureus IAM12544, S. cerevisiae IAM4512, S. ludwigii IAM12243 and silica NH-DM1020 immobilized chitosan) S. pombe IMA4863) was adsorbed and their adsorption rate was measured. As an aqueous medium, 50 mM cysteine + 10 mM MOPS was used.

  FIG. 4 shows the results obtained.

  From the results shown in FIG. 4, each bacterial cell showed a high adsorption rate, and thus it was found that it can be used for a wide variety of bacterial species.

(Measurement example 2)
Three types of IR spectra were measured: silica NH-DM1020, silica NH-DM1020 bonded with glutaraldehyde, and silica NH-DM1020 bonded with chitosan using glutaraldehyde as a crosslinking agent.

  The IR spectrum is shown in FIG.

  From this result, it can be seen that the deacetylation degree of the bound chitosan is low.

(Measurement Example 3)
The effect of microorganisms ( E. coli and B. subtilis ) on the difference in salt (NaCl) concentration in the aqueous suspension containing chitosan immobilized on silica prepared in Example 1 was examined.

Sample A: salt concentration 0M in water suspension
Sample B: 0.5M salt concentration in water suspension
Sample C: 1M salt concentration in water suspension
In sample A, all microorganisms were alive, but in sample B, E. coli was alive, but B. subtilis was dead, and in sample C, E. coli , B. subtilis was both strains co-dead state.

  From these facts, it is considered that chitosan in each sample is immobilized on silica in the state and molecular structure as shown in the schematic diagram of FIG.

1-1 is a graph showing the absorbance value of each fraction obtained in Example 2. FIG. FIG. 1-2 is a photomicrograph of two fractions showing the peaks of FIG. 1-1. FIG. 2-1 is a graph showing the absorbance value of each fraction obtained in Example 3. FIG. 2-2 is a photomicrograph of two fractions showing the peaks of FIG. 2-1. FIG. 3-1 is a graph showing the absorbance value of each fraction obtained in Example 4. Fig. 3-2 is a photomicrograph of two fractions showing the peaks of Fig. 3-1. FIG. 4 is a graph showing the adsorption rate of each bacterial cell. FIG. 5 is an IR spectrum. Fig. 6 is a schematic diagram of coated chitosan immobilized on silica.

Claims (4)

  A method for separating a specific microorganism from a sample containing one or more kinds of microorganisms in the form of viable cells and / or concentrating the sample with amino groups. A first contact step in which the coated chitosan is contacted in an aqueous medium in the presence of an SH group-containing compound, and the adsorption rate and desorption rate of the specific microorganism with the amino group-coated chitosan are A method for separating and / or concentrating microorganisms utilizing the fact that they are different from those of other microorganisms.   Microbe-adsorbed chitosan produced by adsorbing the specific microorganism to the amino group-coated chitosan by the first contact step, and a desorbing agent capable of desorbing the specific microorganism from the microorganism-adsorbed chitosan into the aqueous medium The separation and / or concentration method according to claim 1, further comprising a second contact step of contacting the first and second contact steps.   The separation and / or concentration method according to claim 2, further comprising a separation step of separating a specific microorganism detached in the aqueous medium.   A kit for separating a specific microorganism from a sample containing two or more types of microorganisms from other microorganisms in a living cell form, wherein the kit can adsorb the specific microorganism contained in a container. A kit comprising a base-coated chitosan and a desorbing agent capable of desorbing the specific microorganism from the amino group-coated chitosan adsorbed by the specific microorganism.

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