JP6153461B2 - Microbial recovery method - Google Patents

Description

  The present invention relates to a method for recovering a target microorganism in a sample containing acidic fermented milk, a method for determining the quality of a sample using the recovery method, and a method for evaluating the activity of the target microorganism.

  Conventionally, collection of various microorganisms has been required in a wide range of fields such as food and beverage inspection, clinical inspection, and environmental inspection. For example, fermented dairy products such as yogurt use various microorganisms such as lactic acid bacteria, bifidobacteria and yeast, and are widely eaten and eaten with the expectation of effects such as intestinal regulation. Therefore, it is necessary to recover microorganisms from fermented milk products in a high yield and in a alive state.

  Along with the development of genetic engineering, many studies have focused on the physiological effects of useful microorganisms and their metabolites. Recently, research into the mechanisms of physiological effects of microorganisms by examining the state and active ingredients of useful microorganisms in foods and drinks has been progressing. Among them, not only live bacteria but also dead bacteria have various physiological functions. It has been found to be effective. In related research, it may be necessary to collect more than a certain amount of microorganisms in the experimental protocol. For example, using techniques such as lectin microarrays, polysaccharides and sugar chains present on the surface of microorganisms after collection may be used. This is the case when analyzing the added bacterial cell component or analyzing the protein present on the surface of the recovered microorganism using a technique such as sugar microarray. In addition, in order to accurately and easily measure microorganisms, it is necessary to provide samples with a high degree of purification with reduced contaminants that hinder the experiment. There has been a demand for a method for recovering microorganisms in a specimen in a high yield and reflecting the state in the specimen, since it cannot be used as it is.

  As a method for recovering microorganisms, for example, a sample, that is, a food, drink, cosmetics, pharmaceutical, or the like is diluted with an appropriate solvent, applied to an agar medium containing components suitable for the growth of microorganisms, and cultured for a predetermined period of time for proliferation. There is a method in which colonies of collected microorganisms are collected and, if necessary, further concentrated and cultured to increase the concentration, and then the microorganisms are collected using means such as centrifugation.

  However, with this method, only microorganisms that can form colonies on the medium can be detected, and it is impossible to recover dead bacteria, and the agar medium is collected from the agar medium because the environment differs from that in the sample. There is a problem that it is unclear whether or not the microorganisms reflected reflect the state in the original specimen. In addition, since the sample often contains impurities that make the medium turbid, detection may be inaccurate at the stage of detecting colonies that appear on the agar medium after culturing.

  As a means for solving these problems, for the specimen containing emulsion particles and microorganisms, the emulsion particles are made finer than the size of the microorganisms, and then filtered to remove the finely divided emulsion particles and collect the microorganisms. A method (Patent Document 1) is known. However, these methods require cumbersome operations, are expensive to collect, and have a low degree of purification of the specimen after treatment, so that a sufficient amount of microorganisms cannot be collected. There is a problem that it is difficult to use for the analysis of the surface layer structure and the validity confirmation of the specimen. It was also difficult to recover viable bacteria while maintaining the activity of the microorganisms.

JP 2006-94848 A

  Therefore, an object of the present invention is to provide a method for recovering microorganisms in a sample simply, at low cost, in a high yield and with a high degree of purification. Another object of the present invention is to provide a method for recovering the microorganism with high survival rate while maintaining the activity of the microorganism.

  As a result of diligent research to solve the above-mentioned problems, the present inventors have determined that a sample containing acidic fermented milk is adjusted to pH 5.4 or higher, a step of centrifuging the sample, and a sample containing the sample or the target microorganism. By using a method that includes the step of adding macromolecular organic matter degrading enzyme to the sample and decomposing the recovery inhibitor, the microorganism can be easily and highly purified while reflecting the state of the sample regardless of whether the microorganism in the sample is alive or dead. The present invention has been completed by finding that it can be recovered in a high yield with a high yield, and that when the microorganism contains a living microorganism, it can be recovered while maintaining the activity such as the survival rate and colony forming ability.

That is, the present invention is a method for recovering a target microorganism from a specimen containing acidic fermented milk, and includes the following steps a) to c), and step b) is performed after step a): A method for recovering microorganisms is provided.
a) adjusting the pH of the sample solution to 5.4 or higher,
b) a step of centrifuging the specimen, and c) a step of adding a macromolecular organic substance-degrading enzyme to the fraction containing the specimen or the target microorganism to decompose the collection inhibitor.

  The present invention also provides a sample quality determination method characterized by recovering the target microorganism using the recovery method and determining the quality of the sample using the recovered microorganism.

  Furthermore, the present invention provides a method for evaluating the activity of a target microorganism, wherein the target microorganism is recovered using the recovery method, and the activity of the recovered target microorganism is evaluated.

  According to the recovery method of the present invention, it is possible to recover live and dead microorganisms as target microorganisms in a sample with simple, low cost, high yield and high purity while reflecting the state in the sample. it can. In addition, when the microorganism contained in the specimen is a living microorganism, the microorganism can be recovered in a high yield while maintaining the activity while being alive. Therefore, the sample obtained using the recovery method can accurately and easily perform the quality determination of the original specimen and the activity evaluation of the recovered microorganisms. This is useful when evaluating the activity of microorganisms.

  The present invention includes a step of adjusting a sample containing acidic fermented milk to a pH of 5.4 or higher, a step of centrifuging the sample, and decomposing the collection inhibitor by adding a macromolecular organic substance-degrading enzyme to the fraction containing the sample or the target microorganism. Through this step, the collection inhibitory factor present in the specimen is reduced, and more target microorganisms are collected while reflecting the state in the specimen.

  In the present invention, “fermented milk” is not particularly limited as long as it is a fermented product containing milk-derived components. For example, animal milk such as cow's milk, goat milk, sheep milk, horse milk, reduced milk from powdered milk or skimmed milk powder. In addition, fermented products obtained by adding and fermenting lactic acid bacteria, bifidobacteria, yeast, etc. to a medium containing milk components such as cream, processed products, diluted products, processed products, etc. It is not limited to fermented milk specified by a ministerial ordinance (Ministerial Ordinance on Milk, etc.) regarding ingredient specifications.

  The “specimen containing acidic fermented milk” of the present invention (hereinafter sometimes referred to as “specimen”) refers to a fermented milk itself or a composition containing fermented milk having a pH lower than 5.4. Further, the pH of the specimen is preferably 5.3 or less, more preferably 3.0 to 5.2, and particularly preferably 3.4 to 5.2. Examples of the form of the sample include foods and drinks, cosmetics, pharmaceuticals, etc., foods and drinks or cosmetics are preferable, and foods and drinks are particularly preferable. Among the specimens, for example, fermented milk and dairy lactic acid bacteria beverages such as milk ordinances usually contain live bacteria and dead bacteria, and processed foods that have been heat-sterilized contain dead bacteria. Only included. The recovery method of the present invention can be used for specimens containing either or both live and dead bacteria, but is particularly suitable for those containing live bacteria. In addition, in this invention, a living microbe means the microbe which has colony formation ability. In addition, dead bacteria refer to bacteria that do not have colony-forming ability, and include not only dead bacteria but also bacteria that have lost colony-forming ability and have membrane stability and enzyme activity.

  In the present invention, the “target microorganism” is not particularly limited. For example, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus coriniformis, Lactobacillus gasseri, lacto Bacillus zeae, Lactobacillus johnsonii, Lactobacillus delbrukki Subspecies. Delbrukki, Lactobacillus delbrukki Subspecies. Lactobacillus bacteria such as Bulgaricus, Streptococcus bacteria such as Streptococcus thermophilus, Lactococcus lactarus, Lactococcus plantarum, Lactococcus raffinolactis and other Lactococcus bacteria, Leuconostoc mesenteroides, Leucono Stock Mecenteroides Subspecies. Lactobacillus such as Cremoris, Leuconostoc lacticus, etc., or Enterococcus faecalis, Enterococcus facium, etc., lactic acid bacteria, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacteria Bifidobacterium such as Um longum, Bifidobacterium animaris, Bifidobacterium adrecentis, Bifidobacterium angularum, Bifidobacterium catenuratum, Bifidobacterium pseudocatenulatum Genus bacteria and yeasts such as Saccharomyces cerevisiae, Saccharomyces cerevisiae, etc., among which the recovery rate of target microorganisms, viability of viable bacteria, and colony-forming ability of bacteria , Preferably lactic acid bacteria in terms of the search for the physiological effects possessed by the bacteria, in particular Lactobacillus bacteria are preferred. In addition, these microorganisms may contain 1 type, or 2 or more types.

  In the present invention, the “recovery inhibiting factor” is a kind of contaminants originally contained in the specimen, and inhibits separation of the microorganism from other contaminants in the separation and recovery of the target microorganism from the specimen. Carbohydrates and high molecular organic substances. There are cases where the collection inhibitory factor acts directly on the target microorganism to prevent its separation and recovery, or the presence of the collection inhibitory factor causes impurities to remain in the fraction of the target microorganism, resulting in a reduction in the degree of purification. The collection inhibitor is not particularly limited as long as it is a factor that physically and chemically inhibits the separation and recovery, and includes impurities such as proteins, carbohydrates, lipids, polysaccharides, fruit-derived components, etc. present in the sample. More specifically, examples include proteins, carbohydrates, lipids, and polysaccharides.

  As the “step of adjusting the pH of the specimen to 5.4 or higher” (pH adjusting step) of the present invention, for example, a method of adding a pH adjusting agent such as NaOH or a buffer to the specimen to adjust the pH to 5.4 or higher. Can be mentioned. In the research leading to the present invention, it has been suggested that milk protein adheres to target microorganisms as casein micelles in fermented milk, thereby hindering recovery of the target microorganisms by centrifugation or the like. Generally, when the pH is increased, the casein micelles in the fermented milk disappear from the aggregation of the casein micelles, and each casein micelle changes to a state of floating in the solution. Therefore, the casein micelles can be recovered by centrifugation. become unable. For this reason, it has been expected that the target microorganism adhered to the casein micelles also floats in the solution by the pH adjustment step, and the recovery rate of the target microorganism is further reduced. However, surprisingly, by performing the pH adjustment step, only the milk protein that is a recovery inhibitory factor is transferred to the supernatant after centrifugation, and the target microorganism in the sample can be efficiently recovered by the separation step described later. Became clear. This is considered that the adhesion between the casein micelle and the target microorganism was destroyed in the pH adjustment step, but the details are unknown. From the viewpoint of improving the recovery rate of target microorganisms, viability of live bacteria, and purification, the pH of the specimen is preferably pH 5.4 to pH 10, more preferably pH 5.8 to pH 10, and pH 6 to pH 8. It is particularly preferable to do this. The pH adjuster used for pH adjustment is not particularly limited, and a buffer solution such as a phosphate buffer solution such as NaOH or calcium phosphate can be used, and NaOH is particularly preferable from the viewpoint of increasing the recovery rate of the target microorganism.

  The “step of centrifuging a specimen” (separation step) of the present invention is performed by centrifugation. From the viewpoint of the recovery rate and survival rate of microorganisms, the centrifugation is preferably performed at 4 ° C. to 10 ° C. at 3000 × g or more, particularly 7000 to 8000 × g, for about 8 to 30 minutes. The separation step can be applied as it is to the sample subjected to pH adjustment or enzyme treatment, but in order to increase the removal efficiency of water-soluble components such as carbohydrates present in the sample, a solvent (diluent) is used. It is preferable to carry out after adding and diluting the specimen. The ratio of dilution is not particularly limited, but it is preferable to add a solvent so as to dilute the specimen 2 to 10 times, preferably 3 to 6 times. Examples of the diluent include water, peptone physiological saline, peptone water, PBS (phosphate buffer), Ringer's solution, physiological saline, glycine (Glycine), 2- (N-morpholino) ethanesulfonic acid (Mes), N- (2-acetamido) iminodiacetic acid (ADA), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), tris (hydroxymethyl) amino-methane (Tris), imidazole (Imidazole) 2- (cyclohexylamino) propanesulfonic acid (CHES), 3- (cyclohexylamino) propanesulfonic acid (CAPS), sodium / potassium phosphate (Na / K Phosphate), bis (2-hydroxyethyl) amino-tris It is possible to use an aqueous solution containing (hydroxymethyl) -methane (Bis-Tris) and 3-morpholinopropanesulfonic acid (MOPS). it can. These solvents may contain trehalose, sodium chloride, glutamic acid, arginine, monolauric acid polyoxyethylene sorbitan (Tween 20) and the like. When a buffer solution is used for pH adjustment in the pH adjustment step of the sample solution, pH adjustment and dilution can be performed simultaneously by adding the buffer solution itself as a diluent.

  In the step (enzyme treatment step) of adding the macromolecular organic matter-degrading enzyme to the fraction containing the sample or the target microorganism separated through the separation step of the present invention and decomposing the recovery inhibitor (the enzyme treatment step), A macromolecular organic matter-degrading enzyme is added to the fraction containing the microorganism to decompose the recovery inhibitor remaining in the fraction containing the target microorganism. In addition, a solvent may be appropriately added depending on the concentration of the target microorganism or the specimen. As the macromolecular organic matter degrading enzyme, when the recovery inhibitor is a protein, the proteolytic enzyme is used. When the recovery inhibitor is a lipid, a lipolytic enzyme such as lipase or phospholipase is used, and the recovery inhibitor is a polysaccharide. In some cases, polysaccharide-degrading enzymes such as cellulase, amylase, and pectinase can be used. The enzyme treatment may be performed near the optimum temperature of the enzyme to be used, and 37 to 40 ° C. is preferable for a proteolytic enzyme. As the solvent for the enzyme treatment, PBS, water, peptone physiological saline, peptone water, Ringer's solution, physiological saline and the like can be used.

  The proteolytic enzyme may be appropriately selected according to the type of protein in the specimen, and is preferably an enzyme that does not inhibit the growth of the target microorganism. Specific examples of proteolytic enzymes include protease, peptidase, papain, trypsin, chymotrypsin, pronase, etc., and any proteolytic enzyme that exhibits an optimum pH in the alkaline region, neutral region or acidic region. A proteolytic enzyme that exhibits an optimum reaction pH in the neutral region is preferred.

  It is preferable to use a proteolytic enzyme, in particular, a milk proteolytic enzyme, as the high molecular organic substance degrading enzyme because of its high purity, viability of the target microorganism, and maintenance of colony forming ability. The treatment conditions in the case of using milk proteolytic enzyme are not particularly limited, but the pH at the time of treatment is preferably in the range of pH 5.4 to pH 10, for example, protease, more preferably pH 5.8 to pH 10, and pH 6 to pH 8. A range is particularly preferred. In addition, the amount of enzyme added is preferably 0.15 U or more per gram of specimen, more preferably 0.15 to 1.5 U, and particularly preferably 0.15 to 0.3 U. The treatment time is preferably 5 to 30 minutes, particularly preferably 5 to 10 minutes. Here, the titer (U) indicates the activity (enzyme titer) of the enzyme involved in the reaction of 1 μmol of the substrate per minute at the optimum pH and temperature of the enzyme reaction. The activity of liberating 1 μmol of tyrosine per minute at the optimum pH and temperature of the enzyme reaction as a substrate. Further, in order to increase the efficiency of the enzyme treatment, it is preferable to concentrate the fraction containing the specimen or the target microorganism separated through the separation step 2 to 10 times, particularly 2 to 5 times, and perform the enzyme treatment. The amount of enzyme added in this case may be calculated from the amount of the original sample. For example, a solvent was added to a fraction containing the target microorganism obtained by subjecting a 10 g sample to the separation step to 2 g. In some cases, it is preferable to use an enzyme of 1.5 U or more. The enzyme reaction can be stopped by using normal means such as cooling or inactivating the enzyme by heat treatment. However, in order to maintain the fungal activity and prevent the survival rate from decreasing, the sample is cooled and the enzyme reaction is stopped. It is preferable to do. Further, in the present invention, it has been found that the recovery rate of viable bacteria is significantly increased by performing the enzyme treatment. It was feared that the enzyme acts on the cell membrane of the target microorganism and the survival of the fungus is inhibited, but surprisingly, by carrying out this enzyme treatment step, many viable bacteria are recovered, and the live bacteria in the specimen It became possible to collect the bacterial cells in a state reflecting the proportion of dead bacteria. In addition, the recovery method of the present invention is very useful in that each cell can be separated and a sample that can be easily analyzed in relation to the surface layer structure of the bacterium can be obtained.

  In the separation step and the enzyme treatment step, a step of washing and collecting the target microorganism (washing and collecting step) is performed at a desired stage by adding a solvent to the target microorganism that has passed through the separation step or the enzyme treatment step and washing it. These steps can be used in appropriate combination depending on the properties of the sample and the recovery inhibitory factor. By performing the washing and recovery treatment, contaminants such as proteins and sugars can be sufficiently removed. The pH of the cleaning solution used for cleaning is not particularly limited, but a pH of 3 to 10 can be used, and pH 6 to 10 is particularly preferable. Specifically, peptone saline, peptone water, PBS, Ringer's solution, physiological saline, Glycine, Mes, ADA, HEPES, Tris, Imidazole, CHES, CAPS, MOPS, Na / K Phosphate, Bis-Tris, etc. An aqueous solution containing one or more of Glycine, Mes, ADA, HEPES, Tris, Imidazole, CHES, CAPS, MOPS, Na / K Phosphate and Bis-Tris can be used as a cleaning solution. It is preferable to use it as a washing solution because the amount of recovered protein is high and the survival rate is high when the target microorganism is a living microorganism. From the viewpoint of maintaining the activity of the target microorganism and maintaining the surface layer structure, Imidazole, CHES, CAPS It is particularly preferable to use an aqueous solution containing one or more of Bis-Tris and MOPS as a cleaning solution. From the same viewpoint, the additive to the solvent is preferably trehalose, sodium chloride, glutamic acid, arginine, or polyoxyethylene sorbitan monolaurate (Tween 20), and particularly preferably trehalose or sodium chloride. As a specific example from the washing to the recovery of the target microorganism, for example, there is a method in which the specimen after the enzyme treatment step is diluted about 2 to 10 times with a solvent and centrifuged under the same conditions as in the separation step to recover the target microorganism. Can be mentioned. When a solvent is added after centrifugation and washing and recovery are performed, it is preferable to add a solvent to be a washing solution and perform centrifugation twice or more. Examples of the method for collecting the target microorganism include centrifugation and capture by an antigen-antibody reaction between the antibody and the target microorganism.

  The pH adjustment step, the separation step, and the enzyme treatment step can be used in appropriate combination as described above except that the separation step is performed after the pH adjustment step. Further, the separation step does not need to be performed immediately after the pH adjustment step, and during that time, the treatment such as the enzyme treatment step may be performed at the same time, but from the viewpoint of the efficiency of the enzyme treatment and the viability of viable bacteria The treatment step is preferably performed after the pH adjustment step and the separation step. Further, as described above, in the separation step and the enzyme treatment step, the washing and recovery step can be performed at a desired stage. For example, after adjusting the pH of the specimen, the target microorganism is separated by centrifugation, the separated target microorganism is subjected to an enzyme treatment, and then the solution after the enzyme treatment is washed and recovered. The pH adjuster in the specimen And a method of separating the target microorganism by centrifugation after adding an enzyme-degrading enzyme and polymer treatment, and washing and recovering the separated target microorganism, adjusting the pH of the specimen, and then subjecting the target microorganism to centrifugation And the like, and subjecting the separated target microorganisms to washing and recovery treatment, subjecting the obtained target microorganisms to enzyme treatment, and further washing and recovery methods. In addition, from the viewpoint of improving the degree of purification, maintaining the survival rate of the target microorganism, maintaining colony-forming ability, etc., after adjusting the pH of the sample solution, the target microorganism and the collection inhibitor are separated by centrifugation, and the fraction containing the target microorganism is obtained. It is preferable to subject the sample to an enzyme treatment, and a method of repeating the washing and collecting treatment a plurality of times after the enzyme treatment is more preferred. More specifically, after adjusting the pH of the specimen containing acidic fermented milk to 5.4 or more, the target microorganism is separated by centrifugation, and after the milk protein-degrading enzyme is allowed to act on the separated target microorganism, It is particularly preferred that the solution after the enzyme treatment is washed and recovered.

  The sample quality determination method of the present invention is, for example, for recovering a fermented milk product by using the present invention to collect microorganisms and measuring (quantitatively) the viable count of the sample after treatment by an agar plate culture method or the like, or When verifying the usefulness of the fermented dairy product by measuring the total number of bacteria including viable and dead bacteria by DAPI staining, does the sample satisfy the criteria set based on the number of viable and total bacteria? To determine the presence or number of contaminating microorganisms in a sample, to verify the storage state and deterioration of a sample from the ratio of live and dead bacteria, etc. Can be used. Samples that have undergone the pH adjustment process of fermented dairy products and the decomposition inhibitory factor decomposition process have sufficiently reduced contaminants that make the medium turbid. It becomes easy, and even a relatively small colony can be distinguished. As a result, the culture time is shortened and the accuracy of discrimination at the stage of detecting colonies is increased. In other words, compared to the case where microorganisms are detected by an agar plate culture method using a specimen having a relatively large amount of contaminants as in the past, the target microorganism in the specimen can be detected quickly and with high sensitivity. It can be suitably used in a method for judging the quality of foods and drinks containing live bacteria such as specimens, particularly fermented milk products containing useful microorganisms. The method for measuring the number of viable bacteria is not particularly limited, and a commonly used agar plate method or the like may be used as appropriate. Detection of the growing colonies of the target microorganism may be performed visually or by using a commercially available stereomicroscope or digital optical microscope, but the use of a digital optical microscope is preferred in terms of facilitating detection of minute colonies.

  The target microorganism activity evaluation method of the present invention uses, for example, the present invention from a specimen such as a fermented milk product containing useful microorganisms, and the target microorganism is recovered and the surface layer structure of the microorganism is evaluated using an electron microscope or the like. When determining the active ingredient that contributes to its physiological activity from the microorganism, when determining whether or not the surface structure of the cell body has been mutated by the antigen-antibody reaction using the antibody, the colony can be obtained by a normal culture method. When assessing the ability to form (CFU), when assessing membrane stability or form retention using a fluorescent dye emigration method, etc., when assessing changes in physiological activity intensity due to the ratio of viable and dead bacteria, It can be used for evaluating the difference between components contained in live bacteria and dead bacteria. In addition, for example, when it is desired to perform the activity evaluation as described above separately for live bacteria and dead bacteria, it is used by using the enzyme activity of the bacterial cells, for example, esterase activity, as an index, or for determining the survival of the bacteria. It is also possible to separate live bacteria killed by a staining method into a sample. The target microorganism recovered through the method of the present invention can be recovered in a high yield regardless of whether it is live or dead, compared to the case of using conventional recovery means, and because it does not pass through a medium or the like, it is the target in the product. Since microorganisms can be recovered as they are, and their effects on the surface layer structure are small and the activity before the treatment is maintained, they are preferable as specimen samples used for the activity evaluation method.

  Hereinafter, the content of the present invention will be described in more detail with reference to examples, but the present invention is not limited by these.

Test Example 1 Effect of washing liquid on pH adjustment and separation treatment on fermented milk products Yakult (non-fat milk solid content 3.1%, milk fat content 0.1%, pH 3.5, manufactured by Yakult Honsha Co., Ltd.) Was added dropwise with 20% NaOH (final concentration 0.24%) while stirring with a stirrer to adjust the pH to 6.7 to 6.9. Next, distilled water was added so that the whole became 325 mL (5 times amount), and the mixture was rapidly stirred to prepare a sample for centrifugation. Dispense the sample into a 30 mL centrifuge tube and centrifuge (7000 × g, 10 minutes, 4 ° C .: use centrifuge GRX-220 (TOMY) (hereinafter, all test examples and examples use the same machine) ), And the supernatant was removed with an aspirator. After washing the lactic acid bacteria (Lactobacillus casei YIT9029 (FERM-BP-1366)), each washing solution shown in Table 1 was added and suspended, and the same centrifugation was performed to remove the supernatant (this The operation was repeated three times). The collected cells were suspended in 6 mL of each washing solution to obtain test samples, and the total number of bacteria (DAPI method) was measured as follows. In addition, the sugar mass and protein mass in the supernatant recovered by centrifugation were measured as follows. In addition, the same centrifugal treatment as described above was performed on a sample (pH 3.5) that was not adjusted for pH.

Measurement of total bacterial count by DAPI staining (DAPI method)
DAPI staining was used to determine the total bacterial count of the test sample. The test sample was added to a micro slide glass for counting at 10 μL / well, sufficiently dried and fixed, then treated with 100% EtOH for 5 minutes and then sufficiently dried. Next, 4.6 μL / well of Vecta Shield with DAPI (Vector) was added to each well, covered with a cover glass, and the number of bacteria was measured with a fluorescence microscope (OLYMPUS BX51) (eyepiece 10 ×, objective) Lens 100 times). The total number of bacteria by the DAPI method was determined by measuring 12 visual fields in one well for one sample to obtain an average value, and further measuring this for 3 wells to obtain the average value.
Measurement of Neutral Sugar Mass by the Phenol Sulfate Method The supernatant after separation of the cells and the neutral saccharide in the supernatant after the washing operation were quantified by the phenol sulfate method. 100 μL of the sample was put in a glass test tube, and 100 μL of 5% (w / v) aqueous phenol solution was added and stirred, and then 500 μL of concentrated sulfuric acid was added and rapidly stirred vigorously for 10 seconds. After standing at room temperature for 20 minutes or more, absorption at a wavelength of 490 nm was measured with an ARVO ^™ × 3 Multilabel Reader using an ELISA microplate. A standard curve was prepared using a 1% glucose solution.
Measurement of protein mass by Bradford method In order to confirm the protein removal rate, the protein mass of the supernatant after cell separation and washing was measured by the Bradford method. After adding 160 μL of 4-fold diluted protein assay (Bio-Rad CBB G-250 dye) to 40 μL of the sample on a microplate, absorption at a wavelength of 595 nm was measured with an ARVO ^™ × 3 Multilabel Reader. A standard curve was prepared using a 0.2% BSA solution.
Both the sugar mass and the protein mass were measured twice to obtain an average value. Of the supernatants obtained by a total of four centrifugation operations (one at the time of bacterial cell separation and three at the time of washing), the fourth (final) supernatant was recovered as supernatant 1.

  As a result, the total number of bacteria was able to be recovered by 50% or more regardless of which cleaning solution was used, but more bacteria were recovered especially by using peptone water, 1/4 strength Ringer's solution, and PBS. (Table 2). As a result of measuring the sugar mass in the supernatant, it was confirmed that the saccharides could be removed by using any washing solution. However, in the 1/4 strength Ringer's solution and physiological saline, it was 0 in the supernatant 1 (final supernatant). It was found that these two types of cleaning solutions are suitable for removing carbohydrates (Table 3). Regarding protein removal, the difference in the protein contained in the supernatant 1 between the washing solutions was several times, and the difference depending on the washing solution was small (Table 4). In addition, since the protein mass of peptone water was 7.4 μg / mL, the correction value excluding the protein quantitative value was shown for this solution. From these results, it was confirmed that any washing solution can be used as the solvent, and that peptone water, 1/4 strength Ringer's solution, and PBS are preferable from the viewpoint of recoverability of bacteria. In addition, in the sample that was not adjusted in pH, the bacterial cells did not settle even after centrifugation, and the lactic acid bacterial cell fraction could not be obtained.

Test Example 2 Effect of pH adjuster on pH adjustment and separation treatment on fermented dairy products Add 65 mL of Yakult to a sterilized container and stir with a stirrer, 20% NaOH (final concentration 0.24%), or 0.1M phosphoric acid Potassium solution (pH 7.2) was added drop by drop individually and the pH was adjusted as described in Table 5. Distilled water was added to the adjusted solution so that the total was 325 mL (5 times the amount), and the mixture was rapidly stirred to prepare a sample for centrifugation. The sample was dispensed into a 30 mL centrifuge tube, centrifuged (8,000 × g, 10 minutes, 4 ° C.), and the supernatant was removed with an aspirator. 6 mL of 1/4 strength Ringer's solution was added to and suspended in the lactic acid bacteria after collection from 30 mL of the above 5-fold diluted sample, and centrifuged in the same manner to remove the supernatant (this operation was repeated three times). Repeated). 6 mL of 1/4 Ringer's solution was added and suspended in the collected bacterial cells to prepare test samples. Using this sample, the total number of bacteria by DAPI staining (DAPI method) was measured. In addition, using the supernatant recovered by centrifugation, the neutral sugar mass by the phenol-sulfuric acid method and the protein mass by the Bradford method were measured in the same manner as in Test Example 1, and carbohydrates contained in the supernatant for a total of 4 times And the total amount of protein was determined. In addition, the test was performed by two persons and the average value was calculated | required.

  As a result, the ratio of the total number of collected lactic acid bacteria to the specimen itself (Yakult) (recovered total number of bacteria) was compared with the case where 0.1 M potassium phosphate solution was used as a pH adjuster. The case was as high as about 71%. The recovered amounts of carbohydrate and protein were almost the same (Table 5). Therefore, it was confirmed that both 0.1 M potassium phosphate solution and NaOH can be used as the pH adjuster, and that NaOH is more preferable in terms of the recovery rate of the total number of bacteria.

Example 1 Effect of enzyme treatment on fermented milk product (1) Preparation of sample to be subjected to enzyme treatment Add 65 mL of Yakult to a sterilized container and add 20% NaOH (final concentration 0.24%) drop by drop while stirring with a stirrer The pH was adjusted to 6.7 to 6.9. Distilled water was added to the adjusted solution so that the total amount was 325 mL (5 times volume), and the mixture was rapidly stirred, and dispensed into a 30 mL centrifuge tube and centrifuged (7,000 × g, 10 minutes). 4 ° C). The bacterial cell fraction obtained from 325 mL of the 5-fold diluted sample was suspended in 13 ml (concentrated 5 times with respect to the specimen) of PBS, and used as a sample for enzyme treatment. The enzyme treatment reaction was stopped by freezing the cell solution.

(2) Influence of enzyme concentration and reaction time For 100 μL of bacterial cell solution, final concentrations of 25, 50, 100, and 250 μg / g (0.75 to 7.5 U per 1 g of bacterial cell solution (from 0.15 U to 1 per 1 g of specimen) Protease P “Amano” 3DS was added as shown in Table 6 within the range of 5 U)). Moreover, the sample which does not add an enzyme as a control was also created and the following operation was performed similarly to the sample which added the enzyme. For the enzymatic reaction, a microplate for a thermal cycler was used, and the enzymatic reaction was performed at 37 ° C. for 5 minutes in the thermal cycler. The solution after the enzyme reaction was centrifuged at 7000 × g for 10 minutes at 4 ° C., and then the absorbance at 280 nm of the centrifuged supernatant was measured as an index of increase in the amount of protein and amino acid collected in the supernatant. Moreover, the supernatant collected in the precipitate fraction containing microbial cells was added with an amount of distilled water and resuspended, and the absorption (turbidity) at 660 nm was measured. The digestibility (enzyme reaction rate) was determined from the following formula from the turbidity of the precipitate before and after the enzyme treatment. ARVO ^™ × MV Multilabel Reader was used for measuring absorbance and turbidity.
Digestibility (%) = (Turbidity before enzyme treatment (T0) −Turbidity after enzyme treatment (T)) / T0 × 100

  As a result, when the enzyme concentration is 25 μg / g (0.15 U / g of specimen) or more, the absorbance at 280 nm and the digestibility increase, the separation of the protein that is a collection inhibitor and the bacterial cell proceeds, and the protein is transferred to the supernatant. (Table 6).

(3) Effects of enzyme reaction temperature and enzyme treatment on bacterial activity To 100 μL of the bacterial cell solution, protease P “Amano” 3DS was added so that the final concentrations were 25, 50, and 100 μg / g. Moreover, the sample which does not add an enzyme as a control was also created and the following operation was performed similarly to the sample which added the enzyme. The enzyme treatment was performed at a reaction temperature of 37 ° C. or 40 ° C. for 5 minutes. The solution after the enzyme reaction was centrifuged at 7000 × g for 10 minutes at 4 ° C., and the absorbance at 280 nm of the centrifuged supernatant was measured in the same manner as in (2) above. In addition, for the purpose of confirming whether or not the activity after the enzyme treatment is maintained based on the surface layer structure of the bacterial cell, using the following method as an index, whether or not the bacterial cell after the enzyme treatment retains the specific antibody reactivity, Antibody reactivity was examined.

Measurement of antibody reactivity by ELISA method The precipitated fraction containing the cells after centrifugation is suspended in an ELISA buffer (Carbonate Buffer), washed by centrifugation (7,000 × g, 4 ° C., 10 minutes), and Carbonate. Resuspended in Buffer. The number of bacteria per well of the 96-well immunoplate was 2.2E + 07 cells. L8 antibody (Lactobacillus casei YIT9029 strain-specific antibody) was used as the primary antibody, peroxidase-conjugated antibody was used as the secondary antibody, and SHIGMA FAST o-phenylenediamine dihydrochoride tablet sets (SHIGMA) was used as the substrate for the color reaction. After the color development reaction, the color development reaction was stopped with sulfuric acid, and then the absorbance at 490 nm was measured. The absorbance was 1.0 or more, and L8 antibody was positive. As a control positive for L8 antibody reaction, pure cultured cells of YIT9029 strain cultured in MRS medium were used.

  As a result, even when the reaction temperature (37 ° C., 40 ° C.) and the enzyme concentration (25-100 μg / g) were different, the absorbance increase rate at 280 nm was equivalent to 1.4-1.5. In addition, the L8 antibody reaction of the microbial cells collected under each enzyme treatment condition was positive, and it was confirmed that the L8 antibody reactivity, that is, no influence on the surface layer structure of the microbial cells was observed even when the enzyme treatment was performed (Table 7). ). In addition, the bacterial cells in the specimens that had been subjected to enzyme treatment showed higher antibody binding than those that had not been treated with enzyme. This also suggests that impurities adhering to the surface layer of the cells were removed by the enzyme treatment. The absorbance of the pure culture of the YIT9029 strain as a control was 3.7, which was positive.

(4) Effect of Enzymatic Reaction on Viable Cell Survival Rate Protease P “Amano” 3DS is added to 100 μL of the bacterial cell solution so that the final concentration is 25 μg / g (0.15 U per 1 g of the sample). Using a microplate, the enzyme reaction was carried out at 40 ° C. for 5 minutes or 30 minutes. Moreover, the microbial cell solution which did not perform an enzyme process and was left to stand for 5 minutes or 30 minutes at 40 degreeC was also prepared. These four solutions were centrifuged at 7000 × g for 10 minutes at 4 ° C., and the lactic acid bacterial cell fraction after the separation was suspended in a ¼ strength Ringer's solution, and subjected to the same centrifugation treatment (this operation was repeated 3 times). Repeated). Suspension was made with the same washing solution to a total of 100 μL to prepare a test sample, and the total number of bacteria (DAPI method) was measured in the same manner as in Test Example 1 as follows. Further, in the same manner as in Test Example 1, the sugar mass and protein mass in the supernatant collected by centrifugation were measured. As in Test Example 1, the fourth (final) supernatant was described as supernatant 1.

Measurement of viable cell count using BCP medium A test sample was applied to a BCP medium (Nissui Pharmaceutical Co., Ltd.) to determine the viable cell count. 1 / 100,000 dilution of Yakult and 1 / 10,000 dilution of each sample after centrifugation are smeared on a BCP plate medium using a spiral plater, and after aerobic culture at 37 ° C. for 3 days The number of colonies was measured with a colony counter (Color QCount 530). For the viable cell count (CFU) in the BCP medium, the average value of two plates was obtained for one sample.

  The number of viable bacteria in the case of the enzyme treatment showed a high numerical value, which was about 10 times higher than that in the case of no enzyme treatment. In addition, when the enzyme treatment was performed, the ratio of the viable cell count to the total cell count (CFU / DAPI) was equivalent to the result of the specimen itself (Yakult). From this, it was found that the bacterial cells could be recovered without changing the ratio of viable and dead bacteria in the sample by performing the enzyme treatment (Table 8). Regarding the removal of carbohydrates and proteins, in the final supernatant (supernatant 1) after the enzyme treatment, the sugar is completely removed, and the protein is also contained in the final supernatant compared to the sample not subjected to the enzyme treatment. The amount was very small, and it was suggested that carbohydrates and proteins can be removed with fewer washings by using enzyme treatment. From the above results, it has been found that passing through the separation step and the enzyme treatment step after the pH adjustment step is effective in removing milk proteins and carbohydrates and efficiently recovers live bacteria.

Example 2 Effect of cell washing liquid on fermented milk products (1) Examination of washing liquid 1
Using the OptiSol ^™ Protein Solubility Screening Kit 200 (Dilyx Biotechnologies, LLC) kit, the washing solution shown in Table 9 was added to a 96-well microplate, and the influence of each washing solution on the removal of proteins and the survival rate of the target microorganism was examined. In the same manner as in Example 1 (1), a bacterial cell solution to be subjected to enzyme treatment was prepared, and enzyme reaction was performed at an enzyme concentration of 25 μg / g (0.15 U per 1 g of specimen) at 40 ° C. for 5 minutes. did. To 20 μL of this enzyme-treated bacterial cell, 80 μL of each washing solution was added and suspended (using a 96-well PCR plate), and after centrifugation at 7000 × g for 10 minutes at 4 ° C., the supernatant (about 80 μL) was recovered. To the bacterial cell fraction, 80 μL of the same solution was added and suspended, and the same centrifugation was performed. For suspension of the cells recovered by the second centrifugation, a 1/4 Ringer solution was used instead of each washing solution. The amount of protein recovered in each centrifugal supernatant was measured by the Bradford method to determine the total amount. Next, the number of viable cells was measured with BCP medium for the cells recovered after washing twice, and the relative recovery rate (%) of each solution was calculated when the recovery rate by 1/4 Ringer's solution was 100%. .

The results are shown in Table 10. Judging from the fact that the number of viable bacteria and the amount of recovered protein were high, Glycine, Mes, ADA, HEPES, Tris, Imidazole, CHES, CAPS, MOPS-NaCl, Na / K Phosphate-NaCl, Bis- Tris-Treha l ose, was recognized as MOPS-Treha l ose, Imidazole- Treha l ose, Na / K Phospahte-Arg / Glu, and Glycine-Tween 20 is suitable for the cleaning liquid. Moreover, pH of these preferable washing | cleaning liquids was 3-10.

(2) Examination of cleaning solution 2
Using the cleaning solutions in Table 11, the effects of each cleaning solution on the removal of proteins and the survival rate of target microorganisms were examined. Using a 1.5 mL tube, the treatment was carried out at 10 times the amount (1). That is, 800 μL of each washing solution was added to 200 μL of the enzyme-treated bacterial cells, suspended, and centrifuged at 7000 × g for 10 minutes at 4 ° C., and the supernatant (about 800 μL) was recovered. After 800 μL of the same solution was added to the cell fraction and suspended, the same centrifugation treatment was applied to recover the supernatant. The obtained microbial cells were suspended using 1/4 Ringer's solution. Thereafter, the viable cell count was measured in the same manner as in (1), and the L8 antibody reactivity was examined in the same manner as in Example 1 (3). Further, the sugar mass and protein mass of the centrifugal supernatant were measured in the same manner as in Test Example 1. The number of viable bacteria in the table is an average value of two measurements. Consequently, protein recovery of height, the survival rate of live bacteria height, from the viewpoint of maintaining the activity of the target microorganisms, Imidazole, CHES, CAPS, Bis -Tris-Trehalose, MOPS-Treha l ose, Imidazole -Treha l ose was the best. Moreover, pH of these preferable washing | cleaning liquids was 6-10.

Example 3 Effect of pH during pH adjustment and enzyme treatment While stirring 65 mL of a commercially available fermented milk product (Yakult), 20% NaOH was added to adjust the pH as shown in Tables 12 and 13 (divided into two steps). A similar experiment was conducted). Sterile water was added so that the total was 325 mL (5 times volume), centrifugation was performed (7000 × g, 15 minutes, 4 ° C.), and the supernatant was removed with an aspirator. The obtained bacterial cell fraction is diluted in 13 mL of PBS (pH is the same as when the bacterial cells are recovered), and the enzyme (protease P “Amano”) is added to a final concentration of 25 μl / mL (0.15 U). , At 40 ° C. for 5 minutes. To the enzyme-treated solution, 52 mL of a CAPS solution (pH is the same as at the time of bacterial cell collection) was added and suspended, and centrifuged at 7000 × g for 15 minutes at 4 ° C., and the supernatant was removed with an aspirator (this work) Was repeated three times). The cells were suspended in PBS and used as a sample, and the protein was measured by the Bradford method. Moreover, about what adjusted pH to 5.4 or more, the total number of bacteria by DAPI, the number of living bacteria by a BCP culture medium, and the amount of neutral sugars by the phenol-sulfuric acid method were measured. In the protein measurement, the supernatant collected in the separation step before the enzyme treatment is defined as the supernatant 1, and the total of the supernatants washed three times after the enzyme treatment is defined as the supernatant 2. In the carbohydrate measurement, the supernatant after the enzyme treatment is treated. The supernatant after washing 1 and 2 was designated as supernatant 3, and the supernatant after washing 3 times after enzyme treatment was designated as supernatant 4.

  As a result, it was confirmed that by setting the pH of the sample to 5.4 or more, many proteins migrate to the supernatant during separation treatment and can be removed from the specimen. In particular, at pH 5.8 to 10.0, the protein could be recovered at 90 mg / mL or more, and at pH 6.0 to 10.0, 150 mg / mL or more could be recovered, and the protein could be efficiently removed (Table 12, 13). In addition, at pH 5.4 to 10.0, it was confirmed that the saccharide contained in the supernatant became almost 0 after three washings, and the saccharide was sufficiently removed (Tables 14 and 15). In the range of pH 5.4 to 8.0, both the total number of recovered bacteria and the number of viable bacteria showed high numerical values exceeding 1.12E + 10 (Tables 16 and 17). Overall, it was recognized that adjusting the specimen in the pH range of 6.0 to 8.0 during pH adjustment and enzyme treatment was the best.

  According to the method for recovering a microorganism of the present invention, live and dead microorganisms as target microorganisms in a sample are recovered with simple, low cost, high yield and high purity, while reflecting the state in the sample. be able to. In addition, when the microorganism contained in the specimen is a living microorganism, the microorganism can be recovered in a high yield while maintaining the activity while being alive. Therefore, the sample obtained using the recovery method can accurately and easily perform the quality determination of the original specimen and the activity evaluation of the recovered microorganisms. This is useful when evaluating the activity of microorganisms.

Claims (14)

A target microorganism recovery method from the analyte (pH 5.3 or less) containing acidic fermented milk, comprising the following a) to c) of the process, step b) is facilities after step a), and, step c) method for recovering microorganisms characterized by facilities Succoth after step a).
a) adjusting the pH of the specimen to 5.4 to 10.0 using a pH adjuster;
b) a step of centrifuging the specimen, and c) a step of adding a macromolecular organic substance-degrading enzyme to the fraction containing the specimen or the target microorganism to decompose the collection inhibitor.   The method of claim 1, wherein step a) is followed by step b) and then step c).   The method according to claim 1 or 2, wherein the pH adjuster is NaOH or a phosphate buffer.   The method according to any one of claims 1 to 3, wherein the pH of the sample after pH adjustment is 6.0 to 8.0.   The method according to any one of claims 1 to 4, wherein the specimen containing the acidic fermented milk is a food or drink or a cosmetic, and the recovery inhibitor contains milk protein.   The method according to any one of claims 1 to 5, further comprising a washing and collecting step of washing and collecting the target microorganism.   The washing liquid used for the washing and recovery treatment is glycine, 2- (N-morpholino) ethanesulfonic acid (Mes), N- (2-acetamido) iminodiacetic acid (ADA), N-2-hydroxyethylpiperazine-N '-2-ethanesulfonic acid (HEPES), tris (hydroxymethyl) amino-methane (Tris), imidazole, 2- (cyclohexylamino) propanesulfonic acid (CHES), 3- (cyclohexylamino) propanesulfonic acid (CAPS) , 3-morpholinopropanesulfonic acid (MOPS), sodium / potassium phosphate (Na / K Phosphate), and bis (2-hydroxyethyl) amino-tris (hydroxymethyl) -methane (Bis-Tris) 7. The method of claim 6, comprising one or more.   The washing liquid used for the washing and recovery treatment is imidazole, 2- (cyclohexylamino) propanesulfonic acid (CHES), 3- (cyclohexylamino) propanesulfonic acid (CAPS), bis (2-hydroxyethyl) amino-tris ( The method according to claim 6, comprising one or more selected from hydroxymethyl) -methane (Bis-Tris) and 3-morpholinopropanesulfonic acid (MOPS).   The method according to any one of claims 1 to 8, wherein the macromolecular organic matter degrading enzyme is a proteolytic enzyme.   The method according to claim 9, wherein the proteolytic enzyme is used at 0.15 U or more per 1 g of the specimen.   The method according to claim 1, wherein the target microorganism is a lactic acid bacterium.   The method according to any one of claims 1 to 11, wherein the target microorganism contains viable bacteria.   A sample quality determination method comprising: recovering the target microorganism using the method according to any one of claims 1 to 12, and determining the quality of the sample using the recovered target microorganism.   A method for evaluating the activity of a target microorganism, comprising collecting the target microorganism using the method according to any one of claims 1 to 12 and evaluating the activity of the collected target microorganism.