WO2020003120A1

WO2020003120A1 - Culture and/or enumeration of hiochi and hiochi-natured bacteria and kits useful for the same

Info

Publication number: WO2020003120A1
Application number: PCT/IB2019/055339
Authority: WO
Inventors: Takayuki Suda; Shoko Iwasaki
Original assignee: 3M Innovative Properties Company
Priority date: 2018-06-29
Filing date: 2019-06-25
Publication date: 2020-01-02
Also published as: JP2021529521A; CN112352043A

Abstract

A method that allows for culturing, for example in the same culture plate, hiochi and/or hiochi-natured bacteria. A method that allows for enumerating, for example in the same culture plate, hiochi and/or hiochi-natured bacteria. A kit comprising a buffer useful for such methods and one or more culture plates useful for such methods.

Description

CULTURE AND/OR ENUMERATION OF HIOCHI AND HIOCHI-NATURED BACTERIA

AND KITS USEFUL FOR THE SAME

BACKGROUND

Hiochi bacteria (sometimes referred to as hi-ochi or hi-ochi kin) are a type of lactic acid bacteria that cause spoilage of alcoholic beverages, such as sake. Hiochi -natured bacteria are lactic acid bacterial that are related to hiochi bacteria in that they also cause spoilage of alcoholic beverages, such as sake. Both hiochi and hiochi-natured bacteria are characterized by their ability to grow in high alcohol

concentrations, such as those found in wine and sake.

SUMMARY

A method of preparing a culture of hiochi or hiochi-natured bacteria, can comprise admixing a sample comprising hiochi or hiochi-natured bacteria with a buffer to form an inoculation mixture and inoculating a culture plate with the inoculation mixture. The method is characterized in that the buffer comprises phosphate, citrate, ethanol, and mevalonic acid.

A method of enumerating hiochi or hiochi-natured bacteria, can comprise

admixing a sample comprising hiochi or hiochi-natured bacteria with a buffer, the buffer comprising a phosphate, a citrate, ethanol, and mevalonic acid to form an inoculation mixture, inoculating a culture plate with the inoculation mixture to form an inoculated culture plate, and counting the number of colonies of hiochi or hiochi-natured bacteria on the culture plate. Like the method of preparing a culture, the method of enumerating is characterized in that the buffer comprises phosphate, citrate, ethanol, and mevalonic acid.

A kit can comprise one or more culture plates and a buffer. Like the method of culturing and the method of enumerating, the kit is characterized in that the buffer comprises phosphate, citrate, ethanol, and mevalonic acid.

DETAILED DESCRIPTION

Throughout this disclosure, singular forms such as“a,”“an,” and“the” are often used for convenience; however, the singular forms are meant to include the plural unless the singular alone is explicitly specified or is clearly indicated by the context. When the singular alone is called for, the term “one and only one” is typically used.

Some terms in this disclosure are defined below. Other terms will be familiar to the person of skill in the art, and should be afforded the meaning that a person of ordinary skill in the art would have ascribed to them. Terms such as“common,”“typical,” and“usual,” as well as“commonly,”“typically,” and“usually” are used herein to refer to features that are often employed in the description and, unless specifically used with reference to the prior art, are not intended to mean that the features are present in the prior art, much less that those features are common, usual, or typical in the prior art.

The term“sake” is used to refer to a type of alcoholic beverage typically made from rice and associated with Japan and Japanese cuisine, although today sake is not necessarily manufactured in Japan; in modem times sake is manufactured in a variety of other places as well, including the United States. “Sake” includes all types of alcoholic beverages that are commonly referred to as sake in the English language, including seishu (clear sake), doburoku (unfiltered sake), nigori (cloudy sake), futsu-shu (ordinary sake), tokutei meisho-shu (specially designated sake), junmai daiginjo-shu (pure rice, very specially brewed sake), daiginjo-shu (very specially brewed sake), junmai ginjo-shu (pure rice, specially brewed sake), ginjo-shu (specially brewed sake), tukubestsu junmai-shu (special pure rice sake), tokubetsu honjozo-shu (special genuine brew sake), junmai-shu (pure rice sake), honjozo-shu (genuine brew sake), namazake (unpasteurized sake), genshu (undiluted sake), koshu (aged sake), taruzake (sake ached in wooden barrels, usually of sugi, i.e., Japanese cedar), shiboritate (freshly pressed sake), fukurozuri (drip sake), tobingakoi (sake pressed into large containers), amazake (sweet-style sake), jizake (locally brewed sake), kuroshu (sake made from brown rice), teseihaku-shi (sake made with highly- polished rice), toso (sake infused with a medicinal powder, such as tososan), and the like.

Hiochi bacteria are a type of lactic acid bacteria that can grow in environments having high ethanol contents, in some cases as much as 25% ethanol or even more. Typical hiochi bacteria are of the genus Lactobacillus, and include the species homohiochii and all sub-species thereof. Hiochi-natured bacterial are lactic acid bacteria that are not considered in the art to be true hiochi bacteria, but nonetheless can grow in high-ethanol environments like those that support hiochi bacteria. Hiochi-natured bacteria are also of the genus Lactobacillus, and include, among others, at least those subspecies of the Lactobacillus species casei, hilgardii, and casei that can grow in high alcohol environments.

Hiochi and hiochi-natured bacteria can be responsible for spoilage of alcoholic beverages.

Particularly, hiochi and hiochi-natured bacteria are problematic for alcoholic beverages produced from rice, but they can also spoil other alcoholic beverages, for example those that use fungi in the production process. This includes grape-based alcoholic beverages such as wines, because many wine grapes are intentionally affected by so-called“noble rot,” (also known by the French terms“pourriture” and “pourriture noble,”) which is a desirable form of a grey fungus, typically Botrytis cinerea. Other alcoholic beverages such as miju (Chinese rice wine), cheongju (Korean rice wine), awamori (a rice wine liquor traditionally produced in Okinawa), shochu (rice liquor), and the like can also be spoiled by hiochi or hiochi natured bacteria. Despite having the common characteristic of growing in high alcohol environments, culturing samples in such a way so that hiochi bacteria, hiochi-natured bacteria, or both can be cultured in a single culture plate is a problem. A related problem is finding one test that can enumerate colonies of hiochi bacteria, hiochi-natured bacteria, or both on one culture plate. Current methods do not permit culturing or enumerating of hiochi and hiochi-natured bacteria together in a single thin-film culture plate with ready to use media. Currently, an agar culture plate must be prepared by the user for the culture (as opposed to the convenience of a ready-to-use thin film plate). This is problem because it takes more operator time to prepare an agar culture plate and there is more of a chance for operator error when an agar culture plate must be prepared each time as opposed to using a ready-to-use thin-film culture plates.

Another problem relates to the amount of time that is required to test for hiochi and hiochi-natured bacteria. While hiochi and hiochi-nature bacteria can both be detected using agar plates, a long time, such as seven to ten days, is required to culture and detect both hiochi and hiochi-natured bacteria using known methods.

This disclosure addresses these problems by providing a buffer for admixing with a sample containing hiochi bacteria or hiochi-natured bacteria, which can then be inoculated on a thin-film culture plate. When the buffer as described herein is used, both hiochi and hiochi-natured bacteria can be cultured together in the same thin-film culture plate. Typically, the thin-film culture plate is one that is available under the PETRIFILM™ brand of culture plates from 3M Company (St. Paul, MN), and most commonly a PETRIFILM™ brand LAB thin-film culture plate. When the buffer as described herein is used, both hiochi and hiochi-natured bacteria can be enumerated on the same plate. Thus, by practicing the enumeration method as disclosed herein, the practitioner can determine, with a single enumeration, whether the sample contains hiochi or hiochi-natured bacteria. What is more, the process of enumerating the hiochi or hiochi-natured bacteria, and particularly hiochi-natured bacteria, can be significantly faster, sometimes as little as three days as opposed to seven or more days, than previously known methods.

This disclosure also solves the problem by providing kits containing culture plates and the buffer as described herein, which facilitates a user culturing or enumerating hiochi or hiochi-natured bacteria.

Thus, this disclosure provides, for the first time, the ability to culture hiochi and hiochi-natured bacteria using a ready-to-use media or thin-film culture plate. Because either one of hiochi or hiochi-natured can cause spoilage of beverages, such as sake, being able to culture and detect both at the same time is a significant advantave.

Except where otherwise specified, it is not required that the method distinguish between hiochi and hiochi-natured bacteria, because in the case of a food or beverage sample the presence of either one or a combination of the two at unacceptable levels indicates spoilage of the food or beverage. Nonetheless, it is possible to distinguish between hiochi and hiochi-natured bacteria in some cases. A method of preparing a culture of hiochi or hiochi matured bacteria can include admixing a sample comprising hiochi or hiochi-natured bacteria with a buffer to form an inoculation mixture and inoculating a culture plate with the inoculation mixture. The method is characterized in that the buffer has the components and pH as described herein. The method can further comprise an enumerating step of counting the number of colonies of the hiochi or hiochi-natured bacteria on the culture plate.

A method of enumerating a culture of hiochi or hiochi-natured bacteria can include admixing a sample comprising hiochi or hiochi-natured bacteria with a buffer to form an inoculation mixture, inoculating a culture plate with the inoculation mixture to form an inoculated culture plate, and counting the number of colonies of hiochi or hiochi-natured bacteria on the inoculated culture plate. The method is characterized in that the buffer has the components and pH as described herein.

In either the method of culturing or enumerating hiochi or hiochi-natured bacteria, the sample can be any sample that contains, or may contain, hiochi or hiochi-natured bacteria. The sample may be from a food or beverage product, but may also be an environmental sample, for example, from a surface of a manufacturing facility that produces food or beverage products. Generally, when the sample is from a food or beverage, it is an alcoholic beverage. Most commonly, the alcoholic beverage is one that is brewed or distilled from rice, which includes those brewed or distilled from mixtures of rice and other grains. Sake is the most common beverage that is used as a sample, although miju (Chinese rice wine), chcongju (Korean rice wine), awamori (a rice wine liquor traditionally produced in Okinawa), shochu (a rice liquor), grape wine, and the like can also be used as a sample any of the methods described herein. When the sample is an environmental sample, it is typically obtained by swabbing one or more surfaces, such as work surfaces, surfaces of equipment, or the like, in a facility that makes alcoholic beverages.

In either the method of culturing or enumerating hiochi or hiochi-natured bacteria, or in the kit, the buffer comprises at least one buffering agent that provides a pH of 4.0 to 6.0 upon addition to the culture plate, and which is preferably a combination of phosphate and citrate, as well as ethanol and mevalonic acid. Each of these components must be present; eliminating any one of these components will make the buffer ineffective for culturing both hiochi and hiochi-natured bacteria. Thus, it is surprising that only a buffer containing all of these elements can be used to culture, enumerate, or culture and enmerate hiochi or hiochi-natured bacteria in the same culture plate.

The buffer can be at any pH at which hiochi and hiochi-natured bacteria can grow. Typically, after admixing the sample with the buffer the sample is quickly inoculated onto a culture plate. Thus, the pH of the buffer is typically sufficient to provide, after inoculation of the culture plate, a culture plate having an appropriate pH where the hiochi and hiochi-natured bacteria can grow. In such cases, the pH of the buffer will depend on the components of the culture plate. In many cases, the buffer has a pH sufficient such that, after inoculation, the inoculated culture plate has a pH of 4.0 - 6.0, such as , 4.0 or greater, 4.1 or greater, 4.2 or greater, 4.3 or greater, 4.4 or greater, 4.5 or greater, 4.6 or greater, 4.7 or greater, 4.8 or greater, 4.9 or greater, 5.0 or greater, 5.1 or greater, 5.2 or greater, 5.3 or greater, 5.4 or greater, 5.5 or greater, 5.6 or greater, 5.7 or greater, 5.8 or greater, or even 5.9 or greater. In such cases, the buffer has a pH sufficient such that, after inoculation, the inoculated culture plate has a pH of 6.0 or less, 5.9 or less, 5.8 or less, 5.7 or less, 5.6 or less, 5.5 or less, 5.4 or less, 5.3 or less, 5.2 or less, 5.1 or less, 5.0 or less, 4.8 or less, 4.7 or less, 4.6 or less, 4.5 or less, 4.4 or less, 4.4 or less, 4.3 or less, 4.2 or less, or even 4.1 or less. The buffer particularly has a pH that provides the inoculated culture plate with a pH of 4.5-5 5, and most particularly 4.8-5.2.

In many cases, the buffer itself has a pH of 3.5-6.0, such as 3 5 or greater, 3.6 or greater, 3.7 or greater, 3.8 or greater, 3.9 or greater, 4.0 or greater, 4.1 or greater, 4.2 or greater, 4.3 or greater, 4.4 or greater, 4.5 or greater, 4.6 or greater, 4.7 or greater, 4.8 or greater, 4.9 or greater, 5.0 or greater, 5.1 or greater, 5.2 or greater, 5.3 or greater, 5.4 or greater, 5.5 or greater, 5.6 or greater, 5.7 or greater, 5.8 or greater, or even 5.9 or greater. In such cases, the buffer has a pH sufficient such that, after inoculation, the inoculated culture plate has a pH of 6.0 or less, 5.9 or less, 5.8 or less, 5.7 or less, 5.6 or less, 5.5 or less, 5.4 or less, 5.3 or less, 5.2 or less, 5.1 or less, 5.0 or less, 4.9 or less, 4.8 or less, 4.7 or less, 4.6 or less, 4.5 or less, 4.4 or less, 4.4 or less, 4.3 or less, 4.2 or less, 4.1 or less, 4.0 or less, 3.9 or less, 3.8 or less, 3.7 or less, or even 3.6 or less. Mcllvaine buffer can be employed to achieve the appropriate pH by methods known to the artisan.

When the buffer includes citrate and phosphate, the citrate and phosphate can be used in any ratio that permits growth of hiochi or hiochi matured bacteria. While other buffers can be employed, a buffer including citrate and phosphate provides faster growth and detection of hiochi and hiochi matured bacteria as opposed to, for example, phosphate buffer alone. Thus, while the disclosure is not limited to buffers that include both citrate and phosphate, such buffers can be advantageous. Typical ratios are determined by using a concentration of citrate and phosphate as described herein to achieve the desired pH, also described herein. A ratio of 11 :9 (citrate to phosphate) is typical.

The citrate and phosphate concentration can be any concentration that permits growth of hiochi or hiochi-natured bacteria. Typically the phosphate is employed in an amount of 0.05 M or more and 0.5 M or less, such as 0.05 M or more, 0.075 M or more, 0.1 M or more, 0.125 M or more, 0.15 M or more, or 0.175 M or more, 0.2 M or more, 0.225 M or more, 0.25 M or more, 0.275 M or more, 0.3 M or more,

0.35 M or more, 0.4 M or more, 0.45 M or more, 0.5 M or less, 0.45 M or less, 0.35 M or less, 0.325 M or less, 0.3 M or less, 0.275 M or less, 0.25 M or less, 0.225 M or less, 0.2 M or less, 0.175 M or less, 0.15 M or less, 0.125 M or less, 0.1 M or less, or 0.075 M or less. The citric acid is typically employed in an amount of 0.025 M or more and 0.5 M or less, such as 0.025 M or more, more, 0.075 M or more, 0.1 M or more, 0.125 M or more, 0.15 M or more, or 0.175 M or more, 0.2 M or more, 0.225 M or more, 0.25 M or more, 0.275 M or more, 0.3 M or more, 0.35 M or more, 0.4 M or more, 0.45 M or more, 0.5 M or less, 0.45 M or less, 0.35 M or less, 0.325 M or less, 0.3 M or less, 0.275 M or less, 0.25 M or less, 0.225 M or less, 0.2 M or less, 0.175 M or less, 0.15 M or less, 0.125 M or less, 0.1 M or less, or 0.075 M or less. The phosphate can include any phosphate, such as phosphate salts, hydrogen phosphate salts (which are considered phosphate for the purposes of this disclosure), and the like. The counterions of the phosphate can be any cations, so long as the resulting phosphate is soluble in the buffer, and are typically alkali or alkali earth metal ions, most commonly sodium or potassium. Depending on the pH, the phosphate can be a hydrogen phosphate or a unprotonated phosphate or a combination of hydrogen phosphate and unprotonated phosphate. The citrate can include any citrate, such as citric acid or citrate salts. Typically, at the pH values used, the citrate will be predominantly in the form of one or more citrate salts, although it is also possible to have some or all of the citrate in the form of citric acid. When all or part of the citrate is present as a citrate salt, any counterion of the citrate salt can be used so long as the resulting salt is soluble in the buffer. Most commonly alkali or alkali earth metal ions, particularly sodium or potassium, are employed as counterions of the citrate salt.

The ethanol can be present in the buffer in any concentration suitable for growth of hiochi and hiochi-natured bacteria. Typical concentrations of ethanol in the buffer, in terms of % by volume, are from 3% to 30%, such as 3% or greater, 4% or greater, 5% or greater, or 6% or greater, and also such as 30% or less, 25% or less, 20% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, or 8% or less. Most commonly, a concentration of 7% ethanol is used.

The mevalonic acid can be present in the buffer in any concentration suitable for growth of hiochi and hiochi-natured bacteria. Typical concentrations of mevalonic acid are 0.5 ppm to 50 ppm, such as 0.5 ppm or greater, 1 ppm or greater, 2 ppm or greater, 3 ppm or greater, 4 ppm or greater, and also such as 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 9 ppm or less, 8 ppm or less, 7 ppm or less, or 6 ppm or less. Most commonly, a concentration of 5 ppm is used.

Other components can be included in the buffer, although they are not required unless otherwise specified. When employed, the most common other component is sodium chloride, which can be used in a concentration sufficient to provide an ionic strength that is appropriate for the growth of microbes, and particularly lactic acid bacteria. When employed, the amount and concentration of sodium chloride will depend on the amount of the other solutes in the buffer. Nutrients, vitamins, and the like, can also be employed as components of the buffer, however none of these are required unless otherwise specified.

Because thin-film culture plates are typically dry, or if not they have only a very small amount of liquid, there is little concern that the buffer will be diluted when a thin-film culture plate is used. Thus, unlike with agar plates, a pre-made buffer can be reliably used with thin-film culture plates without the need to be concerned about dilution of the buffer by hydration media as in agar culture plates.

The sample can be admixed with the buffer in any suitable ratio, including ratios known in the art for art-recognized methods of culturing hiochi or hiochi-natured bacteria. Typically, the sample is admixed in a vol/vol ratio of sample: buffer that is 1 : 1 to 1 :20, such as 1:5 to 1: 15, such as l :7 to 1: 12 In either the method of culturing or the method of enumerating hiochi or hiochi-natured bacteria, inoculation of the culture plate can be performed by any means in the art, and the details of how to inoculate a culture plate will be known to a person of ordinary skill in the relevant art. How the inoculation is performed, including the volume of inoculation mixture to be used, the location on the culture plate in which the inoculation media is to be placed, and the like, will depend on the particular culture plate used.

In either the method of culturing or the method of enumerating hiochi or hiochi-natured bacteria, or in the kit, the thin-film culture plate can be any thin-film culture plate suitable for growing lactic acid bacteria, such as those of the genus Lactobacillus, and does not have to be specially designed for hiochi or hioch-natured bacteria so long as the buffer as described herein is employed. Generally speaking, a variety of thin-fdm culture plates are known in the art; some thin-film culture plates are disclosed, for example, in WO2017019345, US4565783, WO1993012218, as well as elsewhere in the art. The thin- film culture plate that is most commonly used with this disclosure is the PETRIFILM™ brand lactic acid bacteria (LAB) count plate (3M Company, St. Paul, MN, USA), although other thin-film culture plates can also be used. Regardless of the type of culture plate used, in the methods as described herein, the same culture plate can be used to culture or enumerate hiochi and hiochi-natured bacteria.

Either the method of culturing or the method of enumerating can further comprise a step of exposing the inoculated culture plate to elevated temperature. The elevated temperature can be any temperature that facilitates the growth of lactic acid bacteria, such as those of the genus Lactobacilli and particularly hiochi and hiochi-natured bacteria. Exemplary temperatures are 25^° C to 35^° C, such as 30^° C. The culture plate can be exposed to this temperature for a period of time sufficient to allow for the growth of the hiochi or hiochi-natured bacteria. An exemplary period of time is 2 to 14 days, such as 2 or more days, 3 or more days, 4 or more days, 5 or more days, 6 or more days, 7 or more days, 8 or more days, 9 or more days, 10 or more days, 11 or more days, 12 or more days, or even 13 or more days. The period of time can also be 14 or fewer days, 13 or fewer days, 12 or fewer days, 11 or fewer days, 10 or fewer days, 9 or fewer days, 8 or fewer days, 7 or fewer days, 6 or fewer days, 5 or fewer days, 4 or fewer days, or even 3 or fewer days. Typical periods of time are 5-9 days, and most typically, 7 days. Periods of time longer than 14 days or shorter than 2 days may also be used depending on the requirements of the user, the particular sample being cultured, the culture plate being used, and the like. Neither the elevated temperature nor any particular period of time is necessarily required in all cases. With regard to the enumeration method, and the embodiments of the culturing method that also include an enumeration step, it is sufficient that the hiochi or hiochi-natured bacteria that are present in the culture plate are sufficiently detectable to be enumerated.

Enumeration, either as part of the enumeration method or as part of those culturing methods that also have an enumeration step, can be accomplished by any means known in the art. Culture plates can be enumerated using the human eye and manual counting by techniques known to the artisan. Alternatively, a plate reader, such as those commercially available from 3M Company, St. Paul, MN, USA, may be used, in which case the user can follow the instructions for the plate reader that is employed. Any plate reader capable of enumerating the culture plate that is employed may be used.

A kit for culturing one or both of hiochi and hiochi-natured bacteria can comprise one or more culture plates, which can be any of the culture plates as described above, and the buffer as described herein. The culture plates are thin film plates, an example of which is Petrifilm™ brand LAB plates, among others.

Examples

Bacterial Strains

The Lactobacillus strains listed in Table 1 were obtained from the National Research Institute of Brewing (Hiroshima, Japan) or the National Institute of Technology and Evaluation (Kisarazu, Japan) and individually incubated in SI semi-solid medium at 30 °C for 3-7 days. Individual inoculums were prepared by serially diluting each culture sample with one of the buffers selected from Table 2.

Following the serial dilutions, the final concentration of each inoculum was about 1-150 colony forming unit (cfu) counts per 1 mL

Table 1. Bacterial Strains used in Examples

SI powder media was obtained from the Brewing Society of Japan (Tokyo, Japan).

Mevalonic acid was contained in the media.

Mevalonic acid was obtained from the Sigma- Aldrich Corporation (St. Louis, MO).

Citric acid, Na HPO . and Ethanol (200 proof) were obtained from the Wako Pure Chemical Corporation (Osaka, Japan).

Citrate-phosphate buffer (Mcllvaine buffer) was prepared by mixing Nail IPOr (0.2 M) and citric acid (0.1 M) in a 9: 11 ratio (volume/volume). The pH of the citrate-phosphate buffer was about 4.5.

The compositions of each buffer used to form an inoculation mixture are described in Table 2. Table 2. Composition of Buffers used in Inoculation Mixtures of the Examples

Example 1.

Four inoculation mixtures containing a single Lactobacillus strain were prepared by serially diluting each of the Lactobacillus strains (A-D in Table 1) with Buffer 1 (Table 2). Each inoculation mixture (1 mL) was added to a separate PETRIFILM Lactic Acid Bacteria Count Plate (3M Company, St. Paul, MN) according to the manufacturer’s instructions and incubated at 30 °C for 2, 3, 7, or 10 days. Following the addition of the inoculum mixture, the hydrated culture medium had a pH of about 5 (as measured by a pH meter and probe available from HORIBA, Let (Kyoto, Japan)). At the end of the incubation period, colony forming units (cfu) were counted by visual inspection. The cfu per milliliter (ciii/mL) count of the original (undiluted) sample was then calculated by adjusting the colony count from the plate by the number and volume of serial dilutions. The results are reported in Table 3 as the mean cfu/mL count (n=2) at each incubation time point.

In Table 8, the calculated ratio of the cfu/mL count obtained by the method of Example 1 for each Lactobacillus strain relative to the corresponding cfu/mL count obtained by the method of Comparative Example 1 is reported as a percentage (equation for ratio calculation: [(cfu/mL obtained by method of Example l)/(cfu/mL obtained by method of Comparative Example 1)] x 100).

Comparative Example 1. SI agar Plates

Four inoculation mixtures containing a single Lactobacillus strain were prepared by serially diluting each of the Lactobacillus strains (A-D in Table 1) with phosphate buffered saline (PBS, 3M Corporation, St. Paul, MN). SI agar media was prepared by mixing 5 g of SI powder media with 90 mL of distilled water and 1 g of agar powder. The mixture was heated in a bath of boiling water until all of the agar melted. After the heating, 10 mL of ethanol was added aseptically. Each inoculation mixture (1 mL) was inoculated to a petri dish. The SI agar media was then poured into the petri dish. The petri dish was covered and anaerobically incubated at 30 °C for 2, 3, 7, or 10 days using ANEROPACK-ANERO in a rectangular jar (Mitsubishi Gas Chemical, Tokyo, Japan). At the end of the incubation period, the white colony forming units (cfu) were counted by visual inspection. The cfu per milliliter (cfu/mL) count of the original (undiluted) sample was then calculated by adjusting the colony count from the plate by the number and volume of serial dilutions. The results are reported in Table 4 as the mean cfu/mL count (n=2) at each incubation time point.

Example 2.

The same procedure as described in Example 1 was followed with the exception that Buffer 2 was used to prepare the inoculation mixture, instead of Buffer 1. The results are reported in Table 5 as the mean cfu/mL count (n=2) at each incubation time point.

Example 3.

The same procedure as described in Example 1 was followed with the exception that Buffer 3 was used to prepare the inoculation mixture, instead of Buffer 1. The results are reported in Table 6 as the mean cfu/mL count (n=2) at each incubation time point.

Example 4.

The same procedure as descnbed in Example 1 was followed with the exception that Buffer 4 was used to prepare the inoculation mixture, instead of Buffer 1. The results are reported in Table 7 as the mean cfu/mL count (n=2) at each incubation time point.

Example 5.

The same procedure as described in Example 1 was followed with the exception that Buffer 5 was used to prepare the inoculation mixture, instead of Buffer 1. The results are reported in Table 8 as the mean cfu/mL count (n=2) at each incubation time point.

Table 3. Colony Counts for the Method of Example 1 (2, 3, 7 and 10 Day Incubations)

Table 4. Colony Counts for the Method of Comparative Example 1

(2, 3, 7and 10 Day Incubations)

Table 5. Colony Counts for the Method of Example 2 (2, 3, 7and 10 Day Incubations)

Table 6. Colony Counts for the Method of Example 3

(2, 3,7and 10 Day Incubations)

Table 7. Colony Counts for the Method of Example 4 (2, 3, 7and 10 Day Incubations)

Table 8. Colony Counts for the Method of Example 2 (2, 3, 7and 10 Day Incubations)

Table 9. Calculated Ratio of Colony Counts Obtained for the Method of Example 1 Relative to the Method of Comparative Example 1 (Presented as a Percentage)

Claims

What is claimed is:

1. A method of culturing hiochi or hiochi-natured bacteria, the method comprising

admixing a sample comprising hiochi or hiochi-natured bacteria with a buffer to form an inoculation mixture; and

inoculating a thin-fdm culture plate comprising nutritional media with the inoculation mixture to form an inoculated culture plate;

characterized in that the buffer comprises, ethanol and mevalonic acid, and has a pH that is sufficient to provide a pH of 4.0-6.0 in the inoculated culture plate with a pH of 4.0-6.0.

2. A method of enumerating hiochi or hiochi-natured bacteria, the method comprising

culturing hiochi or hiochi-natured bacteria according to claim 1, and

counting colonies of hiochi or hiochi-natured bacteria on the culture plate.

3. The method of any of the preceding claims, wherein the buffer is further characterized as comprising citrate and phosphate.

4. The method any of claims 2-3, further comprising the step of culturing the inoculated culture plate at a temperature of 25^° C to 35^° C for 2 to 14 days, the step of culturing the inoculated culture plate taking place before the step of counting the number of colonies on the culture plate.

5. The method of any of the preceding claims, wherein the buffer has a pH of 4.5-5.5.

6. The method of any of the preceding claims, wherein the method is a method of culturing or enumerating both hiochi and hiochi-natured bacteria.

7. The method of claim 5, wherein the sample comprises both hiochi and hiochi-natured bacteria.

8. The method of any of the preceding claims, wherein the sample is an environmental sample.

9. The method of any of claims 1-8, wherein the sample is a food and beverage sample.

10. The method of claim 9, wherein the beverage is sake.

11. The method of any of claims 3-10, wherein the ratio of citrate salt to phosphate salt in the buffer is about 11 :9.

12. The method of any of the preceding claims, wherein the buffer comprises 0.5 ppm to 50 ppm mevalonic acid

13. The method of any of the preceding claims, wherein the buffer comprises 3% by volume to 30% by volume ethanol.

14. The method of any of the preceding claims, wherein the buffer comprises 5 ppm mevalonic acid.

15. The method of any of the preceding claims, wherein the buffer comprises 7% by volume ethanol.

16. A kit for culturing or enumerating one or both of hiochi or hiochi-natured bacteria, the kit comprising one or more thin-film culture plates, the culture plates comprising nutritional media that facilitate the growth lactic acid bacteria; and

a buffer;

characterized in that characterized in that the buffer comprises, ethanol and mevalonic acid, and has a pH that is sufficient to cause the pH of the nutritional media to be 4.0-6.0 after adding the buffer to the nutritional media.

17. The kit of claim 16, wherein the buffer comprises 0.5 ppm to 50 ppm mevalonic acid.

18. The kit of any of claims 16-17, wherein the buffer comprises 3% by volume to 30% by volume ethanol.

19. The kit of any of claims 16-18, wherein the buffer has a pH of 4.5-5.5.

20. The kit of any of claims 16-19, wherein the buffer contains citrate and phosphate.