JP2012223134A - Method for measuring viable cell count - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique for accurately measuring a viable cell count of such as lactic acid bacteria and bifidobacteria contained in an oil and fat composition.SOLUTION: The method for measuring a viable cell count includes preparing a powdered cell suspension by mixing the oil and fat composition containing powdered cells with a liquid oil and fat which is a liquid in a temperature condition for growing cells, subsequently preparing a diluent for measurement by diluting the powdered cell suspension with a water-soluble solvent, and measuring the cell count on a medium after adding the diluent for measurement on the medium to culture the cells.

Description

The present invention relates to a method for measuring the number of viable bacteria in an oil and fat composition containing a microbial powder (bacterial powder) such as lactic acid bacteria and bifidobacteria.
The present invention also relates to a method for preparing a bacterial powder suspension used for measuring the number of viable bacteria in an oil or fat composition containing bacterial powder.

Bifidobacteria and lactic acid bacteria are widely known as useful intestinal bacteria. In particular, there have been many reports on the physiological significance of bifidobacteria, producing organic acids such as lactic acid and acetic acid in the intestine, and inhibiting the growth of harmful bacteria, producing vitamins, and stimulating immunity Etc. have been clarified (Non-Patent Document 1).
Therefore, these bifidobacteria and lactic acid bacteria are used as probiotics in various foods such as fermented milk such as yogurt or powdered foods. In recent years, these bacteria have been processed into capsules, tablets, microcapsules and the like, and are widely used as health foods or supplements.

  Various methods have been reported to date to measure the viable count of bifidobacteria, lactic acid bacteria, or other microorganisms in foods and supplements, but samples such as physiological saline, distilled water, or buffer solutions can be used. After suspending in an aqueous solvent and further diluting with the same aqueous solvent, the diluted solution is mixed or smeared on an appropriate agar medium such as a standard agar medium or BCP-added plate count medium, and the bacteria are cultured. A method for measuring the number of bacteria (colony number) on a medium is common (Patent Document 1).

On the other hand, since bifidobacteria and lactic acid bacteria are highly sensitive to heat, low pH, moisture, etc., when producing supplements using these bacteria as probiotics, the number of viable bacteria in the product should be reduced. In order to maintain or to make the preparation enteric, a technique of coating the fungus powder with fats and oils or encapsulating it has been employed (Patent Document 2).
Many of the oils and fats used in these technologies have a relatively high melting point of 40 ° C. or higher and are solid under temperature conditions during distribution and storage.

International Publication No. 2009/157316 Pamphlet JP 2005-68094 A

Edited by Tomokazu Mitsuoka, "Study on Bifidobacteria", Japan Bifidobacteria Center, 1994

Accurately measuring the number of viable bacteria in foods and supplements is essential to maintain and guarantee product quality.
However, in the case of products such as supplements formulated using fats and oils having a relatively high melting point as described above, there is a problem that the number of viable microorganisms contained in the product cannot be accurately measured.

  That is, as described above, when measuring the viable count of microorganisms, it is usually necessary to prepare a diluted solution by suspending a sample in an aqueous solvent such as physiological saline or a buffer solution. However, when an oil or fat composition is mixed with an aqueous solvent, if the temperature of the aqueous solvent is low, the oil or fat contained in the oil or fat composition is solidified, and the bacterial powder in the oil or fat composition is not extracted into the aqueous solvent. Therefore, the exact viable count could not be measured.

On the other hand, when the temperature of the aqueous solvent is raised to a high temperature such as 45 ° C. or higher and the powder contained in the oil / fat composition is melted and an attempt is made to extract the bacterial powder into the aqueous solvent, the oil / fat begins to melt and the bacterial powder becomes aqueous. Since the number of bacteria begins to decrease under the influence of the temperature of the aqueous solvent from the point of contact with the solvent, there is still a problem that the exact number of bacteria cannot be measured.
In addition, in such a method, it is necessary to keep the aqueous solvent for suspension at a high temperature, so it takes time to prepare for the count of bacteria, and increases the difficulty in processing many samples. There is also a problem.

  In the case of a supplement containing bifidobacteria or lactic acid bacteria, the quality of the product cannot be guaranteed and communicated to consumers unless the viable count of bifidobacteria and lactic acid bacteria contained in the supplement can be accurately measured. Furthermore, if so-called miscellaneous bacteria such as general bacteria, molds and yeasts in the product cannot be accurately measured, hygienic quality control or quality inspection cannot be performed sufficiently, which is a problem.

  This invention is made | formed in view of the said situation, and it aims at providing the technique which measures correctly the number of living bacteria of lactic acid bacteria, bifidobacteria, and other microbes contained in an oil-and-fat composition.

As a result of intensive studies in view of the problems of the prior art, the present inventors have melted an oil and fat composition containing a fungal powder (melt) with liquid oil and fat that are liquid under the growth temperature conditions of the fungus. It was found that by mixing to prepare a bacterial powder suspension and diluting it with an aqueous solvent, the bacteria can be efficiently dispersed in the aqueous solvent. And by adding the diluted solution prepared in this way to the medium and culturing the bacteria, the number of viable bacteria can be accurately measured, and the measurement accuracy of the number of viable bacteria in the oil and fat composition can be improved. I found out that I can do it.
In addition, by controlling the temperature of the aqueous solvent for diluting the bacterial powder suspension within the range of the growth temperature of the bacteria, it is possible to suppress the death of the bacteria when the bacterial powder suspension is suspended in the aqueous solvent, It was found that the viable count could be measured more accurately.

The present invention for solving the above problems is a method for measuring the number of viable bacteria contained in an oil and fat composition, wherein the melted oil and fat composition containing a fungal powder is liquid under the growth temperature conditions of the fungus. A step of preparing a bacterial powder suspension by mixing with fats and oils, a step of diluting the bacterial powder suspension with an aqueous solvent to prepare a measurement diluent, and adding the measurement diluent to the medium, A step of culturing the fungus.
By using the method of the present invention, it is possible to efficiently disperse the bacteria in the oil and fat composition in an aqueous solvent having a temperature lower than the melting point of the oil and fat used in the oil and fat composition containing the bacterial powder (for example, room temperature). The measurement accuracy of the number of viable bacteria in the oil and fat composition can be increased.

In a preferred embodiment of the present invention, the temperature of the aqueous solvent is within the range of the growth temperature of the fungus.
As a result, after the bacterial powder suspension is mixed with the aqueous solvent, the bacteria are immediately subjected to the growth temperature condition, so that the killing of the bacteria can be suppressed and the measurement accuracy of the viable cell count can be further increased.

  The method for measuring the number of viable bacteria of the present invention is suitable for the measurement of the number of viable bacteria in an oil or fat composition containing an oil that is solid under the growth temperature conditions of the fungus. As described above, since such an oil and fat composition cannot be easily suspended in an aqueous solvent having a temperature within the growth temperature range of the bacteria, the problem that the number of viable bacteria cannot be accurately measured has been significant. is there.

In a preferred embodiment of the present invention, the mixing ratio of the oil / fat composition containing the fungus powder and the liquid oil / fat may be as follows.
Oil and fat composition containing fungus powder: Liquid oil and fat = 2: 1 to 1: 1000
By setting it as such a mixing ratio, the bacterial powder turbid liquid which is easy to disperse | distribute to an aqueous solvent can be prepared.

In a preferred embodiment of the present invention, the aqueous solvent contains a surfactant such as polysorbate.
Thereby, dispersion | distribution to the aqueous | water-based solvent of a bacterial powder suspension becomes easy, and the measurement precision of viable count can be improved further.

In the preferable form of this invention, melting | fusing point of the fats and oils used for the fat and oil composition containing the said fungus powder is 40 degreeC or more.
The method of the present invention is particularly useful for the measurement of bacteria in an oil or fat composition using such a high melting point oil or fat.

  The viable cell count measurement method of the present invention is particularly useful for an oil and fat composition containing a powder of bifidobacteria or lactic acid bacteria. This is because bifidobacteria and lactic acid bacteria are susceptible to the effects of temperature and pH, and therefore, particularly accurate measurement of the number of viable bacteria is required.

The present invention also relates to a method for preparing a bacterial powder suspension for measuring the number of viable bacteria contained in an oil and fat composition. The method for preparing a bacterial powder suspension according to the present invention is to prepare a powdered powder suspension by mixing a melted oil-and-fat composition containing bacterial powder with a liquid oil that is liquid under the growth temperature conditions of the bacteria. Features.
According to the preparation method of the present invention, it is possible to prepare a bacterial powder suspension that can be easily mixed with an aqueous solvent used for measuring the number of viable bacteria.

If the viable count method of the present invention is used, the viable count of the fungi contained in the oil and fat composition can be accurately measured. For example, it is possible to collect bacteria contained in products such as health foods and supplements containing useful bacteria formulated with fats and oils in high yield, and it is possible to accurately measure the number of viable bacteria in products It becomes.
In addition, by using the method for preparing a bacterial powder suspension of the present invention, it is possible to prepare a bacterial powder suspension that can be easily mixed in an aqueous solvent used in the measurement of the bacterial count used to measure the number of viable bacteria. Preparation of a diluent for measurement is facilitated. As a result, it is expected to improve the accuracy and efficiency of the measurement of the number of viable bacteria for quality inspection and quality control of foods and supplements.

It is process drawing which shows one form of the viable count method of this invention.

Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention.
In the present specification, the percentage is expressed by mass unless otherwise specified, except for the survival rate of the bacteria.

The oil and fat composition to be a sample (specimen) of the method for measuring the viable cell count of the present invention comprises a fungus powder and fats and oils.
Bacterial powder means powdered bacterial cells by freeze drying or the like.
In the present invention, the bifidobacteria and lactic acid bacteria powdered by freeze-drying or the like are described as bifidobacteria powder and lactic acid bacteria powder. In addition, powdered bacteria such as bifidobacteria and lactic acid bacteria may be collectively referred to as bacterial powder, and powdered fungi such as yeast and mold may be referred to as fungal powder.
In addition, bacterial powder such as bifidobacteria powder and lactic acid bacteria powder, and fungal powder may be collectively referred to as fungus powder or microbial powder.
The bacterial powder is produced by a usual method. Taking the production method of bifidobacteria powder as an example, it can be produced by the following method.

GAM liquid medium (manufactured by Nissui) supplemented with glucose (Nacalai Tesque) at a rate of 2% is inoculated with Bifidobacterium longum ATCC BAA-999 (obtained from ATCC), 37 Culturing is carried out at 20 ° C. for 12 hours, and after completion of the culture, cells (wet cells) are collected from the culture solution by centrifugation. A sample is prepared by dispersing the wet cells in an aqueous solution of skim milk powder adjusted to a concentration of 10% at a cell solid content concentration of 10%. The pH of the prepared sample was adjusted to 7.0 with a 10% sodium hydroxide solution (manufactured by Nacalai Tesque), and then freeze-dried at 20 ° C. for 24 hours using a freeze dryer (manufactured by Kyowa Vacuum). Then, the cells after lyophilization can be pulverized to obtain a Bifidobacterium powder having a concentration of 1.0 × 10 ¹¹ CFU / g.

There are no particular limitations on the bacteria constituting the bacterial powder contained in the oil or fat composition, that is, the bacteria whose viable cell count can be measured according to the present invention.
For example, lactic acid bacteria such as bacteria belonging to the genus Bifidobacterium (hereinafter sometimes referred to as bifidobacteria), bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Lactococcus, bacteria belonging to the genus Streptococcus, Examples include fungi and the like.

As bacteria belonging to the genus Bifidobacterium, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium pseudolongum, and Bifidobacterium pseudo A bacterium selected from catenuratam (Bifidobacterium pseudocatenulatum) and the like can be exemplified.
For example, Bifidobacterium longum subsp. Longum ATCC BAA-999, Bifidobacterium longum subsp. Infantis LMG 23728, Bifidobacterium breve LMG 23729, Bifidobacterium breve MCC1274 (FERM BP-11175), Bifidobacterium pseudolongum NITE SD 00053, etc. be able to.
Among the above strains, ATCC BAA-999 is from American Type Culture Collection (ATCC), LMG 23728, LMG 23729 is from BELGIAN CO-ORDINATED COLLECTIONS OF MICRO-ORGANISMS (BCCM), NITE SD 00053 is an independent administrative corporation product. Each can be obtained from the Evaluation Technology Infrastructure.
Among the above strains, Bifidobacterium breve MCC1274 was commissioned on August 25, 2009, to the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan). Deposited under the number FERM BP-11175.

The bacteria belonging to the genus Lactobacillus are selected from Lactobacillus delbrueckii subsp. Lactis, Lactobacillus reuteri, Lactobacillus acidophilus, etc. Can be exemplified.
Examples of bacteria belonging to the genus Lactococcus include Lactococcus lactis and Lactococcus lactis subsp. Lactis.
Examples of bacteria belonging to the genus Streptococcus include Streptococcus thermophilus.
For example, Lactococcus lactis subsp. Lactis MCC866 (FERM BP-10746), Lactobacillus acidophilus NITE SD 00054, etc. can be used to measure the number of viable bacteria.
Among the above strains, NITE SD 00054 is generally available from the National Institute of Technology and Evaluation Technology.
Among the above strains, Lactococcus lactis subsp. Lactis MCC866 was established on December 1, 2006 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1st Street, Tsukuba, Ibaraki, Japan). In addition, it is deposited under the deposit number FERM BP-10746.

The fats and oils contained in the fat and oil composition are not particularly limited, and examples thereof include fats and oils that are solid at room temperature and are usually used in preparations containing fungal powder.
The method for measuring the viable cell count of the present invention is suitable for measuring the viable cell count of an oil or fat composition using a relatively high melting point fat that is solid under the growth temperature conditions of the fungus to be measured. The growth temperature conditions of the bacteria vary depending on the bacterial species and strains, but the upper limit of the growth temperature of bifidobacteria and lactic acid bacteria is usually about 40 to 55 ° C.
Furthermore, the method for measuring the viable cell count of the present invention is suitable for measuring the viable cell count of an oil and fat composition using a solid fat under the culture temperature conditions (near the optimum growth temperature) of the fungus described below. The culture temperature conditions of the bacteria vary depending on the bacterial species and strains, but the culture temperature conditions for bifidobacteria and lactic acid bacteria are usually about 25 to 42 ° C.
As melting | fusing point of the fats and oils used for a fat and oil composition, Preferably, it is 40 degreeC or more. Although the upper limit of melting | fusing point is not specifically limited, Usually, it is about 80 degreeC, Preferably it is about 70 degreeC.

In this invention, melting | fusing point of fats and oils can be calculated | required with the following measuring methods.
That is, the melting point temperature can be determined by the establishment of the Japan Oil Chemists' Society, the Standard Oil and Fat Analysis Test Method (I) 1996 edition, 2.2.4.2-1996, and the measuring method of the rising melting point. Specifically, an oil and fat sample is melted and filtered with dry filter paper, and a sample in which one end of a capillary tube having an inner diameter of 1 mm, an outer diameter of 2 mm or less, a length of 50 to 80 mm and open at both ends is completely melted (usually 60 ° C.) ), Fill the sample to a height of about 10 mm, quickly solidify with ice pieces, etc., and leave it at 10 ° C. or lower for 24 hours or on ice for 1 hour to prepare a sample-prepared capillary tube.
This sample-prepared capillary tube is immediately brought into close contact with the lower part of a thermometer (length: 385-390 mm, mercury bulb length: 15-25 mm, 0.2 ° C. scale) by a rubber ring or a suitable method, Align.
The thermometer with the capillaries in close contact is immersed in a beaker (with an internal volume of 500 to 1000 mL) filled with water having a temperature about 20 ° C. lower than the expected melting point, and the lower part of the thermometer is placed at a depth of 30 mm below the water surface. .
The water in the beaker is first heated to increase by 2 ° C./min while stirring in an appropriate manner. After reaching 10 ° C. below the expected melting point, heat at 0.5 ° C./min.
The temperature at which the sample begins to rise in the capillary is defined as the melting point (rising melting point).

As fats and oils constituting the fat and oil composition, those usually used in solid preparations containing bacteria such as supplements and medicines can be mentioned without particular limitation. Examples are given below.
・ Product name: TP-9 (NOF Corporation) Melting point 65-69 ° C
・ Product name: RHPI (Taiyo Yushi Co., Ltd.) Melting point 60.5 ℃
Sample name: S1044 (Taiyo Yushi Co., Ltd.) Melting point 53.5 ° C
Sample name: S1043 (Taiyo Yushi Co., Ltd.) Melting point 49.9 ° C
Sample name: S0182 (Taiyo Yushi Co., Ltd.) Melting point 46.4 ° C

  The dosage form of the oil and fat composition to which the method for measuring the viable cell count of the present invention can be applied is not particularly limited. Granules obtained by coating the bacterial powder on the granules with fat and oil, Tablets coated with the above, powdered fats and oils powdered by spray cooling after mixing fungus powder and fats, capsules filled with hard capsules and soft capsules after mixing fungus powders and fats and the like.

Hereinafter, each process of the viable count method of the present invention will be described.
FIG. 1 is a process diagram of the viable cell count measuring method of the present invention.

<Preparation of bacterial powder suspension>
In the viable cell count measurement method of the present invention, the melted oil / fat composition is mixed with liquid oil / fat to prepare a bacterial powder suspension. The fat composition can be melted by heating to a temperature equal to or higher than the melting point of the fat used in the fat composition.
Any liquid oil may be used as long as it is liquid under the growth temperature conditions of the bacteria to be measured for viable cell counts.
The upper limit of the growth temperature of the bacteria to be measured for the number of viable bacteria is, for example, GAM liquid medium (produced by Nissui) supplemented with glucose (produced by Nacalai Tesque) at a rate of 2%, reduced skim milk powder (Morinaga Milk Industry) Made by dissolving 8% to 12% of the non-fat dry milk medium, or the activated non-fat dry milk medium or the like obtained by adding nutrient components such as yeast extract and peptone to the non-fat dry milk medium as appropriate. Culture is performed at a temperature, and the upper limit of the growth temperature of each bacterium can be determined from a change in pH or a change in the number of bacteria.
The growth temperature conditions of the bacteria vary depending on the bacterial species and strains, but the upper limit of the growth temperature of bifidobacteria and lactic acid bacteria is usually about 40 to 55 ° C. Below, the upper limit of the growth temperature of various bifidobacteria is illustrated.
・ Bifidobacterium longum subsp. Longum ATCC BAA-999: 45 ℃
・ Bifidobacterium breve LMG 23729: 48 ℃
・ Bifidobacterium breve MCC1274 (FERM BP-11175): 42 ℃
・ Lactococcus lactis subsp. Lactis MCC866 (FERM BP-10746): 45 ° C
・ Lactobacillus acidophilus NITE SD 00054: 50 ° C

Among these, liquid oils and fats are preferably those that are liquid under the culture temperature conditions (near the optimum growth temperature) of the bacteria to be measured for the number of viable bacteria.
The culture temperature condition of the bacteria varies depending on the bacterial species and strains, but in the case of bifidobacteria and lactic acid bacteria, it is usually about 25 to 42 ° C.

Also from this point of view, liquid oils and fats that are liquid at room temperature (for example, about 20 to 30 ° C.) (melting point below room temperature) are particularly easy to use. Examples are given below.
・ Product name Soybean oil (manufactured by Nacalai Tesque) Liquid at room temperature (melting point 22-31 ° C)
・ Product name Edible corn oil (manufactured by J-Oil Mills) Liquid at room temperature ・ Product name Salad oil (manufactured by J-Oil Mills) Liquid at room temperature ・ Product name Olive oil (manufactured by J-Oil Mills) Liquid at room temperature Product name Edible sunflower oil (manufactured by J-Oil Mills) Liquid at room temperature / Product name "CY-2" (manufactured by Taiyo Yushi Co., Ltd.) Liquid at room temperature (freezing point -12 to 0 ° C)
・ Product name "MCD-25" (manufactured by Taiyo Yushi Co., Ltd.) Liquid at room temperature (freezing point -5 ° C)
(The freezing point can be determined by the freezing point measurement method (Schkoff method) published in the standard oil and fat analysis test method (Japan Oil Chemists' Society).)

The mixing ratio of the oil-and-fat composition containing the bacterial powder and the liquid oil and fat is not particularly limited as long as both are sufficiently mixed and a bacterial powder turbid liquid can be prepared. As a range of such a mixing ratio, the following ratio can be used as a guide.
Oil and fat composition containing fungus powder: Liquid oil and fat = 2: 1 to 1: 1000

<Preparation of diluent for measurement>
Subsequently, the obtained bacterial powder suspension is diluted with an aqueous solvent to prepare a measurement diluent. As the aqueous solvent, a physiological saline solution, a buffer solution, or the like that has been conventionally used as a diluent when measuring the number of viable bacteria can be used without particular limitation.
In this step, the temperature of the aqueous solvent is preferably maintained within the range of the growth temperature of the bacterium to be measured for the number of viable bacteria. As a result, when the above-mentioned bacterial powder suspension is mixed with an aqueous solvent and the bacterial powder is extracted into the aqueous solvent, the bacteria can be immediately brought to the growth temperature condition and the decrease in the number of viable bacteria can be suppressed. Can do. Further, from the above viewpoint, it is more preferable that the temperature of the aqueous solvent is maintained at the culture temperature (near the optimum growth temperature) of the bacterium to be measured for the viable cell count.

  When the bacterial powder suspension is mixed with an aqueous solvent, the temperature of the bacterial powder suspension is preferably within the range of the growth temperature of the bacteria whose number of viable cells is to be measured, and further the culture temperature of the bacteria (optimal growth). It is preferable that the temperature is maintained. As a result, when the bacterial powder suspension is mixed with an aqueous solvent, the temperature change of the aqueous solvent is suppressed, and it becomes easy to immediately put the bacteria at the growth temperature, and more reliably suppress the decrease in the number of viable bacteria. Can do.

The aqueous solvent preferably contains a surfactant. By using a surfactant, mixing of the bacterial powder suspension with an aqueous solvent can be facilitated, and dispersion of the bacteria into the aqueous solvent can be made more reliable.
A preferred example of the surfactant is polysorbate. Polysorbate is a product obtained by condensing ethylene oxide with sorbitan fatty acid ester (polyoxyethylene sorbitan fatty acid ester).
The number of ethylene oxides constituting the polysorbate is usually about 20 molecules.
As the fatty acid constituting the polysorbate, a saturated or unsaturated fatty acid having 12 to 18 carbon atoms is generally used. Specific examples of the fatty acid include lauric acid, stearic acid, palmitic acid, oleic acid and the like.
As the polysorbates, commercially available products can be used. Examples of such commercially available products include Tween (registered trademark) 20, Tween 40, Tween 60, Tween 65, Tween 80, and the like.
Tween 20 is also referred to as polysorbate 20 and polyoxyethylene sorbitan monolaurate.
Tween 40 is also referred to as polysorbate 40, polyoxyethylene sorbitan monopalmitate.
Tween 60 is also referred to as polysorbate 60 and polyoxyethylene sorbitan monostearate.
Tween 65 is also referred to as polysorbate 65 and polyoxyethylene sorbitan tristearate.
Tween 80 is also referred to as polysorbate 80 and polyoxyethylene sorbitan monooleate.

  In the aqueous solvent, the concentration of the surfactant is preferably 0.05% or more, and more preferably 0.5% or more. By setting it as such a range, a microbe suspension can be disperse | distributed efficiently. The upper limit of the surfactant concentration can be 30% as a guide. Considering economic efficiency, it is preferable to make it 2% or less.

  The aqueous solvent may appropriately contain components that can be added to an aqueous solvent that is generally used for measuring the number of viable bacteria.

<Culture>
The measurement diluent prepared as described above is added to the medium, and the bacteria are cultured. Culture can be performed according to a conventional method. Examples of the method of adding to the medium include a method of pouring with the medium (pour method), a method of applying to the medium (flat plate coating method), and the like.
As a medium to be used, a known medium can be appropriately selected according to the type of bacteria to be measured. Culture conditions can also be set as appropriate according to the type of bacteria to be measured. In the case of bifidobacteria, it may be cultured under anaerobic conditions usually at 25 to 42 ° C. for several days. In the case of lactic acid bacteria, the culture is usually performed at 25 to 42 ° C. for several days under either aerobic conditions or anaerobic conditions.

<Measurement of the number of bacteria>
By measuring the number of bacteria (colony number) on the medium after culturing the bacteria, the number of viable bacteria contained in the measurement diluent can be measured, and based on this, the live bacteria in the oil / fat composition A number can be calculated. These methods are the same as the conventional method for measuring the number of viable bacteria.

<Method for preparing bacterial powder suspension>
The method for preparing a bacterial powder suspension of the present invention is used for the above-mentioned measurement of the number of viable bacteria, and the melted fat composition is mixed with a liquid fat that is liquid under the growth temperature conditions of the fungus. A bacterial powder suspension is prepared. The description of the method for measuring the viable cell count of the present invention is applied as it is to the preferred mode of preparing the bacterial powder suspension.

  Hereinafter, the present invention will be described in more detail using examples, but the present invention is not limited to these examples.

[Example 1]
The purpose of Example 1 is to examine the measurement accuracy (recovery rate) of the viable cell count of bacteria contained in the oil and fat composition measured by the viable cell count measuring method of the present invention.

(Test method)
After heating 9 g of fats and oils having a melting point of 46.4 ° C. (Taiyo Yushi Co., Ltd., oil sample No. S0182) to a temperature of 47 ° C. and melting to form a liquid, 100 billion (10 ¹¹ ) pieces per gram Bifidobacterium breve MCC1274 (FERM BP-11175) containing Bifidobacteria powder B-3 (manufactured by Morinaga Milk Industry Co., Ltd.) was added and mixed uniformly to obtain a Bifidobacterium-containing oil and fat composition. Obtained.
After 1 g of the melt of the above fat composition was mixed with 9 g of a liquid soybean oil (manufactured by Nacalai Tesque) having a melting point of 22 to 31 ° C. to prepare a bacterial powder suspension, Dilution buffer kept at 0 ° C. (aqueous solvent: 4.5 g potassium dihydrogen phosphate, 6.0 g disodium hydrogen phosphate, 0.5 g L-cysteine hydrochloride monohydrate, 0.5 g Tween®) 80, 1,000 mL of distilled water) was added to 9 g and sufficiently suspended.
This suspension was further diluted 10-fold with the same buffer according to a conventional method to obtain a 10 8th diluted solution.
This diluted solution was mixed with a reinforced Clostridia agar medium (Reinforced Clostridial Agar Medium: manufactured by OXOID), seeded in a petri dish, solidified, then subjected to anaerobic culture at 37 ° C. for 3 days, and the number of colonies was counted. The recovery rate from the number of bacteria at the time of addition was calculated.
In addition, after suspending 1 g of bifidobacteria-containing high-melting-point oil composition as a target in 9 g of the dilution buffer kept at 25 ° C., 37 ° C., 42 ° C., 45 ° C., and 60 ° C. Then, the number of bacteria was measured, and the recovery rate (ratio of the number of measured bacteria after culture to the number of inoculated bacteria in the medium) was determined.

(Test results)
The obtained bacterial count results are shown in Table 1 below.
As shown in Table 1, when the bifidobacteria powder high melting point fat composition is added directly to a dilution buffer (aqueous solvent) at 25 ° C. and 37 ° C., the fat solidifies and is uniformly suspended in the dilution buffer. Therefore, the number of bacteria could not be measured.
Moreover, when diluting with a dilution buffer kept at 60 ° C. so as not to solidify the fats and oils, most of the bifidobacteria were killed due to its high temperature, and the recovery rate was 10% or less.
Furthermore, the recovery rate was 50% when the incubation temperature of the dilution buffer was set to 45 ° C., and 62% when the temperature was set to 42 ° C. With these recovery rates, although the recovery of bacteria was enhanced, it was not a result of ensuring sufficient accuracy in the measurement of the number of bacteria.
Thus, it is considered that a sufficient recovery rate could not be obtained because the temperature sensitivity of the bifidobacteria was high, or the bifidobacteria were not sufficiently dispersed in the dilution buffer due to partial solidification of the fats and oils.

On the other hand, when the Bifidobacterium powder high-melting-point oil-fat composition is suspended in soybean oil having a melting point of 22-31 ° C. and then diluted with a dilution buffer kept at 37 ° C., it is uniform even at a low temperature of 37 ° C. As a result of suspending the fats and oils to disperse the bifidobacteria and then culturing, it was observed that the number of bifidobacteria could be measured with a high recovery rate of 90% or more.
That is, when measuring the viable count of bifidobacteria contained in a solid fat composition at room temperature containing a high melting point fat, by suspending the melted fat composition in a low melting point fat such as soybean fat It was found that the viable count of bifidobacteria contained in the solid fat composition can be accurately measured.

  The method for measuring the number of viable bacteria according to the present invention can be used for quality control and quality inspection of health foods and supplements prepared from fats and oils of useful bacteria such as bifidobacteria and lactic acid bacteria.

Claims (9)

A method for measuring the number of viable bacteria contained in an oil and fat composition,
A step of preparing a suspension of powdered powder by mixing the melted fat and oil composition containing the powdered powder with a liquid fat that is liquid under the growth temperature conditions of the fungus,
Diluting the bacterial powder suspension with an aqueous solvent to prepare a diluent for measurement;
Adding the measurement diluent to the medium and culturing the bacteria;
A method for measuring the number of viable bacteria, comprising a step of measuring the number of bacteria on the medium after culturing.   The method for measuring the viable cell count according to claim 1, wherein the temperature of the aqueous solvent is within the range of the growth temperature of the fungus.   The method for measuring the viable cell count according to claim 1 or 2, wherein the fat used in the fat composition containing the fungus is solid under the growth temperature conditions of the fungus.   The mixing ratio of the oil-and-fat composition containing the fungus powder and the liquid oil is according to any one of claims 1 to 3, wherein the oil-and-fat composition containing fungus powder: liquid oil and fat = 2: 1 to 1: 1000. Method for measuring the number of viable bacteria.   The method for measuring the viable cell count according to any one of claims 1 to 4, wherein the aqueous solvent contains a surfactant.   The viable cell count measurement method according to claim 5, wherein the surfactant is polysorbate.   The method for measuring the viable cell count according to any one of claims 1 to 6, wherein the oil and fat used in the oil and fat composition containing the fungus powder has a melting point of 40 ° C or higher.   The method for measuring the number of viable bacteria according to any one of claims 1 to 7, wherein the fungus powder is a powder powder of bifidobacteria or lactic acid bacteria. A method of preparing a bacterial powder suspension for measuring the number of viable bacteria contained in an oil and fat composition,
A method for preparing a bacterial powder suspension, which comprises mixing a melted fat and oil composition containing a bacterial powder with a liquid fat that is liquid under the growth temperature conditions of the bacteria to prepare a bacterial powder suspension.

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