JP3946313B2 - Selective medium for Bifidobacterium - Google Patents

Description

【００４１】
（注） Ｂ：ビフィドバクテリウム Ｌ：ラクトバチルス
Ｅ：エンテロコッカス Ｓ：ストレプトコッカス
（表５においても同じ）
【００４２】
実施例１
比較例１で用いたＴＯＳ培地およびＢＬ培地、ならびにＴＯＳ培地にプロピオン酸ナトリウム（１.５％）を含有させた本発明の選択培地により、ビフィドバクテリウム菌５種２６株、および乳酸菌５株（比較例１においてＴＯＳ培地上にコロニーを形成したもの）の培養を行なった。培養条件は、培養を混釈培養により行なったほかは比較例１の場合と同様にした。
【００４３】
その結果は表５のとおりで、プロピオン酸ナトリウムを添加したＴＯＳ培地では乳酸菌の増殖がほぼ完全に抑制され、しかもビフィドバクテリウム菌はＢＬ培地とほとんど同等の水準で増殖することが確認された。
【００４４】
【表５】
各種培地におけるビフィドバクテリウム菌および乳酸菌の増殖

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a selective medium for measuring the number of Bifidobacterium in foods and drinks, pharmaceuticals and the like containing Bifidobacterium and lactic acid bacteria.
[0002]
In this specification, in addition to a medium that is ready to be used for bacterial culture, it is called a culture medium in the sense that it contains a mixture or set of medium constituents other than water for sterilization after dissolution in water. .
[0003]
[Prior art]
Bifidobacterium is a useful enteric bacterium that is closely related to health for both infants and adults. For this reason, many foods and beverages and Bifidobacterium preparations containing Bifidobacterium have been commercialized so that Bifidobacterium can be easily ingested on a daily basis.
[0004]
Some of these Bifidobacterium-containing foods and drinks contain only Bifidobacterium, but those that take the form of fermented milk also contain lactic acid bacteria in relation to the Ordinance of the Ministry of Health and Welfare regarding dairy products. There are many things.
[0005]
In the production and sale of foods and drinks containing both Bifidobacterium and lactic acid bacteria, the number of Bifidobacterium is distinguished from lactic acid bacteria and the exact number of viable bacteria is measured for quality control. It will be necessary.
[0006]
To measure the viable count of Bifidobacterium, inoculate the specimen dilution into a normal medium and culture, and count the Bifidobacterium colonies selected based on the color and morphology of the resulting colonies. Although there is a method, this method not only requires a high degree of skill for colony identification but also has a problem that the accuracy is low when the number of Bifidobacterium is small.
[0007]
On the other hand, inoculate the sample in a medium (so-called selective medium) that allows Bifidobacterium to preferentially grow without substantially growing lactic acid bacteria, and cultivate the number of Bifidobacterium from the total number of colonies. There is a way to know. As an example of the selective medium, a medium in which a growth inhibitor of lactic acid bacteria is added to a medium in which both Bifidobacterium and lactic acid bacteria can form a colony, for example, an MGLP medium added with lithium chloride and penicillin ( Food hygiene magazine, Vol. 23, pp. 39-44), RB medium supplemented with sodium propionate and lithium chloride (Journal of Microbiological Methods, vol.27, 33 ■ 43) is known. In the seed selective medium, the growth of not only lactic acid bacteria but also some Bifidobacterium bacteria tends to be suppressed, and accurate measurement values could not be obtained.
[0008]
In the selective medium for Bifidobacterium described in Japanese Patent Publication No. 4-29359, the general formula Gal- (Gal) n-Glc (provided that Gal Is a galactose residue, Glc is a glucose residue, n represents an integer of 1 to 4), and is an enzyme protein hydrolyzate having a molecular weight of 5,000-10,000 and sugar-free casein hydrolyzate using amino acid as a main component achieves the desired selectivity. However, even in this selective medium, it was confirmed as a result of the experiment that expanded the lactic acid bacteria strains to be tested that some of the many lactic acid bacteria strains could grow. In addition, this selective medium has very strict requirements on the nitrogen source to be used, and accurate measurement values cannot be obtained if a readily available nitrogen source such as yeast extract is employed.
[0009]
[Problems to be solved by the invention]
Therefore, the object of the present invention is to selectively and vigorously grow only Bifidobacterium from a sample in which lactic acid bacteria and Bifidobacterium are mixed, thereby enabling accurate measurement of the number of Bifidobacterium. An object of the present invention is to provide a selective medium for Bifidobacterium having a high degree of selectivity.
[0010]
Another object of the present invention is a selective medium for Bifidobacterium which is not only excellent in enabling a high degree of selective growth but also has a stable selectivity and a highly reliable measurement result. Is to provide.
[0011]
[Means for Solving the Problems]
The selective medium for Bifidobacterium according to the present invention has the general formula Gal- (Gal) n-Glc (where Gal is a galactose residue, Glc is a glucose residue, and n is an integer of 1 to 4, respectively) (Hereinafter referred to as galactooligosaccharide) as an essential carbon source, propionic acid or a salt thereof (preferably a sodium salt) as a growth inhibitor for lactic acid bacteria, and carbon other than the above oligosaccharide In the case of containing a source, the carbon source, nitrogen source and other auxiliary components are selected from those which do not substantially promote the growth of lactic acid bacteria in anaerobic culture using this medium.
[0012]
In addition, in this specification, in addition to components that do not promote the growth of lactic acid bacteria regardless of the amount of addition, those that show the effect of growing lactic acid bacteria only when the addition amount is large are those in which the effect of promoting the growth of lactic acid bacteria does not substantially appear In the meaning including the said component in the case of using by, it is called "thing which does not promote growth of lactic acid bacteria."
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Propionic acid or a salt thereof (hereinafter referred to as propionic acids) used as a growth inhibitory component of lactic acid bacteria in the selective medium of the present invention is used in combination with lithium chloride to suppress the growth of lactic acid bacteria in the selective medium as described above. However, it has been confirmed that by itself, the growth inhibition of lactic acid bacteria is incomplete and may promote the growth. However, when propionic acids are added to a medium using galactooligosaccharide as a carbon source for Bifidobacterium (hereinafter referred to as TOS medium), the growth of lactic acid bacteria is almost completely suppressed by the cooperative action of both. It has been found that sufficiently satisfactory selectivity is achieved.
[0014]
The present invention has succeeded in remarkably improving the selectivity of TOS medium based on the above novel findings.
Hereinafter, the composition of the selective medium of the present invention will be described in detail.
[0015]
Galactooligosaccharide used as an essential carbon source for Bifidobacterium is a transfer oligosaccharide obtained by treating lactose with β-galactosidase produced by Aspergillus oryzae, and its composition and production method are special. This is described in detail in Japanese Patent Publication No. 58-20266. Since impurities containing glucose or galactose at a high rate as impurities derived from the production method allow growth of lactic acid bacteria, it is desirable to use those purified to a purity of 99.5% or more.
[0016]
The selective medium of the present invention can contain other sugars such as xylose, arabinose, and raffinose that can be assimilated by Bifidobacterium (but not assimilated by lactic acid bacteria) together with galactooligosaccharide, if necessary. . For example, there are Bifidobacteria that are very rare but difficult to grow in a medium containing only galactooligosaccharide as a carbon source. If the presence of such Bifidobacteria cannot be ignored, all such as xylose The measurement accuracy of the number of Bifidobacterium can be improved by using a monosaccharide that is easily assimilated by Bifidobacterium.
[0017]
The required concentration of propionic acids to be added as a lactic acid bacteria growth inhibitor varies depending on the basic composition of the TOS medium, but is generally in the range of 4.5 to 20 g / l. The inhibitory effect of propionic acids on the growth of Bifidobacterium on the TOS medium appears at a concentration of about 30 g / l or more. Therefore, the selective medium of the present invention reliably suppresses the growth of lactic acid bacteria. Therefore, it is possible to contain propionic acids up to about twice the above required amount.
[0018]
As propionic acids to be contained in the medium, alkali metal salts are easy to use, and sodium salts are particularly preferred because they are readily available.
[0019]
The use of galactooligosaccharides and propionic acids to suppress the growth of lactic acid bacteria is a necessary condition in the present invention, but is not a sufficient condition. Even when the above two components are used, if any of carbon sources other than galactooligosaccharides, nitrogen sources, and other auxiliary components promotes the growth of lactic acid bacteria, some lactic acid bacteria are prevented from growing to a significant level. I can't. For example, sorbitan higher fatty acid ester-based nonionic surfactant Tween, which is commonly used in Bifidobacterium culture media, grows a significant number of lactic acid bacteria in the culture medium of the present invention using galactooligosaccharides and propionic acids in combination. enable. Therefore, in selecting medium components other than galactooligosaccharides and propionic acids, it is necessary to use suspicious substances after confirming that there is no action that enables the growth of lactic acid bacteria.
[0020]
Yeast extracts, various protein hydrolysates, amino acid mixtures, etc. often used as nitrogen sources for this type of medium are not stable in quality, and small amounts of them often promote the growth of lactic acid bacteria. When used in a large amount, it often promotes the growth of lactic acid bacteria. Therefore, when these nitrogen sources are used, it is desirable to use the highest quality one within the range that does not affect the growth of lactic acid bacteria.
[0021]
Ammonium sulfate is a nitrogen compound that is particularly effective as a nitrogen source for Bifidobacterium bifidum.
[0022]
In addition, sulfur-containing amino acids such as cysteine hydrochloride and magnesium salts such as magnesium sulfate are extremely effective as auxiliary components for the selective medium of the present invention.
[0023]
Since the pH of the medium is desirably maintained at 6.7 to 7.0 until the end of the culture, it is desirable that the medium is sufficiently added with an effective buffer such as potassium phosphate.
[0024]
The standard composition of the selective medium according to the present invention (% by weight per medium containing water) is as follows.
[0025]
Galactooligosaccharide 0.5-3.0
Xylose 0-3.0
Propionic acids 0.5-3.0
Tripty case peptone (BBL, USA) 1.0-2.0
Yeast extract 0.05-0.1
Ammonium sulfate 0.005-0.5
Cysteine hydrochloride monohydrate 0.03-0.1
Magnesium sulfate heptahydrate 0.01-0.04
Monopotassium phosphate / dipotassium phosphate 0.1 / 0.16 to 0.4 / 0.64
Agar 1.0-1.5
[0026]
There is no need for a special method for preparing the selective medium according to the present invention and using it for the measurement of the number of Bifidobacterium, and an anaerobic culture such as a gas pack method or a steel wool method may be performed by a conventional method.
[0027]
Hereinafter, the present invention will be described with reference to experimental examples. The galactooligosaccharide used in each experiment was manufactured according to Example 1 described in the above Japanese Patent Publication No. 58-20266, and 99.7% It consists of 3-6 saccharides, the rest being lactose, galactose and glucose.
[0028]
Comparative Example 1
Using RB medium, TOS medium and BL medium having the following composition, Bifidobacterium 5 species 25 strains and lactic acid bacteria 12 species 19 strains were cultured under the following conditions.
[0029]
Culture conditions: Incubate anaerobically at 37 ° C. for 72 hours. The strain is smeared on the surface.
[0030]
RB medium: A known selective medium for Bifidobacterium containing raffinose as the only sugar and additionally containing sodium propionate and lithium chloride as lactic acid bacteria growth inhibitors.
[0031]
TOS medium: A selective medium for Bifidobacterium containing galactooligosaccharide as the only sugar.
[0032]
BL medium: A standard medium in which both Bifidobacterium and lactic acid bacteria can grow.
[0033]
Details of the medium composition are shown in Tables 1 to 3.
[0034]
[Table 1]
RB medium composition (all figures are in 1000 ml medium)
Casein sodium 5.0g
Yeast extract 5.0g
Sodium thioglycolate 0.5g
1% bromcresol purple 15.0ml
Hydrochloric acid 0.5g
Raffinose 7.5g
L-cysteine hydrochloride 0.5g
Lithium chloride 3.0g
Sodium propionate 15.0g
Agar 18.0g
40.0 ml of salt solution
pH 6.7 ± 0.1
(Note) Salt solution composition: MgSO ₄ 0.2 g / l, CaCl ₂ 0.2 g / l, K ₂ PO ₄ 1.0 g / l,
KH ₂ PO ₄ 1.0 g / l, NaHCO ₃ 10.0 g / l, NaCl 2.0 g / l
[0035]
[Table 2]
TOS medium (unit:% by weight)
Tripty case peptone (BBL, USA) 5.0
Yeast extract 0.1
Monopotassium phosphate 0.3
Dipotassium phosphate 0.48
Ammonium sulfate 0.3
Cysteine hydrochloride monohydrate 0.05
Magnesium sulfate heptahydrate 0.02
Galactooligosaccharide 1.0
Agar 1.5
[0036]
[Table 3]
BL Medium Beef Extract 3g
Proteose peptone 10g
Tripty case peptone (BBL, USA) 5g
Phyton (BBL, USA) 3g
Yeast extract 5g
Liver extract 150ml
Glucose 10g
Soluble starch 0.5g
10 ml of solution A
Solution B 5ml
Tween80 1g
Agar 15g
10 ml of L-cysteine hydrochloride monohydrate (5% solution)
Horse blood 50ml
765 ml of purified water
[0037]
(Note) Solution A: 25 g of KH ₂ PO _{4 and} 25 g of K ₂ HPO ₄ dissolved in 250 ml of purified water.
Solution B: MgSO ₄ · 7H ₂ O 10 g, FeSO ₄ · 7H ₂ O 0.5 g, NaCl 0.5 g, MnSO ₄
A solution obtained by dissolving 0.337 g in 250 ml of purified water.
[0038]
After completion of the culture, the number of colonies on the medium was counted. For each strain, Table 4 shows the number of bacteria on various media when the number of bacteria on the BL medium is 100.
[0039]
In the RB medium, colonies of Lactobacillus salivarius were formed, and the growth of some Bifidobacterium such as Bifidobacterium bifidum was suppressed. In TOS medium, Bifidobacterium colonies were well formed, but some lactic acid bacteria such as Lactobacillus casei also formed colonies.
[0040]
[Table 4]
Growth of Bifidobacterium and lactic acid bacteria in various media

[0041]
(Note) B: Bifidobacterium L: Lactobacillus E: Enterococcus S: Streptococcus (same in Table 5)
[0042]
Example 1
By using the TOS medium and BL medium used in Comparative Example 1 and the selective medium of the present invention in which sodium propionate (1.5%) was contained in the TOS medium, Bifidobacterium 5 species 26 strains and lactic acid bacteria 5 strains (Comparative example 1 in which colonies were formed on TOS medium) was cultured. The culture conditions were the same as in Comparative Example 1 except that the culture was performed by pour culture.
[0043]
The results are shown in Table 5. It was confirmed that the growth of lactic acid bacteria was almost completely suppressed in the TOS medium supplemented with sodium propionate, and that Bifidobacterium grew at almost the same level as the BL medium. .
[0044]
[Table 5]
Growth of Bifidobacterium and lactic acid bacteria in various media

Claims (2)

General formula Gal- (Gal) n-Glc
(In the formula, Gal represents a galactose residue, Glc represents a glucose residue, and n represents an integer of 1 to 4)
Containing 1.0 % by weight as an essential carbon source,
Contains 1.5 % by weight of sodium propionate as a growth inhibitor of lactic acid bacteria,
A selective medium for measuring the number of Bifidobacterium in a substance containing Bifidobacterium and lactic acid bacteria, characterized by containing ammonium sulfate, cysteine hydrochloride and magnesium sulfate as auxiliary components. 2. The selective medium according to claim 1, wherein xylose is contained as the carbon source when a carbon source other than oligosaccharide is contained.