JPS60114186A - Selective medium for bifidobacterium mold - Google Patents

Abstract

PURPOSE:To measure accurately the number of live molds of Bifidobacterium in a drink, etc. containing lactic acid bacteria, by using a medium which does not substantially multiply Bifidobacterium molds and lactic acid bacteria as a basic medium, adding a specific nutritive component which assimilates Bifidobacterium molds but not lactic acid bacteria to the basic medium. CONSTITUTION:An oligosaccharide shown by the formula Gal-(Gal)n-Glc (Gal is galactose residue; Glc is glycose residue, n is 1-4) and further xylose are added to a basic agar medium containing a nutrition demanding component except saccharide and a nonnutritive auxiliary component of Bifidobacterium mold, a protein hydrolyzate with an enzyme (hydrolyzate of casein or gelatin) having 5,000-10,000mol.wt. and Trypticase pepton (trade name) as a nitrogen source, and no growth regulating component of lactic acid bacteria, but not multiplying substantially lactic acid bacteria in anaerobic cultivation.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a selective medium for measuring the number of Bifidobacterium bacteria in foods, drinks, medicines, and other items containing Bifidobacterium bacteria and 7L acid bacteria. be.

Bifidobacterium has been noted as one of the useful intestinal bacteria that is related to the health of both infants and adults, and for this reason, in recent years fermented milk containing live Bifidobacterium has been developed. Many products and live bacteria preparations have been commercialized.

Are there any foods or drinks containing Bifidobacterium that contain only Bifidobacterium?Waka'1υ does not contain only Bifidobacterium due to the Ministry of Health and Welfare Ordinance regarding dairy products. Many also contain lactobacillus or lactic acid coccus (in the Meisen 1st era, it is called cicada sterilization to include the two swords of these bacteria). When manufacturing and selling foods and drinks containing Bifidobacterium and lactic acid bacteria, it is necessary to measure the viable counts of Bifidobacterium and lactic acid bacteria for quality control purposes. Selective medium 1 used in such cases, ie, a medium capable of selectively growing only specific bacteria, for Bifidobacterium, has not been very good in the past. In other words, conventional selective media for Bifidobacterium have the disadvantage of giving lower measured values than non-selective media.
When foods and beverages containing Bifidobacterium 1v4 have been stored for a long time after being manufactured, they may appear in the public eye. In addition, both had the disadvantage that the medium composition was complex and high in 1+IIi.

3) on a conventional selective medium for Bifidobacterium.
The disadvantage of measurement values as described in item 1 is that neither conventional selective media nor selective agents for suppressing the production of lactic acid IY4,
This is probably because it contains antibiotics, propionic acid, nalidixic acid, lithium chloride, etc., and is completely harmless to Bifidobacterium bacteria. Therefore, in order to improve the selective culture performance of Bifidobacterium 13 bacteria, it is necessary to develop a history of selective agents since no harmful selective agents have been found for Bifidobacterium bacteria. It seems necessary to completely abolish the use of this method and to determine the selective culturing ability by other means.

As a result of various studies from this viewpoint, the present inventors found that Bifidobacterium 11 bacteria contain almost everything necessary as a medium component or lack certain essential nutritional components, so that Bifidobacterium and lactic acid bacteria Bifidobacteria can be grown by using a medium that does not significantly grow, and by adding the above-mentioned specific Eigo ingredients that are assimilated by Bifidobacterium but not by Lactic Acid Bacteria. We came up with the idea of creating a selective medium 11) that does not inhibit the growth of P. aeruginosa, and as a result of further research, we have completed the present invention.

The present invention consists of two inventions, the first of which is a selective medium,
Contains nutritional components other than sugar groups of Bifidobacterium bacteria and non-nutritional supplement components, and the nitrogen source is a protein hydrolyzate with chlorine with a molecular weight of 5 + (10C
1 to 10, 0 (1 (l) and trypticase peptone (trade name), and does not contain lactic acid bacteria reproduction-inhibiting ingredients, but does not significantly grow 2L acid bacteria in anaerobic culture. On a certain basal agar medium, the general formula G a
l -(Gal)n-Glc (However, in the formula, Gal is a galactose residue, Glc is a glucose residue, and l is 1-
, 1, respectively) are added thereto.

Further, the selective medium according to the second aspect of the present invention is obtained by further adding xylose to the selective medium according to the first aspect of the present invention (However, in this specification, the selective medium is a medium that is ready to be used immediately for culturing bacteria. In addition, it is called a medium in the sense that it includes water V, a mixture of other medium constituents, or cent, which is used for culture after sterilization by lysis in water.
υ1 does not proliferate means that no colonies are formed at all in petri dish culture, or even if they are formed, the diameter is approximately 0.5 mm.
This means that the cells are allowed to proliferate only to the extent that only so-called pinpoint colonies, which are less than Il)m, are formed. ), thus, in the present invention, In@- is selected as a specific nutritional component to be added to the basal medium that does not allow the growth of lactic acid bacteria. The requirements for sugar added at the edge are strict;
When added to the basal medium, half of all Bifidobacterium bacteria are assimilated, and half of all 7L acid bacteria are assimilated by the above oligo 11. ! J is extremely excellent in terms of selective assimilation, and a mixture of oligosaccharides and xylose is superior to J. In addition, the basal medium is
The basic property is that lactic acid bacteria do not proliferate to a large extent, and Bifidobacterium also hardly proliferates, or when sugars of the above q and 'i are added, - This results in a medium in which Bifidobacterium can actively grow. Therefore, the selective medium according to the present invention not only enables the selective cultivation of Bifidobacterium, but also guarantees vigorous growth of Bifidobacterium comparable to non-selective medium. Then, the number of viable Bifidobacterium bacteria in foods and drinks containing Bifidobacterium and lactic acid bacteria can be measured more accurately than before.

Hereinafter, the constituent components of the selective medium according to the present invention will be explained in detail.

The selective medium according to the invention requires no frost! As a source, it is a protein hydrolyzate produced by an enzyme with a molecular weight of 5.0.
00~], 0,000 (hereinafter referred to as protein hydrolyzate) and trypticase peptone.

These sources (nitrogen) are important because they allow selective growth of Bifidobacterium.

For example, acid 1:j extract, meat extract, liver extract, etc. are substances often used as nitrogen sources in media for Bifidobacterium painting;
When only the bipedal oligosaccharide is added, the increase in η11 of 7L acid bacteria increases by 6'1. Therefore, as a nitrogen source, we use pond nitrogen compounds as a nitrogen source (2), especially as a nitrogen source.
When using a nitrogen compound, it must be thoroughly confirmed that it does not have an adverse effect on the selective growth of Bifidobacterium before use. f
Examples of nitrogen compounds that can be used for jl include ammonium salts and sulfur-containing amino acids such as cysteine, cystine, and menaoine. Is the protein hydrolyzate also Ij?”
Even conditions limited to binary oligosoil11 are useful for the vigorous growth of Hyphidobacterium, and when an amino acid mixture such as an acid hydrochloride of protein is used instead of this nitrogen source, This may result in poor growth of Fidobacterium bacteria (some Bifidobacterium bacteria do not substantially grow).

Protein hydrolysates can be made from proteins such as 111. Care must be taken when using foods that use potatoes (especially those that contain carbohydrates as impurities) as they may allow the growth of lactic acid bacteria. Casein and gelatin are highly pure or easily obtained, and can be used as raw materials for hydrolysis.
, y is preferable. Any enzyme may be used to prepare the protein hydrolyzate as long as it gives a high yield of the hydrolyzate in the above molecular weight range, that is, peptite.
The product name is Hann, a Kyoei Bussan product). Note that even if the hydrolyzate contains peptides or amino acids outside the above molecular weight range, it is not necessary to remove them and the hydrolyzate may be used as is.

Trypticase peptone is a casein hydrolyzed IIT commercially available from BBL, USA. The main components are amino acids and contain no sugar (the main reason why trypticase peptone is so good as a nitrogen source for selective media is that it contains no sugar at all. Amino acid mixtures other than case peptone that do not contain sugar, even if BBL's acidicase peptone or gelysate peptone are used instead of trypticase peptone, there is no problem in terms of selectivity. When trypticase peptone is used, the most vigorous growth of Bifidobacterium 19 is observed.) In addition, trypticase is considered to be a source of vitamins as well as a nitrogen source.

Limited 41 in the selection Jjt area according to the first aspect of the present invention
．． ``Bipedal Orinia'' $1.-, which is a source of Ji, is an oligosaccharide obtained by treating lactose with β-lactosigase produced by Aspergillus oryzae, and its composition and production are The method is described in detail in 1, Te Kosho 58-2 (No. 1000, No. 11i). In addition to this oligosaccharide, Bifidobacterium or 1
,? A large number of oligosaccharides, including 2, 3 and 3 oligosaccharides, are known, but 111
``All of these options are insufficient in terms of tonnage utilization (see @Experiment Example 2). In the case of Ji:JJ of the first invention in which the most sugar is only this oligosaccharide, the Biphytoha'acterium l investigated by the present inventors
'l1111 bacteria (Biphytohycterium longumno\TCC157 (1'7 strain only formed pinpoint colonies, but a small number of such 1) strains do not assimilate. is not a problem at all unless the strain contained in the thrift assay (E), and the above strains also assimilate xylose well, so the medium of the second invention containing oligosaccharides and xylose is completely Bifidobacterium that does not form colonies is considered to be non-existent in nature.

The preferred blending amount (percent coverage per water-containing medium) of the "-2 essential component in the selective medium according to the present invention is as follows.

Protein hydrolyzed fish: t), 5~2.0 Trypticase peptone: (1,,')~2.02.0 Oligosaccharide: 5~
3. () Xylose 20.5 to 3.0 In addition to agar, which is included as an essential component, the components of the selective medium according to the present invention do not enable the growth of lactic acid bacteria in the presence of the above-mentioned essential components. As long as you can't mix the ingredients of pressure relief. Examples include various types of j
Examples include metal salts, reducing agents (for example, cysteine), surfactants (for example, Tween 80), and the like.

The appropriate 11H of the medium is 6.7 to 7.0.

The method for preparing the selective medium according to the present invention and using it to increase the number of Bifidobacterium bacteria to 1llllW does not require any special equipment, and anaerobic culture such as the gas back method or steel wool method can be carried out using conventional methods. ).

Hereinafter, the present invention will be explained with reference to experimental examples. The oleifsugar used in each experiment was produced according to the above-mentioned Example described in Japanese Patent Publication No. 58-20266. 99
．． 7% consists of 3 to 6 and 11 types, the rest is lactose,
〃Lactose and glucose. Also, the 1.1 base Jl that does not contain sugar used in the feeding test? The earth has the following composition (add water to make a total of 117 to 10()OI++
I; pi-1 is 6.7 to 7.0).

Basal medium A: casein hydrolyzate x l 1og tributicase peptone] (1g auxiliary component x 3) Basal medium B: gelatin hydrolyzate 10g trypticase peptone 10g Supplementary supplement method 3 Basic Jaji X (for control) : Yeast extract 1 (1 g Trypticase peptone 10 g Auxiliary ingredient'3 81 Proteomics 77-g in 10% casein soda solution)
Ze (Hankyu) (manufactured by Eibusan) is added with 10% casein, p
1-17, 0, lyophilized after being allowed to react at 50°C for 2 hours.

Components with a molecular weight of 5,000 to 10,000 account for about 65%.

×2 A 10% aqueous gelatin solution was treated in the same manner as the above casein soda solution and 1: blown. Components with a molecular weight of 5,000 to 10,000 account for about 70%.

” (NJ-1,) So, 3 g, Ql acid sodium 3 g
, 2HP0゜2 g, K112PO4] g, L-E
/X Tin hydrochloride 1g, Tween 801g, agar 1
5I! 715 compounds.

For the utility test of Experimental Examples 1 and 3, Bifidobacterium and lactic acid bacteria were inoculated with 0.5% yeast extract No. At c,
・18-7211: This is a cultured bald solution that has been coated with a cloth and subjected to 4R dilution using a conventional method. In the culture test, anaerobic culture was performed for Bifidobacterium, and aerobic culture was used for 7L acid bacteria.

Experimental Example 1 When the 11 strains of Bifidobacterium used in Experimental Example 2 described later were cultured with a basal medium that does not contain sugar, 3
No colonies were formed on any of the species' basal f media.

In addition, the 19 strains of lactic acid bacteria used in Experimental Example 2 described later were
16 strains formed colonies and 1 strain formed a pinpoint colony, but only 5 strains formed pinpoint colonies in basal medium A and 3 strains in basal medium 13.

Experimental example 2 Olicosaccharide, lactulose (90% purity), raffinose (100% purity) or xylose (special grade reagent)
11 strains of Bifidobacterium and 19 strains of Lactic Acid Bacteria were added to basal medium A with 1% (wt% added to the basal medium, hereinafter referred to as the interval) or 1% xylose added to oligosaccharide-added ljl base. The results of culturing were as shown in Tables 1 and 2.

From the diagram, it can be seen that only when both oligosaccharides and xylose are used as sugars, there is complete selectivity for the growth of all Bifidobacterium bacteria and no growth of all lactic acid bacteria. In addition, a medium using only oligosaccharides,
Although the l\TCC15707 strain is difficult to proliferate, it exhibits sufficient selectivity for practical use.

When basal medium B was used instead of basal medium A, the same results as above were obtained.

Experimental Example 3 The number of Bifidobacterium bacteria in a 48-hour culture solution was measured using a medium prepared by adding 1% oligosaccharide to the basal medium I3 and a medium prepared by adding 1% xylose to this medium. In addition, the number of bacteria was measured for the same culture solution using a BL agar medium (non-selective medium) for measuring Bifidobacterium bacteria, and the measured values were compared. The results are shown in Table 3, and the medium containing only oligosaccharide and A'rCC]
It can be seen that, except for the combination with the 5707 strain, the selective medium according to the present invention gives quantitative values that are the same as those of the non-selective medium.

Also, when basal medium A is used instead of basal medium H, the above result is not the same (see the results of Hill 11).

Experimental Example 4 50 parts by weight of the sample for measuring the number of Bifidobacterium 'J11 in Experimental Example 3 was mixed with 50 parts by weight of the lactic acid bacteria culture obtained in the same manner, and syrup (sugar) was added to 70 parts by weight of the mixed liquid. Fermented milk was prepared by mixing 30 parts by weight (containing 15% and 15% glucose). The number of Bifidobacterium bacteria in this fermented milk was determined immediately after production and during 10 sips.
Measurements were taken after storage at C. The selected substrate of the present invention used for the measurement was basal medium B with 1% each of oligosaccharide and xylose added. Also, for use against Jjil, Island II
Selective medium of I et al. (Food T'lj Journal, J 8, 537-54
6; composition as shown below) was used.

The results are shown in Table 4. Is there any difference in the measured values for fermented milk immediately after production when using the selective medium of the present invention and when using the monthly medium? The selective medium of the invention gave clearly higher localization.When the selective medium of the present invention was used, bottle point colonies of lactic acid bacteria were observed in the first two cases in Table 4, but Bifidobacterium Bacterial colonies are much larger than that (diameter ratio = + (;'
(there are two), so the two could be easily separated.

Kuriman Inferior Ferment 1iJ Extract 5g, Tween 801 L-Cystine 0.3g, Lactose ()tζ, Tryptose 3g, Tributicase Peptone 10. J, phosphate-potassium
3g, dipotassium phosphate 3g, ammonium citrate 2g, magnesium sulfate 57.5 mε, ferrous sulfate 3
4018, manganese sulfate 12 (LnE, agar 15g,
Basic soil 10001.1 consisting of distilled water 1000+nl
1, selective agent solution (Sorg propionate 3 () 2g paromomycin sulfate 100+n2; neomycin sulfate
・↓00 l1llis Add water to 6 g of trilithium huride to make a total amount of 10 (to make 1 ml) 501111.

Experimental Example 5 Bifidobacterium and lactic acid i'lj were separately cultured (1.) (cultivated, collected, freeze-dried, and then combined with iR at a ratio of 1:1). Mixed seedlings were produced.

Regarding the above seedlings, the total number of strokes was measured using BL medium (manufactured by Yoneken Kagaku), and the number of Bifidobacterium bacteria was determined using the selective medium according to the present invention (basal medium A with 1% each of oligosaccharides and xylose added). The number of lactic acid bacteria was determined using 13cP-added plate agar, which is a selective medium for lactic acid bacteria.
Measurement was made using a base (manufactured by Yoneken Kagaku). The results are shown in Table 5.

In each measurement example, the sum of the number of Bifidobacterium bacteria and the number of Lactic Acid bacteria almost matched the total number of strokes, which was achieved by the selective medium of the present invention! It can be seen that it is necessary to accurately separate and quantify the Bifidobacterium bacteria in the final 8 bacteria.

Claims (6)

[Claims] (1) Contains nutritional auxiliary components other than sugars of Bifidobacterium bacteria and non-nutritional supplementary components, and the nitrogen source is an enzymatic protein hydrolyzate with a molecular weight of 5,000 to 1
0, 01) 0 and tributicase peptone (trade name), and does not contain a component that inhibits the live Tj of 7L acid bacteria, but does not substantially cause the growth of lactic acid bacteria in anaerobic culture. , the general formula Ga1-(G
al) n-Glc (in the formula, Gal is a lactose residue, Glc is a glucose residue, and 11 represents a number of 1° to 4, respectively). , a selective medium for measuring the number of Bifidobacterium in a substance containing Bifidobacterium and lactic acid bacteria. (2) The selective medium according to claim 1, wherein the protein hydrolyzate is a casein or gelatin hydrolyzate. (3) When the hydrolyzate of casein or gelatin is a hydrolyzate of P-a-teoliquidase (trade name) or an animal hydrolyzate thereof 3'l'Claim fir, item 2, item 4
・K selective medium. (4) Contains nutritional components and non-nutritional supplements other than sugars of Bifidobacterium, and the nitrogen source is an enzymatic protein hydrolyzate with a molecular weight of 5.0 (1
0 to 10,000 and trypticase peptone (trade name), and does not contain lactic acid bacteria bio-t'-7 inhibitory components or does not substantially cause lactic acid bacteria to grow in anaerobic culture. The general formula Ga1 is added to the accelerated agar medium.
-(Gal)n-Glc (in the formula, Gal is a galactose residue, Glc is a glucose residue, and +1 is an integer from 1 to 4, respectively) and xylose are added to the bifidobacterium. A selective medium for measuring the number of Bifidobacterium in a substance containing P. aeruginosa and lactic acid bacteria. (5) The selective medium according to item 4, wherein the protein hydrolyzate is a casein or gelatin hydrolyzate. (6) The selective medium according to claim 5, which is a hydrolyzate of casein or gelatin, proteoliquiphagase (trade name), or an animal hydrolyzate thereof.