JPH01257476A - Bifidobacterium-breve and food and drink - Google Patents

Abstract

PURPOSE:To obtain Bifidobacterium.breve (FERM P-9967) having a high survival ratio in an acidic medium in a high dissolved oxygen concentration, such as fermented milk. CONSTITUTION:Bifidobacterium.breve YIT4006 (FERM P-752) is inoculated into a skin milk powder culture medium containing an yeast extract and cultured at 37 deg.C until pH attains 4.7-4.8. The resultant milk culture is added to a skin milk powder adjusted to pH 4.5 and air is then blown thereinto to saturate oxygen (about 11ppm). The obtained milk is preserved at 10 deg.C. Colonies are subsequently separated from plates in which viable cell number attains <=10<3>/ml. Preservation in the above-mentioned acidic milk saturated with the oxygen is carried out for each colony to select colonies with delayed deterioration in viable cell number. The afore-mentioned separation and preservation are repeated to afford the aimed strain having >=1% survival ratio when preserved in an acidic milk in 4.2ppm initial dissolved oxygen concentration and pH 4.2 for 4 days.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to Bifidobacterium breve, which has good survival in food and beverages, and food and beverages containing living Bifidobacterium bacteria. It is something.

[Conventional technology]

Bifidobacterium is a predominant bacterium in the intestinal flora and is known to have various physiological effects that help maintain human health. As its usefulness has become generally recognized, various foods and drinks containing Bifidobacterium have become commercially available. Several Bifidobacterium bacteria suitable for this purpose have already been provided (Japanese Patent Publications No. 59-53031, No. 61-19220).
No. etc.).

However, since Bifidobacterium is essentially an anaerobic bacterium and is sensitive to acids, it is not easy to make it exist in foods and drinks even if the above-mentioned particularly selected bacteria are used. do not have. In other words, Bifidobacterium not only needs to be maintained in an anaerobic state during cultivation, but also in a state cut off from oxygen from the time the food or drink containing the bacterium is manufactured until it is consumed. If the dissolved oxygen concentration is high, most bacteria will die in a short period of time.

The influence of dissolved oxygen is particularly noticeable when the pH of the food or drink is low.

As a means to prevent the number of viable Bifidobacterium bacteria in foods and drinks from decreasing due to oxygen and acids, it is necessary to use oxygen-blocking containers, as well as using strains with improved acid resistance. Methods such as adding substances effective to improve survival have been adopted, but these are not sufficient.

Regarding the improvement of oxygen tolerance of Bifidobacterium,
Until now, research has been carried out mainly to alleviate the requirements for strict anaerobic conditions during culture, and little attention has been paid to improving oxygen tolerance at the stage of producing products such as fermented milk. Not known.

[Problem to be solved by the invention]

The present inventors believe that it is necessary to consider separately the anaerobic conditions necessary for the growth process of Bifidobacterium and the anaerobic conditions necessary for the survival of Bifidobacterium in storage conditions, Furthermore, we thought that Bifidobacterium bacteria with improved oxygen tolerance during the growth process may not necessarily have good oxygen tolerance even in storage conditions, and as a result of confirming various Bifidobacterium bacteria, we found that The following findings were obtained.

That is, ordinary Bifidobacterium bacteria such as Bifidobacterium brepem TCC15fi9B, 15700 were suspended in acidic milk at pH 4.2 and temperature 10°C, and under anaerobic conditions with an initial dissolved oxygen concentration of 0.2 ppm. The survival rate after storage for 4 days was as high as 5% or more. In a similar storage test, when the initial dissolved oxygen concentration was increased, the survival rate was lower than that under anaerobic conditions by more than 3 orders of magnitude. In addition, it has good oxygen tolerance during the growth process,
Bifidobacterium breve YIT4006 (Feikoken Jokyo No. 752, Japanese Patent Publication No. 59-53031), which can be cultured under aerobic conditions, also showed similar results.

From the above findings, even normal Bifidobacterium
Under anaerobic conditions, it can survive sufficiently at a low pH of about 4.2 (that is, it has acid resistance), and it can maintain a high viable count for a long period of time in acidic milk containing Bifidobacterium. It was found that in order to maintain this level, Bifidobacterium must not only have acid tolerance but also a high degree of oxygen tolerance (in storage conditions).

Therefore, the present invention aims to provide Bifidobacterium that has good survival when present in foods and drinks such as acidic milk.

Another object of the present invention is to provide food and drink products that can maintain the number of viable Bifidobacterium bacteria at a meaningfully high level for a long period of time by containing Bifidobacterium bacteria that have good survival in the above-mentioned acidic milk, etc. It's about doing.

[Means to solve the problem]

The present invention, which has succeeded in achieving the above object, has a survival rate of 1% when live bacteria are suspended in acidic milk with an initial dissolved oxygen concentration of 4.2 ppm and a pH of 4.2 and kept at 10°C for 4 days.
Bifidobacterium breve (Bifidobacterium breve) characterized by the above
), and Bifidobacterium breve YI, which has particularly excellent characteristics among the Bifidobacterium bacteria.
T411S! (Feikoken Bacteria Serial No. 9967), as well as food and drink products containing these bacteria.

The Bifidobacterium breve YIT40S2 provided by the present invention was created as follows.

Bifidobacterium breve YIT4008 (Feikoken Jokyo No. 752) was inoculated into a 10% skim milk powder medium (containing 1% yeast extract) at 37°C, pH 4.7-4.
Incubate until 8. DH this milk culture with lactic acid.
1 to acidic milk consisting of 10% skim milk powder adjusted to 4.5.
Add %. Then, air was blown at 10°C to saturate the mixture with oxygen (approximately 119P). Dispense this into test tubes, seal with butyl rubber stoppers, and store at 10°C. Modified R over time
The number of viable bacteria was measured using Bosa agar medium, and the number of viable bacteria was 1.
03/ Colonies are separated from the plate that has become layer 1 or less, and each colony is preserved in the above-mentioned oxygen-saturated acidic milk, and isolated colonies whose viable bacterial count decreases slowly are selected.

Colonies are then re-separated from plates of 1 G"/weight or less and stored in oxygen-saturated acidic milk. By repeating this process more than 20 times, survival in oxygen-saturated acidic milk gradually improves. Among them, Bifidobacterium breve YIT showed the most excellent properties.
4O5! There is.

Bifidobacterium breve YIT4052 (hereinafter sometimes referred to as YIT40S2) has the following mycological properties.

■Taxonomic properties When observed using an optical microscope, it shows rod-shaped or branched pleomorphism, and Durham staining is positive. When cultured anaerobically on BL blood agar medium, white, shiny colonies were formed. In addition, milk culture produces lactic acid and acetic acid, which coagulate milk. catalase activity, gelatin liquefaction,
Nitrate reduction, indole production, and hydrogen sulfide production were all negative.

Sugar fermentability is positive for lipose, mannose, fructose, galactose, sucrose, maltose, cellobiose, lactose, melibiose, raffinose, meletitose, salicin, arabinose, xylose, trehalose, mannitol, sorbitol, inulin,
Starch is negative.

From the above properties, YI74052 is BerHey's
Manual of Systematic B! ct
It was identified as Bifidobacterium breve with reference to eriolBy, Tol, 2 (+986).

While YI740S2 has the general properties of Bifidobacterium breve, as will be described later, it has oxygen resistance that is not found in known Bifidobacterium breve, and this resistance can be achieved by subculture. It will not be lost even if you repeat it.

■ Comparison of resistance to acid and oxygen Bifidobacterium was inoculated into a milk medium prepared by adding 0.1% yeast extract to 10% skimmed milk, and the DH was incubated at 37°C.
The cells were cultured to 4.7 to 4.8. The obtained culture solution was added at 1% to 10% skimmed milk whose PL was adjusted to 4.2 with lactic acid and mixed. Nitrogen, a mixed gas of nitrogen and oxygen, or air was blown into this to remove dissolved oxygen. Concentration 0.2-10゜7
Set to ppm. Each bacterial cell suspension with a different dissolved oxygen concentration was dispensed into a test tube whose gas phase had been replaced with the gas used to set the dissolved oxygen concentration, and after being sealed with a butyl rubber stopper, it was incubated at 10°C.
It was stored for 4 days. The dissolved oxygen concentration is BECKMA
NQxyHe++Mu-alyser Model 02
60 (Oxygen electrode: Measured at 10°C using eH5S.

The results of the above storage test performed on YIT4052 and four comparative Bifidobacterium breve strains are as follows:
Shown in Tables 1 to 5. Note that the survival rate is the ratio of the number of viable bacteria after 4 days of storage to the number of viable bacteria at the start of storage, expressed as a percentage.

Table I 7174052 strain Table 2 ATCC1560 strain Table 3 MuTCC15700 strain Table 4 YIT4DO6 strain Table 5 YIT4010 strain Bifidobacterium breve YIT40Sl not only has excellent oxygen tolerance during storage, but also has the influence of oxygen during culture. It is easy to cultivate on an industrial scale. Moreover, the flavor of the culture is also good. Therefore, this strain is extremely advantageous as a strain for producing fermented products such as fermented milk in which live Bifidobacterium bacteria are ingested.

〔Example〕

1 milk medium containing 15% skim milk powder and 0.1% yeast extract
The cells were sterilized by heating to 15°C for 15 minutes, inoculated with YI74052, and cultured at 37°C until the medium pH reached 4.6.

After cooling with water, the culture was homogenized using a homogenizer. Meanwhile, a milk medium containing 12% skim milk powder was heated to 115℃ for 15 minutes.
Sterilize by heating for 1 minute, inoculate with Streptococcus thermophilus, and incubate at 370 ml until medium l11 (=4.3
The cells were cultured at C, cooled with water, and then homogenized. Further, a 30% sucrose solution was sterilized by heating to 100° C. for 30 minutes, and then cooled on ice to obtain a syrup.

The above YIT40S2 culture, Streptococcus thermophilus culture and syrup were mixed at a ratio of 1:3:1 to obtain liquid fermented milk. P immediately after producing this fermented milk
H was 4.5 and dissolved oxygen concentration was 7.8 ppm.

After putting this fermented milk into a test tube and sealing it with a butyl rubber stopper or covering it with an aluminum cap,
When stored at 10°C, the number of viable YIT4052 bacteria after one week was 1.4xlO'/ml (survival rate 18%) in all cases.

〔Effect of the invention〕

Since the Bifidobacterium according to the present invention has extremely high oxygen tolerance in acidic media,
It has good survivability when added to acidic foods and beverages such as fermented milk, and is therefore suitable for producing foods and beverages characterized by containing live Bifidobacterium bacteria. Using this bacteria not only slows down the decline in the number of viable bacteria due to oxygen that inevitably gets mixed into the product, but also reduces fluctuations in the number of viable bacteria depending on the amount of oxygen mixed in, making it possible to increase the number of viable bacteria more than before. It becomes possible to easily provide consumers with products that have a high bacterial count and are stable.

Claims (4)

[Claims] (1) Bifidobacteria characterized by having a survival rate of 1% or more when live bacteria are suspended in acidic milk with an initial dissolved oxygen concentration of 4.2 ppm and a pH of 4.2 and kept at 10°C for 4 days. Bifidobacterium breve
reve). (2) Bifidobacterium breve YIT4052
(Feikoken Bibori No. 9967). (3) Bifidobacterium according to claim 1 or 2;
Food and drink containing Breve. (4) The food or drink according to claim 3, wherein the food or drink is fermented milk.