JP3469881B2 - Method for preparing heat-resistant lactic acid bacteria Bacillus coagulans and roux containing the lactic acid bacteria - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a spore-forming lactic acid bacterium Bacillus coagulans (Bacillus coagul) capable of forming spores having more excellent heat resistance than conventional ones.
ans) C001, a method for preparing a heat-resistant lactic acid bacterium, a food containing the heat-resistant lactic acid bacterium, a drug, in particular, a roux such as curry roux and roux for stew, and a cooked food using the roux.

2. Description of the Related Art Lactic acid bacteria are useful bacteria in the intestine, and it is known that if they are taken in foods or the like, that is, if lactic acid bacteria can be carried to the intestines in a living state, they are healthy. However,
There are the following problems in putting a lactic acid bacterium into a food to put it into practical use and bring it into the intestine in a living state.

Lactic acid bacteria have no inherent heat resistance and die during heat treatment during food production or cooking. When the food contains a large amount of water, the added lactic acid bacteria start fermentation during storage and die. Since heat resistance is improved when spores are formed in lactic acid bacteria, a method for producing a preparation using spores of lactic acid bacteria is known (Japanese Patent Publication No. 36-18248). However, even when the preparation produced by the method described in this publication is used in the production of curry roux, lactic acid bacteria are killed during the heat treatment for cooking the curry roux (heat treatment at about 98 ° C. for about 20 to 70 minutes). Will end up. Thus, the conventional method has not been able to obtain lactic acid bacteria with high heat resistance or spores with high heat resistance.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a spore-forming lactic acid bacterium with high heat resistance. Another object of the present invention is to provide a method for preparing a spore-forming lactic acid bacterium with high heat resistance. Another object of the present invention is to provide foods, pharmaceuticals, especially roux, which contain spore-forming lactic acid bacteria having high heat resistance. Another object of the present invention is to provide a cooked food using roux containing spore-forming lactic acid bacteria having high heat resistance.

[0004]

According to the present invention, a lactic acid bacterium with high heat resistance can be obtained by culturing a spore-forming lactic acid bacterium in a medium containing reducing sugar, yeast extract, manganese salt and / or calcium salt. It was made based on the knowledge. That is, the present invention is positive in the production of acid from xylose and mannitol, respectively,
The lactic acid bacterium Bacillus coagulans (Bacillus coagulans) has a survival rate of 70% or more after being left for a minute.
ulans) C001 is provided. The present invention also provides a method for preparing a thermostable lactic acid bacterium Bacillus coagulans, which comprises culturing spore-forming lactic acid bacterium Bacillus coagulans in a medium containing reducing sugar, yeast extract, manganese salt and / or calcium salt. .

The present invention also provides that before culturing in the above medium,
Provided is a method for preparing heat-resistant lactic acid bacterium Bacillus coagulans, which comprises growing spore-forming lactic acid bacterium Bacillus coagulans in a liquid medium containing a yeast extract and glucose. The present invention further provides foods, pharmaceuticals, and roux, which are characterized by containing heat-resistant lactic acid bacterium Bacillus coagulans. The present invention further provides a cooked food product using the roux.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The lactic acid bacterium Bacillus coagulans C001 of the present invention has the following mycological properties. (1) Morphology of bacteria Cell size: Diameter less than 0.9 μm Cell shape: Rod-like motility: None Spore formation: Yes

[0007] (2) Physiological properties   Attitude toward oxygen: Facultative and anaerobic, grows under aerobic and anaerobic conditions   Without reducing nitrates   VP test: positive   Starch hydrolysis: Negative   Gelatin liquefaction: Negative   Lecithinase activity: negative   Citrate availability: negative   Gas production from glucose: negative   Acid production from sugars               Xylose: Positive               Mannitol: positive               Glucose: positive   Heat resistance: The survival rate of the bacterium after leaving the spores at 102 ° C for 15 minutes                             70% or more

Regarding the heat resistance, it is more preferable that the survival rate after the above-mentioned standing is 80% or more, preferably 90% or more. Along with this, the survival rate after standing at 98 ° C. for 60 minutes is 60% or more, preferably 70% or more, and more preferably 80% or more. The heat resistance is based on that in an aqueous system, more specifically in a phosphate buffer. still,
The heat resistance was evaluated by the TDT tank method. That is, the sample was placed in a TDT tank and kept at a constant temperature, the sample in the tank was sampled with time (for example, every 10 minutes), and the viable cell count in the sample was measured by using a BCP plate count agar medium. It was measured by culturing at 48 ° C. for 48 hours.
In addition, a minitech test was conducted on the difference in mycological properties between the known bacteria B. coagulans (IFO No. 12583: indicated as A in the table below) and B. coagulans C001 (indicated as B in the table below). An experiment based on the law was conducted. The results are as follows.

[0009]

[Table 1] A B Microscopic observation of bacteria with a width of 0.9 μm or more − − Presence or absence of anaerobic growth Puncture by test tube ＋ ＋ Presence of GAS Observation of bubbles in test tube − − Lecithinase activity Observation with medium − − Starch degradability Iodine reaction after culturing in starch-containing medium − − Gelatin liquefaction − − Citrate utilization Minitech disc use − − Use of xylose Use of mini tech disc- ＋ Mannitol availability Minitech disk use- + Glucose utilization capacity Minitech disk use ＋ ＋ Nitrate reduction ability Use of Minitech disc − − Foge soup Roscauer reaction with minitech disc ＋ ＋

From the above test results, Bergey's Manual of
B. coagulans C001 based on Systematic Biology
However, the lactic acid bacterium Bacillus coagulans (Bacillus coagul
ans) was confirmed. In addition, the above Bergey's Manual
It is described that, regarding the availability of sugars of Bacillus bacterium, arabinose, xylose, and mannitol show different availability depending on the strain. The lactic acid bacterium Bacillus coagulans C
001 has been deposited as FERM BP-5958 on June 3, 1997 at the Institute of Industrial Science and Technology for Industrial Technology, Ministry of International Trade and Industry.

The B. coagulans C001 of the present invention is a lactic acid bacterium Bacillus coagulans which is positive in the production of acids from xylose and mannitol, for example, a bacterium isolated from a spore-forming lactic acid bacterium (Lachris bacterium) preparation manufactured by Sankyo Co., Ltd. , Reducing sugar, yeast extract, manganese salt, and / or calcium salt. Preferable examples of the manganese salt include manganese sulfate and manganese chloride, and examples of the calcium salt include calcium chloride, calcium carbonate, and calcium phosphate. In addition, the mycological properties of the above isolated Lacrisium were as follows: lactic acid bacterium Bacillus coagulan
s) Same as C001, but with respect to heat resistance, the survival rate of the bacterium after leaving it at 98 ° C for 60 minutes was 0.8%, and the survival rate of the bacterium after leaving its spores at 102 ° C for 15 minutes was 1.3%. %Met.

[0012] As the above-mentioned medium, reducing sugar is 0.05 to 0.4% by weight (particularly preferably 0.1% by weight) and yeast extract is 0.05%.
˜2 wt% (particularly preferably 0.5 wt%), manganese salt 5 to 500 ppm (particularly preferably 50 ppm) and / or calcium salt 5 to 500 ppm (particularly preferably 50 ppm), with the balance being water. Liquid media are preferred. Further, glucose is preferable as the reducing sugar, and it is preferable to contain both a manganese salt and a calcium salt. The culture temperature is preferably 30 to 65 ° C, more preferably 45 to 55 ° C, and most preferably about 50 ° C. The culture time of the bacterium is preferably 20 to 72 hours, more preferably 20 to 48 hours, and most preferably about 24 hours.

In the present invention, the heat resistance of the spore-forming lactic acid bacterium can be improved in this way, and the spore-containing heat-resistant lactic acid bacterium thus obtained can be used.
Furthermore, it is preferable from a commercial point of view that the cells are grown in a liquid medium containing a yeast extract and glucose before being cultured in the above medium to impart heat resistance. As a medium used for growth, yeast extract is 0.05 to 1% by weight (particularly preferably 0.5% by weight) and glucose is 0.05 to 1% by weight.
A liquid medium containing (particularly preferably 0.5% by weight) and the balance being water is preferable. The culture temperature is preferably 30 to 65 ° C, more preferably 45 to 55 ° C, and most preferably about 50 ° C. The culture time of the bacterium is 6 to 24 hours, preferably 8 to 12 hours.

In the present invention, the above-mentioned spore-resistant, heat-resistant lactic acid bacterium is contained in foods, pharmaceuticals and roux, but it is particularly preferable to contain it in the form of spores. Examples of foods containing spore-resistant heat-resistant lactic acid bacteria include noodles, seasonings (including seasoning bases for various dishes), confectionery, and the like, in addition to the following roux. Examples of roux include curry roux, stew roux, hayashirou, hashed beef roux, beef stroganofurufu, and the like. These products may be in the form of solid, powder, granules, paste, liquid or the like.

The following method is exemplified as a method for producing the roux containing the spore-resistant thermostable lactic acid bacterium of the present invention.
First, wheat flour and edible oil and fat are heated and mixed to obtain wheat flour roux. The heating conditions in this case can be exemplified by a temperature of 100 to 130 ° C. and a time of 30 to 60 minutes. To the obtained flour roux, flour, corn starch, salt, granulated sugar,
Powdered raw materials such as milk powder, various seasonings, various flavors, and various extracts, various pastes, various flavor oils, liquid raw materials such as emulsifiers are added and mixed, and heated to about 85 to 120 ° C and stirred (heating and stirring step ) And get Rou. The raw material composition and the like are appropriately adjusted depending on the final form of roux such as solid, powder, granule, paste and liquid. For example, when the final form of roux is solid, edible fats and oils will use hardened oil, and the roux obtained will be appropriately filled in a container and cooled to obtain a molded roux. Become.

Taking the case of the above-mentioned solid roux as an example, the spore-resistant thermostable lactic acid bacterium of the present invention is added at any stage before the heating and stirring step. Therefore, it may be added to any of the above raw materials, or may be added at the time of mixing each of the above raw materials, but in order to maintain a high survival rate of spore lactic acid bacteria, a powder raw material that is less in contact with water Is preferably added to. Further, in order to avoid damage of lactic acid bacteria due to heating during production, it is preferable to add it as late as possible in the heating step. In this way, a solid roux containing spore lactic acid bacteria can be obtained. The amount of the spore-resistant thermostable lactic acid bacterium of the present invention added to the roux can be arbitrary, but it is preferably added in an amount of about 0.0005 to 0.005% by weight based on the entire roux.

The present invention can further provide a cooked food using the above roux. Examples of such cooked foods include curry rice, curry stew, curry sauce, stew, hayashi rice, hashed beef, beef stroganoff and the like. As described above, in the present invention, various foods containing spore-resistant thermophilic lactic acid bacteria, pharmaceuticals, such as intestinal medicine, may be provided, but when the foods are in the following forms, the function of the present invention Is achieved more effectively. That is, foods, etc., have a pH of 7 or less and water activity.
It is desirable that it has a water activity of 0.88 or less and a pH of 4 or less and a water activity of 0.96 or less (both at 35 ° C.).
This is because the fermentation of lactic acid bacteria during storage can be suppressed and the lactic acid bacteria can be maintained in the product until eating. Also, foods and the like to be heated at the time of feeding, for example, in the case of foods using the roux, noodles, seasoning base for cooking, etc., suppressing the death of lactic acid bacteria during the heating, more lactic acid bacteria It becomes possible to eat while living.

[0018]

Industrial Applicability According to the present invention, a spore-forming lactic acid bacterium having higher heat resistance than conventionally known lactic acid bacterium is provided. Therefore,
By using this spore-resistant heat-resistant lactic acid bacterium, foods, pharmaceuticals,
The proportion of lactic acid bacteria that are killed during heat treatment or heat sterilization during the production of roux is low, and foods containing lactic acid bacteria in a living state can be efficiently prepared. Further, when the final food is cooked with this roux, the lactic acid bacterium is rarely killed, so that a cooked food capable of transporting the lactic acid bacterium to the intestines in a living state can be easily prepared. Next, the present invention will be described with reference to examples.

[0019]

Examples Example 1 1) Preparation of inoculum The following components were dissolved in 1 liter of water to prepare a culture medium for growth. Yeast extract 5 g Glucose 5 g 10 ml of the above medium was dispensed into a 100 ml Erlenmeyer flask and sterilized, and then spore-forming lactic acid bacterium (Lachris bacterium) manufactured by Sankyo Co., Ltd.
1 platinum loop inoculation of B. coagulans isolated from the formulation, 50
Shake culture for 12 hours at ℃, 400ml of the above medium
Was dispensed into a 2 liter flask, the sterilized one was subcultured with all the above-mentioned culture, and the one that was shake-cultured at 50 ° C. for 12 hours was used as an inoculum.

2) Addition of heat resistance The following components were dissolved in 1 liter of water and used as a heat-resistant addition medium. Yeast extract 5 g Glucose 1 g CaCl ₂ 50 ppm MnSO ₄ 50 ppm 25 liters of a medium having the above composition was prepared in a jar fermenter, and after sterilization, all of the above inoculum was subcultured at 50 ° C. for 20 to 48 hours, Culture with aeration and agitation was performed (aeration condition 25 liter / min, agitation condition
100-300 rpm). The heat resistance of the obtained spore-resistant heat-resistant lactic acid bacterium Bacillus coagulans was evaluated by the following method. That is, the viable cell count (number) of the lactic acid bacteria in a phosphate buffer at 102 ° C. was measured with time by the TDT tank method (Table 1 and FIG. 1, the viable cell count at the start of the measurement is 6.2
× 10 ³ pieces).

Example 2 15 parts by weight of wheat flour and 39 parts by weight of hardened oil were added in 12 minutes for 30 minutes.
The mixture was heated and mixed so as to reach 5 ° C to obtain flour roux.
Separately, 2 parts by weight of wheat flour, 10 parts by weight of corn starch, 8 parts by weight of salt, 10 parts by weight of granulated sugar, and 10 parts by weight of curry powder were powder-mixed, and the spore-resistant heat resistance obtained in Example 1 was added thereto. 4.4 × 10 ^{7 of} lactic acid bacteria (for 1 g of roux)
A powder raw material was obtained by adding and mixing. Next, 2 parts by weight of the powder raw material and 2 parts by weight of various kinds of pastes, 2 parts by weight of various kinds of pastes, various kinds of seasonings, various flavors, various flavor oils and emulsifiers are mixed with the above flour roux, and the mixture is heated at 105 ° C. for 30 minutes. The mixture was heated and stirred under conditions such that the temperature reached the above temperature to obtain a paste-like curry roux. Then, it was filled in a suitable container and cooled to 25 ° C. to obtain a solid curry roux containing spore-resistant heat-resistant lactic acid bacteria.

The spore lactic acid bacteria in the obtained solid curry roux
The number of viable bacteria was measured to be 2.5 x 10 ⁷ (For 1 g of roux
It was). In addition, the number of viable bacteria is measured by BCP plate
Incubate at 45 ℃ for 48 hours using CountAgar medium
went. Add the above curry roux to the TDT tank
The viable cell count (number) of the lactic acid bacteria at
Measured (Table 2 and Figure 2, showing the number of bacteria per 1g of curry
). The number of viable bacteria at the start of measurement is 2.6 x 10³ It was an individual.

Comparative Example 1 The heat resistance of the spore-forming lactic acid bacterium manufactured by Sankyo Co., Ltd. used in Example 1 was measured by the same method as in Example 1 (Table 1 and FIG. 1, viable cell count at the start of measurement). 7.8 × 10 ² pieces). Comparative Example 2 A solid curry roux was produced in the same manner as in Example 2, using the spore-forming lactic acid bacterium manufactured by Sankyo Co., Ltd. used in Example 1. The viable cell count of spore lactic acid bacteria in roux was measured to be 2.5.
The number was × 10 ⁴ (relative to 1 g of roux). In addition, the viable cell count of lactic acid bacteria during heating was measured by adding water to and heating the roux above (Table 2 and FIG. 2, viable cell count at the start of measurement 2.5).
× 10 ³ ). All of the above measurements were performed according to the method of Example 2.

[0024]

[Table 2] Table 1 (Number of viable bacteria in a phosphate buffer at 102 ° C) Elapsed time (minutes) Bacteria obtained in Example 1 Bacteria of Comparative Example 3 6200 380 6 6400 207 9 6800 99 12 6100 10 15 6100 5 18 4730 21 3530 24 1730 27 670 30 360

[0025]

[Table 3] Table 2 (Number of viable cells in curry at 98 ° C) Elapsed time (minutes) Bacteria obtained in Example 2 Bacteria of Comparative Example 2 0 2560 2500 5 2680 2600 10 3060 2120 20 2650 2170 30 2910 1400 40 3200 670 50 2760 210 60 2210 20

[Brief description of drawings]

FIG. 1 shows the time-dependent changes in the viable cell count (number) of the lactic acid bacterium in the phosphate buffer at 102 ° C. of the bacterium obtained in Example 1 (the bacterium of the present invention) and the bacterium of Comparative Example 1.

FIG. 2 shows the time-dependent changes in the viable cell count (number) of the lactic acid bacterium in the 98 ° C. curry of the bacterium obtained in Example 2 (the bacterium of the present invention) and the bacterium of Comparative Example 2.

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Claims (4)

(57) [Claims] 1. Reducing sugar, yeast extract, manganese salt and / or
Alternatively, the spore-forming lactic acid bacterium Bacillus coagulans is cultured in a medium containing a calcium salt.
A method for preparing a thermostable lactic acid bacterium Bacillus coagulans, wherein the survival rate of the bacterium after standing at 02 ° C for 15 minutes is 70% or more. 2. The spore-forming lactic acid bacterium Bacillus coagulans is grown in a liquid medium containing yeast extract and glucose, and then reducing sugar, yeast extract, manganese salt and / or
Alternatively, it is cultivated in a medium containing calcium salt, and the survival rate of the bacterium is 70% after being left at 102 ° C. for 15 minutes.
The method for preparing the heat-resistant lactic acid bacterium Bacillus coagulans as described above. 3. A heat-resistant lactic acid bacterium is prepared by the method according to claim 1 , and the heat-resistant lactic acid bacterium obtained below is added to the raw material.
A method for producing a food , which comprises adding . 4. Heat resistance according to the method of claim 1 or 2.
Prepare the lactic acid bacterium and add the heat-resistant lactic acid bacterium obtained in the next step to the raw material.
A method for producing roux, which comprises adding .