JPH0669367B2 - Method of culturing lactic acid bacteria - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for culturing a lactic acid bacterium, which is widely used in fermented foods, lactic acid bacterium beverages, processed milk foods, etc., at a high concentration.

BACKGROUND ART Conventionally, in order to culture lactic acid bacteria, a method of culturing while adjusting the medium in a culture tank has been performed according to the method of microbial culture method. A high-concentration culture system has been proposed which consists of separating bacteria and their inhibitors through a filtration membrane for the purpose of removal.

As such a culturing system, a method of reducing the concentration of metabolites produced by culturing by substituting a substrate with a backwashable filter incorporated in a culturing tank for culturing microorganisms (JP-A-58- 47485), an apparatus for continuously culturing by passing the culture solution through a cylindrical filter to separate metabolites and bacteria, and circulating the culture solution containing the bacterial cells to a culture tank (JP-A No. 62-138184) and the like are known.

However, when the above-mentioned known culturing technique is used for culturing lactic acid bacteria, there is a problem with the filtration and extraction method that removes metabolites that are inhibitors of lactic acid bacteria through a filtration membrane (filter), resulting in high concentration culture of lactic acid bacteria Is limited.

That is, in culturing lactic acid bacteria, lactose is supplemented to the medium in addition to various vitamins as sugar, but lactic acid bacteria utilize lactose to produce lactic acid as a metabolite, and this lactic acid is lactic acid bacteria with low acid resistance. It becomes an inhibitory factor on the growth of. Therefore, it is necessary to prevent the concentration of lactic acid from increasing during the culture of lactic acid bacteria. However, in the culture method for filtering known inhibitors, the concentration of lactic acid bacteria is about 10 ⁹ to 10 ¹⁰ (cfu / ml). Culture was the limit.

In addition, in the conventional filtration membrane used when performing filtration culture of lactic acid bacteria, sterilization with a bactericide was performed to prevent deterioration due to heat, but it is impossible to sterilize and long-term culture is performed. It was difficult to completely prevent contamination in the process.

The present invention, in the culture of lactic acid bacteria using a filtration membrane, by controlling the sugar concentration of the medium to a specific range, by suppressing the concentration of lactic acid produced in the culture solution below a certain level, It is an object of the present invention to provide a method for culturing lactic acid bacteria at a high concentration reaching the number of lactic acid bacteria of 10 ¹¹ (cfu / ml).

The present invention will be described in detail below.

Means for Solving the Problems In the present invention, sugar (lactose) in a medium is used for culturing lactic acid bacteria.
It is important to adjust the concentration to 1 to 1.5% by weight,
With this adjustment, the amount of lactic acid produced in the culture solution is always 10 g.
It is possible to increase the concentration of lactic acid bacteria obtained by culturing to a high concentration of 10 ¹¹ (cfu / ml).

As the lactic acid bacterium in the present invention, any lactic acid bacterium used in the production of fermented foods, lactic acid bacterium beverages, milk processed foods and the like can be used without particular limitation.

Further, the present invention, during the culture of lactic acid bacteria, while separating and removing the inhibitory substances of lactic acid bacteria and other wastes generated in the medium using a filtration membrane as a lactic acid bacterium culture solution, culturing a culture solution containing lactic acid bacterium bodies It is also characterized in that it is circulated to the tank and, on the other hand, the culture is carried out while supplementing with a new medium whose sugar concentration is adjusted to the above range.

Hereinafter, the culture method according to the present invention will be described with reference to FIG. 1 which illustrates an apparatus used for the culture method.

In FIG. 1, reference numeral 1 is a culture tank in which an agitator 11 is provided.
The bottom of the culture tank 1 is in communication with the lower part of the filtration membrane 3 by a pipe, and the upper part of the filtration membrane 3 is in communication with the upper part of the culture tank 1 by a pipe. In addition, a line for replenishing a fresh medium is connected to the upper part of the culture tank 3. In the figure, 4 is a pump for removing the inhibitory substance of the bacteria through the filtration membrane, 5 is a level meter for controlling the level of the medium in the culture tank, and 6 is a pump for supplying the medium. 7 shows a pump for supplying the neutralizing agent to the medium, 8 shows a supply port of nitrogen gas, and 9 shows a discharge port of nitrogen gas. Reference numeral 10 represents an outlet for the inhibitor and other wastes removed through the filtration membrane.

In order to culture lactic acid bacteria using the apparatus configured as described above, first, a new medium in which the sugar concentration is adjusted to 1 to 1.5% by weight is charged, and the lactic acid bacteria are inoculated and cultured with stirring. During this period, the pH of the medium is controlled by appropriately adding a neutralizing agent while observing the pH meter. Since lactic acid in the medium is produced with the progress of the culture and inhibition against lactic acid bacteria is expressed, the medium is passed through the filtration membrane 3 via the pump 2 and the medium containing lactic acid is separated from the lactic acid bacteria via the pump 4 to obtain 10 On the other hand, the medium containing lactic acid bacteria (the lactic acid bacteria are not discharged by the filtration membrane) is returned to the culture tank 1 while being discharged. At this time, in the culture tank, the level meter 5 supplies a fresh medium in an amount corresponding to the outflow of the medium through the pump 6. The culture tank 1 is kept at a constant temperature by a thermometer.

In the above-mentioned culture operation, it is necessary to change the culture supply rate in the increasing direction with time after the lapse of time from the start of culture when the inhibition phenomenon due to lactic acid production appears.

By this operation, it is possible to cope with an increase in the required amount of nutrients, sugars, etc. as well as an increase in the number of viable bacteria, and to quantitatively suppress the increase in lactic acid produced.

If the medium supply rate is increased in accordance with the increase in the viable cell count, the culture efficiency is good and the culture time can be shortened. Therefore, if the lactose concentration is 1.5% or less, the decrease in the growth rate due to lactic acid production can be prevented, but if the concentration is too low, the rate of multiplication will be small and the culture time will be long. Culture at lactose concentration in the range of ~ 1.5%,
Prevent the inhibition by lactic acid and maintain a state that does not affect the multiplication rate.

The device used in the present invention is preferably equipped with a filtration membrane made of an organic substance such as vinylidene difluoride and a circulation line for the medium, until the inhibition phenomenon by lactic acid produced by the culture in the culture tank appears. The lactic acid bacteria are cultured only in the medium in the culture tank 1, and the filtration membrane 3 and the circulation line can be heat-sterilized during this period. Then, after the inhibition phenomenon due to lactic acid has appeared, the circulation line is operated to remove the inhibitory substances mainly lactic acid and waste products in the filtration membrane 3, and a fresh medium corresponding to the amount of liquid removed here is prepared. The lactic acid bacterium-containing medium, which operates so as to be supplied to the culture tank 1 while not permeating through the filtration membrane 3, is returned to the culture tank by the circulation line.

Here, since the growth concentration of lactic acid bacteria increases with the lapse of culture time, the amount of the removed medium in the filtration membrane 3 and the amount of fresh medium supplied to the culture tank increase with time.

The lactic acid bacterium in the present invention is not particularly limited, and any lactic acid bacterium widely used in conventionally known fermented foods, lactic acid bacterium beverages, processed milk foods and the like can be used. Such lactic acid bacteria include lactic acid bacteria belonging to the genus Bifidobacterium, the genus Lactobacillus or the genus Streptococcus, which are commercially available and easily available.

EFFECTS OF THE INVENTION As described above, according to the present invention, the problem of contamination found in the conventional filtration culture is solved, while on the other hand, it is possible to culture without suppressing the inhibition phenomenon by lactic acid and affecting the multiplication rate. Even with the same capacity of treatment equipment, the number of cultures per unit volume can be increased, which can also contribute to reduction of manufacturing cost (capable of obtaining 10 ¹¹ cfu / ml) and equipment cost.

Further, in the present invention, since a filtration membrane made of an organic substance that can be sterilized by high temperature heating is used, it is possible to sterilize by steam including a circulation line, and it is possible to completely prevent sterilization and contamination. There is an advantage that management is also simplified.

The present invention will be specifically described below with reference to examples.

Example 1 This example shows the case of culturing using Buffybacterium (Bifidobacterium longum SBT-2933 R) as the lactic acid bacterium.

When the culture medium 1 and bifidobacteria were added to the culture tank 1 to start the culture, growth inhibition by lactic acid began to appear from about 4 hours, so the inhibitor removal line using the filtration membrane was operated.

Medium composition: lactose 1.25% yeast extract 1.00% polypeptone 1.00% dipotassium phosphate (K ₂ HPO ₄ ) 0.5% potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0.1% sodium L-ascorbate 0.1% of the medium The supply amount of fresh medium to the culture tank 1 and the outflow amount from the filtration membrane were controlled so that the lactose concentration was always 1.25% by weight. The growth number of Bifidobacterium reached 10 ¹¹ cfu / ml 8 hours after the start of culture.

In addition, FIG. 2 shows the time-dependent state of the number of bacteria. The changes in the amounts of lactose and lactic acid in the medium at this time are as shown in Fig. 3. Lactose is temporarily deficient, but lactic acid, which is a metabolite of Bifidobacterium, exceeds 10 g / l. It is recognized that there is no.

The supply rate (dilution) of fresh medium from the start of filtration to completion of the culture 4.5Hr ^-1 from 0.4hr ^-1 with increasing lactic acid bacteria
Continuously changed until. The relationship between the change in lactose concentration in the medium and the amount of lactic acid produced and the cell yield is shown in the table below.

As shown in this table, it is desirable to suppress the concentration range of lactose to 1 to 1.5 in order to suppress the production of lactic acid and increase the cell yield.

[Brief description of drawings]

FIG. 1 shows a schematic view of an apparatus used in the present invention. In Fig. 1, 1 ... Culture tank 3 ... Filtration membrane 4 ... Pump for discharging inhibitor and waste products 6 ... Pump for supplying fresh medium 11 ... Stirrer

Claims (4)

[Claims] 1. When culturing a lactic acid bacterium, the sugar concentration of the medium is adjusted to 1 to 1.5% by weight, and an inhibitor of the bacterium produced in the medium by the culturing is passed through a filtration membrane arranged in communication with the culture tank. It is characterized in that the culture broth, which has been removed by removing the inhibitor, is circulated to the culture tank, and at the same time, the culture tank is replenished with a fresh medium of a liquid amount corresponding to the portion removed through the filtration membrane. Method for culturing lactic acid bacteria. 2. The lactic acid bacterium is a bifidobacteria (1).
The method for culturing lactic acid bacteria according to. 3. The method for culturing lactic acid bacteria according to claim 1, wherein the sugar concentration of the medium is a lactose concentration. 4. The method for culturing lactic acid bacteria according to claim 1, wherein the filtration membrane is composed of vinylidene difluoride.