JPS62208293A - Production of multiplication growth factor of mold of bifidobacterium - Google Patents

Abstract

PURPOSE:To produce a multiplication growth factor of mold of Bifidobacterium in a short time in high yield, by cultivating yeast belonging to the genus Bullera, capable of producing galactooligosaccharide from lactose in a medium containing lactose as a carbon source at <=fixed pH. CONSTITUTION:Yeast such as Bullera singularis YIT8243 (FERM P-8677), etc., belonging to the genus Bullera, capable of producing galactooligosaccharide at pH4.5 is precultivated in a medium containing lactose. The yeast is cultivated in a medium containing only lactose as a carbon source, yeast essence, peptone, etc., as a nitrogen source, an inorganic salt, etc., kept at pH3-7. After removal of the mold, the culture solution is decolored with an ion exchange resin, adsorbed on an active carbon column and eluted with an aqueous solution of ethanol to give the aimed galactooligosaccharide.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a Bifidobacterium growth promoting factor using lactose as a raw material.

j Many substances are known as Bifidobacterium growth-promoting factors, that is, substances that have the effect of promoting the growth of Bifidobacterium. Oligosaccharide Ga1-(Ga
l) n-Glc (in the formula, Gal is a galactose residue, Glc is a glucose residue, and Glc is an integer from 1 to 4, respectively; Japanese Patent Publication No. 58-20266) is a bifidobacterium in the large intestine. It is attracting attention as being particularly effective in promoting bacterial growth. However, the method that the present inventors first proposed as an industrial method for producing a Bifidobacterium growth promoting factor consisting of the above-mentioned oligosaccharides (hereinafter referred to as lactooligosaccharides), namely, the method using β-galactosidase produced by Aspergillus oryzae. Although the method of acting on lactose has various advantages,
There was room for improvement in two points: the yield of galactooligosaccharides relative to lactose and the lactooligosaccharide content of the reaction product.

On the other hand, Japanese Patent Application Laid-open No. 60-251896 discloses that Cryptococcus microorganisms are cultured in a lactose-containing medium and lactooligosaccharide is added to the culture. A method for producing galactooligosaccharides is disclosed, which involves collecting lactooligosaccharides from a culture after F-accumulation, and this method is said to be free from the drawbacks of the method using β-lactosidase.

However, this production method not only requires long-term cultivation for 4 to 6 days, but also requires the use of Crybutofuccus microorganisms to assimilate lactose, resulting in a hydrolysis reaction of some lactose and the resulting production of galactose. During the transfer reaction and the production of lactose, part of the lactose that should be transferred to lactose is consumed by the microorganism, and therefore the yield of lactose to lactose cannot be very high. That is the problem.

Also, although it was not carried out with any interest as a method for producing a Bifidobacterium growth promoting factor, it was published in Canadian Journal of Chemistry 42. p, 134
1 (1964), yeast, Sporobolomyces
Spolo-bolomyces s
Ingularis; however, it is currently classified as a member of the genus Brera) is cultured in a medium containing lactose and yeast extract,
A research report has been published in which lactooligosaccharide was obtained from culture. According to this, when using the above-mentioned yeast, it is important to adjust the initial pH value of the culture medium, and the pH should be adjusted to 3.7.
When it was set to 5, oligosaccharide formation was observed, but the initial p.
It is said that when H exceeds 4, no oligosaccharide was produced.

According to additional tests by the present inventors, when the initial 1) H is set to about 3.75, the pH becomes 3 or less after the middle stage of culture. Moreover, in such a low pH region, yeast growth must be extremely slow, and even with this production method, high yields cannot be expected even if the culture is continued for a long time. Furthermore, if this method can only be carried out in a small area with low r+H, which is close to the limit for yeast growth, there are many difficulties in terms of culture technology in order to carry out this method industrially.

Problems to be Solved by the Invention In view of the fact that all conventional production methods have the above-mentioned drawbacks, the present invention aims at producing lactooligosaccharide in a shorter time and reducing the reaction product lactooligosaccharide. We aim to find a method for preparing lactooligosaccharides with a high sugar content and easy separation and purification of lactooligosaccharides, thereby making it possible to obtain highly purified Bifidobacterium growth promoting factors at a lower cost than before. This is the purpose.

In the method for producing the Bifidobacterium growth promoting factor of the present invention, which has succeeded in achieving the above object, the lactose rise is lactooligosaccharide, that is, the general formula Ga1 (Gal). n-Glc
(However, in the formula, Gal represents a lactose residue, Glc represents a glucose residue, and Glc represents an integer from 1 to 4.)
A yeast of the genus Brera that has the ability to produce the oligosaccharide represented by It is characterized by collecting.

The manufacturing method of the present invention will be described in detail below.

As a specific example of the Brella yeast that can be used in the production method of the present invention, Brella singularis (Bullera singularis)
singularis) YrT8243. This strain was originally Brella singularis ATCC241, which the present inventors received from ATCC in 1982.
93, but it is currently deposited with the Microbial Technology Research Institute with the above name (Feiko Kenzono No. 8677).
. Its main mycological properties are as follows.

A. Growth in various media ■ Wort liquid medium: When cultured at 20°C for 3 days, the cells were oval to elongated, (2,5-3,5)umX(4,5-
7.0) IJ+m.

Multipolar budding, forming a ring-shaped film, the medium becomes cloudy, and a precipitate is formed.

■ Wort agar medium: Colonies are milky white, shiny, smooth and viscous after 1 month at 20°C.

■ Slide culture: Does not form pseudohyphae on potato extract agar medium.

B. Formation of ascospores is not observed in a general sporulation medium.

C0 extruded spore form I&: not observed when cultured on wort agar or cornmeal agar.

D. Physiological properties ■ Optimum growth conditions: pH 4-6. Temperature 28℃ ■ Growth range: pH 3-8. Temperature 20-29℃■ Nitrate assimilation:
Not assimilated.

■ Production of carotenoids: Not produced.

■ Production of starch-like substances: Not produced.

■ Vitamin requirement: Will not grow in vitamin-deficient medium.

■ Growth in 50% glucose yeast extract medium: No growth 1°■ Growth at 30°C: No growth.

E, assimilability for each carbon source L-7 Rabinose - D-ribose - D-xylose - D-glucose + D-galactose - L-Rhamnose - Maltose - Shita sugar - Lactose 10′ Cellobiose + Trehalose + Raffinose − Soluble starch - Elitrit - Inoshira F - D-man knit + D-gluconate Succinate Citrate Note that it has no fermentability for sugars.

It goes without saying that any strain other than the above-mentioned strains having the ability to produce lactooligosaccharide at pH 4 or 5 can be used in the production method of the present invention. As mentioned above, there is a research report in 1964 in which galactooligosaccharides were obtained using Sporobolomyces singieralis, which is currently considered to belong to the genus Brera, but the strain used there had a pH of 4. If the yeast exceeds this level, it will not be able to produce oligosaccharides, and in this respect it is different from the Brera yeast adopted in the present invention.

〃In the case of producing Bifidobacterium growth promoting factor consisting of lactooligosaccharide using microorganisms”
The advantage of the ``Brella yeast that has the ability to produce lactose'' is that this yeast grows by assimilating the former of lactose and glucose produced by hydrolysis of some lactose, so it is not used in the culture. It has the property of not assimilating galactose and therefore does not consume galactose for its own sake. "The ability to produce lactooligosaccharides" makes it possible to carry out the cultivation process of Brera yeast in a weakly acidic region of pH 4 or higher where yeast can actively proliferate. By being fully utilized, a high rate of transfer reaction from lactose to lactooligosaccharides can be achieved in a short time, and the culture can
It has a high lactooligosaccharide content.

When carrying out the production method of the present invention, “pH 4,
In 5, "Yeast of the genus Brera that has the ability to produce lactooligosaccharides" (hereinafter simply referred to as yeast of the genus Brera) is one that has been pre-cultured in a lactose-containing medium for 1 to 3 days to enhance the lactosyl transfer ability of the hedron. Although it is desirable that there be one, it may be pre-cultured in a lactose-free medium.

The medium for main culture preferably contains only lactose as a carbon source. Although the coexistence of other carbon sources is advantageous for the growth of yeast, it is not necessary even if it is used, since it reduces the lactooligosaccharide content of the culture and burdens the purification process. It should be kept to a minimum. The lactose concentration of the medium is preferably about 2 to 25%. At higher concentrations, particularly above about 30%, yeast growth slows down and the rate of lactooligosaccharide production also decreases. There are no particular restrictions on other medium components, including nitrogen sources such as yeast extract, peptone, corn steep liquor, and meat extract, phosphates, magnesium salts, and vitamins.
Those commonly used as yeast medium components can be appropriately contained.

The pH of the medium must be kept within the range of 3 to 7, suitable for yeast growth, throughout the period of culture. A particularly preferred pH is 3.5-6, and it is desirable that at least the majority of the cultivation period be carried out in this pH range.
Even if the pH is below 3, lactooligosaccharides will be produced as long as there is a growing yeast of the genus Brera, but the production rate will be extremely slow. As a method to maintain the medium pH between 3 and 7, the initial pH of the medium should be adjusted so that the pH is 3 or higher even at the end of the culture.
A method of keeping 9H sufficiently high (usually it is necessary to make it 4 or more), a method of controlling 9H so that it does not become less than 3 by occasionally adding dilute alkali or buffer solution during culture, or a method of controlling these two methods. There are ways to use them together.

The culture temperature may be within the range of about 20 to 29°C, but
A temperature around 8°C is most suitable for yeast growth and lactooligosaccharide production. Since yeast of the genus Brera grows only under aerobic conditions, it is necessary to perform shaking culture or aerated agitation culture.

When culturing is carried out under the above conditions, lactose in the medium is consumed and gradually decreases, and accordingly, the bacterial cell concentration and the concentration of V (galacto-oligosaccharides, mainly trisaccharides) increase. However, the oligosaccharide concentration begins to gradually decrease after a certain point. 〃The composition of lactooligosaccharides also changes with the passage of culture time, and the ratio of oligosaccharides of tetrasaccharides or higher begins to decrease around the time when the oligosaccharide concentration reaches its maximum. The culture time required to achieve the highest lactooligosaccharide yield varies depending on the culture conditions (particularly the pH of the medium), but for example, culture is performed at 27°C with a lactose concentration of 10% and a pH controlled at 3.75. In this case, it will take 2 to 3 days. The ratio of monosaccharides, glucose and galactose, to the total sugars accumulated in the medium is about 2% at most throughout the culture period.

After terminating the culture at an appropriate stage, the face pieces are separated by filtration or centrifugation. The isolated bacterial cells can be used repeatedly for culture if the isolation is performed aseptically. To collect lactooligosaccharides from the culture after bacterial cell isolation, for example, decolorization using activated carbon or ion exchange resin,
After desalting, it is passed through an activated carbon column to adsorb galactooligosaccharides, then eluted with an aqueous ethanol solution, and the eluate is concentrated under reduced pressure to form a syrup, or the concentrated liquid is further powdered by spray drying or freeze drying. Bye.

1 ■Ω The production method of the present invention utilizes the yeast of the genus Brera, which has many advantageous properties as mentioned above, while actively multiplying, and is therefore more effective than any of the conventional methods using microorganisms. In addition, not only the required culture time is short, but also the yield of lactooligosaccharide is high, and it is easy to achieve a yield of 60 to 70% based on lactose. In addition, since almost no glucose or galactose is accumulated in the medium and a culture with a high ratio of lactooligosaccharides to the total sugar content is obtained, purification after culturing is easy.

The production method of the present invention also provides a method for producing yeasts of the genus Brera, which grow actively.
Although the culture is carried out in the H region, the culture process is different from the known method in which the culture is carried out at pH 4, 5 under conditions that are not favorable for the growth of yeast of the genus Brera, which does not have the ability to produce lactooligosaccharides. It has the advantage of being easy to manage and producing a culture with a stable composition.

Therefore, according to the present invention, a high quality Bifidobacterium growth promoting factor can be produced more easily and at a lower cost than before.

The present invention will be described below with reference to Examples. In addition, the quantitative determination of lactooligosaccharide in the Examples was performed by high performance liquid chromatography. Moreover, "bacterial cell growth rate" directly indicates the absorbance at a wavelength of 660 nm measured for the culture solution using a spectrophotometer. However, if the measured value of absorbance becomes 0.6 or more, the proportional relationship with the bacterial cell concentration is lost, so in that case, measure the culture solution diluted so that the absorbance is 0.6 or less. The value obtained by multiplying the measured value by the dilution factor is displayed.

Example 1 Lactose 10%, yeast extract 0.3%, phosphate-potassium 0
．． 1% magnesium sulfate, 0.05% magnesium sulfate, pH 5.0 liquid medium 8e was placed in a 10e shear, and 20 bacterial suspensions of Brella singularis YIT8243 were precultured in the same medium.
0el was added and cultured at 27°C with continued aeration and stirring. Figure 1 shows changes in the medium composition during culture. The production of lactooligosaccharide reaches its maximum at 62 hours, and at this time, the yield of lactose to lactose is 70%, and the total yield of lactose, including monosaccharides, is 70%. The ratio of lactooligosaccharide in sugar was 87%.

Further, 500 ml of the culture solution after 62 hours was taken out, centrifuged to remove bacterial cells, 2.5 g of powdered activated carbon was added to the supernatant, stirred, and filtered to obtain a transparent sugar solution. This sugar solution is passed through an activated carbon column to adsorb sugars, and then
Glucose, galactose, and some disaccharides were eluted by flowing water in C. Further, 5% ethanol was flowed to remove the remaining disaccharides, and then 50% ethanol was flowed to elute the lactooligosaccharides. The eluate was concentrated under reduced pressure, filtered, and lyophilized to yield a white powder of 30. I
g was obtained. This powder is Ga1-(β1-+4)-
23.6 g of Gai(β1-+4)-Glc, Ga1(
β1-4)-Gal-(β1-4)-Gal(β1-+
4) It contained 6.0 g of -Glc and 0.5 g of lactose.

Example 2 Medium 5e having the same composition as used in Example 1 except that the pH was adjusted to 3.75 was placed in a 10e jar, and 100 ml of a bacterial solution of Brella singularis YIT8243 pre-cultured in the same medium was added. 27℃ with continuous aeration and stirring
It was cultured in During the culture, monitor the medium pH and control the pH fluctuation by dropping hydrochloric acid or potassium hydroxide aqueous solution.
．． It was kept within the range of 75±0.1.

Table 1 shows the changes over time in the bacterial cell growth rate and the concentration of lactooligosaccharide produced in the medium in this culture and in the same culture except that the pH was not controlled.

Table 1: 72 hours after the start of culture, the culture solution was taken out, centrifuged to remove the bacterial cells, 20g of powdered activated carbon was added to the supernatant, stirred, filtered, and further filtered through an ion exchange resin column (strongly acidic cation exchange resin). and a strongly basic ion exchange resin) to obtain a transparent sugar solution 10e. This sugar solution was concentrated under reduced pressure to 500ml and filtered to obtain a viscous sugar solution.

The ratio of lactooligosaccharide to the total amount of sugar in this sugar solution was 91%.

Example 3 In the case of culturing while controlling the pH in the same manner as in Example 2, the effect of the set value of culture medium pH on the maximum yield of lactooligosaccharide was investigated. The results are shown in Table 2.

Table 2 Medium pH Maximum galactooligosaccharide yield (%2.0
0 2.5 0 3.0 61 3.5 68 3.75 67 4.0 - 67 4.5 68 S, 0 69 6.0 66 7.0 58 8.0 0 Example 4 Lactose 10%, yeast 100ml liquid medium with 0.75% extract
1 was placed in a 500 ml Erlenmeyer flask, and Brella singularis YIT, which had been precultured and washed with physiological saline, was added to the flask.
Inoculate a certain amount of 8243 cells. Thereafter, it is incubated at 27°C using a rotating shaking culture *a (pH control is not performed).

When the above culture was carried out by variously changing the initial pH of the medium, the maximum yield of lactooligosaccharide and the pH at which the maximum yield was reached were as shown in Table 3.

Table 3 Initial pH of medium Maximum oligosaccharide yield (%) p when maximum yield is reached
H2, OXi −2・5 ×1 −3.0
30,4 2.43.5
41.1 2.63.75
45,8 2.74, OS 2
．． 4 3.04.5 60.
2 3.35.0 59.9
3.45.5 56,7
3.66.0 60,6
3.86.5 58,1
4.27.0 54,5
4.68.0 *2
-9.0 *1
-×1 Bacteria do not grow and oligosaccharides are not produced *2 Bacteria grow very slowly and hardly any oligosaccharides are produced

[Brief explanation of drawings]

FIG. 1 is a graph showing changes in culture medium composition over time in Example 1.

Claims (3)

[Claims] (1) From lactose, the general formula Gal-(Gal)n-Glc(
However, in the formula, Gal represents a lactose residue, Glc represents a glucose residue, and n represents an integer from 1 to 4, respectively). A method for producing a Bifidobacterium growth promoting factor, which comprises culturing in a medium containing lactose while maintaining the pH of the medium at 3 to 7, and collecting the oligosaccharide from the culture. (2) The manufacturing method according to claim 1, wherein the culture is carried out within a pH range of 3.5 to 6. (3) Brella singularis YIT824 as yeast
3 (KAIKOKEN BIYORI NO. 8677).