JP3072670B2 - Method for producing galactooligosaccharide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing galactooligosaccharides. Galactooligosaccharides have a function as a growth promoter for bifidobacteria, and therefore have high utility as a functional food material and as a feed additive for preventing diarrhea and loose stool in livestock.

[0002]

2. Description of the Related Art As a method for producing galactooligosaccharides by using lactose as a raw material and reacting with an enzyme or a microorganism, a filamentous fungus Aspergillus oryzae is used.
ae) using β-galactosidase (JP-B-58)
-20266), a method using β-galactosidase of bacteria Bacillus circulans (Agric. Biol. Chem., Vol. 48, p. 3053, 1984).
Years), genus Lipomyces, Rhodotorra
torula), Kluyveromyces,
There is a method using a quiescent cell of yeast such as the genus Debaryomyces (JP-A-63-185373). Also, the yeast Cryptococcus lorentii (Crypt
It is also known that ococcus laurentii) is cultured in a medium containing lactose to produce and accumulate galactooligosaccharides in the culture solution (Fermentation Engineering, Vol. 66, p. 225, 1988).

However, in the method using β-galactosidase or quiescent cells, glucose produced as a by-product during the reaction inhibits the galacto-oligosaccharide production reaction, so that the galacto-oligosaccharide production yield is reduced to 30 to 40%. There is.

In the method of culturing yeast in a medium containing lactose and producing and accumulating galacto-oligosaccharides in the culture solution, the production of galacto-oligosaccharides by glucose is required to assimilate the by-product glucose into the cells and remove them from the reaction system. Inhibition is avoided, but lactose causes growth inhibition of bacterial cells if lactose is added to the medium at a high concentration from the beginning of culture when the bacterial cells are not sufficiently grown. Therefore, in this case, there is a drawback that a high concentration of lactose cannot be charged into the medium, and as a result, the yield of galacto-oligosaccharide increases but the accumulated concentration decreases.

[0005] Furthermore, the present inventors have cultured yeasts belonging to the genus Sterigmatomyces, Rhodotorula or Silobasidium and having the ability to produce galactooligosaccharides from lactose. A method for producing and accumulating galactooligosaccharides in a culture solution at a high concentration and a high yield by continuing the culture has been developed (Journal of the Japanese Society of Agricultural Chemistry, vol. 63, p. 309, 1989).
Year). However, also in this method, since the yeast extract and peptone having a high amino nitrogen content are contained as a component of the medium in a large amount, not only the cost of the medium is increased, but also these remain as impurities in the culture solution. Even when galacto-oligosaccharides can be produced at high concentration and high yield, there is a drawback that isolation and purification of galacto-oligosaccharides after cultivation are not easy and purification cost is high. Furthermore, since industrial waste liquid rich in organic nitrogen compounds is discharged, it is desirable to minimize the use of organic nitrogen compounds in the culture medium from the viewpoint of environmental protection. However, simply reducing the amount of peptone or yeast extract in the medium does not result in yeast cells having high galacto-oligosaccharide production ability, and galacto-oligosaccharides cannot be efficiently produced. Development of a method for producing galactooligosaccharides has been desired.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a medium having a low concentration of organic nitrogen compounds, that is, a medium having an amino nitrogen content of 0.01% or less, and having a high accumulation and a high yield.
An object of the present invention is to provide a novel production method for obtaining galactooligosaccharides with high purity.

[0007]

Means for Solving the Problems In view of the above-mentioned circumstances, the present inventors have conducted intensive studies and, as a result, belonged to the genus Sterigmatomyces, Rhodotorula, Silobasidium or Lipomyces, from lactose to galactooligosaccharides. Microorganisms having the ability to produce nitrocellulose with an amino nitrogen content of 0.01%
By culturing in a lactose-containing liquid medium to which iron ions, copper ions or zinc ions are added and a significant amount of galacto-oligosaccharide is produced and accumulated in the culture solution, and furthermore, the cells grown on the medium are They have a high galacto-oligosaccharide-producing activity and found that when they act on lactose or a lactose-containing substance in an aqueous medium, a considerable amount of galacto-oligosaccharide is produced and accumulated, thereby completing the present invention.

[0008] That is, the present invention relates to a microorganism belonging to the genus Sterigmatomyces, Rhodotorula, Silobasidium or Lipomyces and having the ability to produce galactooligosaccharides from lactose, having an amino nitrogen content of 0.01%.
A method for producing a galacto-oligosaccharide, which comprises culturing in a lactose-containing liquid medium to which iron ions, copper ions or zinc ions have been added, producing and accumulating galacto-oligosaccharides in the culture solution, and collecting the resultant. To provide.

Further, the present invention relates to a microorganism belonging to the genus Sterigmatomyces, Rhodotorula, Silobasidium or Lipomyces and having the ability to produce galacto-oligosaccharide from lactose, which has an amino nitrogen content of 0.01%. The following are cultured in a liquid medium to which iron ions, copper ions or zinc ions are added, and the grown cells are allowed to act on lactose or a lactose-containing substance in an aqueous medium to produce and accumulate galactooligosaccharides. It is intended to provide a method for producing a galactooligosaccharide, which is characterized by being collected.

As the microorganism used in the present invention, any microorganism can be used as long as it belongs to the genus Sterigmatomyces, the genus Rhodotorula, the genus Silobasidium or the genus Lipomyces and has the ability to produce galactooligosaccharides from lactose. Specific examples include the following. Sterigmatomyces eliviae (Steri gm atomyces)
elviae) CBS8119 Sterigmatomyces eliviae (Steri gm atomyces)
elviae) CBS5922 Sterigmatomyces eliviae (Steri gm atomyces)
elviae) CBS8120 Sterigmatomyces eliviae (Steri gm atomyces)
elviae) CBS8121 Rhodotorula minuta CBS4
407 Rhodotorula minuta CBS2
177 Rhodotorula marina IFO1
432 Rhodotorula marina CBS5
695 Shirobashidiumu Magnum (Sirobasidium m a gnum)
CBS6803 Shirobashidiumu Magnum (Sirobasidium m a gnum)
CBS6804 Shirobashidiumu Magnum (Sirobasidium m a gnum)
CBS6805 Shirobashidiumu Magnum (Sirobasidium m a gnum)
CBS6806 Lipomyces stark ey i JC
M5995 Lipomyces stark ey i JC
M5996 Lipomyces stark ey i JC
M5997 Lipomyces stark ey i JC
M5998

As a medium for culturing these microorganisms, carbon sources such as glucose, sucrose and lactose; inorganic nitrogen sources such as ammonia and ammonium sulfate; inorganic salts such as phosphate, magnesium salt and potassium salt; A liquid medium appropriately containing vitamins such as thiamine, paraaminobenzoic acid, etc. is used. Further, as a substitute for vitamins, organic nutrients such as yeast extract containing the same can be used. In this case, the addition concentration of the yeast extract may be 0.1% or less, and the amino nitrogen content in the medium is 0.01% or less.

The most important part in the present invention is to add a trace amount of metal ion, iron ion, copper ion or zinc ion to such a medium having an amino nitrogen content of 0.01% or less. Addition concentration of trace metal ions in the case of iron ions, 0.0 as FeSO ₄ · 7H ₂ O
1 to 1000 mg / l, in the case of copper ions, CuSO _4.
If the 5H ₂ O 0.01~100mg / l, of zinc ion, 0.1～1000M as ZnSO ₄ · 7H ₂ O
g / l.

As a method for producing and accumulating galacto-oligosaccharides in a culture using the above-mentioned medium, lactose or a lactose-containing substance as a raw material is added to the culture before or during the start of the culture, and the culture is performed. Just do it. In this case, if lactose is added to the medium at a high concentration from the initial stage of culture when the cells are not sufficiently grown, lactose causes growth inhibition of the cells, so lactose should be added after the cells have been grown to some extent. Is preferred. As a culture method, the pH of the culture solution is controlled to be in the range of 4.0 to 9.5 under aerobic conditions, and
Incubate at 40 ° C for 12 hours to 5 days. By culturing in this way, galacto-oligosaccharides are highly accumulated in the culture solution,
Obtained in high yield and high purity.

Since the cells grown by culturing using the above-mentioned medium have high galacto-oligosaccharide-producing activity, the cells are separated from the culture by centrifugation, ultrafiltration membrane or the like. It can be separated and acted on lactose or a lactose-containing substance in an aqueous medium to produce and accumulate galactooligosaccharides. In this case, the reaction may be carried out under aerobic conditions using the same medium as described above, while performing the growth and metabolism of the microbial cells, or the reaction may be carried out under static conditions. Further, these microbial cells can be immobilized on a carrier and used in the reaction as immobilized growth cells.

In the above reaction, the concentration of lactose or a lactose-containing substance in the aqueous medium is not particularly limited, but lactose is 100 to 700 g / l, preferably 25 to 500 g / l.
Range. The reaction temperature is usually 20 to 40 ° C., preferably 25 to 30 ° C. when the reaction is carried out while performing the growth and metabolism of the microbial cells. Is carried out usually at 40 to 90C, preferably 50 to 70C. The reaction pH is in the range of 2 to 8, preferably 3 to 7. Thus, by performing the reaction for 2 hours to 10 days, galacto-oligosaccharide can be obtained with high accumulation, high yield, and high purity in the reaction solution.

When the reaction is carried out under aerobic conditions while the growth and metabolism of the microbial cells are carried out, it is possible to prepare cells having high galacto-oligosaccharide-producing ability simultaneously with the production of galactooligosaccharides. The subsequent cells can be repeatedly used for the galactooligosaccharide production reaction.

From the culture solution after the completion of the culture or the reaction solution after the completion of the reaction, the bacterial cells are separated, if necessary, and then subjected to chromatography such as ion-exchange resin, gel filtration, activated carbon adsorption, etc., to convert the galactooligosaccharide. Can be purified.

The galactooligosaccharide thus obtained is mainly composed of trisaccharide and tetrasaccharide, and its binding mode is 3
Sugar is Galβ1 → 4Galβ1 → 4Glc, 4 sugars are Ga
1β1 → 4Galβ1 → 4Galβ1 → 4Glc (Gal indicates a galactose residue, Glc indicates a glucose residue).

[0019]

The present invention will be described in more detail with reference to the following examples. In the examples, the amount of galacto-oligosaccharide, residual lactose and by-product glucose was determined by high performance liquid chromatography (pump: Model 655, manufactured by Hitachi, Ltd., detector: Model SE-51, manufactured by Showa Denko, column: Sh manufactured by Showa Denko).
Odex-801, column temperature: 80 ° C., solvent: water, flow rate: 0.7 ml / min).
(Retention time: galactooligosaccharide trisaccharide: 16.1 minutes, galactooligosaccharide tetrasaccharide: 15.0 minutes, lactose: 17.8 minutes, glucose: 21.0 minutes)

[0020] Example 1 Table medium 1 are shown the composition (amino nitrogen content in the medium 0.0035%) to _{FeSO 4 · 7H 2 O, CuSO} 4 ·
5H ₂ O and ZnSO ₄ · 7H ₂ O, respectively 15 mg /
The resulting mixture was dispensed into a 500 ml flask, and sterilized by heating at 115 ° C. for 15 minutes. Sterigmatomyces eliviae CBS8119, Rhodotorula minuta CBS4407, and Silobasidium obtained by previously culturing on a Malt extract agar medium at 30 ° C. for 3 days.
Inoculate one platinum loop of the cells of Magnum CBS6803 or Lipomyces starky JCM5995,
C., shaking culture for 3 days. Then 20g in each flask
Lactose was added, and the culture was further continued.

[0021]

[Table 1]

Table 2 shows the growth amount of the cells immediately before lactose input and the amount of galactooligosaccharide produced and accumulated in the culture solution 70 hours after lactose input. As a comparative control, FeSO
_{4 · 7H 2 O, CuSO 4} · 5H 2 O and ZnSO ₄ · 7H ₂
When the same culture was performed in a medium without adding O,
_{4 · 7H 2 O, CuSO 4} · 5H 2 O and ZnSO ₄ · 7H ₂
10 g of polypeptone (manufactured by Nippon Pharmaceutical) instead of O
1 and the results when the yeast yeast extract S (manufactured by Nippon Pharmaceutical Co., Ltd.) was added at 9 g / l and cultured in a medium having an amino nitrogen content of 0.1% were also shown. The growth of the cells was determined by measuring the absorbance at 562 nm of a 26-fold dilution of the culture solution.

[0023]

[Table 2]

As is clear from the results, as compared with the case of culturing in a medium having an amino nitrogen content of 0.1%, a medium having an amino nitrogen content of 0.01% or less has an iron ion,
The amount of galacto-oligosaccharide accumulated in the culture solution obtained without the addition of copper ions and zinc ions was reduced, but by adding iron ions, copper ions and zinc ions to the medium and culturing, almost the same amount was obtained. Galactooligosaccharides were produced and accumulated in the culture solution. In addition, the amount of bacterial cell growth immediately before lactose input was not significantly different between the case where iron ion, copper ion and zinc ion were added and the case where iron ion, copper ion and zinc ion were not added. It can be seen that the effect is not to promote cell growth but to increase the galacto-oligosaccharide-forming activity of the grown cells.

[0025] Example 2 Table medium 3 shows the composition of each metal ion (amino nitrogen content 0.0035% in the medium) FeSO ₄ · 7H
15 mg / l of each of ₂ O, CuSO ₄ .5H ₂ O, and ZnSO ₄ .7H ₂ O was added alone or in combination, and 50 ml was dispensed into a 500 ml flask and 115 ml.
Heat sterilization at 15 ° C. for 15 minutes. One platinum loop of the cells of Sterigmatomyces eliviae CBS8119 obtained by previously culturing on a Malt extract agar medium at 30 ° C. for 3 days was inoculated, and cultured with shaking at 30 ° C. for 3 days. After completion of the culture, the cells were collected by centrifugation and washed once with 100 mM phosphate buffer (pH 6.0). The wet cells were suspended in an equal volume of the same buffer to obtain an enzyme cell solution.

[0026]

[Table 3]

Next, 5 ml of each enzyme cell solution was added to 45 ml of 100 mM phosphate buffer (pH 6.0) in which 10 g of lactose was dissolved.
The reaction was carried out at 60 ° C. for 2 hours.
Table 4 shows the amount of galactooligosaccharide produced and accumulated in the reaction solution.

[0028]

[Table 4]

As is clear from this table, the galacto-oligosaccharide-forming activity of the grown cells increased by culturing in a medium to which iron ions, copper ions, and zinc ions were added alone or in combination. Although the addition of each metal ion alone is effective, a more preferable result is obtained by adding them in combination.

The medium FeSO ₄ · 7H ₂ O having compositions shown in Example 3 in Table 5, CuSO
₄ · 5H ₂ O, and the concentration added alone ZnSO ₄ · 7H ₂ O, respectively, by dispensing 50ml min to 500ml flask 1
The mixture was sterilized by heating at 15 ° C. for 15 minutes. Sterigmatomyces eliviae CBS in this medium in the same manner as in Example 2.
8119 was cultivated, and each enzyme cell fluid was prepared to perform a galactooligosaccharide production reaction. The amount of galactooligosaccharide produced and accumulated in the reaction solution by the reaction at 60 ° C. for 2 hours was as shown in Table 6.

[0031]

[Table 5]

[0032]

[Table 6]

Example 4 A medium having the composition shown in Table 7 was used in the same manner as in Example 2 to obtain Sterigmatomyces eliviae CBS5922,
CBS8120, CBS8121, Rhodotorula minuta CBS4407, CBS2177, Rhodotorula Marina IFO1432, CBS5695, Silobasidium magnum CBS6803, CBS6804, CB
S6805, CBS6806, Lipomyces starky JCM5995, JCM5996, JCM5997, and JCM5998 were prepared. Using this, a galacto-oligosaccharide production reaction was carried out in the same manner as in Example 2 at 60 ° C. for 2 hours. For comparison,
_{FeSO 4 · 7H 2 O, CuSO} 4 · 5H 2 O, ZnSO 4
· 7H performed similarly cultured in a medium of any additive-free ₂ O, and subjected to reaction using ferments body fluid prepared. The results are shown in Table 7.

[0034]

[Table 7]

[0035]

[Table 8]

As is clear from these results, the amount of galacto-oligosaccharides produced by the obtained cells was remarkably improved by culturing each of the strains by adding iron, copper and zinc metal ions to the medium. .

Example 5 A medium having the composition shown in Table 9 was added to a 500 ml flask in a volume of 50 m.
and sterilized by heating at 115 ° C. for 15 minutes. Sterigmatomyces eliviae CBS8119 previously obtained by culturing the mixture on a Malt extract agar medium at 30 ° C. for 3 days.
Was inoculated with one loopful of the cells and cultured with shaking at 30 ° C. for 3 days. After completion of the culture, the cells were collected by centrifugation,
Washed once with M phosphate buffer (pH 6.0). The cells were suspended in an equal volume of the same buffer to obtain an enzyme cell solution.

[0038]

[Table 9]

A substrate solution having the composition shown in Table 10 was prepared, and 50
Dispense 45 ml into a 0 ml Sakaguchi flask at 115 ° C, 1
Heat sterilization for 5 minutes. To this was added 5 ml of the above enzyme cell solution, and the mixture was shaken at 30 ° C. to perform a galactooligosaccharide formation reaction. The pH was adjusted to 6.0 with hydrochloric acid or aqueous potassium hydroxide solution every 12 hours.

[0040]

[Table 10]

After reacting for 70 hours, the cells were collected by centrifugation and washed once with 100 mM phosphate buffer (pH 6.0). The cells were suspended in an equal volume of the same buffer, and this was successively repeated as an enzyme cell solution to carry out a reaction.

Table 7 shows the sugar composition of the reaction solution when the galactooligosaccharide formation reaction was repeated every 70 hours. In the method of the present invention, no decrease in galacto-oligosaccharide-producing ability was observed even when yeast cells were repeatedly used.

[0043]

[Table 11]

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ identification code FI (C12P 19/00 C12R 1: 645) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12P 19/00-19 / 64 A23L 1/30 C12N 1/00-1/38 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A microorganism belonging to the genus Sterigmatomyces, Rhodotorula, Silobasidium or Lipomyces and having the ability to produce galactooligosaccharides from lactose, having an amino nitrogen content of 0.01% or less; A method for producing galactooligosaccharides, comprising culturing in a lactose-containing liquid medium to which iron ions, copper ions or zinc ions have been added, producing and accumulating galactooligosaccharides in the culture solution, and collecting the galactooligosaccharides. 2. A microorganism belonging to the genus Sterigmatomyces, Rhodotorula, Silobasidium or Lipomyces and having the ability to produce galactooligosaccharides from lactose, having an amino nitrogen content of 0.01% or less; Culture in a liquid medium supplemented with iron ions, copper ions or zinc ions, and allowing the grown cells to act on lactose or lactose-containing substances in an aqueous medium to produce and accumulate galacto-oligosaccharides, and to collect these. A method for producing a galactooligosaccharide.