Abstract:
A galactose transfer product is prepared by a process of allowing a microorganism capable of producing a galactose transfer product of the formula: (Gal) n  --R, wherein Gal represents a galactose residue, n represents an integer of 1 to 4 and R represents a galactose receptor to act on a combination of lactose or a galactose donor and a galactose receptor; and collecting the galactose transfer product produced.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a method for producing a galactose transfer product. 
     2. Description of the Background 
     In recent years, attention has been directed to compounds which contain a galactose residue, especially transfer products of sugar (especially lactose), which can be used as a factor for the proliferation of Bifidobacteria (BIFIDUS, Vol. 2, No. 2, 1989). In addition, a change in physical properties such as increased solubility, or the like is noted in transfer products to sugar alcohols, nucleosides, alcohols, and the like. These transfer products are of high utilization value in their application to drugs, and the like. 
     A method which is known for producing galactose transfer products uses β-galactosidase of E. coli. In the method the galactose residue is transferred to fructose or N-acetylglucosamine (Biotechnology Letters, 9, 243 (1987)) or the galactose residue is transferred to nucleosides such as adenosine, or the like. (Journal of Agricultural Association, Japan, 48, 605 (1979)). However, these methods have the disadvantage that the yield of transfer products production is poor. Another method for producing galactose transfer product (galacto-oligosaccharide), wherein the galactose donor is lactose and the galactose receptor is lactose, is known in which the β-galactosidase of Aspergillus oryzae is used (Published Examined Japanese patent application no. 58-20266). However, the method is not very practical since the yield of galacto-oligosaccharide is poor. A need therefore continues to exist for an improved method of producing a galactose transfer product. 
     SUMMARY OF THE INVENTION 
     Accordingly, one object of the present invention is to provide a method of preparing a galactose transfer product in enhanced yields which is industrially acceptable. 
     Briefly, this object and other objects of the invention as hereinafter will become more readily apparent can be attained in a process of preparing a galactose transfer product by allowing a microorganism capable of producing a galactose transfer product of the formula: (Gal) n  --R, wherein Gal represents a galactose residue, n represents an integer of 1 to 4 and R represents a galactose receptor to act on a combination of lactose or a galactose donor and a galactose receptor; and 
     collecting the galactose transfer product produced. 
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     In the investigation leading to the present invention a survey was made for microorganisms which are capable of producing and accumulating galactose transfer product from lactose or a galactose donor such as o-nitrophenyl-β-D-galactopyranoside, or the like, and a galactose receptor such as sugar, a sugar alcohol, nucleosides, alcohols or derivative thereof, in a high yield at a high rate of accumulation. As a result, it has been found that microorganism strains belonging to the genus Rhodotorula, the genus Sterigmatomyces, the genus Cryptococcus, the genus Geotrichum, the genus Apiotrichum, the genus Corynebacterium, the genus Bacillus, the genus Flavobacterium, the genus Rhizobium or the genus Sirobasidium can produce galactose transfer products from lactose or a galactose donor such as o-nitrophenyl-β-D-galactopyranoside, or the like and a galactose receptor such as sugar, a sugar alcohol, a nucleoside, an alcohol or derivatives thereof, at an extremely high rate of accumulation and yield. 
     Specific examples of the microorganisms include i Rhodotorula minuta IFO 879, Sterigmatomyces elviae AJ 14199 (FERM BP-2586,FERM P-10001), Cryptococcus laurentii IFO 609, Geotrichum amycelicum AJ 14196 (FERM P-10071), Apiotrichum humicola ATCC 14438, Corynebacterium michiganense ATCC 492, Bacillus megaterium AJ 1272 (FERM P-3747), Flavobacterium aurantianum AJ 2462 (FERM.P-10069), Rhizobium meliloti AJ 2823 (FERM P-8197) and Sirobasidium magnum CBS 6803. 
     For the culturing of these microorganisms, any nutrient source is usable as long as it can be assimilated by the microorganism. Appropriate culture media can be formulated with, for example, carbohydrates such as glucose, sucrose, and the like; alcohols such as ethanol, glycerol and the like; organic acids such as acetic acid, propionic acid, and the like; carbon sources such as soybean oil, and the like or a mixture thereof; nitrogen-containing inorganic or organic nutrient sources such as yeast extract, peptone, meat extract, corn steep liquor, ammonium sulfate, ammonia, and the like; inorganic nutrient sources such as phosphates, magnesium, iron, manganese, potassium, and the like; vitamins such as biotin, thiamine, and the like. For culturing, the pH of the nutrient medium should be within the range of from 4.0 to 9.5 and culturing is carried out aerobically at a temperature ranging from 20° to 40° C. for 12 hours to 5 days. 
     In order to produce the galactose transfer product, the culture medium is supplemented with lactose or galactose donors such as o-nitrophenyl-β-D-galactopyranoside, or the like and galactose receptors such as sugar, sugar alcohols, nucleosides, alcohols and derivatives thereof, or the like at the initial stage of culturing or during the culturing. Alternatively, the galactose transfer product may be produced using resting microorganisms. 
     A method which uses resting microorganisms simply employs a culture solution as is. Another method comprises isolating cells by centrifugation, or an equivalent technique, resuspending the cells in phosphate buffer or equivalent, further adding lactose or a galactose donor such as o-nitrophenyl-β-D-galactopyranoside, or the like and a galactose receptor such as sugar, sugar alcohol, a nucleoside, alcohol or derivative thereof, to the suspension and then reacting these ingredients. The microorganisms may be viable cells or the cells may have been subjected to treatment with acetone or may have been subjected to freeze drying. Furthermore, the microorganism may also have been immobilized on a carrier or it may have been used in a bioreactor utilizing an ultrafiltration membrane. 
     In the reaction in which the galactose transfer product is formed from lactose or a galactose donor such as o-nitrophenyl-β-D-galac-topyranoside, or the like and a galactose receptor such as sugar, a sugar alcohol, a nucleoside, an alcohol or derivatives thereof, the amounts of lactose or the-galactose donor and the galactose receptor employed are not particularly limited, but the amount is in a range of 0.5 to 70 wt. %, preferably in a range of 1 to 30 wt. %, when calculated as lactose. When o-nitrophenyl-β-D-galactopyranoside, the amount ranges from 0.2 to 10 wt. %, preferably 0.5 to 2 wt. %. When calculated as the sugar, sugar alcohol, nucleoside, alcohol or derivatives thereof, the amount ranges from 0.1 to 50 wt. %, preferably 1.0 to 30 wt. %. The reaction is carried out at a temperature of generally 20° to 70° C., preferably 25° to 65° C. at a pH range of from 2 to 10, preferably  3 to 7, for 2 hours to 10 days. 
     After the reaction is completed, if necessary, the cells are removed, and the galactose transfer product is isolated from the reaction solution by a technique using a exchange resin, by gel filtration, by adsorption with activated charchoal, by chromatography or the like. The galactose transfer product can then be purified. 
    
    
     Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified. 
     Lactose and galactose transfer product were quantitatively analyzed for by determination of the peak area of a high performance liquid chromatograms (pump: Model 655 manufactured by Hitachi Ltd., detector: SE-51 manufactured by Showa Denko Co., Ltd.; column: Shodex-S801; solvent:water). 
     EXAMPLE 1 
     Into a flask of a 500 ml volume was charged 50 ml of a medium (pH 7.0) containing 1.0 g/dl of lactose, 1.0 g/dl of glycerol, 1.0 g/dl of yeast extract, 1.0 g/dl of polypeptone, 0.5 g/dl of (NH 4 ) 2  SO 4 , 0.3 g/dl of K 2  HPO 4 , 0.1 g/dl of KH 2  PO 4  and 0.05 g/dl of MgSO 4 .7H 2  O. The medium was then sterilized at 115° C. for 15 minutes. A plurality of such media were each inoculated with a platinum loop of one of the microorganism strains of Rhodotorula minuta IFO 879, Sterigmatomyces elviae AJ 14199 (FERM BP-2586, FERM P-10001), Cryptococcus laurentii IFO 609, Geotrichum amycelicum AJ 14196 (FERM P-10071), Apiotrichum humicola ATCC 14438 and Sirobasidium magnum CBS 6803,  each of which had been cultured in a malt extract agar medium at 25° C. for 2 days, and Corynebacterium michiganense ATCC 492, Bacillus megaterium AJ 1272 (FERM P-3747), Flavobacterium aurantianum AJ 2462 (FERM P-10069) and Rhizobium meliloti AJ 2823 (FERM P-8197), which had been cultured in a bouillon agar medium at 30° C. for 24 hours. The inoculated media were then cultured with shaking at 30° C. for 2 days in the cases of cultures of IFO 879, AJ 14199 (FERM BP-2586, FERM P-10001), IFO 609, AJ 14196 (FERM P-10071), ATCC 14438 and CBS 6803, and for 24 hours in the cases of cultures of ATCC 492, AJ 1272 (FERM P-3747), AJ 2462 (FERM P-10069) and- AJ 2823 (FERM P-8197). 
     The bacterial cells were resuspended in 50 ml of substrate solution (in 50 mM phosphate buffer, pH 7.0) supplemented with 2.5 g of lactose and 5 g each of the sugars shown in Table 1. Reaction was allowed to proceed at 50° C. for 24 hours. The amounts of galactose transfer product in each of the reaction solutions are shown in Table 1. 
     
                                           TABLE 1__________________________________________________________________________Amount of Galactose Transfer Product of Each Sugar (total (GAL)n - R)(g/dl)__________________________________________________________________________                                         Rham-      Lactulose           Raffinose                Melibiose                     Galactose                           Ribose                               Arabinose                                     Xylose                                         nose__________________________________________________________________________Rhodotorula      1.7  1.2  1.2  1.4   1.5 1.2   1.0 0.8minuta IFO 879Sterigmatomyces      1.8  0    1.3  1.4   1.6 1.2   1.1 0.8elviae AJ 14199(FERM BP-2586)(FERM P-10001)Cryptococcus      1.2  0    0    0.3   0.4 0     0.3 0laurentii IFO 609Geotrichum 1.4  0.4  0.9  0.2   0.2 0.2   0   0amycelicum AJ 14196(FERM P-10071)Apiotrichum      1.1  0.3  0.7  0.2   0.2 0     0   0humicolaATCC 14438Sirobasidium magnum      1.6  0.5  1.3  1.4   1.2 1.1   1.0 0.6CBS 6803Corynebacterium      1.0  0.6  0.8  0.2   0.3 0.6   0.8 0michiganensisATCC 492Bacillus megaterium      0.8  0.1  0.3  0.2   0.3 0     0   0AJ 1272(FERM P-3747)Flavobaterium      0.8  0.4  0.4  0.2   0.3 0.5   0.6 0.1aurantianum AJ 2462(FERM P-10069)Rhizobium meliloti      0.8  0.5  0.8  0.2   0.2 0.7   0.7 0.2AJ 2823(FERM P-8197)__________________________________________________________________________           2-Deoxy-  N-acetyl-   α-Methyl-                                       α-Methyl-           glucose                Sorbose                     glucosamine                            Fructose                                 glucoside                                       mannoside__________________________________________________________________________Rhodotorula     0.7  0.9  0.4    0.3  0.5   0.4minuta IFO 879Sterigmatomyces 0.8  1.0  0.5    0.2  0.6   0.5elviae AJ 14199(FERM BP-2586)(FERM P-10001)Cryptococcus    0.2  0.2  0      0.1  0     0laurentii IFO 609Geotrichum      0.2  0.3  0      0.1  0     0amycelicum AJ 14196(FERM P-10071)Apiotrichum     0.2  0.3  0      0    0     0humicolaATCC 14438Sirobasidium magnum           0.5  0.8  0.3    0.3  0.3   0.3CBS 6803Corynebacterium 0.3  0.3  0      0.1  0     0michiganensisATCC 942Bacillus megaterium           0.2  0.2  0      0    0     0AJ 1272(FERM P-3747)Flavobaterium   0.3  0.3  0      0    0     0aurantianum AJ 2462(FERM P-10069)Rhizobium meliloti           0.4  0.3  0      0    0     0AJ 2823(FERM P-8197)__________________________________________________________________________ 
    
     EXAMPLE 2 
     The cells were prepared in a manner similar to the procedure of Example 1. The prepared cells were resuspended in 50 ml of substrate solution (in 50 mM phosphate buffer, pH 7.0) supplemented with 2.5 g of lactose and 5 g each of the sugar alcohols and alcohols shown in Table 2. Reaction was allowed to proceed at 50° C. for 24 hours. The amounts of galactose transfer product in the reaction solutions are shown in Table 2. 
     
                       TABLE 2______________________________________Amount of Galactose Transfer Product to Each SugarAlcohol and Alcohol (total (GAL)n - R) (g/dl)         Ino- Sor-   Mann-         Gly-         sitol              bitol  itol    Xylitol                                   cerol______________________________________Rhodotorula     2.2    2.1    2.3   1.2   0.9minuta IFO 879Sterigmatocymes elviae           2.4    2.2    2.4   1.3   0.8AJ 14199 (FERM BP-2586)(FERM P-10001)Cryptococcus    0.6    0.5    0.5   1.0   0.6laurentii IFO 609Geotrichum amycelicum           0.4    0.4    0.5   0.4   0.2AJ 14196 (FERM P-10071)Apiotrichum humicola           0      0.1    0.1   0.3   0.2ATCC 14438Sirobasidium magnum           2.0    2.0    2.1   1.0   0.7CBS 6803Corynebacterium 0.2    0.3    0.2   0.3   0.4michiganensis ATCC 492Bacillus megaterium           0      0.1    0.1   0.2   0.2AJ 1272 (FERM P-3747)Flavobaterium aurantianum           0.2    0.2    0.2   0.3   0.4AJ 2462 (FERM P-10069)Rhizobium meliloti           0      0.1    0.2   0.3   0.5AJ 2823 (FERM P-8197)______________________________________ 
    
     EXAMPLE 3 
     The cells were prepared in a manner similar to the procedure of Example 1. The prepared cells were resuspended in 10 ml of substrate solution (in 50 mM phosphate buffer, pH 7.0) supplemented with 0.5 g of lactose and 1.0 g each of the nucleosides shown in Table 3. Reaction was allowed to proceed at 50° C. for 24 hours. The amounts of galactose transfer product in the reaction solutions are shown in Table 3. 
     
                                           TABLE 3__________________________________________________________________________Amount of Galactose Transfer Product to Each Nucleoside (total (GAL)n -R) (g/dl)                                   2&#39;-Deoxy-         Adenosine               Guanosine                     Inosine                         Cytidine                              Uridine                                   adenosine__________________________________________________________________________Rhodotorula   1.8   0.3   2.6 2.6  2.6  2.4minuta IFO 879Sterigmatomyces elviae         1.9   0.2   2.7 2.6  2.6  2.3AJ 14199 (FERM BP-2586)(FERM P-10001)Cryptococcus  1.3   0     1.5 1.6  1.5  1.4laurentii IFO 609Geotrichum amycelicum         1.2   0.1   0.6 1.0  0.9  0.6AJ 14196 (FERM P-10071)Apiotrichum humicola         0.4   0.2   0.5 0.3  0.3  0.3ATCC 14438Sirobasidium magnum         1.7   0.1   2.4 2.5  2.4  2.2CBS 6803Corynebacterium         1.3   0.1   1.0 1.3  1.1  0.4michiganensis ATCC 492Bacillus megaterium         0.6   0.1   0.5 0.2  0.3  0.2AJ 1272 (FERM P-3747)Flavobaterium aurantianum         1.2   0.2   0.8 1.2  1.0  0.6AJ 2462 (FERM P-10069)Rhizobium meliloti         1.1   0.2   0.9 1.1  0.8  0.5AJ 2823 (FERM P-8197)__________________________________________________________________________ 
    
     EXAMPLE 4 
     The cells were prepared in a manner similar to the procedure of Example 1. The prepared cells were resuspended in 10 ml of substrate solution (in 50 mM phosphate buffer, pH 7.0) supplemented with 0.15 g of o-nitrophenyl-β-D-galactopyranoside and 1.0 g each of the nucleosides shown in Table 4. Reaction was allowed to proceed at 50° C. for 20 hours. The amounts of galactose transfer product in the reaction solutions are shown in Table 4. 
     
                                           TABLE 4__________________________________________________________________________Amount of Galactose Transfer Product to Each Nucleoside (total (GAL)n -R) (g/dl)                                   2&#39;-Deoxy-         Adenosine               Guanosine                     Inosine                         Cytidine                              Uridine                                   adenosine__________________________________________________________________________Rhodotorula   0.7   0.2   1.3 1.3  1.3  1.3minuta IFO 879Sterigmatomyces elviae         0.7   0.2   1.4 1.4  1.4  1.3AJ 14199 (FERM BP-2586)(FERM P-1001)Cryptococcus  0.4   0.1   0.8 0.9  0.8  0.2laurentii IFO 609Geotrichum amycelicum         0.4   0.1   0.3 0.5  0.4  0.1AJ 14196 (FERM P-10071)Apiotrichum humicola         0.1   0.1   0.2 0.1  0.1  0.2ATCC 14438Sirobasidium magnum         0.6   0.2   1.2 1.3  1.3  1.3CBS 6803Corynebacterium         0.6   0.1   0.6 0.5  0.6  0.2michiganensis ATCC 492Bacillus megaterium         0.3   0.1   0.2 0.1  0.1  0.1AJ 1272 (FERM P-3747)Flavobaterium aurantianum         0.5   0.2   0.4 0.6  0.5  0.3AJ 2462 (FERM P-10069)Rhizobium meliloti         0.5   0.1   0.5 0.6  0.4  0.3AJ 2823 (FERM P-8197)__________________________________________________________________________ 
    
     EXAMPLE 5 
     The cells of Rhodotorula minuta IFO 879 were prepared in a manner similar to the procedure of Example 1. The prepared cells were resuspended in 50 ml of substrate solution (in 50 mM phosphate buffer, pH 7.0) supplemented with 2.5 g of lactose and 5 g inosine. Reaction was allowed to proceed at 50° C. for 48 hours. The amounts of galactose transfer product in the reaction solution are shown in Table 5. 
     
                       TABLE 5______________________________________Amount of Galactose Transfer Products Based on Inosine (g/dl)Number of Galac-      Number of Galactose                     Number of Galactosetose Residue: 1      Residue: 2     Residue: 3 or more______________________________________1.4        0.8            0.4______________________________________ 
    
     EXAMPLE 6 
     Into a flask of a 500 ml volume was charged 50 ml of a medium (pH 7.0) containing 10.0 g/dl of lactose, 2.0 g/dl of glycerol, 0.05 g/dl of yeast extract, 0.5 g/dl of (NH 4 ) 2  SO 4 , 0.3 g/dl of K 2  HPO 4 , 0.1 g/dl of KH 2  PO 4  and 0.005 g/dl of MgSO 4 .7H 2  O. The medium was then sterilized at 115° C. for 15 minutes. 
     Each of several samples of the medium was inoculated with a platinum loop of Rhodotorula minuta IFO 879, Sterigmatomyces elviae FERM P-10001 or Sirobasidium magnumCBS 6803, which had been cultured in a malt extract agar medium at 25° C. for 2 days. Each inoculated medium was then cultured with shaking at 30° C. for 4 days. The amount of the galacto-oligosaccharide product produced in each of supernatants in the culture solutions is shown in Table 6. 
     
                       TABLE 6______________________________________     Residual            Produced Galacto-oligosaccharide     Lactose            Trisaccharide                        Tetrasaccharide     (g/dl) (g/dl)      or more (g/dl)______________________________________Rhodotorula 0.8      4.2         2.6minuta IFO 879Sterigmatomyces       2.1      6.7         0elviaeFERM BP-2586FERM P-10001Sirobasidium       1.8      5.2         1.9magnum CBS 6803______________________________________ 
    
     EXAMPLE 7 
     Into a flask of a 500 ml volume was charged 50 ml of a medium (pH 7.0) containing 1.0 g/dl of lactose, 2.0 g/dl of glycerol, 1.0 g/dl of yeast extract, 1.0 g/dl of polypeptone, 0.5 g/dl of (NH 4 )SO 4 , 0.3 g/dl of K 2  HPO 4 , 0.1 g/dl of KH 2  PO 4  and 0.05 g/dl of MgSO 4 .7H 2  0. The thus prepared medium was sterilized at 115° C. for 15 minutes. 
     Each of several samples of the medium was then inoculated with a platinum loop of Rhodotorula minuta  IFO 879, Sterigmatomyces elviae FERM P-10001 or Sirobasidium magnum CBS 6803, which had been cultured in a malt extract agar medium for 2 days. Each inoculated medium was then cultured with shaking at 30° C. for 4 days. After completion of the culturing, the bacterial cells were collected from each medium by centrifugation and were then washed once with the same amount of physiological saline as the culture solution from which the cells had been collected. 
     The cells were resuspended in 50 ml of 22 g/dl of lactose solution (in 50 mM phosphate buffer, pH 7.0). Reaction was allowed to proceed at 50° C. for 2 days. The sugar composition of each reaction solution is shown in Table 7. 
     
                       TABLE 7______________________________________     Residual            Produced Galacto-oligosaccharide     Lactose            Trisaccharide                        Tetrasaccharide     (g/dl) (g/dl)      or more (g/dl)______________________________________Rhodotorula 9.6      8.4         1.7minuta IFO 879Sterigmatomyces       10.5     9.3         0elviaeFERM BP-2586FERM P-10001Sirobasidium       11.2     8.0         0.6magnum CBS 6803______________________________________ 
    
     EXAMPLE 8 
     Into a fermenter of a 5 liter volume was charged 2 l of a medium of pH 7.0 containing 1.0 g/dl of lactose, 2.0 g/dl of glycerol, 1.0 g/dl of yeast extract, 1.0 g/dl of polypeptone, 0.5 g/dl of (NH 4 ) 2  SO 4 , 0.3 g/dl of K 2  HPO 4 , 0.1 g/dl of KH 2  PO 4  and 0.05 g/dl of MgSO 4 .7H 2  O. Media prepared in this manner were inoculated with a sample of Rhodotorula minuta IFO 879, Sterigmatomyces elviae FERM P-10001 or Sirobasidium magnum CBS 6803. Each inoculated medium was then aerobically cultured at 30° C. for 36 hours with agitation at 500 rpm in an aerial amount of 1/2 vvm. Each culture solution was centrifuged to isolate the bacterial cells, which were used as an enzyme source. Then the following steps were performed. 
     Step (1): The resulting cells were suspended in 2 l of 36 g/dl of lactose solution (in 50 mM phosphate buffer, pH 6.0). Reaction was allowed to proceed at 30° C. while performing aerial agitation. 
     Step (2): After reaction for 3 days, the reaction solution was subjected to recycling filtration through an ultrafiltering polysulfone membrane (manufactured by Amicon Inc.) until the outer solution became 1000 ml. Then, the amount of the inner solution, the concentration of lactose and pH were adjusted to 2 l, 36 g/dl and 6.0, respectively. The operation of step (2) was repeated 4 times. The concentration of galacto-oligosaccharide product in each outer solution obtained by this operation is shown in Table 8. Ultrafiltration was carried out under a mean filtering pressure of 1 kg/cm 2  at a temperature of 30° C. 
     In each embodiment of the example, the mean filtering pressure and temperature of ultrafiltration were the same. 
     
                       TABLE 8______________________________________Concentration of Galacto-oligosaccharide in Outer Solution (g/dl)     Number of     Operation             First  Second  Third Fourth______________________________________Enzyme Source:Rhodotorula       21.6   22.6    21.2  21.3minuta IFO 879Sterigmatomyces   21.4   21.2    20.8  20.6elvaieFERM BP-2586FERM P-10001Sirobasidium      19.5   19.3    19.8  18.9magnum CBS 6803______________________________________ 
    
     Having now fully described the invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.