Process for preparing D-mannitol

A D-glucose solution is added molybdic acid compound thereto and heated at a temperature of 110.degree.-160.degree. C with pH 2.0-4.5 to perform epimerizing reaction of D-glucose, and thereby 30-36%, based on the D-glucose, of D-mannose is formed. To the epimerized mixture thus obtained is further added glucose-isomerase to perform enzymatic isomerizing reaction of D-glucose remaining in the epimerized mixture, thereby 46% based on the remaining D-glucose is converted into D-fructose. The epimerized mixture or enzymatically isomerized mixture is subjected to catalytic hydrogenation under high pressure, and thereby is produced D-mannitol at high yield based on the initial D-glucose.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a process for preparing D-mannitol. More 
particularly, the invention relates to a process for preparing D-mannitol, 
which comprises isomerization(s) of D-glucose to convert a portion of the 
D-glucose into D-mannose alone or into D-mannose and D-fructose, followed 
by catalytic hydrogenation of the resulting mixture of sugars under high 
hydrogen pressure. 
D-mannitol has recently come into wide use as the materials for medicines, 
foodstuffs and chemical products. Generally, D-mannitol occurs 
spontaneously in plant bodies, especially in marine plants. D-mannitol is 
also formed chemically through hydrogenation of D-mannose or D-fructose. 
The theoretical formation ratio of D-mannitol from D-mannose through the 
hydrogenation is 100%, while that of D-mannitol from D-fructose is 50% 
because of half mole of D-fructose used being transformed to D-sorbitol. 
(In this specification and claims, both "%" and "part" are on the dried 
matter basis). 
2. The Prior Art 
D-mannitol is currently prepared chemically in such a manner as 100 part 
sucrose is inverted (i.e., hydrolyzed) to form a mixture of 52 part 
D-glucose and 52 part D-fructose and the mixture is hydrogenated to 
transform it to a mixture of D-sorbitol and D-mannitol followed by 
crystallization of D-mannitol to separate therefrom. However, according to 
this process, formation ratio of D-fructose from the sucrose used is 52% 
at most, so that that of D-mannitol by the hydrogenation reduces, from the 
above-mentioned theoretical formation ratio (i.e., 50%) of D-mannitol from 
D-fructose, necessarily to 26% or below, resulting in the yield of 
crystalline D-mannitol being about 17% based on the initial material, 
because of a considerable amount of D-mannitol remaining inevitably in the 
mother liquor at the crystallizing step. 
In connection with the above, it is known that D-glucose is isomerized to 
convert a portion of it into D-fructose and/or D-mannose in the presence 
of an enzyme or base. The chemical formula of such isomerization of 
D-glucose is represented as below. (Among these isomerizing reactions of 
sugars, such ones as the conversion of D-glucose into D-mannose are 
specifically or restrictedly defined as epimerization.) 
##STR1## 
Known examples of the isomerization of D-glucose are as follows: When 
glucose-isomerase is added to an aqueous solution of 100 part D-glucose 
and the resultant mixture is warmed at a temperature of 65.degree. C for 3 
days, there is formed an equilibrium mixture of 54 part D-glucose and 46 
part D-fructose. There is further, when a saturated lime solution 
containing 100 part D-glucose is held at a temperature of 35.degree. C for 
5 days, formed an equilibrium mixture of 63.5 part D-glucose, 31.0 part 
fructose, 2.5 part D-mannose and 3.0 part impurities. As for the latter 
process, rise in the reaction temperature results in lowering of the 
formation ratio of D-fructose. 
Furthermore, it has been disclosed lately in Angew. Chem., 83(23), 967 
(1971) as well as specification of Czechoslovakian patent No. 149,051 that 
an equilibrium mixture of 75 part D-glucose and 25 part D-mannose is 
obtained without any formation of D-fructose, when 1 part molybdic acid is 
added to an aqueous solution of low concentration (i.e., about 17%) of 100 
part D-glucose and the resultant mixture is heated at a temperature of 
70.degree.-100.degree. C for 5-10 hours with pH 7.0 or below (any 
adjustment of pH values of the reaction systems is not however conducted 
in the examples of these literatures, while about pH 5 has been exhibited 
by the trace-experiments of such examples carried out by us). 
However, the equilibrium mixture(s) of D-glucose, D-fructose and/or 
D-mannose obtained by the isomerizations or epimerization, of D-glucose, 
as mentioned above, are all, from the content(s) of D-fructose and/or 
D-mannose thereof, expected to be, when submitted to the preparation of 
D-mannitol, only equivalent to or below the said mixture of 52 part 
D-glucose and 52 part D-fructose formed by the current chemical process, 
in point of the D-mannitol formation ratio therefrom based on the initial 
material. 
SUMMARY OF THE INVENTION 
We studied the isomerizing reaction of D-glucose, especially the 
epimerizing reaction of D-glucose according to the process of 
Czechoslovakian patent and have found that when this epimerization 
reaction is conducted under the conditions of a low pH and a high 
temperature, that is, of pH 2.0-4.5 and temperature of 
110.degree.-160.degree. C, the D-mannose formation ratio based on the 
D-glucose used is improved remarkably (e.g., to the extent of 30-36%) 
beyond the critical composition ratio of D-glucose and D-mannose (75 : 25) 
obtained by the said conventional process. Under such our reaction 
conditions as above, the epimerization reaction can also be effected with 
D-glucose solution of high concentration and a little amount of molybdic 
acid compound. Furthermore, the reaction period thereof is very short, for 
instance, less than 40 minutes. 
Thus, the mixture of D-glucose and D-mannose obtained in the above is 
subjected to the catalytic hydrogenation, thereby is formed D-mannitol at 
a high ratio such as 30-36% based on the initial D-glucose, which results 
in such a high yield of crystalline D-mannitol as 25-30%. 
OBJECTS OF THE INVENTION 
An object of the invention is accordingly to provide a process for 
preparing D-mannitol from D-glucose at high yield. 
Another object of the invention is to provide a process for preparing 
D-mannitol from D-glucose with a simplified operation. 
Still another object of the invention is to provide a process for preparing 
D-mannitol from D-glucose with a short time operation. 
The other objects and advantages of the invention will be apparent from the 
detailed explanation of the invention described hereinafter.

DESCRIPTION OF THE INVENTION 
Referring now to the drawings, FIG. 1 is a graph illustrating the 
relationship between the D-mannose formation ratio based on the D-glucose 
used and the reaction period, when to a 60% D-glucose aqueous solution is 
added ammonium molybdate in an amount of 0.2% based on the D-glucose and 
the resulting mixture is heated at a temperature of 130.degree. C, but 
with pH value varying from 1 to 7, and 
FIG. 2 is a graph similarly illustrating the relationship of D-mannose 
formation ratio-reaction period, when the mixture of 60% D-glucose aqueous 
solution and 0.2% of ammonium molybdate as above is adjusted to pH 3.0 
with dilute sulfuric acid and heated at a reaction temperature varying 
from 95.degree. C to 160.degree. C. 
According to the first step (i.e., epimerizing reaction of D-glucose) of 
the process of the present invention, a mixture of a D-glucose aqueous 
solution and molybdic acid compound is adjusted to pH 2.0-4.5, preferably 
to 3.0-4.0, and heated at a temperature of 110.degree.-160.degree. C, 
thereby the reaction is performed within a short time, e.g., about 40 
minutes, at high formation ratio of D-mannose based on the D-glucose 
material used. The reaction is further performed effectively even when a 
high concentration solution of D-glucose (e.g., 40-80%) and a little 
amount of molybdic acid compound are employed. 
Epimerization reaction of the present process as above will be probably 
promoted by the effect of co-operation (or synergism) of pH with 
temperature, of the reaction system, to give high formation ratio of 
D-mannose. With rise in the reaction temperature in the range of 
110.degree.-160.degree. C, the formation ratio of D-mannose increases and 
the reaction period is shortened, proportionally thereto. In FIG. 2, for 
instance, the formation ratio of D-mannose amounts to about 32% in about 
30 minutes at a reaction temperature of 120.degree. C; to about 34% in 
about 7 minutes at 140.degree. C; and to about 36% in about 4 minutes at 
160.degree. C, respectively. However, in case the temperature exceeds 
160.degree. C, the reaction system becomes deep in color by decomposition 
of the sugars contained therein. 
The required time for the epimerization reaction of the present process is 
so markedly shortened that the application of a continuous reaction system 
is readily attained to this reaction. For instance, a D-glucose solution 
added molybdic acid compound thereto is pumped continuously under pressure 
into a reaction pipe having a predetermined length from an end of the pipe 
and heated under pressure while it flows through the reaction pipe, 
thereby the finished solution is discharged continuously from the other 
end of the pipe. 
Values of pH of the epimerization reaction system of the present process 
also affect the formation of D-mannose remarkably, as can be seen from 
FIG. 1. When pH value exceeds 4.5 there appears lowering of the D-mannose 
formation ratio, while with pH below 2.0 it is observed that the solution 
becomes deep in color by decomposition of sugars. In order to adjust pH 
value of the reaction system, is used either inorganic acid such as 
hydrochloric acid, sulfuric acid or phosphoric acid, or organic acid such 
as oxalic acid. 
According to the process of epimerizing reaction of D-glucose described in 
specification of said Czechoslovakian patent, the reaction system was, as 
mentioned previously, heated at a temperature below 100.degree. C for 5-10 
hours, presumably with pH 5-7. Therefore, it will be understood that 
combination of the conditions of the epimerization reaction of the present 
process, that is, the combination of low pH of 2.0-4.5, high reaction 
temperature of 110.degree.-160.degree. C and short reaction period such as 
4-40 minutes constitutes a novel feature that has never been known. 
Molybdic acid compound used for the epimerization reaction of this process 
includes ammonium molybdate, sodium molybdate, potassium molybdate, 
molybdenum trioxide, molybdic acid, and the like. With increase in the 
amount of molybdic acid compound to be added, the reaction velocity 
becomes larger while the D-glucose material is more decomposed by 
side-reactions. Favourable amount of the molybdic acid compound used for 
the epimerization reaction of the present process is however only 
0.05-0.20% relative to the initial D-glucose which corresponds to about 
1/20 to 1/5 the amount of molybdic acid used for the conventional process. 
(In case of using however such D-glucose material containing much 
impurities as hydrol described hereinafter, the amount (%) of molybdic 
acid compound is calculated on the overall dried matter of the D-glucose 
material). Such a reduction of the amount of molybdic acid compound (i.e., 
catalyst) depends exclusively on the great difference between the 
conditions of the epimerization reaction of the present process and those 
of the conventional process. Reduction of the amount of the catalyst used 
is also advantageous in that the refining treatment thereafter becomes 
more facile. 
The D-glucose material for the epimerization reaction of the present 
process, includes crystalline D-glucose, starch hydrolyzate, so-called 
hydrol which is the concentrate of starch hydrolyzate from which 
crystalline D-glucose has been separated, and the like. As to an impure 
material of D-glucose such as hydrol, it is preferable to use that 
Dextrose Equivalent (hereinafter abridged to DE) of which is above 80. 
Finished solution of the epimerization reaction (or epimerized mixture) 
thus obtained is yellow or light brown in color and contains, for 
instance, 30-36%, relative to the D-glucose used, of D-mannose. The 
epimerized mixture is, for example, decolorized with a little amount of 
active carbon, and further refined by ion exchange resin treatment to give 
a colorless and transparent solution. This refined solution exhibits pH 
5.0-7.0. 
According to the second step of the process of the present invention, the 
epimerized mixture or its refined solution is subjected to the catalytic 
hydrogenation reaction under high pressure. For example, the refined 
solution is adjusted to have 50-70% dried matter, added thereto a 
hydrogenating catalyst such as nickel catalyst (e.g., Raney nickel) or 
platinum group catalyst (e.g., platinum, ruthenium or palladium) and 
heated, while agitated, at a temperature of 110.degree.-160.degree. C for 
1.0-2.5 hours under a hydrogen pressure of 100-200 kg./cm.sup.2. Dried 
matter composition of the finished solution of the hydrogenating reaction 
(or hydrogenated mixture) thus obtained is, when crystalline D-glucose 
have been employed as the material for the epimerization reaction, for 
instance, 70-64% D-sorbitol and 30-36% D-mannitol. When the epimerized 
mixture which is not refined undergoes the catalytic hydrogenation, the 
amount of the hydrogenating catalyst to be used is increased a little, 
thereby is obtained the D-mannitol formation ratio almost similar to that 
obtained by the epimerized mixture having been refined. 
The hydrogenated mixture is, for example, filtered to remove the catalyst, 
decolorized with active carbon, treated with ion exchange resins, and 
concentrated in vacuo followed by cooling to crystallize out D-mannitol. 
The yield of crystalline D-mannitol amounts, for instance, to 25-30% based 
on the initial D-glucose, which yield is superior greatly to that by the 
conventional process, that is, about 17%. 
Furthermore, when such an impure D-glucose material as hydrol containing 
oligosaccharides is used for the epimerization reaction of the present 
process, the subsequent (or later) hydrogenation reaction is 
advantageously effected by employing the reaction conditions of low pH 
such as 2-3 and high temperature such as 160.degree.-180.degree. C, 
thereby hydrogenation involving hydrolyzation, of the oligosaccharides, 
occurs so that the viscosity of the hydrogenated mixture formed lowers 
resulting in improvement in the yield of crystalline D-mannitol. 
Next, we further studied the possibility of enzymatic isomerization of the 
D-glucose which coexists with D-mannose in the epimerized mixture of the 
present process having been described above. We have firstly tested 
whether the isomerizing reaction to be caused by glucose-isomerase is not 
inhibited by the molybdic acid compound which has remained inevitably in 
the epimerized mixture. We have further tested the influence of the 
glucose-isomerase added thereto on the equilibrium formed between 
D-glucose and D-mannose in the epimerized mixture. 
As the result of the above studies, we have found that such enzymatic 
isomerization is, when the amount of the molybdic acid compound in the 
epimerized mixture has been below 0.4% relative to the dried matter of 
this mixture, effected normally so as to about 46%, at its maximum, based 
on the D-glucose remaining in the epimerized mixture be converted into 
D-fructose without being influenced by the coexisting D-mannose. To the 
contrary, in case the amount of the molybdic acid compound exceeds 0.4%, 
there appears reduction of the D-fructose formation ratio and extension of 
the period, of the isomerization reaction. Therefore, in other words, the 
enzymatic isomerization reaction of the present process can be conducted 
effectively by employing the epimerized mixture without any refining 
treatment of it but adjustment of pH, when the amount of molybdic acid 
compound in the epimerized mixture has been below 0.4%. This results in an 
advantage of the present process that the operation is simplified 
significantly. 
The enzymatic isomerization reaction of the present process is, for 
instance, carried out in such a manner as the epimerized mixture is 
adjusted to pH 6.5-8.5 and to have 50-65% dried matter and added a 
predetermined amount of glucose-isomerase thereto followed by warming the 
resultant aqueous mixture at a temperature of 65.degree.-75.degree. C for 
48-72 hours, while agitated gently and maintained at pH 6.5-8.5. Finished 
solution of the isomerization reaction (or isomerized mixture) is, for 
example, filtered to separate the enzyme, treated with active carbon and 
further with ion exchange resins to give a colorless, transparent 
solution. Refined solution obtained exhibits pH 5.0-7.0 and its dried 
matter composition is, when crystalline D-glucose have been employed as 
the material for the initial epimerization reaction, for instance, 39-35% 
D-glucose, 31-29% D-fructose and 30-36% D-mannose. 
Isomerization reaction of the present process may also be conducted by 
using the so-called immobilized enzyme system. For instance, 
glucose-isomerase is adsorbed to a suitable carrier, with which is filled 
a column having a predetermined length, and the epimerized mixture is made 
to flow continuously through the column at a suitable rate, thereby the 
remaining D-glucose in the epimerized mixture is isomerized enzymatically 
while has been retained in the column. 
To the glucose-isomerase to be used for the enzymatic isomerization 
reaction of the present process, are available all enzyme preparations of 
this kind being on the market, which have been prepared from the cultured 
materials of the microorganisms belonging to genus Streptomyces, 
Pseudomonos, Aerobacter, and the like. With increase in the amount of 
glucose-isomerase used, the reaction velocity of the isomerization becomes 
larger and the formation ratio of D-fructose is improved. For the 
industrial application however, it is preferable to use 0.5-2.0%, relative 
to the dried matter of the D-glucose material for the epimerization 
reaction, of an enzyme preparation having about 1,000 GIU/g. of enzyme 
activity, if 1 GIU is defined to represent such an enzyme activity that 
produces 1 mg. of D-fructose at a temperature of 70.degree. C in 60 
minutes. 
The refined solution of the isomerized mixture can be subjected to the 
catalytic hydrogenation in the similar manner to that applied to the 
refined solution of the epimerized mixture described previously. For 
instance, the refined solution of the present isomerized mixture is 
adjusted to have about 50-70% dried matter, and heated, while agitated, 
under hydrogen pressure in the presence of a hydrogenating catalyst. 
Composition of the dried matter of the hydrogenated mixture thus obtained 
is, when crystalline D-glucose have been used as the material for the 
epimerization reaction, for instance, 54-50% D-sorbitol and 46-50% 
D-mannitol. On refining and concentrating the hydrogenated mixture, 
crystalline D-mannitol is obtained at such a high yield as 40-45% based on 
the initial D-glucose. 
According to the process of the present invention, respective compositions 
of the epimerized mixture, the enzymatically isomerized mixture and the 
hydrogenated mixture, are determined quantitatively by means of gas liquid 
chromatography which has been recently applied widely to the separating 
estimation of components in a mixture of sugars and/or sugar alcohols. 
D-sorbitol and D-mannitol in the hydrogenated mixture of the present 
process are both estimated effectively after have been converted into 
hexaacetyl ester thereof. However, D-glucose and D-mannose in the 
epimerized mixture as well as D-glucose, D-mannose and D-fructose in the 
isomerized mixture are difficult to estimate separately, because the peaks 
either of them or of their derivatives on gas chromatograms have 
interfered with each other. Therefore, the compositions of such sugar 
mixtures are counted backward from the respective quantities of D-sorbitol 
and D-mannitol estimated by gas chromatography after these sugar mixtures 
having once been hydrogenated, applying the respective theoretical 
formation ratio(s) of D-sorbitol and/or D-mannitol from D-glucose, 
D-mannose or D-fructose mentioned previously. 
Dried matter of the D-glucose materials containing much impurities, such as 
hydrol, refers to that having been dried in vacuo at a temperature of 
95.degree.-100.degree. C for 16 hours. 
A flowsheet of the reaction steps of the present invention using 
crystalline D-glucose as the material is illustrated as below: 
##STR2## 
The following Examples serve to illustrate the invention without limiting 
it in any way: 
EXAMPLE 1 
3.3 kg. of crystalline D-glucose (with 9% crystal water content; 
hereinafter the same) being on the market is dissolved in 1.7 kg. of 
deionized water. The resulting solution is added thereto 6.0g. 
(corresponds to 0.20% relative to the D-glucose) of ammonium molybdate 
(NH.sub.4).sub.6 .multidot.Mo.sub.7 O.sub.24 .multidot.4H.sub.2 O, 
adjusted to pH 2.5 with a dilute sulfuric acid and heated in an autoclave 
of 10 liter-capacity, while agitated, at a temperature of 115.degree. C 
for 40 minutes, to perform the epimerizing reaction of the D-glucose. 
Epimerized mixture obtained exhibits pH 2.2 and is light yellow in color. 
After cooled, this mixture is decolorized with a little amount of active 
carbon, and passed in turn through two columns which have been filled 
respectively with 200 ml. of cation exchange resin (H.sup.+) (Trademark 
"Amberlite IR-120"; hereinafter the same) and with 400 ml. of anion 
exchange resin (OH.sup.-) (Trademark "Amberlite IRA-68"; hereinafter the 
same). Effluent discharged from the second column (or refined solution) 
exhibits pH 6.2, and is adjusted to have 60% dried matter. 
600 g. of the refined and adjusted solution as above is taken out, added 15 
g. of 5%-Ru-carbon (manufactured by Nippon Engelhard Co.,Ltd.) thereto, 
and heated in an autoclave of 1 liter-capacity, while agitated, at a 
temperature of 130.degree. C for 120 minutes under a hydrogen pressure of 
150 kg./cm.sup.2, to perform the hydrogenating reaction of sugars. 
Hydrogenated mixture obtained is filtered to remove the catalyst, 
decolorized with active carbon, and further refined by passing through two 
columns filled with 50 ml. of cation exchange resin (H.sup.-) and with 100 
ml. of anion exchange resin (OH.sup.-), respectively. Composition of the 
refined solution is estimated quantitatively by gas chromatography using 
an apparatus of SHIMAZU GC-4A type. Outline of the procedures is as 
follows: 
(a) Preparation of sample: A predetermined amount of the refined solution 
is evaporated and dried in vacuo to remove its moisture completely, added 
acetic acid anhydride and pyridine thereto followed by heating the 
resultant mixture under reflux for an hour to convert the sugar alcohols 
into hexaacetate esters thereof. To this hexaacetate esters is added a 
predetermined amount of di-2-ethylhexyl adipate as the internal standard, 
to thereby prepare a sample to be injected in the gas chromatograph. 
(b) Operating parameters: Column consisting of stainless steel pipe of 
3m.times.3mm.phi., which is packed with 1.5%QF-1 on Chromosorb W of 60-80 
mesh; Column temp. 200.degree. C; Detector temp. 250.degree. C; Carrier 
gas flow 70 ml./min.; H.sub.2 flow 50 ml./min.; Air flow 1.0 liter/min. 
(c) Preparation of standard curve: Groups of samples of accurately weighed 
pure D-sorbitol and D-mannitol are respectively acetylated and gas 
chromatographed in the same manner as has been mentioned above, and 
thereby are prepared the standard curves illustrating the relations 
between peak areas on the respective gas chromatograms and the 
corresponding weights of the sugar alcohols used. 
(d) Estimation of sugar alcohols: The sample prepared under item(a) 
undergoes gas chromatography with the paramaters under item(b), and 
respective peak areas on a gas chromatogram are measured, followed by 
comparing these values of areas with those on the standard curves prepared 
under item(c), thereby are determined the quantities of D-sorbitol and 
D-mannitol in the sample. 
As the result of the above, it is revealed that the formation ratio of 
D-mannitol in the hydrogenated mixture is 31.2% based on the initial 
D-glucose while that of D-sorbitol is 68.8%. 
The above refined solution of the hydrogenated mixture is concentrated in 
vacuo to a syrup, which is then cooled to crystallize out D-mannitol, the 
melting point of the crystals being 166.2.degree. C. The yield is 88.6 g., 
corresponding to 24.6% based on the initial D-glucose. 
EXAMPLE 2 
An aqueous solution of crystalline D-glucose is prepared in the same way as 
in Example 1. To this solution is added 4.5 g. (0.15% relative to the 
D-glucose) of ammonium molybdate, and the resulting solution is adjusted 
to pH 3.5 with a dilute sulfuric acid followed by heating it in an 
autoclave of 10 liter-capacity, while agitated, at a temperature of 
120.degree. C for 30 minutes. Epimerized mixture obtained exhibits pH 3.2 
and is light yellow in color. 
500 g. of the epimerized mixture is taken out, and undergoes the refining 
treatment in the similar manner to that applied to the epimerized mixture 
in Example 1. Refined solution obtained is subjected to the catalytic 
hydrogenation in the similar manner to that described hereinafter in this 
Example, and D-mannitol formed therein is estimated quantitatively in the 
same way as in Example 1. As the result, it is revealed that the formation 
ratio of D-mannitol in the hydrogenated mixture is 31.8% based on the 
initial D-glucose, said value being able to be regarded as showing on the 
other hand the formation ratio of D-mannose in the epimerized mixture. 
The remainder of the epimerized mixture is adjusted to pH 7.0 with a dilute 
sodium hydroxide solution, added thereto 30 g. of an enzyme preparation 
with Trademark "Glucose-Isomerase Nagase" (manufactured by Nagase & 
Co.,Ltd.; with enzyme activity of 1,000 GIU/g.), and warmed, while 
agitated gently and maintained at pH 6.5-7.0 during the reaction, at a 
temperature of 70.degree. C for 50 hours, to perform the enzymatic 
isomerizing reaction of the D-glucose remaining in the epimerized mixture. 
Isomerized mixture obtained is filtered to separate the enzyme, decolorized 
with active carbon, and passed through two columns filled respectively 
with 200 ml. of cation exchange resin (H.sup.+) and with 400 ml. of anion 
exchange resin (OH.sup.-). Refined solution obtained exhibits pH 6.2 and 
is adjusted to have 60% dried matter. 
550 g. of the refined and adjusted solution as above is taken out, added 20 
g. of Raney nickel thereto, and the resulting mixture is heated in an 
autoclave of 1 liter-capacity, while agitated, at a temperature of 
120.degree. C for 120 minutes under a hydrogen pressure of 150 
kg./cm.sup.2. 
Hydrogenated mixture obtained is, in the same manner as in Example 1, 
filtered and refined followed by the quantitative estimation of sugar 
alcohols by gas chromatography, and thereby it is revealed that the 
formation ratio of D-mannitol based on the initial D-glucose is 46.4% 
while that of D-sorbitol is 53.6%. The above refined solution is 
concentrated in vacuo to crystallize out D-mannitol, the melting point of 
the crystals being 166.0.degree. C. The yield is 133.0 g., corresponding 
to 40.3% on the initial D-glucose. 
On the other hand, the formation ratio of D-fructose in the enzymatic 
isomerized mixture is counted backward as 29.2%, from the values of 46.4% 
of D-mannitol formation ratio in the hydrogenated mixture and 31.8% of 
D-mannose formation ratio in the epimerized mixture. 
EXAMPLE 3 
3.8 kg. of crystalline D-glucose on the market is dissolved in 1.2 kg. of 
deionized water. The resultant solution is added 3.5 g. (0.10% relative to 
the D-glucose) of sodium molybdate Na.sub.2 MoO.sub.7 .multidot.2H.sub.2 O 
thereto, adjusted to pH 3.5 with a dilute sulfuric acid, and heated in an 
autoclave of 10 liter-capacity, while agitated, at a temperature of 
130.degree. C for 15 minutes. Epimerized mixture exhibits pH 3.2 and is 
light yellow in color. 
The said mixture is adjusted to pH 7.0, added 35 g. of "Glucose-Isomerase 
Nagase" thereto, and warmed, while agitated gently and maintained at pH 
6.5-7.0 during the reaction, at a temperature of 65.degree. C for 64 
hours. Isomerized mixture obtained undergoes the refining treatment in the 
similar manner to that applied to the isomerized mixture in Example 2. 
Refined solution exhibits pH 6.2 and is adjusted to have 60% dried matter. 
600 g. of the refined and adjusted solution is taken out, added 20 g. of 
Raney nickel thereto, and heated, while agitated, in an autoclave of 1 
liter-capacity at a temperature of 130.degree. C for 120 minutes under a 
hydrogen pressure of 150 kg./cm.sup.2. Hydrogenated mixture is, in the 
same way as in Example 1, filtered and refined followed by the 
quantitative estimation of sugar alcohols, thereby it is revealed that 
D-mannitol formation ratio based on the initial D-glucose is 47.5%. The 
refined solution is concentrated in vacuo to crystallize out D-mannitol, 
the melting point of the crystals being 165.8.degree. C. The yield is 
147.6 g., which corresponds to 41.0% on the initial D-glucose. 
EXAMPLE 4 
Aqueous solution of crystalline D-glucose which is prepared in the same way 
as in Example 1 is added 3.0g. (0.10% relative to the D-glucose) of 
ammonium molybdate thereto, and adjusted to pH 3.2 with a dilute sulfuric 
acid. The resulting solution is pumped continuously with a plunger pump, 
at a rate of 20 ml./min., into a stainless steel reaction pipe of 0.8 cm. 
internal diameter and 3m. length (i.e. 150ml.-capacity) provided with a 
pressure gauge as well as a discharge valve, and heated, while the 
solution flows through the pipe, at a temperature of 140.degree. C under a 
pressure of 3.0 kg./cm.sup.2, to thereby perform the continuous 
epimerization reaction of D-glucose. Retaining time of the material 
solution in the pipe is about 7.5 minutes. Finished solution (or 
epimerized mixture) is discharged continuously from the discharge valve, 
which exhibits pH 2.9 and is slightly colored. The epimerized mixture is 
adjusted to pH 6.0 with a dilute sodium hydroxide solution and to have 60% 
dried matter. 
550 g. of the mixture adjusted as above is taken out and subjected to the 
catalytic hydrogenation in the similar manner to that to the refined 
isomerized mixture in Example 3. The hydrogenated mixture obtained is 
refined, followed by the quantitative estimation of D-mannitol, and 
thereby it is revealed that the formation ratio of D-mannitol amounts to 
33.4% based on the initial D-glucose. The refined solution is concentrated 
in vacuo to crystallize out D-mannitol, the melting point of the crystals 
being 165.8.degree. C. The yield is 87.5 g., which corresponds to 26.5% on 
the initial D-glucose. 
EXAMPLE 5 
Aqueous D-glucose solution prepared in the same way as in Example 1 is 
added 1.5 g. (0.05% relative to D-glucose) of ammonium molybdate thereto 
and adjusted to pH 3.5 with dilute sulfuric acid. The resulting solution 
undergoes the continuous epimerization reaction of D-glucose, by employing 
the same apparatus as in Example 4, while adopting reaction conditions of 
a temperature of 160.degree. C, solution pumping rate of 35 ml./min., 
retaining time of the solution of about 4.3 minutes and a pressure in the 
reaction pipe of 6.2 kg./cm.sup.2. Epimerized mixture discharged 
continuously from the reaction pipe exhibits pH 3.1 and is slightly brown 
in color. This mixture is adjusted to pH 6.0 and to have 60% dried matter. 
550 g. of the mixture adjusted as above is taken out and subjected to the 
catalytic hydrogenation in the similar manner to that to the refined 
isomerized mixture in Example 3, but using 30 g. of Raney nickel. 
Hydrogenated mixture is refined, followed by the estimation of its 
D-mannitol formation ratio, thereby it is revealed such ratio amounts to 
35.4% based on the initial D-glucose. The refined solution is concentrated 
in vacuo to crystallize out D-mannitol, the melting point of the crystals 
being 165.6.degree. C. The yield is 93.1 g., corresponding to 28.2% based 
on the D-glucose. 
EXAMPLE 6 
4.12 kg. of crystalline D-glucose on the market is dissolved in 0.88 kg. of 
deionized water. The solution is added 3.8 g. (0.10% relative to the 
D-glucose) of ammonium molybdate thereto and adjusted to pH 3.2 with 
dilute sulfuric acid. The resulting solution undergoes the continuous 
epimerization reaction with the same apparatus and manner as in Example 4. 
Epimerized mixture discharged exhibits pH 2.9 and is slightly brown in 
color. This mixture is adjusted to pH 6.2 and to have 60% dried matter. 
550 g. of the mixture adjusted is taken out and subjected to the catalytic 
hydrogenation in the similar manner to that to the refined isomerized 
mixture in Example 3, but employing reaction period of 150 minutes. 
Hydrogenated mixture obtained is refined, followed by the estimation of 
its D-mannitol formation ratio, and thereby it is revealed such ratio 
amounts to 33.0% based on the initial D-glucose. This refined solution is 
concentrated in vacuo to crystallize out D-mannitol, the melting point of 
the crystals being 165.7.degree. C. The yield is 85.2 g., which 
corresponds to 25.8% based on the initial D-glucose. 
EXAMPLE 7 
5.0 kg. of starch hydrolyzate (DE 97; 40% water content) is added 6.0g. 
(0.20% relative to the dried matter of the starch hydrolyzate) of sodium 
molybdate thereto, adjusted to pH 3.2 and heated, while agitated, in an 
autoclave of 10 liter-capacity at a temperature of 120.degree. C for 35 
minutes. Epimerized mixture exhibits pH 2.9 and is slightly brown in 
color. 
500 g. of the mixture is taken out, and submitted to the determination of 
D-mannose formation ratio, applying correspondingly the determinating 
manner of D-mannose formation ratio in the epimerized mixture in Example 
2, thereby it is revealed such ratio is 29.8% based on the dried matter of 
the starch hydrolyzate used. 
The remainder of the epimerized mixture is adjusted to pH 6.6, added 50 g. 
of "Glucose-Isomerase Nagase" thereto, and warmed at a temperature of 
70.degree. C for 48 hours, while agitated gently and maintained at pH 
6.5-7.0 during the reaction. Isomerized mixture obtained undergoes the 
refining treatment in the similar manner to that to the isomerized mixture 
in Example 2. Refined solution exhibits pH 6.2 and is made to have 60% 
dried matter. 
600 g. of the refined and adjusted solution is subjected to the catalytic 
hydrogenation in the similar manner to that to the refined isomerized 
mixture in Example 3. After refining, the hydrogenated mixture reveals 
that the D-mannitol formation ratio is 42.7% based on the dried matter of 
the starch hydrolyzate used. This refined solution is concentrated in 
vacuo to crystallize out D-mannitol, the melting point of the crystals 
being 166.2.degree. C. The yield is 124.0 g., which corresponds to 34.4% 
based on the dried matter of the starting material. 
On the other hand, the D-fructose formation ratio in the enzymatic 
isomerized mixture is counted backward as 25.8% based on the dried matter 
of the starting material, from the values of 42.7% of D-mannitol formation 
ratio in the hydrogenated mixture and 29.8% of D-mannose formation ratio 
in the epimerized mixture. 
EXAMPLE 8 
5.0 kg. of starch hydrolyzate having the same composition as in Example 7 
is added 6.0g. (0.20% relative to the dried matter of the starch 
hydrolyzate) of ammonium molybdate thereto, adjusted to pH 3.0, and 
heated, while agitated, in an autoclave of 10 liter-capacity at a 
temperature of 140.degree. C for 10 minutes. Epimerized mixture exhibits 
pH 2.7 and is slightly brown in color. The mixture undergoes the refining 
treatment in the similar manner to that to the epimerized mixture in 
Example 1. Refined solution exhibiting pH 6.2 is adjusted to have 60% 
dried matter. 
600 g. of the refined and adjusted solution is subjected to the catalytic 
hydrogenation in the similer manner to that to the refined isomerized 
mixture in Example 3, but using 20 g. of 2.5%Ru2.5%Rh-carbon as 
hydrogenating catalyst. Dried matter composition of the hydrogenated 
mixture obtained is 31.6% mannitol, 67.9% sorbitol and 0.5% impurities. 
Refined solution of the mixture is concentrated in vacuo to crystallize 
out D-mannitol, the melting point of the crystals being 166.1.degree. C. 
The yield is 86.2 g., corresponding to 23.9% based on the dried matter of 
the starting material. 
EXAMPLE 9 
5.0 kg. of hydrol (DE 91; 40% water content) is added 6.0 g. (0.20% 
relative to the dried matter of the hydrol) of ammonium molybdate thereto, 
adjusted to pH 3.0 and heated, while agitated, in an autoclave of 10 
liter-capacity at a temperature of 140.degree. C for 10 minutes. 
Epimerized mixture exhibits pH 2.7 and is slightly brown in color. 
500 g. of the mixture is taken out, and submitted to the determination of 
D-mannose formation ratio, applying correspondingly the determinating 
manner of D-mannose formation ratio in the epimerized mixture in Example 
2, thereby it is revealed such ratio is 27.2% based on the dried matter of 
the hydrol used. 
The remainder of the epimerized mixture is adjusted to pH 6.8, added 50 g. 
of "Glucose-Isomerase Nagase" thereto, and warmed at a temperature of 
65.degree. C for 48 hours, while agitated gently and maintained at pH 
6.5-7.0 during the reaction. The isomerized mixture undergoes the refining 
treatment in the similar manner to that to the isomerized mixture in 
Example 2. Refined solution exhibits pH 6.2 and is adjusted to have 60% 
dried matter. 
600 g. of the refined and adjusted solution is taken out, added 30 g. of 
Raney nickel thereto, adjusted to pH 3.0 with a dilute sulfuric acid, and 
heated, while agitated, in an autoclave of 1 liter-capacity at a 
temperature of 175.degree. C for 120 minutes under a hydrogen pressure of 
150 kg./cm.sup.2. Hydrogenated mixture is refined, followed by the 
estimation of D-mannitol formation ratio thereof, and thereby it is 
revealed such ratio is 39.1%. The refined solution is concentrated in 
vacuo to crystallize out D-mannitol, the melting point of the crystals 
being 165.5.degree. C. The yield is 108.6 g., which corresponds to 30.2% 
based on the dried matter of the starting material. 
On the other hand, from the values of 39.1% of D-mannitol formation ratio 
in the hydrogenated mixture and 27.2% of D-mannose formation ratio in the 
epimerized mixture, the D-fructose formation ratio in the isomerized 
mixture is counted backward as 23.8% based on the dried matter of the 
hydrol used. 
EXAMPLE 10 
5.0 kg. of hydrol having the same composition as in Example 9 is added 
6.0g. (0.20% relative to the dried matter of the hydrol) of molybdenum 
trioxide MoO.sub.3 thereto, adjusted to pH 2.5 with a dilute hydrochloric 
acid, and heated, while agitated, in an autoclave of 10 liter-capacity at 
a temperature of 130.degree. C for 20 minutes. Epimerized mixture exhibits 
pH 2.1 and is slightly brown in color. The mixture is adjusted to have 60% 
dried matter, while its pH is not adjusted. 
600 g. of the mixture adjusted is taken out, added 100 g. of a catalyst 
with trademark "Stabilized Nickel Catalyst G-49B" (manufactured by Nissan 
Girdler Catalyst Manufacturing Co.,Ltd.) thereto, and heated, while 
agitated, in an autoclave of 1 liter-capacity at a temperature of 
180.degree. C for 120 minutes under a hydrogen pressure of 150 
kg./cm.sup.2. Dried matter composition of the hydrogenated mixture is 
26.7% mannitol, 71.3% sorbitol and 2.0% impurities. Refined solution of 
the hydrogenated mixture is concentrated in vacuo to crystallize out 
D-mannitol, the melting point of the crystals being 165.7.degree. C. The 
yield is 65.3 g. corresponding to 18.1% based on the dried matter of the 
hydrol used.