Method of improving intestinal floras

A .beta.-glucooligosaccharide-containing composition comprising at least one material selected from food, drink and medicine, and at least one selected from a glucooligosaccharide comprising at least one .beta.-1,6 bond and a reduced product thereof. A method of improving intestinal floras, comprising administering to a human or animal for ingestion a physiologically effective amount of at least one selected from a glucooligosaccharide comprising at least one .beta.-1,6 bond and a reduced product thereof. The ingestion of the .beta.-glucooligosaccharide and/or the reduced product thereof can bring about promotion of the selective growth of useful bacteria such as Bifidobacteria and lactic acid bacteria, inhibition of the growth of harmful bacteria or putrefactive bacteria, and hence improvement in the intestinal floras.

FIELD OF THE INVENTION 
The present invention relates to a composition such as food, drink or 
medicine which contains a .beta.-glucooligosaccharide and/or a reduced 
product thereof, and a method of improving intestinal floras, using a 
.beta.-glucooligosaccharide and/or a reduced product thereof as an active 
ingredient. 
BACKGROUND OF THE INVENTION 
In the manufacture of food and drink of various kinds, a variety of 
saccharides such as sucrose, corn starch syrup, glucose, maltose and 
high-fructose corn syrup have been used as sweeteners. However, these 
saccharides, which comprise glucose, fructose or a sugar formed of 
.alpha.-glucosidic linkage of glucose and are digested in living orgnaisms 
and accumulated as caloric sources, have the problem that an excessive 
intake thereof may cause corpulence or may bring about adult diseases such 
as diabetes. 
A non-caloric synthetic sweetener such as Aspartame has been also 
developed. Since, however, synthetic sweeteners are not natural products, 
there is anxiety about the safety to human bodies. 
In the recent trend of consumption of food and drink, people avoid 
sweetness more than ever, and tend not to be satisfied if food and drink 
is seasoned only with a sweetener. 
On the other hand, in recent years, intestinal floras (the aggregate of 
bacteria) is known to concern the health of humans, and there is an 
increasing interest in the intestinal floras. For example, Bifidobacteria 
are one of main bacterial species that constitute human intestinal floras, 
and is known, for example, to inhibit growth of putrefactive bacteria or 
pathogenic bacteria, thus playing a variety of useful physiological roles 
in humans or animals. The Bifidobacteria may decrease or disappear because 
of various diseases or aging, and hence it has been variously attempted to 
increase Bifidobacteria in intestines. 
As food or medicine suited for such a purpose is known to include, for 
example, yoghurt containing Bifidobacteria, powder of Bifidobacteria, and 
oligosaccharides capable of growing Bifidobacteria. Of these, the 
oligosaccharides capable of growing Bifidobacteria are of current 
interest. Reported as those having the effect of growing Bifidobacteria 
are fructooligosaccharide, soybean oligosaccharide, konjak 
oligosaccharide, isomaltooligosaccharide, galactooligosaccharide, and so 
forth. Part of these has been already made commercially available as 
health food materials. 
Lactic acid bacteria (or Lactobacilli) are also known from old times as 
useful bacteria that affect intestinal floras. The lactic acid bacteria, 
like Bifidobacteria, are also considered to play roles to inhibit 
intestinal growth of putrefactive bacteria. Thus, viable lactic acid 
bacteria are mixed in an intestinal regulator, or drinks containing lactic 
acid bacteria are commercially available. 
The effect of promoting the growth of Bifidobacteria, attributable to the 
oligosaccharides described above, is caused by the action of 
Bifidobacteria such that it can decompose the oligosaccharides to utilize 
them as nutrient sources although most other bacteria can not decompose 
the oligosaccharides, and consequently the Bifidobacteria selective grow. 
These oligosaccharides, however, are not necessarily selectively utilized 
only by Bifidobacteria, and there exist bacteria other than 
Bifidobacteria, that utilize the oligosaccharide for their growth. It 
depends on the kinds of oligosaccharides what sorts of bacteria can 
utilize the respective oligosaccharides and what sorts of bacteria can not 
utilize them. Under the existing circumstance, this can not be made clear 
unless experiments are actually tried. 
The intestinal floras are comprised of a great number of bacteria living 
together in intestines. Even when the Bifidobacteria have become 
temporarily predominant in intestines, its influence may readily change. 
Thus, it is preferred to promote the growth of not only Bifidobacteria but 
also other useful bacteria such as lactic acid bacteria in order to stably 
obtain the effect of improving intestinal floras. In other words, the 
influence of lactic acid bacteria and so forth may also be increased 
together with that of Bifidobacteria, so that the effect of inhibiting the 
growth of putrefactive bacteria such as Welch bacilli (Clostridium 
perfringens) can be obtained in a stable state. 
Researches hiterto made on the improvement in intestinal floras by the use 
of oligosaccharides have been mainly focused only on the action of 
promoting the growth of Bifidobacteria. Under existing circumstances, 
studies have not been made so much on the effect on other useful bacteria 
such as lactic acid bacteria. 
With regard to the lactic acid bacteria, viable lactic acid bacteria have 
been used in intestinal regulators or drinks. These, however, are not used 
for the purpose of improving the growth environment of lactic acid 
bacteria in intestines. Hence, even if the influence of lactic acid 
bacteria is temporarily increased by ingestion of living bacteria, no 
stable effect of improving intestinal floras can be expected since their 
influence may readily change because of the presence of other bacteria 
having better adapted themselves to the environment. 
SUMMARY OF THE INVENTION 
Accordingly, an object of the present invention is to provide a composition 
such as food and drink that can suppress the amount of caloric intake and 
yet has the taste of a new type. 
Another object of the present invention is to provide a composition such as 
food, drink or medicine having the effect of improving intestinal floras. 
Still another object of the present invention is to provide a new use of 
.beta.-glucooligosaccharides that have been little utilized in an 
industrial scale. 
To achieve the above objects, the present inventors made studies on various 
saccharides to examine their effect of imparting a taste and effect of 
improving intestinal floras. As a result, they have found that 
.beta.-glucooligosaccharides are non-caloric, have a pleasant bitter 
taste, and also have an excellent effect of improving intestinal floras. 
Thus, they have accomplished the present invention. 
From one aspect, the present invention is a 
.beta.-glucooligosaccharide-containing composition comprising at least one 
material selected from food, drink and medicine, and at least one selected 
from a .beta.-glucooligosaccharide and a reduced product thereof. 
From another aspect, the present invention is a method of improving 
intestinal floras, comprising having a living body ingest a 
physiologically effective amount of at least one selected from a 
.beta.-glucooligosaccharide and a reduced product thereof. 
The .beta.-glucooligosaccharide herein referred to is a saccharide obtained 
by .beta.-1,6-glucosidic bond and/or .beta.-1,4-glucosidic bond of 
glucose. The reduced product thereof is obtained by catalytic reduction 
(or hydrogenation) of a .beta.-glucooligosaccharide. These have a much 
higher safety to human bodies than synthetic sweeteners. 
The .beta.-glucooligosaccharide also has an appropriate bitter taste. The 
reduced product thereof loses the bitter taste to render a good and mild 
sweet taste. Thus, they can be added to food and drink and so forth to 
impart good various tastes that have not been given by conventional 
sweeteners. 
In addition, the .beta.-glucooligosaccharide and the reduced product 
thereof have a low calorie content since they are not digestible in living 
organisms, and hence it is possible to prevent the corpulence or adult 
diseases that may be caused by an excessive caloric intake. 
As will be evident from the experimental results described later, the 
.beta.-glucooligosaccharide and the reduced product thereof are not only 
well utilized by Bifidobacteria but also well utilized by lactic acid 
bacteria. Moreover, harmful bacteria such as Welch bacilli (Clostridium 
perfringens) as well as other putrefactive bacteria can not utilize these 
oligosaccharides and can not grow. 
Thus, ingestion of the .beta.-glucooligosaccharide and/or the reduced 
product thereof can bring about promotion of the selective growth of 
useful bacteria such as Bifidobacteria and lactic acid bacteria, 
inhibition of the growth of harmful bacteria or putrefactive bacteria, and 
hence improvement in the intestinal floras.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention will be described below in detail by giving preferred 
embodiments. 
The .beta.-glucooligosaccharide used in the present invention can be 
readily produced in a high yield by, for example, allowing a 
.beta.-glucosidase originating from a variety of microorganisms to act on 
glucose and/or a .beta.-glucooligosaccharide so that an ultimate function 
of the condensation and transglycosylation action possessed by the 
.beta.-glucosidase can be exhibited at its maximum. 
This production process will be detailed here. Any .beta.-glucosidase 
originating from a variety of microorganisms can be used as the 
.beta.-glucosidase. For example, preferably used are enzymes originating 
from microorganisms such as mold fungi Trichoderma viride, Trichoderma 
reesei, Trichoderma koningii, Aspergillus niger and Penicillium 
frequentans; wood-rotting fungi Polypolus tulipiferae, Chrysosprium 
liqnorum and Shizophyllum commune; and bacteria Pseudomonas fluorescens 
var. cellulosa, Cellulomonas uda, Clostridium thermocellum and 
Ruminococcus albus. These microorganisms are all known in the art, and are 
readily available for the preparation of the enzymes. 
As a substrate, at least one of D-glucose and .beta.-glucooligosaccharide 
can be used. Here, the .beta.-glucooligosaccharide serving as a substrate 
refers to cellobiose, gentiobiose, or a gentiooligosaccharide having a 
higher degree of polymerization than these. When the 
.beta.-glucooligosaccharide is used as a substrate, a 
.beta.-glucooligosaccharide with a higher degree of polymerization can be 
obtained by the present enzymatic reaction. In a particularly preferred 
embodiment, at least one selected from glucose, cellobiose and gentiobiose 
is used as the substrate. 
The .beta.-glucosidase may be made to act on glucose and/or a 
.beta.-glucooligosaccharide, whereby a .beta.-glucooligosaccharide of 
various types such as cellobiose, sophorose, laminaribiose, gentiobiose, a 
4-O-.beta.-D-gentiooligosyl-D-glucose and a 
6-O-.beta.-D-gentiooligosyl-D-glucose can be obtained. Here, the 
4-O-.beta.-D-gentiooligosyl-D-glucose refers to 
4-O-.beta.-D-gentiobiosyl-D-glucose, 4-O-.beta.-D-gentiotriosyl-D-glucose, 
or a saccharide having a higher degree of polymerization than these. The 
6-O-.beta.-D-gentiooligosyl-D-glucose also refers to 
6-O-.beta.-D-gentiobiosyl-D-glucose (gentiotriose), 
6-O-.beta.-D-gentiotriosyl-D-glucose (gentiotetraose), or a saccharide 
having a higher degree of polymerization than these. 
These reaction products may vary depending on the enzymes used. When 
glucose or cellobiose is used as a substrate, the above 
.beta.-glucooligosaccharide of various types tends to be produced as a 
mixture of several kinds. When gentiobiose is used as a substrate, only a 
gentiooligosaccharide such as 6-O-.beta.-D-gentiobiosyl-D-glucose or 
6-O-.beta.-D-gentiotriosyl-D-glucose tends to be formed as a reaction 
product. 
Conditions for the enzymatic reaction will be described below. There are no 
particular limitations on the concentration of the substrate. In usual 
instances, it may preferably be in the range of from 1 to 90% (solid 
content/volume), and more preferably from 5 to 80% (solid content/volume). 
The higher the concentration of enzyme based on the substrate is, the 
better. In usual instances, the substrate may preferably be used in a 
concentration of not less than 100 mg per gram of the substrate. The 
reaction may be carried out under optimum temperature and pH conditions 
for the enzymes used. In usual instances, the reaction may preferably be 
carried out at a temperature of from 30.degree. to 80.degree. C., and at a 
pH of approximately from 3 to 8. Reaction time may be so set as to be the 
time during which a sufficient amount of the desired 
.beta.-glucooligosaccharide can be produced and accumulated. In usual 
instances, the reaction may suitably be carried out approximately for 2 to 
72 hours. The reaction may be carried out by adding the enzyme to the 
substrate. Alternatively, the reaction may be carried out by a continuous 
reaction method in which the enzyme is absorbed on a suitable immobilizing 
agent to form an immobilized enzyme and the resulting immobilized enzyme 
is used. The reaction product thus prepared may further be fractionated by 
various methods so that .beta.-glucooligosaccharides of various kinds can 
be respectively separated and purified. 
In the present invention, it is possible to use a reduced product of the 
.beta.-glucooligosaccharide thus obtained. The reduced product can be 
obtained by subjecting a .beta.-glucooligosaccharide to catalytic 
reduction (or hydrogenation). Such a treatment is a treatment method known 
in the production of sugar alcohols. 
The .beta.-glucooligosaccharide-containing composition of the present 
invention can be obtained by adding the .beta.-glucooligosaccharide and/or 
the reduced product thereof to a material such as food, drink or medicine 
in the course of producing such materials. 
In this way, the .beta.-glucooligosaccharide and/or the reduced product 
thereof may be added to food, drink or medicine, so that it is possible to 
impart an approprately bitter taste or a mild sweet taste which have been 
not attained by conventional saccharides, thus bringing about the effect 
of improving tastes. Since the .beta.-glucooligosaccharide and the reduced 
product thereof are not digestible in living organisms, a dietary effect 
can also be obtained. In addition, since the .beta.-glucooligosaccharide 
and the reduced product thereof are rich in moisture retention, they are 
also effective as a humectant, an anti-crystallization agent, or an agent 
for imparting gloss, body or the like. 
In the present invention, the .beta.-glucooligosaccharide and/or the 
reduced product thereof is/are also utilized as a substance for improving 
intestinal floras. More specifically, the .beta.-glucooligosaccharide 
and/or the reduced product thereof may be ingested as health foods or 
medicines as it is, or may be added to a material such as food, drink or 
medicine, so that the growth of Bifidobacteria and lactic acid bacteria in 
intestines can be promoted, the growth of harmful bacteria can be 
inhibited and thus the intestinal floras can be kept in a good condition. 
In the composition of the present invention, only the 
.beta.-glucooligosaccharide and/or the reduced product thereof may be 
added as a taste improver. Since, however, a sweet taste tends to be a 
little short, the .beta.-glucooligosaccharide and/or the reduced product 
thereof may be used in combination with one or more kinds of other 
sweeteners as exemplified by sucrose, corn syrup, glucose, maltose, 
high-fructose corn syrup, honey, sorbitol, maltitol, lactitol, 
L-aspertylphenylalanine methyl ester (Aspartame), saccharin, glycyrrhizin, 
and stevioside. 
In the present invention, the food and drink to which the 
.beta.-glucooligosaccharide and/or the reduced product thereof is/are 
added includes various seasonings as exemplified by soy sauce, mayonnaise, 
dressing, vinegar, an instant Chinese food mix, soup for tempura (fried 
food), sauce, catchup, gravy for grilled meat, curry roux, an instant stew 
mix, an instant soup mix, an instant broth mix, a composite seasoning, and 
mirin (sweet sake for seasoning). It also includes all sorts of food and 
drink or favorite food as exemplified by Japanese-style confections such 
as arare (rice-cake cubes), mochi (rice cakes), manju (bean-jam buns), 
uiro (sweet rice jelly), an (bean paste or jam), yokan (sweet bean jelly), 
jelly, castella (sponge cake), and Japanese candies; Western-style cakes 
such as bunds, biscuits or crackers, cookies, pies (or tarts), pudding, 
butter cream, cream puffs, sponge cake, doughnuts, chocolate, chewing gum, 
caramels, and hard candies; ices such as ice cream, and sherbet; fruits 
preserved in syrup; syrups such as ice molasses; pastes such as flour 
paste, peanut paste, and fruit paste; processed fruits such as jam, 
marmalade, syrupped fruits, and sweetmeats; pickles such as fukujin-zuke 
(sliced vegetalbes pickled in soy sauce), senmai-zuke (pickled turnips), 
and rakkyo-zuke (pickled scallions); meat products such as ham, and 
sausage; fish products such as kamaboko (boiled fish paste), and chikuwa 
(fish paste hollow-rods); all sorts of dilicacies; tsukudani (food boiled 
down in soy sauce); alcohol such as beer, liqueur, and sake (rice wine); 
drinks or soft drinks (or aerated drinks) such as coffee, cocoa, juice, 
carbonated drink, drink preparations, lactic acid drink, and lactic acid 
bacteria drink; and instant food and drink such as instant juice, and 
instant coffee. 
The .beta.-glucooligosaccharide and/or the reduced product thereof may also 
be mixed with other biologically active substances such as dietary fiber, 
lactic acid bacteria, Bifidobacteria, and vitamins to give health foods or 
medicines. 
When added to food, drink or medicine, the .beta.-glucooligosaccharide 
and/or the reduced product thereof may preferably be in an amount of from 
0.5 to 50% by weight, and more preferably from 1.0 to 30% by weight, in 
order to sufficiently obtain the effect of improving tastes or improving 
intestinal floras as stated above. 
The present invention will be described below in greater detail by giving 
Examples. 
EXAMPLE 1 
Production of .beta.-glucooligosaccharide 
(1) In 200 ml of a 0.2M acetate buffer (pH 3.5), 5 g of a crude cellulase 
preparation "Meiselase" (trade name; a product of Meiji Seika Kaisha Ltd.) 
originating from mold fungi Trichoderma viride was dissolved. The solution 
was then subjected to column chromatography using Amberlite CG-50. FIG. 1 
shows the elution patterns thus obtained. In FIG. 1, the line with black 
triangles indicates amylase activity; the line with white circles, 
Avicel-saccharification activity; the line with black circles, 
CMC-saccharification activity; the line with "x", .beta.-glucosidase 
activity; and the line with dots, amount of protein. In this way, a 
fraction Peak II showing a strong cellulase activity and a fraction Peak 
III showing a strong .beta.-glucosidase activity were separated. 
The fraction Peak III was collected and further subjected to gel filtration 
chromatography using Bio-Gel P-60 to obtain the elution patterns as shown 
in FIG. 2. In FIG. 2, the line with white circles indicates 
Avicel-saccharification activity; the line with black circles, 
.beta.-glucosidase activity; the line with black triangles, 
CMC-saccharification activity; and the line with dots, amount of protein. 
From the resulting eluate, a fraction having .beta.-glucosidase activity 
was collected. 
Next, the above .beta.-glucosidase-active fraction was subjected to 
isoelectric focusing using an LKB 110 ml column to obtain the purified 
.beta.-glucosidase (FIG. 3). In FIG. 3, the line with white circles 
indicates Avicel-saccharification activity; the line with black circles, 
.beta.-glucosidase activity; the line with black triangles, 
CMC-saccharification activity; and the line with dots, amount of protein. 
The isoelectric focusing was carried out using a carrier Ampholite with pH 
4 to 6, and under electrophoresis conditions of 5 mA, 600 V (at the time 
of start) to 1.5 mA, 1,500 V (at the time of completion). From the 
resulting eluate, a fraction having .beta.-glucosidase activity was 
collected. The final purified enzyme preparation was thus obtained. 
The purified enzyme thus obtained gave a single protein band as a result of 
polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE. The resulting 
enzyme, as shown in FIG. 3, has a potent .beta.-glucosidase activity, and, 
even though very weak, the enzyme also shows both Avicel-and 
CMC-saccharification activities. 
The enzyme activity was measured by carrying out the reaction under 
conditions of pH 5.0 and a temperature of 30.degree. C., using 
p-nitrophenyl .beta.-D-glucosidase (.beta.-PNPG). One unit of enzyme 
activity was defined as the amount of enzyme that catalyses the liberation 
of reducing sugar equivalent to 1.0 .mu.mol of D-glucose from the 
substrate per min. under the above conditions. 
(2) To 300 g of D-glucose, the purified .beta.-glucosidase in the above was 
added in a an amount of 5.8.times.10.sup.5 unit (500 ml) (glucose: about 
60% w/v), and the reaction was carried out at pH 5.0 and a temperature of 
60.degree. C. After completion of the reaction, the reaction mixture was 
heated at 100.degree. C. for 5 minutes to stop the reaction, decolored 
with activated carbon according to a conventional method, and purified by 
deionization, followed by concentration to a solid content of 72% (w/w) 
under reduced pressure. 
After a column with a jacket of 2 cm in inner diameter and 120 cm in length 
(60.degree. C.) was packed with a cationic ion-exchange resin "Dowex 99" 
(Na.sup.+ type; a product of Dow Chemical Co., the reaction product 
saccharide solution thus obtained was loaded so as to give a solid content 
of from 5 to 7% (w/v) based on the amount of resin, and fractionated at a 
space velocity (SV.hr.sup.-1) of 0.35 to obtain a gentiooligosaccharide 
fraction (OS-1). Saccharide composition of this fraction was analyzed by 
high-performance liquid chromatography to obtain the results as shown in 
Table 1. The analysis by the high-performance liquid chromatography was 
carried out under the following conditions: 
Column: SCR-101, manufactured by Shimadzu Corporation 
Detector: A differential refractometer 
Column temperature: 55.degree. C. 
Column flow rate: 0.8 ml/min 
The above operation was carried out 10 times to give about 40 g of 
gentiooligosaccharide (OS-1). The product was freeze-dried and formed into 
powder. 
(3) The above gentiooligosaccharide (OS-1) was further fractionated in the 
same manner as in the above to carry out preparation. A fraction of 
.beta.-glucodisaccharide (F-2), a fraction of .beta.-glucotrisaccharide 
(F-3) and a fraction of .beta.-glucotetra(or more)saccharide (F-4) were 
thus obtained. The saccharide composition of these fractions were analyzed 
by high-performance liquid chromatography to obtain the results as shown 
in Table 1. 
In the foregoing, the .beta.-glucodisaccharide refers to a disaccharide 
comprising a .beta.-glucoside bond, as exemplified by cellobiose, 
sophorose, laminaribiose, or gentiobiose. The .beta.-glucotrisaccharide 
refers to 4-O-.beta.-D-gentiobiosyl-D-glucose, 
6-O-.beta.-D-gentiobiosyl-D-glucose (gentiotriose), etc. The 
.beta.-glucotetra(or more)saccharide refers to 
4-O-.beta.-D-gentiotriosyl-D-glucose or a saccharide having a higher 
degree of polymerization than it, 6-O-.beta.-D-gentiotriosyl-D-glucose or 
a saccharide having a higher degree of polymerization than it. 
TABLE 1 
______________________________________ 
(Saccharide composition of OS-1, F-2, F-3, F-4) 
OS-1 F-2 F-3 F-4 
______________________________________ 
Fructose (F): 3.1 0 0 0.2 
Glucose (G): 11.8 1.2 0.5 2.7 
.beta.-glucodisaccharide (G.sub.2): 
52.8 93.6 0 0 
.beta.-glucotrisaccharide (G.sub.3): 
22.1 5.1 90.3 0.3 
.beta.-glucotetra(or more)- 
10.2 0.1 9.2 96.8 
saccharide (G.sub.4): 
______________________________________ 
EXAMPLE 2 
Tests on Utilization by Intestinal Bacteria 
Utilization of each oligosaccharide by intestinal bacteria was tested in 
the following way: 
(1) Strains Tested 
Used were 17 strains of Bacteroides, 20 strains of Bifidobacterium, 26 
strains of Clostrudium, 6 strains of Eubacterium, 5 strains of 
Fusobacterium, 6 strains of Peptstreptococcus, 9 strains of Lactobacillus, 
5 strains of Enterococcus, 5 strains of Escherichia coli, and 21 strains 
of others, i.e., 120 strains in total. 
(2) Test Groups 
1. Control (no carbohydrate added) 
2. Glucose 
3. Meioligo-P (trade name; a commercially available fractooligosaccharide; 
a product of Meiji Seika Kaisha, Ltd. 
4. OS-1 (a .beta.-glucooligosaccharide fraction) 
5. F-2 (a .beta.-glucodisaccharide fraction) 
6. F-3 (a .beta.-glucotrisaccharide fraction) 
7. F-4 (a .beta.-glucotetra(or more)saccharide fraction) 
In the foregoing, Meioligo-P has the following saccharide composition: 
Sucrose (GF) . . . 4% by weight 
1-Kestose (GF2) . . . 35% by weight 
Nystose (GF3) . . . 50% by weight 
1-Fractosylnystose (GF4) . . . 11% by weight 
(3) Culture Medium 
To a semi-fluid agar medium of a Pepton-Yeast-Fildes solution (PYF), each 
saccharide of the test groups was added so as to finally give a 
concentration of 0.5%, and the medium was used after autoclave 
sterilization at 115.degree. C. for 20 minutes. 
In the foregoing, the RYF semi-fluid agar medium has the following 
composition: 
______________________________________ 
Trypticase (BBL) 10.0 g 
Yeast estract (Difco) 10.0 g 
Fildes solution 40.0 ml 
Salts solution 40.0 ml 
L-systin hydrochloride monohydrate 
0.5 g 
Agar 1.5 g 
Deionized water 920 ml 
______________________________________ 
In the forefoing, the salts solution has the following composition: 
______________________________________ 
CaCl.sub.2, anhydrous 0.2 g 
MgSO.sub.4 0.2 g 
K.sub.2 HPO.sub.4 1.0 g 
KH.sub.2 PO.sub.4 1.0 g 
NaHCO.sub.3 10.0 g 
NaCl 2.0 g 
Deionized water 1,000 ml 
______________________________________ 
(4) Test Method 
Freezed strains for the test were each streak-cultured in a BL agar medium 
to obtain isolated colonies. This operation was carried outed twice to 
obtain pure-cultured strains. A BL agar plate used was obtained by adding 
5% of equine defibrinated blood produced by Kohjin Co., Ltd., to a BL agar 
medium produced by Nissui Chemical Industries, Ltd. Culture was carried 
out using an anaerobic incubator manufactured by Sanyo-Forma Co. 
The strains tested thus obtained by pure culture were inoculated on a 
Fildes solution-added GAM broth culture medium (obtained by adding 0.4% of 
a Fildes solution to a GAM broth produced by Nissui Chemical Industries, 
Ltd), and subcultured by anaerobic culture at 37.degree. C. for 24 hours 
using an anaerobic incubator manufactured by Sanyo-Forma Co. 
The resulting culture solution was inoculated in 1.5 ml of the above test 
culture medium in an amount of 0.03 ml, using an automatic multi-strain 
inoculating apparatus "MD-120", manufactured by LIFETEC CO., and cultured 
at 37.degree. C. for 4 days (96 hours) under anaerobic conditions. 
Thereafter, the pH was measured. After inoculation, the inoculated strain 
solution was inspected whether or not contamination or poor growth 
occurred, and those pertinent thereto were removed from data. The 
anaerobic culture was carried out using the anaerobic incubator 
manufactured by Sanyo-Forma Co., and in an atmosphere of a mixed gas of 
10% of CO.sub.2, 10% of H.sub.2 and the balance of N.sub.2. The pH was 
measured and the data were processed, using "BIS-120", manufactured by 
LIFETEC CO. 
(5) Judgement on Utilization 
A decreae in the pH value of the culture was measured, and the occurrence 
or strength of utilization was judged. Judgement was made according to the 
following criteria: 
-: pH 6.0 or more: 
.+-.: pH 5.5 to less than 6.0 
+: pH 5.0 to less than 5.5 
++: pH 4.5 to less than 5.0 
+++: less than pH 4.5 
(6) Test Results 
Test results obtained are shown in Table 2. Table 2 shows the following: 
(i) With regard to Bacteroides, all the test groups show substantially the 
same value of utilization. 
(ii) With regard to Bifidobacterium, all the test groups show a strong 
value of utilization as a whole. With regard to Bifidobacterium bifidum, 
no utilization is seen on Meioligo-P, F-3 and F-4, but utilization is 
confirmed on OS-1 and F-2 which contain .beta.-glucodiaccharide. 
(iii) With regard to Clostridium, utilization is seen on glucose in many 
cases, but no utilization is seen on Meioligo-P, OS-1, F-2, F-3 and F-4 in 
almost all cases. 
(iv) With regard to Eubacterium, Fusobacterium, Pepststreptococcus and 
Escherichia coli, no utilization is seen on Meioligo-P, OS-1, F-2, F-3 and 
F-4 in many cases. 
(v) With regard to Lactobacillus, no utilization in seen on Meioligo-P in 
many cases, but utilization is seen on OS-1, F-2, F-3 and F-4 in 
considerably many cases. In particular, with regard to Lactobacillus 
casei, no utilization is seen at all on Meioigo-P, but a strong value of 
utilization can be seen on OS-1, F-2, F-3 and F-4. 
It is clear from the above results that OS-1, F-2, F-3 and F-4 comprising a 
.beta.-glucooligosaccharide are well utilized by not only Bifidobacteria 
but also lactic acid bacteria, and can be utilized with difficulty by 
other pathogenic bacteria or putrefactive bacteria. Hence, the ingestion 
of these .beta.-glucooligosaccharides is seen to enable promotion of the 
selective growth of Bifidobacteria and lactic acid bacteria, inhibition of 
the growth of pathogenic bacteria or putrefactive bacteria, and 
improvement in the intestinal floras. 
The same results as the above were obtained also when reduced 
gentiooligosaccharides were used. 
TABLE 2 
__________________________________________________________________________ 
(Test results on utilization) 
Tested Meioligo 
Bacteria strain 
Control 
Glucose 
P OS-1 
F-2 F-3 F-4 
__________________________________________________________________________ 
Bacteroides asaccharolyticus 
GAI#25260 
- - - - - - - 
Bacteroides bivius 
GAI#5518 
- ++ - ++ ++ + + 
Bacteroides distasonis 
GAI#5462 
- + .+-. + + .+-. 
.+-. 
Bacteroides fragilis 
GAI#5524 
- + + ++ ++ + + 
Bacteroides fragilis 
GAI#5562 
- ++ + ++ ++ ++ + 
Bacteroides fragilis 
R-18 - ++ + ++ ++ ++ + 
Bacteroides intermedius 
46 
Bacteroides ovatus 
JCM 5824 
- ++ + ++ ++ ++ + 
Bacteroides ovatus 
CIFL N0029 
- ++ ++ +++ ++ + + 
Bacteroides thetaiotaomicron 
GAI#5628 
- + + ++ ++ + + 
Bacteroides thetaiotaomicron 
VI-98 - + + ++ ++ ++ + 
Bacteroides uniformis 
GAI#5466 
- + + + + + + 
Bacteroides vulgatus 
GAI#5460 
- ++ .+-. + + .+-. 
.+-. 
Bacteroides vulgatus 
R-16 - ++ - + + + + 
Bacteroides vulgatus 
B-25 - ++ + + + + .+-. 
Bacteroides vulgatus 
B-84 - ++ + + + + .+-. 
Bacteroides vulgatus 
V-114 - ++ .+-. +++ + .+-. 
.+-. 
Bifidobacterium adolescentis 
CIFL N0035 
- +++ ++ +++ +++ +++ +++ 
Bifidobacterium adolescentis 
CIFL N0037 
- +++ +++ +++ +++ +++ +++ 
Bifidobacterium adolescentis 
CIFL N0038 
- +++ +++ +++ +++ +++ +++ 
Bifidobacterium adolescentis 
CIFL N0042 
- +++ +++ +++ +++ +++ +++ 
Bifidobacterium adolescentis 
CIFL N0046 
- +++ +++ +++ +++ +++ +++ 
Bifidobacterium adolescentis 
aE194a 
- +++ +++ +++ +++ +++ +++ 
Bifidobacterium animalis 
CIFL N0040 
- +++ + +++ +++ +++ +++ 
Bifidobacterium bifidum 
R-2 - +++ - +++ ++ - - 
Bifidobacterium bifidum 
aE318 - +++ - ++ ++ - - 
Bifidobacterium breve 
bs46 - +++ ++ +++ +++ +++ +++ 
Bifidobacterium breve 
as50 - +++ ++ +++ +++ +++ +++ 
Bifidobacterium breve 
IV-14 - +++ ++ +++ +++ +++ +++ 
Bifidobacterium breve 
IV-19 - +++ ++ +++ +++ +++ +++ 
Bifidobacterium infantis 
S-12 - +++ +++ +++ +++ +++ +++ 
Bifidobacterium infantis 
CIFL N0044 
- +++ ++ +++ +++ +++ +++ 
Bifidobacterium longum 
CIFL N0036 
- +++ + +++ +++ +++ +++ 
Bifidobacterium longum 
CIFL N0044 
- +++ + +++ +++ +++ +++ 
Bifidobacterium longum 
aE194b 
- +++ ++ +++ +++ +++ +++ 
Bifidobacterium longum 
R-3 - +++ ++ +++ +++ ++ ++ 
Bifidobacterium longum 
M101-2 
- +++ + +++ +++ +++ ++ 
Clostridium butyricum 
GAL#7503 
- +++ ++ ++ ++ ++ ++ 
Clostridium butyricum 
Rw23 - +++ ++ +++ +++ ++ ++ 
Clostridium cadaveris 
XI-10 - + - - - - - 
Clostridium clostridioforme 
GAI#5458 
- + .+-. .+-. 
.+-. 
- - 
Clostridium clostridioforme 
R-14 - + - - - - - 
Clostridium difficile 
GAI#10038 
- .+-. - - - - - 
Clostridium difficile 
V-6 - .+-. - - - - - 
Clostridium difficile 
GAI#10042 
- .+-. - - - - - 
Clostridium difficile 
GAI#10037 
- ++ - - - - - 
Clostridium histlyticum 
Ccm5943 
- - - - - - - 
Clostridium innocuum 
GAI#5472 
- +++ - .+-. 
- - - 
Clostridium novyi (Type A) 
GAI#5614 
- ++ - - - - - 
Clostridium paraputrificum 
V-96 - ++ - + + + + 
Clostridium paraputrificum 
R-13 - +++ - + + - - 
Clostridium paraputrificum 
R-78 - ++ - + + + + 
Clostridium perfringens 
GAI#5526 
- ++ - .+-. 
.+-. 
.+-. 
.+-. 
Clostridium perfringens 
R-11 - + - .+-. 
.+-. 
.+-. 
.+-. 
Clostridium perfringens 
B-165-16 
- + - .+-. 
.+-. 
.+-. 
.+-. 
Clostridium perfringens 
B-3-10 
- + - .+-. 
.+-. 
.+-. 
.+-. 
Clostridium perfringens 
C-01 - + - .+-. 
.+-. 
.+-. 
.+-. 
Clostridium ramosum 
V-8 - +++ .+-. ++ ++ ++ .+-. 
Clostridium ramosum 
C-00 - +++ .+-. ++ ++ ++ .+-. 
Clostridium septicum 
GAI#7502 
- ++ - .+-. 
.+-. 
- - 
Clostridium tertium 
GAI#5618 
- ++ - + + .+-. 
.+-. 
Clostridium sordellii 
GAI#5612 
- .+-. - - - - - 
Clostridium sporogenes 
GAI#5562 
- - - - - - - 
Eubacterium aerofaciens 
R-6 - +++ .+-. - .+-. 
- - 
Eubacterium aerofaciens 
S-12 - +++ .+-. + + - - 
Eubacterium lentum 
R-7 - - - - - - - 
Eubacterium limosum 
R-9 - +++ - .+-. 
- - - 
Eubacterium limosum 
V-60 - ++ - .+-. 
- - - 
Eubacterium nitritogenes 
R-8 - +++ - .+-. 
.+-. 
- - 
Fusobacterium mortiferum 
GAI#5442 
- + - - - - - 
Fusobacterium necrophrum 
GAI#5634 
- .+-. - - - - - 
Fusobacterium russii 
GAI#0317 
- .+-. - - - - - 
Fusobacterium varium 
GAI#5566 
- .+-. - - - - - 
Fusobacterium varium 
R-25 - .+-. - - - - - 
Megamonas hypermegas 
R-15 - +++ ++ ++ ++ + - 
Mitsuokella multiacida 
VI-71 - +++ +++ +++ +++ +++ + 
Mitsuokella multiacida 
VI-70 - +++ +++ +++ +++ +++ + 
Peptostreptococcus asacccharolyticus 
GAI#2356 
- - - - - - - 
Peptostreptococcus asacccharolyticus 
R-22 - - - + + - - 
Peptostreptococcus magnus 
GAI#5528 
- - - - - - - 
Peptostreptococcus micros 
GAI#5540 
- - - - - - - 
Peptostreptococcus prevotii 
R-23 - - - .+-. 
- - - 
Peptostreptococcus productus 
X-45 - +++ + ++ ++ ++ ++ 
Propionibacterium acnes 
GAI#5648 
- ++ - - - - - 
Propionibacterium granurosum 
R-20 - ++ - + .+-. 
.+-. 
- 
Veillonella parvura 
GAI#5602 
- - - - - - - 
Veillonella parvura 
R-10 - - - - - - - 
Citrobacter diversus 
CIFL A0016 
- ++ - + + - - 
Citrobacter freundii 
CIFL A0015 
- ++ - + .+-. 
- - 
Enterobacter aerogenes 
CK121-1 
- + + + + .+-. 
- 
Enterobacter cloacae 
CIFL A0001 
- + - .+-. 
.+-. 
- - 
Enterococcus faecalis 
CIFL A0013 
- +++ - ++ ++ .+-. 
+ 
Enterococcus faecalis 
L-220 - +++ ++ ++ ++ - - 
Enterococcus faecalis 
ST-201 
- +++ ++ ++ ++ ++ + 
Enterococcus faecium 
L-225 - +++ ++ ++ ++ - - 
Enterococcus faecium 
ST-101 
- +++ ++ ++ ++ - - 
Escherichia coli CIFL A0008 
- ++ - .+-. 
.+-. 
- - 
Escherichia coli I-3 - + - - - - - 
Escherichia coli O-1 - ++ - - - - - 
Escherichia coli M-1 - ++ - - - - - 
Escherichia coli U-1 - +++ .+-. .+-. 
+ - - 
Klebsiella pneumoniae 
CIFL A0003 
Klebsiella pneumoniae 
CK46 (1) 
- + + + + + - 
Lactobacillus acidophilus 
I-61 - +++ ++ +++ +++ ++ + 
Lactobacillus acidophilus 
I-68 - +++ + +++ +++ +++ +++ 
Lactobacillus casei 
I-139 - +++ - +++ +++ .+-. 
- 
Lactobacillus casei 
II-8 - +++ - +++ +++ +++ +++ 
Lactobacillus fermentum 
CIFL A0066 
- ++ - .+-. 
.+-. 
- - 
Lactobacillus fermentum 
JCM 1173 
- +++ - .+-. 
+ - - 
Lactobacillus gasseri 
JCM 1131 
- +++ +++ +++ +++ + + 
Lactobacillus salivarius 
I-117 - + - .+-. 
- - - 
Lactobacillus salivarius 
I-108 - +++ +++ ++ .+-. 
- - 
Morganella morganii 
ME138-1 
- + - - - - - 
Proteus mirabilis ME14-(2) 
- + .+-. - - - - 
Proteus vulgaris CIFL A0011 
- + .+-. .+-. 
- - - 
Serratia marcescens 
CIFL A0007 
- + .+-. .+-. 
.+-. 
- - 
Staphyrococcus aureus 
CIFL A0012 
- ++ + + - - - 
Staphyrococcus epidermidis 
CIFL A0018 
- ++ .+-. - - - - 
Streptococcus haemolyticus 
CK6-2 - ++ .+-. .+-. 
- - - 
Streptococcus pyrogenes 
CIFL A0017 
- ++ - + .+-. 
- - 
__________________________________________________________________________ 
EXAMPLE 3 
Preparation of Hard Candies 
In 500 ml of an aqueous 50% sucrose solution, 100 g of a concentrated 
solution of the gentiooligosaccharide having a solid content of 72% (w/w), 
obtained in (2) of Example 1 was dissolved with heating, and then the 
solution was concentrated under reduced pressure with heating until it 
came to have a water content of not more than 2%. In the resulting 
concentrate, 5 g of citric acid and small amounts of lemon perfume and 
colorant were mixed, and the mixture was molded by a conventional method 
to give hard candies. 
The resulting products were confirmed to be hard candies having a well 
harmonized taste of sweetness with bitterness, which was a novel taste. 
EXAMPLE 4 
Preparation of Lactic Acid Drink 
After 500 g of skin milk was sterilized with heating at 80.degree. C. for 
20 minutes, the sterilized product was cooled to 40.degree. C., followed 
by addition of 15 g of a starter to carry out fermentation at 35.degree. 
to 37.degree. C. for 12 hours. Subsequently, the fermented product was 
homogenized, to which 50 g of a concentrated solution of the 
gentiooligosaccharide having a solid content of 72% (w/w), obtained in (2) 
of Example 1, 50 g of sucrose and 100 g of an high-fructose corn syrup 
were added, followed by sterilization at a temperature kept at 80.degree. 
C. After cooling, a small amount of perfume was added, and then the 
product was bottled. 
The resulting product was confirmed to be a lactic acid drink having a well 
harmonized taste of flavor and bitterness with sourness. 
EXAMPLE 5 
Preparation of a Soft Drink 
To 500 ml of lemon juice squeezed from fresh lemons, 10 g of sucrose, 20 g 
of high-fructose corn syrup and 5 g of gentiooligosaccharide powder (OS-1) 
obtained in Example 1 were added, which were thoroughly mixed using a 
mixer, followed by filtration. Subsequently, the product was sterilized at 
a temperature kept at 70.degree. C., thereafter cooled, and then bottled. 
The resulting product was confirmed to be a soft drink having a well 
harmonized taste of bitterness with sourness. 
EXAMPLE 6 
Preparation of Cookies 
A base was prepared by mixing 50 g of weak flour, 30 g of margarine, 25 g 
of an egg, 0.5 g of baking powder, 25 g of first-grade white sugar, 10 g 
of gentiooligosaccharide powder (OS-1) obtained in Example 1 and 10 g of 
water. The base was baked at 170.degree. C. for 10 minutes according to a 
conventional method to give a product. 
The resulting product was confirmed to be cookies having a pleasant bitter 
taste and improved in flavor. 
EXAMPLE 7 
Preparation of a Jelly 
Using 36 g of gelatin, 84 g of first-grade white sugar, 28 g of the 
concentrated solution of gentiooligosaccharide with a solid content of 72% 
(w/w), obtained in (2) of Example 1, 420 g of wine and 413 g of water, a 
jelly was prepared according to a conventional method. More specifically, 
the gelatin was previously swelled with water used in an amount of 1/2 of 
the prescribed amount, and the first-grade white sugar and the 
gentiooligosaccharide syrup were added to the remaining water, which were 
dissolved with heating and then boiled. The swelled gelatin was added 
thereto, and the mixture was again boiled. The resulting solution was 
cooled with ice water, the wine was added when the temperature of the 
solution fell to 50.degree. C., and then the solution was further cooled. 
When the solution became viscous, the viscous solution was dividedly 
poured into cups. The cups were covered up, followed by solidification in 
a refrigerator kept at 5.degree. C. to give a product. 
The present product had a pleasant bitterness, rendering the taste of a 
high grade.