Abstract:
The present invention provides a method for promoting the proliferation of microorganisms of the genus Bifidibacterium comprising contacting said microorganisms with a growth promoting effective amount of an isomalto-oligosaccharide.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a method and an agent for promoting the proliferation of intestinal bifidobacteria using isomalto-oligosaccharides as an active substance. 
     2. Discussion of Background 
     Bifidobacteria, which belong to the genus Bifidobacterium, are known to be useful microorganisms inhabiting the human intestines. They have an important role in maintaining human health by preventing the increase of microorganisms which produce harmful substances, such as amines and ammonia, in the intestines. 
     In general, sugars are known nutrients for bifidobacteria as well as for noxious microorganisms, and sugars which are utilized only by bifidobacteria are little known. Additionally, it cannot be presumed from the properties of a sugar, such as molecular structure, whether the sugar will be utilized selectively by bifidobacteria or not. 
     The bifidobacteria proliferating action of sugars such as lactulose, raffinose, stachyose is well-known. Lactulose has found practical use in food. However, these sugars are consumed not only by bifidobacteria but also by the microorganisms which produce harmful substances in the intestines. 
     The bifidobacteria proliferating action of palatinose, an isomer of sucrose, and of fructo-oligosaccharides which consist of one sucrose unit and from one to four fructose units is also known. They are reported as useful bifidobacteria proliferating agents since they are utilized selectively by the bifidobacteria (Japan Kokai Nos. 91193/1982 and 53834/1984). 
     The inventors have made the surprising discovery that isomalto-oligosaccharides exhibit selective bifidobacteria proliferating action. 
     SUMMARY OF THE INVENTION 
     It is an object of this invention to provide a new bifidobacteria proliferation promoting substance which is selectively utilized by bifidobacteria and not by noxious microorganisms. The inventors have made the surprising discovery that isomalto-oligosaccharides are selectively utilized by bifidobacteria and not by noxious microorganisms. 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The terms &#34;isomalto-oligosaccharides&#34; or &#34;isomalto-sugars&#34; are used herein as a general term for the oligosaccharides having branched structure and containing the α-D-(1→6) linkage of a glucose unit in the molecules. Isomaltose, isomaltotriose, panose, isomaltotetraose, and isomaltopentaose are typical sugars in this group, and are used alone or as mixtures in this invention. 
     They are prepared, for example, by reacting a sugar condensating enzyme such as glucoamylase with an aqueous solution of glucose with a concentration at least 50% by weight glucose (Japan patent application No. 245470/1984 and 61248/1985), or by reacting a sugar transfer enzyme such as α-glucosidase with an aqueous solution of maltose, preferably with β-amylase and a starch debranching enzyme such as pullulanase (Japan patent application No. 58483/1985). 
     A syrup containing about 40% isomalto-sugar (to solid) can be obtained by the above methods. However it is possible to increase the content to over 60% by selecting the conditions appropriately. Sugars other than isomalto-sugars present in the syrup are mainly glucose and maltose. 
     A typical composition of isomalto-oligosaccharide in the syrup thus obtained is 20 to 30% isomaltose, 10  to 15% panose, isomaltotriose and other trioses, and 5 to 10% isomaltotetrasose and saccharides which consist of four or more glucose units. 
     A syrup containing mainly saccharides consisting of three or more glucose units can also be produced by reacting a sugar transfer enzyme with the starch which has been hydrolyzed by a starch debranching enzyme and α-amylase (Japan Patent application No. 53017/1985). The isomalto-sugar composition of the product is from 10 to 25% isomaltose, 20 to 35% panose, isomaltotriose and other trioses, and from 5 to 15% isomaltotetrasose and saccharides consisting of four or more glucose units. 
     The isomalto-oligosaccharide syrup thus obtained can be further subjected to the following treatments in order to increase the concentration of isomalto-sugars to about 90% or more relative to the solid. 
     (1) Concentrating and cooling the syrup slowly to crystallize out glucose, etc., and thereby remove them from the syrup. 
     (2) Adding sodium chloride into the syrup to crystallize out a glucose-sodium chloride complex and thereby remove them from the syrup. 
     (3) Adding an organic solvent such as alcohols, acetone, etc., into the syrup to precipitate sugars othe than isomalto-oligosaccharides and thereby remove them. 
     (4) Removing sugars other than isomalto-oligosaccharides from the syrup by chromatographic separation techniques. 
     (5) Removing sugars other than isomalto-oligosaccharides from the syrup by yeast fermentation. 
     The sugar composition of concentrated isomalto-oligosaccharides thus obtained are as follows: 
     
                       TABLE 1______________________________________                     Isomalto-sugar                     comprising three or          Isomaltose more glucose unitsPrincipal component          (%)        (%)______________________________________Isomaltose     40˜70                     25˜35Panose, isomaltotriose          15˜30                     40˜50and other triosesIsomaltotetraose and          10˜20                     15˜20saccharides consisting offour or more glucose units______________________________________ 
    
     These isomalto-sugar syrups are further treated, if necessary, by gelfiltration or an ion exchange resin column to fractionate the sugars by the number of glucose units. Fractions consisting of isomalto-disaccharides, mainly isomaltose; isomalto-trisaccarides, mainly panose and isomaltotriose; and isomalto-tetrasaccarides, mainly isomaltotetraose, can be obtained separately, and they are also active as bifidobacteria proliferating agents. 
     The isomalto-sugar syrup or the separated fractions may further be powdered by well-known methods such as, for example, spray drying and adsorbing on a carrier. 
     The bifidobacteria proliferation promoting action of the isomalto-oligosaccharides was examined as follows. 
     Several kinds of microorganisms including those in the genus Bifidobacterium were incubated in sterilized culture media consisting of peptone yeast-Fildes solution (hereinafter referred to as &#34;PYF medium&#34;) into which were added 0.5 weight % of the sugars tested. 
     1. Culture Medium 
     The composition of PYF medium (pH 7.2) is as follows: 
     
         ______________________________________Pancreatic digest of casein                  10      grams(BBL Trypticase peptone made byBecton Dickinson and Company)Yeast extract (made by Difco company)                  5       gramsFildes solution        40      millilitersSalts solution         40      millilitersCaCl.sub.2 (anhydrous) 0.2     gramsMgSO.sub.4             0.2K.sub.2 HP0.sub.4      1.0KH.sub.2 PO.sub.4      1.0NaHCO.sub.3            10.0NaCl                   2.0Pure water             1,000   millilitersL-Cysteine.HCl.H.sub.2 O                  0.5     gramsPure water             920     milliliters______________________________________ 
    
     Fildes solution is prepared by mixing the following components, digesting the mixture for one night in a bath maintained at 55° C., adding 12 milliliters of 20% aqueous solution of NaOH, and adjusting the pH at 7.6 by NaOH. 
     
         ______________________________________Physiological saline 150      milliliters(0.85% aqueous solution of NaCl)Concentrated hydrochloric acid                6        millilitersHorse blood          50       millilitersPepsine, 1/10,000 aqueous solution                1        gram(Difco Co.)______________________________________ 
    
     2. Method and Result 
     Fresh microorganisms incubated on agar plate culture media were innoculated at 10 8  cfu (colony formation unit) per each test tube of PYF medium, in which the sugars examined were added at a concentration of 0.5 weight %. The test tubes were incubated at 37° C. for 48 hours under anaerobic conditions by the steelwool method. The absorbance of the resultant liquid at 650 nanometers was determined and the consumability of the tested sugars by the microorganisms was calculated by the following equation: ##EQU1## RG: rate of growth OD: optical density 
     The results are shown in Table 2, in which the signs represent the following RG values. 
     ++=RG≧75 
     +=50 ≦RG&lt;75 
     ±=25 ≦RG&lt;50 
     -RG &lt;25 
     
                                           TABLE 2__________________________________________________________________________                          90% Isomal-Microorganisms      Glucose           Maltose                Lactose                     Raffinose                          mixt.*                              tose                                  Triose**                                       Tetraose***__________________________________________________________________________Bifidobacterium      ++   ++   ++   ++   ++  ++  ++   ++adolescentis E298Bifidobacterium      ++   ++   ++   ++   ++  ++  ++   ++infantis I-10-5Bifidobacterium      ++   ++   ++   ++   ++  ++  ++   ++breve SIBifidobacterium      ++   ++   ++   ++   ++  ++  ++   ++longum E194Lactobacillus      ++   +    ++   -    -   -   -    -acidophilus ATCC4356Clostridium      ++   ++   ++   -    -   -   -    -paraputrificumClostridium SP2      ++   +    ± -    -   -   -    -Bacteroides vulgatus      ++   ++   ++   +    -   ±                                  -    -Bacteroides ovatus      ++   ++   ++   +    -   -   -    -Escherichia coli      ++   ++   ± ++   -   -   -    -Peptostreptococcus      +    -    -    ++   -   -   -    -anaerobius X-36Fusobacterium varium      ++   -    -    -    -   -   -    -VI-43__________________________________________________________________________ *The 90% mixture contains 90% of isomaltooligosaccharides to the solid an the composition is as follows: Isomaltose 50% Isomaltotrisaccharides (isomaltotriose, panose, etc.) 25% Other isomaltosugars 15% Sugars other than isomaltosugars (mainly glucose and maltose) 10% **Over 85% (to the solid) of triose mainly panose and isomaltotriose. ***Over 85% (to the solid) of tetraose mainly isomaltotetraose. 
    
     The isomalto-sugars as well as the other sugars are consumed by the microorganisms of the genus bifidobacteria. However, most microorganisms other than bifidobacteria do not consume the isomalto-sugars, as is seen in Table 2. Thus, it is apparent that the isomalto-sugars are consumed selectively by the bifidobacteria. 
     Raffinose is not consumed by some of the microorganisms, such as the genus Clostridium or the genus Fusobacterium, while other sugars such as glucose and lactose are consumed by all microorganisms. However, Raffinose is consumed by Escherichia coli and the genus Bacteroides which are the microorganisms which produce harmful substances in the intestine. The selectivity of Raffinose is apparently lower than the selectivity of isomalto-sugar for bifidobacteria. 
     The isomalto-sugars are selective and efficient bifidobacteria proliferating agents. The isomalto-sugars may be used singly, as a mixture with foodstuffs, as a raw material or with a pharmaceutically acceptable diluent in the forms such as, for example, a powder, granule, tablet, sugar-coated tablet, capsule, suspension, solution, emulsion or ampoule. As an active ingredient, the isomalto-sugars are present from about 0.1 to 100% by weight in the proliferating agent which is taken orally or parenterally according to this invention. 
     The amount suitable for oral intake of the proliferating agent is in the range from about 0.01 to about 2.0 grams/kg of body weight per day, preferably 0.1 to 1.5 g/kg/day, although it varies depending on individual differences. 
     Bifidobacteria may be admixed with the proliferating agent in order to increase the proliferation promoting action. For this purpose, dried cells of the microorganisms belonging to the genus Bifidobacterium are used at about 30 to 70 parts by weight to about 70 to 30 parts by weight of the isomalto-oligosaccharides solid. 
     Vitamins, i.e. pantetheine, pantothenic acid, and riboflavin are also known as essential substances for the growth of Bifidobacterium, and therefore, their use together with the isomalto-oligosaccharides is effective to increase the growth of Bifidobacterium in the intestines. Suitable amounts of these vitamins are from about 1/100 to 1/300 to the weight of the isomalto-oligosaccharides. 
     Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limitlng thereof. 
    
    
     EXAMPLE 1 
     Bread was made by straight process with the following composition: 
     
         ______________________________________Composition    Control        Test______________________________________Wheat flour    500       gr       500  grSucrose        100                5055% fructose syrup          33                 33Isomalto-sugar**          0                  66Whole egg      50                 50Shortening     30                 30Dried yeast    10                 10Yeast food     0.5                0.5Salt           3                  3Water          217                201______________________________________ *Moisture 25%. &#34;Sunfruct&#34; made by Sanmatsu Kogyo Co. **Moisture 24.2% and about 50% of isomaltooligosaccharides to the solid. 
    
     Dough was prepared by the following steps: 
     (1) Activate yeast prior to use by mixing warm water with the mixture of dried yeast and a small amount of sucrose. 
     (2) Mix sugar including isomalto-sugar syrup, egg, yeast food, salt and half of the required flour in a mixing bowl, and knead the mixture at 160 rpm for 1 minute. 
     (3) Add yeast and knead at 160 rpm for 3 minutes. 
     (4) Add the rest of the flour and knead at 160 rpm for 1 minute and then at 230 rpm for 3 minutes. 
     (5) Add shortening and knead at 230 rpm for 4 minutes to obtain the dough. 
     Fermentation and baking were carried out as follows: 
     (1) Place the dough in a thermo-hygrostst at 29° C. temperature and 90 to 100% relative humidity for 2 hours. 
     (2) Punch (degas) the dough and place it in the same conditions for 1 hour. 
     (3) Punch ag in and further ferment it in the same conditions for 20 minutes. 
     (4) Cut the dough into 50 gram pieces. 
     (5) Bench at 30 to 31° C. for 20 minutes. 
     (6) Proof at 36 to 37° C. for 60 minutes. 
     (7) Bake at 180° C. for 10 minutes. 
     The moisture content of the bread using the isomalto-oligosaccharides (&#34;test&#34;) was stable during the preservation and staling was considerably retarded. Taste, texture of the crumbs, etc. were also excellent as compared to the bread of the &#34;control&#34;. 
     EXAMPLE 2 
     Fifty parts of spray dried isomalto-oligosaccharides containing about 90% isomalto-sugar were admixed with 5 parts of magnesium stearate, 25 parts of corn starch and 20 parts of lactose and made into a tablet. The composition of isomalto-oligosaccharides was as follows: 
     
         ______________________________________Isomaltose           28%Panose and Isomaltotriose               45Isomaltotetraose and others               17______________________________________ 
    
     EXAMPLE 3 
     (1) The bread prepared in Example 1 was fed to 20 male and female volunteers ages between 25 and 35 years old. The volunteers were divided into two groups, and the bread &#34;control&#34; was fed to the first group and the bread &#34;test&#34; to the other. The daily intake of isomalto-oligosaccharides was about 6 grams for the &#34;test&#34; group. The feeding continued for 4 weeks. Feces samples were collected from the 20 persons at the beginning and the final days of the period, and the numbers of microorganisms in the samples were determined. 
     It was observed that the number of microorganisms belonging to the genus Bifidobacterium increased about 100 times by comparing the samples from the beginning and the final days of the &#34;test&#34; group, while the number was unchanged in the &#34;control&#34; group. The numbers of the microorganisms other than bifidobacteria such as the genus Clostridium, Escherichia coli, etc. on the final day were relatively lower than the beginning in the &#34;test&#34; group. 
     (2) The tablet prepared in Example 2 was fed to 10 male vounteers ages between 65 and 80 years old, with a daily intake of 20 grams of isomalto-sugar, for 60 days. The numbers of microorganisms in the feces collected from all the members were determined every 5  days from the beginning of the feeding. An increase in the number of bifidobacteria during the whole period was observed for 7 persons, and for three members of this group the number of bifidobacteria was very high. No increase was observed for one person, while a very slight increase was observed for two of the remaining three persons. 
     Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the intended claims, the invention may be practiced otherwise than as specifically described herein.