The effects of pullulan (PR-5) intake were examined in thirteen healthy human volunteers . Pullulan (10 g/day) was orally administrated at lunch for fourteen days . Administrated pullulan was fully digested in the intestinal tracts, including large intestine, and not detected in feces. Daily fecal weight was increased by 33%, and fecal pH level was decreased by the intakes of PR-5. These results indicated that pullulan was available for human intestinal bacteria on growth as nutrients, and for the substrate of anaerobic fermentation in human large intestine. Fecal populations of Bifidobacterium were increased in five of six subjects by the PR-5 intakes. As a result, the ratio of Bi fidobacterium in human fecal microflora was transformed: 11.9% before intake; 21.9% after intake. No significant differences were observed in blood composition, including total-cholesterol level and blood nressure. These findings suggested that orally administrated pullulan functions as dietary fiber, and improves human intestinal environments.

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The α-amylase usually contained in commercial Rhizopus glucoamylase preparations was isolated and purified to an electrophoretically pure state. The optimum pH of the enzyme was around 5.0-5.2 The enzyme was stable over a pH range from 3.0 to 8.0 where the stability was not affected by the presence of EDTA. On the other hand, the enzyme was sensitive at temperatures higher than 50°C, and highly sensitive to mercuric ions. The action pattern on and specificity for starch of Rhiz, α-amylase considerably differed from those of Taka amylase. In the specificity, Rhiz. α-amylase seemed to be rather similar to bacterial α-amylase of saccharifying type. On the other hand, Rhiz. α-amylase showed negligible activity in degrading raw corn starch, but accelerated the hydrolysis of starch by Rhiz. glucoamylase. This acceleration mechanism of Rhiz. α-amylase remains unsolved. However, the a-amylase was observed to make wider the holes being bored by glucoamylase on starch granules and the implication of the results was discussed.

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The nucleotide sequence of alkalophilic Bacillus oligo-1, 6-glucosidase gene was determined . The coding sequence on a 3.1 kb DNA fragment in pMC21 showed an open reading frame of 1527 by encoding 509 amino acids. The amino acid sequence from position 2 to 11 corresponds exactly to the NH2-terminal sequence of the purified enzyme protein from strain F5. The molecular weight (60, 142) of the protein derived from 1524 by open reading frame coincided with the molecular weight (60, 000) of the parent strain. On the other hand, the molecular weight of purified oligo-1, 6-glucosidase produced by E. coli HB101 carrying plasmid pMC21 showed 65, 000 by SDS-PAGE. The enzymatic properties, i. e., pH and temperature optima, pH and thermal stabilities, and substrate specificities, were almost the same as those obtained from an alkalophilic Bacillus sp. (strain F5).

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Samples of eleven varieties of chestnut (Castanea crenata SIEB, et ZUCC.), early fruit (Tanzawa, Ibuki, Yamato-wase, Izumo, and Fukunami), intermediate fruit (Tsukuba), and late fruit (Ginyose, Imakita, Otomune, Taziri-ginyose, and Ishizuchi), were harvested at each differing stage during the period of June to October, 1986. Each slice of the chestnut endosperm was observed under polarized-light microscope and scanning electron microscope (SEM) and the properties of starch granules were examined. The results obtained were as follows 1) By polarized-light microscopy, the particle size of starch granules was found to increase during the early developmental stages ; 2) By SEM, starch granules in the slice of the chestnut endosperm were found to be slender ellipsoid and chestnut shape ; 3) In the particle size distribution study on the starch granules, the average size of chestnut starch granules was found to increase during the early stage of fruit development. The most common particle size of the granules was 10-20μm except for the Taziri-ginyose harvested on September 22; 4) X-ray diffractograms of the starches were Ca, C, or B types in response to the environmental temperature during the chestnut fruit development.

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Samples of eleven varieties of chestnut (Castanea crenata SIEB. et ZUCC.) were harvested at different stages of development and the properties of starch granules prepared from the chestnut were examined. The results obtained were as follows 1) The susceptibility of chestnut starch granules to hog pancreatin was relatively high and tended to be higher in the later stages of development than in the earlier stages, except for the Imakita variety, under the 24-hr reactions. Considering all varieties of chestnut, the difference in susceptibility between more-susceptible starch granules and less-susceptible ones to the action of pancreatin was about 15% with 3-hr and 24-hr reaction. 2) By SEM, we observed oval and spherical granules and slender granules like chestnut shape, and erosion on the surface of the starch granules attacked by pancreatin. 3) The initiation temperature for gelatinization of chestnut starches in the earlier stage and in the later stage of development was 66-70°C and 55-61°C, respectively, as judged on photopastegraphy, and was 67-70°C and 57-60°C, respectively, as judged on differential scanning calorimetry (DSC) . It tended to be lower in the later stages of development than in the earlier stages. 4) The amylose content of chestnut starches, determined by amperometric iodine titration method, was 15-20% and increased in the early stages of fruit development until August 25 for Tanzawa variety and until September 8 for others. The amylose content of intermediate and late fruits tended to be lower than that of early fruits. 5) The amylose content of chestnut (for Tanzawa, Tsukuba and Ginyose) starches, determined by the enzyme-chromatographic method, tended to increase with development.

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Maltooligosaccharides (G₃-G₆) have been used as reagents for research and clinical reagents for the determination of serum amylase activity. G₅ has been used as a nutrient for calorie deprivation. G₅ as well as G₃, G₄, and G₆ are all highly soluble and produce clear viscous solutions which are palatable. Therefore, these compounds may potentially be of use as superior nutrients for infants and the aged. The price of pure maltooligosaccharides is, however, extremely high, because of the difficulties encountered in producing each in a pure state . Several reports have been published on the production of G₂, G₃, G4, G₅, etc., from starch. G₂ production from starch has been widely studied using plant β-amylases ; G₃-forming amylaseswere discovered in various bacteria. Among the maltooligosaccharide (G₃-G₅)-forming amylases, it was reported that a G₄-forming amylase was produced by Pseudomonas stutzeri, but no other bacteria which would produce G ₄-forming amylases have yet been reported. Pseudomonas saccharophila was found to produce that enzyme. Detail on this enzyme will be reported in this journal in the near future. G₅-forming amylases and a G₅-producing bacterium were reported to form mainly G₅, with considerable amounts of other oligosaccharides such as G₆, G₇, G₂, G₃, and G₄, during the reaction and cultivation. The authors tried to find a novel enzyme which forms G₅, without any other saccharides at some stage of the reaction. A novel microorganism which produces such an enzyme has been discovered.

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