The goal of the present study was to produce and characterize resistant starch-containing preparations with different susceptibilities to pancreatic α-amylase, and compare the growth of selected strains of Bifidobacterium when prepared resistant starches with different susceptibility to continued pancreatic α-amylase digestion are used as the main source of carbohydrate.
RS-containing starch preparations were obtained by rehydration (at 1% or 2% starch concentration for 16 hours) in the presence of variable levels of isoamylase (0, 10 or 20U/g of starch) of an ethanol precipitate of either common corn starch (CCS) or high-amylose maize starch (HAMS) that had been molecularly dispersed in 0.5N NaOH. Aspects of physical structure such as crystallinity, helicity, and thermal properties were analyzed using wide-angle X-ray diffraction (WXRD), solid-state 13C CP/MAS NMR, and differential scanning calorimetry (DSC). The molecular composition of the RS-containing preparations was analyzed by size exclusion chromatography (SEC) and high performance size exclusion chromatography (HPSEC). The molecular composition of the RS obtained from the RS-containing preparations was also analyzed by HPSEC.
When the ethanol precipitate was rehydrated in the absence of isoamylase, the RS content was not enhanced for the CCS preparations, but this treatment did enhance RS levels somewhat for the HAMS preparations. HPSEC profiles of fully debranched starch obtained by this rehydration treatment of the ethanol precipitates of CCS and HAMS were similar to the profiles of the corresponding CCS and HAMS starting materials, indicating proportional coprecipitation of amylose and amylopectin from dispersion. The lack of crystallinity in the WXRD spectra, the sharpness of the C4 peak (∼82 ppm) in the NMR spectra, and the broadness of the endotherm at <140°C in the DSC thermograms suggested the presence of amorphous amylose-amylose double helices and mixed double helices between amylose and amylopectin.
For CCS, the sample rehydrated at 2% with 20U isoamylase (2%R-20U) gave the highest starch recovery (48%) and the highest proportion of RS (18%). Therefore, to compare the nature of the preparations obtained, the CCS and HAMS samples rehydrated at 2% starch concentration with 20U isoamylase were used in further studies. For these two samples, the effect of particle size and thermal treatment on RS content was investigated. The proportion of RS after thermal treatment was independent of particle size, about 14% for the CCS sample and 23% for the HAMS sample. Molecular composition of the RS indicated a greater proportion of longer double helices in the thermally treated RS than in RS. This outcome was attributed to melting of the shorter chains during the thermal treatment and to double helical association of the then mobile longer chains during the thermal treatment or during cooling.
Because a primary objective of the present thesis was to explore whether bifidobacterium strains could degrade RS, every effort was made to reduce the amylolytic activity associated with the recovered RS without losing integrity of the RS structure. The most effective treatment in reducing the residual amylolytic activity was 24 h incubation at 37°C of the recovered RS (without ethanol precipitation) in 0.1% EDTA solution. Although this EDTA treatment reduced the residual amylolytic activity of the RS recovered in the supernatant by more than 95%, the total level of residual pancreatic α-amylase associated with the EDTA treated RS could not be quantified.
Finally, the putative RS from HAMS, HMT, CCS 2%R-20U and HAMS 2%R-20U was recovered, exposed to the EDTA treatment, and used as the main carbohydrate source in MRS medium to evaluate the growth of three strains of Bifidobacterium (B. choerinum ATCC 27686, B. infantis ATCC 15697, and B. pseudolongum ATCC 25526) each of which had been shown to partially degrade RS-containing starch according to prior screening. For all three strains, the putative RS from HMT, having the lowest residual susceptibility to pancreatic α-amylase digestion, had the least favorable effect on viable cell counts. This outcome indicates that the susceptibility of RS to further digestion may influence the ability of specific Bifidobacterium strains to utilize the RS for growth in vitro . (Abstract shortened by UMI.)