Screening and selection of Bifidobacterium strains isolated from human feces capable of utilizing resistant starch.

BACKGROUND: Resistant starch (RS) has been studied for its ability to serve as a substrate for the microbiota present in the human large intestine and for its beneficial physiological effects. The aim of this study was to screen and select novel strains of lactic acid bacteria (LAB) in the genus Bifidobacterium isolated from human fecal samples for further application as probiotics relying on their utilization of RS3, a prebiotic. RESULTS: LAB were isolated from human fecal samples, based on their ability to utilize RS3 as a carbon source. Consequently, two LAB were identified as Bifidobacterium adolescentis based on morphological, physiological and biochemical properties, and molecular biological analysis. The RS3-utilizing ability of these isolates was shown by the rapid decrease in pH of RS3-MRS media and by the pinhole traces on the surface of RS3 particles. Isolated B. adolescentis JSC2 was shown to be negative for ß-glucuronidase, suggesting that it would be safe for human use, and was found to be tolerant towards the acidic, bile-salt environment. CONCLUSION: This synbiotics approach of B. adolescentis JCS2, an RS-utilizing probiotics, coupled with RS utilization, is expected to enhance RS utilization in the food industry and be beneficial for the promotion of human health. © 2018 Society of Chemical Industry.

Effects of charge distribution and degree of methylesterification of pectin emulsifier on bioaccessibility of curcumin incorporated in nanoemulsions.

The objectives of this study were to elucidate the effects of degree of methyl esterification (DM) and charge distribution of pectin on the stability of emulsions and to analyze bioaccessibility of curcumin incorporated in emulsions stabilized by pectins. Three commercial pectins, CP72 (DM72), CP50 (DM50), and CP7 (DM7), were used. MP50 (DM50) with consecutive demethylesterified galacturonic acid residues was prepared from CP72 via demethylesterification to induce different charge distributions. Emulsions containing curcumin were prepared and were stored for 30 days. The CP72 and CP50 emulsions remained relatively stable for 30 days. However, MP50 and CP7 were less effective at forming stable emulsions. When the pectin emulsions passed through each phase of the simulated gastrointestinal tract (GIT), the CP72 and CP50 emulsions retained their initial droplet structures after in vitro mouth and gastric digestion, whereas the MP50 and CP7 emulsions exhibited gel-like clusters, although the gel-like formation of MP50 was distinct from that observed in CP7. MP50 emulsion showed a high degree of final lipid digestion and high bioaccessibility of curcumin while CP72 emulsion displayed a low degree of final lipid digestion. CP50 exhibited low bioaccessibility of curcumin, which might have been contributed by its fast lipid digestion profiles.

Determining the gelation temperature of ß-lactoglobulin using in situ microscopic imaging.

Evolution of microstructure during heat-induced gelation of ß-lactoglobulin (ß-LG) was investigated in situ using confocal laser scanning microscopy at various gel-preparation conditions: pH=2, 5, and 7; protein content=5, 10, and 15%; and salt (NaCl) content=0, 0.1, and 0.3 M. The number and area of evolving ß-LG clusters were observed as a function of time and temperature and the data were fitted to a log-normal model and sigmoid model, respectively. The gelation temperature (Tgel) of the ß-LG system was determined from both the number (Tgel/N) and total area (Tgel/A) of ß-LG clusters versus temperature data. The range of Tgel/N and Tgel/A values for all the cases was 68 to 87°C. The effect of pH was the most dominant on Tgel/N and Tgel/A, whereas the effects of ß-LG and salt contents were also statistically significant. Therefore, the combined effect of protein concentration, pH, and salt content is critical to determine the overall gel microstructure and Tgel. The Tgel/N and Tgel/A generally agreed well with Tgel determined by dynamic rheometry (Tgel/R). The correlations between Tgel/N and Tgel/A versus Tgel/R were 0.85 and 0.72, respectively. In addition, Tgel/N and Tgel/A values compared well with Tgel/R values reported in the literature. Based on these results, Tgel/N determined via in situ microscopy appears to be a fairly good representative of the traditionally measured gelation temperature, Tgel/R.

Modeling of in vitro digestion behavior of corn starches of different digestibility using modified log of slope (LOS) method.

This study aimed to further improve the previously described first-order equation representing in vitro digestion of starch by extensively explaining modified log of slope (LOS) plot method. Hydrolysis curves of various starches were analyzed using original and/or modified LOS plot methods. Some starches showed significant differences in the results from the two methods; specifically, the modified method better described the digestive behavior of starch with various digestion properties, supported by higher determination coefficient values and better estimation of the digestibility data over digestive phase. The digestion parameters obtained from the modified method provided multiple types of information, including amount and digestion rate of each starch fraction (rapidly digestible, slowly digestible, and resistant starch), supporting the concept of digestible fraction classification. Therefore, the modified LOS plot method described here can be applied as an effective tool for analyzing and describing the multi-scale in vitro digestion behavior of starch.

Isothermal and temperature-cycling retrogradation of high-amylose corn starch: Impact of sonication on its structural and retrogradation properties.

In this study, the effects of sonication and temperature-cycled storage on the structural properties and resistant starch content of high-amylose corn starch were investigated. Sonication induced a partial depolymerization of the molecular structures of amylopectin and amylose. Sonication treatment induced the appropriate structural changes for retrogradation. Although the relative crystallinity of sonicated starch was lower than that of non-sonicated starch, sonicated starch after retrogradation showed much higher relative crystallinity than non-sonicated starch. Regardless of sonication treatment, temperature-cycled storage resulted in a higher degree of retrogradation than isothermal storage, but the rate of retrogradation was greater in sonicated starch than in non-sonicated starch, as supported by retrogradation enthalpy, the Avrami constant, and relative crystallinity. The highly developed crystalline structure in sonicated starches due to retrogradation was reflected by the large amount of resistant starch.

Amylosucrase-modified waxy potato starches recrystallized with amylose: The role of amylopectin chain length in formation of low-digestible fractions.

This study examined the contribution of amylose to the organization of each starch fraction in recrystallized starch. Amylosucrase (AS)-modified waxy potato starches with different branch chain lengths were completely solubilized with amylose (3:1 ratio) and recrystallized at 4 °C for 48 h. The content of rapidly digestible starch and resistant starch (RS) showed linear change with degree of AS modification, while slowly digestible starch (SDS) did not. The changes in structural characteristics were tracked according to serial removal of each fraction. Results from iodine binding property, branch chain length, X-ray diffraction, and thermal property analysis indicated that branch chain length of amylopectin determined the length of the amylose-amylopectin double helix and the mobility of amylose and that formation of SDS or RS could be induced by controlling the length of amylopectin chains. These findings could be used for production of customized starches with specific digestive properties for health benefits.

Structural characteristics of low-digestible sweet potato starch prepared by heat-moisture treatment.

The objectives of this study were to optimize the conditions for the preparation of sweet potato starch containing a high proportion of slowly digestible starch (SDS) by heat-moisture treatment (HMT), and to investigate the structural properties of SDS and resistant starch (RS) fractions isolated from this modified starch. Response surface methodology evaluation of considering temperature, time, and moisture content was conducted to determine the HMT condition for maximizing SDS yield. Maximum SDS content (50.9%) was obtained after 8.6 h at 113 °C with 22.8% moisture level. Scanning electron micrographs of RS fractions displayed growth ring structure and hollow regions. X-ray diffraction patterns of SDS + RS and RS fractions were not changed by HMT. Gelatinization enthalpy of the SDS + RS fraction was decreased more than that of the RS fraction. The results indicated that the SDS fraction consisted of less perfect crystallites and amorphous regions, whereas the RS fraction was composed mostly of crystalline regions.

Impact of antioxidant on the stability of ß-carotene in model beverage emulsions: Role of emulsion interfacial membrane.

The effect of the thickness and density of droplet interfacial membrane on the chemical stability of ß-carotene in emulsions was investigated, and its impact on the effectiveness of oil-soluble antioxidants to retard ß-carotene degradation was examined. ß-Carotene was incorporated into the emulsions stabilized by PEGylated emulsifiers having various-sized hydrophilic groups. In the presence of oxidative stresses (pH, iron ions, and radicals in this study), it was observed that the interfacial thickness was relevant to the stability of ß-carotene encapsulated into emulsion droplets. Particularly, iron-mediated carotene degradation was effectively retarded in the emulsions having a thin interfacial membrane than ones with a thick interfacial membrane. The interfacial denseness also affected ß-carotene stability but its ability to retard ß-carotene degradation was influenced by the interfacial thickness. Although ß-carotene degradation rate decreased upon the addition of oil-soluble antioxidants, its antioxidant activity depended on what prooxidant promoted the degradation of ß-carotene in the emulsions.

Complexation of Amylosucrase-Modified Waxy Corn Starch with Fatty Acids: Determination of Their Physicochemical Properties and Digestibilities.

In this study, starch-lipid complexes were prepared using normal corn starch (NC) and amylosucrase-modified waxy corn starch (ASWC) with myristic acid (C14:0) and palmitic acid (C16:0). The amylosucrase modification elongated branch chains in waxy corn starch leading to an increase of apparent amylose content (29.7%) similar to that of NC (29.0%). The X-ray diffraction of starch-lipid complexes revealed a V-type pattern, a clear indication of complex formation. The ability of the ASWC to complex with fatty acids was greater than that of NC. Interestingly, the changes in relative crystallinity, thermal parameters, and digestion properties according to the complexation showed opposite patterns in NC and ASWC. This study found that the structure of ASWC contributes to the formation of starch-fatty acid complexes and suggested that the ASWC can be preferred over NC in a delivery system. PRACTICAL APPLICATION: Amylopectin has been considered to be incapable of forming complexes with fatty acids due to its short chain length and steric hindrance. Through this study, an appropriate enzymatic modification of the molecular structures of waxy starches could make a complexation of waxy starches with fatty acids possible. The findings of this study suggest a promising perspective for utilization of waxy starch as a carrier material of lipophilic molecules.

Antioxidative and hypocholesterolemic activities of water-soluble puerarin glycosides in HepG2 cells and in C57 BL/6J mice.

Puerarin is an isoflavone derived from Kudzu roots and has antioxidant and hypocholesterolemic effects; however, its insolubility often limits its biological availability in vivo. Using a novel transglycosylation process, the solubility of puerarin glycosides was increased >100-fold, but it was not known whether these modified puerarin glycosides maintained biological activities. We found that water-soluble puerarin glycosides fully maintained antioxidant activities compared with puerarin assessed by radical scavenging activity, reducing power assay, superoxide dismutase activity, and non-site-specific hydroxyl radical scavenging activity. Both puerarin and its glycosides also significantly reduced low-density lipoprotein (LDL) oxidation. Mice fed with puerarin glycosides (0.1% w/w) showed significantly reduced plasma total cholesterol levels, thus, we further investigated their hypocholesterolemic mechanisms by assessing several key gene expressions both in vitro and in vivo. Puerarin and its glycosides induced multiple changes in hepatic cholesterol metabolism. The LDL receptor promoter activity was increased dose-dependently in puerarin glycosides-treated HepG2 cells. Accordingly, the expression of LDL receptor mRNA and protein were also significantly increased in HepG2 cells and mouse livers. The transcription and translation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase were down-regulated both in vitro and in vivo. The cholesterol 7alpha-hydroxylase (CYP7A1) mRNA levels were not affected in vitro but significantly up-regulated in the mouse livers. Collectively, our results show that puerarin and its glycosides are biologically fully active isoflavone and have antioxidant and hypocholesterolemic effects in HepG2 cells and in C57BL/6J mice. In the livers, hypocholesterolemic effects of puerarin glycoside may be achieved by multiple mechanisms including increasing LDL uptake, reducing cholesterol biosynthesis, and possibly enhancing cholesterol degradation.

Backscattered electron imaging for reduced charging of moisturized corn starch granules: implications for versatile imagery of hygroscopic powder specimens.

Charging artifacts and surface features of corn starch granules were investigated by scanning electron microscopy. Three types of industrial waxy corn starch granules with different levels of moisture content (0, 10.3, and 24.2%) were prepared and subjected to both secondary electron imaging and backscattered electron imaging. There were no significant charging artifacts in secondary electron images at 3 or 5 kV. However, imaging at higher magnifications and accelerating voltages much lower than 3 kV ranging from 0.1 to 1 kV did not show well-resolved structures. At higher accelerating voltages than 5 kV, charging was manifested as excessive brightness at specific areas and alteration of bright and dark lines in the direction of the raster pattern in secondary electron images of all the types of specimens tested. As the accelerating voltage increased up to 30 kV in secondary electron images, the charging also increased. Meanwhile, no charging was detected in all the backscattered electron images taken at different accelerating voltages. As the accelerating voltage increased in backscattered electron images, the resolution increased with less depth of focus. Consistent results were found in all the types of corn starch granules assayed in this study. These results suggest that a simple and rapid morphological analysis of moisturized starches can be performed by backscattered electron imaging without considerable heat drying of starches. Concomitantly, it allows for imposing a higher accelerating voltage to ensure better image resolution, facilitating morphological characterization of diverse starch granules as they are in native states.

Purification and characterization of branching specificity of a novel extracellular amylolytic enzyme from marine hyperthermophilic Rhodothermus marinus.

An extracellular enzyme (RMEBE) possessing alpha- (1-->4)-(1-->6)-transferring activity was purified to homogeneity from Rhodothermus marinus by combination of ammonium sulfate precipitation, Q-Sepharose ion-exchange, and Superdex- 200 gel filtration chromatographies, and preparative native polyacrylamide gel electrophoresis. The purified enzyme had an optimum pH of 6.0 and was highly thermostable with a maximal activity at 80 degrees . Its half-life was determined to be 73.7 and 16.7 min at 80 and 85 degrees , respectively. The enzyme was also halophilic and highly halotolerant up to about 2 M NaCl, with a maximal activity at 0.5M. The substrate specificity of RMEBE suggested that it possesses partial characteristics of both glucan branching enzyme and neopullulanase. RMEBE clearly produced branched glucans from amylose, with partial alpha-(1-->4)-hydrolysis of amylose and starch. At the same time, it hydrolyzed pullulan partly to panose, and exhibited alpha-(1-->4)-(1-->6)-transferase activity for small maltooligosaccharides, producing disproportionated alpha-(1-->6)-branched maltooligosaccharides. The enzyme preferred maltopentaose and maltohexaose to smaller maltooligosaccharides for production of longer branched products. Thus, the results suggest that RMEBE might be applied for production of branched oligosaccharides from small maltodextrins at high temperature or even at high salinity.

Influence of emulsion interfacial membrane characteristics on Ostwald ripening in a model emulsion.

Ostwald ripening is a major destabilization mechanism for emulsions containing flavor oils with relatively high water solubilities. Emulsions with different oil phase compositions were prepared that were stabilized by polyoxyethylene alkyl ether-type emulsifiers with differently sized hydrophilic and hydrophobic groups. Emulsions prepared using only orange oil were highly unstable to Ostwlad ripening during storage. When emulsifier concentration was increased, Ostawald ripening in emulsions containing emulsifiers with small hydrophilic groups was inhibited, while size increment of droplets in emulsions containing emulsifiers with large hydrophilic groups was not. Droplet enlargement was effectively inhibited by incorporating corn oil into the oil phase prior to homogenization. However, the concentration of corn oil required to inhibit Ostwald ripening varied depending on the structural characteristics and concentrations of the emulsifiers present. These results could have important implications for the selection of emulsifiers to improve the physical stability of orange oil emulsions for use in the food and beverage industries.

Oxidative stability of extracts from red ginseng and puffed red ginseng in bulk oil or oil-in-water emulsion matrix.

BACKGROUND: Explosive puffing can induce changes in the chemical, nutritional, and sensory quality of red ginseng. The antioxidant properties of ethanolic extracts of red ginseng and puffed red ginseng were determined in bulk oil and oil-in-water (O/W) emulsions. METHODS: Bulk oils were heated at 60°C and 100°C and O/W emulsions were treated under riboflavin photosensitization. In vitro antioxidant assays, including 2,2-diphenyl-1-picrylhudrazyl, 2,2'-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid, ferric reducing antioxidant power, total phenolic content, and total flavonoid content, were also performed. RESULTS: The total ginsenoside contents of ethanolic extract from red ginseng and puffed red ginseng were 42.33 mg/g and 49.22 mg/g, respectively. All results from above in vitro antioxidant assays revealed that extracts of puffed red ginseng had significantly higher antioxidant capacities than those of red ginseng (p < 0.05). Generally, extracts of puffed red and red ginseng had high antioxidant properties in riboflavin photosensitized O/W emulsions. However, in bulk oil systems, extracts of puffed red and red ginseng inhibited or accelerated rates of lipid oxidation, depending on treatment temperature and the type of assay used. CONCLUSION: Although ethanolic extracts of puffed red ginseng showed stronger antioxidant capacities than those of red ginseng when in vitro assays were used, more pro-oxidant properties were observed in bulk oils and O/W emulsions.

Molecular cloning and characterization of trehalose biosynthesis genes from hyperthermophilic archaebacterium Metallosphaera hakonesis.

The trehalose (alpha-D-glucopyranosyl-[1,1]-alpha-D-glucopyranose) biosynthesis genes MhMTS and MhMTH, encoding a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively, have been cloned from the hyperthermophilic archaebacterium Metallosphaera hakonesis. The ORF of MhMTS is 2,142 bp long, and encodes 713 amino acid residues constituting a 83.8 kDa protein. MhMTH is 1,677 bp long, and encodes 558 amino acid residues constituting a 63.7 kDa protein. The deduced amino acid sequences of MhMTS and MhMTH contain four regions highly conserved for MTSs and three for MTHs that are known to constitute substrate-binding sites of starch-hydrolyzing enzymes. Recombinant proteins obtained by expressing the MhMTS and MhMTH genes in E. coli catalyzed a sequential reaction converting maltooligosaccharides to produce trehalose. Optimum pH of the MhMTS/MhMTH enzyme reaction was around 5.0 and optimum temperature was around 70 degrees C. Trehalose-producing activity of the MhMTS/ MhMTH was notably stable, retaining 80% of the activity after preincubation of the enzyme mixture at 70 degrees C for 48 h, but was gradually abolished by incubating at above 85 degrees C. Addition of thermostable 4-alpha-glucanotransferase increased the yield of trehalose production from maltopentaose by 10%. The substrate specificity of the MhMTS/MhMTH-catalyzed reaction was extended to soluble starch, the most abundant maltodextrin in nature.

Preparation and characterization of the inclusion complexes between amylosucrase-treated waxy starch and palmitic acid.

Amylosucrase-treated waxy corn starch (AS) was produced to extend the chain length of amylopectin to a great extent in comparison to its native chain length. An amylopectin-palmitic acid (PA) complex was prepared by heat-treating (121°C) a starch/PA mixture and its subsequent further incubation (95°C, 24 h); moreover, its structure and digestibility were studied. Unmodified waxy starch could not complex at all, whereas elongation due to amylosucrase modification allowed amylopectin to form a complex with PA to a small extent. Complexation between AS and PA caused a decrease in relative crystallinity. The AS-PA complex displayed an endothermic peak representing type I inclusion complexes rather than type II complexes. The formation of complexes did not significantly affect the in vitro digestibility maintaining the low digestibility of AS resulting from extremely small amounts of complexes and the type of complex.

Water-soluble genistin glycoside isoflavones up-regulate antioxidant metallothionein expression and scavenge free radicals.

Genistin has antioxidant activities; however, its insolubility in water often limits its biological availability in vivo. Using a novel transglycosylation process, the solubility of genistin glycosides was increased 1000 to 10000-fold, but it was not known whether these modified genistin glycosides maintained antioxidant activity. We found that both genistin and its glycosides similarly up-regulated the transcription of several metallothionein (MT) antioxidant genes (MT1A, MT2A, MT1E, and MT1X), as well as the glucose 6-phosphate dehydrogenase (G6PD) gene in HepG2 cells. This gene induction was mediated by the sequestration of zinc in the cytosol, which up-regulated the metal-responsive transcription factor-1 (MTF-1) that induced MT gene expression. Although not as effective as ascorbic acid, genistin glycosides possessed slightly greater reducing power than genistin. We concluded that genistin and genistin glycosides have a direct antioxidant effect and an indirect antioxidant effect, perhaps via induction of MT by activity of MTF-1.

PCR identification of ruminant tissue in raw and heat-treated meat meals.

To control the spread of bovine spongiform encephalopathy in cattle through contaminated animal feedstuffs, screening of feed products is essential. We designed five pairs of primers to identify specifically raw and heat-treated tissue from cattle, sheep, goat, deer, and ruminants in general. A forward common primer was designed based on a conserved DNA sequence in the mitochondrial 12S rRNA-tRNA(val)-16S rRNA gene, and reverse primers were designed to hybridize with a species-specific DNA sequence for each species considered. All primers were developed to create a specific PCR product small enough (less than 200 bp) to be suitable for heat-treated material. To evaluate the effect of heat treatment, a severe sterilization condition (133 degrees C at 300 kPa for 20 min) was chosen. Species-specific amplicons were obtained from all types of heat-treated meat meals. Analysis of laboratory-contaminated vegetable meals revealed that the detection limit of the assay was 0.05% for each species analyzed. This PCR-based analysis can be used as a routine method for detecting banned animal-derived ingredients in raw and heat-treated feedstuffs.

Low digestion property of amylosucrase-modified waxy adlay starch.

Structural and digestion properties of amylosucrase-modified waxy adlay starch were investigated. The unique reaction of amylosucrase caused a decrease and an increase in the proportion of short chains and long chains, respectively, via attachment of glucosyl units to the non-reducing ends of branch chains. The in vitro digestion profile of amylosucrase-modified starch revealed that elongated branch chains were the main reason for high contents of slowly digestible and resistant starches due to formation of a more perfect crystalline structure via easy association between elongated branch chains. The glucose response in mice after consumption of amylosucrase-modified starch was similar to the response for commercial resistant starch with a gradual increase followed by a gradual decrease in blood glucose concentrations over a prolonged time. Both in vitro and in vivo tests were used to verify increased resistance to digestive enzymes caused by amylosucrase modification.

Production of an in Vitro Low-Digestible Starch via Hydrothermal Treatment of Amylosucrase-Modified Normal and Waxy Rice Starches and Its Structural Properties.

We investigated dual modification of normal and waxy rice starch, focusing on digestibility. Amylosucrase (AS) was applied to maximize the slowly digestible and resistant starch fractions. AS-modified starches were adjusted to 25-40% moisture levels and heated at 100 °C for 40 min. AS-modified starches exhibited a B-type crystalline structure, and hydrothermal treatment (HTT) significantly (p < 0.05) increased the relative crystallinity with moisture level. The thermal transition properties of modified starches were also affected by the moisture level. The contents of rapidly digestible starch fraction in AS-modified normal and waxy starches (43.3 ± 3.9 and 18.1 ± 0.6%) decreased to 13.0 ± 1.0 and 0.3 ± 0.3% after HTT, accordingly increasing the low digestible fractions. Although the strengthened crystalline structures of AS-modified starches by HTT were not stable enough to maintain their rigidity under cooking, application of AS and HTT was more effective in waxy rice starch than normal rice starch when lowering digestibility.