Development of Slowly Digestible Starch Derived α-Glucans with 4,6-α-Glucanotransferase and Branching Sucrase Enzymes.

Previously, we have identified and characterized 4,6-α-glucanotransferase enzymes of the glycosyl hydrolase (GH) family 70 (GH70) that cleave (α1→4)-linkages in amylose and introduce (α1→6)-linkages in linear chains. The 4,6-α-glucanotransferase of Lactobacillus reuteri 121, for instance, converts amylose into an isomalto/malto-polysaccharide (IMMP) with 90% (α1→6)-linkages. Over the years, also, branching sucrase enzymes belonging to GH70 have been characterized. These enzymes use sucrose as a donor substrate to glucosylate dextran as an acceptor substrate, introducing single -(1→2,6)-α-d-Glcp-(1→6)- (Leuconostoc citreum enzyme) or -(1→3,6)-α-d-Glcp-(1→6)-branches (Leuconostoc citreum, Leuconostoc fallax, Lactobacillus kunkeei enzymes). In this work, we observed that the catalytic domain 2 of the L. kunkeei branching sucrase used not only dextran but also IMMP as the acceptor substrate, introducing -(1→3,6)-α-d-Glcp-(1→6)-branches. The products obtained have been structurally characterized in detail, revealing the addition of single (α1→3)-linked glucose units to IMMP (resulting in a comb-like structure). The in vitro digestibility of the various α-glucans was estimated with the glucose generation rate (GGR) assay that uses rat intestinal acetone powder to simulate the digestive enzymes in the upper intestine. Raw wheat starch is known to be a slowly digestible carbohydrate in mammals and was used as a benchmark control. Compared to raw wheat starch, IMMP and dextran showed reduced digestibility, with partially digestible and indigestible portions. Interestingly, the digestibility of the branching sucrase modified IMMP and dextran products considerably decreased with increasing percentages of (α1→3)-linkages present. The treatment of amylose with 4,6-α-glucanotransferase and branching sucrase/sucrose thus allowed for the synthesis of amylose/starch derived α-glucans with markedly reduced digestibility. These starch derived α-glucans may find applications in the food industry.

New View on Dietary Fiber Selection for Predictable Shifts in Gut Microbiota.

Dietary fibers can be utilized to shape the human gut microbiota. However, the outcomes from most dietary fibers currently used as prebiotics are a result of competition between microbes with overlapping abilities to utilize these fibers. Thus, divergent fiber responses are observed across individuals harboring distinct microbial communities. Here, we propose that dietary fibers can be classified hierarchically according to their specificity toward gut microbes. Highly specific fibers harbor chemical and physical characteristics that allow them to be utilized by only a narrow group of bacteria within the gut, reducing competition for that substrate. The use of such fibers as prebiotics targeted to specific microbes would result in predictable shifts independent of the background microbial composition.

Milk glucosidase activity enables suckled pup starch digestion.

UNLABELLED: ᅟ: Starch requires six enzymes for digestion to free glucose: two amylases (salivary and pancreatic) and four mucosal maltase activities; sucrase-isomaltase and maltase-glucoamylase. All are deficient in suckling rodents. OBJECTIVE: The objective of this study is to test (13)C-starch digestion before weaning by measuring enrichment of blood (13)C-glucose in maltase-glucoamylase-null and wild-type mice. METHODS: Maltase-glucoamylase gene was ablated at the N-terminal. Dams were fed low (13)C-diet and litters kept on low (13)C-diet. Pups were weaned at 21 days. Digestion was tested at 13 and 25 days by intragastric feeding of amylase predigested (13)C-α-limit dextrins. Blood (13)C-glucose enrichment was measured by gas chromatography combustion isotope ratio mass spectrometry (GCRMS) using penta-acetate derivatives. RESULTS: Four hours after feeding, blood (13)C-glucose was enriched by 26 × 10(3) in null and 18 × 10(3) in wild-type mice at 13 days and 0.3 × 10(3) and 0.2 × 103 at 25 days (vs. fasting p = 0.045 and p = 0.045). By jejunal enzyme assay, immunohistochemistry, or Western blots, there was no maltase activity or brush border staining with maltase-glucoamylase antibodies at 13 days, but these were fully developed in the wild-type mice by 25 days. In 13-day null mice, luminal contents were stained by maltase-glucoamylase antibodies. Lactating the mammary gland revealed maltase-glucoamylase antibody staining of alveolar cells. Reverse transcription/polymerase chain reaction (RT/PCR) of lactating glands revealed a secreted form of maltase-glucoamylase. CONCLUSIONS: (1) (13)C-α-limit dextrins were rapidly digested to (13)C-glucose in 13-day mice independent of maltase-glucoamylase genotype or mucosal maltase activity. (2) This experiment demonstrates that a soluble maltase activity is secreted in mouse mother's milk which enables suckling pup starch digestion well before brush border enzyme development. (3) This experiment with (13)C-α-limit dextrins needs to be repeated in human breast fed infants.

Nutritional evaluation of high-digestible sorghum for pigs and broiler chicks.

Two experiments were conducted to evaluate the nutritional quality of 2 varieties of Purdue high-digestible sorghum (PHD1 and PHD2) and a normal sorghum, compared with corn, in diets of pigs and broiler chicks. In Exp. 1, 12 pigs (average BW, 55 kg) fitted with ileal T-cannula were fed 4 diets containing 946 g of corn or sorghum (PHD1, PHD2, and normal) per kg in a 2-period crossover design (i.e., each pig received 2 diets over a 2-wk period with 6 pigs per dietary treatment) to determine apparent ileal or total tract digestibility of nutrients and energy. There was no difference in the ileal or total tract digestibility of DM, energy, P, Ca, or N among dietary treatments. In Exp. 2, a total of 192 broiler chicks were grouped by weight into 8 blocks of 4 cages each with 6 chicks per cage, and cages were assigned randomly to 1 of the 4 dietary treatments within each block. Chicks were fed corn-soybean meal (SBM) or sorghum-SBM diets for 21 d to determine apparent total tract retention and then switched to diets containing 935 g of the corresponding corn or 1 of the 3 sorghum varieties per kg for 7 d to determine apparent ileal digestibility and total tract retention. Apparent ileal digestibilities of DM and P, as well as energy, were not different in chicks fed diets containing 935 g of corn or 1 of the 3 sorghum varieties per kg. However, apparent total tract retention of DM, energy, and N in chicks fed corn was greater (P < 0.05) than those fed 1 of the 3 sorghum varieties. Although the apparent ME content of corn was greater than PHD1 and normal sorghum (P < 0.01), it was not different from PHD2 sorghum. There was no difference in apparent total tract retention of DM between chicks fed the corn-SBM and PHD-SBM diets, but it was greater (P < 0.05) in chicks fed the corn-SBM diet than those fed the normal sorghum-SBM diet. Apparent total tract retention of N in chicks fed the PHD1-SBM diet was lower (P < 0.05) than in those fed the corn-SBM diet but greater (P < 0.05) than in chicks fed the normal sorghum-SBM meal diet. No difference in the apparent ME content between the corn-SBM and PHD2-SBM diets was observed, but it was greater (P < 0.05) for the corn-SBM diet than the PHD1- or normal sorghum-SBM diet. Weight gain, feed intake, and feed efficiency were not different in chicks fed the corn-SBM or sorghum-SBM diets. Sorghum could serve as a substitute for corn in cereal grain-SBM diets for pigs and broiler chicks.

A highly digestible sorghum mutant cultivar exhibits a unique folded structure of endosperm protein bodies.

The endosperm of a sorghum mutant cultivar, with high in vitro uncooked and cooked protein digestibilities, was examined by transmission electron microscopy and alpha-, beta-, and gamma-kafirins (storage proteins) were localized within its protein bodies. Transmission electron microscopy micrographs revealed that these protein bodies had a unique microstructure related to high protein digestibility. They were irregular in shape and had numerous invaginations, often reaching to the central area of the protein body. Protein bodies from normal cultivars, such as P721N studied here, with much lower uncooked and cooked digestibilities are spherical and contain no invaginations. Immunocytochemistry results showed that the relative location of alpha- and beta-kafirins within the protein bodies of the highly digestible genotype were similar to the normal cultivar, P721N. Gamma-kafirin, however, was concentrated in dark-staining regions at the base of the folds instead of at the protein body periphery, as is typical of normal cultivars. The resulting easy accessibility of digestive enzymes to alpha-kafirin, the major storage protein, in addition to the increased surface area of the protein bodies of the highly digestible cultivar appear to account for its high in vitro protein digestibility.

Elongation factor 1 alpha concentration is highly correlated with the lysine content of maize endosperm.

Lysine is the most limiting essential amino acid in cereals, and for many years plant breeders have attempted to increase its concentration to improve the nutritional quality of these grains. The opaque2 mutation in maize doubles the lysine content in the endosperm, but the mechanism by which this occurs is unknown. We show that elongation factor 1 alpha (EF-1 alpha) is overexpressed in opaque2 endosperm compared with its normal counterpart and that there is a highly significant correlation between EF-1 alpha concentration and the total lysine content of the endosperm. This relationship is also true for two other cereals, sorghum and barley. It appears that genetic selection for genotypes with a high concentration of EF-1 alpha can significantly improve the nutritional quality of maize and other cereals.

Effect of dietary fiber and starch on fecal composition in preschool children consuming maize, amaranth, or cassava flours.

Metabolic balance studies were carried out in young children fed diets based on maize, amaranth, or cassava flours and in corresponding casein controls. Dietary fiber intakes were 22.2, 20.5, and 9.0 g/day for the maize, amaranth, and cassava groups, respectively. Fecal energy losses at least doubled in all test diets when compared with the corresponding controls and could generally be accounted for by recovered fiber in the feces. Fecal starch was also a significant contributor to fecal energy in the cassava group. All cassava fiber was recovered in the feces, whereas only 48.4 and 16.3% were recovered from ingested maize and amaranth. 2,6-Diaminopimelic acid (DAPA), an indicator of bacterial mass, was highest in the cassava group. Expired breath hydrogen was highest for those consuming maize or cassava. Resistant starch may have been responsible for the high DAPA and breath hydrogen values in the cassava group.

Distribution of B-6 vitamers in human milk during a 24-h period after oral supplementation with different amounts of pyridoxine.

The relative distribution of B-6 vitamers, separated by reverse-phase liquid chromatography, was examined in human milk during a 24-h period after supplementation with 2.5 or 15 mg pyridoxine hydrochloride. Consistently, pyridoxal (PL) was the predominate vitamer and the most responsive to vitamin B-6 intake. During 3-8 h after supplement ingestion, PL, pyridoxal phosphate, and pyridoxamine concentrations were significantly higher than at other times examined. In the first two periods after supplementation, PL as a percentage of total vitamin B-6 was slightly but significantly higher in milk from the group supplemented with 15 mg than from the group supplemented with 2.5 mg. With the exception of PL, the distribution of B-6 vitamers, expressed as percent of total vitamin B-6, was similar for the two supplemented groups at all times examined. Percentage PL of total vitamin B-6 in milk was approximately 25% lower in unsupplemented than in supplemented women.

A micromethod for determination of plasma pyridoxal phosphate and its use in assessment of storage stability of the vitamer.

A widely used macromethod employing tyrosine apodecarboxylase for measurement of pyridoxal phosphate (PLP) concentration in 0.5-1.0-ml plasma was modified to a microscale utilizing 0.1-ml plasma. Mean PLP levels in 12 plasma samples were 160.6 +/- 32.8 pmol/ml (mean +/- SD) when analyzed by the macromethod, and were not significantly different compared to those obtained by the micromethod (158.4 +/- 28.2 pmol/ml). Results of the two methods were significantly correlated (r = +0.97, p less than 0.001). Plasma PLP concentrations of 11 samples determined by the micromethod (means = 151.8 +/- 30.0 pmol/ml) were similar and significantly correlated (r = +0.95, p less than 0.001) to levels measured in the same samples 1-2 years earlier (means = 145.1 +/- 26.2 pmol/ml). This suggests that plasma PLP content of the samples was stable for up to 2 years of storage when the micromethod was utilized for analysis. The strong significant correlation between macro- and micromethods attests that the micromethod is a reliable alternative to the macromethod. The micromethod is useful in instances where only small samples of plasma are available for measurement of PLP.

Improving the in vitro protein digestibility of sorghum with reducing agents.

We have shown in previous reports that cooked sorghum protein is less digestible than other cooked cereal proteins. The pepsin-indigestible proteins in sorghum were found to be mainly prolamin proteins. Cooking sorghum in the presence of 2-mercaptoethanol increased protein digestibility (in vitro with pepsin or trypsin/chymotrypsin) to a level comparable with other cereals. At a concentration of 100 mM, other reducing agents (dithiothreitol, sodium bisulfite, and L-cysteine) were equally effective in improving sorghum digestibility. When maize was cooked in the presence of 2-mercaptoethanol, protein digestibility increased 5% compared to 25% for sorghum. Cooking barley, rice, and wheat with 2-mercaptoethanol had no significant effect on protein digestibility. The addition of reducing agents appears to prevent the formation of protein polymers linked by disulfide bonds.

Digestibility and utilization of protein and energy from Nasha, a traditional Sudanese fermented sorghum weaning food.

Whole grain sorghum flour was fermented into Nasha, a traditional Sudanese food, and freeze-dried or drum-dried. It was cooked and fed to convalescent malnourished infants and small children as 61% of total diet calories and all of 6.4% protein calories, with (Lys+) and without lysine supplementation to 3% of protein. Apparent absorptions of nitrogen were 73 +/- 5 and 74 +/- 6% of intake, significantly (P less than 0.01) less than those from preceding (Cas-1, 86 +/- 3%) and following (Cas-2, 85 +/- 3%) isonitrogenous casein diets. Apparent retentions of nitrogen from Nasha (26 +/- 10%) were significantly lower than those from Lys + (34 +/- 9%, P less than 0.05), Cas-1 (35 +/- 11%, P less than 0.01) or Cas-2 (49 +/- 9%, P less than 0.01). Retentions from Cas-2 were higher than those from Cas-1 or Lys + (P less than 0.01). Fecal wet and dry weights were higher (P less than 0.02) during both Nasha diets and Cas-2 than during Cas-1. Fecal energy and carbohydrate were significantly (P less than 0.01) higher from either Nasha diet than from either casein diet; fecal fat was not different. Two children received drum-dried Nasha without further cooking; digestibilities were not different from those of the cooked product but biological value was much lower. When properly cooked and consumed along with small amounts of a good source of lysine, Nasha is a satisfactory weaning food.