Raffinose/D-Galactose Assay Kit

Procedures for the measurement of starch, starch damage (gelatinised starch), resistant starch and the amylose/amylopectin content of starch, β-glucan, fructan, glucomannan and galactosyl-sucrose oligosaccharides (raffinose, stachyose and verbascose) in plant material, animal feeds and foods are described. Most of these methods have been successfully subjected to interlaboratory evaluation. All methods are based on the use of enzymes either purified by conventional chromatography or produced using molecular biology techniques. Such methods allow specific, accurate and reliable quantification of a particular component. Problems in calculating the actual weight of galactosyl-sucrose oligosaccharides in test samples are discussed in detail.

An American Association of Cereal Chemists/AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measurement of total starch in a range of cereal grains and products. The flour sample is incubated at 95 degrees C with thermostable alpha-amylase to catalyze the hydrolysis of starch to maltodextrins, the pH of the slurry is adjusted, and the slurry is treated with a highly purified amyloglucosidase to quantitatively hydrolyze the dextrins to glucose. Glucose is measured with glucose oxidase-peroxidase reagent. Thirty-two collaborators were sent 16 homogeneous test samples as 8 blind duplicates. These samples included chicken feed pellets, white bread, green peas, high-amylose maize starch, white wheat flour, wheat starch, oat bran, and spaghetti. All samples were analyzed by the standard procedure as detailed above; 4 samples (high-amylose maize starch and wheat starch) were also analyzed by a method that requires the samples to be cooked first in dimethyl sulfoxide (DMSO). Relative standard deviations for repeatability (RSD(r)) ranged from 2.1 to 3.9%, and relative standard deviations for reproducibility (RSD(R)) ranged from 2.9 to 5.7%. The RSD(R) value for high amylose maize starch analyzed by the standard (non-DMSO) procedure was 5.7%; the value was reduced to 2.9% when the DMSO procedure was used, and the determined starch values increased from 86.9 to 97.2%.

Despite the commercial availability of gluten-free (GF) products, numerous nutritional, sensory, and textural limitations have been brought to the attention of the baking industries. This study aimed at the characterization of four GF flours (pregelatinized rice, pearl millet, common buckwheat, and soy protein isolate) for their nutritional and functional properties. Protein and starch were the major components in soy protein isolate and pregelatinized rice, respectively, whereas buckwheat and millet contained the highest amount of dietary fiber. Free phenolic compounds and antioxidant activity were at the highest levels in buckwheat flour followed by soy protein isolate. Likewise, all investigated ingredients varied greatly in their physicochemical properties. Based on single-ingredient baked-model, the effect on the texture and volumetric profiles of bread was reported, distinguishing GF ingredients in four different clusters with different characteristics. Accordingly, the four GF ingredients were combined to create a composite GF bread with acceptable textural properties approaching to those of a typical wheat bread. These findings might be regarded as a basis to design further innovative recipes and combinations using these raw GF ingredients.

A fermentation protocol including selected lactic acid bacteria has been applied to defatted durum wheat germ, resulting from the oil extraction, to produce a nutritionally valuable ingredient for bread production. An integrated approach was used to evaluate the microbiological, nutritional, technological, and sensory properties of the fermented ingredient and the corresponding fortified bread. The fermentation led to a significant increase of the concentration of free amino acids (3-times) and decrease of the phytic acid (50%) and raffinose (93%) contents. The bread fortified with the sourdough-fermented defatted wheat germ could be labelled as source of fiber (3.3 g/100 g of bread) and source of protein (15.4% of the energy value was provided by proteins), according to the Regulation EC No. 1924/2006. When the fermented ingredient was used, the free amino acids concentration was 80% higher and the glycemic index lower (84 vs 95) than the control bread. Although final volume, hardness and chewiness of bread fortified with the fermented ingredient were similar to those of the control bread, an easier fracturability was found probably due to the high content of dietary fibers and acidity. Sensory analysis showed that fermented defatted wheat germ conferred perceptible acidic odor and taste to the bread.

To lower the risk of obesity, diabetes, and other related diseases, the WHO recommends that consumers reduce their consumption of sugars. Here, we propose a microbiological method to reduce the sugar content in red beet juice, while incurring only slight losses in the betalain content and maintaining the correct proportion of the other beet juice components. Several yeast strains with different metabolic activities were investigated for their ability to reduce the sugar content in red beet juice, which resulted in a decrease in the extract level corresponding to sugar content from 49.7% to 58.2%. This strategy was found to have the additional advantage of increasing the chemical and microbial stability of the red beet juice. Only slight losses of betalain pigments were noted, to final concentrations of 5.11% w/v and 2.56% w/v for the red and yellow fractions, respectively.

Fresh pasta is subjected to rapid spoilage, mainly due to the metabolic activity of bacteria, yeasts, and especially molds, which negatively affect the sensorial characteristics and the safety of the product. In this work, chickpea flour was fermented with selected lactic acid bacteria, characterized in terms of the antifungal activity, and used to fortify fresh semolina pasta. Pasta was characterized and subjected to a long period of storage after being artificially inoculated with Penicillium roqueforti. Conventional fresh semolina pasta, produced with or without calcium propionate addition, was used as a reference. The water/salt-soluble extract from chickpea sourdough exhibited antifungal activity towards a large spectrum of molds. Its purification led to the identification of ten potentially active peptides. Besides the high content of dietary fibers (4.37%) and proteins (11.20%), nutritional improvements, such as the decrease of the antinutritional factors concentration and the starch hydrolysis index (25% lower than the control) and the increase of the protein digestibility (36% higher than the control), were achieved in fresh pasta fortified with the chickpea sourdough. Inhibition of the indicator mold growth during a 40-day storage period was more effective than in pasta added to calcium propionate.

Aiming at meeting recent consumers requirements in terms of high nutritional value and functional foods, the cereal food industry has proposed the use of legumes as wheat substitutes due to the high contents of proteins with high biological value and dietary fibers. Nevertheless, legumes contain several anti-nutritional factors which may limit the bio-availability of several nutrients. In this study, an integrate biotechnological approach, combining a thermal treatment (“gelatinization”) and fermentation with selected lactic acid bacteria, was set-up in order to improve the functional and nutritional quality of red and yellow lentils, white and black beans, chickpeas and peas flours. Gelatinization carried out at pilot-plant level on legume grains before milling, affected the nutritional properties of the flours by the increase of protein digestibility, resistant starch formation, the decrease of trypsin inhibitors, although negatively affecting the antioxidant activity. Fermentation with Lactobacillus plantarum MRS1 and Lactobacillus brevis MRS4 further enhanced the nutritional properties of processed legume flours through the increase of free amino acids concentration and protein digestibility, the degradation of phytic acid, condensed tannins and raffinose, and the decrease of the trypsin inhibitory activity and starch hydrolysis index. Moreover, fermentation also contributed to the increase of the radical scavenging activity of both raw and processed legumes.

Soymilk is a good source of proteins and health-promoting isoflavones, but it contains oligosaccharides that cause flatulence. Fermenting it with probiotic bacteria may reduce the oligosaccharides and enhance its health benefits. The present study determined the growth of different lactic acid bacteria (LAB) in soymilk obtained from soybean varieties grown in Rwanda and the effect of fermentation on oligosaccharides that cause flatulence (stachyose, raffinose and verbascose), and antioxidant activity of fermented soybean milk. After fermentation at 30°C for 24 hours, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus cremoris and Lactobacillus casei attained around 8 log CFU/ml, which is sufficient for probiotic effects. However, only L. reuteri, L. brevis and L. plantarum caused sufficient drop in pH and increase in viscosity characteristic of a good fermented product. Soymilk from different soybean varieties did not show significant differences in the growth of these three LAB. These LAB reduced content of oligosaccharides and total polyphenols, but increased antioxidant activity in soymilk, which translate into health benefits of fermented soybean products.

A biotechnological approach including enzymatic treatment (protease and xylanase) and lactic acid bacteria fermentation has been evaluated to enhance the nutritional value of semolina pasta enriched with hemp, chickpea and milling by-products. The intense (up to circa, (ca.) 70%) decrease in the peptide profile area and (up to two-fold) increase in total free amino acids, compared to the untreated raw materials, highlighted the potential of lactic acid bacteria to positively affect their in vitro protein digestibility. Fermented and unfermented ingredients have been characterized and used to fortify pasta made under pilot-plant scale. Due to the high contents of protein (ca. 13%) and fiber (ca. 6%) and according to the Regulation of the European Community (EC) No. 1924/2006 fortified pasta can be labelled as a “source of fiber” and a “source of protein”. The use of non-wheat flours increased the content of anti-nutritional factors as compared to the control pasta. Nevertheless, fermentation with lactic acid bacteria led to significant decreases in condensed tannins (ca. 50%), phytic acid and raffinose (ca. ten-fold) contents as compared to the unfermented pasta. Moreover, total free amino acids and in vitro protein digestibility values were 60% and 70%, respectively, higher than pasta made only with semolina. Sensory analysis highlighted a strong effect of the fortification on the sensory profile of pasta.

Seed germination begins the growth phases of plants and its rate is affected not only by plant hormones, including abscisic acid (ABA), gibberellin (GA) and brassinosteroids (BRs), but also by environmental factors. In this study, we searched for additional chemical reagents that affect seed germination, using the det2-1 and ga1-3 mutants that showed reduced seed germination due to defective BR- or GA- biosynthesis, respectively. We found that the reducing reagent dithiothreitol (DTT) specifically enhanced seed germination of det2-1 compared with that of ga1-3. To further investigate the underlying molecular mechanism for this phenomenon, we identified AtGOLS1 as a differentially expressed gene in germinating seeds treated with DTT by GeneFishing analysis. AtGOLS1 encodes a galactinol synthase, critical for the first step in raffinose family oligosaccharides synthesis during seed maturation. We observed that expression of AtGOLS1 decreased when conditions were favorable for seed germination. We also determined that the seed germination rate was faster in T-DNA knockout atgols1 mutant and transgenic plants transformed with an RNA interference construct targeting AtGOLS1 compared with wild type plants. The double mutant of det2-1 and atgols1 also suppressed the reduced seed germination of the det2-1. Taken together, our results suggest that AtGOLS1 acts as a negative regulator in seed germination.

The present study was conducted to investigate how fermentation (FE) and enzymatic treatment (ET) of pea affect standardized ileal digestibility (SID) of nutrients in diets with peas as the only protein source, and evaluate the consequences of inclusion of different pea products in turkey diets (100 g/kg) on growth performance and bird health. For FE process, Pisum sativum L. was mixed with water (1:1) containing 4.9 × 10⁸ Bacillus subtilis and licheniformis spores/kg pea (BIOPLUS 2B^®, Chr. Hansen, Denmark). The prepared dough was fermented for 48 h at 30°C. For ET, the dough water contained three enzymes, AlphaGal™ (α-galactosidase − Kerry, USA), RONOZYME^® ProAct and VP (protease and pectinases, respectively − DSM, Switzerland). The dough (500 g/kg DM) was incubated for 24 h at 30°C. Both processes reduced α-galactosides, phytate, trypsin inhibitor activity and resistant starch in peas. For standardized ileal digestibility (SID) assay, 288 turkeys were assigned to 24 pens and received four experimental diets including native (NP), fermented (FEP) and enzymatically treated peas (ETP) as well as a N-free diet (all supplemented with vitamins and minerals). The ETP had better SID of protein, Glu, Phe and Val compared with FEP and NP. Enzymatic treatment of pea also improved standardized ileal digestibility of Ala, Gly, His, Ilu, Leu and Lys (P ≤ 0.05), however digestibility of these nutrients in fermented pea were similar to other two types of pea (P > 0.05). Both processes drastically improved ileal digestibility of starch (P ≤ 0.05). For performance trial, 960 turkeys were allocated into 60 pens and received 4 different diets consisted of a basal mash wheat-SBM diet (CON) and there experimental diets which were prepared by inclusion of each pea products NP (NPD), FEP (FEPD) and ETP (ETPD) in the basal diet at the rate of 100 g/kg. The experiment lasted 105 d. In general, in the most time periods of the performance trial, birds received ETPD or FEPD diets showed better growth performance than those fed NPD diet, while birds in ETPD group displayed similar performance to those fed CON diet. At the end of the trial, birds fed CON and ETPD diets had the best FCR and birds which received NPD diet had the worst one (P ≤ 0.05). Birds in ETPD group showed the best footpad dermatitis score and turkeys in the NPD group had the worst score (P ≤ 0.05). The footpad dermatitis scores for turkeys in CON and FEPD groups were identical and considerably different from those in ETPD and NPD groups (P ≤ 0.05). In conclusion, both processes could improve the nutritional quality of pea by reduction in ANF and increasing ileal starch digestibility. Furthermore, ET process considerably improved SID of protein and AAs in pea. Inclusion of ETP in turkey diets (100 g/kg) demonstrated neither positive nor negative impact on growth performance, while it remarkably improved footpad dermatitis score. The present data shows the feasibility of these processes, particularly ET, for improving the nutritional quality of pea as a protein source for turkey diets.

The present study examined the impacts of native, fermented or enzymatically treated peas (Pisum sativum L.) inclusion in broiler diets, on growth performance and nutrient digestibility. For the fermentation process, Madonna pea was mixed with water (1/1) containing 2.57×10⁸ Bacillus subtilis (GalliPro^®) spores/kg pea and then, incubated for 48 h at 30°C. For the enzymatic treatment process, the used water for dough production contained three enzymes, AlphaGal^TM (α-galactosidase), RONOZYME^® ProAct and VP (protease and pectinases respectively – DSM, Switzerland) and the pea dough incubated for 24 h at 30°C. Nine corn-wheat-soybean diets were formulated by supplying 10%, 20% and 30% of the required CP with either native, fermented or enzymatically treated peas. Performance was recorded weekly and at the end of the experiment (day 35), apparent ileal digestibility (AID) of CP, amino acids (AA), crude fat, starch, Ca, P and K were determined. Data were subjected to ANOVA using GLM procedure with a 3×3 factorial arrangement of treatments. Both processes reduced α-galactosides, phytate, trypsin inhibitor activity and resistant starch in peas. Increasing levels of pea products up to 300 g/kg diet, reduced BW gain and feed intake (P≤0.05). Broilers fed diets containing enzymatically treated pea had the best feed conversion ratio at day 35. Different types of pea product and their inclusion levels had no effect on AID of all nutrients. The interaction between type of the pea products and inclusion levels was significant for AID of starch. For native pea diets, 10% group showed similar AID of starch to 20% native pea but it had higher AID than 30% native pea. For fermented and enzymatically treated groups, all three levels displayed similar AID of starch. In conclusion, enzymatic treatment and fermentation could improve the nutritional quality of pea. Inclusion of enzymatically treated pea in broiler diets could improve broiler performance compared with other pea products while, it displayed neither positive nor negative impact on nutrient digestibility. The present findings indicate the feasibility of these processes, particularly enzymatic treatment, for improving the nutritional quality of pea as a protein source for broiler nutrition.

Background: Dietary prevention strategies are increasingly recognized as essential to combat the current epidemic of obesity and related metabolic disorders. The purpose of the present study was to evaluate the potential prebiotic effects of indigestible carbohydrates in Swedish brown beans (Phaseolus vulgaris var. nanus) in relation to cardiometabolic risk markers and appetite regulating hormones. Methods: Brown beans, or white wheat bread (WWB, reference product) were provided as evening meals to 16 healthy young adults in a randomised crossover design. Glucose, insulin, appetite regulatory hormones, GLP-1, GLP-2, appetite sensations, and markers of inflammation were measured at a following standardised breakfast, that is at 11 to 14 h post the evening meals. Additionally, colonic fermentation activity was estimated from measurement of plasma short chain fatty acids (SCFA, including also branched chain fatty acids) and breath hydrogen (H₂) excretion. Results: An evening meal of brown beans, in comparison with WWB, lowered blood glucose (−15%, p<0.01)- and insulin (−16%, p<0.05) responses, increased satiety hormones (PYY 51%, p<0.001), suppressed hunger hormones (ghrelin −14%, p<0.05), and hunger sensations (−15%, p= 0.05), increased GLP-2 concentrations (8.4%, p<0.05) and suppressed inflammatory markers (IL-6 −35%, and IL-18 −8.3%, p<0.05) at a subsequent standardised breakfast. Breath H₂ (141%, p<0.01), propionate (16%, p<0.05), and isobutyrate (18%, p<0.001) were significantly increased after brown beans compared to after WWB, indicating a higher colonic fermentative activity after brown beans. Conclusions: An evening meal with brown beans beneficially affected important measures of cardiometabolic risk and appetite regulatory hormones, within a time frame of 11–14 h, in comparison to a WWB evening meal. Concentrations of plasma SCFA and H₂ were increased, indicating involvement of colonic fermentation. Indigestible colonic substrates from brown beans may provide a preventive tool in relation to obesity and the metabolic syndrome.

Background and Aims: To date, most floral nectarins (nectar proteins) are reported to function in nectar defence, particularly for insect-pollinated outcrossing species. We compared nectarin composition and abundance in selfing common tobacco (Nicotiana tobaccum) with outcrossing ornamental tobacco plants to elucidate the functional difference of nectarins in different reproductive systems. Methods: Common tobacco (CT) nectarins were separated by SDS-PAGE and the N terminus of the most abundant nectarin was sequenced via Edman degradation. The full-length nectarin gene was amplified and cloned from genomic DNA and mRNA with hiTail-PCR and RACE (rapid amplification of cDNA ends), and expression patterns were then investigated in different tissues using semi-quantitative reverse transcriptase PCR. Additionally, high-performance liquid chromatography and enzymatic analyses of nectar sugar composition, and other biochemical traits and functions of the novel nectarin were studied. Key Results: The most abundant nectarin in CT nectar is an acidic α-galactosidase, here designated NTα-Gal. This compound has a molecular mass of 40 013 Da and a theoretical pI of 5•33. NTα-Gal has a conserved α-Gal characteristic signature, encodes a mature protein of 364 amino acids and is expressed in different organs. Compared with 27 other melliferous plant species from different families, CT floral nectar demonstrated the highest α-Gal activity, which is inhibited by D-galactose. Raffinose family oligosaccharides were not detected in CT nectar, indicating that NTα-Gal does not function in post-secretory hydrolysis. Moreover, tobacco plant fruits did not develop intact skin with galactose inhibition of NTα-Gal activity in nectar, suggesting that NTα-Gal induces cell-wall surface restructuring during the initial stages of fruit development. Conclusions: α-Gal was the most abundant nectarin in selfing CT plants, but was not detected in the nectar of strictly outcrossing sister tobacco species. No function was demonstrated in antimicrobial defence. Therefore, floral nectarins in selfing species maintain their functional significance in reproductive organ development.

This study investigated the enzymatic treatment of soy slurry using Viscozyme L to hydrolyze the carbohydrates. The optimum temperature of Viscozyme L action was 55°C. The increase of glucose and galactose content in tofu (1.36 and 0.19 g/100 g, respectively) confirmed the Viscozyme activity on soy slurry when compared to the control. The treated tofu had more total phenolics than the control (173 and 161 mg gallic acid equivalents/100 g freeze-dried tofu, respectively) and higher antioxidant activity by the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and 1,1-diphenyl-2-picryhydrazyl,2,2-diphenyl-1-picryhydrazyl radical tests. Total reducing sugar (glucose equivalents) content in treated tofu was approximately four times higher than that in the control under the optimum conditions (30 Fungal Beta-Glucanase units/10 g solids, 55°C, 30 min). The tofus differed in the sensory analysis for soy odor and surface uniformity, but there was no preference for one over the other.