D-Glucose Assay Kit (GOPOD Format)

A simple method for measurement of the amylolytic activity of malt has been developed and fully evaluated. The method, termed the Maltose Value (MV) is an extension of previously reported work. Here, the MV method has been studied in detail and all aspects of the assay (sample grinding and extraction, starch hydrolysis, maltose hydrolysis and determination as glucose) have been optimised. The method is highly correlated with other dextrinising power methods. The MV method involves extraction of malt in 0.5% sodium chloride at 30°C for 20 minutes followed by filtration; incubation of an aliquot of the undiluted filtrate with starch solution (pH 4.6) at 30°C for 15 min; termination of reaction with sodium hydroxide solution; dilution of sample in an appropriate buffer; hydrolysis of maltose with a specific α-glucosidase; glucose determination and activity calculation. Unlike all subsequent reducing sugar methods, the maltose value method measures a defined reaction product, maltose, with no requirement to use equations to relate analytical values back to Lintner units. The maltose value method is the first viable method in 130 years that could effectively replace the 1886 Lintner method.

Background: The level of available carbohydrates in our diet is directly linked to two major diseases; obesity and Type II diabetes. Despite this, to date there is no method available to allow direct and accurate measurement of available carbohydrates in human and animal foods. Objective: The aim of this research was to develop a method that would allow simple and accurate measurement of available carbohydrates, defined as non-resistant starch, maltodextrins, maltose, isomaltose, sucrose, lactose, glucose, fructose and galactose. Method: Non-resistant (digestible) starch is hydrolysed to glucose and maltose by pancreatic α-amylase and amyloglucosidase at pH 6.0 with shaking or stirring at 37°C for 4 h. Sucrose, lactose, maltose and isomaltose are completely hydrolyzed by specific enzymes to their constituent monosaccharides, which are then measured using pure enzymes in a single reaction cuvette. Results: A method has been developed that allows the accurate measurement of available carbohydrates in all cereal, vegetable, fruit, food, and feed products, including dairy products. Conclusions: A single-laboratory validation was performed on a wide range of food and feed products. The inter-day repeatability (%RSDr) was <3.58% (w/w) across a range of samples containing 44.1 to 88.9% available carbohydrates. The LOD and LOQ obtained were 0.054% (w/w) and 0.179% (w/w), respectively. The method is all inclusive, specific, robust and simple to use. Highlights: A unique method has been developed for the direct measurement of available carbohydrates, entailing separate measurement of glucose, fructose and galactose; information of value in determining the glycemic index of foods.

Most commonly used methods for the measurement of starch in food, feeds and ingredients employ the combined action of α‐amylase and amyloglucosidase to hydrolyse the starch to glucose, followed by glucose determination with a glucose oxidase/peroxidase reagent. Recently, a number of questions have been raised concerning possible complications in starch analytical methods. In this paper, each of these concerns, including starch hydrolysis, isomerisation of maltose to maltulose, effective hydrolysis of maltodextrins by amyloglucosidase, enzyme purity and hydrolysis of sucrose and β‐glucans have been studied in detailed. Results obtained for a range of starch containing samples using AOAC Methods 996.11 and 2014 .10 are compared and a new simpler format for starch measurement is introduced. With this method that employs a thermostable α-amylase (as distinct from a heat stable α-amylase) which is both stable and active at 100°C and pH 5.0, 10 samples can be analysed within 2 h, as compared to the 6 h required with AOAC Method 2014.10.

Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.

It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

Parboiled rice is preferably consumed in many countries due to its nutritional superiority and lower starch digestibility. Parboiling affects rice cooking quality, starch digestibility and phytic acid which affects minerals bioavailability. Cooking quality was improved in parboiled brown (PB) and parboiled milled (PM) rice. Parboiling has significantly (P < 0.05) reduced glycemic index in both PB and PM rice with a proportionately increase in resistant starch. After milling, the phytic acid (PA) and Fe were reduced significantly (P < 0.05), however, parboiling further reduced PA but increased Fe content and bioavailability in PM rice due to its inward diffusion. Zn content was lower in PB and PM rice due to its outward movement during parboiling. The impact of Zn retention on its bioavailability was insignificant in parboiled rice as non-parboiled rice. This study provides better insights on rice parboiling as a method to reduce starch digestibility and improve mineral bioavailability which could be beneficial for diabetics and malnourished population.

Batch cultivation of recombinant bacteria in shake flasks typically results in low cell density due to nutrient depletion. Previous studies on high cell density cultivation in shake flasks have relied mainly on controlled release mechanisms. Here, we report a true fed-batch strategy to achieve high cell density of recombinant E. coli in shake flasks in 24 h by feeding a mixture of glycerol and yeast extract with a syringe pump. Feed composition and feed rate were obtained by cybernetic model-based, multi-objective optimization. Model parameters were estimated from time-course measurement of substrate, biomass, and dissolved oxygen levels. The optimized process yielded 20.7 g dry cell weight/L, in agreement with the model prediction. Volumetric protein productivity improved by 10-34-fold compared to batch cultivation with 2.8-fold further improvement when the fed-batch process was replicated in a 3 L bioreactor. The process has significance in the routine laboratory cultivations and in scaleup studies.

The starch digestion processing of whole grain foods is associated with its health benefits in improving insulin resistance. This study modified the digestibility of whole quinoa flour (WQ) via heat-moisture treatment (HMT), HMT combined with pullulanase (HMT+P), HMT combined with microwave (HMT+M), and HMT combined with citric acids (HMT+A), respectively. Results showed that all the treatments significantly increased (p < 0.05) the total dietary fiber (TDF) content, amylose content, and resistant starch (RS) content, however, significantly decreased (p < 0.05) the amylopectin content and rapidly digestible starch (RDS) content of WQ. HMT+P brought the highest TDF content (15.3%), amylose content (31.24%), and RS content (15.71%), and the lowest amylopecyin content (30.02%) and RDS content (23.65%). HMT+M brought the highest slowly digestible starch (SDS) content (25.09%). The estimated glycemic index (eGI) was respectively reduced from 74.36 to 70.59, 65.87, 69.79, and 69.12 by HMT, HMT+P, HMT+M, and HMT+A. Moreover, a significant and consistent reduction in the heat enthalpy (ΔH) of WQ was observed (p < 0.05), after four treatments. All these effects were caused by changes in the starch structure, as evidenced by the observed conjunction of protein and starch by a confocal laser scanning microscope (CLSM), the decrease in relative crystallinity, and transformation of starch crystal.

In this study, starch citrates were synthesized by superheated steam (SS) treatment without changing the granule structure of starch. The degree of substitution (DS) of starch citrates prepared in present study was ranged from 0.015 to 0.064. Fourier transform infrared spectroscopy confirmed that citric acid (CA) and starch were successfully esterified with an absorption peak appeared at 1735 cm⁻¹. In vitro digestion results revealed that the content of resistant starch (RS) was significantly increased by citric acid esterification, which was attributed to the cross-linked structure of starch and the inhibition of gelatinization and swelling. According to the physicochemical properties analysis, the pasting temperature of rice starch citrates were significantly increased by citric acid treatment, while the swelling power and gelatinization enthalpy were significantly decreased and the short- and long-range molecular order of the starch was disrupted after the esterification reaction. These results indicated that it is a feasible method to synthesize starch citrate by superheated steam to increase resistant starch, and it could be used as an alternative approach for preparing starch citrate to conventional hot air treatment.

Reusing biocatalysts and repeated-batch process are promising approaches for the reduction of time and costs of a fermentation process. The present study investigated the use of α-amylase, glucoamylase, and cells of Saccharomyces cerevisiae immobilized on bacterial cellulose (BC) in repeated-batch simultaneous saccharification and fermentation (SSF) process for ethanol production from cassava pulp (CP). Ratio between the immobilized α-amylase and glucoamylase, and concentration of CP were optimized to achieve high CP saccharification efficiency, and the immobilized enzymes and cells were reused for four times in five consecutive batches of SSF. The optimum ratio between immobilized α-amylase and glucoamylase, and CP concentration were 75:25 by mass, and 60 g/L, respectively, yielding 0.74 g_glucose/g_CP (80% hydrolysis efficiency). Repeated-batch SSF gave an overall ethanol yield of 0.28 g/g_CP, and an ethanol production rate of 0.27 g/h, whereas a batch SSF gave a yield of 0.3 g_ethanol/g_CP and a production rate of 0.036 g_ethanol/h. The results reported in this study revealed the applicability of BC as a supporting material for enzymes and cells immobilizations, and the feasibility of the repeated-batch SSF process in ethanol fermentation.

Background: Interest on the use of short rotation willow as a lignocellulose resource for liquid transport fuels has increased greatly over the last 10 years. Investigations have shown the advantages and potential of using Salix spp. for such fuels but have also emphasized the wide variations existing in the compositional structure between different species and genotypes in addition to their effects on overall yield. The present work studied the importance of tension wood (TW) as a readily available source of glucose in 2-year-old stems of four Salix clones (Tora, Björn, Jorr, Loden). Studies involved application of a novel approach whereby TW-glucose and residual sugars and lignin were quantified using stem cross sections with results correlated with HPLC analyses of milled wood. Compositional analyses were made for four points along stems and glucose derived from enzyme saccharification of TW gelatinous (G) layers (G-glucose), structural cell wall glucose (CW-glucose) remaining after saccharification and total glucose (T-glucose) determined both theoretically and from HPLC analyses. Comparisons were also made between presence of other characteristic sugars as well as acid-soluble and -insoluble lignin. Results: Preliminary studies showed good agreement between using stem serial sections and milled powder from Salix stems for determining total sugar and lignin. Therefore, sections were used throughout the work. HPLC determination of T-glucose in Salix clones varied between 47.1 and 52.8%, showing a trend for higher T-glucose with increasing height (Björn, Tora and Jorr). Using histochemical/microscopy and image analysis, Tora (24.2%) and Björn (28.2%) showed greater volumes of % TW than Jorr (15.5%) and Loden (14.0%). Total G-glucose with enzyme saccharification of TW G-layers varied between 3.7 and 14.7% increasing as the total TW volume increased. CW-glucose measured after enzyme saccharification showed mean values of 41.9-49.1%. Total lignin between and within clones showed small differences with mean variations of 22.4-22.8% before and 22.4-24.3% after enzyme saccharification. Calculated theoretical and quantified values for CW-glucose at different heights for clones were similar with strong correlation: T-glucose = G-glucose + CW-glucose. Pearson’s correlation displayed a strong and positive correlation between T-glucose and G-glucose, % TW and stem height, and between G-glucose with % TW and stem height. Conclusions: The use of stem cross sections to estimate TW together with enzyme saccharification represents a viable approach for determining freely available G-glucose from TW allowing comparisons between Salix clones. Using stem sections provides for discrete morphological/compositional tissue comparisons between clones with results consistent with traditional wet chemical analysis approaches where entire stems are milled and analyzed. The four clones showed variable TW and presence of total % G-glucose in the order Björn > Tora > Jorr > Loden. Calculated in terms of 1 m³, Salix stems Tora and Björn would contain ca. 0.24 and 0.28 m³ of tension wood representing a significant amount of freely available glucose.

Background: Salix (willow) species represent an important source of bioenergy and offer great potential for producing biofuels. Salix spp. like many hardwoods, produce tension wood (TW) characterized by special fibres (G-fibres) that produce a cellulose-rich lignin-free gelatinous (G) layer on the inner fibre cell wall. Presence of increased amounts of TW and G-fibres represents an increased source of cellulose. In the present study, the presence of TW in whole stems of different Salix varieties was characterized (i.e., physical measurements, histochemistry, image analysis, and microscopy) as a possible marker for the availability of freely available cellulose and potential for releasing D-glucose. Stem cross sections from different Salix varieties (Tora, Björn) were characterized for TW, and subjected to cellulase hydrolysis with the free D-glucose produced determined using a glucose oxidase/peroxidase (GOPOD) assay. Effect of cellulase on the cross sections and progressive hydrolysis of the G-layer was followed using light microscopy after staining and scanning electron microscopy (SEM). Results: Tension wood fibres with G-layers were developed multilaterally in all stems studied. Salix TW from varieties Tora and Björn showed fibre G-layers were non-lignified with variable thickness. Results showed: (i) Differences in total % TW at different stem heights; (ii) that using a 3-day incubation period at 50 °C, the G-layers could be hydrolyzed with no apparent ultrastructural effects on lignified secondary cell wall layers and middle lamellae of other cell elements; and (iii) that by correlating the amount of D-glucose produced from cross sections at different stem heights together with total % TW and density, an estimate of the total free D-glucose in stems can be derived and compared between varieties. These values were used together with a literature value (45%) for estimating the contribution played by G-layer cellulose to the total cellulose content. Conclusions: The stem section-enzyme method developed provides a viable approach to compare different Salix varieties ability to produce TW and thus freely available D-glucose for fermentation and biofuel production. The use of Salix stem cross sections rather than comminuted biomass allows direct correlation between tissue- and cell types with D-glucose release. Results allowed correlation between % TW in cross sections and entire Salix stems with D-glucose production from digested G-layers. Results further emphasize the importance of TW and G-fibre cellulose as an important marker for enhanced D-glucose release in Salix varieties.

Maitake (Grifola frondosa) is a medicinal mushroom known for its peculiar biological activities due to the presence of functional components, including dietary fibers and glucans, that can improve human health through the modulation of the gut microbiota. In this paper, a Maitake ethanol/water extract was prepared and characterized through enzymatic and chemical assays. The prebiotic potential of the extract was evaluated by the growth of some probiotic strains and of a selected probiotic consortium. The results revealed the prebiotic properties due to the stimulation of the growth of the probiotic strains, also in consortium, leading to the production of SCFAs, including lactic, succinic, and valeric acid analyzed via GC-MSD. Then, their beneficials effect were employed in evaluating the vitality of three different healthy and tumoral colorectal cell lines (CCD841, CACO-2, and HT-29) and the viability rescue after co-exposure to different stressor agents and the probiotic consortium secondary metabolites. These metabolites exerted positive effects on colorectal cell lines, in particular in protection from reactive oxygen species.

Gluten-free (GF) biscuits based on buckwheat, sorghum and lentil flours were produced in an industrial plant. These flours are very little exploited in commercial products. In addition, since there is growing attention on glycaemic index (GI) of cereal based products. Sucrose replacers, mainly maltitol and inulin, and type 2 resistant starch (RS) were used to substitute sucrose and flours, respectively, in the view of reducing GI of biscuits. Ingredients were used in amounts established on the basis of previous experiments and European Regulations on nutritional claims. This study aimed at the evaluation of the effect of maltitol, inulin and RS on physico-chemical and sensory characteristics of biscuits. In particular, glucose released by sucrose and starch hydrolysis during in vitro digestion was placed under the spotlight. The use of sucrose contributed to the reduction of in vitro glucose release and consequently of predicted glycaemic index (pGI) with values decreasing from 84 to 67. Despite some slight differences in the sensory profile of the biscuits, reformulated products had a reduced friability and sweetness, and increased adhesiveness and nuts odour, all the formulations scored well above the threshold of acceptability (from 6.5 to 7.6) by consumers used to eat biscuits made with alternate flours.

Hot-extrusion 3D printing (HE-3DP) is an emerging technique to create customized food with desired structure and properties. This research is focused on the effect of pre-printing gelatinization degree on the structure and digestibility of rice and wheat starch gels after HE-3DP. The results show that the lamellar, crystalline and helical structures were destroyed, the compactness of nano-aggregates formed decreased significantly, the content of semi-bound water in the gel system increased, and the content of bound water decreased, and these changes were more significant with an increasing gelatinization degree. Interestingly, the ideal printability and digestion resistance of the printed materials could be obtained with the pre-printing gelatinization degrees of rice and wheat starches being 50% and 40%, respectively. Overall, the pre-printing gelatinization degree has been found to be an important factor in regulating the printability and digestibility of starch during HE-3DP.

In the present study, pigmented and non-pigmented maize grains cultivated in Turkey were used to produce functional baked snacks. According to the results, the highest DPPH, ABTS and FRAP antioxidant activity was determined in maize snacks produced by pigmented whole maize grains. The highest total dietary fibre content was determined in blue maize snacks as 26.88 g/100 g, followed by yellow and red maize snacks with 23.35 and 20.28 g/100 g, respectively. The total anthocyanin content of blue and red maize snacks was determined as 49.60 and 4.51 mg CGE/kg; however, it could not be detected in yellow maize snacks. The lowest rapidly available glucose (11.07 g/100 g) and the highest slowly available glucose (16.26 g/100 g) were also obtained in blue maize snack. Snack produced by blue maize grains was classified in low GI foods, while yellow and red maize snacks were classified as medium GI food. This result showed that anthocyanins are more effective in lowering eGI of maize snacks than dietary fibres and phenolics.

Plasma-activated water (PAW) production and use is an emerging technology for enhancing product safety, extending shelf-life and quality retention, and promoting sustainable processing. At present, it has generated considerable attention for applications to starch and flour modification. This work presents an innovative approach to wheat flour (WF) modification using PAW and heat-moisture treatment (HMT), and compares this approach with distilled water (DW) treatment. As expected, PAW and HMT promoted flour granule clustering, increasing particle size. These treatments accelerated molecular interactions between wheat starch and non-starch components (e.g. proteins and lipids), which eventually increased resistant starch (RS) content. Addition of modified flour (30 g) to WF positively affected its rheological properties, and closely bound water content of the dough. The gluten protein network structure in the dough suffered varying degrees of damage. In conclusion, our results showed that PAW and HMT may provide a novel beneficial method for modifying wheat flour during food processing to obtain viscoelastic wheat flour products with nutritional functions.

Winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch. ex Poir.) are widely consumed in the world. Empirical evidences showed that their cooked texture was related to the starch content and probably starch properties. In this study, starches were extracted from 2 winter squash cultivars (Yinli and Heili) and 1 pumpkin cultivar (Miben) popularly cultivated in China. The mature fruits of three cultivars contained 1.05-6.47% (fresh weight) starch with different granule sizes (d (0.5) 9.51-15.18 μm). Starches from winter squash and pumpkin had polyhedral and spherical shape granules with amylose content varied from 21.35% to 30.17%. All starches exhibited B-type crystalline structure. The relative crystallinity ranged from 26.15% to 31.31%, and the IR absorbance ratios of 1022/995 cm⁻¹ and 1045/1022 cm⁻¹ ranged from 0.759 to 0.806 and from 0.565 to 0.607, respectively, among 3 cultivars. SAXS pattern indicated 9.59-9.80 nm D_Bragg for these starches. The M_w was highest (5.03 × 10⁷ g/mol) in Miben starch but lowest (3.64 × 10⁷ g/mol) in Heili starch. All starches had identical amylopectin branch chain distributions but varied in average chain length and f_b1 and f_b2 chains. The gelatinization temperature (T_o, T_p, T_c) and enthalpy change (ΔH) were lowest in Heili starch and highest in Miben starch. The peak, trough, final and setback viscosities varied from 4468 to 6266 cP, 3116 to 4259.5 cP, 4075 to 6073 cP, and 787 to 1813 cP, respectively. All starch granules showed strong resistance to enzymatic hydrolysis. Yinli and Miben starches had higher RS content in starch pastes and retrograded gels than Heili starch.

Effects of degree of milling on starch digestibility of cooked rice during (in vitro) small intestine digestion were investigated. By fitting starch digestograms to the logarithm of slope plot and combination of parallel and sequential digestion kinetics model, two starch fractions with distinct digestion rate constants were identified. Results from scanning electronic microscope and confocal laser scanning microscope showed that the rapidly digestible starch fraction (RDF) was mainly composed of gelatinized starch, while the slowly digestible starch fraction (SDF) was consisted of relatively intact starch granules, protein matrix encapsulated starch and starch-protein binary complex. The cooked rice with milling treatment had more loosely packed and larger network cells compared to that for brown rice. Consequentially, the RDF content was decreased, while that for SDF was increased by the milling treatment. These results could help the rice processing industry to develop healthy rice products with desirable starch digestibility.