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.

This study investigates freeze-dried okra seed flour (OSF) as a functional ingredient to attenuate postprandial blood glucose rise by creating lower glycemic index (GI) rice noodles. Secondary objectives are postprandial gut hormone responses, noodle satiety, and sensory evaluation. Twenty healthy individuals consumed control rice noodle (CON), 10 % okra seed fortified noodle (OKN), 20 % OKN and glucose reference drinks on separate visits. 20 % OKN significantly lowered the postprandial glucose and insulin peaks compared to CON. Compared to CON, there was a 20-point reduction of GI value for 20 % OKN to 64. All noodles produced similar responses in postprandial ghrelin and glucagon-like peptide-1 (GLP-1). OKN were found to be more satiating than control noodles given the same amount of available carbohydrates. Sensory evaluation revealed that OSF can be added to noodle to create sensorially acceptable products, though ratings for sensory characteristics decreased with increased fortification.

The effects of dairy and plant protein addition on microstructural characteristics and in vitro gastro-small intestinal starch digestion characteristics of Chinese steamed breads (CSBs) were studied. Breads containing rennet casein (RC) and a mixture of soy protein isolate and milk protein concentrate (SM) at two different levels (RC I, RC II; SM I, SM II) were prepared. Microstructural characteristics of the undigested and digested control (100% wheat flour) bread and high protein steam bread (HPCSB) versions were compared through scanning electron microscopy. The compact microstructure of HPCSBs displayed a network of proteins wrapped around starch granules and had fewer air cells compared to the control. The addition of both proteins influenced the microstructure of HPCSBs, which in turn affected their textural and starch digestion properties. The in vitro starch digestion of control CSB and HPCSBs confirmed that the addition of proteins is capable of lowering the starch hydrolysis (%). The highest starch hydrolysis was observed for the control wheat bread, followed by SM1 > RC I > SM II and RC II at the end of the small-intestinal digestion. The estimated glycaemic indices (eGI) for all HPCSBs were statistically lower than the control CSB. In comparison to control CSB, the microstructure of HPCSBs appeared more irregular, less porous, and compact during gastric and small intestinal digestion.

The present investigation was aimed to study functional properties, antioxidant activity and in-vitro digestibility characteristics of brown and polished flours obtained from four rice cultivars (SR-4, K-39, Mushq Budij and Zhag) of Kashmir. Brown rice flours had higher total dietary fibre (3.08%-3.68%), oil absorption (116.0%-139.0%), emulsion capacity (4.78%-9.52%), emulsion stability (87.46%-99.93%) and resistant starch content (6.80%-9.00%) than polished flours. However, polished flours presented greater water absorption (102.0%-122.0%), foaming capacity (8.00%-13.63%), apparent amylose (19.16%-22.62%), peak (2260.0-2408.0 cP), trough (1372.0-1589.0 cP) and breakdown (714.0-978.0 cP) viscosities than their brown counterparts. Brown rice flours depicted highest total phenolic content (4.40-6.40 mg GAE/g) and inhibition of lipid peroxidation (19.50%-33.20%). However, equilibrium starch hydrolysis percentage (C∞) and predicted glycemic index of brown rice flours were lower than their polished counterparts. Among rice cultivars, brown Zhag flour had the highest total dietary fibre (3.68%), emulsion capacity (9.52%), emulsion stability (99.93%), resistant starch (9.00%), DPPH radical scavenging activity (85.45%) and inhibition of lipid peroxidation (33.20%), respectively. Emulsion capacity and emulsion stability were positively correlated with protein content of rice flours. However, peak, trough, breakdown and setback viscosities were negatively correlated with protein and fat contents of rice flour. The present investigation will be helpful in identifying nutritive role of rice flours from studied cultivars in human diet.

Background and Objectives: The present study investigated dough rheology, textural, sensory and digestibility characteristics of barley flour added leavened flat bread called "Taftaan". The objective was to increase the consumption of fiber through the addition of barley flour in taftaan as it is a frequently consumed Pakistani flat bread. Findings: The study showed that fiber content increased significantly but water absorption decreased owing to the poor quality of gluten network. Rheological analysis showed insignificant difference in elastic and viscous characteristics of dough after adding barley flour. Textural analysis revealed that breads containing barley flour were stiffer and less elastic. However, sensory panelists were unable to discriminate between taftaan breads made with (0–15)% barley flour. Interestingly, it was also observed that predicted glycemic index of control taftaan bread was found to be 83.14 which was significantly reduced to 70.10 by addition of 20% barley flour. Conclusion: Thus, barley flour addition in taftaan upto 15% can significantly reduce predicted glycemic index of flat bread without affecting sensory characteristics. Significance and Novelty: There is limited work done on barley flour added taftaan which is a frequently consumed bread of Asia. Therefore, barley flour added taftaan can increase consumption of both soluble (β-glucan) and insoluble fiber in daily diet.

Low-GI biscuits are commonly produced using whole-grain flour, bran, or soluble dietary fibers, giving an undesirable texture. New low-GI biscuits containing dietary fibers and with improved palatability were formulated by substituting 60% of wheat flour (WF) with a native starch (ST) and 15% of WF with a resistant dextrin (RD), a source of dietary fibers. The botanical source of ST was common buckwheat (Fagopyrum esculentum Moench). Biscuits were also made with a single substitution by ST or by RD at the same level for comparison. The firmness of the biscuits was increased with the single substitution by RD due to its small average molecular size and high hygroscopicity, while it was decreased with the single substitution by ST. The double substitution by ST and RD not only produced the texture with the lowest firmness and brittleness, but also led to the lowest in vitro starch digestion rate and total starch digestibility. The human trial confirmed that the biscuits with the double substitution had a low GI of 47. The results indicated the additive or synergistic effects of ST and RD on the properties of the biscuits, demonstrating that low-GI biscuits can be produced with a substantial dietary fiber content without jeopardizing their palatability.

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.

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.