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.

Traditional noodle samples (erişte) were supplemented with hull-less barley and lentil flours as the source of β-glucan and protein at different ratios and their cooking quality, phenolic content, antioxidant capacity and estimated GI values were evaluated. The estimated GI of control erişte produced from wheat flour was the highest (74.7), while GI of those supplemented with 15%, 30%, 45% barley or lentil flour were 68.7%, 66.0%, 61.2% and 67.5%, 63.8%, 60.6%, respectively. GI values of mixtures of barley and lentils flours (Mix-1-4 samples) were lower (58.9-61.0). All noodles supplemented with barley and/or lentil flours had medium GI values. The erişte samples supplemented with 45% hull-less barley flour and Mix erişte samples meet the requirements of FDA health claim (0.75 g β-glucan per serving). Protein content of control sample was 16.30%, while those supplemented with lentil flour had higher protein contents (18.15%-22.36%). Hence, noodle samples supplemented with 30% and 45% lentil flour can be labeled as “high protein” and all other noodle samples can be labeled as “source of protein” according to EC Regulation because calories which can be received from proteins per serving are > 20% and > 12%, respectively. Significant increases were also observed in phenolic contents and antioxidant capacities of erişte samples supplemented with barley/lentil flours.

Today, there is considerable interest in creating artificial microbial consortia to solve various biotechnological problems. The use of such consortia allows for the improvement of process indicators, namely, increasing the rate of accumulation of target products and enhancing the conversion efficiency of the original substrates. In this work, the prospects for creating artificial consortia based on anaerobic sludge (AS) with cells of different yeasts were confirmed to increase the efficiency of methanogenesis in glucose- and glycerol-containing media and obtain biogas with an increased methane content. Yeasts of the genera Saccharomyces, Candida, Kluyveromyces, and Pachysolen were used to create the artificial consortia. Their concentration in the biomass of consortium cells was 1.5%. Yeast cells were used in an immobilized form, which was obtained by incorporating cells into a cryogel of polyvinyl alcohol. The possibility of increasing the efficiency of methanogenesis by 1.5 times in relation to the control (AS without the addition of yeast cells) was demonstrated. Using a consortium composed of methanogenic sludge and yeast cells of the genus Pachysolen, known for their ability to convert glycerol into ethanol under aerobic conditions, the possibility of highly efficient anaerobic conversion of glycerol into biogas was shown for the first time. Analysis of the metabolic activity of the consortia not only for the main components of the gas phase (CH₄, CO₂, and H₂) and metabolites in the cell culture medium, but also for the concentration of intracellular adenosine triphosphate (ATP), controlled by the method of bioluminescent ATP-metry, showed a high level of functionality and thus, prospects for using such consortia in methanogenesis processes. The advantages and the prospect of using the developed consortia instead of individual AS for the treatment of methanogenic wastewater were confirmed during static tests conducted with several samples of real and model waste.

The thermal process is the predominant method in chestnuts industry. However, it presents nutritional paradox due to high glycemic index (GI) conflicting with low-GI dietary guidelines. This study evaluated four conventional cooking methods systematically, revealing their striking differences in expected glycemic index (eGI). eGI values of moist-heat processed chestnuts (boiled: 81.40; steamed: 86.67) were higher than dry-heat chestnuts (baked: 69.47; fried: 69.85). Through structure analysis, texture profile analysis and ¹H low-field nuclear magnetic resonance (LF-NMR), the mechanisms of eGI differences were elucidated. Structural and texture in baked/fried samples demonstrated superior textural integrity, correlating with restricted starch accessibility. Furthermore, LF-NMR results shown that immobilized water (A21) in fresh chestnut (4.23) decreased after cooked, and A21 in moist-heat processed chestnuts (boiled: 2.25; steamed: 1.11) were higher than dry-heat counterparts (fried: 1.07; baked: 0.89), while free water (A22) increased inversely (boiled: 32.36; steamed: 27.34; fried: 22.02; baked: 20.18) when compared with fresh chestnut (14.15), indicating that the presence of immobilized water could block the binding of enzymes and starch, thereby slowing down the digestion. These results indicated that water phase transitions would be the predominant determination of chestnut GI. These findings provide valuable insights for designing low-GI chestnut products through targeted processing optimization.

Brown rice arouses great attention due to the enriched nutrients, but the unpleasant texture caused by bran layer limits consumer acceptance. To date, although individual enzyme or fermentation treatment has been developed to modify the rice bran, the feasibility of the combination method and co-effects on whole brown rice have not been investigated. In this study, brown rice treated with cellulase and Lactobacillus plantarum P-8 together (CAF-BR) was compared with samples individually treated with cellulase (E-BR) or fermentation (F-BR), in terms of cellulase activity, textural attributes, and variations in the main component of brown rice. The results showed CAF-BR had higher cellulase activity and less glucose in the hydrolysate than E-BR, reflecting a cooperative effect on rice bran hydrolysis. Moreover, CAF-BR showed a significantly lower hardness and higher stickiness, compared to E-BR and F-BR. This was consistent with the most ruptured bran of CAF-BR observed by scanning electron microscopy, and the correspondingly highest amount of water absorption ratio, which possibly explained the decreased hardness of CAF-BR. In addition, the leachates from CAF-BR possessed the most starch content and the highest content of large molecular weight amylopectin among all samples, which was highly associated with an increased stickiness. Ultimately, this study extends the knowledge about the feasibility of combining enzymatic treatment with fermentation for modifying the wholegrain brown rice, providing a theoretical basis of the cooperative effects on improving the textural properties.

Galloyl-based polyphenols including gallic acid (GA) and tannic acid (TA) were used to form complexes with high-amylose maize starch (HAMS). The relationship among complex structure, starch digestibility in vitro, and postprandial glycemic response, hormone concentration and satiety in mice were investigated. Both GA and TA interacted with starch through noncovalent bonds and formed complexes with V-type crystalline structure. The polyphenol loading content in HAMS-TA was higher than HAMS-GA, probably because TA has more galloyl moieties. In vitro digestion revealed HAMS-TA/GA complexes had lower digestion rate (k) and proportion of digestible starch (C_∞) than control samples, attributing to their higher crystallinity and inhibition on α-amylase. HAMS-TA had lower k and C_∞ than HAMS-GA, resulted from its higher polyphenol loading content. In vivo digestion in mice indicated complexes had lower glycemic response, higher concentrations of GLP-1 and PYY than control groups. They also showed good ability in decreasing glycemic elevation after a subsequent meal and enhancing satiety. HAMS-TA exhibited better performance than HAMS-GA. The in vitro and in vivo results are in good agreements, suggesting the potential ability of starch-galloyl-based polyphenol complexes, especially HAMS-TA complex, in preventing type-2 diabetes and obesity. These would provide guidance for developing starchy foods with functional properties.

Short-term germination has been applied to different conventional and unconventional legume seeds to increase nutritional values, beneficial health effects, and techno-functional characteristics for diverse food applications. Nonetheless, the potential impact on black turtle beans (BTBs) is limited in literature. This study examines the compositional, bioactivity, functional, pasting, thermal, and color characteristics of BTBs over a 72 h germination period. On a time-dependent basis, germination increased (p ≤ 0.05) α-amylase activity, protein, insoluble, soluble, and total dietary fiber, resistant starch, minerals, vitamins (B₁, B₂, B₆ and C), as well as the majority of the essential amino acids, protein digestibility, phytochemicals, and antioxidant activity of BTB flour. Germination caused significant reductions in the level of crude fat, total starch, digestible starch, and antinutritional compounds (tannin, phytic acid, and trypsin inhibitors). Germination also modified techno-functional attributes (functional, pasting, thermal, and color characteristics) of BTB flour. Germination also caused interactions with some functional groups, as demonstrated by the FTIR (Fourier transform infrared) spectroscopy. This study revealed that germination could serve as an affordable and natural means to enhance the functionality of BTB flour by improving its nutritional quality, bioactivity, and techno-functional characteristics for the formulation of new food ingredients that meet the current food requirements of the people. PRACTICAL APPLICATION: Short-term germination improved the nutritional value, physicochemical attributes, bioactivity, and techno-functional characteristics of black turtle bean flours. The germinated black turtle bean flour could serve as a novel ingredient for the development of nutritious foods with potential health benefits.

Although it is well known that both retrogradation and presence of tannic acid can reduce starch digestibility, their combined impact on starch digestibility is not clear. To this end, wheat starch was added with varying contents of tannic acid, and their digestibility was evaluated after different retrogradation periods. Notably, starch digestibility was significantly increased after the addition of 5 % and 10 % tannic acid, while decreased when 20 % tannic acid was added. Mechanistically, it was found that tannic acid at low concentrations (5 % - 10 %) promoted starch digestibility by inhibiting starch intermolecular interactions and reducing its long-range ordering during retrogradation. In comparison, tannic acid at high concentrations (i.e., 20 %) reduced starch digestibility, primarily through increasing the free tannic acid content and inhibiting enzymatic activities of both pancreatin and amyloglucosidase. Collectively, these results revealed, for the first time, a competitive inhibition mechanism between tannic acid and retrogradation on starch digestibility, which could be applied in the future to develop staple foods with slow starch digestibility.

The structural interactions between starch and rice proteins play a pivotal role in shaping the textural and nutritional properties of rice noodles. This study systematically explored the multi-scale interactions between fractionated rice starch particles (classified by mesh size) and rice proteins during gel network formation, with a particular focus on their combined effects on rheological behavior and enzymatic digestibility. Results demonstrated that rice proteins significantly reduced starch pasting viscosity (from 3160 cP to 1628 cP) while enhancing shear stability, especially in systems containing smaller starch granules (<75 μm). Microstructural analysis revealed dual stabilization mechanisms: (1) hydrogen bond-mediated protein-starch adhesion (FTIR peak at 3285 cm⁻¹), which preserved gel elasticity, and (2) the formation of V-type amylose-protein complexes (XRD peak at 20° 2θ), which improved structural stability and increased resistant starch content (from 20.0% to 37.0%). Notably, the interplay between starch particle size and rice proteins primarily influenced overall digestive outcomes rather than initial hydrolysis rates. Sub-200-mesh starch particles exhibited a 24.9% faster digestion within the first 30 min; however, in the presence of rice proteins, total digestibility was reduced by 13.55%, underscoring the regulatory role of protein-starch interactions in modulating enzymatic accessibility. These findings highlight that rice proteins influence starch digestion not by directly inhibiting enzymatic reactions but by introducing steric hindrance that restricts enzyme access. The synergistic effects between proteins and starch components provide a novel strategy for developing texturally resilient rice noodles with a reduced glycemic response, offering valuable insights for the formulation of functional rice-based products.

Polysaccharides were the promising candidates for the development of selenium-enriched health-promoting foods. However, there was a lack of information regarding the synthesis of selenylated starch granules using straightforward methods and the impact of selenium decoration on starch properties. This study introduced a process for creating selenylated starch granules by heating a water-limited ethanol solution containing starch granules, acetic acid, and selenic acid. The results demonstrated that selenylated starch granules could be synthesized by introducing selenium to the hydroxyl groups on the starch chains. The selenylated starch granules produced through this method retained the same morphology as native starch but exhibited a smaller molar mass, less ordered structures, increased solubility, significantly lower viscosity, and reduced digestibility. Compared to selenylated starch synthesized in water, starch modified in the water-ethanol system yielded more intact granules, achieved a higher yield (91.9% versus 88.0%), contained more selenium (1.3 mg/g starch versus 0.9 mg/g starch), and displayed more ordered structures (including helical, crystalline, and lamellar structures). Additionally, these granules showed higher viscosity, lower solubility, and reduced digestibility. These findings paved the way for a promising approach to synthesize selenylated starch granules.

The incorporation of artichoke bracts, a by-product of artichoke processing, into pasta formulations represents an innovative approach to enhancing the nutritional and functional properties of this staple food while promoting environmental sustainability. This study aimed to evaluate the impact of artichoke powder (AP) enrichment (10% w/w replacement of semolina) on the technological, nutritional, antioxidant, and sensory properties of pasta. The enriched pasta (P-AP) was compared to control pasta (P-CTR) through comprehensive physicochemical analyses, including cooking performance, polyphenol characterization, and in vitro digestion. Polyphenol analysis revealed that chlorogenic acid, dicaffeoylquinic acids, and flavonoids accounted for 87% of total identified phenolic compounds in P-AP. Despite a 42% reduction in free polyphenols due to cooking, in vitro digestion revealed a 47% increase in total identified polyphenols, attributed to the release of bound polyphenols. Antioxidant assays (DPPH, ABTS, and FRAP) confirmed a significantly higher antioxidant capacity in P-AP compared to P-CTR. Additionally, P-AP exhibited a lower predicted glycemic index (pGI = 56.67) than the control (pGI = 58.41), a beneficial feature for blood glucose regulation. Sensory analysis highlighted distinct differences between samples, with P-AP showing stronger vegetal, artichoke, and legume-like notes, as well as higher intensity in bitterness and astringency. While panelists rated P-CTR higher in overall liking, enriched pasta maintained acceptable sensory characteristics. These findings support the valorization of artichoke by-products in pasta production, demonstrating their potential to enhance nutritional quality and functional properties while contributing to a circular economy.