Sucrose/D-Fructose/D-Glucose Assay Kit

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.

Cherry kernels occur in significant amounts as waste material during the processing of fruits. However, their subsequent use is limited due to the presence of cyanogenic glycosides, which are potentially dangerous to human health. In this study, the application of pulsed electric fields (PEF) was investigated as pre-treatment to improve the debittering process and to facilitate the degradation of cyanide precursors, naturally present in cherry kernels. Diverse PEF treatments were carried out at constant electric field strength of E = 2.2 kV/cm and specific energy input was varied between 10 and 50 kJ/kg, varying the number of pulses. Two different debittering procedures were performed with a common incubation time 0-20 h at 40°C: a) incubation of whole kernels in deionized water; b) incubation of whole kernels without water stored in air at 80% relative humidity. HPLC analysis was used to examine the kinetics of the amygdalin and HCN contents. In both debittering methods, the PEF-treated samples with the highest intensity (2.2 kV/cm, 50 kJ/kg) showed higher and faster detoxification efficiency for the investigated compounds as compared to the untreated sample. In particular, the PEF treated samples incubated with water showed a reduction in the amygdalin and HCN contents of up to 86% (up to 72% of the raw material content.). Moreover, the PEF pre-treatment led to comparable efficiency in amygdalin reduction in both debittering processes: 86% reduction for the incubation with water and 81% for the incubation without water. Consequently, the combined application of PEF and the debittering process including incubation without water has remarkable potential as an industrial application due to its inherent reduced water consumption, and therefore, diminished wastewater management issues. A further advantage of this process is the minimizing of sugar loss typically occurring during the debittering through soaking.

Bacterial Cellulose (BC) is a biopolymer with numerous applications. The growth of BC-producing bacteria, Komagataeibacter intermedius, could be stimulated by Dekkera bruxellensis, however, the effect on BC yield needs further investigation. This study investigates BC production and biochemical changes in the K. intermedius-D. bruxellensis co-culture system. D. bruxellensis was introduced at various concentrations (10³ and 10⁶ CFU/mL) and inoculation times (days 0 and 3). BC yield was ~24% lower when D. bruxellensis was added at 10³ CFU/mL compared to K. intermedius alone (0.63 ± 0.11 g/L). The lowest BC yield was observed when 10³ CFU/mL yeast was added on day 0, which could be compromised by higher gluconic acid production (10.08 g/L). In contrast, BC yields increased by ~88% when 10⁶ CFU/mL D. bruxellensis was added, regardless of inoculation time. High BC yield might correlate with faster sugar consumption or increased ethanol production when 10⁶ CFU/mL D. bruxellensis was added on day 0. These results suggest that cell concentration and inoculation time have crucial impacts on species interactions in the co-culture system and product yield.

Propionic acid bacteria (PAB) are a source of valuable metabolites, including propionic acid and vitamin B12. Propionic acid, a food preservative, is synthesized from petroleum refining by-products, giving rise to ecological concerns. Due to changing food trends, the demand for vitamin B12 has been expected to increase in the future. Therefore, it is necessary to look for new, alternative methods of obtaining these compounds. This study was conducted with an aim of optimizing the production of PAB metabolites using only residues (apple pomace, waste glycerine, and potato wastewater), without any enzymatic or chemical pretreatment and enrichment. Media consisting of one, two, or three industrial side-streams were used for the production of PAB metabolites. The highest production of propionic acid was observed in the medium containing all three residues (8.15 g/L, yield: 0.48 g/g). In the same medium, the highest production of acetic acid was found - 2.31 g/L (0.13 g/g). The presence of waste glycerine in the media had a positive effect on the efficiency of propionic acid production and P/A ratio. The concentration of vitamin B12 obtained in the wet biomass of Propionibacterium freudenreichii DSM 20271 ranged from 90 to 290 µg/100 g. The highest production of cobalamin was achieved in potato wastewater and apple pomace, which may be a source of the precursors of vitamin B12 - cobalt and riboflavin. The results obtained show both propionic acid and vitamin B12 can be produced in a more sustainable manner through the fermentation of residues which are often not properly managed.

The artichoke (Cynara cardunculus L. subsp. scolymus Hegi) is an intriguing source of indigestible sugar polymers such as inulin-type fructans. In this study the amount and the quality of inulin-type fructans have been evaluated in the cultivated artichoke (Cynara cardunculus L. subsp. scolymus Hegi) as well as in the wild cardoon ( C. cardunculus var. sylvestris L. Fiori). The expression pattern of the genes codifying for the two key enzymes sucrose:sucrose 1-fructosyltransferase (1‐SST) and fructan 1‐fructosyltransferase (1‐FFT), involved in fructans biosynthesis, have been also evaluated both in capitula (heads) and rhizomes. The results showed that the inulin-type fructans were much higher in the heads of the wild cardoon than artichoke, one together with a higher expression of the two key genes fructans codifying enzymes. A conspicuous level of inulin-type fructans was found also in the rhizome, supporting the significant role of these compounds in the storage and in protection from cold and/or winter stress. The results indicate that wild cardoon could be a good and suitable crop for inulin production as well a sustainable agri-food system in Mediterranean marginal areas.

In this study, twelve strains of acetic acid bacteria (AAB) belonging to five different genera were tested for their ability to produce levan, at 70 and 250 g/L of sucrose concentration, respectively. The fructan produced by the bacterial strains was characterized as levan by NMR spectroscopy. Most of the strains produced levan, highlighting intra- and inter-species variability. High yield was observed for Neoasaia chiangmaiensis NBRC 101099^T, Kozakia baliensis DSM 14400^T and Gluconobacter cerinus DSM 9533^T at 70 g/L of sucrose. A 12-fold increase was observed for N. chiangmaiensis NBRC 101099^T at 250 g/L of sucrose concentration. Levan production was found to be affected by glucose accumulation and pH reduction, especially in Ko. baliensis DSM 14400^T. All the Gluconobacter strains showed a negative correlation with the increase in sucrose concentration. Among strains of Komagataeibacter genus, no clear effect of sucrose on levan yield was found. Results obtained in this study highlighted the differences in levan yield among AAB strains and showed interdependence between culture conditions, carbon source utilization, and time of incubation. On the contrary, the levan yield was not always related to the sucrose concentration.

The use of biomass for the production of energy and higher added value products is a topic of increasing interest in line with growing environmental concerns and circular economy. Mesoporous material Sn-In-MCM-41 was synthesized for the first time and used as a catalyst for the transformation of sugars to methyl lactate (ML). This catalyst was characterized in depth by various techniques and compared with Sn-MCM-41 and In-MCM-41 catalysts. In the new Sn-In-MCM-41 material, both metals, homogeneously distributed throughout the mesoporous structure of MCM-41, actuate in a cooperative way in the different steps of the reaction mechanism. As a result, yields to ML of 69.4 and 73.9% in the transformation of glucose and sucrose were respectively reached. In the case of glucose, the ML yield 1.5 and 2.6 times higher than those of Sn-MCM-41 and In-MCM-41 catalysts, respectively. The Sn-In-MCM-41 catalyst was reused in the transformation of glucose up to four cycles without significant loss of catalytic activity. Finally, life cycle assessment comparison between chemical and biochemical routes to produce ML allowed us to conclude that the use of Sn-In-MCM-41 reduces the environmental impacts compared to Sn-MCM-41. Nevertheless, to make the chemical route comparable to the biochemical one, improvements in the catalyst and ML synthesis have to be achieved.

Grape phylloxera (Daktulosphaira vitifoliae) can infest both roots and leaves of Vitis species. In commercial vineyards planted with Vitis vinifera scions grafted on rootstocks, grape phylloxera infestation is generally limited to root feeding. Vineyards are, however, increasingly subjected to vineyard-wide foliar infestations that last throughout the growing season. While some vineyards are affected by the infestation pressure of external leaf-feeding populations, other annually affected V. vinifera vineyards do not have these in their vicinity. Much is known about the damage potential of grape phylloxera root feeding; however, data on how phylloxera leaf infestation affects V. vinifera grapevines in commercial vineyards are lacking. This study, therefore, aimed to assess whether grapevine growth and yield are affected due to leaf infestation as it occurred in three commercial vineyards in the study area. Treatments were based on phylloxera leaf infestation and additional defoliation. Single-leaf carbon acquisition was measured with gas exchange analyses on healthy and galled leaves. Pruning weight and internode length were measured to assess the effect of leaf infestation and the effect of plant growth and vigour on leaf gall outbreaks. Yield quantity and quality were measured, and grapes were vinified for sensory analyses. Furthermore, using enzymatic analyses, non-structural carbohydrates were analysed in perennial wood. A significant decrease in sugar content in grapes (10 %) and starch reserves in perennial wood (11 %) was found in the most heavily infested vineyard. Grape must of infested plants in another vineyard furthermore showed a significantly higher level of titratable acid (7.5 %). Significant infestation effects seen in one vineyard were not significant in the other two vineyards. No significant differences were seen for carbon acquisition, harvest quantity, wine sensory analysis, pruning weight or internode length. The overall effect of phylloxera leaf infestation in the studied vineyards was, therefore, marginal. Grapevine vigour did not differ between infested vines, insecticide-sprayed vines, and vines on which no leaf infestation outbreaks took place. By analysing phylloxera leaf infestation under field conditions, these preliminary results form a basis for future long-term field studies about phylloxera leaf feeding on Vitis vinifera within the context of other biotic and abiotic plant stresses.

Shading has been used to produce high-quality lettuce (Lactuca sativa) in locations where production conditions are not optimal for this cool-season crop. To learn what additional benefits shading provides if heat-tolerant cultivars are used and to understand the effects of shading on growth, sensory quality, chemical content, and transcriptome profile on heat-tolerant lettuce, we grew two romaine lettuce cultivars with and without shading using 50% black shadecloth in 2018 and 2019. Shading reduced plant leaf temperatures, lettuce head fresh weights, glucose and total sugars content, and sweetness, but not bitterness, whereas it increased lettuce chlorophyll b content compared with unshaded controls. Transcriptome analyses identified genes predominantly involved in chlorophyll biosynthesis, photosynthesis, and carbohydrate metabolism as upregulated in unshaded controls compared with shaded treatments. For the tested cultivars, which were bred to withstand high growing temperatures, it may be preferable to grow them under unshaded conditions to avoid increased infrastructure costs and obtain lettuce deemed sweeter than if shaded.

Starch deposited in the endosperm of cereal seeds serves as a source of food and animal feed, and as a substrate for bioethanol production. To gain insights into the molecular mechanisms underlying genetic variation in seed size and seed starch content, this study investigated transcriptional regulation of starch biosynthesis genes during seed filling in two wheat genotypes that exhibit contrasting phenotype in seed size. Our data showed that variation in starch accumulation during seed filling is closely associated with modulations in the expression patterns of specific starch biosynthesis genes including TaAGPL1, TaAGPS2, TaGBSSI, TaSSI, TaSSIV, TaSBEIIa, TaISA1, and TaISA3, as well as alterations in the activity of AGPase, GBSS, and SS enzymes. Consistently, the genotype that produces larger seeds that are characterized by a higher seed starch content generated higher amounts of fermentable sugars and bioethanol before and after fermentation, respectively. Since the amount of phenol per seed dry weight is higher in the genotype that produces smaller seeds, the prevalence of lower starch to bioethanol conversion efficiency in this genotype, despite the higher amount of glucose generated per bioavailable starch, suggests that the phenolic compounds interfere with the fermentation process and thereby affecting the bioethanol yield.

Floral nectar, an important resource for pollinators, is inhabited by microbes such as yeasts and bacteria, which have been shown to influence pollinator preference. Dynamic and complex plant-pollinator-microbe interactions are likely to be affected by a rapidly changing climate, as each player has their own optimal growth temperatures and phenological responses to environmental triggers, such as temperature. To understand how warming due to climate change is influencing nectar microbial communities, we incubated a natural nectar microbial community at different temperatures and assessed the subsequent nectar chemistry and preference of the common eastern bumble bee, Bombus impatiens. The microbial community in floral nectar is often species-poor, and the cultured Brassica rapa nectar community was dominated by the bacterium Fructobacillus. Temperature increased the abundance of bacteria in the warmer treatment. Bumble bees preferred nectar inoculated with microbes, but only at the lower, ambient temperature. Warming therefore induced an increase in bacterial abundance which altered nectar sugars and led to significant differences in pollinator preference.

Drought is one of the main abiotic stresses, which affects plant growth, development, and crop yield. Plant response to drought implies carbon allocation to sink organs and sugar partitioning between different cell compartments, and thereby requires the involvement of sugar transporters (SUTs). Among them, the early response to dehydration six-like (ESL), with 19 members in Arabidopsis thaliana, form the largest subfamily of monosaccharide transporters (MSTs) still poorly characterized. A common feature of these genes is their involvement in plant response to abiotic stresses, including water deficit. In this context, we carried out morphological and physiological phenotyping of A. thaliana plants grown under well-watered (WW) and water-deprived (WD) conditions, together with the expression profiling of 17 AtESL genes in rosette leaves. The drought responsiveness of 12 ESL genes, 4 upregulated and 8 downregulated, was correlated to different water statuses of rosette leaves. The differential expression of each of the tandem duplicated AtESL genes in response to water stress is in favor of their plausible functional diversity. Furthermore, transfer DNA (T-DNA) insertional mutants for each of the four upregulated ESLs in response to water deprivation were identified and characterized under WW and WD conditions. To gain insights into global sugar exchanges between vacuole and cytosol under water deficit, the gene expression of other vacuolar SUTs and invertases (AtTMT, AtSUC, AtSWEET, and AtβFRUCT) was analyzed and discussed.

Persimmon fruit (Diospyros kaki Thunb.) production suffers great losses (15-20%) due to the inefficient overripening process. In order to valorise this waste, a compositional characterization of different fruit stages of the residues was done, and immature fruit was selected due to its high pectin and very high polyphenol content. A 3-level full factorial design was carried out to study the effect of temperature (70-95°C) and low pH (0.5-1.5), on yield, degree of esterification, carbohydrate constituents, phenolic content and antioxidant capacity of the extracted pectin, and a complete polyphenol profile (UPLC-MS/MS) was performed on selected extracts. All responses could be accurately adjusted to the models (R² > 80; lack of fit). Pectin yield, phenolic compounds and antioxidant activity ranged from 1.4 to 4.5%, 53.3 ± 2.27 to 111.7 ± 9.74 mg GAE/g pectin and 0.29 ± 0.01 to 2.77 ± 0.04 TEAC (Trolox μmol/mg pectin), respectively. A strong pectin-polyphenol interaction was found, which significantly enhanced acid resistance of both the pectin and polyphenol constituents, with optimum yield and polyphenol content at pH 1 and 95°C. These new pectin-based ingredients might have a great potential as functional foods or natural food ingredients enhancing the quality and shelf-life.

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.

High yielding ruminant livestock require high nutritive value in forage for maintenance, growth and production. Climate change has been documented as impacting on the nutritive value of forage plants. Therefore, the aim of this study was to investigate the impact of increased temperature in combination with CO₂-enhancement on the nutritive value of orchard grass (cocksfoot; Dactylis glomerata L.), as a C3 model plant, widespread in mountainous permanent grassland plant communities. Functional traits and forage quality of orchard grass were investigated both under ambient (C0T0) and under simulated, future climate conditions (C2T2) with increased temperature (+3°C) and enhanced CO₂ concentration (+300 ppm) under field conditions. Plant samples were taken from each of the three growths over a period of three consecutive years and numerous functional properties and forage quality parameters were determined. Special attention was paid to the determination of water-soluble carbohydrates (WSC), crude protein (CP), non-protein N (NPN) and metabolizable energy (ME) as possible indicators for different climatic conditions. It is hypothesized that (i) functional traits and (ii) forage quality of orchard grass are altered by increased temperature and higher CO₂ concentration. The results showed a negative impact of C2T2 compared to C0T0 on tiller height (54.4 v. 70.6 cm) and weight (2.42 v. 3.22 g) as average over cuts and years. The NPN content was lower in C2T2 (312 g/kg CP) compared to C0T0 (339 g/kg CP). In contrast, the WSC content was higher in C2T2 (90.3 g/kg DM) compared to C0T0 (82.5 g/kg DM). Both ME content and digestibility were increased in C2T2 (9.18 ME/kg DM and 68.2%) compared to C0T0 (8.86 ME/kg DM and 65.4%). Concluding, under increased temperature and enhanced CO₂, both functional traits and certain nutrients and their fractions appear to change in orchard grass.

As the global desire for natural remedies derived from botanicals increases, the pressure on plant populations and biodiversity intensifies. Therefore, to conserve biodiversity as a valuable genetic and biochemical resource, sustainable utilisation and commercial production should be prioritised. Myrsine africana L. (MA), has recently been found to possess significant cosmeceutical properties, such as elastase inhibition (anti-wrinkle) and anti-tyrosinase (skin even tone) activity. However, this species is relatively slow growing, recalcitrant to adventitious root (AR) development, and has slightly insufficient bioactivity in raw extracts. These factors reduce the economic feasibility of producing this commercially valuable indigenous species. Consequently, this may enhance wild harvesting of this species, placing pressure on wild populations. Manipulation of light is a common practice in plant production to exploit plant growth and development, as light quantity and quality effectively influence the primary and secondary metabolism of plants. Therefore, the current study aimed to investigate the influence of selected photoselective shade net on vegetative growth and metabolites of MA shoot material. Results displayed significantly enhanced growth (p < 0.001) under green (50 % density), black (50 %) and red (80 %) shade net in comparison to the control (cultivation under full sun) and inhibited growth under blue (50 %) shade net. Shade net effectively influenced starch and soluble carbohydrate content. Furthermore, significantly higher elastase inhibition was observed under green and red shade net treatments in comparison to the control in autumn, with IC₅₀ values of 18.59 μg/mL, 19.28 μg/mL and 37.93 μg/mL, respectively. In addition, bioactivity was significantly higher in autumn (p < 0.001) under green, red and control treatments. It can be concluded that photoselective shade net may be used to enhance plant growth and bioactivity of MA.

In sweet cherry (Prunus avium L.), infection by Candidatus Phytoplasma pruni results in small fruit with poor color and taste, rendering the fruit unmarketable. Yet, the disease pathology is poorly understood, particularly at the cultivar level. Therefore, in this study we examined the physiological effects of Ca. P. pruni infection across a range of cultivars and locations within eastern Washington. We found that infection could be separated into early and established stages based on pathogen titer, that correlated with disease severity, including fruit size, color, and sugar and metabolite content. Furthermore, we also observed that the effects of early-stage infections were largely indistinguishable from healthy, uninfected plants. Cultivar and location-specific disease outcomes were observed with regards to size, color, sugar content, and citric acid content. This study presents the first in-depth assessment of X-disease symptoms and biochemical content of fruit from commercially grown sweet cherry cultivars known to be infected with Ca. P. pruni.

Most Brazilian fruits are highly perishable but in frozen state would have the potential to be developed into a variety of commercially viable products. Pitanga and araza were investigated to determine the effect of composition on the freezing behavior using differential scanning calorimetry (DSC). The effect of low molecular weight compounds on the ice melting temperature T_m was investigated for: 1. Whole fruit pulp (WP); 2. Soluble fractions (SF) isolated by centrifugation from WP; and 3. Simulated systems (SS) prepared only with sugars (glucose, fructose and sucrose) and organic acids (citric, malic, and tartaric acids) as they occur in WP. Ice melting temperatures (T_m) were determined for WP, SF and SS over the range of concentrations between 10 wt. % (T_m = -0.19 °C for both for araza and pitanga) and 40 wt. % T_m = -5.0 °C and -7.05 °C for araza and pitanga, respectively). The T_m data could be fitted using the Chen equation, (C.S. Chen, J. Food Sci. 50 (1985) 1158-1162) for both fruits for WP, SF and SS. Deviations between the predictions of the Chen equation and T_m data are observed for the highest concentration studied (40 wt. %); these deviations can be minimized by fitting the parameters of the Chen equation to the experimental data rather than calculating them based on the molecular properties and composition of the system. We observe that the sugars have the highest impact on the melting behavior, with, in the concentration ranges investigated, a limited effect of organic acids. Using the lever rule, the weight fraction of ice is calculated from ice melting curves as a function of concentration and temperature. Both then ice melting line and the ice fractions are important in developing formulations have the desired freezing behavior as well as determining the processing and storage conditions in the frozen state.

Watermelon is the second largest fruit crop worldwide, with great potential to valorise its rind waste. An experimental design was used to model how extraction parameters (temperature, pH, and time) impact on the efficiency of the process, purity, esterification degree, monosaccharide composition and molar mass of watermelon rind pectin (WRP), with an insight on changes in their structural properties (linearity, branching degree and extraction severity). The models for all responses were accurately fitted (R² > 90%, lack of fit p ≥ 0.05) and experimentally validated. At optimum yield conditions, WRP yield (13.4%), purity (540 µg/g galacturonic acid) and molar mass (106.1 kDa) were comparable to traditional pectin sources but showed a higher branching degree with longer galactan side chains and a higher protein interaction. Harsher conditions (pH 1) generated purer homogalacturonan fractions with average molar masses (80 kDa) at the expense of yield, while mild extraction conditions (pH ≥ 2) produced highly branched entangled pectin structures. This study underlines novel compositional features in WRP and the possibility of producing novel customized pectin ingredients with a wider potential application scope depending on the targeted structure.