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.

Microoxygenation (MOX) is used to improve wine colour and sensory quality; however, limited information is available for Pinot noir wines and wines with different initial phenolic content. In this study, MOX was applied to two Pinot noir wines, with either a low or a high phenolic content, at two doses (0.50 and 2.11 mg/L/day) for 14 days. With the sterile filtration applied, acetaldehyde formation during MOX was very low, supporting the influence of yeast on acetaldehyde production during MOX. The MOX dosage rate did not significantly affect colour development, while the Pinot noir wine with higher phenolics benefited more from MOX, significantly increasing colour intensity and SO₂ resistant (polymeric) pigments. However, these changes did not guarantee colour stability, as a final SO₂ addition (100 mg/L) largely erased the improvement to colour in all wines. This could be due to the lower acetaldehyde formation, thus less ethyl-bridged stable pigments resistant to SO₂ bleaching. MOX also decreased the flavan-3-ols and anthocyanin monomers, which differed between the two Pinot noir wines, reflecting the initial phenolic content. Lastly, MOX generally increased the measured tannin concentration and affected the proportion of tannin subunits, with a decrease in tannin mass conversion and proportion of (-)-epigallocatechin extension units. Some of these changes in phenolic compounds could potentially increase astringency, suggesting that MOX should be applied to Pinot noir and other low phenolic wines with caution.

Continuous cell-to-cell contact between different species is a general feature of all natural environments. However, almost all research is conducted on single-species cultures, reflecting a biotechnological bias and problems associated with the complexities of reproducibly growing and controlling multispecies systems. Consequently, biotic stress due to the presence of other species remains poorly understood. In this context, understanding the effects of physical contact between species when compared to metabolic contact alone is one of the first steps to unravelling the mechanisms that underpin microbial ecological interactions. The current technologies to study the effects of cell-to-cell contact present disadvantages, such as the inefficient or discontinuous exchange of metabolites when preventing contact between species. This paper presents and characterizes a novel bioreactor system that uses ceramic membranes to create a “multi-membrane” compartmentalized system whereby two or more species can be co-cultured without the mixing of the species, while ensuring the efficient sharing of all of the media components. The system operates continuously, thereby avoiding the discontinuities that characterize other systems, which either have to use hourly backwashes to clean their membranes, or have to change the direction of the flow between compartments. This study evaluates the movement of metabolites across the membrane in co-cultures of yeast, microalgae and bacterial species, and monitors the movement of the metabolites produced during co-culturing. These results show that the multi-membrane system proposed in this study represents an effective system for studying the effects of cell-to-cell contact in microbial consortia. The system can also be adapted for various biotechnological purposes, such as the production of metabolites when more than one species is required for such a process.

Stevioside (ST) is currently considered as a highly-demanded natural and zero-caloric replacer of sucrose with several health-promoting properties. Nonetheless, its bitter aftertaste limits its use in the food industry. Herein, glucosyl steviosides were synthesized using primarily a food-grade lactic acid bacteria, Leuconostoc kimchii dextransucrase and conversion yield (%) was 40.3%. A glucose moiety was transferred stereo-selectively to ST by α-1,6-linkage and this is the first report about obtaining rebaudioside A (Reb-A) like glucosyl stevioside-2 (STG-2). Glucosyl steviosides revealed greatly improved stability up to 120°C and remained stable over 32.1% and 58.12% in the pH (1.4) compared with 30.55% of ST. Moreover, the glucosylated steviosides improved the stability, reaching 95% after 30 days and Reb-A like compound (STG-2) especially exhibited higher stability in commercial beverages. Furthermore, the glucosyl steviosides showed over 1.92- and 2.24-fold decreases than that of enzymatically modified ST in the glucose generation rate test.

Wines from warm(ing) climates often contain excessive ethanol but lack acidity. The yeast Lachancea thermotolerans can ameliorate such wines due to partial conversion of sugars to lactic acid during alcoholic fermentation. This study compared the performance of five L. thermotolerans strains in two inoculation modalities (sequential and co-inoculation) to Saccharomyces cerevisiae and un-inoculated treatments in high sugar/low acidity Merlot fermentations. The pH and ethanol levels in mixed-culture dry wines were either comparable, or significantly lower than in controls (decrease of up to 0.5 units and 0.90% v/v, respectively). The analysis of volatile compounds revealed marked differences in major flavour-active yeast metabolites, including up to a thirty-fold increase in ethyl lactate in certain L. thermotolerans modalities. The wines significantly differed in acidity perception, alongside 18 other sensory attributes. Together, these results highlight the potential of some L. thermotolerans strains to produce ‘fresher’ wines with lower ethanol content and improved flavour/balance.

Given the pharmacological properti es and the potential role of kynurenic acid (KYNA) in human physiology and the pleiotropic activity of the neurohormone melatonin (MEL) involved in physiological and immunological functions and as regulator of antioxidant enzymes, this study aimed at evaluating the capability of Saccharomyces cerevisiae EC1118 to release tryptophan derivatives (dTRPs) from the kynurenine (KYN) and melatonin pathways. The setting up of the spectroscopic and chromatographic conditions for the quantification of the dTRPs in LC-MS/MS system, the optimization of dTRPs’ production in fermentative and whole-cell biotransformation approaches and the production of dTRPs in a soybean-based cultural medium naturally enriched in tryptophan, as a case of study, were included in the experimental plan. Variable amounts of dTRPs, with a prevalence of metabolites of the KYN pathway, were detected. The LC-MS/MS analysis showed that the compound synthesized at highest concentration is KYNA that reached 9.146 ± 0.585 mg/L in fermentation trials in a chemically defined medium at 400 mg/L TRP. Further experiments in a soybean-based medium confirm KYNA as the main dTRPs, whereas the other dTRPs reached very lower concentrations. While detectable quantities of melatonin were never observed, two MEL isomers were successfully measured in laboratory media.

Potato chips are among the highest contributors to the dietary intake of acrylamide, a potent neurotoxin and likely carcinogen in heat-processed foods. The present study aimed to determine the effects of frying conditions and additive treatments on reducing sugars, asparagine levels, and acrylamide formation in fried potato chips. Three commercially important chipping cultivars (Atlantic, Snowden, and Vigor) were tested using different frying times (3, 5, and 7 min) and temperatures (160, 170, 180, and 190°C). Acrylamide formation in chips was found to be cultivar-specific and increased with increasing frying time and temperature. The acrylamide levels were significantly lower in chips processed at high temperatures for short frying times than in those processed at low temperatures for long frying times. In all cultivars, acrylamide synthesis was accompanied by significant decreases in the levels of reducing sugars and asparagine. The cultivar exhibiting the lowest acrylamide levels in the processed potato chips was Snowden, while the conditions most conducive to acrylamide formation in all cultivars were frying at 190°C for 7 min. Using a 7 min frying time, decreasing the frying temperature from 190 to 160°C mitigated the acrylamide formation in potato chips processed from Atlantic, Snowden and Vigor by 84, 67, and 78%, respectively. We also examined the effects of additives, such as 1% acetic acid, 1% citric acid, 0.1 M sodium chloride, 0.1 M calcium chloride, 0.5% ascorbic acid, or 1 M l-glycine, during the blanching of potato slices prior to frying. Blanching in distilled water led to the greatest decreases (19-59%) in acrylamide formation in all cultivars. Our findings provide the basis for new processing strategies to mitigate acrylamide formation and improve the quality of chips from these, and possibly other, potato cultivars.

Prebiotics are increasingly examined for their ability to modulate the neonate gut microbiota of livestock, and products such as inulin are commonly added to milk replacer used in calving. However, the ability of specific members of the bovine neonate microbiota to respond to inulin remains to be determined, particularly among indigenous lactobacilli and bifidobacteria, beneficial genera commonly enriched by inulin. Screening of Bifidobacterium and Lactobacillus isolates obtained from fresh feces of dairy calves revealed that lactobacilli had a higher prevalence of inulin fermentation capacity (58%) than bifidobacteria (17%). Several Ligilactobacillus agilis (synonym Lactobacillus agilis) isolates exhibited vigorous growth on, and complete degradation of, inulin; however, the phenotype was strain specific. The most vigorous inulin-fermenting strain, L. agilis YZ050, readily degraded long-chain inulin not consumed by bifidobacterial isolates. Comparative genomic analysis of both L. agilis fermenter and nonfermenter strains indicated that strain YZ050 encodes an inulinase homolog, previously linked to extracellular degradation of long-chain inulin in Lacticaseibacillus paracasei, that was strongly induced during growth on inulin. Inulin catabolism by YZ050 also generates extracellular fructose, which can cross-feed other non-inulin-fermenting lactic acid bacteria isolated from the same bovine feces. The presence of specific inulin-responsive bacterial strains within calf gut microbiome provides a mechanistic rationale for enrichment of specific lactobacilli and creates a foundation for future synbiotic applications in dairy calves aimed at improving health in early life.

Hydroxytyrosol is well known for its potent antioxidant activity and anticarcinogenic, antimicrobial, cardioprotective and neuroprotective properties. Main food sources are olive oil (formed from the hydrolysis of oleuropein) and wine. One possible explanation to its origin in wines is the synthesis from tyrosol, which in turn is produced from the Ehrlich pathway by yeasts. This work aims to explore the factors that could increase the content as the strain of yeast, the initial tyrosine concentrations as precursor and the effect of synthetic and sterilized natural grape musts. Alcoholic fermentations in synthetic must showed that hydroxytyrosol is produced by all the yeast strains under study. Commercial Saccharomyces cerevisiae yeasts were those which produced higher concentrations, being the Red Fruit strain the biggest producer (6.12 ng/mL). Once the strain was selected, alcoholic fermentations were performed in synthetic must, with different tyrosine concentrations. The amount of hydroxytyrosol did not increase in a proportional way as tyrosine does. On the other hand, higher concentrations of hydroxytyrosol were obtained in natural grape musts (10.46 ng/mL) than in synthetic must (4.03 ng/mL). This work confirms the capacity of winemaking yeasts to produce the bioactive hydroxytyrosol.

In Latin, ‘pulcherrima’ is a superlative form of an adjective that translates as beautiful. Apart from being ‘the most beautiful’ yeast, Metschnikowia pulcherrima has a remarkable potential in production of wines with lower ethanol content. The oenological performance of six M. pulcherrima strains was hereby tested in sequential cultures with Saccharomyces cerevisiae. The best-performing strain MP2 was further characterised in fermentations with different S. cerevisiae inoculation delays in both white grape juice and Chemically Defined Grape Juice Medium (CDGJM). The analysis of main metabolites, undertaken prior to sequential inoculations and upon fermentation completion, highlighted metabolic interactions and carbon sinks other than ethanol in MP2 treatments. Depending on the inoculation delay, MP2 white wines contained between 0.6% and 1.2% (v/v) less ethanol than the S. cerevisiae monoculture, with even larger decreases detected in the CDGJM. The MP2 treatments also contained higher concentrations of TCA cycle by-products (i.e. fumarate and succinate) and glycerol, and lower concentrations of acetic acid. The analysis of volatile compounds showed increased production of acetate esters and higher alcohols in all MP2 wines, alongside other compositional alterations arising from the S. cerevisiae inoculation delay.

A biotechnological protocol to produce a focaccia (a typical Italian flat bread) without bakers' yeast addition and with reduced salt was developed, to meet the current needs of the consumer. Based on its leavening capability, the Leuconostoc citreum strain C2.27 was selected to be used as a starter instead of the baker's yeast and inoculated in a liquid sourdough (type-II) for the production of the “yeast-free” focaccia. The addition of different NaCl concentrations and the replacement of the salt with food grade seawater were evaluated, and the capability of the selected strain to affect technological, nutritional and sensory features of the focaccia investigated. A significant improvement of the nutritional characteristics of the focaccia was observed compared to the control (leavened with bakers' yeast and added with NaCl 1.5 g/100 g) using 0.7 g/100 g of salt in the form of NaCl or seawater. Besides the reduced Na content (66% lower than the control), focaccia with seawater also showed a higher content of Ca²⁺ and Mg²⁺ (ca. 36% and 53%, respectively), and the lowest predicted glycemic index compared to the other experimental focaccia.

FODMAPs are fermentable oligo-, di, and monosaccharides and polyols, which can have adverse effects on human health. Especially in individuals with gastrointestinal disorders such as irritable bowel syndrome, ingestion of FODMAPs may trigger or aggravate symptoms like abdominal pain, bloating, or diarrhea. Our objectives were to investigate the FODMAP content in the flour of 21 wheat varieties and in their 42 yeast-leavened breads prepared with a short (110 min) or a long dough fermentation (24 h), the latter according to a typical baker's recipe. Fructan, glucose, fructose and excess fructose were measured using enzymatic kits. On average, the breads of both dough fermentations had a FODMAP content reduced by > 65% compared to that of the whole grain wheat flour. Average FODMAP content of both dough fermentations was 0.22 g per 100 g fresh bread. FODMAP content tended to be lower in the long compared to the short dough fermentation, however, the difference was not statistically significant. We also found that the 21 wheat varieties differed up to five times in their potential to form FODMAPs in bread. In conclusion, the choice of a low fructan wheat variety coupled with long dough fermentation with sourdough and yeast is best to minimize FODMAPs in breads.

Aim: A Saccharomyces uvarum isolate was assessed for its ability to metabolize acetic acid present in juice and during the fermentation of partially dehydrated grapes. The impact on other yeast metabolites was also compared using an S. uvarum isolate and an S. cerevisiae wine yeast. The upper limit of fruit concentration that allowed the S. uvarum isolate to ferment wines to < 5 g/L residual sugar was defined. Methods and results: Cabernet franc grapes were partially dehydrated to three different post-harvest sugar targets (24.5 °Brix, 26.0 °Brix, and 27.5 °Brix) along with non-dehydrated grapes (21.5 °Brix control). Musts from all treatments were vinified with either the S. uvarum isolate CN1, formerly identified as S. bayanus, or S. cerevisiae EC1118. All wines were successfully vinified to less than 5 g/L residual sugar. Fermentation kinetics between the two yeasts were similar for all wines other than 27.5 °Brix, where CN1 took three days longer. During fermentation with CN1, acetic acid peaked on day two, then decreased in concentration, resulting in final wine acetic acid lower than that measured on day two. Wines fermented with EC1118 showed an increase in acetic acid over the time-course of fermentation. Significantly lower wine oxidative compounds (acetic acid, acetaldehyde and ethyl acetate) and higher glycerol resulted in wine produced with CN1 in comparison to EC1118. Both yeasts produced comparable ethanol at each Brix level tested. Further studies showed that CN1 lowered acetic acid seven-fold from 0.48 g/L in juice to 0.07 g/L in wine whereas EC1118 reduced acetic acid to 0.18 g/L. Conclusions: The autochthonous S. uvarum yeast isolate successfully fermented partially dehydrated grapes to < 5 g/L sugar up to 27.5 ºBrix. The consumption rate of acetic acid was faster than its production during fermentation, resulting in low acetic acid, acetaldehyde and ethyl acetate in wine in comparison to a commercial S. cerevisiae yeast while consistently producing higher glycerol. Significance and impact of the study: The S. uvarum yeast isolate can metabolize acetic acid during fermentation to significantly lower acetic acid, ethyl acetate and acetaldehyde in wine. It can also reduce acetic acid by seven-fold from the starting juice to the finished wine, which could have potential application for managing sour rot arising in the vineyard or during the dehydration process in making appassimento-style wines.

Biomass has recently been extensively exploited as an attractive alternative to the scarce fossil fuels and has been widely used for the production of fine chemicals. The present work defines an integrated approach for high yield of 5-hydroxymethylfurfural (HMF) from inulin via a two-step process. Initially, insolubilization of inulinase from Aspergillus niger on chitosan-coated magnetic nanoparticles (cMNP) was achieved, and under optimal conditions, hydrolysis of 5% inulin by immobilized inulinase (2.5 U/mg) led to fructose release of 35.8 g/L at pH 6.0 and temperature 60°C in 3 h. Subsequently, the fructose was dehydrated to HMF in the presence of TiO₂-magnetic silica spheres (TiO₂-MSS). Upon optimizing the process parameters, HMF yield of 96.58% was achieved with 15% fructose in 15 min at 140°C with a TiO₂-MSS load of 10% (w/w). Concerning the operational stability of the immobilized biocatalyst/catalyst, the immobilized inulinase was recycled up to 10 cycles of inulin hydrolysis with 9.2 g/L of fructose released at the 10th cycle. TiO₂-MSS also showed high operational stability and were recycled up to 10 cycles of HMF production with 5.4 g/L of HMF produced at 10th cycle. The mass balance for inulin to HMF was performed which showed the efficiency of the process.

Despite significant efforts recently made to improve the recovery yield of food pigments from natural sources, the development of green and sustainable biotechnological approaches is currently under investigation. Within the context of circular economy, food wastes represent a cheap source for the recovery of valuable compounds including food ingredients. In this study, a conservative approach consisting in a tailored enzyme-assisted extraction protocol for the recovery of carotenoid-containing chromoplasts from unsold tomatoes in which lycopene is stable and protected against oxidation, has now been developed. A tailored enzymatic mix based on polygalacturonase, pectin lyase, cellulase and xylanase, has been designed taking into account the polysaccharide composition of the tomato cell wall. The optimal process conditions for enhancing the recovery of carotenoids from tomatoes i.e.; temperature, pH, enzymatic mix of total dosage and processing time, have been investigated. The suitable temperature and pH identified by the RSM analysis, were found to be: 45-55°C at pH 5-5.5. The treatment carried out using the total dosage of 25 U/g for 180 min was the most convenient for maximizing the recovery yield [about 4.30 ± 0.08 (mg_Lyc/Kg_tomato)/U as carotenoid-containing chromoplasts and about 5.43 ± 0.04 (mg_Lyc/Kg_tomato)/U as total carotenoids].

Waxy rice starch was hydrolyzed by β-amylase and then modified by octenyl succinic anhydride (OSA) to prepare octenyl succinate β-limit dextrin (OSA β-limit dextrin). Subsequently, structure, digestibility and emulsifying capacity of OSA β-limit dextrin were measured. The FT-IR analysis confirmed formation of OSA β-limit dextrin. When the addition of OSA was 1.0%, 2.0% and 3.0%, the DS of OSA β-limit dextrin was 0.006, 0.011 and 0.019, respectively. The molecular weight, resistant starch content and emulsifying capacity of OSA β-limit dextrin was higher than β-limit dextrin and increased with the increasing of DS. In addition, the solubility of OSA β-limit dextrin reached 100%. The emulsion prepared by OSA β-limit dextrin was stable at pH 3.0-7.0 and became unstable at pH 8.0-11.0 or after addition of NaCl. These results suggested that OSA β-limit dextrin was safe soluble resistant dextrin with emulsifying ability, which might be used in functional food and beverage products.

Wines produced from red interspecific hybrid grape cultivars (Vitis spp.) typically have lower tannin concentrations than wines produced from vinifera cultivars, which can be attributed to the lower concentration of tannins and higher concentration of tannin-binding proteins of interspecific cultivars. Tannin in wines produced from hybrid cultivars may be increased by blending hybrids with vinifera. We hypothesized that blending of grapes prior to fermentation (cofermentation) would result in final wine tannin concentrations lower than those predicted from the individual components due to protein-tannin binding, but that this effect would be absent from monovarietal wines blended postfermentation. To evaluate this hypothesis, a high tannin V. vinifera cultivar (Cabernet Sauvignon) was blended with an interspecific hybrid (Marquette) at different ratios either before (cofermentation) or after fermentation over a two-year period. The tannin and protein concentrations of the wines were measured by methyl cellulose precipitation assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. Tannin and protein concentrations in blended wines were compared to values predicted from the linear combination of the two monovarietal wines. Cofermented blends with a high proportion of Marquette had up to 25% lower tannin than predicted, but observed and predicted tannin concentrations did not differ for most cofermentations and postfermentation blends. However, protein concentrations for many of the blends-especially from cofermentation-were lower than predicted values (>50% in some cases). Loss of protein due to adsorption to tannin was well modeled by a Freundlich adsorption isotherm.

In the present work, three Spanish local varieties of Prunus avium (L.), as well as two foreign varieties were studied. The content of total phenols, flavonoids, anthocyanins, glucose and fructose of methanolic extracts from ripe fruits of each variety were analysed. A phytochemical profile of these cultivars was performed by UHPLC-qTOF-MS. The employed chromatographic method allowed a clear and rapid separation of the three main phenolic compound groups present in the extracts: hydroxycinnamic acids, anthocyanins and flavonoids. In addition, the extracts DPPH radical scavenging ability, as well as their capacity to affect xanthine/xanthine oxidase system, were determined. Finally, variations in ROS intracellular concentrations in HepG2 cell line cultures treated with cherry extracts were measured through DCFH-DA assay. All extracts showed a significant inhibitory effect on the xanthine/xanthine oxidase system. Differences between in vitro and in cell culture results evidence the interaction among the phenolic compounds of the extract.

Novel Shiraz red wine products enriched with Ganoderma lucidum (GL) extract, a traditional Asian medicinal mushroom, were developed and characterized. GL extract was added at different levels prior to and after primary fermentation to investigate its impact on the juice fermentation kinetics, and the chemical composition and sensory properties of the resulting wines. The fermentation kinetics of red grape juice were not significantly different between ferments. Basic chemical analyses plus headspace solid-phase micro-extraction (HS-SPME), gas chromatography-mass spectrometry (GC-MS), and a rate-all-that-apply (RATA) (n = 65) sensory panel were used to investigate the influence of GL extract additions on wine composition and sensory characteristics. Of the 54 sensory attributes assessed, 39 significantly differentiated the wines. A clear separation between GL wine treatments was evident with PLS regression, where specific volatiles were correlated with relevant sensory attributes that dominated the wines. These products could be promising for emerging wine markets.