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.

Sugar beet pulp (SBP), sugar beet molasses (SBM) and unfermented grape marcs (UGM) represent important waste in the agro-food sector. If suitably pre-treated, hexose and pentose sugars can be released in high quantities and can subsequently be used by appropriate cell factories as growth media and for the production of (complex) biomolecules, accomplishing the growing demand for products obtained from sustainable resources. One example is vitamin B₉ or folate, a B-complex vitamin currently produced by chemical synthesis, almost exclusively in the oxidized form of folic acid (FA). It is therefore desirable to develop novel competitive strategies for replacing its current fossil-based production with a sustainable bio-based process. In this study, we assessed the production of natural folate by the yeast Scheffersomyces stipitis, investigating SBM, SBP and UGM as potential growth media. Pre-treatment of SBM and SBP had previously been optimized in our laboratory; thus, here we focused only on UGM pre-treatment and hydrolysis strategies for the release of fermentable sugars. Then, we optimized the growth of S. stipitis on the three media formulated from those biomasses, working on inoculum pre-adaptation, oxygen availability and supplementation of necessary nutrients to support the microorganism. Folate production, measured with a microbiological assay, reached 188.2 ± 24.86 μg/L on SBM, 130.6 ± 1.34 μg/L on SBP and 101.9 ± 6.62 μg/L on UGM. Here, we demonstrate the flexibility of S. stipitis in utilizing different residual biomasses as growth media. Moreover, we assessed the production of folate from waste, and to the best of our knowledge, we obtained the highest production of folate from residual biomasses ever reported, providing the first indications for the future development of this microbial production process.

Breadfruit (Artocarpus altilis) is a traditional fruit tree of 15–30 m tall in Oceania. The species is a staple crop for food security in the tropics. Tree loss from tropical windstorms, together with transition toward high-density planting has driven an interest in the dwarf phenotype of the species. Information on dwarfing rootstocks for breadfruit is currently limited. The aim of this study was to assess the performance of breadfruit growth with lakoocha (Artocarpus lakoocha) as rootstocks. We compared the phenotype of breadfruit trees on lakoocha rootstocks with those on self-graft and non-graft within 21 months after grafting. These led to the discovery of a rootstock-induced dwarf trait in breadfruit species. Breadfruit scions on lakoocha rootstocks displayed a reduction in tree height, stem thickness, and internode length, with fewer branches and leaves, resulting in about 32% of the standard height at the end of 21 months after grafting. These suggest lakoocha rootstocks have the potential to control breadfruit tree vigor. Non-structural carbohydrate analysis showed the composite trees exhibited lower hexose concentration in both scion stems and roots, but higher sucrose level in scion stems, and higher starch level in roots. The significance of these parameters in rootstock dwarfing is discussed.

The indigenous vineyard mycobiota contribute both to wine quality and vineyard sanitary status. Wines made from same grape variety but from different geographical locations are appreciated for their diversity. Because no information on indigenous mycobiota of Croatian grapevines is available, the aim of the present study was to start filling this knowledge gap by characterizing the indigenous mycobiota of Maraština variety. The use of metataxonomic approach has enabled the identification of 25 different fungal genera present on Maraština grape berries collected from 11 vineyards located within the Croatian coastal winegrowing region of Dalmatia (Northern Dalmatia, Dalmatian hinterland, Central and Southern Dalmatia). The substantial regional and local scale differences in their distribution were observed. Overall, Aureobasidium was the dominant genus followed by Cladosporium and Metschnikowia. Botrytis and Plenodomus were associated with the vineyards located in Central and Southern Dalmatia, whereas Pichia was associated with Northern Dalmatia vineyards. The largest abundance of Buckleyzyma, Cladosporium, Eremothecium, Fusarium, Papiliotrema, and Rhodotorula was observed in Dalmatian hinterland. Moreover, data suggested that climate conditions and soil type partially influenced the distribution of fungal communities. The local-scale differences emerged also for the physicochemical characteristics of fresh musts. The high malic acid content supported the development of Metschnikowia, and inhibited Fusarium growth, whereas a positive correlation between Erysiphe and pH values was observed. Sporobolomyces and Cystobasidium were negatively associated with high glucose concentration. The revealing of Maraština indigenous mycobiota provided information on the members of fungal community negatively influencing the grapevine sanitary status as well as those which could be employed in disease biocontrol.

The study aimed to investigate the effect of modification of Fagopyrum esculentum Moench sprouts by probiotic yeast strain on their nutritional value related to protein and starch. For this purpose, an attempt was also made to 1) evaluate the digestibility of protein and starch and 2) examine anti-nutritional factors that may affect the digestibility of the indicated macronutrients. Probiotic-rich sprouts were characterized by the highest content of total protein, free amino acids and peptides, resistant starch, and total free sugars. In addition, the amino acid profile of buckwheat sprouts changed compared to seeds. Modified buckwheat sprouts were characterized by a higher content of sulfur amino acids compared to seeds and control sprouts. The highest methionine content was found in sprouts rich in probiotics, 4.65 ± 0.29 mg/g 16gN. The results show also that the modification reduced protein and starch digestibility by 7.4% and 4.2%, respectively, compared to the seed. The digestibility may have been influenced by anti-nutritional compounds contained in the raw material, i.e. condensed tannins, phenols and α-amylase inhibitor. The presented research gives a new direction for the use of the raw material of buckwheat sprouts.

Recovery of bioactive compounds from biowaste is gaining more and more interest in circular economy models. The oilseed cakes are usually insufficiently exploited by most technologies since they represent valuable matrices abundant in proteins, minerals, and phytochemicals, but their use is mostly limited to feed ingredients, fertilizers or biofuel production. This study was thus focused on the exploration of new valorization pathways of oilseed cakes by subcritical water, representing a safe and economic alternative in the creation of value chains. Pumpkin, hemp, and flax seed cakes were treated with subcritical water in nitrogen and carbon-dioxide atmospheres, as well as in nitrogen atmosphere with the addition of acid catalyst. The degradation of carbohydrate fraction was studied by quantifying sugars and sugar degradation products in the obtained extracts. The extracts obtained under different conditions were further compared chemically with respect to total phenols and flavonoids, as well as to the content of individual phenolic compounds. Furthermore, the effects of subcritical water treatment conditions on antioxidant, antiradical and cytotoxic properties of thus obtained extracts were defined and discussed.

Food resources in arid habitats are important for the survival of native fauna, especially where resources provide water or key nutrients during dry periods. However, food resource plants can be susceptible to grazing species or may not be suitable for revegetating arid areas. Pimelea microcephala subsp. microcephala (P.m.microcephala) is an Australian dioecious shrub bearing bright fruit that are likely to attract birds. The plant is also noted for its production of simplexin, a compound toxic to mammals. The aims of our study were 1) to assess the nutrient and simplexin content of P.m.microcephala fruits, and 2) to confirm the identity of native avian species that consume the fruits. With both pieces of information, we could then determine the conservation utility of P.m.microcephala. Combining chemical analysis of fruit nutrients with field observations, we found that ripe fruits contain 60.5% water, 2.8% sugar, and potent antioxidants, which would likely be of nutritional benefit to consuming frugivores. The fruits also contain high levels of the toxin simplexin, which comprised 3.6% of ripe fruits by weight. We identified eight bird species interacting with P.m.microcephala, with at least five of these consuming ripe fruits. Our study demonstrates the potential for P.m.microcephala to contribute to revegetation and provide a food resource for arid zone birds, whilst being protected from grazing by the presence of simplexin. Further studies are needed to establish the species’ absolute significance in terms of fruits as a source of water and nutrients to arid zone bird diets.

In wine fermentation, improved wine complexity and sensorial properties can arise from the use of non-Saccharomyces yeast. Generally less alcohol tolerant, such strains often do not finish fermentation, therefore requiring a second inoculation with the more robust Saccharomyces cerevisiae, usually added on Day 3. This sequential approach affords non-Saccharomyces time to make an impact before being overtaken by S. cerevisiae. However, two inoculations are inconvenient; therefore the identification of a slow growing S. cerevisiae strain that can be used in a single co-inoculation with the non-Saccharomyces yeast is highly attractive. In this study we investigated the use of the naturally occurring ‘loss of function’ SER1 variant, identified in a Sake yeast, for the purposes of carrying out co-inoculated wine fermentations. The SER1-232(G > C; G78R) change was introduced into the commonly used wine strain, EC1118, via CRISPR/Cas9 editing. In a chemically defined grape juice medium, the SER1(G78R) mutant grew and fermented more slowly and increased acetic acid, succinic acid and glycerol concentrations. Simultaneous inoculation with the slower-growing mutant with a Metschnikowia pulcherrima or Lachancea thermotolerans strain in sterile Sauvignon blanc juice resulted in differences in sensorial compounds, most likely derived from the presence of non-Saccharomyces yeasts. The EC1118 SER1 (G78R) mutant completed fermentation with M. pulcherrima, MP2, and in fact improved the viability of MP2 compared to when it was used as a monoculture. The SER1 (G78R) mutant also promoted both the growth of the SO₂-sensitive L. thermotolerans strain, Viniflora® Concerto™, in a juice high in SO₂ and its subsequent dominance during fermentation. In co-fermentations with wild-type EC1118, the Concerto™ population was substantially reduced with no significant changes in wine properties. This research adds to our understanding of the use of a novel slow-growing S. cerevisiae yeast in wine fermentations co-inoculated with non-Saccharomyces strains.

For the first time, we report differences in gene and protein expression in potato (Solanum tuberosum L.) tubers possessing the T- or D-type cytoplasm. Two F1 diploid reciprocal populations, referred to as T and D, were used. The pooling strategy was applied for detection of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) in tubers consisting of extreme chip colour after cold storage. RNA and protein bulks were constructed from contrasting phenotypes. We recognized 48 and 15 DEGs for the T and D progenies, respectively. DEPs were identified in the amyloplast and mitochondrial fractions. In the T-type cytoplasm, only 2 amyloplast-associated and 5 mitochondria-associated DEPs were detected. Of 37 mitochondria-associated DEPs in the D-type cytoplasm, there were 36 downregulated DEPs in the dark chip colour bulks. These findings suggest that T- and D-type of cytoplasm might influence sugar accumulation in cold-stored potato tubers in different ways. We showed that the mt/nucDNA ratio was higher in D-possessing tubers after cold storage than in T progeny. For the D-type cytoplasm, the pt/nucDNA ratio was higher for tubers characterized by dark chip colour than for those with light chip colour. Our findings suggest that T- and D-type cytoplasm might influence sugar accumulation in cold-stored potato tubers in different ways.

Viognier is a warm climate grape variety prone to loss of acidity and accumulation of excessive sugars. The yeast Lachancea thermotolerans can improve the stability and balance of such wines due to the partial conversion of sugars to lactic acid during alcoholic fermentation. This study compared the performance of five L. thermotolerans strains in co-inoculations and sequential inoculations with Saccharomyces cerevisiae in high sugar/pH Viognier fermentations. The results highlighted the dichotomy between the non-acidified and the bio-acidified L. thermotolerans treatments, with either comparable or up to 0.5 units lower pH relative to the S. cerevisiae control. Significant differences were detected in a range of flavour-active yeast volatile metabolites. The perceived acidity mirrored the modulations in wine pH/TA, as confirmed via “Rate-All-That-Apply” sensory analysis. Despite major variations in the volatile composition and acidity alike, the varietal aromatic expression (i.e., stone fruit aroma/flavour) remained conserved between the treatments.

Grass-clover silage constitutes a large part of ruminant diets in Northern and Western Europe, but the impact of silage quality on methane (CH₄) production is largely unknown. This study was conducted to identify the quality attributes of grass silage associated with variation in CH₄ yield. We expected that silage nutrient concentrations and silage fermentation products would affect CH₄ yield, and that these factors could be used to predict the methanogenic potential of the silages. Round bales (n = 78) of grass and grass-clover silage from 37 farms in Norway were sampled, incubated, and screened for in vitro CH₄ yield, i.e. CH₄ production expressed on the basis of incubated organic matter (CH₄-OM) and digestible OM (CH₄-dOM) using sheep. Concentration of indigestible neutral detergent fiber (iNDF) was quantified using the in situ technique. The data were subjected to correlation and principal component analyses. Stepwise multiple regression was used to model methanogenic potential of silages. Among all investigated silage composition variables, neutral detergent fiber (aNDFom) and water-soluble carbohydrate (WSC) concentrations obtained the greatest correlations to CH₄-OM (r = −0.63 and r = 0.57, respectively, P < 0.001), while concentration of iNDF negatively correlated with CH₄-OM (r = −0.48, P < 0.001). In vivo organic matter digestibility (OMD) and concentration of ammonia-N (NH₃-N) in silages were also correlated to CH₄-OM (r = 0.44 and r = −0.32, P < 0.001 and P < 0.01, respectively). The stepwise regression using CH₄-OM as response variable included aNDFom, WSC, iNDF, silage propionic acid and pH in descending order. The stepwise regression using CH₄-dOM as response variable included WSC, aNDFom and iNDF in descending order. Among in vitro rumen short chain fatty acids (SCFA), molar proportion of butyrate was the most prominent in increasing CH₄-OM and CH₄-dOM (r = 0.23 and r = 0.36, P < 0.05 and P < 0.01, respectively), while molar proportion of propionate was the most prominent SCFA in reducing CH₄-OM and CH₄-dOM (r = −0.23 and r = −0.26, respectively, P < 0.05). Regression models that account for silage quality attributes can be used to predict CH₄ yield from silages with a coefficient of determination (R²) between 0.33 (CH₄-dOM) and 0.65 (CH₄-OM). In conclusion, concentration of WSC increased in vitro CH₄-OM and CH₄-dOM, while concentration of aNDFom and iNDF decreased CH₄-OM and CH₄-dOM in grass silages.

In this investigation, the combinations of exogenous pretreatment (melatonin or vitamin C) and contact ultrasound-assisted air drying were utilized to dry broccoli florets. To understand the influences of the studied dehydration methods on the conversion of glucoraphanin to bioactive sulforaphane in broccoli, various components (like glucoraphanin, sulforaphane, myrosinase, etc.) and factors (temperature and moisture) involved in the metabolism pathway were analyzed. The results showed that compared with direct air drying, the sequential exogenous pretreatment and contact ultrasound drying shortened the drying time by 19.0-22.7%. Meanwhile, contact sonication could promote the degradation of glucoraphanin. Both melatonin pretreatment and vitamin C pretreatment showed protective effects on the sulforaphane content and myrosinase activity during the subsequent drying process. At the end of drying, the sulforaphane content in samples dehydrated by the sequential melatonin (or vitamin C) pretreatment and ultrasound-intensified drying was 14.4% (or 26.5%) higher than only air-dried samples. The correlation analysis revealed that the exogenous pretreatment or ultrasound could affect the enzymatic degradation of glucoraphanin and the generation of sulforaphane through weakening the connections of sulforaphane-myrosinase, sulforaphane-VC, and VC-myrosinase. Overall, the reported results can enrich the biochemistry knowledge about the transformation of glucoraphanin to sulforaphane in cruciferous vegetables during drying, and the combined VC/melatonin pretreatment and ultrasound drying is conducive to protect bioactive sulforaphane in dehydrated broccoli.

Dextransucrases released by certain lactic acid bacteria form glucose polymers with predominantly α-1,6-linkages and may be exploited biotechnologically for the tailored production of polysaccharides with application potential. Despite releasing two closely related dextransucrases, previous studies showed that water kefir borne Liquorilactobacillus (L.) hordei TMW 1.1822 and L. nagelii TMW 1.1827 produce different amounts of polysaccharides with distinct particle sizes (molecular weight and radius of gyration) and molecular architectures. To investigate where these differences originate and thus to provide deeper insights into the functionally diverse nature of polysaccharide formation during water kefir fermentation, we constructed two variants of the L. nagelii dextransucrase-a full-length enzyme and a truncated variant, devoid of a C-terminal glucan-binding domain that reflects the domain architecture of the L. hordei dextransucrase-and applied them at various enzyme concentrations to form dextran over 24 h. The full-length enzyme exhibited a high activity, forming constant amounts of dextran until a four-fold dilution, whereas the truncated variant showed a gradual decrease in activity and dextran formation at an increasing dilution. The application of the full-length enzyme resulted in higher average particle sizes compared to the truncated variant. However, the dilution of the enzyme extracts also led to a slight increase in the average particle size in both enzymes. Neither the domain architecture nor the enzyme concentration had an impact on the structural architecture of the dextrans. The presented results thus suggest that the comparatively higher processivity of the L. nagelii dextransucrase is predominantly caused by the additional C-terminal glucan-binding domain, which is absent in the L. hordei dextransucrase. The average particle size may be influenced, to some extent, by the applied reaction conditions, whereas the structural architecture of the dextrans is most likely caused by differences in the amino acid sequence of the catalytic domain.

The present work investigated the impact of ultrafiltration (UF) and nanofiltration (NF) membranes on the recovery and fractionation of polyphenolic compounds and polysaccharides from Sangiovese and Cabernet Sauvignon wine lees. A laboratory-made flat-sheet membrane in cellulose acetate (CA400-38) was used in the UF treatment of Sangiovese wine lees; three laboratory-made flat-sheet membranes in cellulose acetate (CA316, CA316-70, CA400-22) and a polyamide commercial membrane (NF90) were used in the NF treatment of Cabernet Sauvignon wine lees. All membranes were characterized in terms of hydraulic permeability and rejection toward references solutes; the performances of the membranes were measured in terms of productivity, fouling index, cleaning efficiency and retention toward target compounds. Experimental results indicated that all UF and NF membranes were effective in separating target compounds rejecting more than 92% of polysaccharides with polyphenols preferentially permeating through the membrane. The UF membrane rejected more than 40% of total polyphenols; rejections toward non-flavonoids and flavonoids were less than 25% and 12.5%, respectively. The laboratory-made NF membranes exhibited higher permeate flux values (of the order of 11–12 L/m²h) in comparison with the commercial NF membrane, despite the observed differences in the retention of specific solutes. Among the prepared membranes the CA316 showed a total rejection toward most part of non-flavonoids and flavonoids. The experimental results support the use of UF and NF processes in a sequential design to fractionate and refine phenolic compounds from winery sludge for the production of concentrated fractions with high antioxidant activities.

The effect of autohydrolysis (AH) temperature (165°C, 195°C, 225°C) on the structure, purity, and recovery yield of the cellulose residue isolated after additional alkaline and bleaching steps from wheat straw, was investigated. The processes were quantified for mass yields in the different steps and for antioxidants and sugars release during AH. AH at 195°C allowed for the highest cellulose residue yield (83.5%) with purity (~70%) and structure similar to the other residues. FTIR and XRD analyses showed straw cellulose (SC) with a type II polymorphism and crystallinity index increasing with AH temperature. SC obtained at the end of the entire fractionation process (SC-195°C) starting from AH residue-195°C was tested as a reinforcing agent in different percentage (0, 2 and 5% by weight) in poly(butylene adipate-co-terephthalate) (PBAT) films. The Young's modulus of the films increased by ~17% with 5 wt% cellulose, while tensile strength and elongation at break decreased.

Bread is a high glycemic index (GI) food with high amounts of readily digestible carbohydrates. Fucoidan refers to a group of sulfated polysaccharides isolated from brown seaweed that has been gaining traction for its many functional properties, including its ability to inhibit starch hydrolases. In this study, fucoidan was added into bread to lower the glycemic index of bread. Fucoidan fortification at 3.0% reduced the starch digestion rate of baked bread by 21.5% as compared to control baked bread. This translated to a 17.7% reduction in the predicted GI (pGI) with 3.0% of fucoidan. Fucoidan was retained in the bread after baking. Although the in vitro bioavailability of fucoidan was negligible, the in vitro bioaccessibility of fucoidan was high, at 77.1–79.8%. This suggested that although fucoidan may not be absorbed via passive diffusion, there is potential for the fucoidan to be absorbed via other modes of absorption. Thus, there is a potential for the use of fucoidan as a functional ingredient in bread to reduce the glycemic potential of bread.

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.