Enzymatic modification of stevioside by cell-free extract of Gibberella fujikuroi

Stevioside is a natural sweetener obtained from the leaves of Stevia rebaudiana. It is a glycoside of steviol and other glycosides of the same aglycone are also found in the plant. Stevioside is usually the major component in commercial products, but it is not the one with the best organoleptic properties. It has a bitter aftertaste and, for this reason, attempts have been made in order to modify its molecule. In this work, the commercial and purified stevioside were modified by hydrolytic enzymes from Gibberella fujikuroi. A screening was carried out on six strains of the fungus in order to select the most active. The production of the enzymes by the fungi was induced by its culture in a medium containing stevioside as the sole carbon source and the enzymatic extract was then used in the experiments. The products obtained were analyzed by HPLC-UV and HPLC-MS/MS. The results showed a significant increase in the concentration of rebaudioside A in the final product, which has better organoleptic properties than stevioside.     

甜菊醇糖苷生物合成关键基因的导入和鉴定分析

甜叶菊（Stevia rebaudiana Bertoni）生产的甜菊醇糖苷因具有高甜度、低热能、不参与人体内代谢兼具保健功能等特点,被誉为最有发展前途的新糖源。从甜叶菊叶片克隆了甜菊醇糖苷生物合成途径中的关键基因SrUGT85C2、SrUGT91D2m和SrUGT76G1,构建植物基因过量表达载体,以单独或组合的形式将这些基因导入到甜叶菊中,获得转基因植株。与野生型对照植株相比,单独导入SrUGT85C2的转基因植株中甜菊醇单糖苷含量提高,总糖苷、莱包迪苷A含量及占比没有明显变化;单独导入SrUGT91D2m的转基因植株中甜菊醇单糖苷含量显著降低,而甜菊醇双糖苷含量显著增加;单独导入SrUGT76G1的转基因植株中,总糖苷含量显著提高,莱包迪苷A含量达到10%以上,比对照提高了2倍,而甜菊糖苷含量减少了一半。3个基因组合同时导入的转基因甜叶菊植株与单独导入SrUGT76G1的转基因甜叶菊植株类似,其总糖苷、莱包迪苷A含量及其占比均显著提高。这些结果为以后通过分子生物学技术来调控甜菊醇糖苷生物合成关键基因的表达,培育莱包迪苷A含量高的高品质甜叶菊新品系提供了理论依据和技术方法。     

A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni)

The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 ara<>ispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia.     

In vitro screening of probiotic lactic acid bacteria and prebiotic glucooligosaccharides to select effective synbiotics

Probiotics and prebiotics have been demonstrated to positively modulate the intestinal microflora and could promote host health. Although some studies have been performed on combinations of probiotics and prebiotics, constituting synbiotics, results on the synergistic effects tend to be discordant in the published works. The first aim of our study was to screen some lactic acid bacteria on the basis of probiotic characteristics (resistance to intestinal conditions, inhibition of pathogenic strains). Bifidobacterium was the most resistant genus whereas Lactobacillus farciminis was strongly inhibited. The inhibitory effect on pathogen growth was strain dependent but lactobacilli were the most effective, especially L. farciminis. The second aim of the work was to select glucooligosaccharides for their ability to support the growth of the probiotics tested. We demonstrated the selective fermentability of oligodextran and oligoalternan by probiotic bacteria, especially the bifidobacteria, for shorter degrees of polymerisation and absence of metabolism by pathogenic bacteria. Thus, the observed characteristics confer potential prebiotic properties on these glucooligosaccharides, to be further confirmed in vivo, and suggest some possible applications in synbiotic combinations with the selected probiotics. Furthermore, the distinctive patterns of the different genera suggest a combination of lactobacilli and bifidobacteria with complementary probiotic effects in addition to the prebiotic ones. These associations should be further evaluated for their synbiotic effects through in vitro and in vivo models.     

Glucosylation of Steviol and Steviol-Glucosides in Extracts from Stevia rebaudiana Bertoni

To evaluate and characterize stevioside biosynthetic pathway in Stevia rebaudiana Bertoni cv Houten, two enzyme fractions that catalyze glucosylation of steviol (ent-13-hydroxy kaur-16-en-19-oic acid) and steviol-glucosides (steviol-13-O-glucopyranoside, steviolbioside and stevioside), utilizing UDP-glucose as the glucose donor, were prepared from the soluble extracts of S. rebaudiana leaves. Enzyme fraction I, passed through DEAE-Toyopearl equilibrated with 50 millimolar K-phosphate pH 7.5, catalyzed the glucosylation to steviol and 19-O-methylsteviol, but not to iso-steviol and 13-O-methylsteviol, indicating that 13-hydroxyl group of the steviol skeleton is glucosylated first from UDP-glucose to produce steviol-13-O-glucopyranoside. Enzyme fraction II, eluted from the DEAE-Toyopearl column with 0.15 molar KCI, catalyzed the glucose transfer from UDP-glucose to steviol-13-O-glucopyranoside, steviolbioside and stevioside, but not to rubusoside (13, 19-di-O-glucopyranoside) and rebaudioside A. The reaction products glucosylated from steviol-13-O-glucopyranoside, steviolbioside and stevioside were identified to be steviolbioside, stevioside and rebaudioside A, respectively. These results indicate that in the steviol-glucoside biosynthetic pathway, steviol-13-O-glucopyranoside produced from the steviol glucosylation is successively glucosylated to steviolbioside, then to stevioside producing rebaudioside A.     

NMR studies of the conformation of the natural sweetener rebaudioside A

Rebaudioside A is a natural sweetener from Stevia rebaudiana in which four β-d-glucopyranose units are attached to the aglycone steviol. Its ¹H and ¹³C NMR spectra in pyridine-d₅ were assigned using 1D and 2D methods. Constrained molecular dynamics of solvated rebaudioside using NMR constraints derived from ROESY cross peaks yielded the orientation of the β-d-glucopyranose units. Hydrogen bonding was examined using the temperature coefficients of the hydroxyl chemical shifts, ROESY and long-range COSY spectra, and proton-proton coupling constants.     

In vitro Effects of Prebiotics and Synbiotics on Apis cerana Gut Microbiota

This study aimed to investigate in vitro effects of the selected prebiotics alone, and in combination with two potential probiotic Lactobacillus strains on the microbial composition of Apis cerana gut microbiota and acid production. Four prebiotics, inulin, fructo-oligosaccharides, xylo-oligosaccharides, and isomalto-oligosaccharides were chosen, and glucose served as the carbon source. Supplementation of this four prebiotics increased numbers of Bifidobacterium and lactic acid bacteria while decreasing the pH value of in vitro fermentation broth inoculated with A. cerana gut microbiota compared to glucose. Then, two potential probiotics derived from A. cerana gut at different dosages, Lactobacillus helveticus KM7 and Limosilactobacillus reuteri LP4 were added with isomalto-oligosaccharides in fermentation broth inoculated with A. cerana gut microbiota, respectively. The most pronounced impact was observed with isomalto-oligosaccharides. Compared to isomalto-oligosaccharides alone, the combination of isomalto-oligosaccharides with both lactobacilli strains induced the growth of Bifidobacterium, LAB, and total bacteria and reduced the proliferation of Enterococcus and fungi. Consistent with these results, the altered metabolic activity was observed as lowered pH in in vitro culture of gut microbiota supplemented with isomalto-oligosaccharides and lactobacilli strains. The symbiotic impact varied with the types and concentration of Lactobacillus strains and fermentation time. The more effective ability was observed with IMO combined with L. helveticus KM7. These results suggested that isomalto-oligosaccharides could be a potential prebiotic and symbiotic with certain lactobacilli strains on A. cerana gut microbiota.     

Efficient enzymatic production of rebaudioside A from stevioside

Stevioside and rebaudioside A are the chief diterpene glycosides present in the leaves of Stevia rebaudiana. Rebaudioside A imparts a desirable sweet taste, while stevioside produces a residual bitter aftertaste. Enzymatic synthesis of rebaudioside A from stevioside can increase the ratio of rebaudioside A to stevioside in steviol glycoside products, providing a conceivable strategy to improve the organoleptic properties of steviol glycoside products. Here, we demonstrate the efficient conversion of stevioside to rebaudioside A by coupling the activities of recombinant UDP-glucosyltransferase UGT76G1 from S. rebaudiana and sucrose synthase AtSUS1 from Arabidopsis thaliana. The conversion occurred via regeneration of UDP-glucose by AtSUS1. UDP was applicable as the initial material instead of UDP-glucose for UDP-glucose recycling. The amount of UDP could be greatly reduced in the reaction mixture. Rebaudioside A yield in 30?h with 2.4?mM stevioside, 7.2?mM sucrose, and 0.006?mM UDP was 78%.     

Purification and some properties of a beta-glucosidase from Flavobacterium johnsonae

Flavobacterium johnsonae was isolated as a microorganism that produced a beta-glucosidase with hydrolytic activity of beta-glucosyl ester linkages in steviol glycosides. The enzyme was purified to homogeneity from a cell-free extract by streptomycin treatment, ammonium sulfate fractionation, and column chromatographies on S-Sepharose and phenyl-Toyopearl. The molecular mass of the purified enzyme was about 72 kDa by SDS-PAGE. An isoelectric point of pI 8.8 was estimated by isoelectric focusing. The enzyme was most active at pH 7.0, and was stable between pH 3.0 and 9.0. The optimum temperature was 45 degrees C, and the enzyme was stable below 35 degrees C. The enzyme hydrolyzed glucosyl ester linkages at site 19 of rebaudioside A, stevioside, and rubusoside, although it could not degrad beta-glucosidic linkages at site 13 of rebaudioside B or steviol bioside. The enzyme acted on aryl beta-glucosides such as p-nitrophenyl beta-glucoside, phenyl betaglucoside, and salicin, and glucobioses such as sophorose and laminaribiose. The enzyme activity on Rub was inactivated completely by Hg2+, and reduced by Fe3+, Cu2+, p-chloromercuric benzoate, and phenylmethylsulfonyl fluoride (residual activity; 67.9-84.8%). The pNPG hydrolysis was also inactivated to almost the same degrees. Kinetic behaviors in the mixed substrate reactions of rebaudioside A and steviol monoside, and of steviol monoglucosyl ester and phenyl beta-glucoside suggested the glucosidic and glucosyl ester linkages were hydrolyzed at a single active site of the enzyme.     

Dermal bioactives from lactobacilli and bifidobacteria

Lactobacilli and bifidobacteria are the most common genera of probiotics with documented potentials on gut health. Recent studies have suggested that such potentials can be extended beyond gut well-being, such as that of dermal health. Our present study aimed to evaluate the production of bioactives that are essential for skin defense, such as lipoteichoic acid, peptidoglycan, hyaluronic acid, sphingomyelinase, lactic acid, acetic acid, and diacetyl, from lactobacilli and bifidobacteria grown in milk. All strains studied showed the presence of LTA in the cell wall fraction, with higher amounts from Lactobacillus rhamnosus FTDC 8313 and Bifidobacterium longum BL 8643 than other strains studied. Meanwhile, all strains studied showed equal concentrations of cell wall peptidoglycan. Our results showed that all strains studied were capable of producing hyaluronic acid, with higher production by lactobacilli than bifidobacteria. Production of diacetyl was more prevalent from strains of lactobacilli, while bifidobacteria produced higher amounts of acetic acid. Strains of lactobacilli and bifidobacteria studied also produced acid and neutral sphingomyelinase, an enzyme that generates ceramides and subsequent development of physical barriers in the stratum corneum. Our current findings show that bioactive and inhibitive extracts are produced from the fermentation of lactobacilli and bifidobacteria in milk, with potentials for dermal applications.     

In vitro evaluation of the fermentation properties and potential prebiotic activity of Agave fructans

Aims: This study was carried out to evaluate in vitro the fermentation properties and the potential prebiotic activity of Agave‐fructans extracted from Agave tequilana (Predilife). Methods and Results: Five different commercial prebiotics were compared using 24‐h pH‐controlled anaerobic batch cultures inoculated with human faecal slurries. Measurement of prebiotic efficacy was obtained by comparing bacterial changes, and the production of short‐chain fatty acids (SCFA) was also determined. Effects upon major groups of the microbiota were monitored over 24 h incubations by fluorescence in situ hybridization. SCFA were measured by HPLC. Fermentation of the Agave fructans (Predilife) resulted in a large increase in numbers of bifidobacteria and lactobacilli. Conclusions: Under the in vitro conditions used, this study has shown the differential impact of Predilife on the microbial ecology of the human gut. Significance and Impact of the Study: This is the first study reporting of a potential prebiotic mode of activity for Agave fructans investigated which significantly increased populations of bifidobacteria and lactobacilli compared to cellulose used as a control.     

Growth of infant fecal bacteria on commercial prebiotics

Fecal bacteria from 33 infants (aged 1 to 6 months) were tested for growth on commercial prebiotics. The children were born vaginally (20) or by caesarean section (13). Bifidobacteria, lactobacilli, gram-negative bacteria, Escherichia coli, and total anaerobes in fecal samples were enumerated by selective agars and fluorescence in situ hybridization. The total fecal bacteria were inoculated into cultivation media containing 2 % Vivinal® (galactooligosaccharides—GOS) or Raftilose® P95 (fructooligosaccharides—FOS) as a single carbon source and bacteria were enumerated again after 24 h of anaerobic cultivation. Bifidobacteria dominated, reaching counts of 9–10 log colony-forming units (CFU)/g in 17 children born vaginally and in seven children delivered by caesarean section. In these infants, lactobacilli were more frequently detected and a lower number of E. coli and gram-negative bacteria were determined compared to bifidobacteria-negative infants. Clostridia dominated in children without bifidobacteria, reaching counts from 7 to 9 log CFU/g. Both prebiotics supported all groups of bacteria tested. In children with naturally high counts of bifidobacteria, bifidobacteria dominated also after cultivation on prebiotics, reaching counts from 8.23 to 8.77 log CFU/mL. In bifidobacteria-negative samples, clostridia were supported by prebiotics, reaching counts from 7.17 to 7.69 log CFU/mL. There were no significant differences between bacterial growth on Vivinal® and Raftilose® P95 and counts determined by cultivation and FISH. Prebiotics should selectively stimulate the growth of desirable bacteria such as bifidobacteria and lactobacilli. However, our results showed that commercially available FOS and GOS may stimulate also other fecal bacteria.     

Effect of nitrogen and phosphate on in vitro growth and metabolite profiles of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> Bertoni (Asteraceae)

The commercialization of Stevia rebaudiana Bertoni (Asteraceae) extracts as a natural sweetener is driving interest in the use of in vitro propagation systems as an alternative source of steviol glycosides. Out of this suite of chemicals, stevioside is the most abundant but rebaudioside A is the sweetest. We established an in vitro propagation method from germinated seedlings on a Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium with aims to study the effects of nitrogen and phosphate on the growth and metabolite profiles of S. rebaudiana plants. Generally, NH₄NO₃ is supplied at a concentration of 20.61 mM in MS medium and together with 18.79 mM KNO₃, provide nitrogen to in vitro growing plants. In this study, we used a range of 0.3–72.1 mM NH₄NO₃ and 9.4–65.8 mM KNO₃ and generated six different media with altered nitrogen. Similarly, six different concentrations of KH₂PO₄, ranging from 0.6 to 4.4 mM were tested for the phosphate treatments and the control medium had 1.25 mM KH₂PO₄. By reducing the nitrogen and phosphate levels to half, respectively, this led to the tallest plants. Increasing concentrations of nitrogen in the medium significantly lowered the amount of rebaudioside A as plants on the control medium accumulated 270 mg g^?1 rebaudioside A compared to those that were on a medium with 3.5 times the nitrogen supply (30 mg g^?1 rebaudiose A). Steviol increased with increasing nitrogen available to the microplants. The highest levels of stevioside (740 mg g^?1) quantified was linked to microplants on a medium with half the phosphate concentration. To further assess changes to the metabolomic profiles of treated microplants, LC–MS/MS was used in combination with multivariate statistical analyses. Two distinct clusters were revealed after principal component analysis. Steviol hydrate, stevioside hydrate and rebaudioside A contributed significantly to the separation of phosphate-treated plants from those with variable nitrogen concentrations. Chlorogenic acid and its derivatives were linked to changing phosphate concentrations. The clustering suggests different molecular mechanisms at play that are affected by nitrogen and phosphate supply which serve to alter secondary metabolic flux, resulting in different chemical profiles.     

A Human Volunteer Study on the Prebiotic Effects of HP-Inulin—Faecal Bacteria Enumerated Using Fluorescent In Situ Hybridisation (FISH)

An in vivo study was carried to determine the effect of HP-inulin, a high-molecular-weight fraction of chicory-derived inulin, on the human gut microflora composition. Ten healthy volunteers were allowed a free-living diet whereby they also ingested 8 g/d of maltodextrin for 14 days and this was followed by 8 g/d HP-inulin for 14 days. Nine of the ten volunteers completed the trial. The trial was conducted in a double blind manner and faeces were collected periodically such that predominant groups of gut bacteria i.e. total bacterial populations, Bacteroides spp., Bifidobacterium spp., Clostridium perfringens/histolyticum sub-group and lactobacilli/enterococci could be enumerated. To overcome difficulties with culture-based techniques, the bacteria were enumerated using fluorescent in situ hybridisation (FISH). A small but statistically significant increase in bifidobacteria was observed when data from the volunteers were pooled. Similarly, a statistically significant increase was observed in clostridial numbers, although the magnitude of change in this bacterial group was about ten times less than that seen with bifidobacteria. HP-inulin intake had little or no effect on numbers of total bacteria,Bacteroides spp., or lactobacilli and enterococci present in the gut microflora of the volunteers. This study has confirmed the prebiotic nature of HP-inulin. However, in this trial the effects were most marked in those volunteers with low starting levels of bifidobacteria—indicating that there may be a relationship between prebiotic effect and initial bifidobacterial numbers.     

Enrichment of the rebaudioside A concentration in Stevia rebaudiana extract with cyclodextrin glycosyltransferase from Bacillus licheniformis DSM 13

Stevia rebaudiana is a sweet herbaceous perennial plant, which is frequently used in the preparation of plant-based sweeteners. The demand for such sweeteners continues to increase due to purposeful nutrition and modern-day metabolic syndromes. More than 20 types of steviol glycosides provide a sweet taste, which are more than 300 times sweeter than sucrose. They are formed of two main components, namely stevioside and rebaudioside A. Only a handful of studies have dealt with Stevia rebaudiana leaf extracts, the conversion of pure stevioside into the preferred rebaudioside A is more common. The aim of this study was to enrich the rebaudioside A content of Stevia rebaudiana leaf extract using enzymatic bioconversion by applying fermented cyclodextrin glycosyltransferase from Bacillus licheniformis DSM13. Two differently processed plant materials, namely dried and lyophilized Stevia rebaudiana plants, were extracted and compared. Following the bioconversion, the rebaudioside A content was on average doubled. The maximum increase was fivefold with a 70–80% conversion of the stevioside.     

In Vitro Fermentation of Breast Milk Oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri

It has been proposed that human milk oligosaccharides (HMO) function as a prebiotic for bifidobacteria, yet this activity has not been adequately investigated. In this study, Bifidobacterium infantis was shown to ferment purified HMO as a sole carbon source, while another gut commensal, Lactobacillus gasseri, did not ferment HMO. Our results support the hypothesis that HMO selectively amplify bacterial populations in the infant intestine.     

Metabolism of prebiotic products containing beta(2-1) fructan mixtures by two Lactobacillus strains

The capacity of two probiotic strains, isolated from human breast milk, to use several beta(2-1) fructan mixtures as carbon and energy source in in vitro cultures has been tested. Results showed that both strains, Lactobacillus gasseri CECT5714 and Lactobacillus fermentum CECT5716, reached higher growth levels on culture media containing fructooligosaccharide mixtures produced by enzymatic synthesis, compared to those obtained by inulin hydrolysis. Furthermore, the shortest beta(2-1) fructan, kestose, was the only prebiotic compound in the mixtures significantly metabolized in all growth media tested. Analysis of short-chain fatty acid production showed no correlation between the fatty acid profile produced and the carbon source used in each experiment. These data could serve to select appropriate beta(2-1) fructans to be used as prebiotics for L. gasseri CECT5714 and L. fermentum CECT5716 and to design suitable symbiotic food products containing the mentioned lactobacilli.     

In vitro comparison of the prebiotic effects of two inulin-type fructans

Faecal cultures were used to compare the prebiotic effects of a new fructan containing high solubility inulin (HSI) and of a well-established prebiotic containing oligofructose (OF) with a negative control (CT). Changes in the intestinal microbiota, pH, ammonia, volatile organic acids and lactic acid were monitored during incubation. Molecular techniques for microbial enumeration indicated that both HSI and OF led to a significant increase in bifidobacteria (P< or =0.05) and lactobacilli (P< or =0.05) compared to the control. Significant changes in the pH and levels of ammonia with both inulin-type fructans were observed, as well as higher levels of acetic, lactic and formic acids (P< or =0.05). The fermentative metabolism appeared to be faster on OF than on HSI. Both OF and HSI showed clear prebiotic effects, but had differences in fermentation kinetics because of to the different degree of polymerization (DP). This study provides proof for the prebiotic effectiveness of HSI, and shows that inulin-type fructans with higher DP might have a prolonged bifidogenic effect, thus could extend the saccharolytic metabolism and low pH to the distal parts of the colon.     

甜菊糖苷的应用及生物合成研究进展*

甜菊糖苷是一种从甜叶菊叶片中提取的高甜度、零热量甜味剂,可用作食品添加剂。近年来,甜菊糖苷在国内外市场需求量剧增,引起了广泛关注。概述国际上对甜菊糖苷的安全性研究及评价,从经济价值角度分析甜菊糖苷的市场需求及应用前景,总结甜菊糖苷作为食品甜味添加剂的应用以及其在抗糖尿病、抗心脏纤维化、抗菌等保健功能方面的最新研究成果。综述甜菊糖苷的生物合成研究进展,重点介绍甜菊糖苷的微生物体从头合成以及生物催化低甜度糖苷生成高甜度、口感更优的甜菊糖苷,探讨提高甜菊糖苷产量的关键因素,为高端甜味剂绿色合成工艺的研究与开发提供理论依据。