Transcriptional Comparison Investigating the Influence of the Addition of Unsaturated Fatty Acids on Aroma Compounds During Alcoholic Fermentation

The levels of unsaturated fatty acids (UFAs) in grape must significantly influence yeast metabolism and the production of aroma compounds. In this work, cDNA microarray technology was applied to analyze the transcriptional discrepancies of wine yeast (commercial wine yeast Lalvin EC1118) fermenting in synthetic grape must supplemented with different concentrations of a mixture of UFAs (including linoleic acid, oleic acid, and α-linolenic acid). The results showed that the initial addition of a high level of UFAs can significantly enrich the intracellular UFAs when compared to a low addition of UFAs and further increase the cell population and most volatiles, including higher alcohols and esters, except for several fatty acids. Microarray analyses identified that 63 genes were upregulated, and 91 genes were downregulated during the different fermentation stages. The up-regulated genes were involved in yeast growth and proliferation, stress responses and amino acid transportation, while the repressed genes were associated with lipid and sterol biosynthesis, amino acid metabolism, TCA cycle regulation, mitochondrial respiration, and stress responses. Unexpectedly, the genes directly related to the biosynthesis of volatile compounds did not vary substantially between the fermentations with the high and low UFA additions. The beneficial aromatic function of the UFAs was ascribed to the increased biomass and amino acid transportation, considering that the incorporation of the additional UFAs in yeast cells maintains high membrane fluidity and increases the ability of the cells to resist deleterious conditions. Our results highlighted the importance of UFAs in the regulation of aroma biosynthesis during wine fermentation and suggested that the improvement of the resistance of yeast to extreme stresses during alcoholic fermentation is essential to effectively modulate and improve the production of aroma compounds. A potential way to achieve this goal could be the rational increase of the UFA contents in grape must

Essential Role of Mast Cells in the Visceral Hyperalgesia Induced by T. spiralis Infection and Stress in Rats

Mast cells (MCs) deficient rats (Ws/Ws) were used to investigate the roles of MCs in visceral hyperalgesia. Ws/Ws and wild control (+/+) rats were exposed to T. spiralis or submitted to acute cold restraint stress (ACRS). Levels of proteinase-activated receptor 2 (PAR2) and nerve growth factor (NGF) were determined by immunoblots and RT-PCR analysis, and the putative signal pathways including phosphorylated extracellular-regulated kinase (pERK1/2) and transient receptor potential vanilloid receptor 1 (TRPV1) were further identified. Visceral hyperalgesia triggered by ACRS was observed only in +/+ rats. The increased expression of PAR2 and NGF was observed only in +/+ rats induced by T. spiralis and ACRS. The activation of pERK1/2 induced by ACRS occurred only in +/+ rats. However, a significant increase of TRPV1 induced by T. spiralis and ACRS was observed only in +/+ rats. The activation of PAR2 and NGF via both TRPV1 and pERK1/2 signal pathway is dependent on MCs in ACRS-induced visceral hyperalgesia rats

Screening and verification of late embryogenesis abundant protein interacting with anthocyanidin reductase in grape berries

Anthocyanidin reductase (ANR, EC 1.3.1.77) catalyzes the conversion of anthocyanidins to 2R, 3R-flavan-3-ols in Vitis vinifera grapes. These are basic structural units of proanthocyanidins (PAs). The regulation of PA biosynthesis at protein level is not yet clear. Here, we find a VvANR interaction partner called late embryogenesis abundant protein (LEA), which can interact with VvANR in the yeast two-hybrid (Y2H) system. We verified the interaction between VvANR and Vv-LEA by Y2H and co-immunoprecipitation (CoIP) in yeast in vitro, and using the firefly luciferase complementation imaging (LCI) system in vivo. Additionally, the co-localization of VvANR and VvLEA in Arabidopsis thaliana protoplasts also provids the essential conditions for their interaction. The different expressions of VvANR and VvLEA depended both on the age of the grape berries and on the duration of cold treatment. These findings provide primary evidence for proteinregulation and the potential formation of multi-enzyme complex of VvANR, as well as of the PA biosynthesis.

湿性愈合理论在压疮治疗中的应用

Objective: to explore the effect of moist wound healing theory on pressure sores treatment. Methods: 60 cases of pressure sores were randomly divided into two groups: research group treated with moist wound healing theory of nursing, control group treated with traditional method of nursing and compared the curative effect between the two groups in 8 days. Result: The total effective rate was 93.3% in the research group and it was 40% in the control group. Therefore, the difference is statistically significant (P＜0.05). Healing time difference between the two groups was statistically significant (P <0.05). Conclusion: Moist wound healing theory is an effective treatment method of pressure sores with shorter healing time, and it also reduce the suffering of patients, shorter hospital stays and improve the curative rate of pressure sores, and is worthy of clinical application.目的  探讨应用湿性愈合理论治疗压疮的疗效。方法  将60例压疮患者随机分为观察组和对照组，观察组采用湿性愈合理论护理，对照组采用传统方法护理。8天后对两组压疮进行疗效观察。结果  观察组总有效率为93.3%，对照组总有效率为40%，差异有统计学意义（P＜0.05）。两组治愈时间比较差异有统计学意义（P＜0.05）。结论  临床上运用湿性愈合理论，疗效肯定、治愈时间短，能减轻患者痛苦，缩短住院时间，提高压疮治愈率，是治疗压疮非常有效的方法之一，值得临床推广应用

Accumulation Pattern of Flavonoids in Cabernet Sauvignon Grapes Grown in a Low-Latitude and High-Altitude Region

Particular climate conditions in a low-latitude and high-altitude region endow grape berries with distinctivequality characteristics. So far, few reports have been concerned with the formation of berry flavour in such aregion. This study aimed to investigate the accumulation pattern of flavonoids in Vitis vinifera L. cv. CabernetSauvignon grape berries growing at different altitudes of the highland in southwest China in two consecutivevintages. In addition to the 3-O-monoglucosides and 3-O-acyl monoglucosides of the five main anthocyanidins(delphinidin, cyanidin, peonidin, petunidin and malvidin), some uncommon anthocyanins, such as threediglucosides of anthocyanidins and pelargonidin-3-O-glucoside, were detected in the grape berries. Higheraltitude cultivation greatly promoted the production of anthocyanins and flavonols, particularly cyanidintypeanthocyanins and quercetin-type flavonols from the F3’H branch of the flavonoid biosynthetic pathway.Flavan-3-ols from both branches were comparatively less influenced by vineyard altitude. Vintage in thishigh-altitude region also had a dramatic influence on the accumulation of flavonoids. Most of the anthocyaninand flavonol components were affected more by vineyard altitude than by vintage, whereas the accumulationof flavan-3-ols differed mainly between vintages. The present data will not only improve the understandingof flavonoid accumulation in grapes from a high-altitude region with different climates, but also providepractical guidance for the production of high-quality grapes and wine

Preparation and biological application of antibodies against leucoanthocyanidin reductase and anthocyanidin reductase from grape berry

Proanthocyanidins (PAs) endow wine with the flavor of bitterness and astringency. Both leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) are two key enzymes of PA biosynthesis in grape berries, but the previous studies on these enzymes only focused on the transcriptional expression of these genes. Here, the full-length cDNAs of VvLAR1, VvLAR2 and VvANR, respectively, were cloned from wine grape berries and were then introduced into the pGEX-4T-1 expression vectors, which were highly expressed in Escherichia coli DH5α cells with the induction of the isopropyl-β-D-thiogalactoside (IPTG). The purified fusion proteins were used as the antigens to immunize rabbits, separately. The obtained antiserums were further purified to obtain the immunoglobulin G (Ig G) fractions, which were demonstrated to be capable of specifically immuno-recognizing the VvLAR1, VvLAR2 and VvANR from the crude protein extracts from grape berries with weight masses of approximately 43 kD. The analyses of translational expression of these enzyme genes during berry development and immunohistochemical localization of these proteins, by using the obtained antibodies, showed that a high amount of VvLAR1, VvLAR2 or VvANR was present at the pre-veraison stage and these enzyme proteins were all localized on the outer layer of the berry skin and the vascular bundle, as well as in the inner layer of the seed coat. This work provides an important basis for further studies on PA biosynthesis in grape berries.

Influences of Berry Size on Fruit Composition and Wine Quality of Vitis vinifera L. cv. ‘Cabernet Sauvignon’ Grapes

The heterogeneity of berry heterogeneity is a commonly occurring phenomenon that has a big influence on fruit composition and wine quality. To clarify this relationship, ‘Cabernet Sauvignon’ grapes were collected at harvest from a single vineyard and divided into three categories in two consecutive years: small (≤ 0.75 g), medium (0.76-1.25 g), and large (> 1.25 g). The medium berries were present in the highest frequency, accounting for more than 50% of the berry populations. The standard physicochemical parameters of the fruit were significantly affected by berry size. The relative skin mass and soluble solids contents, as well as total phenolic and anthocyanin concentrations, decreased with the berry size, while the relative seed mass, pH and malic acid content were positively correlated with berry weight. Accordingly, the wine composition also varied with berry size, as the wines made from small berries showed the highestalcohol and residual sugar content. CIELab parameters of the resulting wines showed the small berries were more desirable for making wine with a deeper and more saturated colour. With regard to volatile compounds, berry size showed a limited effect. Only 1-hexanol and laevo-2,3-butanediol showed consistent and significant trends across vintages for wine volatiles, which showed the highest levels in wines made from the small category of berries

The Effect of Cluster Position Determined by Vineyard Row Orientation on Grape Flavonoids and Aroma Profiles of Vitis vinifera L. cv. Cabernet Sauvignon and Italian Riesling in the North Foot of Tianshan Mountains

Vineyard row orientation plays a critical role in determining cluster microclimate. This study aimed tofigure out how cluster positions determined by vineyard row orientation affect grape flavonoids and aromaprofiles of Cabernet Sauvignon and Italian Riesling grapes. Three cluster positions (two canopy sidesand the inner canopy) of NS and EW oriented row Cabernet Sauvignon and NS oriented row ItalianRiesling were selected for the experiment. Microclimate data was monitored around clusters from bothcanopy sides of different row orientations. The south canopy side had higher daytime temperaturesand PAR than the north canopy side in EW row orientation. Flavonoids of grape skins and seeds wereseparated and determined by LC-MS, and aroma compounds of grape must were determined by GC-MS.Results showed that flavanols were affected by orientations, and EW orientation had higher berries skinflavanol concentration than NS orientation. EW-IN berries had fewer glucuronide form flavonols and3’-hydroxylated flavonols than the other two positions in EW orientation. Inner canopy berries had lowerflavonol concentration than other positions in EW orientation of CS and NS orientation of IR. To aromas,C6/C9 were the main compounds significantly affected by row orientations. EW orientation berries hadhigher C6 alcohols concentration such as (E)-3-Hexen-1-ol, (E)-2-Hexen-1-ol and (Z)-3-Hexen-1-ol thanNS orientation, while NS orientation berries had higher C6 aldehyde concentrations such as Hexanal and(E)-2-hexenal than EW orientation. EW-IN berries had more abundant C6/C9 compounds than the othertwo positions. The study provided preliminary scientific evidence for vineyard viticulture practice andharvest strategy

Use of Indigenous Hanseniaspora vineae and Metschnikowia pulcherrima Co-fermentation With Saccharomyces cerevisiae to Improve the Aroma Diversity of Vidal Blanc Icewine

Using novel non-Saccharomyces strains is regarded as an effective way to improve the aroma diversity of wines to meet the expectations of consumers. The non-Saccharomyces Hanseniaspora vineae and Metschnikowia pulcherrima have good aromatic properties useful for the production of table wine. However, no detailed information is available on their performances in icewine fermentation. In this study, simultaneous and sequential fermentation trials of indigenous M. pulcherrima CVE-MP20 or H. vineae CVE-HV11 with S. cerevisiae (SC45) were performed in 50-L fermenters of Vidal icewine, respectively. The results showed that SC45 cofermented with different non-Saccharomyces strains could generate a distinct aroma quality of icewine compared with four S. cerevisiae strain monocultures as evidenced by principal component analysis (PCA). Mixed fermentation of MP20/SC45 produced higher contents of acetate esters and β-damascenone with lower C6 alcohols relative to SC45 monoculture. Interestingly, HV11/SC45 generated the highest amounts of C6 alcohols [(Z)-3-hexen-1-ol and (E)-3-hexen-1-ol], higher alcohols (isobutanol, isopentanol, and 2-phenylethanol), acetate esters (2-phenethyl acetate and isoamyl acetate), cis-rose oxide, β-damascenone, and phenylacetaldehyde. Compared with simultaneous inoculation, sequential inoculation could achieve higher aroma diversity and produce higher intensity of fruity, flowery, and sweet attributes of icewine as assessed by calculating the odor activity values (OAVs). Our results verified the desired enological characteristics of H. vineae strain in icewine fermentation and also demonstrated that using indigenous non-Saccharomyces and Saccharomyces strains is a feasible way to improve aroma diversity of icewine products, which could provide an alternative way to meet the requirement of wine consumers for diversified aromatic quality