Metabolomics and HS-SPME-GC-MS-based analysis of quality succession patterns and flavor characteristics changes during the fermentation of Lycium barbarum and Polygonatum cyrtonema compound wine.

This work set out to investigate how the physicochemical markers, volatiles, and metabolomic characteristics of mixed fermented the fermentation of Lycium barbarum and Polygonatum cyrtonema compound wine (LPCW) from S. cerevisine RW and D. hansenii AS2.45 changed over the course of fermentation. HS-SPME-GC-MS combined with non-targeted metabolomics was used to follow up and monitor the fermentation process of LPCW. In total, 43 volatile chemical substances, mostly alcohols, esters, acids, carbonyl compounds, etc., were discovered in LPCW. After 30 days of fermentation, phenylethyl alcohol had increased to 3045.83 g/mL, giving off a rose-like fresh scent. The biosynthesis of valine, leucine, and isoleucine as well as the metabolism of alanine, aspartic acid, and glutamic acid were the major routes that led to the identification of 1385 non-volatile components in total. This study offers a theoretical foundation for industrial development and advances our knowledge of the fundamental mechanism underlying flavor generation during LPCW fermentation.

Volatilome of brine-related microorganisms in a curd-based medium.

The possible contribution of brine-derived microflora to the sensory attributes of cheese is still a rather unexplored field. In this study, 365 bacteria and 105 yeast strains isolated from 11 cheese brines were qualitatively tested for proteolytic and lipolytic activities, and positive strains were identified by sequencing. Among bacteria, Staphylococcus equorum was the most frequent, followed by Macrococcus caseolyticus and Corynebacterium flavescens. As for yeasts, Debaryomyces hansenii, Clavispora lusitaniae, and Torulaspora delbrueckii were most frequently identified. A total of 38% of bacteria and 59% of yeasts showed at least 1 of the metabolic activities tested, with lipolytic activity being the most widespread (81% of bacteria and 95% of yeasts). Subsequently 15 strains of bacteria and 10 yeasts were inoculated in a curd-based medium and assessed via headspace-solid phase microextraction coupled with gas chromatography-mass spectrometry to determine their volatilome. After a 30-d incubation at 12°C, most strains showed a viability increase of about 2 log cfu/mL, suggesting good adaptability to the cheese environment. A total of 26 compounds were detected in the headspace, carbonyl compounds and alcohols being the major contributors to the volatile profile of the curd-based medium. Multivariate analysis was carried out to elucidate the overall differences in volatiles produced by selected strains. Principal component analysis and hierarchical clustering analysis demonstrated that the brine-related microorganisms were separated into 3 different groups, suggesting their different abilities to produce volatile compounds. Some of the selected strains have been shown to have interesting aromatic potential and to possibly contribute to the sensory properties of cheese.

Production of flavor compounds from rice bran by yeasts metabolisms of Kluyveromyces marxianus and Debaryomyces hansenii.

The aim of this study was to evaluate the biosynthesis of flavor compounds from rice bran by fermentation facilitated by Kluyveromyces marxianus and Debaryomyces hansenii. The growth of both yeasts was assessed by specific growth rates and doubling time. The biosynthesis of flavor compounds was evaluated by gas chromatography-olfactometry (GC-O), gas chromatography-mass spectrometry (GC-MS), and Spectrum™ sensory analysis. The specific growth rate (µ) and doubling time (td) of K. marxianus was calculated as 0.16/h and 4.21h, respectively, whereas that of D. hansenii was determined as 0.13/h and 5.33h, respectively. K. marxianus and D. hansenii produced significant levels of higher alcohols and acetate esters from rice bran. Results showed that K. marxianus can produce 827.27 µg/kg of isoamyl alcohol, 169.77 µg/kg of phenyl ethyl alcohol, and 216.08 µg/kg of phenyl ethyl acetate after 24-h batch fermentation. A significant amount of isovaleric acid was also synthesized by K. marxianus (4013 µg/kg) after the batch fermentation of 96 h. 415.64 µg/kg of isoamyl alcohol and 135.77 µg/kg of phenyl ethyl acetate was determined in rice bran fermented by D. hansenii after 24-h fermentation. Fermented cereals and rose were the characteristic flavor descriptors of the fermented rice bran samples. Rose flavor in fermented rice bran samples was found to be associated with phenyl ethyl alcohol, phenyl ethyl acetate, isoamyl acetate, and guaiacol. Thus, the findings of this study demonstrate that the valorization of rice bran can be achieved with the production of natural flavor compounds by yeast metabolism.

The faecal metabolome and mycobiome in Parkinson's disease.

BACKGROUND: Gut fungal composition and its metabolites have not been assessed simultaneously in Parkinson's disease (PD) despite their potential pathogenic contribution. OBJECTIVE: To evaluate the faecal metabolome and mycobiome in PD by assessing volatile organic compounds (VOCs) and fungal rRNA. METHODS: Faecal VOCs from 35 PD patients and two control groups (n = 35; n = 15) were assessed using gas chromatography and mass spectrometry. DNA was extracted from 44 samples: 18S rRNA gene amplicons were prepared and sequenced. Metabolomics, mycobiome and integrated analyses were performed. RESULTS: Several VOCs were more abundant and short chain fatty acids were less abundant in PD. Hanseniaspora, Kazachstania, uncultured Tremellaceae and Penicillium genera were more abundant, and Saccharomyces less abundant in PD (FDR<0.0007). Torulaspora was associated with PD and two VOCs. CONCLUSION: PD patients had a distinct metabolome and mycobiome suggesting that fungal dysbiosis may contribute to PD pathogenesis.

Investigating Bacterial Volatilome for the Classification and Identification of Mycobacterial Species by HS-SPME-GC-MS and Machine Learning.

Species of Mycobacteriaceae cause disease in animals and humans, including tuberculosis and leprosy. Individuals infected with organisms in the Mycobacterium tuberculosis complex (MTBC) or non-tuberculous mycobacteria (NTM) may present identical symptoms, however the treatment for each can be different. Although the NTM infection is considered less vital due to the chronicity of the disease and the infrequency of occurrence in healthy populations, diagnosis and differentiation among Mycobacterium species currently require culture isolation, which can take several weeks. The use of volatile organic compounds (VOCs) is a promising approach for species identification and in recent years has shown promise for use in the rapid analysis of both in vitro cultures as well as ex vivo diagnosis using breath or sputum. The aim of this contribution is to analyze VOCs in the culture headspace of seven different species of mycobacteria and to define the volatilome profiles that are discriminant for each species. For the pre-concentration of VOCs, solid-phase micro-extraction (SPME) was employed and samples were subsequently analyzed using gas chromatography-quadrupole mass spectrometry (GC-qMS). A machine learning approach was applied for the selection of the 13 discriminatory features, which might represent clinically translatable bacterial biomarkers.

Fungal community succession and volatile compound dynamics in Harbin dry sausage during fermentation.

This study investigated the fungal community succession and volatile compound dynamics of Harbin dry sausage during a twelve-day fermentation using high-throughput internal transcribed spacer amplicon sequencing and headspace solid-phase microextraction gas chromatography-mass spectrometry. Aspergillus pseudoglaucus was found to be the primary species in the sausages during fermentation, whereas Lasiodiplodia theobromae, Alternaria alternata, Aspergillus caesiellus, and Trichosporon asahii were also prevalent. Additionally, a total of 72 volatile compounds were identified in the dry sausages, of which 24 key compounds (odor activity value > 1) dominated flavor development, including 3 aldehydes, 1 ketone, 4 alcohols, 9 esters, 4 alkenes, and 3 other compounds. Furthermore, correlation analysis suggested that most of the core fungi were positively correlated with the key volatile compounds, particularly A. pseudoglaucus, Aspergillus gracilis, Trichosporon caseorum, Debaryomyces hansenii, and T. asahii. Our findings provide novel insights into the fungal ecology and flavor development of Harbin dry sausages.

The Impact of Hanseniaspora vineae Fermentation and Ageing on Lees on the Terpenic Aromatic Profile of White Wines of the Albillo Variety.

Hanseniaspora vineae is a non-Saccharomyces yeast that has a powerful impact on the sensory profile of wines. Its effect on the aromatic profile of non-aromatic grape varieties, such as Albillo Mayor (Vitis vinifera, L), during vinification is a useful biotechnology to improve sensory complexity. Fermentation in steel barrels using Hanseniaspora vineae and sequential inoculation with Saccharomyces cerevisiae have been used to study the formation of terpenes and cell lysis in the production of Albillo white wines. The GC-MS analysis profile shows a significant effect of H. vineae fermentation on the contents of terpenes (≈×3), mainly in linalool (>×3), ß-citronellol (>×4), geraniol (>×2) and α-terpineol (≈×2). The contents of several polyoxygenated terpenes and some volatile phenols with a spicy aroma were increased during fermentation. In summary, Hanseniaspora vineae releases a large number of cell wall polysaccharides during fermentation that affect wine palatability and structure. Hanseniaspora vineae is a powerful bio-tool to enhance the fruitiness, floral notes and freshness in non-aromatic white varieties.

High Voltage Electrical Discharges as an Alternative Extraction Process of Phenolic and Volatile Compounds from Wild Thyme (Thymus serpyllum L.): In Silico and Experimental Approaches for Solubility Assessment.

The objective of this study was to evaluate the potential of green solvents for extractions of bioactive compounds (BACs) and essential oils from wild thyme (Thymus serpyllum L.) using theoretical and experimental procedures. Theoretical prediction was assessed by Hansen solubility parameters (HSPs) and conductor-like screening model for realistic solvents (COSMO-RS), to predict the most suitable solvents for extraction of BACs. An experimental procedure was performed by nonthermal technology high voltage electrical discharge (HVED) and it was compared with modified conventional extraction (CE). Obtained extracts were analyzed for chemical and physical changes during the treatment. Theoretical results for solution of BACs in ethanol and water, as green solvents, were confirmed by experimental results, while more accurate data was given by COSMO-RS assessment than HSPs. Results confirmed high potential of HVED for extraction of BACs and volatile compounds from wild thyme, in average, 2.03 times higher yield of extraction in terms of total phenolic content was found compared to CE. The main phenolic compound found in wild thyme extracts was rosmarinic acid, while the predominant volatile compound was carvacrol. Obtained extracts are considered safe and high-quality source reach in BACs that could be further used in functional food production.

Analysis of phthalate plasticizer migration from PVDC packaging materials to food simulants using molecular dynamics simulations and artificial neural network.

Based on the experimental data of gas chromatography-mass spectrometry, an improved artificial neural network was first established to predict the migration of 2-ethylhexyl phthalate (DEHP) plasticizer from poly(vinylidene chloride) (PVDC) into food simulants (ie., heptane, ethanol and water). The sensitivity analysis indicated that temperature acted as a crucial factor influencing the migration values of DEHP. Then, a combined experimental and molecular dynamic (MD) simulation was performed to understand the migration kinetics and the mechanism of DEHP. Hansen solubility parameters of three component (δd, δp, δh) were simplified into two-component solubility parameters (δvdW, δe), and the tuple was successfully applied to describe the interactions between PVDC and food simulants. The MD results showed that high interaction energy and fractional free volume in PVDC/DEHP/food simulant systems accelerated the migration of DEHP. These fundamental studies would provide significant insights into the migration of environmental contaminants.

A sustainable approach for the extraction of cholesterol-lowering compounds from an olive by-product based on CO2-expanded ethyl acetate.

Olive (Olea europaea) processing results in large amounts of by-products that contain valuable molecules such as phenolic compounds and phytosterols. These molecules have demonstrated to reduce blood cholesterol levels. This work proposes the development of a method to obtain simultaneously phenolic compounds and phytosterols from the olive stone using CO2-expanded liquid extraction. Hansen solubility parameters were employed for the theoretical prediction of the most suitable bio-based solvent to extract target compounds. The Box-Behnken experimental design was employed to select the optimal conditions of pressure (8-25 MPa), the molar fraction of CO2 in ethyl acetate (0.15-0.55), and the temperature (40-80 °C). Extracts showing the highest and the lowest reductions of micellar cholesterol solubility capacity were analyzed by gas chromatography coupled to mass spectrometry to find out the compounds responsible for this activity. Different phenolic compounds, free fatty acids, and phytosterols were identified in the extracts. ß-Sitosterol and, especially, tyrosol and hydroxytyrosol were the compounds that primarily contributed to the reduction of micellar cholesterol solubility capacity.

Gas chromatography-mass spectrometry of sapucainha oil (Carpotroche brasiliensis) triacylglycerols comprising straight chain and cyclic fatty acids.

Sapucainha oil, which may be used to treat leprosy, comprises straight chain and cyclic fatty acids (FA), and triacylglycerols (TAG). The FA and TAG content of the oil sample was analysed using gas chromatography-electron ionisation mass spectrometry (GC-EIMS). FA analysis was performed after derivatisation to fatty acid methyl esters (FAME). For free FA and TAG analysis, the oil sample was dissolved in hexane and injected into a short, high-temperature column, for GC with MS analysis. Free FA and FAME were tentatively identified based on mass spectrum information of their molecular and fragment ions, as well as library matching. Overlapping TAG peaks were deconvoluted based on mass fingerprint data. The FA composition was utilised to predict possible TAG identities. FA residues of TAG were identified based on characteristic fragment ions, such as [M-RCO2]+, [RCO+128]+, [RCO+74]+ and RCO+ where R is the aliphatic hydrocarbon chain. FAME analysis showed that the cyclic FA hydnocarpic (36.1%), chaulmoogric (26.5%) and gorlic (23.6%) acids were the major components. In addition, straight chain FA such as palmitic, palmitoleic, stearic, oleic and linoleic acids were detected. Palmitic, oleic, hydnocarpic, chaulmoogric and gorlic acids were also detected as free FA in the oil sample. Six groups of TAG peaks were eluted from GC at temperatures ≥330 °C. After deconvolution and mass spectrum analysis, each TAG peak group was revealed to comprise 2 to 5 co-eluted TAG molecules; >18 TAG were identified. These TAG consisted of a mix of both cyclic and straight chain FA, but were mostly derived from cyclic FA.

A multi-analytical platform based on pressurized-liquid extraction, in vitro assays and liquid chromatography/gas chromatography coupled to high resolution mass spectrometry for food by-products valorisation. Part 1: Withanolides-rich fractions from goldenberry (Physalis peruviana L.) calyces obtained after extraction optimization as case study.

In this work, a multi-analytical platform that allows obtaining and characterizing high-added value compounds from natural sources is presented, with a huge potential in traditional medicine, natural products characterization, functional foods, etc. Namely, the proposed multi-analytical platform is based on the combination of pressurized liquid extraction (PLE), liquid chromatography (LC) and gas chromatography quadrupole time-of-flight mass spectrometry GC-q-TOF-MS(/MS), in vitro assays and modelling tools for guiding extraction optimization. As case study, goldenberry or cape gooseberry fruit (Physalys peruviana L.) was selected. In particular, the potential of P. peruviana calyces, an important by-product of goldenberry processing, as promising source of bioactive compounds was evaluated. Selection of the most suitable solvent for PLE was based on the Hansen solubility parameters (HSP) approach using 4ß-hydroxywithanolide E (4ßHWE) and withanolide E (WE) as target compounds due to their bioactive potential. A surface response methodology was further applied for the optimization of the PLE parameters: temperature (50, 100 and 150 °C) and solvent composition (% EtOH in the mixture EtOH/EtOAc). The effects of the independent variables on extraction yield, withanolides content (4ßHWE and WE), total phenolic content (TPC), total flavonoids content (TFC) and antioxidant activity (EC50 and TEAC) were evaluated in order to obtain withanolide-rich extracts from P. peruviana calyces. The extract obtained under optimal conditions (at 125 °C and 75% EtOH v/v) exhibited satisfactory extraction yield (14.7%) and moderate antioxidant activity (with an EC50 value of 77.18 µg mL-1 and 1.08 mM trolox g-1), with 4ßHWE and WE concentrations of 8.8 and 2.3 mg g-1, respectively. LC-q-TOF-MS/MS analysis of the extract allowed the quantitation of 4ßHWE and WE and the tentative identification of several other withanolides structures. The obtained results demonstrate the great potential of this multi-analytical approach for developing valorisation strategies of food by-products under sustainable conditions, to obtain bioactive-enriched extracts with potential medicinal or health-promoting properties.

Aroma modulation of Cabernet Gernischt dry red wine by optimal enzyme treatment strategy in winemaking.

Cabernet Gernischt (CG) is a famous Chinese wine grape cultivar, the red wine of which is known for its green trait, especially when produced from grapes cultivated in regions with monsoon climate. To modify CG wine aroma, three enzyme preparations (H. uvarum extracellular enzyme, AR2000, and pectinase) were introduced in different winemaking stages with Saccharomyces cerevisiae. Free and bound aroma compounds in young wines were detected using headspace solid-phase micro-extraction and gas chromatography-mass spectrometry, and aroma characteristics were quantified by trained panelists. Results showed that simultaneous inoculation of enzymes and yeasts improved wine aroma. Partial least-squares regression revealed that the green trait was due mainly to varietal compounds, especially C6 compounds, and could be partly weakened by fermentative compounds. Moreover, H. uvarum enzyme treatments enriched the acid fruit note of CG wine by enhancing the synergistic effect of varietal volatiles and certain fermentative compounds, such as esters and phenylethyls.

Untargeted GC-MS Metabolomics Reveals Changes in the Metabolite Dynamics of Industrial Scale Batch Fermentations of Streptoccoccus thermophilus Broth.

An industrial scale biomass production using batch or fed-batch fermentations usually optimized by selection of bacterial strains, tuning fermentation media, feeding strategy, and temperature. However, in-depth investigation of the biomass metabolome during the production may reveal new knowledge for better optimization. In this study, for the first time, the authors investigated seven fermentation batches performed on five Streptoccoccus thermophilus strains during the biomass production at Chr. Hansen (Denmark) in a real life large scale fermentation process. The study is designed to investigate effects of batch fermentation, fermentation time, production line, and yeast extract brands on the biomass metabolome using untargeted GC-MS metabolomics. Processing of the raw GC-MS data using PARAFAC2 revealed a total of 90 metabolites out of which 64 are identified. Partitioning of the data variance according to the experimental design was performed using ASCA and revealed that batch and fermentation time effects and their interaction term were the most significant effects. The yeast extract brand had a smaller impact on the biomass metabolome, while the production line showed no effect. This study shows that in-depth metabolic analysis of fermentation broth provides a new tool for advanced optimization of high-volume-low-cost biomass production by lowering the cost, increase the yield, and augment the product quality.

Hanseniaspora uvarum prolongs shelf life of strawberry via volatile production.

Gray mold caused by Botrytis cinerea led to severe postharvest losses for strawberry industry. In recent years, some studies have shown that postharvest diseases of strawberry can be controlled by using bacterial, fungal and yeast strains. The yeast strain Hanseniaspora uvarum was shown as an effective antagonist against B. cinerea growth. Here, we further investigated the volatile organic compounds (VOCs) production of H. uvarum and how this could impact on postharvest gray mold control of strawberry. A total of 28 VOCs were detected by GC-MS in the headspace of H. uvarum and strawberry with/without B. cinerea (SI and RSI ≥800). Among these VOCs, 15 VOCs were detected in both conditions, 4 VOCs were H. uvarum and strawberry without B. cinerea and the other 9 VOCs were only detected when B. cinerea was inoculated. Two VOCs, ethyl acetate and 1,3,5,7-cyclooctatetraene, enhanced by inoculation of B. cinerea. In in vitro assay, H. uvarum significantly inhibited mycelial growth and spore germination of B. cinerea via VOCs production. Moreover, in vivo assay showed that H. uvarum reduced B. cinerea infection of strawberry and maintained fruit appearance, firmness and total soluble solids via VOCs production. Collectively, our results showed that H. uvarum VOCs significantly controlled postharvest gray mold of strawberry and prolonged the storage time and shelf life.

Pineapple (Ananas comosus L. Merr.) wine production in Angola: Characterisation of volatile aroma compounds and yeast native flora.

A pineapple vinification process was conducted through inoculated and spontaneous fermentation to develop a process suitable for a quality beverage during two successive vintages in Huambo, Angola. Wines obtained with the conventional Saccharomyces cerevisiae strain, were analysed by gas chromatography, and a total of 61 volatile constituents were detected in the volatile fraction and 18 as glycosidically bound aroma compounds. Concentration levels of carbonyl and sulphur compounds were in agreement with the limited information reported about pineapple fruits of other regions. We report, for the first time in pineapple wines, the presence of significant concentrations of lactones, ketones, terpenes, norisoprenoids and a variety of volatile phenols. Eight native yeast strains were isolated from spontaneous batches. Further single-strain fermentations allowed us to characterise their suitability for commercial fermentation. Three native strains (Hanseniaspora opuntiae, H. uvarum and Meyerozyma guilliermondii) were selected with sensory potential to ferment pineapple fruits with increased flavour diversity. Results obtained here contribute to a better understanding of quality fermentation alternatives of this tropical fruit in subtropical regions.

Effect of yeast assimilable nitrogen on the synthesis of phenolic aroma compounds by Hanseniaspora vineae strains.

In several grape varieties, the dominating aryl alkyl alcohols found are the volatile group of phenylpropanoid-related compounds, such as glycosylated benzyl and 2-phenylethyl alcohol, which contribute to wine with floral and fruity aromas after being hydrolysed during fermentation. Saccharomyces cerevisiae is largely recognized as the main agent in grape must fermentation, but yeast strains belonging to other genera, including Hanseniaspora, are known to predominate during the first stages of alcoholic fermentation. Although non-Saccharomyces yeast strains have a well-recognized genetic diversity, understanding of their impact on wine flavour richness is still emerging. In this study, 11 Hansenisapora vineae strains were used to ferment a chemically defined simil-grape fermentation medium, resembling the nutrient composition of grape juice but devoid of grape-derived secondary metabolites. GC-MS analysis was performed to determine volatile compounds in the produced wines. Our results showed that benzyl alcohol, benzyl acetate and 2-phenylethyl acetate are significantly synthesized by H. vineae strains. Levels of these compounds found in fermentations with 11 H. vineae different strains were one or two orders of magnitude higher than those measured in fermentations with a known S. cerevisiae wine strain. The implications for winemaking in response to the negative correlation of benzyl alcohol, benzyl acetate and 2-phenylethyl acetate production with yeast assimilable nitrogen concentrations are discussed. Copyright © 2016 John Wiley & Sons, Ltd.

Bioflavour production from tomato and pepper pomaces by Kluyveromyces marxianus and Debaryomyces hansenii.

Bioflavours are called natural flavour and/or fragrance compounds which are produced using metabolic pathway of the microorganism and/or plant cells or their enzyme systems with bioengineering approaches. The aim of this study was to investigate bioflavour production from tomato and red pepper pomaces by Kluyveromyces marxianus and Debaryomyces hansenii. Obtained specific growth rates of K. marxianus and D. hansenii in tomato pomace were 0.081/h and 0.177/h, respectively. The bioflavour profile differed between the yeasts. Both yeasts can produce esters and alcohols such as phenyl ethyl alcohol, isoamyl alcohol, isoamyl acetate, phenyl ethyl acetate and isovaleric acid. "Tarhana" and "rose" were descriptive flavour terms for tomato and pepper pomaces fermented by K. marxianus, respectively. Tomato pomace fermented by D. hansenii had the most intense "green bean" flavour while "fermented vegetable" and "storage/yeast" were defined as characteristic flavour terms for pepper pomaces fermented by D. hansenii.

Study of the volatile compounds produced by Debaryomyces hansenii NRRL Y-7426 during the fermentation of detoxified concentrated distilled grape marc hemicellulosic hydrolysates.

The volatile compounds produced by Debaryomyces hansenii NRRL Y-7426 during the fermentation of detoxified concentrated distilled grape marc hemicellulosic hydrolysates was analysed by GC-MS. Thirty-five compounds corresponding to ten groups of volatile compounds: terpenes, higher alcohols, C6 alcohols, aldehydes, volatile acids, acetates, ethyl esters, volatile phenols, sulphur compounds and hydrocarbons were identified. The supplementation with commercial nutrients increased the concentration of 2-phenylethanol, a commercial flavour and fragrance compound, with a rose-like odour, employed in cosmetics and food industries; and other positive compounds to the aroma such as terpenes and ethyl esters. Non-supplemented media produced the highest content in higher alcohols, volatile acids, sulphur compounds and in the majority of hydrocarbons detected, meanwhile supplementation with vinasses hardly produced volatile compounds. Only four volatile compounds contributed directly to the aroma according to the OAVs values higher than 1. Finally, a PCA analysis allowed accounting for 100 % of the variance.