Production Of Volatile Aroma Compounds Research Articles

Molecular cloning, expression, and biochemical characterization of carotenoid cleavage dioxygenase 1 (LbCCD1) from Lycium barbarum

Carotenoid cleavage dioxygenases (CCDs) play a pivotal role in the biosynthesis of volatile apocarotenoids, which have significant industrial applications due to their aromatic and bioactive properties. This study focuses on the molecular cloning and biochemical characterization of the LbCCD1 from Lycium barbarum. The LbCCD1 gene was successfully cloned and heterologously expressed in Escherichia coli and several carotenoids were using as substrates for investigate its specificity by in vitro and in vivo experiment. The LbCCD1 protein was able to cleave a variety of carotenoids including β-carotene, zeaxanthin, astaxanthin, and β-apo-8′-carotenal, at the 9, 10 (9′, 10′) double bond to produce β-ionone, 3‑hydroxy-4-oxo-β-ionone, and 3‑hydroxy-β-ionone, respectively in vitro. LbCCD1 could also cleave zeaxanthin and β-carotene at the 9, 10 (9′, 10′) double bond to produce β-ionone, respectively, in E. coli accumulating carotenoids. Interestingly, LbCCD1 did not exhibit cleavage activity on lycopene either in vivo or in vitro unlike other CCD1 family enzymes.In the previous experiment, it was confirmed that LbCCD1 exhibits cleavage activity towards β-apo-8′-carotenal in vitro, so we used β-apo-8′-carotenal as the substrate for characterizing the enzymatic properties. The expression of LbCCD1 was optimized at such conditions (temperature 24 °C, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of LbCCD1 revealed the optimal activities were at pH 9 and 55 °C. The addition of 10 % ethanol could increase enzyme activity to above 15 %. However, the concentration of Fe2+ has a minimal effect on enzyme activity. The Vmax for β-apo-8′-carotenal was 8.6 U/mg, while the Km was 0.27 mM. To preliminarily verify the potential of LbCCD1 as a biological component for β-ionone production. By introducing LbCCD1 into the β-carotene-high-producing chassis cell and optimizing the conditions (temperature 30 °C, IPTG 0.01 mM, Fe2+ concentration 0.05 mM), the β-ionone yield reached 21.45 mg/L. This study focused on one of the CCDs derived from woody plants, which have been relatively underexplored. It lays the groundwork for expanding the CCD enzyme library, identifying suitable CCDs, and investigating the structure-function relationship of CCDs. Furthermore, it sets the stage for engineering novel CCD genes and developing advanced applications of CCDs as biocatalysts and platforms for synthetic biology. These advancements will enable the efficient production of volatile aroma compounds from carotenoids.

OBSERVATION OF THE SUITABILITY OF SINGLE STRAINS OF STREPTOCOCCUS THERMOPHILUS AND LACTOBACILLUS DELBRUECKII SUBSP. BULGARICUS ISOLATED FROM LOCAL DAIRY SOURCES IN TURKEY AS YOGURT STARTER COMBINATIONS

The present study investigated the suitability of combinations one Streptococcus thermophilus and five Lactobacillus delbrueckii subsp. bulgaricus isolates for industrial yogurt productions. The isolates were selected after a detailed pre-screening procedure based on acidification performances, production of volatile aroma compounds, texture developing properties and sensory evaluations of yogurts made from single cultures. The fermentation profiles of the combinations were investigated by monitoring the small oscillatory rheological parameters and pH reductions during fermentation. The variations in some physical, chemical and organoleptic characteristics of the yogurt samples were also monitored during cold storage at 4 ºC for 21 days. Results indicated that all the combinations reduced the milk acidity to pH 4.6 within 4-5 hour at 43 ºC. Gelation profiles of the samples were close to each other (close tan δ values) with different elastic (G') and loss (G") moduli, indicating that similar chemical bondings took place in gel formation with different levels. All the samples showed a shear-thinning non-Newtonian flow behavior. Total number of starter bacteria was higher than 6 log cfu/mL in each sample after 21 days of storage. The post-acidification rates were also limited (between 4.21-4.37 after 21 days of cold storage). Untrained sensory evaluation panel found no marginal differences between the samples regarding appearance, body and texture, and aroma and flavor of the samples. To conclude, the combinations examined in this study were found to be suitable for commercial yogurt productions.

The Consumption of Amino Acids and Production of Volatile Aroma Compounds by Yarrowia lipolytica in Brewers’ Wort

The yeast Yarrowia lipolytica is well known for its versatile production of metabolites from various substrates, but, although isolated from, e.g., wild-fermented Belgian Sour beers, it is rarely considered a starter culture in fermented beverages. In this study, we aimed to elucidate the ability of Y. lipolytica to ferment brewers’ wort containing iso-α-acid for 7 days at low and high aeration and at 20 °C and 30 °C, with a special focus on amino acid consumption and production of volatile aroma compounds. Y. lipolytica was able to grow in the wort under all four conditions, although the growth was inhibited. Furthermore, it only consumed glucose and fructose, and no ethanol was formed. Moreover, under high aeration conditions, Y. lipolytica consumed 75–80% of the amino acids in the wort. Interestingly, no esters were produced during the fermentations, and only five higher alcohols (1-propanol, 2-methyl-1-propanol, 3-methyl-1-butanol, 3-methyl-3-buten-1-ol, and 2-phenylethanol), two aldehydes (3-methylbutanal and (E)-2-nonenal), two ketones (cyclopentanone and 9-oxabicyclo [6.1.0]nonan-4-one), one fatty acid (3-methyl-butanoic acid), and one benzene derivate (1,2,4-trimethyl-benzene) were produced. These results may contribute to the potential use of Y. lipolytica in a traditional brewery for the production of novel beers; e.g., alcohol-free beer.

Lager Yeast Design Through Meiotic Segregation of a Saccharomyces cerevisiae × Saccharomyces eubayanus Hybrid.

Yeasts in the lager brewing group are closely related and consequently do not exhibit significant genetic variability. Here, an artificial Saccharomyces cerevisiae × Saccharomyces eubayanus tetraploid interspecies hybrid was created by rare mating, and its ability to sporulate and produce viable gametes was exploited to generate phenotypic diversity. Four spore clones obtained from a single ascus were isolated, and their brewing-relevant phenotypes were assessed. These F1 spore clones were found to differ with respect to fermentation performance under lager brewing conditions (15°C, 15 °Plato), production of volatile aroma compounds, flocculation potential and temperature tolerance. One spore clone, selected for its rapid fermentation and acetate ester production was sporulated to produce an F2 generation, again comprised of four spore clones from a single ascus. Again, phenotypic diversity was introduced. In two of these F2 clones, the fermentation performance was maintained and acetate ester production was improved relative to the F1 parent and the original hybrid strain. Strains also performed well in comparison to a commercial lager yeast strain. Spore clones varied in ploidy and chromosome copy numbers, and faster wort fermentation was observed in strains with a higher ploidy. An F2 spore clone was also subjected to 10 consecutive wort fermentations, and single cells were isolated from the resulting yeast slurry. These isolates also exhibited variable fermentation performance and chromosome copy numbers, highlighting the instability of polyploid interspecific hybrids. These results demonstrate the value of this natural approach to increase the phenotypic diversity of lager brewing yeast strains.

Development of A Low-Alcoholic Fermented Beverage Employing Cashew Apple Juice and Non-Conventional Yeasts

Cashew apples are by-products in the production of cashew nuts, which are mostly left to rot in the fields. Cashew apple juice (CAJ), a highly nutritious beverage, can be produced from them. It is rich in sugars and ascorbic acid, but its high polyphenol content makes it bitter and astringent, and therefore difficult to commercialize. The kingdom of fungi contains more than 2000 yeast species, of which only a few species have been studied in relation to their potential to produce aroma compounds. The aim of this research was to develop a new low-alcoholic fermented beverage to valorize cashew apples. For this purpose, a screening was carried out employing non-conventional yeast species and some species of the genus Saccharomyces for comparison, followed by a more detailed study with four selected strains cultured at different conditions. The production of volatile aroma compounds as a function of the presence of oxygen, temperature, and yeast species was investigated. The results showed that the more diverse aroma profiles appeared at 25 °C under anaerobic cultivation conditions, where Saccharomyces cerevisiae WUR 102 and Hanseniaspora guilliermondii CBS 2567 excelled in the synthesis of certain aroma compounds, such as β-phenylethanol and its acetate ester (rose aroma). Further studies are needed to test consumer acceptance of these new products.

One-Step Bioconversion of Fatty Acids into C8-C9 Volatile Aroma Compounds by a Multifunctional Lipoxygenase Cloned from Pyropia haitanensis.

The multifunctional lipoxygenase PhLOX cloned from Pyropia haitanensis was expressed in Escherichia coli with 24.4 mg·L-1 yield. PhLOX could catalyze the one-step bioconversion of C18-C22 fatty acids into C8-C9 volatile organic compounds (VOCs), displaying higher catalytic efficiency for eicosenoic and docosenoic acids than for octadecenoic acids. C20:5 was the most suitable substrate among the tested fatty acids. The C8-C9 VOCs were generated in good yields from fatty acids, e.g., 2E-nonenal from C20:4, and 2E,6Z-nonadienal from C20:5. Hydrolyzed oils were also tested as substrates. The reactions mainly generated 2E,4E-pentadienal, 2E-octenal, and 2E,4E-octadienal from hydrolyzed sunflower seed oil, corn oil, and fish oil, respectively. PhLOX showed good stability after storage at 4 °C for 2 weeks and broad tolerance to pH and temperature. These desirable properties of PhLOX make it a promising novel biocatalyst for the industrial production of volatile aroma compounds.

Production and Characterization of a Distilled Alcoholic Beverage Obtained by Fermentation of Banana Waste (Musa cavendishii) from Selected Yeast

Banana is one of the most important fruits in the Brazilian diet and is mainly consumed naturally. Losses from crop to final consumer are high and estimated in about 30%. The aim of this work was to elaborate a distilled alcoholic beverage from discarded banana and to compare with commercial trademarks. Initially, yeast strains were isolated from banana fruit and characterized by their production of volatile aroma compounds. The highest aroma-producing yeast isolate was identified by ITS-rRNA gene sequencing as Pichia kluyveri. Pasteurized banana pulp and peel was fermented by the selected P. kluyveri at approximately 107 cells/mL. The sugars were converted quickly, and a high ethanol concentration (413 mg/L) was achieved after 24 h of fermentation. The fermented banana must was distilled in a Femel Alambic, and the head, heart and tail fractions were collected. The banana brandy produced had highest concentration of volatile compounds compared to trademarks, such as isoamyl acetate (13.5 mg/L), ethyl hexanoate (0.8 mg/L) and others. The results showed that whole banana must could be a good substrate for fermentation and distillation, and the sensory analysis performed revealed that the produced beverage had good acceptance by the tasters. This study demonstrates the potential of banana as a possible alternative to reduce waste and increase income to farmers.

New Lager Brewery Strains Obtained by Crossing Techniques Using Cachaça (Brazilian Spirit) Yeasts.

The development of hybrids has been an effective approach to generate novel yeast strains with optimal technological profile for use in beer production. This study describes the generation of a new yeast strain for lager beer production by direct mating between two Saccharomyces cerevisiae strains isolated from cachaça distilleries: one that was strongly flocculent, and the other with higher production of acetate esters. The first step in this procedure was to analyze the sporulation ability and reproductive cycle of strains belonging to a specific collection of yeasts isolated from cachaça fermentation vats. Most strains showed high rates of sporulation, spore viability, and homothallic behavior. In order to obtain new yeast strains with desirable properties useful for lager beer production, we compare haploid-to-haploid and diploid-to-diploid mating procedures. Moreover, an assessment of parental phenotype traits showed that the segregant diploid C2-1d generated from a diploid-to-diploid mating experiment showed good fermentation performance at low temperature, high flocculation capacity, and desirable production of acetate esters that was significantly better than that of one type lager strain. Therefore, strain C2-1d might be an important candidate for the production of lager beer, with distinct fruit traces and originating using a non-genetically modified organism (GMO) approach.IMPORTANCE Recent work has suggested the utilization of hybridization techniques for the generation of novel non-genetically modified brewing yeast strains with combined properties not commonly found in a unique yeast strain. We have observed remarkable traits, especially low temperature tolerance, maltotriose utilization, flocculation ability, and production of volatile aroma compounds, among a collection of Saccharomyces cerevisiae strains isolated from cachaça distilleries, which allow their utilization in the production of beer. The significance of our research is in the use of breeding/hybridization techniques to generate yeast strains that would be appropriate for producing new lager beers by exploring the capacity of cachaça yeast strains to flocculate and to ferment maltose at low temperature, with the concomitant production of flavoring compounds.

Mead production: effect of nitrogen supplementation on growth, fermentation profile and aroma formation by yeasts in mead fermentation

Mead is an alcoholic beverage, produced since ancient times, resulting from an alcoholic fermentation of diluted honey by yeasts. When it is produced in a traditional manner, mead producers can encounter several problems related to a lack of essential nutrients, such as available nitrogen. Thus, the aim of this study was to evaluate the effect of nitrogen addition to honey-must on the fermentation performance of two Saccharomyces cerevisiae wine yeasts, QA23 and ICV D47, as well as on the mead composition and production of volatile aroma compounds. A portion of honey-must was supplemented with diammonium phosphate (DAP) to achieve the nitrogen concentration required by yeast to complete alcoholic fermentation. The supplementation with DAP reduced the fermentation length to around 7 days, but not all sugars were fully consumed, suggesting that other factors could be interfering with yeast growth. For both yeasts the specific growth rate and final biomass were higher in musts supplemented with DAP. Mead final composition was similar under the two experimental conditions. Analysis of the volatile profile revealed that the concentrations of the volatile fatty acids and volatile phenols were higher in meads supplemented with DAP. The concentrations of ethyl hexanoate, ethyl octanoate and isoamyl acetate were above their perception threshold and were higher in meads supplemented with DAP, which could contribute to the enhancement of the fruity character. This study could be useful for the optimization of mead production and quality improvement. Copyright © 2015 The Institute of Brewing & Distilling

Function and regulation of yeast genes involved in higher alcohol and ester metabolism during beverage fermentation

The biochemical formation of yeast-derived sensory-active metabolites like higher alcohols and esters determines the different characteristics of aroma and taste in fermented beverages. In yeast fermentation process, a large number of environmental factors affecting the production of volatile aroma compounds are abundant. Factors like substrate composition in fermentation media as well as process parameters influencing these flavor-active metabolites have already been described. These factors can act on the expression of yeast genes involved in aroma metabolism resulting in concentration differences in esters and higher alcohols important for flavor and taste. The understanding of the function of genes involved in biosynthetic pathways of aroma-active substances as well as their regulatory mechanisms is needed to control the production of ester and higher alcohol synthesis to create specific aroma profiles in fermented beverages. This review discusses the known regulation and function of several individual genes (ATF1, ATF2, EEB1, EHT1, BAT1, BAT2 and BAP2) described in fusel alcohol and ester synthesis mainly in S. cerevisiae and S. pastorianus var. carlsbergensis. Also, different factors like oxygen and temperature that allow ester and higher alcohol synthesis to be controlled during yeast fermentation are described.

Effects of Proteus vulgaris growth on the establishment of a cheese microbial community and on the production of volatile aroma compounds in a model cheese

To investigate the impact of Proteus vulgaris growth on a multispecies ecosystem and on volatile aroma compound production during cheese ripening. The microbial community dynamics and the production of volatile aroma compounds of a nine-species cheese ecosystem were compared with or without the presence of P. vulgaris in the initial inoculum. Proteus vulgaris was able to colonize the cheese surface and it was one of the dominant species, representing 37% of total isolates at the end of ripening with counts of 9.2 log(10) CFU g(-1). In the presence of P. vulgaris, counts of Arthrobacter arilaitensis, Brevibacterium aurantiacum and Hafnia alvei significantly decreased. Proteus vulgaris influenced the production of total volatile aroma compounds with branched-chain aldehydes and their corresponding alcohols being most abundant. Proteus vulgaris was able to successfully implant itself in a complex cheese ecosystem and significantly contributed to the organoleptic properties of cheese during ripening. This bacterium also interacted negatively with other bacteria in the ecosystem studied. This is the first time that the impact of a Gram-negative bacterium on cheese microbial ecology and functionality has been described.

Linking gene regulation and the exo-metabolome: A comparative transcriptomics approach to identify genes that impact on the production of volatile aroma compounds in yeast

Background'Omics' tools provide novel opportunities for system-wide analysis of complex cellular functions. Secondary metabolism is an example of a complex network of biochemical pathways, which, although well mapped from a biochemical point of view, is not well understood with regards to its physiological roles and genetic and biochemical regulation. Many of the metabolites produced by this network such as higher alcohols and esters are significant aroma impact compounds in fermentation products, and different yeast strains are known to produce highly divergent aroma profiles. Here, we investigated whether we can predict the impact of specific genes of known or unknown function on this metabolic network by combining whole transcriptome and partial exo-metabolome analysis.ResultsFor this purpose, the gene expression levels of five different industrial wine yeast strains that produce divergent aroma profiles were established at three different time points of alcoholic fermentation in synthetic wine must. A matrix of gene expression data was generated and integrated with the concentrations of volatile aroma compounds measured at the same time points. This relatively unbiased approach to the study of volatile aroma compounds enabled us to identify candidate genes for aroma profile modification. Five of these genes, namely YMR210W, BAT1, AAD10, AAD14 and ACS1 were selected for overexpression in commercial wine yeast, VIN13. Analysis of the data show a statistically significant correlation between the changes in the exo-metabome of the overexpressing strains and the changes that were predicted based on the unbiased alignment of transcriptomic and exo-metabolomic data.ConclusionThe data suggest that a comparative transcriptomics and metabolomics approach can be used to identify the metabolic impacts of the expression of individual genes in complex systems, and the amenability of transcriptomic data to direct applications of biotechnological relevance.

Production of volatile aroma compounds by bacterial strains isolated from different surface-ripened French cheeses

Twelve bacterial strains belonging to eight taxonomic groups: Brevibacterium linens, Microbacterium foliorum, Arthrobacter arilaitensis, Staphylococcus cohnii, Staphylococcus equorum, Brachybacterium sp., Proteus vulgaris and Psychrobacter sp., isolated from different surface-ripened French cheeses, were investigated for their abilities to generate volatile aroma compounds. Out of 104 volatile compounds, 54 volatile compounds (identified using dynamic headspace technique coupled with gas chromatography-mass spectrometry [GC-MS]) appeared to be produced by the different bacteria on a casamino acid medium. Four out of eight species used in this study: B. linens, M. foliorum, P. vulgaris and Psychrobacter sp. showed a high flavouring potential. Among these four bacterial species, P. vulgaris had the greatest capacity to produce not only the widest varieties but also the highest quantities of volatile compounds having low olfactive thresholds such as sulphur compounds. Branched aldehydes, alcohols and esters were produced in large amounts by P. vulgaris and Psychrobacter sp. showing their capacity to breakdown the branched amino acids. This investigation shows that some common but rarely mentioned bacteria present on the surface of ripened cheeses could play a major role in cheese flavour formation and could be used to produce cheese flavours.

Volatile production, quality and aroma-related enzyme activities during maturation of ‘Fuji’ apples

Production of volatile aroma compounds, and quality parameters, were monitored in relation to the activity of lipoxygenase, pyruvate decarboxylase, alcohol dehydrogenase and alcohol o-acyltransferase during maturation of ‘Fuji’ apples. Acetate esters increased during maturation and were quantitatively the most significant compounds contributing to ‘Fuji’ aroma. Increase in acetate ester production was associated with the availability of the necessary alcohol precursors. Principal component analysis revealed a clear separation according to sampling date, which accounted for up to 67% of total variance, and allowed ‘Fuji’ apples used in this study to be classified into three ripeness stages. Overall the results suggest that precursor availability for the related enzymes is a more significant factor than enzyme activity for the development of aroma during on-tree maturation of ‘Fuji’ apples.

Production of volatile aroma compounds by kefir starter cultures

Abstract Production of carbonyl compounds by single-strain cultures, kefir starter (Lactobacillus delbrueckii subsp. bulgaricus HP1+Lb. helveticus MP12+Lactococcus lactis subsp. lactis C15+Streptococcus thermophilus T15+Saccharomyces cerevisiae A13) and kefir grains during fermentation and storage of kefir was studied. The content of carbonyl compounds produced by kefir starter was greater than that produced by kefir grains. The maximum acetaldehyde concentration (18.3 μg g−1) in kefir with starter culture was mainly due to the metabolic activity of Lb. delbrueckii subsp. bulgaricus HP1 isolated from kefir grains. The highest diacetyl production activity was recorded in the starter culture (1.87 μg g−1) and the single-strain culture St. thermophilus T15 (1.62 μg g−1), followed by Lb. helveticus MP12 (0.85 μg g−1) and Lc. lactis subsp. lactis C15 (0.42 μg g−1). The lactobacilli Lb. delbrueckii subsp. bulgaricus HP1 and Lb. helveticus MP12 produced acetone, which was not found in the cocci cultures. The presence of 2-butanone was related to the production ability of Lb. helveticus MP12. In comparison, Lc. lactis subsp. lactis C15 synthesized ethyl acetate more actively than the other single-strain cultures included in the starter. S. cerevisiae A13 produced ethanol and CO2 in amounts (3975 μg g−1; 1.80 g L−1) that lent cultured kefir distinctive flavour and aroma characteristic of authentic kefir.

Volatile Flavour Production by Penicillium caseifulvum

Abstract The production of volatile aroma compounds from the cheese associated fungus Penicillium caseifulvum has been compared to the aroma production by P. camemberti when grown on a liquid cream based medium in Petri dishes. Volatiles were collected by diffusive sampling between day 5 and 10 and between day 10 and 15 and analysed by gas chromatography coupled to mass spectrometry. During both sampling periods similar qualitative aroma profiles could be observed for the two species. In the beginning volatiles like ethanol, acetone, 2-methylpropan-1-ol, 2-pentanone, 3-methylbutan-1-ol, 2-heptanone, 2-nonanone and 2-undecanone were dominant, whereas the metabolism changed later in the growth phase. At this stage unsaturated hydrocarbons like styrene, limonene, β -caryophyllene and other terpenoids, including some possible diterpenes, were major volatiles. The results indicate the potential of P. caseifulvum as a new starter culture for the dairy industry or as a fermentation organism for production of natural cheese flavours by submerge fermentation.