The Monthly Digest – Aromas and flavours – April & May 2018

Aromas and flavours

R & D abstracts – April & May 2018

Biogeneration of aroma compounds (review)

Aroma compounds may be recovered from natural sources, produced by chemical synthesis or by biotechnological means. The latter approach has faced constant growth and it is expected to continue expanding in the future. This study reviews the recent advances in the biogeneration of aroma compounds, focusing on the microbial production (either by de novo synthesis or by biotransformation of widely available substrates) of the most commercially relevant compounds.

Sales A et al (2018) Current Opinion in Food Science (in press)

Production of vanillin from lignin: The relationship between β-O-4 linkages and vanillin yield (research)

Lignin is the renewable source of aromatics in nature, and the conversion of lignin into vanillin is very attractive. However, the vanillin yield is closely related to the resource of lignin and its isolation process, and relatively low yield of vanillin is always obtained from lignin. In order to improve the vanillin yield from lignin, in this work, the relationship between lignin linkages and vanillin yield was explored. Five lignins (kraft lignin, alkali lignin, lignosulfonate, and two enzymatic lignins) and various lignin model compounds (monomers, dimers, and polymers) were characterized by NMR spectroscopy and oxidized to prepare vanillin. Results indicated that the interunit linkages of the lignin had a significant influence on the vanillin yield. The more β-O-4 linkages in lignin, the higher yield of corresponding aldehydes was obtained. It is believed that the vanillin yield can be significantly improved by using lignins with high content of β-O-4 linkages.

Wang Y et al (2018) Industrial Crops and Products 116:116-121

The Monthly Digest – Aromas and flavours – February & March 2018

Aromas and flavours

R & D abstracts – February & March 2018

Biosynthesis and production of sabinene: current state and perspectives (review)

Sabinene is an important naturally occurring bicyclic monoterpene which can be used as flavorings, perfume additives, fine chemicals, and advanced biofuels. Up to now, this valuable terpene is commercially unavailable since there is no applicable manufacturing process. Microbial synthesis can be a promising route for sabinene production. In this review, we summarize knowledge about the metabolic pathway and key enzymes for sabinene biosynthesis. Recent advances that have been made in production of sabinene by microbial fermentation are highlighted. In these studies, researchers have identified the general synthetic pathway of sabinene from simple intermediate metabolites. Sabinene synthases of different origins were also cloned and characterized. Additionally, heterologous systems of the model microbes Escherichia coli and Saccharomyces cerevisiae were constructed to produce sabinene. This review also suggests new directions and attempts to gain some insights for achieving an industrial level production of sabinene. The combination of traditional molecular biology with new genome and proteome analysis tools will provide a better view of sabinene biosynthesis and a greater potential of microbial production.

Cao Y et al (2018) Applied Microbiology and Biotechnology 102:1535-1544

Bioflavoring by non-conventional yeasts in sequential beer fermentations (research)

Non-conventional yeast species have great capacity for producing diverse flavor profiles in production of alcoholic beverages, but their potential for beer brewing, in particular in consecutive fermentations with Saccharomyces cerevisiae, has only poorly been explored. We have screened 17 non-conventional yeast species for production of an appealing profile of flavor esters and phenolics in the first phase of alcoholic fermentation, followed by inoculation with S. cerevisiae to complete the fermentation. For measurement of phenolic compounds and their precursors we developed an improved and highly sensitive methodology. The results show that non-conventional yeast species possess promising potential for enhancement of desirable flavors in beer production. Notable examples are increasing isoamyl acetate (fruity, banana flavor) by application of P. kluyverii, augmenting ethyl phenolic compounds (spicy notes) with Brettanomyces species and enhancing 4-vinyl guaiacol (clove-like aroma) with T. delbrueckii. All Pichia strains also produced high levels of ethyl acetate (solvent-like flavor). This might be selectively counteracted by selection of an appropriate S. cerevisiae strain for the second fermentation phase, which lowers total ester profile. Hence, optimization of the process conditions and/or proper strain selection in sequentially inoculated fermentations are required to unlock the full potential for aroma improvement by the non-conventional yeast species.

Holt S et aal (2018) Food Microbiology 72:55-66

Microbe participation in aroma production during soy sauce fermentation (research)

Soy sauce is a traditional Japanese fermented seasoning that contains various constituents such as amino acids, organic acids, and volatiles that are produced during the long fermentation process. Although studies regarding the correlation between microbes and aroma constituents have been performed, there are no reports about the influences of the microbial products, such as lactic acid, acetic acid, and ethanol, during fermentation. Because it is known that these compounds contribute to microbial growth and to changes in the constituent profile by altering the moromi environment, understanding the influence of these compounds is important. Metabolomics, the comprehensive study of low molecular weight metabolites, is a promising strategy for the deep understanding of constituent contributions to food characteristics. Therefore, the influences of microbes and their products such as lactic acid, acetic acid, and ethanol on aroma profiles were investigated using gas chromatography/mass spectrometry (GC/MS)-based metabolic profiling. The presence of aroma constituents influenced by microbes and chemically influenced by lactic acid, acetic acid, and ethanol were proposed. Most of the aroma constituents were not produced by adding ethanol alone, confirming the participation of yeast in aroma production. It was suggested that lactic acid bacterium relates to a key aromatic compound, 2,5-dimethyl-4-hydroxy-3(2H)-furanone. However, most of the measured aroma constituents changed similarly in both samples with lactic acid bacterium and acids. Thus, it was clear that the effect of lactic acid and acetic acid on the aroma profile was significant.

Harada R et al (2018) Journal of Bioscience and Bioebgineering (2018)

The Monthly Digest – Aromas and Flavours October 2017

Aromas and flavours
R & D abstracts – October 2017

Japan Flavour and Fragrance Materials Association’s (JFFMA) safety assessment of food-flavouring substances uniquely used in Japan that belong to the class of aliphatic primary alcohols, aldehydes, carboxylic acids, acetals and esters containing additional oxygenated functional groups (review)

We performed a safety evaluation using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) of the following four flavouring substances that belong to the class of ‘aliphatic primary alcohols, aldehydes, carboxylic acids, acetals, and esters containing additional oxygenated functional groups’ and are uniquely used in Japan: butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate, 3-hydroxyhexanoic acid and methyl hydroxyacetate. Although no genotoxicity study data were found in the published literature, none of the four substances had chemical structural alerts predicting genotoxicity. All four substances were categorised as class I by using Cramer’s classification. The estimated daily intake of each of the four substances was determined to be 0.007–2.9 μg/person/day by using the maximised survey-derived intake method and based on the annual production data in Japan in 2001, 2005 and 2010, and was determined to be 0.250–600.0 μg/person/day by using the single-portion exposure technique and based on average-use levels in standard portion sizes of flavoured foods. Both of these estimated daily intake ranges were below the threshold of toxicological concern for class I substances, which is 1800 μg/person/day. Although no information from in vitro and in vivo toxicity studies for the four substances was available, these substances were judged to raise no safety concerns at the current levels of intake.

Saito K et al (2017) Food Additives & Contaminants, Part A 34:1474-1484


A review on enzymatic synthesis of aromatic esters used as flavor ingredients for food, cosmetics and pharmaceuticals industries

Many sectors of industry, mainly food, cosmetics and pharmaceutics, have increased their interest in esters due to their flavor property. Flavor esters that possess an aromatic ring in their molecular structure are also known as aromatic esters. These esters are widely found in nature (fruits and plants) and the synthetic (i.e. via chemical) and natural routes (i.e. via direct extraction from nature or via biotechnology) are suitable for their biocatalysis. In this context, from the industrial point of view, enzyme-catalyzed reactions are the most economical approach to reach final green products with no toxicity and no harm to human health. The present article gives an overview of the aromatic esters synthesis, considering the main effects in the reaction media conditions and enzymes used. This review also describes applied trends in enzymatic-catalyzed reactions, pointing alternatives to production, like ultrasound-assisted reactions and process optimization of aromatic esters. Furthermore, this work presents perspectives concerning the biological potential of these esters and recent advances in their encapsulation. Lipases play an important role in the aromatic esters production, with several advantages over synthetic route. Lipase-catalyzed reactions usually follow Ping-Pong Bi-Bi or ternary complex (order Bi-Bi) mechanism. The study of the process parameters and their interaction are very important to understand the system optimization and achieve the maximum reaction yield to scale up. Aromatic esters can present some biological activities, in addition to their fragrances, which increases the interest in the encapsulation of these compounds.

Gomes Almeida A et al (2017) Trends in Food Science and Technology 69 A:95-105

Biotransformation of (+)-carvone and (−)-carvone using human skin fungi: A green method of obtaining fragrances and flavours (research)

The synthesis of optically pure compounds is increasingly in demand among the pharmaceutical, fine chemical and agro-food industries, while the importance of chirality in the activity and biological properties of many compounds has previously been established. The aim of the present study was therefore to evaluate the biotransformation capacities of (+)-carvone and (−)-carvone using the fungi Scolecobasidium sp, three lines of Cladosporium sp, Phoma sp, Aureobasidium sp and Epicoccum sp, all obtained from human skin. The seven fungi evaluated were capable of hydrogenating the activated alkene, followed by the reduction of ketone to chiral alcohol, with conversions between 9.5 and 100%, and with diastereomer excess (d.e.) of over 89% of dihydrocarveol when (+)-carvone was used as a substrate. These results demonstrate that the filamentous fungi of human skin are potential biocatalytic tools for obtaining chiral alcohols.

Dos Santos RAM et al (2017) Biocatalysis and Biotransformation (in press)

Use of non-Saccharomyces yeasts and oenological tannin in red winemaking: Influence on colour, aroma and sensorial properties of young wines (research)

Today, many non-Saccharomyces strains have been verified can be positive for the development of wine anthocyanin and aroma in different fermentation scenarios. Moreover, oenological tannins are widely used in wine industry to improve the colour profile and aroma complexity. The aim of this work is to analyze the fermentation characters of non-Saccharomyces strains and investigate the effects of pre-fermentative addition of oenological tannins on the wine components as well as sensory properties. For this purpose, five selected non-Saccharomyces strains and grape seed tannin were used to carry out the different fermentation trials. As a result, the grape seed tannins were less likely to influence growth kinetics of non-Saccharomyces strains. Schizosaccharomyces pombe has been proved can be effective to reduce the malic acid content while increase the level of vinylphenolic pyranoanthocyanin, which is positive for wine colour stability. Pre-fermentative use of oenological tannin was verified could be beneficial for the wines fermented with non-Saccharomyces regarding the improvement of wine colour, anthocyanin composition and the complexity of volatile compounds. Nevertheless, sensory analysis showed that oenological tannin could be less effective to modify the aroma impression of non-Saccharomyces wines.

Chen K et al (2017) Food Microbiology 69:51-63


The Monthly Digest – Aromas and Flavours September 2017

Aromas and flavours
R & D abstracts – September 2017
Microbial diversity of traditional kefir grains and their role on kefir aroma (research)

Kefir grains consist of rich bacterial and fungal microflora responsible for the production of this traditional fermented milk beverage with unique flavour properties. Here, a pyrosequencing approach was applied for the identification of microbial flora of four kefir grains collected from different regions of Turkey and the volatile compounds in kefir samples produced with these grains were determined. Lactobacillus kefiranofaciens presented in all grains at important levels and EnterobacterAcinetobacterEnterococcus and Pseudomonas spp. were observed in traditional kefir grains. The fungal microflora of kefir grains was dominated by yeast species and Dipodascaceae family was dominant and Saccharomyces cerevisiaepresented in all grains. Other yeast species belonging to KazachstaniaCandidaIssatchenkia and Rhodotorula species were also detected in kefir grains. Volatile compounds of kefir samples were also diverse related to the microbial diversity of kefir grains. This study revealed the rich microflora of Turkish kefir grains and their interactions with the aromatic properties of kefir.

Dertli E and çon HA (2017) LWT Food Science and Technology 85A:151-157


Influence of different yeast/lactic acid bacteria combinations on the aromatic profile of red Bordeaux wine (research)

The typical fruity aroma of red Bordeaux wines depends on the grape variety but also on microbiological processes, such as alcoholic and malolactic fermentations. These transformations involve respectively the yeast Saccharomyces cerevisiae and the lactic acid bacterium Oenococcus oeni. Both species play a central role in red winemaking but their quantitative and qualitative contribution to the revelation of the organoleptic qualities of wine has not yet been fully described. The aim of this study was to elucidate the influence of sequential inoculation of different yeast and bacteria strains on the aromatic profile of red Bordeaux wine. All microorganisms completed fermentations and no significant difference was observed between tanks regarding the main oenological parameters until 3 months’ aging. Regardless of the yeast strain, B28 bacteria required the shortest period to completely degrade the malic acid, compared to the other strain. Quantification of 73 major components highlighted a specific volatile profile corresponding to each microorganism combination. However, the yeast strain appeared to have a predominant effect on aromatic compound levels, as well as on fruity aroma perception. Yeasts had a greater impact on wine quality and have more influence on the aromatic style of red wine than bacteria.

Gammacurta M et al (2017) Journal of the Science of Food and Agriculture


Fed-batch production of vanillin by Bacillus aryabhattai BA03 (research)

Bacillus aryabhattai BA03, a strain isolated in our laboratory, has interesting properties related to the production of natural aromas and flavors. Specifically, we have found that it was able to produce vanillin from ferulic acid (FA). Furthermore, this strain produces high amounts of 4-vinylguaiacol in only 14 h, this being the only intermediate metabolite observed in the process. FA is an inexpensive feedstock for the production of natural value-added compounds when extracted from lignocellulosic wastes. In this study, we optimized the operational conditions (temperature, pH and agitation), medium composition and bioconversion technology (batch or fed-batch) to produce vanillin. In a fed-batch process conducted with just one additional supplementation after 24 h, the maximal concentration of vanillin (147.1 ± 0.9 mg/L) was observed after 216 h (QV = 0.681 mg/L h; YV/fFA = 0.082 mg/mg) after degrading 90.3% FA. In view of our data, we postulate that Bacillus aryabhattai BA03 carries out a decarboxylation of ferulic acid as a metabolic pathway.

Paz A et al (2017) New Biotechnology (in press)


Performance of selected P. fermentans and its excellular enzyme in co-inoculation with S. cerevisiae for wine aroma enhancement (research)

This study evaluated aroma enhancement in dry white wines made via a specific Pichia fermentans strain and its excellular enzyme in co-inoculation with Saccharomyces cerevisiae. The inoculation ratios of two yeasts ranged from 0.1 to 10 and fermentations with pure S. cerevisiae were utilized as control. Esterase activities that expressed as C2-C8 chain substrate specificity were surveyed during mixed fermentation. Finally, wine aroma was analyzed via GC-MS and a trained sensory panel. The results revealed that C2-C8 esterase activities were superior during mixed fermentation to the control. Mixed fermentations significantly increased the contents of acetates, ethyl esters, fatty acids, and numerous other fermentative volatiles; particularly those of medium-chain fatty acids and their corresponding esters, with an increased inoculation ratio of the isolate. Extracellular enzyme treatment highly improved the release of varietal aroma compounds, such as terpenols, C13-norisoprenoids, and C6 compounds. Sensory analysis indicated that the risk of negative earth odor emerged in the wine characterized by strong sweet and acid fruit trait. Therefore, mixed fermentations at yeasts inoculation ratios between 1:4 and 4:1 may provide better fermentation strategies for appropriate wine aroma enhancement.

Ma D et al (2017) LWT – Food Science and Technology 86:361-370


Strains of the Lactobacillus casei group show diverse abilities for the production of flavor compounds in 2 model systems (research)

Cheese flavor development is directly connected to the metabolic activity of microorganisms used during its manufacture, and the selection of metabolically diverse strains represents a potential tool for the production of cheese with novel and distinct flavor characteristics. Strains of Lactobacillus have been proven to promote the development of important cheese flavor compounds. As cheese production and ripening are long-lasting and expensive, model systems have been developed with the purpose of rapidly screening lactic acid bacteria for their flavor potential. The biodiversity of 10 strains of the Lactobacillus casei group was evaluated in 2 model systems and their volatile profiles were determined by gas chromatography-mass spectrometry. In model system 1, which represented a mixture of free AA, inoculated cells did not grow. In total, 66 compounds considered as flavor contributors were successfully identified, most of which were aldehydes, acids, and alcohols produced via AA metabolism by selected strains. Three strains (DPC2071, DPC3990, and DPC4206) had the most diverse metabolic capacities in model system 1. In model system 2, which was based on processed cheese curd, inoculated cells increased in numbers over incubation time. A total of 47 compounds were identified, and they originated not only from proteolysis, but also from glycolytic and lipolytic processes. Tested strains produced ketones, acids, and esters. Although strains produced different abundances of volatiles, diversity was less evident in model system 2, and only one strain (DPC4206) was distinguished from the others. Strains identified as the most dissimilar in both of the model systems could be more useful for cheese flavor diversification.

Stefanovic E et al (2017) Journal of Dairy Science 100:6818-6929


Physiology, ecology and industrial applications of aroma formation in yeast (review)

Yeast cells are often employed in industrial fermentation processes for their ability to efficiently convert relatively high concentrations of sugars into ethanol and carbon dioxide. Additionally, fermenting yeast cells produce a wide range of other compounds, including various higher alcohols, carbonyl compounds, phenolic compounds, fatty acid derivatives and sulfur compounds. Interestingly, many of these secondary metabolites are volatile and have pungent aromas that are often vital for product quality. In this review, we summarize the different biochemical pathways underlying aroma production in yeast as well as the relevance of these compounds for industrial applications and the factors that influence their production during fermentation. Additionally, we discuss the different physiological and ecological roles of aroma-active metabolites, including recent findings that point at their role as signaling molecules and attractants for insect vectors.

Dzialo MC et al (2017) FEMS Microbiology Reviews 41:S95-S128

The Monthly Digest – Aromas and Flavours July & August 2017

Aromas and flavours
R & D abstracts – July & August 2017
Fermentation characteristics and aromatic profile of plum wines produced with indigenous microbiota and pure cultures of selected yeast (research)

Plum (Prunus domestica L.) is one of the most often grown fruit species, which is widespread all over the world. The processing potential of this fruit is not exploited enough. Fruit wines became very popular nowadays because of their nutritional and health-promoting values. Plums are fruit that can give wine with intensive red color and characteristic flavor, which can be compared with the best grape wines. Obtained results would contribute to global acceptance of wine as functional food. An important advantage of fruit wines (concretely plum wine) compared to traditional grape wine is lower alcohol content. Also, plum fruits losses in refrigeration industry could be avoided due to longer shelf life of this fermented beverage.
The aim of this study was to assess and compare fermentation characteristics and aromatic profile of plum wines produced with indigenous microbiota and pure cultures of different selected yeast. Experiments were carried out with plum (Prunus domestica L.) varieties of different fruit ripening times (Čačanska rana, Čačanska lepotica, and Požegača). Wine fermentations were conducted by the activity of indigenous microbiota, commercially available Saccharomyces cerevisiae and Saccharomyces bayanus yeast strains and joint activity of Schizosaccharomyces pombe and S. cerevisiae (sequential inoculation). Statistically significant differences in fermentative characteristics and the content of certain volatile compounds were observed as a result of metabolic activity of various indigenous and/or selected yeasts during fermentation of plum pomace. Minimal duration of fermentation (4 to 5 d) and fastest ethanol production rate (from 12.3 to 15.5 g/L/d) were the characteristics of the studied S. cerevisiae strains. Isobutanol, 3-methyl-1-butanol, 1-heptanol, and 1-octanol were the most prevalent higher alcohols in the tested plum wine samples. The predominant ester in plum wines was ethyl acetate, ethyl lactate, amyl acetate, isoamyl acetate, and ethyl palmitate, esters responsible for the floral and fruity olfactory tones, were also present in large amounts. Also, the use of S. cerevisiae strains resulted in the production of plum wines with better sensory characteristics than ones produced with other investigated yeasts. Obtained results are significant since there is limited data on the compounds responsible for the unique flavor of plum wine, as well as on the impact of different yeast starter cultures application on the overall quality of fruit wines.

Miljic U et al (2017) Journal of Food Science 82:1443-1450


Agrowaste to vanillin conversion by a natural Pediococcus acidilactici strain BD16 (research)

Owing to its flavoring, antimicrobial, antioxidant and anticarcinogenic nature, vanillin is widely used in foods, beverages, perfumes and pharmaceutical products. Ferulic acid (FA) is an important precursor of vanillin which is abundant in cereals like maize, rice and wheat and sugar beet. A major drawback of microbial vanillin production from FA is the degradation and biotransformation of toxic vanillin to other phenolic derivatives. The present study is undertaken to explore microbial vanillin production from FA precursor rice bran by employing vanillin-resistant Pediococcus acidilactici BD16, a natural lactic acid bacteria isolate. Extracellular, intracellular and cellular vanillin dehydrogenase activity was found least, which was minimized vanillin degradation, and the strain resists more than 5 g L−1 vanillin in the medium. A metabolomics approach was followed for the detection of FA, vanillin and other metabolites generated during fermentation of rice bran medium. A metabolic pathway was also predicted for vanillin biosynthesis. Approximately 1.06 g L−1 of crude vanillin was recovered from rice-bran-containing medium and this further offers scope for the industrial utilization of the organism and its genetic manipulation to enhance production of biovanillin.

Chakraborty D et al (2017) Environmental Technology 38:1823-1834


Improvement of aroma and shelf-life of non-alcoholic beverages through cyclodextrins-limonene inclusion complexes (research)

Limonene is a monoterpene flavor compound found in several beverages. However, it easily degrades by oxidation reactions at acidic environment contributing to an undesirable off-flavor. Encapsulation technologies can protect compounds from degradation. This work focuses on the effect of using complexes of limonene with
α-, β-, and γ- and HP-β-cyclodextrins in non-alcoholic beverages to improve flavor and shelf-life stability due to the chemical structure of cyclodextrins. Spray-drying technology was applied to prepare different cyclodextrin/limonene forms, from which the most promising was selected and further applied in simulated lemon juice beverages. Different drying process conditions were tested, namely feed temperature (120, 160, and 180 °C) and setting of prior incubation (temperature, room and 50 °C; time, 0.17 and 24 h). An inlet temperature of 160 °C favored the encapsulation of limonene into resulting nano/microparticles. Moreover, incubation for 24 h enhanced limonene retention for all complexes, especially for β-cyclodextrin/limonene complexes, which achieved 66% of encapsulation efficiency and a 6.25 w/w of limonene load. The β-cyclodextrin/limonene particles which enabled higher load (160 °C, 24 h) presented particle size ranging between 1 and 3 μm and were chosen to undergo an accelerated aging process in a lemon juice
beverage model. This study revealed that the limonene content decreased over time for model and supplemented juice, but decreased less when β- cyclodextrin/limonene particles were added. After 10 days, which mimics 9 months of storage, 40% of complexed limonene remained in the model beverage.

Saldanha do Carmo C et al (2017) Food and Bioprocess Technology 10:1297-1309


Intracellular metabolite profiling of industrial yeast and the synthesis of flavour compounds in beer (research)

Beer quality is largely guaranteed by a rational ratio of different flavour compounds. In this study, we present intracellular metabolic profiles in the light of lager beer flavour compound synthesis on an industrial scale. A total of 62 intracellular metabolites were identified and quantitatively analysed. Among these, amino acid was identified as the most dominant category of metabolites. We also report gene expression profiling of relevant genes encoding six representing amino acids, including serine, alanine, lysine, valine, leucine and asparagine. Nine genes involved in the amino acid synthesis – ALT1, ALT2, ASN1, ASN2, BAT1, SER2, SHM1, SHM2 and LYA1 displayed significantly higher level of expression at various fermentation stages in an industrial lager fermentation. However, concentrations of the recovered amino acids decreased towards the end of fermentation. Our results suggested that the defined amino acids could be assimilated by the yeast to be utilized for synthesis of aroma-active metabolites at different stages in fermentation.

Yin H et al (2017) Journal of the Institute of Brewing (in press)


Great intraspecies diversity of Pichia kudriavzevii in cocoa fermentation highlights the importance of yeast strain selection for flavor modulation of cocoa beans (research)

The use of selected yeasts has recently been proposed as an interesting strategy to improve cocoa bean fermentation and chocolate quality. In this study, a total of 39 cocoa-derived yeast isolates were screened for their capacity to produce volatile aroma compounds in a cocoa pulp simulation medium. The seven highest aroma-producing yeasts were identified by ITS-rRNA gene sequencing as belonging to Pichia kudriavzevii, in spite of exhibiting different metabolic profiles. Repetitive extragenic palindromic (Rep)-PCR genomic fingerprinting approach confirmed this high intraspecies diversity of P. kudriavzevii in cocoa fermentation. Two strains with superior aroma production, namely P. kudriavzevii LPB06 and P. kudriavzevii LPB07, were used in cocoa beans fermentation at laboratory scale. They were able to establish an accelerated fermentation process with efficient yeast growth, sugars consumption and ethanol formation compared to the spontaneous process. The resulting cocoa beans were analyzed by diverse chemical analysis methods, including gas chromatography coupled to mass spectrophotometry (GC-MS), fermentation index and metal and colorimetric analysis. Altogether, the results indicated that inoculated fermentations generated cocoa beans with better color development and richer aroma composition, suggesting that cocoa-associated yeast diversity at strain level can be exploited for flavor modulation of cocoa beans.

Pereira GVM et al (2017) LWT – Food Science and Technology 84:290-297


Microbial diversity of traditional kefir grains and their role on kefir aroma (research)

Kefir grains consist of rich bacterial and fungal microflora responsible for the production of this traditional fermented milk beverage with unique flavour properties. Here, a pyrosequencing approach was applied for the identification of microbial flora of four kefir grains collected from different regions of Turkey and the volatile compounds in kefir samples produced with these grains were determined. Lactobacillus kefiranofacienspresented in all grains at important levels and Enterobacter, Acinetobacter, Enterococcus and Pseudomonas spp were observed in traditional kefir grains. The fungal microflora of kefir grains was dominated by yeast species and Dipodascaceaefamily was dominant and Saccharomyces cerevisiae presented in all grains. Other yeast species belonging to Kazachstania, Candida, Issatchenkia and Rhodotorula species were also detected in kefir grains. Volatile compounds of kefir samples were also diverse related to the microbial diversity of kefir grains. This study revealed the rich microflora of Turkish kefir grains and their interactions with the aromatic properties of kefir.

Dertli E and Çon AH (2017) LWT – Food Science and Technology 85A:151-157

The Monthly Digest – Flavours – May 2017

Aromas and flavours
R & D abstracts – May 2017
Microbial cell factories for the production of terpenoid flavor and fragrance compounds (review)

Terpenoid flavor and fragrance compounds are of high interest to the aroma industry. Microbial production offers an alternative sustainable access to the desired terpenoids independent of natural sources. Genetically engineered microorganisms can be used to synthesize terpenoids from cheap and renewable resources. Due to its modular architecture, terpenoid biosynthesis is especially well suited for the microbial cell factory concept: a platform host engineered for a high flux toward the central C5 prenyl diphosphate precursors enables the production of a broad range of target terpenoids just by varying the pathway modules converting the C5 intermediates to the product of interest. In this review typical terpenoid flavor and fragrance compounds marketed or under development by biotech and aroma companies are given, and the specificities of the aroma market are discussed. The main part of this work focuses on key strategies and recent advances to engineer microbes to become efficient terpenoid producers.

Schempp FM et al (2017) Journal of Agricultural and Food Chemistry (in press)

A review on Lactococcus lactis: from food to factory

Lactococcus lactis has progressed a long way since its discovery and initial use in dairy product fermentation, to its present biotechnological applications in genetic engineering for the production of various recombinant proteins and metabolites that transcends the heterologous species barrier. Key desirable features of this gram-positive lactic acid non-colonizing gut bacteria include its generally recognized as safe (GRAS) status, probiotic properties, the absence of inclusion bodies and endotoxins, surface display and extracellular secretion technology, and a diverse selection of cloning and inducible expression vectors. This have made L. lactis a desirable and promising host on par with other well established model bacterial or yeast systems such as Escherichia coli, Sacharomyces cerevisiae and Bacillus subtilis. In this article, we review recent technological advancements, challenges, future prospects and current diversified examples on the use of L. lactis as a microbial cell factory. Additionally, we will also highlight latest medical-based applications involving whole-cell L. lactis as a live delivery vector for the administration of therapeutics against both communicable and non-communicable diseases.

Song AAL et al (2017) Microbial Cell Factories 16:55

Genetic, enzymatic and metabolite profiling of the Lactobacillus casei group reveals strain biodiversity and potential applications for flavour diversification (research)

The Lactobacillus casei group represents a widely explored group of lactic acid bacteria, characterized by a high level of biodiversity. In this study, the genetic and phenotypic diversity of a collection of more than 300 isolates of the L. casei group and their potential to produce volatile metabolites important for flavour development in dairy products was examined. Following confirmation of species by 16S rRNA PCR, the diversity of the isolates was determined by pulsed-field gel electrophoresis. The activities of enzymes involved in the proteolytic cascade were assessed and significant differences among the strains were observed. Ten strains were chosen based on the results of their enzymes activities and they were analysed for their ability to produce volatiles in media with increased concentrations of a representative aromatic, branched chain and sulphur amino acid. Volatiles were assessed using gas chromatography coupled with mass spectrometry. Strain-dependent differences in the range and type of volatiles produced were evident. Strains of the L. casei group are characterized by genetic and metabolic diversity which supports variability in volatile production. This study provides a screening approach for the knowledge-based selection of strains potentially enabling flavour diversification in fermented dairy products.

Stefanovic E et al (2017) Journal of Applied Microbiology 122:1245-1261

Flavour modulation by bio-processing using flavour forming bacteria strains (patent)

A fermentation of a milk source with Lactococcus lactis subsp. lactis biovar diacetylactis to form a fermented milk product. The fermented milk product has a flavour and aroma. The fermented milk product can be in the form of a powder or a concentrate. The fermented milk product has applications in the food industry.

WO2012085009 A1

Flavour modulation by bio-processing using cream-flavour forming bacteria strains (patent)

A fermentation of a milk source with Lactococcus lactis subsp. lactis diacetylactis strains to form a fermented milk product. The fermented milk product has at least a cream flavour and aroma. The fermented milk product can be in the form of a powder or a concentrate. The fermented milk product has applications in the food industry. A use of these bacterial strains for the manufacture of butter-cream flavouring milk ingredients containing at least one of diacetyl, acetoin and 3,4-dihydroxy-3,4-dimethyl-2,5-hexanedione.

WO2012085010 A1

Flavour modulation by fermenting a milk source for multi-flavour formation with a cocktail of bacteria strains (patent)

A fermentation of a milk source to manufacture a fermented milk product with malty-chocolate-honey-butter-cream flavor and aroma. Fermentation is achieved by addition of Lactococcus lactis subsp. lactis diacetylactis strains. A further bacterium Lactococcus lactis subsp. lactis biovar is also added to the milk source. The milk source comprises amino acids and citrate prior to fermentation.

WO2012085011 A1

Application of Plackett–Burman experimental design for investigating the effect of wort amino acids on flavour-active compounds production during lager yeast fermentation (research)

Aroma-active higher alcohols and esters are produced intracellularly in the cytosol by fermenting lager yeast cells, which are of major industrial interest because they determine aroma and taste characteristics of the fermented beer. Wort amino acid composition and their utilization by yeast during brewer’s wort fermentation influence both the yeast fermentation performance and the flavour profile of the finished product. To better understand the relationship between the yeast cell and wort amino acid composition, Plackett–Burman screening design was applied to measure the changes in nitrogen composition associated with yeast amino acids uptake and flavour formation during fermentation. Here, using an industrial lager brewing strain of Saccharomyces pastorianus, we investigated the effect of amino acid composition on the accumulation of higher alcohols and volatile esters. The objective of this study was to identify the significant amino acids involved in the flavour production during beer fermentation. Our results showed that even though different flavour substances were produced with different amino acid composition in the fermentation experiments, the discrepancies were not related to the total amount of amino acids in the synthetic medium. The most significant effect on higher alcohol production was exercised by the content of glutamic acid, aromatic amino acids and branch chain amino acids. Leucine, valine, glutamic acid, phenylalanine, serine and lysine were identified as important determinants for the formation of esters. The future applications of this information could drastically improve the current regime of selecting malt and adjunct or their formula with desired amino acids in wort.

Yin H et al (2017) Journal of the Institute of Brewing (in press)

Impact of pre-fermentative maceration and yeast strain along with glutathione and SO2 additions on the aroma of Vitis vinifera L. Pošip wine and its evaluation during bottle aging (research)

The aim of the research was characterization and improvement of aroma of white wine Vitis vinifera L. Pošip by application of several enological practices, either individually or in combinations. The effect of pre-fermentative maceration, indigenous yeast strain and antioxidant additions (higher sulfur dioxide and glutathione) was examined, and compared to conventional white wine production: reductive pressing, commercial yeast strain and typical doses of free sulfur dioxide (35 mg/L). The most significant aroma contributors were esters and higher alcohols (fermentation aroma), as well as varietal aroma compounds such as linalool, β-damascenone and thiols (3SH and 3SHA). Furthermore, results showed that pre-fermentative maceration affected the higher concentrations of several aroma compounds, primarily those important for varietal character of wines: terpenes, norisoprenoids and varietal thiols. Fermentation by indigenous yeasts resulted in higher concentrations of different compounds, especially of i-amyl acetate which contributes to desirable fruity, banana-like odors. Furthermore, during 12 months’ aging, most of the aroma compounds significantly decreased, but in wines bottled with antioxidant additions (higher free sulfur dioxide, glutathione and their combination) their slower decrease was observed. The most notable protective effect was observed using the combination of higher free sulfur dioxide (50 mg/L) with glutathione (20 mg/L).

Tomasevic M et al (2017) LWT – Food Science and Technology 81:67-76

Comparison between malolactic fermentation container and barrel toasting effects on phenolic, volatile, and sensory profiles of red wines (research)

Ellagitannin and anthocyanin profiles, woody volatile composition, and sensory properties of wines in which malolactic fermentation (MLF) took place in barrels or stainless steel tanks, have been compared after 12 months of barrel aging. Three different barrel toastings were evaluated. Barrel-fermented wines generally presented 1.2-fold higher total phenolics, whereas tank-fermented wines exhibited 1.1- and 1.2-fold greater total proanthocyanidin and anthocyanin contents, respectively. Concerning ellagitannin composition, the barrel toasting effect seemed to be more important than differences due to MLF container. Certain woody and fruity volatiles varied significantly (p < 0.05) depending on whether MLF occurred in barrels or tanks. Barrel-fermented wines were preferred in the mouth, whereas olfactory preference depended on barrel toasting. This is the first study that evaluates the impact of oak wood during MLF on ellagitannin wine composition, as well as the barrel toasting effect on wine attributes during aging when MLF occurred whether in barrels or in tanks.

Gonzalez-Centeno et al (2017) Journal of Agricultural and Food Chemistry (in press)

Immobilization of a cutinase from Fusarium oxysporum and application in pineapple flavor synthesis (research)

In the present study, the immobilization of a cutinase from Fusarium oxysporum was carried out as cross-linked enzyme aggregates. Under optimal immobilization conditions, acetonitrile was selected as precipitant, utilizing 9.4 mg protein/mL and 10 mM glutaraldehyde as cross-linker. The immobilized cutinase (imFocut5a) was tested in isooctane for the synthesis of short-chain butyrate esters, displaying enhanced thermostability compared to the free enzyme. Pineapple flavor (butyl butyrate) synthesis was optimized leading to a conversion yield of >99% after 6 h, with an initial reaction rate of 18.2 mmol/L/h. Optimal reaction conditions found to be 50 οC, vinyl butyrate:butanol molar ratio 3:1, vinyl butyrate concentration 100 mM and enzyme loading of 11U. Reusability studies of imFocut5a showed that after 4 consecutive runs, the reaction yield reaches the 54% of the maximum. The efficient bioconversion offers a sustainable and environmentally friendly process for the production of “natural” aroma compounds essential for the Food Industry.

Nikolaivits E et al (2017) Journal of Agricultural and Food Chemistry (in press)

High activity and selectivity immobilized lipase on Fe3O4 nanoparticles for banana flavour synthesis (research)

Lipase (E.C. from Thermomyces lanuginosus (TL) was directly bonded, through multiple physical interactions, on citric acid functionalized monodispersed Fe3O4nanoparticles (NPs) in presence of a small amount of hydrophobic functionalities. A very promising scalable synthetic approach ensuring high control and reproducibility of the results, and an easy and green immobilization procedure was chosen for NPs synthesis and lipase anchoring. The size and structure of magnetic nanoparticles were characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The samples at different degree of functionalization were analysed through thermogravimetric measurements. Lipase immobilization was further confirmed by enzymatic assay and Fourier transform infrared (FT-IR) spectra. Immobilized lipase showed a very high activity recovery up to 144% at pH = 7 and 323% at pH = 7.5 (activity of the immobilized enzyme compared to that of its free form). The enzyme, anchored to the Fe3O4 nanoparticles, to be easy recovered and reused, resulted more stable than the native counterpart and useful to produce banana flavour. The immobilized lipase results less sensitive to the temperature and pH, with the optimum temperature higher of 5 °C and optimum pH up shifted to 7.5 (free lipase optimum pH = 7.0). After 120 days, free and immobilized lipases retained 64% and 51% of their initial activity, respectively. Ester yield at 40 °C for immobilized lipase reached 88% and 100% selectivity.

Sarno M et al (2017) Process Biochemistry 56:98-108

The Monthly Digest – Flavours – April 2017

Aromas and flavours
R & D abstracts – April 2017
Role of lactic acid bacteria on the yogurt flavour: a review

Considerable knowledge has been accumulated on the lactic acid bacteria (LAB) that affect the aroma and flavour of yogurt. This review focuses on the role of LAB in the production of flavour compounds during yogurt fermentation. The biochemical processes of flavour compound formation by LAB including glycolysis, proteolysis and lipolysis are summarized, with some key compounds described in detail. The flavour-related activities of LAB mostly depend on the species used for yogurt fermentation and some strategies have been developed to obtain more control of the flavour-forming process. Metabolic engineering can be a powerful tool to reroute the metabolic flux towards the efficient accumulation of the desired flavour compounds with the knowledge of the complex network of flavour-forming pathways and the availability of genetic tools. Further progress made in the omics-based techniques and the use of systems biology approaches are needed to fully understand, control, and steer flavour formation in yogurt fermentation processes.

Chen C et al (2017) International Journal of Food Properties (in press)

Milk-clotting properties of plant rennets and their enzymatic, rheological and sensory role in cheese making: a review

Plant rennets hold an important position amongst various coagulants used in cheese technology. The selection of a suitable plant coagulant is important due to the increasing global demands of cheese alongside reduced supply of calf rennet. Thus, a literature synthesis is presented to investigate recent achievements on their functional properties and enzymatic role in cheese making. Efforts have also been done to compare certain rheological and sensory properties of final products, arising from some plant and animal based rennets. In fact, some coagulants such as actinidin or dubiumin produce cheeses with sensory qualities similar to those produced by animal rennet. Others, like ginger, cucumisin or hieronymain proteases contribute to develop very different textures and flavors, due to excessive proteolytic activity and production of bitter peptides. For milk-clotting enzymes with high non-specific action, several improvement strategies have been developed to produce cheeses with sensory properties close to those of animal rennet. For example, the mixture of coagulants (cardosins/chymosin), the selection of appropriate milk or its ultrafiltration, as well as the increase of salting time of cheese during ripening could be efficient ways to improve texture and reduce bitterness. Concerning cheeses with high yield loss, the whey could be used for a traditional production of whey cheese. To conclude, the selection of appropriate plant rennet with high MCA/PA ratio and the optimization of all coagulation parameters play a central role in manufacturing cheese with superior rheological and sensory properties.

Ben Amira A et al (29017) International Journal of Food Properties (in press)

Flavour-active volatile compounds in beer: production, regulation and control

Many classes of compounds have been shown to play an important role in the development of flavour characteristics of beer. This significantly influences its taste and sensory properties, thus affecting its market performance. Despite the intensive research aimed at unravelling the precise mechanism and regulation of flavour formation in beer, current knowledge remains far from complete. Several reviews are available on the general composition of beer and yeast metabolic pathways involved in the production of volatile compounds in beer. However, a limited amount of work has been reported concerning the influence of some of the beer constituents and other important parameters on beer flavour. This paper reviews the current knowledge of the biochemistry behind flavour formation in beer and discusses the different factors that influence its formation and how it can be controlled during brewery fermentation.

Olaniran AO et al (2017) Journal of the Institute of Brewing (in press)

Aroma recovery of beer flavors by pervaporation through polydimethylsiloxane membranes

Five commercial beers and their corresponding no-alcohol presentations have been analyzed in terms of five aroma compounds: three alcohols and two esters (isobutyl alcohol, ethyl acetate, 2-methyl butanol, 3-methyl butanol, and isoamyl acetate). Substantial reductions of volatile aroma compounds have been encountered in these nonalcoholic beers. Pervaporation of these aromas in order to allow their recovery has been tested through two commercial membranes with polydimethylsiloxane active layers (Pervatech and Sulzer). Total fluxes are higher through the Pervatech membrane while individual aroma ones are similar for both the membranes. Mass recovery is higher for the Pervatech membrane for most of the aromas whereas their enrichment factors are better for the Sulzer membrane. The rest of the beer profile modulates to some extent the general trends referred. Mass recoveries and enrichment factors from 5 to 35% were obtained for the aromas tested. Mass recovery and enrichment factors could be improved by increasing membrane area or time of treatment. Pervaporation is a membrane technique which effectiveness has been proven to separate volatile organic compounds from aqueous mixtures. It could be useful in beer industry in a significant way, helping nonalcoholic beers to improve their organoleptic characteristics. In a near future, by controlling operating parameters involved in the full process, it will be possible to introduce this technique into the factories and produce a tastier and better quality beer by minimizing the lack of valuable aroma compounds.

Paz AI et al (2017) Journal of Food Process Engineering (in press)

Key volatile aroma compounds of lactic acid fermented malt based beverages – impact of lactic acid bacteria strains

This study aims to define the aroma composition and key aroma compounds of barley malt wort beverages produced from fermentation using six lactic acid bacteria (LAB) strains. Gas chromatography mass spectrometry–olfactometry and flame ionization detection was employed; key aroma compounds were determined by means of aroma extract dilution analysis. Fifty-six detected volatile compounds were similar among beverages. However, significant differences were observed in the concentration of individual compounds. Key aroma compounds (flavor dilution (FD) factors ≥16) were β-damascenone, furaneol, phenylacetic acid, 2-phenylethanol, 4-vinylguaiacol, sotolon, methional, vanillin, acetic acid, nor-furaneol, guaiacol and ethyl 2-methylbutanoate. Furthermore, acetaldehyde had the greatest odor activity value of up to 4266. Sensory analyses revealed large differences in the flavor profile. Beverage from L. plantarum Lp. 758 showed the highest FD factors in key aroma compounds and was correlated to fruity flavors. Therefore, we suggest that suitable LAB strain selection may improve the flavor of malt based beverages.

Dongmo NS et al (2017) Food Chemistry 229:565-573

Lactic acid fermentation drives the optimal volatile flavor-aroma profile of pomegranate juice

Pomegranate juice (PJ) fermented with Lactobacillus plantarum C2, POM1, and LP09, unstarted-PJ, and raw-PJ were characterized for the profile of the volatile components (VOC) by PT–GC–MS. Lactic acid fermentation through selected strains enhanced the flavor profile of PJ. Concentrations of desired compounds (e.g., alcohols, ketones, and terpenes) were positively affected, whereas those of non-desired aldehydes decreased. Unstarted-PJ mainly differentiated from fermented PJs for the highest levels of aldehydes and sulfur compounds, and in lesser extent of furans, whereas alcohols, ketones, and alkenes followed by terpenes and benzene derivatives mainly differentiated fermented PJs. As expected, the lowest level of VOC was found in raw-PJ. VOC profile reflected on the sensory features of fermented PJs, unstarted-PJ, and raw-PJ, which were evaluated using a consensus modified flavor profile based on 13 attributes. Fermented PJs were mainly discriminated by the higher intensity of floral, fruity and anise notes than the controls.

Di Cagno R et al (2017) International Journal of Food Microbiology 248:56-62

Biotransformation of α- and β-pinene into flavor compounds

Products that bear the label “natural” have gained more attention in the marketplace. In this approach, the production of aroma compounds through biotransformation or bioconversion has been receiving more incentives in economic and research fields. Among the substrates used in these processes, terpenes can be highlighted for their versatility and low cost; some examples are limonene, α-pinene, and β-pinene. This work focused on the biotransformation of the two bicyclic monoterpenes, α-pinene and β-pinene; the use of different biocatalysts; the products obtained; and the conditions employed in the process.

Vespermann KAC et al (2017) Applied Microbiology and Biotechnology 101:1805-1817

Optimizing bioconversion of ferulic acid to vanillin by Bacillus subtilis in the stirred packed reactor using Box-Behnken design and desirability function

A stirring bioreactor packed with a carbon fiber textiles (FT) biofilm formed by Bacillus subtiliswas used to produce vanillin from ferulic acid. Biofilm formation was characterized by scanning electron microscopy. The interactive effects of three variables on vanillin molar yield (M) and conversion efficiency of ferulic acid (E) were evaluated by response surface methodology (RSM) with a Box-Behnken design (BBD). The optimal conversion conditions with a maximum overall desirability D of 0.983 were obtained by a desirability function. Considering the actual operation, the confirmation tests were performed using the slightly modified optimal conditions (initial ferulic acid concentration 1.55 g/L, temperature 35°C, stirring speed 220 rpm). The results showed that M and E were 57.42 and 93.53%, respectively. This was only 1.03% and 1.87%, respectively, different from the predicted values, confirming the validity of the predicted models. These revealed that the stirred packed reactor could be successfully used in vanillin bioconversion from ferulic acid.

Chen P et al (2017) Food Science and Biotechnology 26 :143-152

Breeding of a sake yeast mutant with enhanced ethyl caproate productivity in sake brewing using rice milled at a high polishing ratio

Sake yeast produces a fruity flavor known as ginjo-ko—which is mainly attributable to ethyl caproate and isoamyl acetate—during fermentation in sake brewing. The production of these flavor components is inhibited by unsaturated fatty acids derived from the outer layer of rice as raw material. We isolated three mutants (hec2, hec3, and hec6) with enhanced ethyl caproate productivity in sake brewing using rice milled at a high polishing ratio from a cerulenin-resistant mutant derived from the hia1 strain, which shows enhanced isoamyl acetate productivity. The hec2 mutant had the homozygous FAS2 mutation Gly1250Ser, which is known to confer high ethyl caproate productivity. When the homozygous FAS2 mutation Gly1250Ser was introduced into strain hia1, ethyl caproate productivity was increased but neither this nor intracellular caproic acid content approached the levels observed in the hec2 mutant, indicating that a novel mutation was responsible for the high ethyl caproate productivity. We also found that the expression of EEB1 encoding acyl-coenzyme A: ethanol O-acyltransferase (AEATase) and enzymatic activity were increased in the hec2 mutant. These results suggest that the upregulation of EEB1 expression and AEATase activity may also have contributed to the enhancement of ethyl caproate synthesis from ethanol and caproyl-CoA. Our findings are useful for the brewing of sake with improved flavor due to high levels of isoamyl acetate and ethyl caproate.

Takahashi T et al (2017) Journal of Bioscience and Bioengineering (in press)

The Monthly Digest – March 2017

Solid-state fermentative production of aroma esters by Myroides sp. ZB35 and its complete genome sequence

Consumers prefer biotechnological food products with high nutritional values and good flavors. Solid-state fermentation is a commonly used technique with a long history. In the present study, Myroides sp. ZB35 was used in solid-state fermentative production of aroma volatiles on a rice medium. Using the headspace solid phase microextraction coupled with gas chromatography–mass spectrometry technique and authentic standards, 22 esters with molecular weight ranging from 102 to 172 were identified. At 192 h, the esters reached a total concentration of 1774 μg/kg. Subsequently, the complete genome of ZB35 was sequenced using the PacBio RS II platform. ZB35 has a single circular chromosome of 4,065,010 bp with a GC content of 34.1% and six putative novel esterase genes were found. ZB35 is the first bacterium here discovered being capable of producing so many kinds of aroma esters. The data revealed here would provide helpful information for further developing this strain as a promising source of aroma esters relevant in food and fragrance industries and the source of novel enzymes with potential usages.

Xiao Z et al (2017) Journal of Biotechnology (in press)


Brewing with 100 % unmalted grains: barley, wheat, oat and rye

Whilst beers have been produced using various levels of unmalted grains as adjuncts along with malt, brewing with 100 % unmalted grains in combination with added mashing enzymes remains mostly unknown. The aim of this study was to investigate the brewing potential of 100 % unmalted barley, wheat, oat and rye in comparison with 100 % malt. To address this, identical brewing methods were adopted at 10-L scale for each grain type by applying a commercial mashing enzyme blend (Ondea® Pro), and selected quality attributes were assessed for respective worts and beers. Different compositions of fermentable wort carbohydrates were observed in the worts (all at ca. 12°P), and in particular oat wort had lower concentration of maltose compared to the others, resulting in the lowest concentration of alcohol in final beer. Moreover, wort made from unmalted grains also showed lower free amino nitrogen and higher viscosity than malt wort. Furthermore, the use of 100 % unmalted grains resulted in a decrease in the levels of colour and brightness, as well as higher alcohols and esters in the final beers. Consequently, the study provides valuable information for exploring beer brewing with 100 % unmalted barley, oat, rye or wheat using exogenously added enzymes. It also helps to understand the process ability by revealing specific needs when manufacturing different type of beers from unmalted grains, potentially paving the way to process optimisation and development of future products.

Zhuang S et al (29017) European Food Research and Technology 243:447-454


Effect of non-Saccharomyces yeasts on the volatile chemical profile of Shiraz wine

This study evaluated the impact that the non-Saccharomyces yeasts, Torulaspora delbrueckii (TD), Lachanceathermotolerans (LT), Pichia kluyveri (PK), Metschnikowia pulcherrima (MP), Candida zemplinina(CZ) and Kazachstania aerobia (KA), in sequential inoculation with Saccharomyces cerevisiae (SC), had on the volatile chemical profile of Shiraz wine. Shiraz grape must was inoculated with monocultures of the non-Saccharomyces yeasts, which were allowed to ferment until 2% v/v ethanol concentration was reached at which point, SC was added to complete the alcoholic fermentation. The control was SC in monoculture. The final wines were subjected to solid phase microextraction-GC × GC-time of flight-MS to evaluate the untargeted volatile metabolite profile of each treatment. Each fermentation produced a unique chemical profile. The LT–SC sequential fermentation was the most significantly different from the control primarily in the ester, alcohol and terpene profile. The KA–SC sequential fermentation had the highest amount of volatile acidity, and the PK–SC sequential fermentation had a relatively high amount of acetaldehyde and a few esters. The MP–SC sequential fermentation also revealed a higher concentration of several esters. The TD–SC sequential fermentation was notable for its lack of a distinct pattern in comparison with that of the other fermentations.Given these characteristics, the LT–SC sequential fermentation showed the most potential for increased chemical complexity of the Shiraz volatile profile.The results demonstrate that there is no clear, singular trend for how different, non-Saccharomycesspecies of yeast – once thought to be wholly unfavourable in wine fermentations – will impact wine chemistry, flavour or quality. Each species presents unique metabolic characteristics, many of which could be beneficial, and their potential application in the wine industry should be considered.

Whitener MEB et al (2017) Australian Journal of Grape and Wine Research (in press) DOI: 10.1111/ajgw.12269


Impact of sequential co-culture fermentations on flavour characters of Solaris wines

Nowadays, the use of non-Saccharomyces yeasts in combination with Saccharomyces cerevisiae is being recognised to enhance the analytical composition of the wines. The aim of this work was to evaluate the influence of indigenous non-Saccharomyces yeasts on the flavour character of wines from the cool-climate grape cultivar Solaris in Denmark. The volatile and non-volatile compounds as well as the sensory properties of wines were evaluated. Solaris wines with Hanseniaspora uvarum sequentially inoculated with S. cerevisiae produced a larger amount of glycerol as well as heptyl acetate and 2-phenylethyl acetate. This co-culture fermentation also produced higher amounts of ethyl acetate and acetic acid, reducing the possibility of its use in winemaking. Three Metschnikowia strains, a M. chrysoperlae strain and two M. fructicola strains, gave a comparable production of volatile compounds. These wines were characterised by several floral and fruity attributes. The Metschnikowia strains turned out to be promising in winemaking from Solaris grapes.

Liu J et al (2017) European Food Research and Technology 243:437-445


Influence of different yeast/lactic acid bacteria combinations on the aromatic profile of red Bordeaux wine

The typical fruity aroma of red Bordeaux wines depends on the grape variety but also on microbiological processes, such as alcoholic and malolactic fermentations. These transformations involve respectively the yeast Saccharomyces cerevisiae and the lactic acid bacteria Oenococcus oeni. Both species play a central role in red winemaking but their quantitative and qualitative contribution to the revelation of the organoleptic qualities of wine has not yet been fully described. The aim of this study was to elucidate the influence of sequential inoculation of different yeast and bacteria strains on the aromatic profile of red Bordeaux wine. All microorganisms completed fermentations and no significant difference was observed between tanks regarding the main oenological parameters until 3 months’ aging. Regardless of the yeast strain, B28 bacteria required the shortest period to completely degrade the malic acid, compared to the other strain. . Quantification of 73 major components highlighted a specific volatile profile corresponding to each microorganism combination. However, the yeast strain appeared to have a predominant effect on aromatic compound levels, as well as on fruity aroma perception. Yeasts had a greater impact on wine quality and have more influence on the aromatic style of red wine than bacteria.

Gammacurta M et al (2017) Journal of the Science of Food and Agriculture (in press) DOI: 10.1002/jsfa.8272


Selected yeasts to enhance phenolic content and quality in red wine from low pigmented grapes

The aim of this work was to enhance—by yeast activity—the quality of red wine produced from black grapes of the Calabrian Gaglioppo variety, used as a model for grapes with reduced synthesis of anthocyanins. Six selected strains of Saccharomyces cerevisiae were used to control winemaking trials. Among the wines, there are significant differences, due to the wine starter used. The following technological parameters were significantly different from strain to strain: total acidity, alcoholic degree, tartaric, malic, lactic, and acetic acid, and free and total SO2; moreover, the following phenolic parameters were significantly different from strain to strain: A420, A520, A620, colour intensity, colour hue, Folin–Ciocalteu index, percentage of DPPH inactivation, total anthocyanins, total polyphenols (A280), total tannins, delphinidin-3-glucoside, cyanidin-3-glucoside, petunidin-3-glucoside, peonidin-3-glucoside, and malvidin-3-glucoside. From a sensory standpoint, significant differences were observed among wine samples during a short bottle aging. Data validate the main role that wine yeast selection plays in enhancing the quality of red wine from low pigmented grape.

Caridi A et al (2017) European Food Research and Technology 243 :367-378


Aroma precursors in grapes and wine: Flavor release during wine production and consumption

Pioneering investigations into precursors to fruity and floral flavors established the importance of terpenoid and C13-norisoprenoid glycosides to the flavor of aromatic wines. Nowadays flavor precursors in grapes and wine are known to be structurally diverse, encompassing glycosides; amino acid conjugates; odorless volatiles; hydroxycinnamic acids and many others. Flavor precursors mainly originate in the grape berry, but also from oak or other materials involved in winemaking. Flavors are released from precursors during crushing and subsequent production steps by enzymatic and non-enzymatic transformations, via microbial glycosidases; esterases; C-S lyases and decarboxylases; and through acid catalysed hydrolysis and chemical rearrangements. Flavors can also be liberated from glycosides and amino acid conjugates by oral microbiota. Hence, it is increasingly likely that flavor precursors contribute to retronasal aroma formation through in-mouth release during consumption, prompting a shift in focus from identifying aroma precursors in grapes to understanding aroma precursors present in bottled wine.

Parker M et al (2017) Journal of Agricultural and Food Chemistry (in press) DOI: 10.1021/acs.jafc.6b05255

Valorization of milk whey for aroma compounds production

The aim of this collaborative study between Eptes and universities in Greece, Germany, and Poland was to investigate the capacity of different yeast strains to grow on cow and goat acid whey, to assimilate different carbon sources and to produce aroma compounds. Twenty different yeasts (with GRAS status) were tested. Two kinds of acid whey, from cows and goats, were characterized in terms of their main chemical components (carbohydrates, organic acids, aminoacids, FAN, total nitrogen) using HPLC techniques and chemical methods. The most effective yeast strains were found to be those belonging to the genera Kluyveromyces, Wickerhamomyces and Saccharomyces. Three K. marxianus strains, one wine strain S. cerevisiae and one strain of W. anomalus were selected based on their ability to grow on whey agar, assimilation profiles and aroma profiles. Their ability to grow on mixed cow and goat acid whey under varying environmental conditions (pH level, supplementation, temperature, oxygen availability) was evaluated.

A selection of 80 samples were evaluated for their flavours profile compositions. The influence of pH, temperature, aeration and supplementation level on the volatile fraction of acid whey fermented with selected yeast strains, i.e. Saccharomyces cerevisiae, Wickerhamomyces anomalus and Kluyveromyces marxianus, was investigated in terms of aroma compounds production. Headspace solid-phase microextraction technique (HS-SPME) coupled to the gas chromatography–mass spectrometry (GC-MS) analysis was applied to characterize volatile compounds of the samples. More than 50 compounds were detected, which belonged to different chemical classes and could come either from different reaction pathways occurring during fermentation or from raw material. The most important compounds were found to be 3-methyl-1-butanol, ethyl alcohol, phenylethyl alcohol, ethyl acetate, ethyl octanoate, 2-phenylethyl acetate, acetic acid, butanoic acid, etc. that contribute to the aroma character of the samples with alcoholic, malty, cheesy, sour, sweet and rose-like notes. In general, the aroma profile of the samples depended on the yeast strain used, and was greatly affected by the temperature and aeration conditions applied during fermentation.

The Monthly Digest – February 2017

Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae

The aim of this study was to investigate the possibility of using tomato and red pepper pomaces for the production eight-carbon volatiles by Trichoderma atroviride and Aspergillus sojae. The fermentation of tomato and pepper pomace-based media by both moulds was conducted in shake flasks and bioreactors. Microbial growth behaviours and fermentation abilities of T. atroviride and A. sojae under both fermentation conditions were followed by microbial counting. The production of flavours from tomato and pepper pomaces by fungal metabolism was determined by gas chromatography–olfactometry, gas chromatography–mass spectrometry and sensory analysis. The results showed that T. atroviride grew faster than A. sojae, and the survival of T. atroviride in the tomato pomace was longer than that of A. sojae. However, T. atroviride grew slower than A. sojae in the pepper pomace. Eight-carbon flavour compounds, including (Z)-1,5-octadien-3-ol, 1-octen-3-ol, (E)-2-octenal and (E)-2-octenol, were produced by T. atroviride and A. sojae from the tomato and pepper pomaces. The highest production levels (265.55 ± 2.79 and 187.47 ± 0.92 μg kg−1) were observed for 1-octen-3-ol in the tomato fermentation by T. atroviride and A. sojae, respectively. The relationships between volatile compounds and their flavour characteristics in tomato and pepper pomaces were analysed using principal component analysis.

Güneser O and Yüceer YK (2017) Journal of Bioscience and Bioengineering (in press)

The role of immobilized rennet on carbon cloth in flavor development during ripening of Gouda cheese

Rennet-free Gouda (RFG) cheese was prepared to investigate the influence of rennet on the non-volatile and volatile profiles of cheese and was characterized by HPLC and GC/MS analyses. Chymosin, a major protease in rennet, was immobilized onto oxidized and chemically modified carbon cloth. The chymosin immobilization efficiency was 60.4%, and the milk-clotting activity used as an index of the stability of the immobilized chymosin decreased by around 20% in 2 weeks. However, the activity was maintained at 70–80% from 2 weeks to 32 weeks and was more stable than that of chymosin solution alone. Non-volatile (organic acids) and volatile profiles of the RFG cheese and rennet-containing normal Gouda cheese were not significantly different during ripening with a few exceptions. Therefore, it can be concluded that cheese flavor is developed by lactic acid fermentation, irrespective of the presence of rennet.

Jeong SH et al (2016) Food Science and Biotechnology 25:1561-1567

Flavor improvement of fermented soy sauce by extrusion as soybean meal pretreatment

Flavor, an important index of soy sauce quality, includes odor and taste which mainly depend on volatile compounds and amino acids. Soy sauce made from extruded mixture of soy meal and flour (EMSF) was employed in fermentation to improve the flavor of final product. Combination of Headspace solid phase microextraction (HS-SPME) and GC-MS was employed to identify the volatile compounds, 40 volatile compounds were identified in soy sauce made from EMSF and 24 volatile compounds in soy sauce made form cooked mixture of soy meal and flour (CMSF). Pyrazines and their derivatives occupied one-third of the total content of volatile compounds. Both samples had the same amino acid number, while total content of amino acids and contents of 15 kinds of amino acids in soy sauce made from EMSF were significantly higher than CMSF. Extrusion had the advantage in low cost and capability of continuous processing, extruded mixture of soy meal and flour were used in soy sauce fermentation. Content of amino acids was improved by extrusion, this pretreatment method also increased volatile compounds of soy sauce and produced more desirable flavor and taste soy sauce.

Zhang DJ et al (2017) Journal of Food Processing and Preservation (in press)

Aroma compounds and characteristics of noble-rot wines of chardonnay grapes artificially botrytized in the vineyard

Aroma characteristics and their impact volatile components of noble-rot wines elaborated from artificial botrytized Chardonnay grapes, obtained by spraying Botrytis cinerea suspension in Yuquan vineyard, Ningxia, China, were explored in this work. Dry white wine made from normal-harvested grapes and sweet wine produced from delay-harvested grapes were compared. Wine aromas were analysed by trained sensory panelists, and aroma compounds were determined by SPME-GC-MS. Results indicated that esters, fatty acids, thiols, lactones, volatile phenols and 2-nonanone increased markedly in noble-rot wines. In addition to typical aromas of noble-rot wines, artificial noble-rot wines were found to contain significant cream and dry apricot attributes. Partial Least-Squares Regression models of aroma characteristics against aroma components revealed that non-fermentative odorants were the primary contributor to dry apricot attribute, especially, thiols, C13-norisoprenoids, lactones, terpenols and phenolic acid derivatives, while cream attribute was dependent on both fermentative and non-fermentative volatile components. Chemical compounds studied in the article: 2-nonanone (PubChem: 13187); ethyl 4-hydroxybenzoate (PubChem: 8434); ethyl isovalerate (PubChem: 7945); α-terpineol (PubChem: 17100); isopentanoic acid (PubChem: 3085367); 3-methyl-3-sulfanyl butanol (PubChem: 520682); 3-sulfanylhexanol (PubChem: 521348); γ-nonalactone (PubChem: 7710); γ-decalactone (PubChem: 12813); phenyl acetic acid (PubChem: 999)

Wang XJ et al (2017) Food Chemistry (in press)

Genetic and phenotypic intraspecific variability of non-Saccharomyces yeasts populations from La Rioja winegrowing region (Spain)

To determine the intraspecific genetic diversity within five non-Saccharomyces yeast species and the diversity in phenotypic characteristic related to their technological properties. Seventy-one non-Saccharomyces yeasts isolated from different fermentations and facilities of the DOCa Rioja (Spain) belonging to five different wine species (Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, Zygosaccharomyces bailii and Williopsis pratensis) were subjected to clonal characterization by RAPD-PCR, which evidenced wide diversity between them. They were also submitted to a screening for some oenological traits related to the improvement of the aroma of the wine and yeast development in musts. Strains within the same species showed different enzyme activities, tolerated different levels of SO2 and possessed different killer phenotypes. These characteristics made them adjust better or worse to specific vinification processes or wine quality criteria.

A significant genetic and phenotypic variation within the non-Saccharomyces species studied was found, which makes necessary to carry out a selection process in each one. Williopsis pratensis, a species that has not been thoroughly explored, may deserve further consideration for oenological applications. Due to the wide range of variation within species, the strains adaptation to the SO2 levels in musts has to be taken into account in selection processes.

  1. González-Arenzana et al (2017) Journal of Applied Microbiology 122:378-388
Effect of sequential fermentations and grape cultivars on volatile compounds and sensory profiles of Danish wines

There has been increasing interest in the use of selected non-Saccharomyces yeasts in co-culture with Saccharomyces cerevisiae. In this work, three non-Saccharomyces yeast strains (Metschnikowia viticola, Metschnikowia fructicola and Hanseniaspora uvarum) indigenously isolated in Denmark, were used in sequential fermentations with S. cerevisiae on three cool-climate grape cultivars Bolero, Rondo and Regent. During the fermentations, the yeast growth was determined as well as key oenological parameters, volatile compounds and sensory properties of finished rosé wines. The different non-Saccharomyces strains and cool-climate grape cultivars produced wines with a distinctive aromatic profile. A total of 67 volatile compounds were identified, including 43 esters, 14 alcohols, 5 acids, 2 ketones, a C13-norisoprenoid, a lactone and a sulphur compound. The use of M. viticola in sequential fermentation with S. cerevisiae resulted in richer berry and fruity flavours in wines. However, the sensory plot showed a more clear separation among wine samples by grape cultivars compared with yeast strains. Knowledge on the influence of indigenous non-Saccharomyces strains and grape cultivars on the flavor generation contributed to producing diverse wines in the cool-climate wine regions.

Liu J et al (2017) Journal of the Science of Food and Agriculture (in press)

Linking wine lactic acid bacteria diversity with wine aroma and flavour

In the last two decades knowledge on lactic acid bacteria (LAB) associated with wine has increased considerably. Investigations on genetic and biochemistry of species involved in malolactic fermentation, such as Oenococcus oeni and of Lactobacillus have enabled a better understand of their role in aroma modification and microbial stability of wine. In particular, the use of molecular techniques has provided evidence on the high diversity at species and strain level, thus improving the knowledge on wine LAB taxonomy and ecology. These tools demonstrated to also be useful to detect strains with potential desirable or undesirable traits for winemaking purposes. At the same time, advances on the enzymatic properties of wine LAB responsible for the development of wine aroma molecules have been undertaken. Interestingly, it has highlighted the high intraspecific variability of enzymatic activities such as glucosidase, esterase, proteases and those related to citrate metabolism within the wine LAB species. This genetic and biochemistry diversity that characterizes wine LAB populations can generate a wide spectrum of wine sensory outcomes. This review examines some of these interesting aspects as a way to elucidate the link between LAB diversity with wine aroma and flavour. In particular, the correlation between inter- and intra-species diversity and bacterial metabolic traits that affect the organoleptic properties of wines is highlighted with emphasis on the importance of enzymatic potential of bacteria for the selection of starter cultures to control MLF and to enhance wine aroma.

Cappello MS et al (2017) International Journal of Food Microbiology 243 :16-27

Effect of mixed yeast starter on volatile flavor compounds in Chinese rice wine during different brewing stages

The volatile compounds of Chinese rice wine fermented by Saccharomyces cerevisiae FC 15 and S. cerevisiae BR 30 and their combination (MIX) were investigated. The results showed that the wines fermented with different starters differed mainly in the numbers of alcohols and esters. Furthermore, the amounts of volatile compounds among three starters were also obviously different. After clarification, the loss of esters was greater than that of alcohols and aldehydes, whereas after sterilization, the loss of aldehydes was the greatest. PCA showed diethyl succinate to be the key flavor component. Moreover, short-chain fatty acid ethyl esters were main flavor substances during fermentation, whereas long-chain fatty acid ethyl esters were typical compounds after clarification and sterilization. Analysis of the ratio of higher alcohols to esters showed that BR 30 wine presented a stronger ester aroma than MIX wine, whereas FC 15 wine exhibited an outstanding alcohol flavor. Sensory evaluation indicated that FC 15 wine was intense in alcohol-aroma, whereas BR 30 wine exhibited high levels of cereal-aroma. Furthermore, both MIX and BR 30 wine were highly assessed in continuation and full body mouth-feel. It means that flavor characteristic of Chinese rice wine could be adjust by combination of yeast starters.

Yang Y et al (2017) LWT – Food Science and Technology 78:373-381

A grapevine cytochrome P450 generates the precursor of wine lactone, a key odorant in wine

Monoterpenes are important constituents of the aromas of food and beverages, including wine. Among monoterpenes in wines, wine lactone has the most potent odor. It was proposed to form via acid-catalyzed cyclization of (E)-8-carboxylinalool during wine maturation. It only reaches very low concentrations in wine but its extremely low odor detection threshold makes it an important aroma compound.

Using LC-MS/MS, we show here that the (E)-8-carboxylinalool content in wines correlates with their wine lactone content and estimate the kinetic constant for the very slow formation of wine lactone from (E)-8-carboxylinalool. We show that (E)-8-carboxylinalool is accumulated as a glycoside in grape (Vitis vinifera) berries and that one of the cytochrome P450 enzymes most highly expressed in maturing berries, CYP76F14, efficiently oxidizes linalool to (E)-8-carboxylinalool.

Our analysis of (E)-8-carboxylinalool in Riesling × Gewurztraminer grapevine progeny established that the CYP76F14 gene co-locates with a quantitative trait locus for (E)-8-carboxylinalool content in grape berries.

Our data support the role of CYP76F14 as the major (E)-8-carboxylinalool synthase in grape berries and the role of (E)-8-carboxylinalool as a precursor to wine lactone in wine, providing new insights into wine and grape aroma metabolism, and new methods for food and aroma research and production.

Ilc et al (2017) New Phytologist

Optimization of the production of 1-phenylethanol using enzymes from flowers of tea (Camellia sinensis) plants

1-Phenylethanol (1PE) can be used as a fragrance in food flavoring and cosmetic industries and as an intermediate in the pharmaceutical industry. 1PE can be synthesized from acetophenone, and the cost of 1PE is higher than the cost of acetophenone. Therefore, it is important to establish an effective and low-cost approach for producing 1PE. Our previous studies found that tea (Camellia sinensis) flowers, which are an abundant and waste resource, contained enzymes that could transform acetophenone to 1PE. In the present study, we extracted crude enzymes from tea flowers and optimized the production conditions of 1PE using response surface methodology. The optimized conditions were an extraction pH of 7.0, a reaction pH of 5.3, a reaction temperature of 55 °C, a reaction time of 100 min, a coenzyme NADPH concentration of 3.75 μmol/mL in the reaction assay, and a substrate acetophenone concentration of 1.25 μmol/mL in the reaction assay. The results provide essential information for future industrial 1PE production using plant-derived enzymes.

Dong F et al (2017) Molecules 22(1):131; doi:10.3390/molecules22010131

Bioengineering of the plant culture of Capsicum frutescens with vanillin synthase gene for the production of vanillin

Production of vanillin by bioengineering has gained popularity due to consumer demand toward vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively. As the ferulic acid precursor and vanillin were found to be the intermediates in the phenylpropanoid biosynthetic pathway of Capsicum species, this work serves as a proof-of-concept for vanillin production using Capsicum frutescens (C. frutescens or hot chili pepper). The cells of C. frutescens were genetically transformed with a codon optimized VpVAN gene via biolistics. Transformed explants were selected and regenerated into callus. Successful integration of the gene cassette into the plant genome was confirmed by polymerase chain reaction. High-performance liquid chromatography was used to quantify the phenolic compounds detected in the callus tissues. The vanillin content of transformed calli was 0.057% compared to 0.0003% in untransformed calli.

Yang Chee MJ et al (2017) Molecular Biotechnology 59:1-8