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

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