Tuning Chocolate Flavor through Development of Thermotolerant Saccharomyces cerevisiae Starter Cultures with Increased Acetate Ester Production

ABSTRACT Microbial starter cultures have extensively been used to enhance the consistency and efficiency of industrial fermentations. Despite the advantages of such controlled fermentations, the fermentation involved in the production of chocolate is still a spontaneous process that relies on the natural microbiota at cocoa farms. However, recent studies indicate that certain thermotolerant Saccharomyces cerevisiae cultures can be used as starter cultures for cocoa pulp fermentation. In this study, we investigate the potential of specifically developed starter cultures to modulate chocolate aroma. Specifically, we developed several new S. cerevisiae hybrids that combine thermotolerance and efficient cocoa pulp fermentation with a high production of volatile flavor-active esters. In addition, we investigated the potential of two strains of two non-Saccharomyces species that produce very large amounts of fruity esters (Pichia kluyveri and Cyberlindnera fabianii) to modulate chocolate aroma. Gas chromatography-mass spectrometry (GC-MS) analysis of the cocoa liquor revealed an increased concentration of various flavor-active esters and a decrease in spoilage-related off-flavors in batches inoculated with S. cerevisiae starter cultures and, to a lesser extent, in batches inoculated with P. kluyveri and Cyb. fabianii. Additionally, GC-MS analysis of chocolate samples revealed that while most short-chain esters evaporated during conching, longer and more-fat-soluble ethyl and acetate esters, such as ethyl octanoate, phenylethyl acetate, ethyl phenylacetate, ethyl decanoate, and ethyl dodecanoate, remained almost unaffected. Sensory analysis by an expert panel confirmed significant differences in the aromas of chocolates produced with different starter cultures. Together, these results show that the selection of different yeast cultures opens novel avenues for modulating chocolate flavor.

[1]  Jan Steensels,et al.  Improving industrial yeast strains: exploiting natural and artificial diversity , 2014, FEMS microbiology reviews.

[2]  N. Rastogi,et al.  Theobroma cacao L., "The Food of the Gods": Quality Determinants of Commercial Cocoa Beans, with Particular Reference to the Impact of Fermentation , 2011 .

[3]  C. Wittmann,et al.  The Key to Acetate: Metabolic Fluxes of Acetic Acid Bacteria under Cocoa Pulp Fermentation-Simulating Conditions , 2014, Applied and Environmental Microbiology.

[4]  P. Dimick,et al.  Chemistry of flavour development in chocolate , 1994 .

[5]  B. Biehl,et al.  Acidification, proteolysis and flavour potential in fermenting cocoa beans , 1985 .

[6]  I. Dunham,et al.  Rapid assessment of S. cerevisiae mating type by PCR. , 1990, Trends in genetics : TIG.

[7]  K. Verstrepen,et al.  Large-scale robot-assisted genome shuffling yields industrial Saccharomyces cerevisiae yeasts with increased ethanol tolerance , 2015, Biotechnology for Biofuels.

[8]  X. Scheldeman,et al.  Diversity of Cacao Trees in Waslala, Nicaragua: Associations between Genotype Spectra, Product Quality and Yield Potential , 2013, PloS one.

[9]  P. Schieberle,et al.  Evaluation of Key Odorants in Milk Chocolate and Cocoa Mass by Aroma Extract Dilution Analyses , 1997 .

[10]  D. Crouzillat,et al.  Chemical composition and flavor of ecuadorian cocoa liquor. , 2002, Journal of agricultural and food chemistry.

[11]  R. Buamah,et al.  The controlled fermentation of cocoa (theobroma cacao L) using yeasts: Enzymatic process and associated physico‐chemical changes in cocoa sweatings , 1999 .

[12]  M. Owusu.  Influence of raw material and processing on aroma in chocolate , 2010 .

[13]  Sonia Collin,et al.  Relationship between procyanidin and flavor contents of cocoa liquors from different origins. , 2004, Journal of agricultural and food chemistry.

[14]  K. Verstrepen,et al.  Production and biological function of volatile esters in Saccharomyces cerevisiae , 2010, Microbial biotechnology.

[15]  T. White.  Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics , 1990 .

[16]  Guy Derdelinckx,et al.  Flavor-active esters: adding fruitiness to beer. , 2003, Journal of bioscience and bioengineering.

[17]  G. Howat,et al.  Cocoa Fermentation , 1954, Nature.

[18]  R. Schwan.  Cocoa Fermentations Conducted with a Defined Microbial Cocktail Inoculum , 1998, Applied and Environmental Microbiology.

[19]  C. L. Ramos,et al.  Dynamic behavior of Saccharomyces cerevisiae, Pichia kluyveri and Hanseniaspora uvarum during spontaneous and inoculated cocoa fermentations and their effect on sensory characteristics of chocolate , 2015 .

[20]  I. S. Pretorius,et al.  Yeast and its Importance to Wine Aroma - A Review , 2019, South African Journal of Enology & Viticulture.

[21]  Peter Schieberle,et al.  Identification of the key aroma compounds in cocoa powder based on molecular sensory correlations. , 2006, Journal of agricultural and food chemistry.

[22]  S. Collin,et al.  Use of gas chromatography-olfactometry to identify key odorant compounds in dark chocolate. Comparison of samples before and after conching. , 2002, Journal of agricultural and food chemistry.

[23]  Anders Blomberg,et al.  Trait Variation in Yeast Is Defined by Population History , 2011, PLoS genetics.

[24]  J. Chun,et al.  Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. , 2012, International journal of systematic and evolutionary microbiology.

[25]  C. Hansen,et al.  Enzyme activities in cocoa beans during fermentation , 1998 .

[26]  K. Verstrepen,et al.  Expression Levels of the Yeast Alcohol Acetyltransferase Genes ATF1, Lg-ATF1, and ATF2 Control the Formation of a Broad Range of Volatile Esters , 2003, Applied and Environmental Microbiology.

[27]  L. Jespersen,et al.  Occurrence and diversity of yeasts involved in fermentation of West African cocoa beans. , 2005, FEMS yeast research.

[28]  K. Verstrepen,et al.  Detailed Analysis of the Microbial Population in Malaysian Spontaneous Cocoa Pulp Fermentations Reveals a Core and Variable Microbiota , 2013, PloS one.

[29]  Julia Kluge,et al.  The Science Of Chocolate , 2016 .

[30]  G. Fleet,et al.  Yeasts are essential for cocoa bean fermentation. , 2014, International journal of food microbiology.

[31]  K. Verstrepen,et al.  Large-Scale Selection and Breeding To Generate Industrial Yeasts with Superior Aroma Production , 2014, Applied and Environmental Microbiology.

[32]  D. Nielsen,et al.  Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation. , 2013, International journal of food microbiology.

[33]  L. De Vuyst,et al.  On-farm implementation of a starter culture for improved cocoa bean fermentation and its influence on the flavour of chocolates produced thereof. , 2012, Food microbiology.

[34]  P. Vandamme,et al.  Dynamics and Biodiversity of Populations of Lactic Acid Bacteria and Acetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans in Ghana , 2007, Applied and Environmental Microbiology.

[35]  B. Biehl,et al.  Cocoa-specific aroma precursors are generated by proteolytic digestion of the vicilin-like globulin of cocoa seeds , 1994 .

[36]  J. Walter,et al.  Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis , 2001, Applied and Environmental Microbiology.

[37]  M. Petersen,et al.  EFFECT OF FERMENTATION METHOD, ROASTING AND CONCHING CONDITIONS ON THE AROMA VOLATILES OF DARK CHOCOLATE , 2012 .

[38]  R. Buamah,et al.  Pure yeast culture fermentation of cocoa (Theobroma cacao L): effect on yield of sweatings and cocoa bean quality , 1997 .

[39]  Mark Fowler,et al.  Matrix effects on flavour volatiles release in dark chocolates varying in particle size distribution and fat content using GC-mass spectrometry and GC-olfactometry. , 2009 .

[40]  C. L. Ramos,et al.  Impact of different cocoa hybrids (Theobroma cacao L.) and S. cerevisiae UFLA CA11 inoculation on microbial communities and volatile compounds of cocoa fermentation. , 2014, Food research international.

[41]  K. Verstrepen,et al.  Parameters Affecting Ethyl Ester Production by Saccharomyces cerevisiae during Fermentation , 2007, Applied and Environmental Microbiology.

[42]  M. Petersen,et al.  Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate , 2014 .

[43]  Fermentation of cacao (Theobroma cacao L.) seeds with a hybrid Kluyveromyces marxianus strain improved product quality attributes. , 2008, FEMS yeast research.

[44]  F. Karst,et al.  Optimisation of interdelta analysis for Saccharomyces cerevisiae strain characterisation. , 2003, FEMS microbiology letters.

[45]  A. Wheals,et al.  The Microbiology of Cocoa Fermentation and its Role in Chocolate Quality , 2004, Critical reviews in food science and nutrition.

[46]  Mark Fowler,et al.  Flavor Formation and Character in Cocoa and Chocolate: A Critical Review , 2008, Critical reviews in food science and nutrition.

[47]  D. Lim,et al.  The contribution of genotype to cocoa (Theobroma cacao L.) flavour , 1994 .

[48]  D. Lane.  16S/23S rRNA sequencing , 1991 .

[49]  J. S. Bonvehí.  Investigation of aromatic compounds in roasted cocoa powder , 2005 .

[50]  E. Lugo-Cervantes,et al.  Identification of predominant yeasts associated with artisan Mexican cocoa fermentations using culture-dependent and culture-independent approaches , 2015, World journal of microbiology & biotechnology.

[51]  Peter Schieberle,et al.  Changes in key aroma compounds of Criollo cocoa beans during roasting. , 2008, Journal of agricultural and food chemistry.

[52]  Kenneth B. Miller,et al.  Cocoa and Coffee , 2007 .

[53]  K. Verstrepen,et al.  Breeding Strategy To Generate Robust Yeast Starter Cultures for Cocoa Pulp Fermentations , 2015, Applied and Environmental Microbiology.

[54]  K. Verstrepen,et al.  Taming wild yeast: potential of conventional and nonconventional yeasts in industrial fermentations. , 2014, Annual review of microbiology.

[55]  G. Fleet,et al.  The effect of lactic acid bacteria on cocoa bean fermentation. , 2015, International journal of food microbiology.

[56]  J. Palmer,et al.  Investigating Deep Phylogenetic Relationships among Cyanobacteria and Plastids by Small Subunit rRNA Sequence Analysis 1 , 1999, The Journal of eukaryotic microbiology.

[57]  P. de Vos,et al.  Yeast diversity of Ghanaian cocoa bean heap fermentations. , 2009, FEMS yeast research.

[58]  C. Kurtzman,et al.  Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene , 1997, Journal of clinical microbiology.

[59]  M. Meilgaard,et al.  Prediction of flavor differences between beers from their chemical composition , 1982 .

[60]  T. Bruns,et al.  ITS primers with enhanced specificity for basidiomycetes ‐ application to the identification of mycorrhizae and rusts , 1993, Molecular ecology.