REVIEW
Food micro-organisms and aromatic ester synthesis
Giulia CRISTIANI, Véronique MONNET*
RÉSUMÉ Synthèse d’esters aromatiques par des micro-organismes alimentaires.
Les esters d’alcools et d’acides gras à courtes chaînes sont des molécules for- tement aromatiques, ayant des seuils de perception bas et qui sont produits par de nombreux micro-organismes au cours de fermentations alimentaires.
Cette production d’esters est le résultat de réactions enzymatiques impliquant soit des estérases soit des acyltransferases. Cependant, l’importance relative de ces enzymes dans la production d’esters et l’influence de la composition des aliments sur la synthèse d’esters sont encore mal connues. L’objectif de cette revue est de faire le point sur l’état de l’art dans ce domaine. Les esté- rases et les lipases sont des enzymes à sérine capables à la fois d’hydrolyser et de synthétiser des liaisons esters. Le sens de la réaction est gouverné par les conditions environnementales et plus particulièrement par la disponibilité de l’eau. Les estérases ont été décrites chez de nombreux micro-organismes et purifiées essentiellement de micro-organismes laitiers. Quelques gènes ont été identifiés et les premiers micro-organismes modifiés pour une activité esté- rase obtenus récemment. Ils vont être utilisés comme outils pour évaluer le rôle des estérases dans la formation d’esters pendant les fermentations.
Les alcools acétyltransférases, présentes chez la levure, synthétisent efficace- ment des esters. Deux gènes codant ces protéines ont été identifiés. Les alcool acétyltransferases sont responsables en grande partie de la production d’esters qui participent de façon importante à la fraction aromatique des bois- sons alcoolisées. Plusieurs auteurs ont montré que la production d’ester était en fait le résultat d’un équilibre entre les activités estérase et alcool acétyl- transférase dans les boissons alcoolisées.
Mots clés : estérase, ester, alcool acétyltransférase, micro-organisme, aliment.
Unité de biochimie et structure des protéines, Institut national de la recherche agronomique, 78352 Jouy-en-Josas cedex, France.
* Correspondence.
monnet@jouy.inra.fr
SUMMARY
Esters of alcohols and short-chain fatty acids are highly aromatic molecules produced during food fermentation by various micro-organisms. This produc- tion is known to be the result of enzymatic reactions involving esterases and alcohol acetyltransferases. However, the relative importance of these enzymes in the ester synthesis process as well as the conditions which favour this syn- thesis are not yet precisely described. The aim of this review is to report the state of the art in the field. Esterases are serine enzymes capable of hydroly- sing or synthesising esters according to the environmental conditions. They have been described in numerous micro-organisms and purified mainly from dairy micro-organisms. A few genes encoding esterases have been identified and the first micro-organisms negative mutants for these activities were recently obtained. They will be used as tools to investigate the role of esterases in the formation of esters during fermentation.
Alcohol acetyltransferases, present in yeasts, are also capable of synthesising esters. Two genes encode alcohol acetyltransferases mainly responsible for the production of esters, which strongly participate in the aromatic note of alcoholic beverages. Several authors have reported that ester production in such beverages is the result of a balance between esterase and alcohol acetyl- transferase activities.
Key-words: esterase, ester, alcohol acetyltransferase, micro-organism, food.
1 - INTRODUCTION
Bacteria, yeasts and moulds participate in the development of food organo- leptic properties by expressing enzymes able to transform substrates into aro- matic compounds. Such micro-organisms are widely used in the food industry to ferment various raw materials. The resulting food products include dairy pro- ducts (fermented milks, yogurts, cheeses), alcoholic beverages (wine, beer, cider, sake), meat products (sausages, hams), fish products (nuoc mam) and plant products (sauerkraut, bread, coffee, cocoa, natto in Japan, sufu in China) (table 1).
Table 1
Main fermented foods and micro-organisms used in fermentation processes (from BOURGEOISand LARPENT, 1996)
Fermented foods Breads and cakes
Beers
Main micro-organisms found in fermented foods
Bacteria: Lactobacillus brevis, casei, delbrueckii, fermentum, plantarum (lactobacilli represent 80% of the total bacteria), Leuconostoc
mesenteroïdes, Pediococcus damnosus Yeasts: Saccharomyces cerevisiae and exigus Candida krusei and tropicalis
Pichia and Hansenula anomala
Yeasts: Saccharomyces cerevisiae and uvarum (ex-carlsbergensis)
Table 1 (continued)
Main fermented foods and micro-organisms used in fermentation processes (from BOURGEOISand LARPENT, 1996)
Fermented foods Wines
Ciders
Alcoholic beverages
Vinegars
Fermented vegetables (Sauerkraut, olives, concombers) Soja based foods (shoyu, miso, tofu and sufu, natto, soja milk)
Cocoa
Coffee
Yogurt
Fermented milks (kefir, buttermilk, viili, …)
Fresh, semi-hard, pressed or mold cheeses
Main micro-organisms found in fermented foods Bacteria: Lactobacillus, Leuconostoc, Pediococcus
Yeasts: Saccharomyces cerevisiae
Yeasts: Saccharomyces cerevisiae and uvarum, Hansenisapora valbyensis, Metschnikowia pulcherrima
Bacteria: Pediococcus sp., Leuconostoc mesenteroïdes, Leuconostoc oenos, Lactobacillus brevis
Bacteria: Lactobacillus delbrueckii, brevis, casei, Leuconostoc mesenteroïdes (whisky), Clostridium saccharobutyricum et butyricum (rum)
Yeasts: Saccharomyces cerevisiae and other species, Schizosaccharomyces (rum)
Acetic bacteria: Acetobacter sp., Gluconobacter
Lactic acid bacteria: Lactobacillus sp., Leuconostoc mesenteroïdes, Pediococcus sp., Enterococcus faecalis, faecium, Lactococcus lactis Bacteria: Tetragenococcus halophilus and Zygosaccharomyces rouxii, Bacillus subtilis and natto
Lactic acid bacteria (for soja milk): Streptococcus thermophilus, Lactococcus lactis, Lactobacillus acidophilus, casei, helveticus, plantarum, Leuconostoc, Bifidobacterium longum.
Fungi: Aspergillus oryzae, Actinomucor elegans, Mucor hiemalis and silvaticus, Rhizopus chinensis
Acetic bacteria: Acetobacter sp., Gluconobacter oxydans
Lactic acid bacteria: Lactobacillus sp., Leuconostoc mesenteroïdes, Pediococcus acidilactici, Lactococcus lactis, Streptococcus thermophilus
Other bacteria: Bacillus sp., Azotomonas insolita, Cellulomonas cellasea, Micrococcus sp., Propionibacterium technicum
Yeasts: Candida krusei, Pichia membranaefaciens, Saccharomyces chevalieri, Candida famata and holmii
Bacteria: Aerobacter, Erwinia, Escherichia, Lactobacillus, Leuconostoc Fungi: Aspergillus, Penicillium, Fusarium
Yeasts: Saccharomyces
Lactic acid bacteria: Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus
Yeasts (for kefir): Saccharomyces cerevisiae and Candida kefir Bacteria: Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus and casei, Bifidobacterium, Lactococcus lactis, Leuconostoc
Lactic acid bacteria: Lactococcus lactis, Streptococcus thermophilus, Lactobacillus casei, delbrueckii, helveticus
Coryneform bacteria: Arthrobacter sp. and Brevibacterium linens Propionibacteria: Propionibacterium sp.
In order to accelerate, intensify or diversify food flavours, to limit the deve- lopment of flavour defects and to innovate in the field of food products, know- ledge of microbial enzymatic systems and how they work is clearly necessary.
In recent years, several studies have made precise inventories of the flavour compounds found in foods i.e. esters, alcohols, amino acids, sulphur com- pounds and aldehydes (BOSSET and GAUCH, 1993; STAHNKE, 1994; FRIEDRICH and ACREE, 1998; KUBICKOVA and GROSCH, 1998; SABLE and COTTENCEAU, 1999), while others have been carried out on the microbial pathways leading to their production (MOLIMARDet al., 1997; MC SWEENEYand SOUSA, 2000; YVON and RIJNEN, 2001). Most aromatic compounds are volatile and are produced in very small, but sufficient amounts to participate in the overall flavour of foods (SCHARPF et al., 1986). Among these compounds, esters are well-represented, are known to contribute significantly to the flavour of foods and are appreciated since they generally carry fruity flavours (FRIEDRICHand ACREE, 1998). Esters are found in fruits, of course, but also in alcoholic beverages, especially in beer, and also in meat and dairy products. Generally, the aromatic note given by esters is appreciated, even if, in some cases, an excess of ester can be perceived as a defect.
The enzymes responsible for ester production in food and the conditions which favour this production have not been clearly identified. However, it is well established that esterases and lipases are capable of hydrolysing and synthesi- sing esters according to the environmental conditions, while acyltransferases participate only in ester synthesis. The aim of this review is to report the state of the art in the field of ester synthesis by food micro-organisms.
Table 1 (continued)
Main fermented foods and micro-organisms used in fermentation processes (from BOURGEOISand LARPENT, 1996)
Fermented foods Fresh, semi-hard, pressed or mold cheeses
Fermented meat products
Fermented fish products (fishes, dressings, nuoc mam)
Main micro-organisms found in fermented foods Yeasts: Kluyveromyces sp., Debaryomyces hansenii, Saccharomyces unisporus, Candida sp., Pichia sp., Kluyveromyces sp., Debaryomyces hansenii, Saccharomyces unisporus, Candida sp., Pichia sp., Yarrowia lipolitica, Clavispora lusitaniae, Trichosporon inkin, Torulospora delbrueckii
Fungi: Penicillium sp., Geotrichum candidum, Mucor miehi Bacteria: Lactobacillus sakei, curvatus and plantarum, Pediococcus pentosaceus and acidilactici, Staphylococcus carnosus and xylosus, Micrococcaceae sp.
Yeasts: Debaromyces hansenii and candida, Rhodotorula rubra Fungi: Penicillium camembertii and nalgiovense, Mucor racemosus Bacteria: Lactobacillus sp., Leuconostoc sp., Pediococcus sp.
2 - SIGNIFICANCE OF ESTERS IN THE AROMATIC FRACTION OF FOODS
2.1 Aromatic properties of esters and perception thresholds
Most esters found in foods carry floral and fruity notes corresponding to pineapple, banana, apricot, pear, rose, honey and wine-like aromas. These molecules derive from the condensation of short or medium chain fatty acids and alcohols produced during sugar fermentation and amino acid catabolism (MOLIMARDand SPINNLER, 1996). When the alcohol is a thiol, the resulting esters are thioesters, which have very low detection thresholds and which are consi- dered to be major aromatic compounds. They are responsible for cooked cab- bage, cheese and chive aromas and give beer its vegetal note. More precisely, a green, floral or pineapple aroma is detected for S-methyl thiohexanoate while thioesters containing a 2-6 carbon chain length are described as “cheesy” (BER- GERet al., 1999b).
Table 2
Aromatic notes and perception thresholds of different esters found in fermented foods (MOLIMARDand SPINNLER, 1996; MOLIMARDet al., 1997; MONTELet al., 1998;
SABLEand COTTENCEAU, 1999).
Detection threshold (1) in water;
Compound Flavour note (2) in oil or butter;
(3) in cheese; (4) in beer 2-phenylethyl acetate floral, rose 0.137 ppm (2); 18.5 ppm (3)
2-phenylethyl butanoate floral, rose, honey ND
2-phenylethyl propanoate floral, 16.8 ppm (3)
3-methylbutyl acetate pear, banana 2 ppb (1)
3-methylbutyl butanoate apricot, pineapple ND
3-phenylbutyl propanoate apricot, pineapple ND
Butyl acetate pineapple 0.066 ppm (1)
Ethyl acetate solvent, pineapple 5 ppm (1); 22 ppm (2)
Ethyl butanoate pineapple, sweet, banana, fragrant 0.13 to 45·104ppb (1); 0.6 ppm (2) Ethyl hexanoate pineapple, banana, apple, powerful 1 ppb (1); 0.85 ppm (2)
Ethyl octanoate apricot, wine ND
Ethyl decanoate fruity ND
Ethyl propanoate pineapple, sweet, solvent 9,9 ppb (1)
Methyl butanoate fruity 0.043 ppm (1)
Methyl hexanoate pineapple, etheral ND
Methyl octanoate green, fruity ND
Methyl decanoate oily, winelike, fruity ND
Propyl butanoate pineapple, banana ND
S-methylthioacetate cooked cauliflower, garlic toast 5 ppb (3)
S-methylthiopropionate cheese, garlic 100 ppb (3)
S-methylthiobutanoate chive, rotten, rancid, rotten cabbage 200 ppb (3)
S-methylthiopentanoate garlic toast ND
S-methylthioisovalerate garlic toast ND
S-methylthiohexanoate cooked vegetable, sulphur, 0.3 ppb (1); 1 ppb (4) ND: not determined.
Esters are volatile molecules found at very low concentrations: from less than 1 ppm (1mg·kg–1) to a few ppb. Nevertheless, and because their thre- sholds are very low, esters are perceived as aroma compounds even at low concentrations (MOLIMARDet al., 1997). The perception thresholds of the most common esters found in foods (table 2) represent the lower compound concen- trations perceived, by sensory analysis, in a sample by more than half of the panel in comparison with a reference sample which does not contain this com- pound (MOLIMARDet al., 1997). The lower thresholds reported in table 2 concern thioesters which are perceived at the ppb level. Among other alcohol esters, ethyl butanoate and 3-methylbutyl acetate are detected with the lowest thre- shold values: 0.13 and 2 ppb in water, respectively. The intensity of ester per- ception is highly dependent on the environmental medium. Perception thresholds of the same esters are lower in water than in oil, cheese or beer. The aromatic note perceived also depends on the ester concentration.
2.2 Importance of esters in food
2.2.1 Esters in dairy products
The chemical analysis of flavour in dairy products is complicated by the heterogeneous nature of milk or cheese which contain lipids, proteins and car- bohydrates. However, methods such as distillation and solvent extraction com- bined with chromatography or head-space analysis make it possible to identify and quantify volatile molecules. Of the aromatic volatile molecules, esters are well represented in dairy products. Two of them, ethyl butanoate and ethyl hexanoate, have been identified and quantified in raw milks from cow, sheep, goat and buffalo. However, heat treatments such as pasteurisation or ultra high temperature sterilisation destroy them. No esters have been found in yogurts while they are produced during the ripening of many cheeses (FRIEDRICH and ACREE, 1998; SABLEand COTTENCEAU, 1999). According to URBACH(1997), ethyl butanoate, ethyl hexanoate, ethyl acetate, ethyl octanoate, ethyl decanoate and methyl hexanoate are the most abundant esters found in cheeses. They are especially abundant in cheeses with complex flora such as soft cheeses in which up to twenty esters have been quantified in the 0.3-0.005 ppm range (SABLE and COTTENCEAU), and in cheeses with long ripening times. Esters are quantitatively and qualitatively important in blue cheeses in which up to fifty- seven esters have been identified (SCHARPFet al., 1986; MOLIMARDet al., 1997).
BOSSETand GAUCH(1993) have analysed the aromatic compounds of six diffe- rent varieties of cheese: Italian Parmigiano Reggiano and Fontina, Spanish Mahon, French Comté and Beaufort, and Swiss Appenzeller. The esters in these six cheeses have been compared (table 3). They are far more abundant and varied in Parmigiano Reggiano cheese which is ripened for 28 months than in Appenzeller which only ripens for 3.5 months. Similarly, ethyl acetate, ethyl butanoate and ethyl hexanoate were identified as aroma volatiles with high odor potency in 3-year-old Cheddar (CHRISTENSENand REINECCIUS, 1995).
However, only a few reports give data on the variations in ester content during cheese ripening. On the 7thday of Camembert cheese ripening, 2-phenyl acetate is the main compound of the aromatic profile at a concentration of 4.6 ppm. This concentration then drops and stabilises at around 1 ppm (ROGER et al., 1988). This observation can be compared to the decrease in esterase
Mahon Fontina Comté Beaufort Appenzeller Parmigianio Reggiano
activity measured in Cheddar after 120 days of ripening even though starter cul- ture population remained high (WEIMER et al., 1997). In Mahon cheese (semi- hard cheese from Minorca) there was a significant decrease in the concentration of all the ethyl esters identified during the 3-month ripening (MULET et al., 1999). At the opposite, the amounts of ethyl, methyl, prolyl and butyl esters, found in Swiss cheese, increased as ripening increased (YANGand MIN, 1994). These two examples demonstrated that ester evolution in cheese is still badly understood.
Esters present in cheeses clearly contribute positively to the overall flavour of cheeses as demonstrated by combined use of gas chromatography and olfactometry (FRIEDRICH and ACREE, 1998). However, in the case of Cheddar cheese, an organoleptic defect due to the excessive production of esters (ten- fold more ethyl butanoate and ethyl hexanoate than in a normal cheese) has been reported (HOSONOet al., 1974).
2.2.2 Esters in fermented meat
Esters present in meat products are produced by micro-organisms but also by muscle and adipose tissues esterases and lipases (MOTILVA and TOLDRA, 1993). In sausages or hams, phenyl acetate gives a floral note while ethyl buta- noate, ethyl acetate, methyl butanoate, ethyl pentanoate, propyl acetate, ethyl 2-methyl propanoate, 2-methyl propyl acetate and 2-methyl butyl acetate give fruity notes (MONTELet al., 1998; TALONet al., 1998; STAHNKE, 1994).
Table 3
Amounts of esters in six varieties of cheeses. Xm represent the average value of peak areas calculated from three independent experiments
(BOSSETand GAUCH, 1993).
Xm
Esters
Butyl acetate 245 268 39
Butyl butanoate 246
Ethyl acetate 802 877 141 209 291 137
Ethyl heptanoate 72 129
Ethyl hexanoate 5454 3617 168 273 137 43
Ethyl octanoate 661 552
Ethyl pentanoate 104
Ethyl propanoate 63 176 856 59 52
Methyl butanoate 203
Methyl hexanoate 401 46
Methyl propanoate 74
Propyl butanoate 118 215 208 40
Propyl hexanoate 29 54 55
STAHNKE(1994) evaluated the contribution of Staphylococcus xylosus to the flavour of dried sausages. She compared the aromatic profiles of sausages fer- mented with S. xylosus to control sausages in which bacteria, yeast and fungi growth was inhibited by adding antibiotics and fungicides. The control sau- sages developed a rancid and unpleasant flavour compared to the sausages fermented with staphylococci. The pleasant flavour found in sausages fermen- ted with S. xylosus is partly due to the presence of fragrant esters produced by S. xylosus or by other micro-organisms, such as lactic acid bacteria, which develop naturally during sausage fermentation. As an example of the diffe- rences observed in the head-space analysis of volatile components, the peak areas corresponding to ethyl acetate divided by total peak areas are 1233 and 29 in S. xylosus fermented sausages and control sausages, respectively. Simi- larly, higher amounts of the following esters: ethyl acetate, ethyl propanoate, ethyl butanoate, ethyl hexanoate and ethyl octanoate have been measured in dry fermented sausages on which Mucor racemosus has been inoculated com- pared to control sausages without Mucor racemosus (BRUNAet al., 2000).
2.2.3 Esters in alcoholic beverages
Production of aromatic esters during the fermentation process of alcoholic beverages is mainly due to yeasts, although slow and limited chemical esterifi- cation reactions (formation of ethyl or diethyl tartrate or malate for example) occur during alcoholic beverage ageing (RIBEREAU-GAYONand PEYNAUD, 1966).
During anaerobic fermentation, Saccharomyces cerevisiae produces medium chain fatty acids and corresponding ethyl esters, which play an important role in alcoholic beverage aroma (BARDIet al., 1998). Acetate esters such as ethyl ace- tate, 3-methylbutyl acetate, and 2-methylpropyl acetate, 3-methylbutyl acetate and ethyl hexanoate are the most abundant (CALDERBANKand HAMMOND, 1994;
DEL CARMEN PLATA et al., 1998; HILARY and PEDDIE, 1989). Esters give vege- table-like flavours to alcoholic beverages: banana, apple, or solvent aroma due to 3-methylbutyl acetate, and rose or honey due to phenylethyl acetate (LEEet al., 1995).
In beer, in addition to the esters mentioned above, numerous flavour active thioesters carrying vegetal notes are produced during fermentation (SCHARPFet al., 1986). After five days fermentation of beer with Saccharomyces cerevisiae, less than 1 µg·L–1 and up to 35 µg·L–1 of S-methylthioacetate, carrying a cooked cauliflower note, were quantified when ale strains and lager strains were used, respectively. This thioester production is stimulated when methanethiol (400 µg·L–1) is added to beer. In these conditions, S-methylthioacetate concen- tration can reach up to 107µg·L–1(WALKERand SIMPSON, 1993).
In wine, although some of the flavour compounds are indigenous to the grape itself, much of the flavour of the finished product also arises from the bio- chemical actions of yeasts and to a lesser extent of the acetic bacteria, Aceto- bacter (RIBEREAU-GAYONand PEYNAUD, 1966). Esters deriving from fermentation and involved in wine aroma are: methyl and ethyl acetates, ethyl hexanoate, ethyl octanoate and ethyl laurate. Only diethyl malonate and ethyl-2-methyl butyrate are significant for differentiating between wine varieties (SCHARPFet al., 1986).
3 - ESTER HYDROLYSIS BY FOOD MICRO-ORGANISMS
Ester synthesis and ester hydrolysis are two reactions which cannot be dis- sociated because the final production of ester results from the balance between hydrolysis and synthesis activities and because lipases and esterases are capable of catalysing both reactions.
3.1 Ester hydrolysis enzymes: esterases and lipases
Ester hydrolysis by food micro-organisms is generally associated with lipase and esterase activities. These enzymes belong to the family of hydrolases and, depending on the environmental conditions, they can catalyse either the condensation of a fatty acid with an alcohol into an ester or the hydrolysis of an ester. Differences between esterases and lipases are attributed to a difference in substrate specificity. Esterases preferentially hydrolyse soluble esters contai- ning short-chain fatty acids (C2 to C8) while lipases are active on emulsified substrates composed of long-chain fatty acids (> C8) (KILCAWLEYet al., 1998).
These enzymes are also involved in the hydrolysis of mono-, di- and tri- glyce- rides (GOBBETTIet al., 1999). They are capable of catalysing two different types of synthesis reactions: common ester synthesis from alcohol and fatty acids and the biotechnologically more important transesterification in which the acyl donor is an ester (JAEGERet al., 1994).
3.2 Detection of esterase activities
The ester hydrolysis activity of esterases and lipases can be detected using natural or synthetic substrates including triacylglycerols, olive oil and chromo- genic substrates (THOMSON et al., 1999 for a review). Triacylglycerols are more similar to the natural substrates encountered by bacterial esterases than the chromogenic substrates. However, the use of the latter, i.e. α- and β-nitrophe- nyl and naphtyl esters, makes possible a coloured visualisation of esterase acti- vities. They are consequently widely used for measuring activities and carrying out zymograms.
3.3 Esterase content of food micro-organisms
The presence of ester hydrolysis activities is common in food micro-orga- nisms. The level of these activities is strain and species dependant. With res- pect to lactic acid bacteria, esterase activity is greater in Streptococcus thermophilus and lactobacilli compared to lactococci (CROWet al., 1994; FORMI- SANOet al., 1974).
The esterase content of food micro-organisms was first screened with zymograms. They generally reveal the presence of several protein bands (up to six) associated with ester hydrolysis activity corresponding to several proteins exhibiting different specificities. Because numerous inventories of ester hydroly- sis activities have been carried out and reported for food bacteria, they cannot be extensively cited. Some of the most recent reports concern lactic acid bac- teria (CROWet al., 1994; KENNEALLYet al., 1998; DE ANGELISet al., 1999), propio-
Table 4 Properties of esterases purified from food micro-organisms Micro-organismFoodBest substratesOptimal InhibitorsActivatorsGene References temperaturesequence Arthrobacter nicotinaeSmear surfaceβ-naphtyl butyrate30°CFe2+, Hg2+,NDNDSMACCHIet al., 2000 ripenedSn2+,Ca2+, cheesesPMSF AcetobacterVinegarα-, β-naphtyl acetateNDNDNDKASHIMAet al., 1999 pasteurianus Esterase 1Yes Esterase 2Yes Brevibacterium linensSurfaceβ-naphtyl esters of C235°CN-ethyl maleimide,DTTNoRATTRAYand FOX, 1997 ATCC9174ripenedto C8 fatty acidspHMB,Cystéine cheesesCd2+, Zn2+, Hg2+EDTA DebaryomycesRoquefortEthyl and methyl esters35°CN-NDNoBESANCONet al., 1995 hanseniicheeseof C2 to C5Bromosuccinimide floraCu2+, Ni2+, Zn2+ Enterococcus faeciumDairy productsp-nitro-phenyl butanoate35°CPMSFNDNoTSAKALIDOUet al., 1994 ACA-DC 237starter LactobacillusDairy productsFatty acid (C2 to C10)30-35°CPMSFCa2+, Mg2+NoGOBBETTIet al., 1997b fermentumDT41starterestersHg2+, Ag2+ LactobacillusDairy productsp-nitrophenyl butyrate35-40°CND3.5% NaClYesFENSTERet al., 2000 helveticusstarterphenyl acetate LactobacillusDairy productsAcetylesters40°CSulfydryl reagentsNDNoOTERHOLMet al., 1971 plantarumstarter LactobacillusDairy productsFatty acid (C2 to C10)35°CPMSFCa2+, Mg2+NoGOBBETTIet al., 1997a plantarum2739starterestersHg2+, Ag2+
Table 4 Properties of esterases purified from food micro-organisms Micro-organismFoodBest substratesOptimal InhibitorsActivatorsGene References temperaturesequence Lactococcus lactisssp.Dairy productsp-nitro-phenyl butanoate45°CPMSFMg2+, Mn2+NoTSAKALIDOUet al., 1992a lactisACA-DC 127starterCu2+, Hg2+ Lactococcus lactisssp.Dairy productsTributyrin and 4-nitro-?NDNDNDYesHOLLANDand COOLBEAR, 1996 cremorisE8starterphenyl butanoate Lactococcus lactisssp.Dairy productsβ-naphtyl butanoic acid55°CPMSFNDYesCHICHet al., 1997 lactis NCDO763starteresterNARDI, personal comm. Mucor miehiSurfaceTributyrin55°CNDNDNoMOSKOWITZet al., 1977 ripened cheeses PropionibacteriaSwiss typeα-naphtyl acetate65°CPMSFNDNoKAKARIARIet al., 2000 freundenreichiicheese flora
nibacteria (DUPUISet al., 1993), staphylococci (TALON and MONTEL, 1997; KEN- NEALLYet al., 1998), Brevibacterium linens (RATTRAYand FOX, 1999).
The purification of esterases leads to results which are not totally in agree- ment with the presence of several esterases suggested by the inventory stu- dies. Indeed, only one esterase has been purified from lactococci and described as the main or unique esterase in this micro-organism (HOLLANDand COOLBEAR, 1996; CHICH et al., 1997). In other micro-organisms, a maximum of two (Hanse- nula mrakii, INOUE et al., 1994; Acetobacter pasteurianus, KASHIMAet al., 1999) or three (Lactobacillus plantarum, GOBBETTI et al., 1997a) different esterases have been described during purification processes. The major properties of the esterases purified from food micro-organisms are reported in table 4. They have mainly been isolated from micro-organisms used in dairy product manufactu- ring. They belong to the serine enzyme group and are optimally active at pH 7-8 and temperatures above 30°C.
The presence of a limited number of esterases has been confirmed in the case of lactococci by genetic data since, of the proteins identified from the sequence of the chromosome of L. lactis, only one protein homologous to known esterases has been identified (BOLOTIN et al., 2001). More recently, the construction of negative mutants for the main L. lactis esterase (FERNANDEZ et al., 2000; NARDI, personal communication) confirmed that the enzyme is respon- sible for most of the ester hydrolysis activity.
Esterases are generally described as intracellular enzymes. However, GOB- BETTI et al. (1997a and 1997b) have described esterases from Lb. fermentum and Lb. plantarum as surface-associated esterases. Similar observations have been made for Brevibacterium linens (EZZAT et al., 1993; SORHAUGand ORDAL, 1974). The available protein sequences of esterases from L. lactis or Acetobac- ter pasteurianus do not possess any typical peptide signal sequence indicating that these esterases are secreted in the extracellular medium (KASHIMA et al., 1999; FERNANDEZet al., 2000; KOEBMANNet al., 2000; NARDI, personal commu- nication).
Studies concerning the conditions for optimal expression of food micro- organism esterases are rare. Esterase activities are maximal during the expo- nential growth phase and generally depend on the nature of the culture medium (UMEMOTOand SATO, 1975; EL SODAet al., 1986; PIATKIEWICZ, 1987). Milk-based media stimulate the production of esterase by lactococci and lactobacilli (PIAT- KIEWICZ, 1987) but inhibit that of St. thermophilus (DE MORAES and CHANDAN, 1982). Media with low glucose content favour lipase synthesis in Lb. curvatus and Carnobacterium piscicola while media rich in tributyrin are inhibitors (PAPON and TALON, 1988). HARPERet al. (1980) also reported that addition of gluthatione increases esterase activity in L. lactis. The most precise report concerns este- rases Est1 and Est2 from Acetobacter pasteurianus which are expressed diffe- rently. The transcription of the est1 gene was indeed induced only when cells were grown in presence of ethanol. In contrast, est2 was repressed in the pre- sence of ethanol (KASHIMAet al., 2000).
4 - ESTER SYNTHESIS BY FOOD MICRO-ORGANISMS
4.1 Ester synthesis capacity
Ester synthesis in food is generally attributed to the activity of micro-orga- nisms (BRUNAet al., 2000). To our knowledge, no non-enzymatic ester synthesis has been reported in foods. However, this spontaneous synthesis cannot be excluded since HELINCK et al. (2000) recently demonstrated that thioesters made up of highly reactive methanethiol and acyl CoA occurred in a buffered solution in absence of any micro-organisms or microbial enzyme. The same authors demonstrated that the amount of S-methylthioacetate synthesised increased in presence of cell-free extracts from Geotrichum candidum but only when the pH value is 7-7.5.
Ester synthesis is much more difficult to detect than ester hydrolysis. Studies reporting such experiments are generally carried out using gas chromatography methods which are not suitable for large scale screenings (HOSONOet al., 1974).
In addition, such methods require the use of extraction techniques such as SPME and liquid-liquid extraction (TALONet al., 1998; LIUet al., 1998; WILKESet al., 2000) which can lead to artefacts and limit the detection threshold.
Studies concerning the overall capacity of food micro-organisms to produce esters have been carried out for both positive and negative food flora. In sau- sage fermentation, all the Staphylococcus species used and tested by TALONet al. (1998) as resting cells, synthesise large amounts of ethyl esters with a prefe- rence for ethyl butanoate. Starter and non starter lactic acid bacteria used in dairy product manufacturing have the same capacity to synthesise ethyl buta- noate as demonstrated by LIUet al. (1998) with resting cells and by AYADet al.
(1999) in milk culture and cheese paste. In L. lactis, strains belonging to the lac- tis and diacetylactis sub-species contained relatively higher enzyme activities for the production of ethyl butanoate and ethyl hexanoate than strains from the cremoris sub-species (HOSONO et al., 1974). The spoilage of milk products by Pseudomonas fragi, characterised by a strong strawberry-like aroma, is also due to the production of 13 fatty acid ethyl esters among 26 different aromatic compounds (CORMIERet al., 1991; RAYMONDet al., 1991).
Several groups of micro-organisms used in dairy products are also capable of synthetising highly aromatic thioesters. Resting or growing cells of coryne- bacteria, micrococcaceae, lactic acid bacteria, Geotrichum candidum and espe- cially B. linens, synthetise thioesters in the presence of methanethiol and fatty acid mixtures (LAMBERET et al., 1997a, 1997b; BLOES-BRETON and BERGERE, 1997, BERGERet al., 1999a).
4.2 Ester synthesis enzymes: esterases and acyltransferases
Two families of enzymes are potentially involved in ester synthesis: the este- rases described above and acyltransferases. Acyltransferases are enzymes that catalyse the transfer of an acyl group from a donor molecule to a receptor mole- cule. Of the transferases known to be involved in ester synthesis, alcohol ace- tyltransferases (AATases) (EC.2.3.1.84) present in yeasts have been the most accurately described. These enzymes catalyse ester synthesis reactions from
an activated fatty acid, generally the acetyl CoA and an alcohol (MOLIMARD et al., 1997). The involvement of other transferases in ester synthesis has not been proved but is possible.
Yeasts are strong producers of esters during alcoholic beverage fermenta- tion and, in this way, contribute to aroma development. Both esterases and alcohol acetyltransferases are involved in this process. In Kluyveromyces fragilis, a constitutive esterase and an inducible alcohol acetyltransferase parti- cipate in ethanol catabolism by producing ethyl acetate. In this yeast, ethanol is catabolised in ethyl acetate, acetate and acetaldehyde. When ethyl acetate concentration is high, the level of residual lactose is low and alcohol acetyl- transferase activity is high whereas, when the level of residual lactose is high, alcohol acetyltransferase activity is low. In Kluyveromyces fragilis, the two enzymes alcohol acetyltransferase and esterase are involved in the synthesis and hydrolysis of ethyl acetate (KALLEL-MHIRIand MICLO, 1993). 3-methylbutyl acetate and ethyl hexanoate are the most important esters conferring to sake its fruity flavor. FUKUDAet al. (1998) demonstrated that it is the balance of Saccha- romyces cerevisiae alcohol acetyltransferase and esterase activities which is important for the accumulation of 3-methylbutyl acetate. The higher the ratio of alcohol acetyltransferase activity to esterase activity, the higher the amount of 3-methylbutyl acetate. To obtain this result, they tested, in small scale sake bre- wing, strains with different numbers of copies of the alcohol acetyltransferase and esterase genes. INOUE et al. (1994) also demonstrated that both esterase and alcohol acetyltransferase are involved in the synthesis of esters by Hanse- nula mrakii and that some strains are capable of growing with ethyl acetate as the sole carbon source. This capacity is probably due to esterase activity which enables Hansenula to grow by hydrolysing ethyl acetate.
Two genes encoding alcohol acetyltransferases have been identified in yeast and named ATF1 and ATF2. The ATF1 gene is the most studied and its expres- sion is higher in anaerobic conditions. It encodes a 61 kDa protein (FUJIIet al., 1994; YOSHIMOTO et al., 1999; KUMARA et al., 1995; KAJIWARA et al., 1997;
HILARYand PEDDIE, 1989).
The ATF2 gene encodes an AATase, which shares 37% of identity with ATF1 (NAGASAWA et al., 1998). The ATF2 protein possesses greater thermal stability and a different affinity for 3-methylbutan-1-ol than ATF1 protein. Otherwise, the ATF2 protein Km for 3-methylbutan-1-ol (the reaction substrate) is higher than the ATF1 protein Km for the same substrate.
4.3 Parameters influencing ester synthesis
Several factors influencing ester synthesis in foods have been identified. The most obvious are the availability of substrates and the level of enzyme activities.
The simplest way to increase the concentrations of substrates is to add them to the food. MORINet al. (1994) have greatly increased the production of ester by Pseudomonas fragi during growth in milk or whey by adding C3-C7 fatty acids to the culture medium. Similarly, adding methanethiol to ale or beer increases the production of S-methylthioacetate during fermentation by Saccharomyces cerevisiae. A more sophisticated way of increasing substrate availability consists in modifying the metabolic pathways of the micro-organisms so as to favour the accumulation of substrates. Yeast strains have been developed to enhance the production of desirable alcohols and consequently of the corres-
ponding esters. Mutants resistant to leucine and phenylalanine analogs, in which there is no more feedback in the biosynthesis pathways of these amino acids, have been selected. They produce keto-acids in excess which lead to higher amounts of 3-methylbutyl and phenylethyl alcohols. The beers fermented with these strains contain higher amounts of ester such as 3-methylbutyl ace- tate and obtain higher sensory scores (LEEet al., 1995).
Selection of strains with high esterase or alcohol acetyltransferase activities is also a way of increasing ester synthesis. LILLYet al. (2000) have constructed Saccharomyces cerevisiae strains used in wine fermentation with enhanced alcohol acetyltransferase activity. The ATF1 gene has been cloned and placed under the control of a strong promoter. The levels of ethyl acetate, 3-methylbu- tyl acetate and 2-phenyl ethylacetate have increased up to 10-fold in wine, demonstrating that flavor profiles of wines can be modified through alcohol ace- tyltransferase overexpression.
In the case of esterases, which can hydrolyse or synthetise esters, the availa- bility and activity of water is a major parameter which influences the direction of the reaction. TALONet al. (1998) indeed observed that a decrease in water activity leads to a decrease in ester hydrolysis by staphylococci. Consequently, adding a drying step to the sausage manufacturing process increases the esterification reaction. Surprisingly, LIU et al. (1998) made a different observation since ester synthesis by lactic acid bacteria was inhibited when water activity was reduced.
Other parameters such as pH, temperature, aeration and NaCl content also influence ester synthesis in foods. The effect of pH is variable from one example to another. Ester synthesis by staphylococci was reduced when pH was decreased from 7 to 5.5 and completely inhibited at pH 5.5 for some strains (TALONet al., 1998). Conversly, a pH decrease from 5.8 to 4 favours ester syn- thesis by lactic acid bacteria and more especially by lactobacilli (LIU et al., 1998). With respect to thioesters, the optimal pH for synthesis is variable from one thioester to another (LAMBERET et al., 1997a, 1997b). No universal rule governs the relationship between temperature and ester synthesis. LIU et al.
(1998) measured a slight decrease in lactic acid bacteria ester synthesis activity when the temperature fell from 30°C to 13°C, the cheese ripening temperature.
Conversely, ester synthesis by yeast increased when the temperature increased from 10°C to 25 °C (CALDERBANKand HAMMOND, 1994).
Three percent NaCl totally inhibits or greatly reduces ethyl butanoate synthe- sis by lactococci and lactobacilli respectively (LIUet al., 1998). GOBBETTI et al.
(1999) observed that esterase/lipase activity of Lb. delbrueckii subsp. bulgaricus was markedly inhibited under cheese-like conditions.
Aeration and the presence of unsaturated fatty acids repress ester synthesis by Saccharomyces cerevisiae. The decrease in alcohol acetyltransferase activity is, in this case, closely related to the repression of ATF1 mRNA. Using a repor- ter gene, FUJI et al., (1997) showed that a 150 bp sequence of the 5’ flanking sequence of the ATF1 gene plays a major role in the repression by aeration and the addition of unsaturated fatty acid.
4.4 Role of ester synthesis in cellular detoxification
The physiological role of esterase is not well known. In the case of Lacto- coccus lactis, FERNANDEZ et al. (2000) demonstrated that the loss of esterase
activity has no effect on the growth of the bacteria in synthetic medium. It is generally admitted that the esterification reaction between alcohols and fatty acids is a mechanism of medium detoxification for toxic alcohol and fatty acid (MOLIMARDet al., 1997).
In Saccharomyces cerevisiae, it has recently been demonstrated that the protein encoded by the ATF2 gene (an alcohol acetyltransferase) is steroid-spe- cific, and esterifies pregnenolone efficiently. Pregnenolone is toxic for yeast and its esterification by the alcohol acetyltransferase ATF2 is a means of detoxifica- tion to decrease the inhibitory action of 3-β-hydroxysteroids on yeast growth (CAUETet al., 1999).
However, BARDIet al. (1998), during fermentation by Saccharomyces cerevi- siae, found no correlation between the concentration of each fatty acid and its ethyl esters. Evidence of a reduction in the toxicity, due to medium-chain fatty acids, via ester synthesis was not demonstrated in this example.
5 - CONCLUSION
Esters of alcohols and short-chain fatty acids are aromatic compounds which significantly participate in the overall flavor of fermented foods. Their pro- duction in foods is mainly the result of enzymatic reactions involving both este- rases or lipases and, when yeasts are involved in the fermentation, alcohol acetyltransferases. Esterases and lipases are capable of both synthetising and hydrolysing esters depending on the environmental conditions, while alcohol acetyltransferases only synthetise esters.
The use of modified micro-organisms, which have lost or which overexpress one of these ester synthetising activities, allow to understand the role of the dif- ferent enzymes in the production of esters in foods. This way has been investi- gated in yeast. The production of esters in alcoholic beverages is the result of a balance between esterase and alcohol acetyltransferase. For other micro-orga- nisms, the first modifications concerning esterases have been recently reported for Lactococcus lactis. The modified micro-organisms will be used as tools to investigate the importance of the enzymatic formation of esters in foods and the role of the different enzymes in this process. Enzymes involved in the pathways leading to alcohol and fatty acids or acyl CoA will be undoubtedly the next tar- gets to increase and control ester production in foods.
ACKNOWLEDGEMENTS
We thank Françoise RUL, Dominique LE BARSand Sandra HELINCKfor criti- cally reading the manuscript and Michèle NARDIfor providing unpublished data.
Received 25 January 2001, accepted 16 May 2001.
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