Lactic Acid Bacteria | Lactic Acid Bacteria in Flavor Development

Lactic Acid Bacteria | Lactic Acid Bacteria in Flavor Development

Lactic Acid Bacteria in Flavor Development T Coolbear, Fonterra Research Centre, Palmerston North, New Zealand B Weimer, University of California, Dav...

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Lactic Acid Bacteria in Flavor Development T Coolbear, Fonterra Research Centre, Palmerston North, New Zealand B Weimer, University of California, Davis, CA, USA M G Wilkinson, University of Limerick, Limerick, Ireland ª 2011 Elsevier Ltd. All rights reserved.

Introduction Lactic acid bacteria (LAB) play a crucial role in the development of flavor (taste, retronasal odor, and orthonasal aroma) of several dairy systems, but because of the time frame over which ripening occurs, aged cheese presents by far the most complex system in terms of both flavor pathways and range of flavor outcomes. Consequently, it is from cheese studies that much of our knowledge of the diversity of flavor pathways of dairy LAB has been derived. However, studies on yogurt have also contributed significantly. The book Improving the Flavor of Cheese edited by Weimer (see ‘Further Reading’) covers flavor formation in cheese extremely well and refers extensively to the role of LAB, while information on LAB-directed flavor formation in yogurt is described within the book Tamime and Robinson’s Yogurt. Science and Technology (see ‘Further Reading’). It is worth noting that enology also provides insights into the diversity of flavor compound formation by LAB, insights that, aspirationally, could be applied to dairy fermentations targeted to the generation of novel all-dairy flavor ingredients, rather than traditional dairy products.

Contextualizing Flavor Generation by LAB The important LAB genera in dairy fermentations are Lactococcus, Lactobacillus, Streptococcus, Leuconostoc, Pediococcus, and Enterococcus. However, for dairy systems, it must be remembered that the LAB are not solely responsible for generating flavor. Flavors can also be generated in products by using rennet (in cheese) and other added enzymes, indigenous milk enzymes and chemical reactions, added microflora other than LAB, raw milk microflora, or derivative enzymes. The levels of both raw milk flora and their enzymes are dependent on how the milk is handled from milking to holding tanks and on the processing of milk (whether or not this includes pasteurization). Flavor molecules can even be passed on from the feed. Flavors generated by the LAB can be primed or otherwise influenced not only by these factors but also by processing and ripening parameters such as pH, salt levels, water activity, and temperature. These parameters


influence the growth, metabolic state, viability, and rate of lysis of the LAB, and the activity and half-life of enzymes released upon lysis. It is worth noting here that there are some misconceptions and dogma regarding the longevity of both LAB and their enzymes in dairy systems. This is especially true in cheese, where the LAB can undoubtedly influence flavor development long after the ability to culture them from the cheese is lost, and subsequently their enzymes, albeit decompartmentalized from any cofactors on eventual lysis of the bacteria, are likely to be far more stable in the high-solids environment than in in vitro aqueous buffer systems. Further influences in cheese can be the presence of other microorganisms, such as surface-ripening microflora, and even the physical dimensions of the cheese and the manner of its packaging. The influences on, and the control points or levers for, flavor generation by LAB in different dairy systems (e.g., cheese vs. yogurt) are therefore very different. Flavor compounds give rise to both positive attributes and defects, depending on absolute and relative levels and on the product in which they are contained and presented. Ultimately, how flavor is defined depends on how it is perceived by the consumer, and so it is not simply which volatile and nonvolatile compounds are present and their levels and interactions that govern flavor but how the compounds are released (and therefore detected by the mouth and nose) on preparation, presentation, and consumption. In this respect, other factors such as composition and texture have a marked impact on flavor perception, and this is further confounded by the wide variation in how individuals perceive flavor. It is not the scope of this article to discuss flavor release and perception, or the interactions between LAB and non-LAB flavor compound pathways, or the impact of manufacturing conditions on flavor development, nor is it to delve into the microbial physiology of the flavor pathways. The scope is to provide an overview of the complexity, the range, and the potential of flavor compound production by LAB, highlight the current level of knowledge of the pathways, and focus on how to harness them to a useful end point. A point to note is that there is often no single standard definition of the flavor of a chemical compound (the perceptions of which often change on dilution or are dependent on presentation) and with this article many readers may take issue with

Lactic Acid Bacteria | Lactic Acid Bacteria in Flavor Development

descriptors used for the different flavor compounds. In working with flavors, one of the initial hurdles is to agree on a single lexicon between collaborators, customers, and sensory panels.

Diversity of Milk as a Raw Material and of LAB as Flavor Factories Although milk can be viewed as having four major components (apart from water) (i.e., caseins, whey proteins, lactose, and milk fat), it is in toto a very complex biological fluid with multiplicity in protein and fatty acid identities and containing a plethora of ‘minor components’ (including proteins, peptides, lipids, sugars, oligosaccharides, glycoconjugates, hormones, vitamins, and minerals). Holistically, milk is an extremely variable substrate, the composition of which not only differs from species to species, but is also influenced by a range of factors such as season, feed, stage of lactation, and animal health. Because some dairy flavor compounds have extremely low flavor thresholds (e.g., readily tasted at concentrations less than 1012 g g1 water), the consequence of such variation in substrate quality on flavor compound production, and therefore the flavor profile of the product, is often enormous. A further complication is that many flavor compounds may well be present in concentrations below their individual taste thresholds, but then act synergistically to deliver a flavor impact detectable by an individual consumer. Some flavors are labeled as ‘characteristic’ of dairy products and illustrative tables have been produced that indicate characteristic flavors and ‘impact compounds’, for example, for different cheeses. However, these can be misleading: flavors as preferred by the consumer reflect a broad, balanced profile with a blend of top, middle, and base notes, rather than single-compound dominance. Furthermore, there is no ‘standard’ product in any category – what is perceived as a quality product to one market region in the globe can often be rejected by another as being unacceptable. For many reasons, therefore, it is not surprising that the overall goals for the dairy industry – consistency in quality flavor delivery, free from flavor defects, with predictability and targeting to consumer preferences – remain elusive. Considering cheese as the model, insights into the potential complexity and diversity of flavor development can be gained by considering the range of cheeses available around the globe, whether produced on an artisanal scale or on an (automated) industrial scale. In addition, within a cheese, there can be literally hundreds of flavor compounds. It is the ability to understand this and subsequently manipulate the relative levels and balance of the myriad components that will help realize the full commercial potential of the fermentations.


In products such as yogurt, there is no ‘ripening’ period and indeed no loss of fermentable substrates (and therefore flavor compound potential) through whey draining. The flavors of the base yogurt matrix (i.e., before the addition of any fruit or other flavorings) essentially depend on a balance of carbonyls delivered by the fermenting LAB. With respect to cheese, however, much discourse on flavor development tends to separate the roles of ‘starter’ and ‘nonstarter’ (NS) LAB – the former being those added primarily to generate lactic acid, and the latter being those that either enter the cheesemaking process in an uncontrolled fashion during manufacture (adventitious NSLAB) or are deliberately added to control and direct the ripening process (adjunct NSLAB). However, starter properties are such that their impact on the development of flavor extends well beyond acid production and indeed contribute to the ‘priming’ of the cheese system in terms of both subsequent rate and direction of flavor development. In this respect, and in terms of taking flavor pathways out of the cheese block and into flavor ingredient fermentations, all LAB, whether starters or adventitious or adjunct nonstarters, can be considered ‘finishers’. Given the complexity of milk as a substrate, flavor production by LAB in dairy systems is, for practical reasons, generally viewed as starting with the breakdown of the predominant carbohydrate, protein, and lipid components of milk. The lactic acid and the various amino and fatty acids that are the predominant products, together with citrate, then become the substrates for further metabolism to many flavor compounds.

Flavor Generation from Carbohydrates by LAB A good summary of the potential for flavor generation from milk carbohydrates by LAB is contained within the article by Wilkinson and Kilcawley (see ‘Further Reading’). Homolactic fermentation of lactose, the main carbohydrate component of milk, gives rise almost exclusively to L-, D-, or DL-isomers of lactic acid (which has a characteristic taste and what consumers often describe as a sour aroma), depending on the LAB, whereas heterolactic fermentation gives both lactic and acetic acids (the latter described as having a sharp, pungent, sour, vinegary odor with a sour, acid taste). When combined with citrate fermentation, this provides the basis for the production of diacetyl (strong butter, caramel), acetoin (sweet, buttery, creamy), acetaldehyde (fruity aroma, pungent, ethereal, green apple, nutty), butane-2,3-diol (fruit, onion), butan-2-one (sharp, butterscotch odor, ether-like, acetone-like, fruity), ethanol (strong alcoholic, medical), formic acid (pungent, vinegar, sour, formyl odors), and CO2. All these compounds have significant flavor impact in themselves. However, these

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compounds can also be metabolized further or become involved in chemical reactions to give rise to other intensely flavored compounds. An example of this is the esterification of ethanol with free fatty acids to give a range of ethyl esters, which have variously fruity, sweet, floral, green, and butterscotch odors. Another example is the involvement of pyruvate as an -keto acid acceptor in amino acid catabolism, which is a major source of flavor compounds in dairy products. One facet of flavor compound production involving carbohydrate metabolism by LAB that is deleterious in some cheeses (e.g., Swiss), but has positive attributes in others (e.g., Mozzarella) and in other dairy-derived products (e.g., dulce de leche) is the Maillard reaction. With galactose-negative LAB, the levels of galactose that accumulate on lactose hydrolysis are such that significant heat-catalyzed browning occurs through the reaction of the carbonyl group of the galactose and the amino group of amino acids. This type of reaction has the potential to yield a wide variety of flavor compounds not catalyzed per se by LAB but illustrating that the deliberate choice of particular LAB for certain fermentations can be a lever to (re)direct flavor formation.

Flavor Generation from Amino Acids by LAB Amino acids are essentially the end points of LABcatalyzed proteolysis of caseins. Certain peptide intermediates of this proteolysis and the amino acids themselves do have associated flavors, such as sweet, brothy, and bitter, but the intensities of these are generally low. While often described as ‘background’ flavors in cheese, they can be the dominant flavors in other dairy systems and by their very nature are often the source of undesirable flavor notes and therefore considered as defects, especially bitterness and, to a lesser extent and in certain applications, brothy notes. The amino acid catabolic pathways of LAB in cheese (and, therefore, utilizing casein protein as substrates) have been well described in the article by Ganesan and Weimer (see ‘Further Reading’). Note that whey protein degradation by LAB is far more limited than that of caseins; only a few reports are available and these essentially pertain to lactobacilli and yogurt systems, so the flavor impact would be expected to be minimal. Amino acids are the substrates for a multiplicity of pathways of flavor compound formation that are not fully clarified, yet provide a high proportion of the most intense flavors that are delivered into dairy products by LAB, in conjunction with nonenzymatic reactions. Through these various pathways, amino acids are decarboxylated (to amines and CO2), transaminated (to new amino acids), deaminated (to -keto acids and ammonia), and desulfated

(to form various sulfur compounds), and further metabolized into a range of short- and branched-chain fatty acids, esters, sulfur compounds, aldehydes, ketones, and lactones that provide a wide range of flavor notes of various intensities. The capability of LAB to metabolize amino acids varies between species and between strains. The known pathways for some of the amino acids have been studied in far more depth than others. Genetic studies suggest the existence of pathways that have not yet been proved active. The group of amino acids with aliphatic side chains includes glycine and alanine, the branched-chain amino acids valine, leucine, and isoleucine, and proline (sometimes referred to as an imino acid, but it does not contain a C¼N double bond). Serine and threonine contain aliphatic hydroxyl side chains. Glycine arises from both protein breakdown and biosynthetically, with 3-phosphoglycerate as a precursor, and its importance in flavor compound generation by LAB lies in single-carbon transfer reactions and its role as a serine precursor (which itself is a precursor of cysteine and methionine). This pathway is induced in lactococci where serine is excreted during carbohydrate exhaustion. Through various precursors, LAB also have the capacity to generate aldehydes and ketones from alanine, which include the important flavor compounds diacetyl and acetaldehyde as well as acetone (solvent, ethereal, apple, pear) and butane-2,3-diol. However, these amino acids are also the precursors for a range of fatty acids such as acetic, propionic (acidic, nutty), butyric (sweaty, butter, cheese, acid, sour-rancid), iso-butyric (sour, acidic), 3-methylbutyric (pungent ‘Roquefort’, rancid, sweaty, putrid), valeric (acidic, sweaty, rancid, sickening, putrid), iso-valeric (sweaty, rancid), caproic (sweaty, goat), and lauric (fatty, coconut) acids. In addition, many of these compounds can be further metabolized to give molecules delivering very different flavor sensations: for instance, esters of valeric acid are fruity, whereas 2-methylpropanal (iso-butyraldehyde) is described as fresh, sweet, fruity, malty, and chocolate-like and 2-methylbutanal as musty, cocoa, coffee, and nutty. The aromatic amino acids, phenylalanine, tryptophan, and tyrosine, can be metabolized by LAB to p-cresol (phenolic, tarry, smoky, medicinal), indole (animal, fecal, mothball), and skatole (animal, fecal, barny), but also to aromatic acids, aromatic aldehydes, and aromatic alcohols that can provide floral (e.g., rosy) and fruity flavor notes. Benzaldehyde (sharp, bitter almond oil odor, sweet cherry taste) is a well-known example of an aromatic aldehyde. The basic amino acid arginine can give rise to putrescine (rotten meat), ammonia (sharp, pungent), and CO2, whereas lysine can be catabolized to fatty acids. The contribution of the third basic amino acid, histidine, to flavor compound generation is yet to be established, although genetic studies have shown its potential to be degraded to glutamate. Glutamate and aspartate have

Lactic Acid Bacteria | Lactic Acid Bacteria in Flavor Development

acidic side chains, negatively charged at physiological pH, whereas their amide derivatives, asparagine and glutamine, respectively, are uncharged. Again, depending on the species and strain, these four amino acids can be catabolized by LAB to acetic, propionic, butyric, iso-butyric, valeric, iso-valeric, and caproic acids. The two sulfur-containing amino acids, cysteine and methionine, give rise to a wide range of volatile sulfur compounds (VSCs) that have a significant impact on flavor formation and give characteristic notes to some aged cheese products. In cheese, VSC production by LAB is limited to mainly methionine catabolism, as S1- and -caseins do not contain cysteine, although there is limited evidence for sulfur fixation. In milk fermentations, the cysteine residues of -casein and whey proteins would potentially be available for conversion into VSCs. Because of their importance in cheese flavor, the biochemistry of VSC production has been the subject of much study. In addition, the breakdown product of methionine, methanethiol, undergoes further conversions with a range of fatty acids, aldehydes, and ketones. Although these pathways are not fully elucidated, the genetic potential of LAB is evident. In terms of flavors derived from sulfur-containing amino acids and their catabolism, cysteine has a sulfury odor, whereas methionine presents an acidic note. Methanethiol presents a decomposing cabbage, garlic odor; methional is described as musty, earthy, cooked potato; and the organic sulfides (dimethyl sulfide, trimethyl sulfide) give sulfury, onion, cabbagy, tomato, green flavor notes. Thioesters (e.g., S-methylthiobutyrate) and thiocarbonyls also give sulfury, garlic, putrid cabbage notes, whereas spermine is ammoniacal. Many of the compounds derived from amino acids have odor descriptors that would imply unpleasant flavors, and indeed, this is the case individually and above certain concentrations. However, it would be more accurate to consider that these compounds reflect the breadth of contributors and the balance of flavors required to provide the overall flavor perception and provide a positive impact both directly and through enhancement of other flavors at low concentrations, but a vulnerability to development of unacceptable ‘off-flavors’ when an imbalance occurs.

Flavor Generation from Milk Fat by LAB Generally, LAB are only weakly lipolytic, but in longterm flavor generation in dairy systems such as in an aged cheese, this is not such a limiting technological feature. However, it can be envisaged that this would be a distinct disadvantage where accelerated ripening of cheese is the target or in any other dairy system where rapid generation of lipolytic flavors is desired – in such cases,


supplementation with lipases from other sources is indeed required. The lipolytic activities of LAB do not include true lipase enzymes that act at the oil–water interface; all such activities described so far are due to soluble esterases. The latter not only catalyze the hydrolysis of fatty acid esters, monoacylglycerides, and diacylglycerides (but not intact milk fat – a lipase substrate), but certain esterases also catalyze an alcoholysis reaction to produce esters from (predominantly) mono- and diacylglycerides and ethanol. Therefore, with milk fat as the substrate, these enzymes generate a range of short-, medium-, and long-chain fatty acids that impart rancid, cheesy, pungent, goaty, fatty, soapy, or waxy flavor notes with relatively high flavor perception thresholds (levels of parts per million). However, the flavor perception thresholds of the esters derived from the corresponding free fatty acids (e.g., ethyl butanoate, ethyl hexanoate) tend to be three orders of magnitude lower and impart fruity flavor notes such as apple, banana, pear, pineapple, and strawberry. Such flavors can mask fatty acid flavor defects, but again an application-dependent balance is required, and fruity flavor notes are often perceived as defects in Cheddar cheese, for example. Nevertheless, the studies of Holland and coworkers are an example of how the outcome of a flavor fermentation can be manipulated by supplementation with an otherwise rate-limiting substrate (in this case ethanol), either through direct addition or by selection of ‘complementary’ LAB.

The Future There is still much to understand about the biochemistry of LAB with respect to flavor compound formation, and the extent to which fermentation systems can be manipulated to produce flavor profiles of interest. Successful industrialization of product manufacture essentially has consistency of product quality as a prerequisite, and scientific understanding underpins the delivery of this consistency. However, the tolerance limits for variation ‘on the supermarket shelf’ are low, which means much tighter control and selection of strains used, with a consequent loss of diversity. Fortunately, there are extensive, untapped pools of genetic diversity in LAB that are found within various culture collections and the artisanal dairy industry. Furthermore, the possibilities for coculturing, albeit between LAB and non-LAB organisms, both bacterial and yeasts, or LAB and selected enzymes, are extensive and offer many options to control flavor composition. In considering manipulation of fermentations, cheese presents some interesting insights, as it is essentially an environment in which the LAB are very much at suboptimal growth conditions and can be considered to be under pH, salt, low temperature, and nutrient starvation

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stresses. Challenging LAB fermentations in different ways may therefore provide the means to not only gain knowledge of the biochemistry of the flavor pathways but also optimize them for targeted flavor delivery. Use of carbohydrate limitation may be a delivery method that potentiates cultures to provide new flavor compounds found in cheese, but not found in the laboratory. For instance, induction of amino acid metabolism during carbohydrate limitation has been shown to produce branched-chain fatty acids not found in milk fat. Also, metabolism of Lactobacillus casei under conditions of lactose starvation, lactose limitation, and nonlimiting lactose (albeit in a semidefined medium, rather than in a dairy stream) results in differences in the identities and relative proportions of both major and minor metabolites produced (although the identities of some components need to be confirmed). Some of the minor components, such as propionic acid (pungent, sour milk, nutty), 3-(methylthio)propionic acid (chocolate, roasted), and

-hexalactone (herbal, coconut, sweet, coumarin), were ubiquitous to all culture conditions. However, production of other compounds, including heptan-2-one (fruity, spicy, sweet, herbal, coconut, woody), nonan-2-one (fresh, sweet, green, weedy, herbal), nonan-2-ol (waxy, green, creamy, citrus), and iso-valeric acid (3-methylbutanoic acid), was not seen in cultures grown with nonlimiting lactose, whereas ethyl lactate (2-hydroxy propionic acid ethyl ester; sharp, tart, fruity, buttery, butterscotch) was identified only under such culture conditions. Furfuryl alcohol (2-furanmethanol; nutty) was identified in cultures containing lactose. With regard to the potential for increasing the diversity of flavors that can be derived from fermentations of LAB, it is interesting to see that studies on both culture collections and more regional and artisanal products are being carried out, although ultimately more needs to be done to establish the key flavor compounds and identify novel flavor compounds. Pertinent ‘artisanal’ examples are the recent studies on Leben, on Pecorino Siciliano cheese, and on a range of dairy and nondairy Lactococcus lactis strains. Leben is made by culturing milk with starters and then churning out the butter, leaving a product with characteristics similar to unsweetened yogurt. A broader range of volatiles was detected in the Leben produced with artisanal strains of Lc. lactis than in the industrial product. In the study on Leben, it was noted that Tunisian consumers prefer the traditionally manufactured product and consider the industrially produced Leben product to have inferior flavor, but no direct correlation between specific flavor compounds and consumer preference was made. Similarly, in Pecorino Siciliano cheeses made with selected artisanal strains, there was a wider range of volatiles produced from a different profile of strains than in the industrial cheeses.

In the study on Lc. lactis strains, aroma compound generation by strains isolated from artisanal raw milk cheeses and industrial starter cultures and nondairy sources was compared. Again, in general, industrial strains presented a lower intensity for aroma descriptors than the other strains, but an aroma potentiation effect was observed with mixtures of strains isolated from different sources. The last observation touches on the potential that coculturing of LAB can present, whether with other LAB strains or with, for example, non-LAB bacteria or yeasts. In cheese, this has been done empirically for millennia, but the deliberate selection of starter and nonstarter strains for targeted cheese ripening based on biochemical understanding of the flavor pathways is comparatively recent. One specific example of combining lactococcal strains to take advantage of the different properties of strains has demonstrated the formation of high levels of branched-chain amino acid derivatives using a strain characterized by its -keto acid decarboxylase and one with high branched-chain aminotransferase activity (see ‘Further Reading’). In ‘crystal-ball gazing’ for the future of flavor production in, or using, dairy systems with LAB, the possibilities for genetic manipulation always need to be considered, but several caveats are immediately relevant. Although LAB can be readily considered to be ‘flavor factories’, and there is potential for those factories to be genetically designed from both homologous and heterologous strategies, two main caveats are consumer acceptance and limitations of milk as a substrate, while the third is application relevance, and the fourth is cost. To illustrate this point, while incorporation of genetic engines for fruit flavor pathways into LAB is possible, present-day consumers may well shy away from any flavors or flavor ingredients thus produced, successful fermentations may require supplementary substrates to be added to the milk (affecting labeling freedom), fruity ingredients based on dairy may have limited application scope, and the cost of the dairy-based ingredients, compared with other flavor sources, may be prohibitive. From a science perspective, how widely fermentations using LAB with dairy substrates can be diversified in terms of flavor generation is an intriguing question: for instance, the potential to deliver new savory, fishy, and chocolate flavors has been seen. However, from a commercial perspective, cost is the most significant driver, and unless an ‘all-dairy’ label can attract a premium for a dairy-based flavor ingredient, or unless such ingredients become more cost-effective than flavors from other sources, the future for LAB flavor fermentations lies within dairy systems that already use their technological properties. In this arena, it is consistency, predictability, targeting, and acceleration that are the drivers of technological success.

Lactic Acid Bacteria | Lactic Acid Bacteria in Flavor Development See also: Cheese: Accelerated Cheese Ripening; Biochemistry of Cheese Ripening; Cheese Flavor; Enzyme-Modified Cheese; Low-Fat and Reduced-Fat Cheese; Microbiology of Cheese; Non-Starter Lactic Acid Bacteria; Overview; Secondary Cultures; Starter Cultures: General Aspects; Starter Cultures: Specific Properties.

Further Reading Amarita F, de la Plaza M, Fernandez de Palencia P, Requena T, and Pelaez C (2006) Cooperation between wild lactococcal strains for cheese aroma formation. Food Chemistry 94: 240–246. Brandsma JB, Floris E, Dijkstra ARD, Rijnen L, Wouters JA, and Meijer WC (2008) Natural diversity of aminotransferases and dehydrogenase activity in a large collection of Lactococcus lactis strains. International Dairy Journal 18: 1103–1108. Crow VL, Coolbear T, Holland R, Pritchard GG, and Martley FG (1993) Starters as finishers: Starter properties relevant to cheese ripening. International Dairy Journal 3: 423–460. Ganesan B, Dobrowolski P, and Weimer BC (2006) Identification of the leucine-to-2-methylbutyric acid catabolic pathway of Lactococcus lactis. Applied and Environmental Microbiology 72: 4264–4273. Ganesan B, Stuart M, and Weimer BC (2007) Carbohydrate starvation causes a metabolically active but nonculturable state in Lactococcus lactis. Applied and Environmental Microbiology 73: 2498–2512. Ganesan B and Weimer BC (2007) Amino acid metabolism in relationship to cheese flavor development. In: Weimer B (ed.) Improving the Flavor of Cheese. Cambridge, UK: Woodhead Publishing Limited. pp. 70–101. Gutierrez-Mendez N, Vallejo-Cordoba B, Gonzalez-Cordova AF, Nevarez-Moorillon GV, and Rivera-Chavira B (2008) Evaluation of aroma generation of Lactococcus lactis with an electronic nose and sensory analysis. Journal of Dairy Science 91: 49–57.


Holland R, Liu S-Q, Crow VL, et al. (2005) Esterases of lactic acid bacteria and cheese flavor: Milkfat hydrolysis, alcoholysis and esterification. International Dairy Journal 15: 711–718. Hussain MA, Rouch DA, and Britz ML (2009) Biochemistry of non-starter lactic acid bacteria isolate Lactobacillus casei GCRL163: Production of metabolites by stationary-phase cultures. International Dairy Journal 19: 12–21. Ko¨nig H, Unden G, and Fro¨hlich J (eds.) (2009) Biology of Microorganisms on Grapes, in Must and in Wine. Berlin; Heidelberg, Germany: Springer-Verlag. Martley FG and Crow VL (1993) Interactions between non-starter microorganisms during cheese manufacture and ripening. International Dairy Journal 3: 461–483. Randazzo CL, Pitino I, de Luca S, Scifo GO, and Caggia C (2008) Effect of wild strains used as starter cultures and adjunct cultures on the volatile compounds of the Pecorino Siciliano cheese. International Journal of Food Microbiology 122: 269–278. Stuart M, Chou L-S, and Weimer BC (1998) Influence of carbohydrate starvation on the culturability and amino acid utilization of Lactococcus lactis ssp. lactis. Applied and Environmental Microbiology 65: 665–673. Tamime AY and Robinson RK (eds.) (2007) Tamime and Robinson’s Yogurt. Science and Technology, 3rd edn. Cambridge, UK: Woodhead Publishing Limited. Weimer BC (ed.) (2007a) Improving the Flavor of Cheese. Cambridge, UK: Woodhead Publishing Limited. Weimer BC (2007b) Genomics and cheese flavor. In: Weimer B (ed.) Improving the Flavor of Cheese. Cambridge, UK: Woodhead Publishing Limited. pp. 219–235. Wilkinson MG and Kilcawley KN (2007) Carbohydrate metabolism and cheese flavor development. In: Weimer B (ed.) Improving the Flavor of Cheese. Cambridge, UK: Woodhead Publishing Limited. pp. 55–69. Ziadi M, Wathelet JP, Marlier M, Hamdi M, and Thonart P (2008) Analysis of volatile compounds produced by 2 strains of Lactococcus lactis isolated from Leben (Tunisian fermented milk) using solid-phase microextraction-gas chromatography. Journal of Food Science 73: S247–S252.