Bacterial diversity and functionalities in food fermentations

Eng. Life Sci. 2012, 12, No. 4, 1–12 Frédéric Ravyts Luc De Vuyst Frédéric Leroy Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Faculty of Sciences and Bio-engineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium 1 Review Bacterial diversity and functionalities in food fermentations Lactic acid bacteria (LAB) play a central role in several food fermentations, producing lactic acid besides other metabolic actions. Popular fermented foods that rely on the use of LAB include fermented meats, sourdoughs, and fermented dairy products. During fermentation, LAB are frequently accompanied by other microorganisms, such as coagulase-negative staphylococci (CNS), yeasts, and filamentous fungi. Whereas fermentation was originally a spontaneous and empiric process, most industrial processes make now use of starter cultures to speed up the fermentation process and standardise the end products and to reduce the risks on misfermentation. A drawback of using commercial starter cultures is their suboptimal selection, which is often solely based on mere technological features. Currently, functional starter cultures are being developed to further optimise the process and to yield additional nutritional, safety, and quality benefits. Specific metabolic properties are being sought for, with a focus on novel, interesting molecules that may, for instance, inhibit undesirable microorganisms, display nutraceutical properties, or contribute to flavour and texture attributes. Keywords: Fermented meats / Fermented milks / Food fermentation / Sourdough / Starter cultures Received: September 27, 2011; revised: December 22, 2011; accepted: January 27, 2012 DOI: 10.1002/elsc.201100119 1 Introduction Fermentation is a biotechnological method historically arisen from the need to preserve food. It can be carried out by filamentous fungi, yeasts, or bacteria, or a combination thereof, which convert fermentable carbohydrates into end metabolites such as organic acids, alcohols, and carbon dioxide [1]. Different foods can be fermented, such as milk, meat, fish, vegetables, and cereals [2]. Lactic acid bacteria (LAB) are of particular interest in fermented foods, because they produce lactic acid as a common metabolic end metabolite [3]. This compound contributes to the prevention of the outgrowth of pathogenic and spoilage microorganisms and thereby prolongs the shelf life of fermented foods. Common food-associated LAB have been granted the ‘generally regarded as safe’ status; food grade genera are Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, OenococCorrespondence: Professor Frédéric Leroy (fleroy@v ub.ac.be), Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Faculty of Sciences and Bio-engineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium Abbreviations: CNS, coagulase-negative staphylococci; LAB, lactic acid bacteria  C 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim cus, Pediococcus, Streptococcus, Tetragenococcus, and Weissella [4]. LAB are divided into three groups according to their carbohydrate metabolism. Obligately homofermentative LAB metabolise hexoses through glycolysis only, yielding solely lactic acid (Fig. 1). Obligately heterofermentative LAB use the phosphoketolase pathway that forms lactate and ethanol or acetate from hexoses and pentoses. Facultatively heterofermentative LAB can degrade hexoses through the glycolysis and pentoses through the phosphoketolase pathway. Next to LAB, other bacterial species are also common in food fermentation. Acetic acid bacteria (AAB) not only contribute to the production of vinegar and cocoa, but are also known for their spoilage capabilities in wine [5]. Coagulase-negative staphylococci (CNS), Kocuria spp., and Micrococcus spp. are found as natural microbiota of fermented meat products next to lactobacilli and pediococci [6]. Besides in meat, CNS can also be found in both hard and soft cheeses, as they are tolerant toward salt and acid [7]. Other bacterial groups that play a role in food fermentations include brevibacteria, corynebacteria, and propionibacteria, which are used in cheese production, and Bacillus subtilis that is used for soybean fermentation [2]. In carbohydrate-rich environments, acidifying LAB frequently grow in association with yeasts that are often lower in numbers. Yeast species of Saccharomyces, Candida, Torula, http://www.els-journal.com 2 F. Ravyts et al. Eng. Life Sci. 2012, 12, No. 4, 1–12 Figure 1. Major pathways active in lactic acid bacteria (LAB). Left panel (based on De Vuyst et al. [10]): carbohydrate metabolism via the glycolysis (homofermentative LAB; lower left) and the phosphoketolase pathway (heterofermentative LAB; lower middle). Upper right panel (based on van Kranenburg et al. [100]): generic conversion of branched-chain amino acids (valine, leucine, and isoleucine), aromatic amino acids (tyrosine, tryptophan, and phenylalanine), and sulphur-containing amino acids (methionine), as initiated by transamination in LAB. Lower right panel (based on Gänzle et al. [23]): arginine deiminase pathway (ADI). Key enzymes involved in these pathways are designated with Roman numbers: I, cell wall-bound fructosyltransferase; II, $-galactosidase; III, maltose phosphorylase; IV, phosphoketolase; V, lactate dehydrogenase; VI, acetate kinase; VII, aminotransferase; VIII, glutamate dehydrogenase; IX, decarboxylase; X, arginine deiminase; XI, ornithine transcarbamoylase; XII, carbamate kinase. Hanseniaspora, Hansenula, and others, can proliferate in these niches causing spontaneous alcoholic fermentation, such as in the spontaneous fermentation of wine and beer [8]. Since the 19th century, the discovery of ‘microorganisms’ enabled the improvement of the production of beer, vinegar, and baker’s yeast by using specific starter cultures [9]. The addition of millions of cells per gram – instead of 100–10,000 cells per gram naturally present on the raw material – to the unfermented material accelerates and guides the fermentation [1]. A drawback of using commercial starter cultures is their suboptimal selection that is often solely based on mere technological features. Usually, the rate and degree of acidification by a LAB strain and its phage resistance are the most important criteria. This limits product diversity and may lead to non-optimally applied microorganisms as starter cultures [1]. 2 Spontaneous food fermentations The outcome of the first spontaneous fermented foods must have been uncertain, depending on the microbiota initially present  C 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim [1]. Optimisation of this unpredictable process was possible through backslopping, during which a small part of a previously successful spontaneous fermentation is used as an inoculum for the next fermentation. After a few refreshments, backslopping leads to a stable microbiota, among which only the best adapted strains will occur [10]. Nowadays, spontaneous fermentations still occur, often as a low-cost alternative to starter cultures, in traditional food fermentations, in developing countries, and for some fermented food products where the exact composition and role of the microbial successions is not fully understood. For example, wine fermentations are often performed naturally to provide a unique regional character. Unfortunately, the fermentation can be too slow or can get stuck with alterations in the wine quality [8]. Backslopping is still applied for sauerkraut, sourdough, and some fermented dairy products [2]. In this review, a focus is set on three economically very important and well-known product groups based on LAB-driven fermentation: fermented dry sausages, sourdoughs, and fermented milk products (Fig. 2). Cheese, another very popular product, was not considered in this study, due to its even wider regional http://www.els-journal.com Eng. Life Sci. 2012, 12, No. 4, 1–12 Bacterial diversity and functionalities in food fermentations 3 Figure 2. Production processes of (A) fermented dry sausage, (B) sourdough bread, and (C) yoghurt. diversity and the fact that its fermentation aspects have already been extensively addressed [2, 11]. 2.1 Fermented dry sausage Together with drying and salting, fermentation is one of the oldest methods to preserve raw meat. Fermented dry sausages are non-heated meat products, mostly made from a mixture of pork meat and fat (Fig. 2). During grinding, additional ingredients such as glucose, lactose, salt, nitrate and/or nitrite, ascorbate, and spices are added. The final mixture is stuffed into casings and is hung vertically to be fermented at temperatures generally between 20 and 30◦ C at a high relative humidity (RH) of 90–95%. During fermentation, the pH decreases due to the action of LAB, making the meat proteins coagulate and generating texture. During maturation, the RH is lowered to 70–80%, which favours the drying and decrease of water activity (aw ). The combination of acidification and aw decrease contributes to the safety of fermented sausages [6]. In spontaneously fermented dry sausages, different species of LAB, CNS, filamentous fungi, and yeasts may be found [6]. Differences in the microbiota lie within technological aspects, including time, temperature, and RH of fermentation and drying. A long fermentation time results in more lactobacilli in the early stages of fermentation with consequent acidity in the flavour profile [12]. The application of long ripening times and a high activity of microorganisms other than LAB, such as CNS, lead to higher levels of volatiles [12]. In raw meat, a presence of LAB in low cell counts of 102 –104 colony forming units per gram of meat (cfu/g) is normal under hygienic circumstances. These LAB grow rapidly and easily dominate the fermentation  C 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim process due to the anaerobic environment of the meat batter, and the presence of NaCl, nitrate, and nitrite. Most common LAB in spontaneously fermented dry sausages are Lactobacillus sakei, Lactobacillus curvatus, and to a lesser extent Lactobacillus plantarum. Next to lactobacilli, pediococci such as Pediococcus pentosaceus and Pediococcus acidilactici, Leuconostoc species such as Leuconostoc gelidum, lactococci such as Lactococcus lactis, Weissella species such as Weissella viridescens, and enterococci such as Enterococcus faecalis and Enterococcus faecium, can be isolated from spontaneously fermented dry sausages [6]. These bacteria, and also other lactobacilli such as Lactobacillus brevis and Lactobacillus rhamnosus, are rarely detected in large amounts compared to L. sakei. This is due to the superior competitiveness of L. sakei, which can be explained by its specialised metabolic repertoire that is well adapted to the sausage environment, including the arginine deiminase (ADI) pathway and the utilisation of nucleosides [13, 14]. Being present in lower cell counts than LAB, CNS play a role in multiple desirable reactions [6]. Important technological properties of CNS are nitrate reductase activity promoting desirable colour development and stabilisation, catalase activity decomposing peroxides to prevent rancidity, and lipolysis and proteolysis generating flavour compounds linked to the conversion of fatty acids and amino acids [12, 15]. In spontaneously fermented sausages, Staphylococcus xylosus and Staphylococcus saprophyticus are commonly present. Nevertheless, S. xylosus may be unable to carry out the fermentation entirely, as the sausage fermentation conditions are not favourable for growth. To a lesser extent, also Staphylococcus succinus and Staphylococcus equorum may play an important role in the ripening process. In general, CNS are poorly competitive in the presence of acidifying microorganisms such as LAB. Most species are only isolated in small quantities from spontaneously fermented sausages. For http://www.els-journal.com 4 instance, Staphylococcus carnosus, a species often recommended and used as commercial CNS starter culture in Europe, is only marginally present in traditional sausage fermentations [16, 17]. In some fermented sausages, particularly those produced in Southern Europe, flavour is also influenced by the development of filamentous fungi and yeasts with proteolytic and/or lipolytic activities [18]. Filamentous fungi, growing on the surface of the product, may also contribute to the stabilisation of the colour through catalase activity, oxygen consumption, and protection against light [18]. The mycobiota of fermented sausages is heterogeneous, but the most predominant mould is Penicillium nalgiovense. Regarding yeasts, Debaryomyces hansenii is often dominating in spontaneously fermented sausages, next to Candida spp. [6]. 2.2 Eng. Life Sci. 2012, 12, No. 4, 1–12 F. Ravyts et al. metabolised via the maltose phosphorylase pathway and the pentose phosphate shunt in heterofermentative LAB species, such as L. sanfranciscensis [23]. Their efficient metabolism coupled to the use of external alternative electron acceptors, together with additional mechanisms such as the ADI pathway, environmental stress responses to overcome acid, high/low temperatures, high osmolarity/dehydration, oxidation, and starvation, increase the competitiveness in the acidic sourdough environment [22, 23, 26]. Besides LAB, a large variety of yeast species is found in sourdough ecosystems. Yeasts are responsible for dough leavening and contribute to flavour formation. Due to the use of baker’s yeast, Saccharomyces cerevisiae is often the most abundant species in bakery sourdoughs. During laboratory fermentations without added baker’s yeast, species such as Candida glabrata and Wickerhamomyces anomalus may prevail [27]. Sourdough Sourdough is a mixture of cereal flour and water that is spontaneously fermented by LAB and yeasts (Fig. 2) [10]. The fermentation of sourdough improves bread texture and flavour, retards staling, and prevents spoilage by moulds and bacteria [19, 20]. As cereal flours are not microbiologically sterile and no heat treatment is applied, addition of water results in the growth of contaminating microorganisms [21]. The bakery environment builds up a house microbiota that may serve as an inoculum for subsequent sourdough fermentations, as adapted LAB are able to survive in consecutive sourdough batches. The stability of a mature sourdough depends on this endogenous microbiota, the metabolic activity of both flour and microorganisms, and technological process parameters such as leavening and storage temperature, fermentation time, pH, redox potential, and dough hydration and yield [22,23]. As a consequence of the heterogeneity of ecological determinants, mature sourdoughs differ in species diversity and metabolic activity, and the most competitive yeasts and LAB will reach numbers above those of the adventitious microbiota. While the cereal type or certain ingredients, such as (added) carbohydrates or salt, favour a certain group of yeasts and/or LAB, a strict correlation can hardly be made between yeasts and/or LAB dominating the fermenting flour/water mixture and the type of sourdough produced [21]. During spontaneous fermentations, obligatory heterofermentative LAB predominate, but also obligatory homofermentative, facultative heterofermentative, and associations of hetero- and homofermentative LAB have been reported [21, 24]. To maintain a more stable product, mother doughs are used as inoculum for the subsequent doughs. These mother doughs are continuously maintained by addition of the desired amount of water and flour according to a defined cycle of preparation. The application of backslopping most often results in the prevalence of obligatory heterofermentative LAB, such as Lactobacillus sanfranciscensis and Lactobacillus pontis [21]. Although sourdough ecosystems are very heterogeneous, they are characterised by a remarkable high stability that may be ascribed to specific metabolic characteristics such as the production of antimicrobial compounds such as organic acids and bacteriocins [25]. The dominance of mainly heterofermentative sourdough LAB is due to their highly adapted carbohydrate metabolism. For instance, maltose, an energy source highly present in dough, is  C 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2.3 Yoghurt and fermented milks Generally speaking, fermented milks are products prepared from milk and/or milk products by the action of specific microorganisms, resulting in a reduction of the pH (Fig. 2). The fermentation can be carried out by (i) LAB, including mesophilic (e.g. cultured buttermilk, Nordic ropy milk), thermophilic (e.g. yoghurt), and probiotic strains (e.g. acidophilus milk), (ii) yeasts and LAB (e.g. kefir, koumiss), or (iii) moulds and LAB (e.g. villi) [28]. Mesophilic LAB, preferring temperatures below 30◦ C, belong to the genera of Lactococcus and Leuconostoc. The microbiota of the thermophilic products, preferring temperatures around 40◦ C, is complex and not always constant; numerous species of LAB such as streptococci and lactobacilli have been found. Probiotic strains belong to species of bifidobacteria, lactobacilli, and enterococci [29]. Products containing yeasts and filamentous fungi are mostly fermented at temperatures around 20◦ C [30]. Due to the high diversity within the group of fermented milk products, a focus will be set in this paper on yoghurts. Even within the group of yoghurts, a large variety exists; differences include type of milk, milk fat level, addition of flavour or fruit, and other ingredients. Also, the type of product can be liquid or semisolid such as for stirred and set yoghurt, respectively. In general, yoghurt production relies upon the fermentation of milk by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus [31]. Legally speaking, the use of the word ‘yoghurt’ implies that the latter two microorganisms are present as live cultures in the product. Both L. delbrueckii subsp. bulgaricus and S. thermophilus are able to grow alone in milk, but when grown together a beneficial effect is obtained, as both species lack parts of the biosynthetic machinery required for de novo synthesis of certain compounds. This cooperative mechanism, results in a higher bacterial growth and a higher production of lactic acid and aroma [32]. Because S. thermophilus is only weakly proteolytic compared to lactobacilli, it heavily relies on the supply of amino acids and peptides provided through the extracellular protease activity of L. delbrueckii subsp. bulgaricus on milk casein [32]. In turn, the production of formic acid, pyruvic acid, folic acid, and carbon dioxide by S. thermophilus stimulates growth of L. delbrueckii subsp. bulgaricus [32]. http://www.els-journal.com Eng. Life Sci. 2012, 12, No. 4, 1–12 3 Directed food fermentations In industrialised countries, the fraction of food products prepared outside the private kitchen is already exceeding 50% [9]. Fermented foods largely belong to this category. As production amounts increase, new issues regarding problems of safety, spoilage, and sensory properties occur. For instance, the use of bulk starter cultures seems to limit product diversity, with a consequent loss of the artisan character of several fermented foods. In an attempt to counterbalance these problems, the use of functional starter cultures has been proposed [1]. Previously, the selection of starter cultures was based on a limited number of properties regarding, for instance, rapid acidification, fast growth, and phage resistance. Besides their basic functions facilitating the early stages of the fermentation, functional starter cultures may improve the fermentation process by contributing to additional sensory, health-promoting, nutritional, and/or bioprotective properties [1]. Ideally, a multifunctional strain is targeted or a combination of different strains combining multiple features can be used. The contribution of extra functionalities can be responsible for the differentiation in quality and value between brands of a certain product [9]. LAB and CNS know a long history of safe use within food preservation and flavour generation; a search for their application as functional bacterial starter cultures may therefore seem evident. 3.1 Fermented dry sausage In the past century, the demand for fermented meats increased and the need for starter cultures emerged. The first trials using acidifying starter cultures were performed by inoculating Lactobacillus onto raw meat by Jensen and Paddock in 1940 [33]. Since, lactobacilli (common in Europe) and pediococci (common in the USA) are used as acidifiers, usually in combination with Gram-positive, catalase-positive cocci (mostly S. xylosus and S. carnosus) [6]. New functional starter cultures, originating from the natural fermentative communities of traditional fermented meats, should lead to improved sausage fermentation with respect to flavour, safety, processing, technology, or health [6, 34]. 3.1.1 Production of antimicrobials Despite the low aw and pH, as well as the presence of curing salt, pathogenic microorganisms such as Listeria monocytogenes, Escherichia coli O157:H7, Salmonella, and Staphylococcus aureus may prevail in fermented sausages [6]. To combat these threats, additional hurdles can be used. For instance, bacteriocins can contribute to the elimination of undesired pathogens such as L. monocytogenes in fermented sausages [35]. In general, the activity of bacteriocins of LAB targets other LAB, contributing to the competitiveness of the producer strain(s), but some bacteriocins are active towards foodborne pathogens. Bacteriocins from LAB offer several desirable properties, as these substances are generally recognised as safe, not active against eukaryotic cells, inactivated by digestive proteases, not influencing the gut microbiota, usually pH- and heat tolerant, often having a bactericidal  C 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Bacterial diversity and functionalities in food fermentations 5 mode of action, and not displaying cross-resistance with antibiotics [36]. Although bacteriocins have only limited inhibitory spectra, several bacteriocinogenic strains have been successfully tested in fermented meats, including enterococci, pediococci, lactobacilli, lactococci, leuconostocs, and CNS [6]. Other antimicrobial compounds may also be successful to produce safer fermented meats [6]. For instance, reuterin produced by Lactobacillus reuteri has a broad spectrum of activity, including fungi, protozoa, Gram-positive bacteria, such as L. monocytogenes, and Gram-negative bacteria, such as Salmonella spp. [37]. Other compounds include nonproteinaceous, low molecular mass, typically hydrophobic, heterocyclic, or aromatic compounds, active towards filamentous fungi and yeasts. Although such compounds may contribute to safety, their use is biased due to extremely low concentrations and due to their production by heterofermentative LAB, such as L. plantarum that also produce carbon dioxide and/or acetic acid [38]. 3.1.2 Flavour production Fermented sausage flavour results from a combination of oxidative and endogenous, bacterial, or even fungal lipolytic and proteolytic enzymatic activities, especially in Southern European type fermented sausages [39,40]. Opportunities exist to improve or diversify flavour by applying carefully selected microorganisms with strong or unique flavour-developing capabilities as functional starter cultures. LAB contribute to flavour due to their carbohydrate metabolism and resulting acidification and usually do not display strong proteolytic and lipolytic activities. CNS are responsible for the major part of the bacterial flavour compounds, due to the breakdown of amino acids resulting from the endogenous proteases [41]. In general, aminotransferases are involved in the conversion of amino acids (branched-chain amino acids, aromatic amino acids, and sulphur-containing amino acids) into the corresponding α-keto acids, followed by decarboxylation into aldehydes (Fig. 1). Amino acids, especially leucine, isoleucine, and valine, are converted into methyl-branched alcohols, aldehydes, and acids, which are linked to fermented dry sausage flavour [12]. For instance, the catabolism of leucine yields 3-methyl1-butanal, 3-methyl-1-butanol, and 3-methylbutanoic acid. Besides the amino acid catabolism, a limited bacterial lipolytic activity has been observed, although both lipolysis and subsequent free fatty acid release are mainly due to tissue lipases [40]. Flavour compounds by CNS may also originate from carbohydrate fermentation, esterase activity, and incomplete lipid β-oxidation. Esterase activity may constitute a relevant technological trait leading to the formation of compounds such as ethyl acetate [42]. β-oxidation may yield compounds such as 2-pentanone, derived from hexanoic acid, and methyl ketones, derived from medium-chain fatty acids [43]. From these, volatile compounds formed through physicochemical lipid oxidation and microbial lipid β-oxidation are more abundant during fast fermentations, while compounds from amino acid catabolism and carbohydrate fermentation are formed mostly when the meat is subjected to a slow fermentation [42]. CNS species and strains differ in their ability to produce flavour compounds [16, 44]. However, it is not fully clear yet http://www.els-journal.com 6 Eng. Life Sci. 2012, 12, No. 4, 1–12 F. Ravyts et al. which species are best suited for flavour enhancement during fermented dry sausage production. For instance, the use of S. xylosus has been mentioned to yield good technological properties, giving a rounder, less acidic taste compared to S. saprophyticus, S. equorum, and S. carnosus [45]. Other authors have suggested that S. carnosus is more related to dry sausage flavour compared to S. xylosus, S. saprophyticus, and S. warneri, as model minces inoculated with S. carnosus contain more methyl ketones and methyl-branched aldehydes and alcohols [12, 41, 46]. Furthermore, the use of S. carnosus could lead to a maturity acceleration, as higher amounts of methyl ketones, methyl-branched aldehydes, and methyl-branched alcohols may be produced [47]. However, depending on the amount of CNS in non-inoculated sausages, starter cultures such as S. equorum and S. succinus do not always result in sausages with improved flavour characteristics [48]. Moreover, a higher specific production rate in liquid medium of 3-methyl-1-butanol production per amount of biomass has been revealed for S. xylosus, S. sciuri, and S. succinus compared to S. carnosus [44]. Inoculation of these species in a Southern European type fermented dry sausage also resulted in a higher 3-methyl-1-butanol production and more appreciated sausages compared to inoculation with S. carnosus [16]. Not only is the choice of CNS important for flavour, but also the applied technology is. For instance, high starting pH values increase flavour compound generation by S. carnosus but decrease flavour release by S. xylosus [49]. Also, increasing the temperature favours the formation of methyl-branched aldehydes and their corresponding alcohols for S. xylosus but not for S. carnosus [50], although the generation of methyl-branched acids increases for both CNS species [49]. Furthermore, sausage ingredients such as curing salts have an effect on the level of volatile compounds. For instance, nitrate may increase or decrease the level of volatile compounds [51]. To enhance flavour compound production by CNS, both the amount of free amino acids and the mechanisms of amino acid conversions must be favoured, as the addition of exogenous proteases or the use of proteolytic starter cultures to increase the amount of free amino acids is not enough to significantly increase aroma compounds [52, 53]. In addition to LAB and CNS, yeasts and filamentous fungi may also contribute to flavour due to proteolytic and lipolytic capabilities [54]. Furthermore, lactate oxidation, conversion of amino acids, and lipid oxidation by these microorganisms may contribute to the sensory quality of the fermented sausages. Especially in Southern-European countries, moulded sausages are part of the typical regional products. When starter cultures are applied, Penicillium in particular is responsible for the seasoning of fermented sausages [54]. Yeasts, such as Debaryomyces species, may also contribute to proteolysis, volatile generation, and esterification [55]. As with bacterial starter cultures, care should be taken when choosing a proper yeast or mould starter culture, because sausage ingredients (e.g. garlic), antifungal compounds produced by LAB (e.g. phenyllactic acid and hydroxyphenyllactic acid), and technological parameters (e.g. temperature and pH) may inhibit growth. Also, metabolic capabilities may differ significantly between strains producing too high or too low concentrations of flavour compounds [54, 56].  C 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 3.1.3 Healthier products Regrettably, consumers often have a negative image about meat products, although several health-promoting factors can be obtained in fermented sausages [57]. A first strategy may be the use of probiotic strains. Probiotic LAB strains have been widely used in dairy products, and recently, attempts have been made to use probiotics in fermented sausages, as they are adequate for the carriage of probiotic bacteria and are usually not heated [58]. LAB strains with probiotic potential can be isolated from spontaneously fermented sausages. Basic requirements include acid and bile salt resistance, antibiotic susceptibility, growth at 37◦ C, and transient adhesion on the intestinal epithelium. Examples include strains from the species L. fermentum, L. pentosus, L. plantarum, L. reuteri, and P. acidilactici [58]. Alternatively, existing probiotic bacterial strains can be applied in meat products [58]. To survive the meat fermentation and drying, the probiotic cells can be microencapsulated in alginate. For instance, a strain of L. reuteri, an inhabitant of the human gastrointestinal tract, has been already applied as a probiotic in fermented sausage through microencapsulation, as a protective strain against infantile colic and Helicobacter pylori infection [59]. However, the effect of probiotic-fermented meats on human health still needs to be proven, since most results are too preliminary and human intervention studies are necessary to prove the envisaged health-promoting property (e.g. immunomodulation) [58]. In addition, several attractive meat-based bioactive substances, such as conjugated linoleic acid (CLA), carnosine, anserine, L-carnitine, glutathione, taurine, and creatine, have been studied for their physiological properties [57]. For instance, CLA possesses potential anti-carcinogenic, anti-atherogenic, anti-cholesterolemic, and immunomodulatory health benefits [60]. Although the production of CLA has already been observed in LAB that could potentially be used in fermented sausages [61], it is unknown whether these physiological doses might have biological effects in humans. Bioactive peptides derived from meat proteins are also a group of promising functional compounds, including for instance the anti-hypertensive, angiotensin I-converting enzyme (ACE) inhibitory peptide [57]. Healthiness of meat products is also related to the absence of toxic compounds. For instance, biogenic amines, such as tyramine and histamine, may appear during sausage fermentation due to microbial decarboxylation of amino acids [62]. LAB can be used as starter cultures to lower the biogenic amine content in fermented sausages. A key requirement in the selection of such LAB strains is the absence of decarboxylase activity on certain amino acids such as tyrosine and histidine. Additionally, the starter cultures should be good acid producers to avoid growth of contaminating non-starter LAB with decarboxylase activity [48]. Mycotoxins are another example of undesired compounds. Both non-mycotoxinogenic and mycotoxinogenic species of filamentous fungi can grow on the surface of fermented sausages. Some filamentous fungi, including Aspergillus ochraceus, Penicillium nordicum, and Penicillium verrucosum, are capable of producing mycotoxins, such as ochratoxin A that forms a potential health risk to the consumer [63]. When being screened as a http://www.els-journal.com Eng. Life Sci. 2012, 12, No. 4, 1–12 potential starter culture, moulds should be nonmycotoxinogenic and competitive towards mycotoxinogenic species from the house microbiota [54]. 3.2 Sourdough Different types of sourdough exist [21]. Type I or traditional sourdoughs are manufactured by continuous backslopping at ambient temperature to keep the microorganisms in an active state. Type II or industrial sourdoughs are one-step or continuous propagation processes characterised by higher incubation temperatures, longer fermentation times, and higher water contents, resulting in a higher acid content. These large-scale sourdough productions result in semi-fluid preparations that are mainly used as dough acidifiers. Type III sourdoughs are dried doughs used as acidifier supplement and aroma carrier for bread production. Commercial available bulk starter cultures aim at standardising the end products by acidifying the dough; new trends tend to develop starter cultures that lead to improved properties [10, 64]. 3.2.1 Production of antimicrobials The bacterial spoilage of bread, known as ropiness, occurs as an unpleasant fruity odour and forms an economic concern in the baking industry. Ropiness is mainly caused by spore-forming bacilli such as B. subtilis, Bacillus licheniformis, and Bacillus cereus, originating from the raw materials. Moreover, Bacillus spp. spores are able to survive the baking process [65]. Bacteriocin production aids LAB to dominate the sourdough ecosystem and to inhibit spore-forming bacilli [10, 66]. In addition, amino acid conversions may provide antifungal compounds. For instance, phenylalanine conversion produces phenyllactic acid that is an antimicrobial agent and therefore of importance in determining the composition of a stable sourdough microbiota [22, 67]. Finally, microbial consortia in sourdough may convert glycerol and lactate to 1,3-propanediol and 1,2propanediol, respectively [68]. The metabolic pathway of 1,3propanediol formation includes reuterin as an intermediate and 1,2-propanediol is further converted to propionate, making the production of these compounds relevant for bread preservation, in particular with respect to fungal contamination [68]. Another promising antimicrobial is reutericyclin, an antibiotic produced by L. reuteri, and active towards Lactobacillus spp., B. subtilis, B. cereus, E. faecalis, S. aureus, and Listeria innocua, but not affecting the growth of Gram-negative bacteria [68]. 3.2.2 Flavour production Proteolysis and subsequent amino acid conversions by sourdough LAB contribute to flavour [10, 23]. Although proteolytic activity by LAB is limited in sourdoughs, acidification through carbohydrate breakdown activates endogenous cereal proteases that liberate peptides and amino acids that can be taken up by the microorganisms present [23]. Amino acid conversions encompass the production of flavour precursors and flavour-active compounds (such as aldehydes and the cor-  C 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Bacterial diversity and functionalities in food fermentations 7 responding alcohols) through transamination/elimination reactions (LAB) or the Ehrlich pathway (yeasts) during sourdough fermentation. In addition, Maillard reactions and Strecker degradation generate aroma during the baking process [19]. Specific dedicated pathways may be involved in flavour formation too. For instance, L. sanfranciscensis and L. reuteri have been found to display glutaminase activity that converts glutamine into glutamic acid. This improves the acid tolerance of these lactobacilli and influences wheat bread flavour [69]. The conversion of arginine into ornithine via the ADI pathway is of special importance during sourdough fermentation in the presence of L. reuteri, L. pontis, L. fermentum, L. brevis, and L. sakei [22, 23, 26]. The ADI pathway enhances the competitiveness of these sourdough LAB species and yields ornithine, a precursor of 2-acetyl-1-pyrroline, which is formed during the baking process and represents the characteristic flavour of baked wheat bread crust [19]. Comparative genomics of enzymes involved in flavourforming pathways from amino acids reveals their presence in lactococci and certain lactobacilli (e.g. glutamate dehydrogenase in the genome of L. plantarum), but their absence in a number of other lactobacilli, such as the type strains of L. brevis and L. reuteri [70]. In addition, experimental evidence on leucine, glutamine, and phenylalanine conversions in sourdough LAB and their relationship with bread flavour exists [23]. However, the presence of some of these (flavour-forming) enzymes can vary between strains from the same species, which explains an additional competitive advantage for sourdough strains possessing this capability, in particular when external electron acceptors are depleted while energy sources are not [71]. Furthermore, this variation is fundamental for the diversity of (artisan) fermented foods, including sourdoughs. 3.2.3 Exopolysaccharide production Bread freshness decreases rapidly during storage as crumb hardness increases. This process, known as staling, lowers consumer acceptance [20]. During sourdough fermentation, LAB produce a number of metabolites, such as organic acids, exopolysaccharides (EPS), and/or enzymes, that positively affect texture and prevent bread staling [72]. The pH drop associated with acid production causes an increase in the activity of flour proteases and amylases, leading to reduced staling and increased quality of the bread [20]. EPS are extracellularly secreted sugar polymers that have the potential to reduce or replace the use of food additives such as hydrocolloids used as bread improvers [73]. Two classes of EPS produced by LAB occur: homopolysaccharides (HoPS), either glucan or fructan polymers, and heteropolysaccharides (HePS), with repeating units composed of galactose, glucose, fructose, and rhamnose [73]. They are produced from sucrose through the activity of extracellular glucansucrases or from sugar nucleotides through intracellular glycosyltransferases, respectively. EPS production by LAB may beneficially affect water absorption of the dough, dough rheology and machinability, dough stability during frozen storage, and loaf volume, as an alternative to expensive addition of plant polysaccharides [74]. http://www.els-journal.com 8 3.2.4 Healthier products As LAB are well adapted to the sourdough environment, cereals can be used as fermentable substrates for the growth of probiotic strains [75]. Moreover, the encapsulation of probiotics can be sustained through cereal constituents, such as starch, to enhance their viability during passage through the gastrointestinal tract. However, multiple technological aspects have to be considered, such as the composition of the raw material, the processing of the raw material, the growth capacity and metabolism of the starter culture, the organoleptic properties and nutritional value of the final product, and the stability of the probiotic strain during storage. Cereals and some of the EPS produced by LAB can be used as sources of non-digestible carbohydrates. Cereals contain, for instance, water-soluble fibres such as β-glucans, arabinoxylans, fructooligosaccharides, and resistant starch [76]. Prebiotic EPS may include levans [77]. These prebiotic properties may selectively stimulate lactobacilli and bifidobacteria present in the colon, promoting beneficial physiological effects [78]. While improving the textural qualities of bread, sourdough fermentation also results in increased availability of bioactive compounds (e.g. lignans, phenolic acids, phytosterols, and tocopherols), reduced phytate content increasing mineral bioavailability, improved usability of the bran fraction that is high in fibres, and low postprandial glucose levels [79]. These nutritional properties may be beneficial towards dental caries, constipation, obesity, colorectal cancer, coronary heart disease, and type-2 diabetes [78]. 3.3 Yoghurt Until the 1950’s, yoghurt production was mostly restricted to communities in the Middle East, the Balkans, India, and Eastern Europe. As the beneficial effect of LAB, common in yoghurts and fermented milks, on human health became of interest, the attitude towards yoghurt consumption has changed [31]. Also, refrigeration and product diversity (e.g. addition of fruits and/or sugar, low-calorie products) opened market opportunities to widely distribute yoghurt products. Nowadays, functional dairy foods and functional dairy starter cultures offer new opportunities, as the natural diversity among LAB has the potential to be exploited beyond mere lactic acid production [31]. As a result, the market of pro- and prebiotics, natural food preservatives, and natural texturisers for dairy application is expanding [1]. 3.3.1 Eng. Life Sci. 2012, 12, No. 4, 1–12 F. Ravyts et al. Production of antimicrobials Naturally produced antimicrobials in fermented milk can be used to replace the addition of chemical preservatives. However, the selection must take into account possible inhibitory effects towards the acidifying starter culture [31]. A wide variety of bacteriocinogenic LAB species can be isolated from fermented milk products, including lactococci, leuconostocs, streptococci, lactobacilli, enterococci, bifidobacteria, and pediococci, and bacteriocins such as nisin and lacticin can be used for dairy preservation [31, 80]. However, not all bacteriocins are successful in yoghurt production. For instance, nisin was ineffective in the prevention of microbial spoilage of minimally processed fruit yoghurts [81].  C 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 3.3.2 Flavour production The proteolysis of milk proteins during milk fermentation generates peptides and amino acids that will contribute to the formation of flavour metabolites [70]. Typical flavour metabolites of plain yoghurt are generated by the starter cultures. Flavour in yoghurt consists mostly of lactic acid, acetaldehyde, and diacetyl [82,83]. The amount of lactic acid largely determines the acceptability of the yoghurt, as too low or too high concentrations will have a negative effect on the usually mild flavour of the product. Lactic acid is the result of homofermentation of lactose, while citrate conversion results in the production of diacetyl below pH 5.5. Acetaldehyde is formed either through pyruvate metabolism or through conversion of threonine [84]. No diacetyl production is obtained in yoghurts prepared with pure cultures of L. delbrueckii subsp. bulgaricus and a larger production is found when a pure culture of S. thermophilus is used compared to a co-culture [83]. In contrast, the production of acetaldehyde is considerably increased during co-culture fermentations [82]. Excess production of acetaldehyde leads to the development of flavour defects [85]. Moreover, strain variability and the effect of the protocooperation will affect the production intensity of flavour compounds throughout the preparation of yoghurts and finally determine consumer acceptability [83]. 3.3.3 Exopolysaccharide production A key sensory attribute of yoghurt production is texture, mostly driven by protein coagulation due to acidification by LAB. EPS are of interest in the dairy industry, as these texture-improving biomolecules can act as ‘natural’ thickener, stabiliser, viscosifier, emulsifier, or gelling agent [73]. This is possible, as EPS form a matrix with the milk proteins, resulting in an improved texture of fermented milks. The polysaccharides xanthan, acetan, and gelan are commercially available but are not favoured, as they are derived from non-food grade microorganisms and result in high production costs [73]. A large variety of LAB, including lactococci, streptococci, lactobacilli, leuconostocs, and pediococci, next to some bifidobacteria, are known to produce HePS [86]. Selection of HePS-producing strains should not only be based on colony ropiness, but also classical selection criteria for starter cultures, including rapid growth, a high acidification rate, and the presence of proteolytic characteristics should be examined [87]. In yoghurt fermentation, HePS production is mainly due to S. thermophilus, but also several strains of L. delbrueckii subsp. bulgaricus are able to produce HePS [86]. Yoghurts fermented with HePS-producing LAB starter cultures show increased mouth thickness, ropiness, and creaminess compared to yoghurts made without these cultures; in addition, yoghurts with HePS-producing cultures have the lowest syneresis and highest gel firmness [88]. Strains of the species S. thermophilus are able to produce a large variety of HePS, which can be subdivided according to their chemical structure, carbohydrate composition, molecular mass, and location [73, 89]. For instance, the monomer composition of HePS produced by S. thermophilus can be subdivided according to the occurrence of galactose, glucose, (N-acetyl)-galactosamine, (N-acetyl)-glucosamine, and rhamnose [87]. The molecular characteristics of the polymers may help to understand the structure–function relationship in a food http://www.els-journal.com Eng. Life Sci. 2012, 12, No. 4, 1–12 matrix and to determine the contribution of different HePS to the thickening of the final products. To do so, the viscofying ability can be investigated by determining the intrinsic viscosity of the HePS in solution [90]. Nevertheless, no clear correlation has been found between acidification rates and milk-clotting abilities of S. thermophilus strains or between acidification rates and HePS production [87]. Furthermore, no clear-cut relationship between the amount of HePS produced and the medium viscosity occurs when different strains are compared. Both size and structure influence the nature of the HePS–protein interactions that are important for the final textural effect of the HePS [88]. Technological parameters are known to influence HePS production during milk fermentations. Incubation temperature, pH, and the available carbohydrates and nitrogen sources may all be of importance [91,92]. Optimal pH conditions for production of HePS are close to pH 6.0 [91], suggesting that maintaining higher pH values will result in increased HePS production, as the exponential growth phase would be extended. Nevertheless, the HePS-producing character can gradually be lowered due to frequent transfer of the culture and prolonged periods of incubation, inducing a spontaneous loss of plasmid-encoded genes in mesophilic strains or DNA rearrangements or deletions in thermophilic strains [73, 86]. As an additional advantage, HePS-producing LAB are believed to offer a more acceptable low-fat fermented milk, due to the HePS masking defects in flavour and mouthfeel. However, lowered acetaldehyde contents have been observed when using HePS-producing LAB in low-fat yoghurts, resulting in a poor flavour [86]. It is important to notice that although certain LAB strains might have the technological properties to be used as starter culture, multiple combinations using different strains to produce yoghurt must be fulfilled to produce a pleasant end product [93]. 3.3.4 Healthier products Dairy products can be divided into basic milk products, addedvalue products, and functional dairy foods [29]. Functional dairy foods provide health benefits beyond their basic nutritional value, including probiotics and prebiotics. In dairy foods, probiotic cultures are usually composed of strains of Bifidobacterium bifidum, Bifidobacterium infantis, and Lactobacillus acidophilus, which do not necessarily contribute to the fermentation [94, 95]. Although some nutritionally beneficial factors are endogenous to milk, others emerge during fermentation processes. For instance, HePS are believed to have hypocholesterolemic, anti-cancer, immunomodulating, and prebiotic abilities [96]. Also, a strain-specific ability to produce folate is present in strains of S. thermophilus [97]. Although milk is a wellknown source of folate, S. thermophilus may increase the folate content by sixfold during fermentation of milk. Several strains of species of lactobacilli, propionibacteria, bifidobacteria, and enterococci are able to form CLA from linoleic acid and thus could be used to increase the CLA level in fermented dairy products such as yoghurt and cheese [98, 99].  C 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Bacterial diversity and functionalities in food fermentations 4 9 Conclusion The production of fermented foods based on acidification by LAB evolved from an ancient, empirical method to improve shelf life of perishable raw materials towards a modern, optimised process to generate end products that are mostly appreciated for their remarkable sensory properties and nutritional value. The introduction of starter cultures has been a landmark in the search for more reliable and uniform production methods. Currently, the search for novel, functional starter cultures opens perspectives [1, 6]. The addition of functional starter cultures affects the overall ecology and composition of the microbial consortia present in the food, ultimately yielding benefits to food producers and consumers. Added value emerges as a result of the altered metabolic impact of the imposed food microbiota, the altered interactions with endogenous enzymes, and/or the influence these changes impose on the food matrix. This added value is mostly related to the perceived food quality (e.g. the development of flavour, texture, and colour, and the use of clean labels), nutritional status (e.g. the inclusion of probiotic or prebiotic elements and the production of nutraceuticals), as well as food safety (e.g. the production of antimicrobials). The detailed mechanisms behind these effects, as outlined in this review, have not always been fully elucidated. The dynamic and spatial complexity of food matrices and the large variability in microbial metabolisms and their regulation, both on species and strain level, hamper optimal selection of starter cultures. An illustrative case is the application of bacteriocin-producing starter cultures, which may be used to replace added chemical preservatives but of which the activity spectrum, production yield, and efficacy is highly variable. Bacteriocins not only target foodborne pathogens, they also may interfere with the background microbiota. The latter may not only result in an enhanced competitiveness of the bacteriocin-producing strains, but it may also affect overall food characteristics in several ways. Also, the in situ production of HePS is not always optimal and may result in low concentrations that may not be sufficient for the desired texture effects. As a result, dedicated research is needed for each of the described functionalities, to further clarify the link between starter cultures, food microbiota, metabolites, food matrix, processing conditions, and impact on the fermented end products. Due to the huge variability in microbial properties, such research is increasingly relying on genomic input [100]. Practical Applications The present review aims at outlining the microbial aspects of fermentation processes for the production of fermented meats, sourdoughs, and fermented milks. These fermented foods are driven by acidification by lactic acid bacteria and are popular foods with important market shares. Besides an overview of the microbial species diversity of these processes and the role of the associated microorganisms in the development of food quality and safety, potential breakthroughs for a further optimisation of the fermentation processes via starter culture innovations are given. http://www.els-journal.com 10 F. Ravyts et al. The authors acknowledge their financial support from the Research Council of the Vrije Universiteit Brussel, in particular the HOA project ‘Artisan quality of fermented foods: myth, reality, perceptions, and constructions’, the Research Foundation-Flanders (FWO) in particular research grant 1514409N, and Flanders’ FOOD. The authors have declared no conflict of interest. 5 References [1] Leroy, F., De Vuyst, L., Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends Food Sci. Technol. 2004, 15, 67–78. [2] Hutkins, R. W. (Ed.), Microbiology and Technology of Fermented Foods, Blackwell Publishing, Iowa 2006. [3] Stiles, M. E., Holzapfel, W. H., Lactic acid bacteria of foods and their current taxonomy. Int. J. Food Microbiol. 1997, 36, 1–29. [4] Wessels, S., Axelsson, L., Hansen, E. B., De Vuyst, L. et al., The lactic acid bacteria, the food chain, and their regulation. Trends Food Sci. Technol. 2004, 15, 498–505. 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