Microbiology of Fermented Dairy Products PDF

Microbiology of Fermented Dairy Products
Luca Cocolin, Paola Dolci, Valentina Alessandria, and Kalliopi Rantsiou, University of Torino, Grugliasco, Torino, Italy
r 2018 Elsevier Inc. All rights reserved.
Introduction
The dairy industry is one of the most relevant in the food sector. It is characterized by a high diversity of products, obtained by
applying different technologies. Especially cheeses can be considered the result of the meeting between tradition and innovation,
since artisanal procedures have been “industrialized” to meet the global consumer demand.
Undoubtedly, microorganisms have an essential role in the dairy sector, especially for products, for which it is necessary to
promote an acidification step in the milk, such as yoghurts and cheeses.
Microbial ecology in dairy products is very diverse and depends from the raw materials (e.g., raw or pasteurized milk), the
processing technology (e.g., curd cooking, stay of the curd in the whey and pressing of the loaf) and the type of product
(e.g., fresh, semi-ripened or ripened cheeses). However, several groups of microorganisms can be generally found in most dairy
products, belonging to lactic acid bacteria (LAB), yeasts and filamentous fungi. LAB are responsible for the conversion of lactose in
lactic acid, resulting in a reduction of milk pH, and they also produce hydrolytic enzymes, active on the protein fraction of the
milk, able to release precursors of aroma compounds. Yeasts and moulds are important for their ability to produce proteolytic and
lipolytic enzymes, which degrade milk proteins and lipids producing important compounds contributing to the final sensory
characteristics.
There is a large body of literature that has described the microbial populations relevant in the dairy sector and there is a general
consensus, in which LAB are divided in starter LAB (SLAB) and non-starter LAB (NSLAB). While the first group is responsible for
the initial acidification of the milk and for the production of proteolytic enzymes, NSLAB possess specific metabolisms able to
produce secondary metabolic compounds, which enrich the organoleptic profile of dairy products, especially yoghurt, butter and
cheese. Yeasts and moulds are not involved in the acidification and they usually participate in the aroma formation through their
enzymatic production. In the case of the molded cheeses, filamentous fungi are also responsible for the typical aspect and
pigmentation of the cheeses.
In this article we will describe these categories of microorganisms, taking into account their role and ecology in the dairy
products. Moreover, we will also discuss the advancements in the understanding of the microbial diversity by using molecular
biology methods, which often have been applied without prior cultivation, thereby avoiding the biases of the traditional
microbiological methods.
Starter Lactic Acid Bacteria
Over the centuries, cheese makers have domesticated fermentation processes empirically, without a deep understanding of the
multiple biochemical reactions governing the sensory qualities of their cheeses. Nowadays, the role of microbiota in terms of
metabolic activities has been mostly understood and many dairy productions are piloted and optimized by using starter cultures to
standardize the fermentation process and reach the targeted properties for the final products.
Natural starter cultures (NSC) are produced daily, at cheese plant, by some form of backslopping and/or by application of
environment selective pressure. Because no actions are used to prevent contamination from cheese making environment and raw
milk, NSC are continuously changing, undefined mixtures of several species and strains of LAB (Parente and Cogan, 2004).
Biodiversity is considered a strength point in NSC and producers of traditional cheeses, as Grana Padano and Parmigiano
Reggiano, accept slight variability in their products as compromise with more peculiar and individual characteristics coming from
their ability to manage wild populations in raw milk and whey starters (Gatti et al., 2014).
The concept of selected starter culture (SSC) is well established in the dairy sector, referring to homofermenting lactic acid
strains, which produce lactic acid giving rise, with added rennet to curd formation. The mesophilic lactococci, mainly Lactococcus
lactis subsp. lactis and Lactococcus lactis subsp. cremoris, generally make up around 90% of a mixed dairy starter population while
thermophilic, mainly Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus helveticus, are employed in
cooked curds because of their heat tolerance. Mesophilic cultures are used in the production of, for example, Cheddar, Gouda,
Blue, Camembert, Edam, while thermophilic cultures are used for high temperature cooked hard cheese such as Emmental,
Gruyere, Grana Padano and Parmigiano Reggiano (Beresford et al., 2001).
As reported above, the homofermentative L. lactis is the species mostly employed as mesophilic starter. The primary function of
this microorganism, when inoculated into vat milk, is to produce acid, reducing the pH of milk to o5.3 in 6 h at 30–371C.
Moreover, L. lactis starter strains can be chosen also for their ability to metabolize citrate, during fermentation process, resulting in
flavor production.
L. lactis is also involved in cheese ripening where its enzymes are involved in proteolysis and conversion of amino acids into
flavor compounds (Beresford et al., 2001). The production of biopeptides by cell envelop-proteinases (CEP) has been studied in L.
Reference Module in Life Sciences doi:10.1016/B978-0-12-809633-8.12108-9 1

2 Microbiology of Fermented Dairy Products
lactis and showed high strain-dependent variability resulting in the production of peptides associated to different aroma
(bitter, sweet, acid etc.) (Thierry et al., 2015). L. lactis contributes to cheese ripening either as intact cells, in early ripening, by
means of its proteolytic enzymatic apparatus, and as autolyzed cells, during late ripening. On the basis of recent insights it has
been hypothesized a role of L. lactis in VNC state, in cheese ripening, (Ruggirello et al., 2016).
In the last years, additional characteristics have been taken into account for the selection of new L. lactis strains to be added to
starter cultures. Bioprotective properties have been studied by different authors. Renes et al. (2014) showed that the use of selected
autochthonous L. lactis cultures reduced the content of biogenic amines in ewe’s milk cheese, while Dal Bello et al. (2012) studied
the ability of bacteriocin producing L. lactis strains to limit Listeria monocytogenes growth in Cottage cheese manufacturing.
Thermophilic starter strains, mainly belonging to S. thermophilus, Lb. delbrueckii subsp. bulgaricus and Lb. helveticus species are
chosen according to their ability to acidify milk in the range of 50–551C. Likewise, other properties related to texture, flavor, health
have been taken into account, for their selection, in the last years.
Fuglsang et al. (2003) studied the production of bioactive peptides in various LAB, and found that Lb. helveticus strains, in vivo
conditions, were able to produce antihypertensive peptides in sufficient amount to efficiently decrease blood pressure.
Stretchabilty has been also widely studied for its importance in the production of cheeses like Mozzarella and Emmental. This
property was correlated, in Lb. helveticus and Lb. delbrueckii subsp. bulgaricus, to a low level of proteolysis accompanied by a low
release of intracellular peptidases into the cheese aqueous phase (Sadat-Mekmene et al., 2013). Richoux et al. (2009) showed that
qualitative more than quantitative aspects of proteolysis were determinants for the development of stretchability.
Finally, some authors investigated the ability of S. thermophilus strains to metabolize galactose. Galactose accumulation in
cheese can favour the growth and CO2 production by undesirable bacteria capable of galactose utilization. Despite most of S.
thermophilus strains are unable to metabolize galactose, Umamaheswari et al. (2014) selected 6 strains Gal þ to be used to reduce
the quantity of residual galactose in yogurt. Mukhurjee and Hutkins (1994) also ascribed the low residual galactose in mozzarella
cheese using S. thermophilus in combination with Lb. helveticus in starter cultures.
Non Starter Lactic Acid Bacteria
Non starter lactic acid bacteria (NSLAB) are important constituents of the cheese microbiota during ripening. Conversely to SLAB,
NSLAB are not involved in the milk acidification process or the curd formation, but are responsible for important biochemical
modifications that take place during ripening of the cheese curd. They play an important role in aroma formation in hard cheeses
while their presence may result detrimental for fresh or soft cheeses that do not undergo ripening. NSLAB most likely originate
from raw milk and are thermo-tolerant enough to survive pasteurization and/or high temperatures used in curd cooking.
Furthermore, they can colonize the dairy environment and equipment, also through biofilm formation, and therefore invo-
luntarily inoculate milk and curd during cheese production.
During cheese ripening, complex and dynamic NSLAB consortia evolve. The kinetics of the different NSLAB populations
depend on parameters such as initial load, environmental conditions (mainly temperature and humidity of ripening, salt
concentration) and other technological aspects such as cooking, stretching of the curd, use of natural starters, ripening time
(Gobbetti et al., 2015). NSLAB mainly consist of species belonging to the Lactobacillus genus. Oftentimes the term mesophilic
lactobacilli is used to designate NSLAB and to distinguish the species mainly found during ripening from the thermophilic,
SLAB responsible for the acidification. Lactobacillus paracasei and Lb. plantarum are considered predominant NSLAB species,
but Lb. rhamnosus, Lb. casei, Lb. fermentum, Lb. parabuchneri, Lb. curvatus, Lb. buchneri, Lb. brevis and Lb. pentosus are important
constituents of NSLAB consortia and frequently isolated from cheeses during ripening. The above mentioned Lactobacillus
species are either facultative or obligate heterofermentative and have the metabolic capability to utilize both hexoses and
pentoses or solely pentoses respectively. Although to lesser extent, other genera of LAB may be part of NSLAB consortia.
Enterococcus spp. naturally occur in artisanal cheeses, mainly produced in Southern Europe (Giraffa, 2003). Enterococcus spp.
are considered controversial microorganisms mainly due to demonstrated antibiotic resistance by members of this group,
capacity to produce biogenic amines and presence of virulence factors within the genome of food associated Enterococcus.
Furthermore, conversely to what has been proven for different Lactobacillus, no direct link between the presence of Enterococcus
and specific modifications (chemical or other) in the product has been shown (Giraffa, 2003). Despite that, certain species,
namely E. faecalis, E. faecium and to a lesser extent E. durans, have been found to dominate the NSLAB microbiota of cheeses
and therefore are believed to contribute to the sensorial characteristics of the product. Pediococcus spp., mainly P. acidilactici
and P. pentosaceus, have also been isolated from a variety of European ripened cheeses. Leuconostoc spp. are used as starter
cultures for the production of Swiss type (for eye formation) and pressed ripened Dutch cheeses and to facilitate cheese
openness in blue veined cheeses (Hemme and Foucaud-Scheunemann, 2004). Consequently, leuconostocs can be isolated
from cheeses, produced with Leuconostoc – Containing starters, during ripening. Furthermore, they are isolated from a variety
of raw-milk, as well as cooked curd and white-brined cheeses (Beresford and Williams, 2004). Isolates from raw milk cheeses
mainly belong to Leuc. mesenteroides ssp, mesenteroides, Leuc. mesenteroides ssp. dextranicum and Leuc. citreum (Hemme and
Foucaud-Scheunemann, 2004).
The cheese components that are potential substrates of NSLAB metabolism during ripening are: residual lactose, lactate and
citrate, triglycerides that undergo lipolysis with liberation of free fatty acids (FFA) subsequently catabolized, and proteins (mainly
the casein matrix) that undergo proteolysis to peptides and free amino acids (FAA) that, in turn, are further degraded
Microbiology of Fermented Dairy Products 3
(McSweeney and Sousa, 2000). The metabolic pathways and enzymatic reactions described above are not exclusively associated to
NSLAB. SLAB, rennet added for the coagulation, the milk itself, as well as exogenous sources, contribute to the biochemical
modifications described during ripening. Residual lactose can be utilized by facultative heterofermentative lactobacilli as an energy
source while citrate can be co-metabolized with sugars, by leuconostocs and mesophilic lactobacilli. The main products of citrate
metabolism are diacetyl (produced in limited amounts), acetoin and 2,3 – butanediol; important flavor compounds for some
cheese varieties. For Dutch cheeses citrate metabolism results in CO2 production that leads to eye formation. Finally, acetate can be
produced from lactose, lactate or citrate metabolism and is considered important flavor compound for many cheeses (McSweeney
and Sousa, 2000). FFA that originate from lipolysis are important flavor compounds, indispensable for the sensorial characteristics
of many types of cheeses. Short chain fatty acids have a direct impact on cheese flavor and are also precursors for the production of
acids, alcohols, aldehydes, esters and thioesters, that further enhance flavor development (McSweeney and Sousa, 2000). Gen-
erally, non-starter lactobacilli, enterococci and pediococci have shown low lipolytic activity although important variability for this
phenotype has been observed (Giraffa, 2003). For hard and semi-hard –type cheeses, casein proteolysis attributable to rennet
enzymes and the microbial proteolytic system is an important biochemical pathway that leads to flavor formation (Smit et al.,
2005). Through proteolysis, LAB are able to satisfy their amino acid requirements. Casein is mainly degraded by chymosin,
endogenous milk enzymes and cell-envelope proteinases of SLAB that liberate peptides and amino acids. NSLAB are mainly
responsible for peptidolysis and liberation of free amino acids. Peptides are hydrolyzed by intracellular peptidases and a wide
variety of peptidolytic specificities has been described in non starter lactobacilli, pediococci and Leuconostoc spp (Beresford and
Williams, 2004). Both peptides and amino acids possess specific flavor characteristics, however it has been underlined that the rate
limiting step in flavor formation is the conversion of amino acids rather than their release through casein degradation (Beresford
and Williams, 2004; Smit et al., 2005). Amino acid conversion ability varies within LAB and these activities are linked to the ability
to synthesize amino acids (Smit et al., 2005). An a-ketoglutarate-dependent transamination reaction is the first step in amino acid
break down. The resultant keto-acids are substrate for enzymatic or chemical reactions that lead to hydro-acids, aldehydes,
carboxylic acids and alcohols; many of them flavor intensive molecules. Branched chain and aromatic amino acid transaminase
activities, that are essential for flavor formation, have been detected in non-starter lactobacilli (Beresford and Williams, 2004). The
amino acid conversion potential of certain non starter lactobacilli has been further confirmed by in silico analysis and comparative
genomics focusing on pathways of flavor formation from amino acids (Liu et al., 2008). Decarboxylation is an alternative catabolic
pathway for the pool of free amino acids that are available to LAB in cheese. Amino acid decarboxylation is believed to play a
crucial role in pH homeostasis and allows microorganisms to respond to low pH stress conditions. The products of amino acid
decarboxylation are CO2 and primary amines. Some of these amines are volatile compounds that contribute to flavor and some
cheese types contain numerous volatile amines (Zuljan et al., 2016). Amino acid decarboxylation reactions in cheese may also
have a food safety implication. Biogenic amines produced by amino acid decarboxylation, when ingested in high quantities, can
cause health problems. The most common biogenic amines found in cheese are tyramine, histamine and putrescine and the main
producers are NS-LAB strains of the Enterococcus and Lactobacillus genera (Zuljan et al., 2016). Production of biogenic amines is
strain dependent; therefore, careful management of the microbiota (both SLAB and NSLAB) throughout production should reduce
the presence of these compounds in cheese.
An interesting trait that is common among LAB is the ability to produce bacteriocins. Bacteriocins have been defined as
bacterially produced, small, heat-stable peptides that are active against other bacteria and to which the producer has a specific
immunity mechanism (Cotter et al., 2005). Bacteriocinogenic LAB have been the focus of intense research since they can
potentially by applied as biopreservation agents, active against foodborne pathogens or spoilage microbes in cheeses. Bacter-
iocinogenic LAB, belonging to Enterococcus and Lactobacillus spp, have been isolated from cheeses and were shown to have
inhibitory activity towards important foodborne pathogens (for example List. monocytogenes, Staphylococcus aureus) and spoilage
agents (for example Clostridium spp. responsible for the late blowing of hard cheeses) (Favaro et al., 2015). The antifungal activity
of several species of Lactobacillus and Enterococcus is also of particular importance since fungal spoilage of dairy products is
common and is currently dealt with using chemical preservatives.
Given the multifaceted attributes of NSLAB that influence the overall quality of the final product, the dairy industry is
particularly interested in controlling the ripening process to achieve desired standards. For this purpose, NSLAB are often added as
secondary/adjunct cultures. Strain selection for the development of such cultures is today sophisticated and takes into
consideration not only technological properties but also safety characteristics, protective and probiotic potential.
Secondary Microbiota
In the definition of cheeses, different microorganisms are involved: longer than LAB population, other groups play fundamental
role in the final character of the different cheeses. They are defined as the secondary microbiota, but of primary importance in the
definition of peculiar characteristics.
The microbial diversity of the cheese depends on both the milk microbiota and on practices of production. This biodiversity
decreases in the core of the cheese where LAB are the prevalent species, but it persists on the surface where numerous species are
detected. The difference between cheeses is due particularly to changes in the dynamics of the microbial population used in the
production. Cheese microbial population still remains difficult to control due to their complex dynamics and to their interactions
(Artini et al., 2015).
The secondary microbiota significantly contributes to cheese characteristics, together with milk composition and starter and
non-starter LAB activity (Bockelmann and Hoppe-Seyler, 2001) they are responsible for color, flavor and texture development
during cheese maturing (Rademaker et al., 2005).
Propionic acid bacteria (PAB) are widely used in Swiss-type cheeses to generate holes and specific flavor notes, and surface
bacteria that contribute to the characteristic flavor and color of smear-rind cheeses. Many of these properties depend on the strain
within a species, and the choice of a selected strain is thus a means to modulate the final cheese properties (Thierry et al., 2015).
The formation of flavor results from the conversion of milk lactose, citrate, caseins and lipids into taste and aroma compounds
during the fermentation of dairy products. Propionibacteria is one of the important class of organisms used for the production of
the Swiss cheese varieties. Swiss type cheese is a generic term for hard cheeses that were initially produced in Emmen valley in
Switzerland and they are easily identifiable by characteristic round regular eyes. In this group of Swiss cheeses are included
Gruyere, Maasdammer, Leerdammer, Emmental etc. Propionibacteria are often added to milk as adjunct cultures, but sometimes
they are indigenous to the raw milk (Thierry et al., 2005).
Propionibacteria are Gram positive, non spore forming, anaerobic or aerotolerant and they possess several metabolic pathway:
one of the most important is the central carbon metabolic pathway through which they use lactate during growth and produce
propionate, acetate and carbon dioxide beside different functional metabolites like B group vitamins, conjugated linolenic acid
(CLA) etc. (Hugenholtz and Smid, 2002). They are used widely as ripening cultures and different studies have also defined them as
potential probiotic bacteria. They are involved also in the food preservation being microorganism able to produce antimicrobial
compounds as bacteriocin and organic acids involved also in the antifungal activity.
Propionibacterium spp. was first discovered in 1906, when the relationship between the presence of Propionibacterium freu-
denreichii and the formation of holes in Swiss Emmental cheese was described. Indeed, Pr. freudenreichii together with Pr. acid-
opropionici are the principally used as an adjunct culture for the production and considered the most industrially important species
(Poonam et al., 2012).
Focusing the attention on their technological attributes, they are mainly used as an adjunct culture for manufacture of Swiss
type cheese where they are responsible for the characteristic flavor and eye formation. Beside this activity, they dominate the cheese
microbiota during the ripening period (Rehn et al., 2011). However, their growth and fermentation rates in cheese depend on the
conditions, in particular, NaCl content of the cheese (Richoux et al., 1998).
Eye formation is the result of CO2 production during their growth, creating holes. On the other hand, flavor production is due
to the metabolism of lactate (produced by LAB), aspartate and amino acid catabolism and fat hydrolysis. Volatile flavor com-
pounds as 3-methylbutanal, 2-methylbutanal, and 3-methylbutanoic acid are produced by the catabolism of branched chain
amino acids, particularly from leucine, isoleucine and valine (Poonam et al., 2012; Thierry and Maillard, 2002). They also produce
FFA through lipolysis of milk fat. Propionibacterium species can also be used in the manufacture of some cheeses without eyes to
enhance flavor formation: they can also modulate the flavor of Cheddar (Fernandez-Espla and Fox, 1998), Raclette and Morbier
cheeses (Thierry and Maillard, 2002; Thierry et al., 2005). The inclusion of PAB as starter affect substantially the volatile profile;
their presence increase the levels of several compounds, such as branched-chain aldehydes, primary alcohols, branched-chain
alcohols, diacetyl, acetoin, ethyl esters, branched-chain acids, and short-chain acids. The aroma formulation is connected to their
inter- and intraspecies diversity in the production of aroma compounds as demonstrated by Yee et al. (2014).
As introduced above, Pr. freudenreichii in particular has a key role in the formation of the typical round holes (eyes).The correct
formation of holes depends on physicochemical factors, such as the presence of nucleation sites for hole development, an
appropriate cheese structure, a massive production of carbon dioxide that induces a local saturation of gas leading to the
formation of holes. During the ripening in the warm room, Pr. freudenreichii produces CO2 from lactate fermentation, thus
producing the main part of the CO2 formed in cheese, the remaining part being formed by facultative heterofermentative
lactobacilli from citrate fermentation and amino acid catabolism (Thierry and Maillard, 2002).
In recent years, Pr. freudenreichii has been evaluated for its probiotic properties and it has been shown that it beneficially
modulates the gut function and physiology (Thierry et al., 2011). Furthermore, Cousin et al. (2016), showed that Pr. freudenreichii
can kill human colon cancer cells acting as a potential protective probiotic against gut cancer both in vivo and in vitro. The
successful application of PAB as probiotics was recently demonstrated with milk fermented by Pr. freudenreichii, which induced
apoptosis of HGT-1 human gastric cancer cells (Cousin et al., 2012). Recently (Angelopoulou et al., 2017) showed that P.
freudenreichii could be used also in the production of Feta cheese.
Many cheeses are characterized by complex surface microbiota. Cheese rind ecosystem is in contact with the external envir-
onment and consequently differs from the cheese core in terms of microbial composition and biochemical characteristics
(Irlinger et al., 2015). The surface microbiota has an important organoleptic impact on cheese thanks to its various enzymatic
activities and its role as a barrier against pathogens and spoilage microorganisms. The structure of cheese rind communities differs
between facilities and describes significant shifts in the microbiota between short- and long-ripened cheeses (Dolci et al., 2009;
Schornsteiner et al., 2014).
During cheese ripening, complex microbial communities, generally referred to as smear, develop on the surface of some types
of cheese. In fact, such cheeses, when exposed to air, naturally tend to develop a smear layer on the surface, typically consisting of
yeasts and bacteria (Chapman and Sharpe, 1990). The composition of this bacteria, yeasts and moulds, depends on the cheese
technologies, environmental conditions, such as temperature, humidity and salt, as well as on the microbiota of the brine and of
the rooms in which cheese is ripened (Fontana et al., 2010). Due to extensive washing of the cheese surfaces with brine during
ripening, bacteria-ripened cheeses are also known as washed rind cheeses and develop a red-brown glistening appearance called
“red smear”. The most widespread cheeses with this characteristic are Tilsiter or Limburger (Dutch production), Baufort and
Munster (French) and Italian cheeses as Taleggio and Fontina (Corsetti et al., 2001; Dolci et al., 2014). In these cheeses, yeasts and
moulds initially dominate the surface post-manufacture because they are acid-tolerant and salt-tolerant. The development of yeasts
during the first few days of ripening, which metabolize the lactate completely into CO2 and H2O, and forming alkaline meta-
bolites, such as ammonia, induces an increase in the surface pH from 5 to 6 (Bonaiti et al., 2004). In addition, the production of
growth factors by yeasts appears to promote the development of a Gram positive, catalase positive, salt-tolerant microbial
communities composed mainly of coagulase-negative cocci (CNC) belonging to genera such as Staphylococcus, Micrococcus
(Bockelmann, 2002) and coryneform bacteria (genera: Brevibacterium, Arthrobacter, Microbacterium and Corynebacterium)
(Bockelmann et al., 2017; Corsetti et al., 2001). Traditionally, growth of the surface microbiota is initiated with the help of mature
cheese, which release part of their surface into the smear tank (Bockelmann, 2002). The disadvantage is that pathogens or
contaminants are also transferred to the smear tank to all the cheeses: the control of smear development on cheese surface is
considered essential during ripening to reduce the risk of surface contamination by spoilage and pathogenic microorganisms
(Bockelmann and Hoppe-Seyler, 2001). The knowledge of the microbial composition is also a prerequisite for the development of
surface starter cultures. The surface starter culture could help in the preservation of spoilage microorganisms.
Generally this smear layer is undesirable in many hard and semi-hard cheeses, but is fundamental for soft and semi soft cheeses
(Bockelmann, 2002). These smear microorganisms play a role in the ripening process, both through the action of proteolytic and
lipolytic enzymes and the formation of many alkaline products that penetrate the body of cheeses.
Brevibacterium linens is one of the important surface microorganism that is present in the smear of surface-ripened cheeses and is
commonly regarded as the organism primarily responsible for the characteristic taste, aroma, and color of surface cheese. B. lines
was the first commercial smear culture and is today sold by all major starter culture suppliers (Bockelmann et al., 2017). The
metabolism and physiology of this microorganism determine its growth on smear surface-ripened cheeses and the effect of such
growth on the characteristics of the cheese (Meile et al., 2008; Motta and Brandelli, 2008).
More in general Brevibacterium spp. is a non-motile, non-spore formed, gram-positive coryneform bacteria that tolerates high
salt concentrations (8%–20%), and is capable of growing in a broad pH range with an optimum at pH 7.0. They also survive
carbohydrate starvation and drying for extended periods. Some strains of Brevibacterium produce distinctive red-orange carotenoid-
type pigments when exposed to light during growth. The color of the colonies varies from orange (B. linens), through gray-white
(B. epidermidis and B. casei) to purple (B. iodinum) (Jones and Keddie, 1986; Motta and Brandelli, 2008). (carotenoids) of the type-
strain is often light dependent.
The interest in B. linens is connected to their ability to produce the self-processing extracellular proteases (Rattray et al., 1995),
high levels of volatile sulfur compounds (Dias and Weimer, 1998a,b), bacteriocin (Motta and Brandelli, 2008), cell membrane-
associated carotenoid pigment, and for their aromatic amino acid metabolism (Arrach et al., 2001).
Other important bacteria involved in the cheese definition and in the surface, are CNC composed mainly by Micrococcus and
Staphylococcus genera. They are considered as positive biota involved in the development of organoleptic characteristic of the end
products thanks to their proteolytic and lipolytic activities.
Among staphylococci, Staphylococcus equorum is one of the more predominant in cheeses. High cell count of staphylococci were
found in brines of several cheese factories, explaining that brines are a natural source for cheese surface (Jaeger et al., 2002).
Except Staph. equorum, other several species as Staph. cohnii, Staphy. epidermidis, Staph. saprophyticus, Staph. xylosus and Staph.
warneri have been isolated from surface ripened cheeses (Bockelmann, 2002; Bockelmann and Hoppe-Seyler, 2001; Rea et al.,
2007).
Staph. equorum was also isolated from milk and some types of cheese (Ercolini et al., 2003) and it was proposed as starter
culture for Swiss semi-hard cheeses (Place et al., 2003) and smear-ripened cheeses (Bockelmann, 2002). Staph. equorum strains were
isolated for their ability to produce bacteriocins and Bockelmann et al. (2017) showed that its cultures would be an ideal adjunct
for cheese factories being able to inhibit List. monocytogenes.
The genus Micrococcus plays also important roles in cheese ripening due to its high proteolytic and lipolytic activities
(Morales et al., 2006).
Yeasts and Moulds
As described above, dairy products are characterized by a high biodiversity in terms of bacterial populations, which are mainly
responsible for the acidification and the production of aromas from proteolytic and lipolytic activities. However the complexity of
these microbial ecosystems is enriched by the presence of eukaryotes, represented by yeasts and moulds (unicellular and multi-
cellular[filamentous] fungi). Compared to the bacteria, the yeast and mould relevant to dairy products are relatively limited and
comprise genera and species able to ferment and assimilate lactose, utilize lactic and citric acids, grow at low temperature and in
presence of high salt concentration, and able to hydrolyze milk proteins and lipids. Kluyveromyces marxianus (anamorph Candida
kefyr), Kluyveromyces lactis, Debaryomyces hansenii (anamorph C. famata), Yarrowia lipolytica (anamorph C. lipolytica), and Sacchar-
omyces cerevisiae are the most isolated yeasts from dairy products (Frohlich-Wyder, 2003). Regarding filamentous fungi, Penicillium
camemberti and P. roqueforti are used as starter cultures for the production of mould fermented cheeses (Haasum and Nielsen,
1998). Moreover, the yeast-like fungus Geotricum candidum is also relevant for Camembert cheese production and some semi-hard
cheeses, in which specific surface smear is developed.
Yeast and moulds are, however, also responsible for spoilage in dairy products. The species may different or the same as the
ones described above, moreover depending on the products from where they are isolated, they can be considered positive
or negative.
Yeasts represent spoilage organisms in the dairy sector due to their ability to grow at low pH and low temperature. They are
mainly involved in blowing of yogurt packages, through their fermentative metabolism, and they are responsible for anomalous
coloration of cheeses (mainly fresh), especially those producing pigments. Like yeasts, adventitious moulds should be considered
spoiling agents of dairy products. They usually grow on the surface of the products due to their need of oxygen, thereby altering
their aspect. However of specific concern is the capability of some species (e.g., Aspergillus flavus) to produce mycotoxins, which
should be considered a risk for human health. The abundance and distribution of spoilage yeasts and moulds differ between
products and within the same type of product manufactures have important role in the contamination. Even different batches of
the same dairy product of the same manufactured can have different levels of spoilage yeasts and moulds (Banjara et al., 2015;
Garnier et al., 2017).
Yeasts in dairy products are relevant to modulate the ecology of the cheese rinds, where they mainly metabolize lactic acid,
thereby allowing the development of a complex microbial ecosystem composed by several bacterial groups, such as Staphylococcus,
Brevibacterium and Corynebacterium (see above) and filamentous fungi. They are present in the raw materials, in the environment
and the brine (smear water) represents an excellent carrier for their spread on the cheese surface. Debaryomyces hansenii, C. versatilis,
K. marxianus, Sacch. cerevisiae, Torulaspora delbrueckii, Trichospomn cutaneum and Y. lipolytica are some of the most relevant species
found in those ecosystems (Larpin-Laborde et al., 2011).
In fresh and short ripening cheeses, yeast ecology is represented by lactose positive species such as K. lactis and K. marxianus. In
mould-ripened cheeses D. hansenii and G. candidurn, as well as K. marxianus and Y. lipolytica dominate the mycobiota
(Frohlich-Wyder, 2003).
In ripened cheeses, they contribute to sensory characteristics through the production of aroma from proteolysis and lipolysis,
and they also play an important technological role in blue-veined cheeses where they produce CO2 from the fermentative
metabolism, which helps to maintain an open structure, allowing oxygen to penetrate in the loaf allowing filamentous fungi
development. Y. lipolytica is recognized to have a great lipolytic activity (Groenewald et al., 2014), releasing fatty acids from
triacylglycerols, that are important precursors of volatile compounds. Proteolysis is also an important role of dairy yeasts. K. lactis,
K. marxianus, D. hansenii, Y. lipolytica, G. candidum and C. catenulata have been all characterized by their strong extracellular
proteolytic and or peptidolytic activity (Baur et al., 2015). Relevant to underline is their role in the brake-down of the bitter
peptides, especially G. candidum (Auberoer et al., 1997).
Regarding the filamentous fungi, as already described above, they are mainly considered as spoilage agents, however some species
are exploited in the dairy sector since they are taking part to the ripening of mould-ripened cheese. Specifically P. camemberti and
P. roqueforti are used as starter cultures for the production of Camembert, Brie, Roquefort and Gorgonzola cheeses.
Penicillium camemberti in Camembert and Brie cheeses is essential for their characteristic appearance and sensory profile, since
low-molecular-weight compounds produced by the fungus contribute significantly to taste. Volatile fatty acids, in particular, are
produced from lipolytic activities important in soft cheeses, such as Camembert, where free fatty acids can reach up to 10% of the
total fatty acids present. P. camemberti produces also proteases, involved as well in the development of the aroma profile. (Abbas
and Dobson, 2011a).
Penicillium roqueforti is used as a fungal starter culture for the production of blue-veined cheeses such as Danablu, Gorgonzola,
Roquefort, and Stilton. The fungus is involved in the production of both proteolytic and lipolytic enzymes. The proteolytic
enzymes soften the curd and produce the desired body in the cheese. The water-soluble lipases hydrolyze the milk fat to free fatty
acids, which contribute to the flavor of Blue cheeses (Abbas and Dobson, 2011b).
Culture-Dependent and Culture-Independent Approaches: What is Changing
Microbial communities in cheeses, at various stages of manufacturing and ripening, have been extensively studied by micro-
biological techniques based on the cultivation of microorganisms on selective media. This approach if allows, on the one hand, to
obtain isolates to be studied for their phenotypic and genotypic characteristics, on the other hand, only a fraction of the
community can be retrieved on cultural media. In fact, traditional agar-based and culture-dependent methods typically reveal the
most commonly occurring microorganisms. These methods often underestimate the less abundant components of microbiota that
could be equally important for cheese ripening and flavor development (Steel et al., 2006). To this regard, Neviani et al. (2009)
optimized an innovative cheese agar medium able to recover minor populations, coming from milk, whey starter and fresh curd,
in long ripened cheeses. Thus, alternative cultural approaches have to be investigated to obtain more reliable pictures of microbial
community in dairy products.
The limits associated to culture-dependent techniques have led, in the last years, to the increasing use of culture-independent
approaches, which depict a more reliable profiling (and also quantification) of microbial communities. Culture-independent
methods overcome most of the cons associated to use of cultural media and they become essential in the study of microbial
populations in physiological stressed state, included microorganisms in a viable but not culturable (VNC) state. Moreover, culture-
independent techniques have shown high potentiality in the study of metabolic and functional genes strictly involved in the
quality and safety of the final products.
Dairy Strains in Viable but not Culturable State
Bacteria in VNC state fail to grow on synthetic substrates on which they would normally grow and develop into colonies, but are
alive and capable of metabolic activity. ATP levels have been found to remain high in VNC cells as also gene expression
(Oliver, 2005). Thus, the role of microorganisms in VNC state has not to be underestimated and their presence has to be carefully
taken into account.
LAB have been demonstrated to enter in VNC state under the stress conditions they can find during fermentation and ripening
processes. In particular, sugar starvation has been recognized as a key stress for LAB (Ganesan et al., 2007). In those conditions,
lactococci switch to the catabolism of amino acids and, potentially, contribute to flavor production during cheese ripening.
Ganesan et al. (2007) have shown that L. lactis IL1403, in carbohydrate starvation, loses the ability to grow on solid media and
expresses genes related to arginine catabolism. Thus, VNC cells, in ripened cheeses, remain intact and continue to metabolize
peptides and amino acids to end products with a potential impact on cheese flavor (Weimer, 2011).
Studies on Swiss-Dutch-type cheeses showed that the physiological state of lactococcal starter cultures affect ripening and flavor
development (Miks-Krajnik et al., 2013). Specifically, L. lactis populations, in VNC state, exhibited different metabolisms than in
logarithmic growth phase and, around 30 days of ripening, their entering in VNC state was correlated to the simultaneously
appearance of the branched-chain fatty acid C5iso.
The presence of L. lactis cells in VNC state has also been hypothesized in some Italian cheeses, by Ruggirello et al. (2014). The
same authors assessed, by culture-independent approaches, the persistence and viability of this microorganism throughout
manufacturing and ripening of model cheeses where L. lactis populations were detected up to the sixth month of ripening
(Ruggirello et al., 2016). On the contrary, from the analysis of the same samples, L. lactis was not found on selective media and this
evidence, together with preliminary results obtained by resuscitation assays, corroborated the hypothesis it entered in a stressed
physiological VNC state.
Community Profiling Methods: From PCR-DGGE to HTS
Since the 90s, methods in molecular microbiology have become a valid support to traditional techniques, in food analysis, and the
trend towards culture-independent approaches have been considered a possible solution to problems associated with selective
cultivation. Denaturing gradient gel electrophoresis (DGGE)-PCR has been, in the last twenty years, the most commonly used
among culture-independent techniques, able to profile the diversity and dynamics of dominant microbial populations in many
matrices, from environmental to food samples, included dairy products (Ercolini, 2004).
Genetic diversity of microbial communities, in dairy products, have been profiled by means of DNA-based protocols by
different authors (Alegrìa et al., 2009; Bonetta et al., 2008; Casalta et al., 2009; Coppola et al., 2001; Ercolini et al., 2003; Flòrez and
Mayo, 2006; Gala et al., 2008; Randazzo et al., 2010). PCR-DGGE technique has been carried out to fingerprint bacterial popu-
lations developing in raw milk cheeses manufactured without the addition of starter cultures, as in the case of Castelmagno PDO
(Dolci et al., 2008, 2010) and Capo Verde cheeses (Alessandria et al., 2010). Moreover, it was also used in the study of the complex
ecosystem of natural fermented Sicilian Ragusano-type cheese, giving useful information for starter culture design and preservation
of artisanal cheese technology (Randazzo et al., 2002). The performance of selected and natural starters was also followed by PCR-
DGGE in studies about whey cultures in Mozzarella cheese (Ercolini, 2004; Guidone et al., 2016) and in Caciocavallo silano
cheese (Ercolini et al., 2008). This technique was also successfully used to determine the geographic origin of cheeses, such as
Fontina, manufactured in alpine farms located at different altitudes (Giannino et al., 2009), Grana Trentino produced in different
factories (Rossi et al., 2012) and Pecorino Crotonese from two areas (Randazzo et al., 2010). Mostly, PCR-DGGE and PCR-TGGE
were performed to follow fate and dynamics of technological microbial populations; in addition, in a few studies, target
microorganisms were related to cheese defects as late blowing (Cocolin et al., 2004; Le Bourhis et al., 2007).
Microbial RNA analysis has been also performed by RT-PCR-DGGE, but to a lesser extent, to obtain a picture of the species
metabolically active at a particular sampling instant (Alessandria et al., 2010; Dolci et al., 2010, 2014; Masoud et al., 2011;
Randazzo et al., 2002; Rantsiou et al., 2008). In particular, it was used to describe microbial communities characterized by a high
complexity, as surface microbiota in Fontina PDO cheese (Dolci et al., 2013). In this study, RT-PCR-DGGE showed a major power
than PCR-DGGE in describing biodiversity of Fontina PDO rind. Thus, the authors proposed that RNA molecules could have been
considered a more informative target than DNA.
In the last years, several next-generation sequencing (NGS) technologies have been developed and applied to the study of dairy
products reaching a high in-depth qualitative and quantitative profiling level, especially if compared to PCR-DGGE technique
mainly focused on dominant populations (Ercolini, 2013).
Alessandria et al. (2016) explored the active microbiota during the manufacturing and ripening of a Grana-like cheese by beta-
diversity analysis of the 16S rRNA sequencing data; Dalmasso et al. (2016) investigated the microbiota of Plaisentif, an artisanal
antiqueue cheese manufactured in the Italian Alps during the violet’s blooming season. In another study, the bacterial diversity
and structure of Mexican Poro cheese was analysed by 454 pyrosequencing in order to gain detailed insight about changes in
bacterial communities associated with the cheesemaking process (Aldrete Tapia et al., 2014). Dolci et al. (2014) investigated
bacterial dynamics, by both RT-PCR-DGGE and high throughput sequencing (HTS), during Fontina PDO cheese manufacturing
and ripening, in relation to the different cow lactation stages, in order to evaluate a possible correlation between microbiota and
phase of lactation. Quigley et al. (2012) carried out HTS to reveal the highly diversity of bacterial populations present in Irish
artisanal cheeses, and detected several genera not previously associated with cheeses, specifically, goats’ milk cheeses. HTS
approach was also performed to investigate the microbial ecosystem of two artisanal smear-ripened cheesemaking plants
(Bokulich and Mills, 2013); in this study, the authors demonstrated how environmental microorganisms dominated the surface
microbiota of washed-rind products even in inoculated cheeses. Dugat-Bony et al. (2015) also collected metagenomic and
metatranscriptomic data for a detailed overview of surface-ripened cheese microbial community, involved in cheese maturation.
Finally, O’Sullivan et al. (2013) reviewed nucleic acid-based approaches to investigate microbial-related cheese quality defects and
underlined the potentiality of NGS in providing greater insight into structural community interactions and metabolic activities.
Lastly, the composition of cheese surface microbiota has been studied by the application of conventional culture-based
analysis, by using culture-independent methods based on direct extraction of DNA/RNA from the matrices and by the application
of HTS technologies. As reported by Irlinger et al. (2015), considering Firmicutes, Staphylcoccus spp. was the most frequently genus
followed by Lactococcus, Enterococcus, Lactobacillus, Streptococcus and among halophilic bacteria Marinilactobacillus and Facklamia. In
the case of Actinobacteria, Brevibacterium, Corynebacterium and Arthrobacter were the most frequent genera followed by Brachy-
bacterium, Microbacterium, Agrococcus and Micrococcus.
Technical issues in culture-independent methods concern nucleic acid extraction: nucleic acid yield, PCR inhibitors as various
substances coming from cheese matrix itself, beside differential PCR amplification, are topics which have been widely reviewed
(Ercolini, 2004; Wintzingerode et al., 1997). For these reasons, protocols have to be accurately optimized and adapted to extract
nucleic acids from all different types of microorganisms and matrices. In particular, many efforts have been made to reach efficient
RNA extraction from dairy matrices (Monnet et al., 2008). Authors agree on the necessity of high quality RNA from cheese as a
prerequisite for reliable gene expression analysis in dairy microorganisms (Ulve et al., 2008; Ruggirello et al., 2014). In ripened
cheeses, the presence of natural constituents such as lipids, proteins, carbohydrates and salt may render extraction very hard
because they might act as PCR inhibitors.
DNA and RNA extraction optimization is fundamental when we move from the qualitative/semi-quantitative approach by
PCR-DGGE, to the quantitative HTS (Quigley et al., 2012). The occurrence of each OUT can be negatively affected by an inefficient
nucleic acid extraction in terms of quantitative estimation and proportions.
Furthermore, the choice of target molecule is critical for the success of any molecular profiling techniques. The target DNA
sequence must include conserved sequences serving as anchors for PCR primers and, at the same time, it must be variable to
differentiate microorganisms belonging to different species and genera (Bokulich and Mills, 2012; Jany and Barbier, 2008).
Bacterial community studies in cheese are based, mainly, on the analysis of 16S rRNA gene and 16 S–23 S intergenic region
(Jany and Barbier, 2008). In particular, different sets of primers targeting the V3 hypervariable region have been extensively used
(Delbès et al., 2007) and allowed identification at species or genus level. Differently, in fungi, the analysis of rRNA genes provides
identification at genus or family level (Anderson and Cairney, 2004) and, thus, fungal internal transcribed spacers (ITS) are
generally chosen as target for fungal community surveys inasmuch provide a greater taxonomic resolution than rRNA genes
(Anderson and Cairney, 2004).
In HTS studies, when species assignment should be the target of the analysis, long sequence reads are required with as much as
possible variable traits of 16S rRNA gene. Ercolini et al. (2012) studied the microbiota involved in the production of water buffalo
mozzarella cheese by amplification of a 500 bp V1-V3 region and they reached identification at species level; differently, Alegria
et al. (2012) used, as target, a 300 bp V5-V6 region to study Osypek cheese and the taxonomic resolution was at genus level.
However, even with long reads, the 16S rRNA gene is not always heterogeneous enough for species discrimination in dairy
bacteria, as observed in a research on Danish cheese where a 450 bp V3-V4 region was the target considered (Masoud et al., 2011).
In addition to the length of the target sequence, another technical issue is the copy number (Bokulich and Mills, 2012). For
example, ribosomal RNA genes vary widely among taxa, skewing quantitative estimates. To circumvent this limit, alternative
single-copy target sites, such as the rpoB gene in bacteria (Renouf et al., 2006), should be taken into account, despite the lack of
representative sequence coverage poses a challenge for widespread implementation.
Quantitative PCR Approach: Detection and Quantification of Dairy Microorganisms and Study of Functional Gene
Expression
In comparison with environmental microbiology, the use of molecular tools applied to the study of population dynamics and
microbial gene expression in food is just beginning (Juste et al., 2008; Postollec et al., 2011). Quantitative PCR (qPCR) has become
extremely popular in food studies, included researches on dairy products. Besides being faster than conventional culture-based
methods, it is also highly sensitive and specific in the detection of microorganisms in dairy matrices; it has been also successfully
applied in the study of sub-dominant populations even in absence of selective enrichment media.
An interesting multiplex qPCR protocol was optimized to simultaneously detect Escherichia coli O157:H7, Salmonella spp. and
List. monocytogenes in milk samples (Omiccioli et al., 2009). Other interesting applications were in the detection and quantification
of technological microorganisms, which positively affect the organoleptic properties of the final products. For example, Zago et al.
(2009) followed sub-dominant populations of E. gilvus in cheese targeting pheS (phenylalanyl-tRNA synthase) gene; other authors
(Furet et al., 2004; Grattepanche et al., 2005) quantified different LAB species in fermented milk, with detection limits between 102
and 103 colony forming units (CFU)/ml, in the presence of other dominant microbial populations. Quantitative PCR was also
applied to study mycelial growth dynamics of P. roqueforti and P. camemberti (Le Dréan et al., 2010) whose presence is fundamental
in mould surface-ripened cheeses. Despite the viable biomass was probably overestimated, in late ripening, the results allowed to
monitor changes in fungal populations. The possibility to follow the growth, in real time, of specific microbial populations, in
mixed dairy communities, opens the way towards industrial applications for the control of fermentation processes.
Beyond cell quantification, the detection of metabolic and functional genes, and also the study of their expression presents a
novel use of qPCR; thus, for example, RT-qPCR is now being increasingly employed to follow microbial activity in dairy products.
The study of the expression of genes related to primary metabolism, or constitutively expressed in most of the cultural conditions,
has been carried out by different authors. For example, Ulve et al. (2008) investigated seven genes of L. lactis inoculated in
ultrafiltrated Cheddar cheeses, and demonstrated metabolic activity of lactococci even after several weeks of ripening. Similar
results were obtained by Ruggirello et al., (2014, 2016) in the study of vitality and persistence, throughout cheese ripening, of
commercial starters of L. lactis in both commercial and model cheeses by studying the expression of the housekeeping tuf gene.
Moreover, the expression of spxB gene, encoding for pyruvate oxidase, was analysed by Sardaro et al. (2016) for monitoring the
metabolically active community of Lb.s casei group involved in different ripening stages. RT-qPCR was also used in the elucidation
of the dynamics, in Emmental cheese ripening, of Lb. paracasei and Pr. freudenreichii (Falentin et al., 2010). The results showed that
Lb. paracasei began to grow in pressed curd and its metabolic activity reached a maximum during the first part of ripening, in cold
room; Pr. freudenreichii began to grow from the beginning of ripening and its activity was maximum at the end of cold ripening
while was stable during the first two weeks in warm room.
Quantitative PCR and RT-qPCR techniques has been applied to the research of metabolically active populations, either spoilage
and pathogenic. For example, Martín et al. (2010) optimized an RT-qPCR protocol, based on lacZ gene, to detect coliforms in cheese,
in a single reaction and within one day. In addition, other authors designed a qPCR protocol aiming to detect and quantify LAB
carrying the histidine decarboxylase (hdcA) gene in milk and cheese (Fernandez et al., 2006). Despite LAB are non-pathogenic,
however, in some environments, certain strains may produce undesirable compounds such as biogenic amines, which are responsible
for food poisoning. Histamine is one of these compounds, resulting from histidine decarboxylation. Finally, a protocol to detect
enterotoxin gene expression in Staph. aureus, artificially inoculated in cheese, was set up by Duquenne et al. (2010).
Thus, RT-qPCR revealed high potentiality in the elucidation of technological microorganisms roles in different stages,
from cheese-making to ripening and, finally, conservation and storage of the final products, considering also the recent
evidences related to the vitality of starter strains in late ripening (Dolci et al., 2010; Flòrez and Mayo, 2006; Rantsiou et al.,
2008; Ruggirello et al., 2014; Ulve et al.2008). Moreover, the impact of the different technological steps and parameters
(temperature, pH, etc.) on the expression of target genes, linked to both primary and secondary metabolisms, could give
additional interesting information. Finally, the detection and quantification of transcripts predicting for the presence of
undesirable molecules and/or pathogenic and spoilage microorganisms could represent an efficient tool for microbiological
control and risk assessment.
Conclusions
As described above, dairy microbiology is characterized by a high level of biodiversity involving different microorganisms, which
interact in the same ecosystem. It is extremely important to understand how those events are taking place in order to have the
possibility to control microbial activities that have a direct impact on the final characteristics of the end products. The use of
molecular methods has given the possibility to better determine the taxonomy of a specific dairy ecosystem, however the
challenges that we are facing at the moment is how to interpret and exploit this new knowledge to improve the safety and the
quality of dairy products. An exiting future is expected for dairy microbiologists, who should be able to take advantage of these
approaches to fully understand microbial diversity, activity and interactions.
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Microbiology of Fermented Dairy Products

Starter Lactic Acid Bacteria

Reference Module in Life Sciences doi:10.1016/B978-0-12-809633-8.12108-9 1

Non Starter Lactic Acid Bacteria

Yeasts and Moulds

Culture-Dependent and Culture-Independent Approaches: What is Changing

Dairy Strains in Viable but not Culturable State

Community Proﬁling Methods: From PCR-DGGE to HTS

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