Microorganisms from raw milk for fermented dairy products
Res. Asst, DVM Bahar ONARAN
Ankara University, Faculty of Veterinary Medicine
Department of Food Hygiene and Technology
• The lactic acid bacteria used in the dairy fermentations can roughly be divided into two groups on the basis of their growth optimum.
• Mesophilic lactic acid bacteria have an optimum growth temperature between 20°C and 30°C and the thermophilic have their optimum
between 30°C and 45°C.
• It is not surprising to discover that the traditional fermented products from sub-tropical countries harbour mainly thermophilic lactic acid bacteria, whereas the products with mesophilic bacteria originate from Western and Northern European countries.
• Most dairy industries use today starter cultures for rapid acidification,
because the relatively small amounts of lactic acid bacteria in raw milk
acidify the milk only slowly.
• These starter cultures are selected and maintained by subcultivation in milk. This method of working has reduced the number of strains present.
• The use of these starter cultures, which is also necessary when
fermenting pasteurised milk, is responsible for a certain uniformity of the products, fermented milks as well as cheeses.
• The introduction of new fermentation techniques has the drawback
that the raw milk floras are in danger of being lost.
• The non-starter lactic acid bacteria in these floras, however, are believed to contain strains, which are essential for producing the characteristic flavours of traditional cheeses.
• Therefore, there is a strong need to study these raw milk floras more closely and to understand their importance in flavour formation.
• These studies may yield strains with promising and useful properties,
which will make them applicable as starters for product innovation.
• In the following sections, several examples of these raw milk microorganisms will be presented.
• The first group to be discussed comprises the lactococci, the most prominent group of mesophilic lactic acid bacteria applied in dairy fermentations.
• Special emphasis will be put on the wild lactococci.
• Another group of mesophilic lactic acid bacteria that will be discussed is that of the lactobacilli, which appear as adventitious flora during the ripening of various cheese types and are designated as non-starter lactic acid bacteria.
• The thermophilic lactic acid bacteria are best known for their role in
yoghurt-type products and as ripening agents in Swiss-type and Italian
cheeses.
• There are now several studies, which show the broad spectrum of strains used for the manufacture of a great variety of products and which point to an interesting pool of potentially useful starters.
• Attention will also be paid to the secondary flora potentially emerging in milk fermentation after the primary acidification phase, particularly to the eucaryotic yeasts and moulds.
• Finally, a synopsis of possible future developments will be presented.
Lactococci
1. Starter cultures
• If raw milk is left at room temperature for some time, a microflora will develop in which mesophilic lactococci generally predominate.
• After maintaining this flora by subculturing it in milk, the number of different strains in the culture will decrease and a culture with only lactococci may eventually emerge.
• These bacteria acidify the milk and, as a consequence, the growth of the other indigenous bacteria is largely inhibited.
• This principle has already been practised in the manufacture of fermented dairy
products for centuries, even before it was known that bacteria were actually involved at all.
• Inoculating the milk with some of the previous day's product was the basis for a
successful fermentation.
Lactococci
1. Starter cultures
• The discovery of the role of lactococci in this success paved the way for their isolation, characterisation and exploitation.
• This started in 1878 (Lister, 1878) and has resulted in the
development of starter cultures for the manufacture of fermented dairy products.
• Their use provides microbiologically safe products with reproducible
organoleptic and structural properties. Both industrial and small-scale
manufacture of fermented dairy products now almost always relies on
industrially prepared starters.
Lactococci
2. Starter functions
• It is interesting to consider that the industrial starters for the majority of cheeses are based on a single species, namely Lactococcus lactis.
• There are many strains of this species employed for the manufacture of various cheese types and, although they exhibit different characteristics, they have several biochemical attributes in common.
• The most important properties are their ability to produce acid in milk and to convert milk protein into flavour components.
• During ripening of cheese, proteolysis is the first biochemical step in the latter process toward the desired flavour and texture.
• Lactococci possess a proteolytic system that, together with other protein-hydrolysing enzymes like chymosin, is responsible for the conversion of casein into peptides and amino acids. Amino acids are the key precursors for the essential cheese flavour.
• They are metabolised by the action of amino acid-converting enzymes to aldehydes, alcohols,
ketones, amines, acids, esters and sulphur-containing compounds, which all contribute to the cheese flavour. In all these conversions, the lactococci are involved.
Lactococci
3. Wild lactococci
• Mesophilic lactococci are generally considered to be associated with the milk environment, but they can also be isolated from other sources.
• Lactococci isolated from artisanal manufacture of fermented dairy products without the application of industrially prepared starter cultures and from non-dairy environments are generally referred to as ‘wild’
lactococci.
• In an international project funded by the European Community, many wild strains of lactic acid bacteria were isolated and partially characterised.
• Initial studies showed that this pool of lactic acid bacteria contained many L. lactis strains, which differ in a number of phenotypic properties from the strains commonly present in industrial starters.
• L. lactis subsp. lactis in the latter are characterised by their ability to hydrolyse arginine, to metabolise a number of sugars and to grow at 40°C and/or in the presence of 4% NaCl.
• Members of the subspecies cremoris, however, are not able to grow under these conditions.
• Although most of the isolated wild lactococci matched this pattern, the phenotypic properties of some strains were not in line with this expected distinction.
• Also, some strains that were phenotypically L. lactis subsp. lactis appeared genotypically L.
lactis subsp. cremoris and vice versa
Lactococci
4. Role in flavour formation
• Wild lactococci isolated from dairy and non-dairy environments were screened in our laboratory for their flavour-producing capacity in milk and in cheese models.
• Several strains exhibited more pronounced sensory characteristics than the reference industrial starter strains. The flavours were described as unusual by the sensory panel for 57 out of the 79 strains examined.
• The unusual flavour profiles were described as cocoa, chocolate, malty, burnt, acid, yeasty, sweet, fruity, etc.
• The production of volatile compounds during growth in milk and cheese paste was examined for some characteristic strains using purge and trap thermal desorption cold-trap gas chromatography mass spectrometry (GC-MS).
• An example of the GC-MS profile of a wild lactococcus strain as compared to that of an industrial strain.
• The relatively high levels of the methyl alcohols and methyl aldehydes containing four or five carbon atoms in the culture with the wild strain were striking.
• These compounds are most likely derived from the branched-chain amino acids, leucine, isoleucine and valine.
Lactococci
4. Role in flavour formation
• The formation of methyl aldehydes in milk by the metabolic activity of L.
lactis subsp. lactis biovar maltigenes has been recognised as the cause of off-flavours in Cheddar cheese.
• On the other hand, 3-methylbutanal has been characterised as an
important volatile compound formed during the ripening of Parmesan and Proosdij-type cheese, which is responsible for a spicy cocoa flavour.
• Apparently, this aldehyde may have positive as well as negative effects on
the sensory perception of a cheese. The contribution of 3-methylbutanal
to the overall perception of the cheese probably depends on the other
volatiles present and the composition of the matrix.
Lactococci
5. Bacteriocins
• Different lactic acid bacteria are able to produce bacteriocins, which are proteinaceous substances with bactericidal activity against microorganisms closely related to the producer strain; this property has been reviewed by several authors.
• Lactic acid bacteria are generally regarded as safe microorganisms and so are their bacteriocins.
• Thus, these bacteriocins can potentially be used to control the growth of spoilage and pathogenic organisms in food.
• Bacteriocin-producing lactococcal strains have been used successfully in
starter cultures for cheesemaking in order to improve the safety and quality
of the cheese
Mesophilic lactobacilli
1. Non-starter lactic acid bacteria
• Although mesophilic lactobacilli are undoubtedly inhabitants of raw milk and the dairy environment, upon acidification of raw milk, they are frequently overgrown by strong acidifiers of the
genus Lactococcus.
• However, they do gain access to the cheesemaking process, because they are often found as secondary flora during the ripening of different cheese varieties.
• This is especially true for raw-milk cheese, but mesophilic lactobacilli are also common in cheese manufactured with modern technologies, using pasteurisation of the milk, defined-strain starters and hygienic processing.
• The starter is responsible for the acidification during the first stages of cheese manufacture and may reach up to 109 colony-forming units (cfu) per gram of cheese.
• During ripening, however, the number of starter cfu generally decreases rather quickly to lower than 107 g−1.
• Non-starter adventitious lactobacilli, which apparently originate from the milk or the environment, grow out subsequently and may reach numbers higher than those of the starter
Mesophilic lactobacilli
2. Role in ripening of cheese
• Since NSLAB dominate the microflora of many long-ripened cheeses, they are believed to contribute to the maturation of cheese.
• The numbers of NSLAB are reported to be higher in Cheddar cheeses made from raw milk than in those from pasteurised milk.
• Differences in flavour between these cheeses, with a more intense flavour in raw milk cheeses, suggest that the indigenous NSLAB play an important role in flavour development.
• Indeed, they have been shown to contribute to the formation of small peptides and amino acids, which are the precursors for the flavour components.
• It thus seems likely that the indigenous NSLAB are at least partly responsible for
this difference.
Mesophilic lactobacilli 3. Adjunct starters
• The observation that the presence of NSLAB in cheese on the one hand leads to a desirable flavour, and on the other hand may induce possible defects or spoilage, makes it a delicate choice for the cheesemaker to use a certain lactobacillus as adjunct starter.
• This strain should be selected with care, because only a limited number of the NSLAB present in cheese combine all the required properties with the concomitant lack of imperfections.
• Adjunct cultures may be defined as those added to cheese for purposes other than acid formation.
• Selected adjunct NSLAB cultures can be added to accelerate ripening and to produce desirable flavour.
• They may eliminate defects by adventitious NSLAB, since they inhibit their outgrowth. Several trials have been done with adjunct lactobacilli in the manufacture of Cheddar cheese.
• In improving the cheese flavour by using strains isolated from raw milk cheese. This improvement was believed to be due to increased formation of amino acids.
• Cheese made from milk inoculated with strains of Lb. plantarum or Lb.
casei subsp. pseudoplantarum received the best gradings.
Thermophilic lactic acid bacteria 1. Fermented milks
• The thermophilic lactic acid bacteria are best known as starters for fermented milks.
• Several varieties of fermented milks originate from countries in Asia Minor and the Balkans, like Armenia, Turkey and Bulgaria.
• These products have emerged from spontaneous acidification of raw milk by indigenous organisms.
• Although these organisms have by no means been exhaustively characterised, they consist largely of thermophilic lactic acid bacteria, probably due to the relatively high incubation temperature determined by the prevailing climate.
• The first description of milk fermentations by these bacteria can be found in the literature of some hundred years ago.
• Several attempts were made at that time to identify the bacteria dominating the flora in yoghurt-like products and they were given the names Bacillus bulgaricus and Diplostreptococcus.
• These spontaneous fermentations of milk into yoghurt have now been developed into microbiologically well- controlled industrial processes.
• The two most frequently used starter bacteria are now classified as Lactobacillus
delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus, generally shortened as Lb.
bulgaricus and S. thermophilus, respectively.
Thermophilic lactic acid bacteria
2. Proteolysis and flavour formation
• The attributes mentioned above will be elaborated in order to evaluate further the potential added value of the thermophilic lactic acid bacteria for yoghurt.
• The starters for yoghurt are known to be responsible for the hydrolysis of milk proteins.
• Especially, Lb. bulgaricus has a highly competent proteolytic system, consisting of an extracellular proteinase and several peptidases.
• The hydrolysis of milk protein by this proteolytic system not only stimulates the growth of the co-cultivated S. thermophilus, but may also liberate interesting peptides from the caseins and serum proteins.
• These proteins are known to include some amino acid sequences which upon liberation from the protein molecule exert a specific biological activity on the physiology of the consumer.
• During the ripening of cheese, the hydrolysis of casein has been shown to liberate such a biologically active peptide, which has the potential to affect the blood pressure by regulating an angiotensin-converting enzyme.
Thermophilic lactic acid bacteria 3. Folic acid production
• An interesting observation for the health of the consumer is the presence of a higher concentration of folic acid in yoghurt than in milk. S. thermophilus is known to produce folic acid during growth in milk, but the extent of production is strain-dependent.
• The amount of folic acid found in cows’ milk ranges from 20 to 60 μg/L, whereas its concentration in yoghurt may be increased depending on the strains used for the fermentation and on the storage conditions to values above 200 μg/L.
• This level also appears to depend on the strain of Lb. bulgaricus used, because the latter organism has been shown to use and to degrade folic acid during its growth.
• It is therefore of utmost importance to select the optimal combination of S.
thermophilus and Lb. bulgaricus strains leading to an organoleptically acceptable yoghurt with a concomitantly increased folic acid concentration.
• The pools of available strains will undoubtedly harbour the desired strains.
Thermophilic lactic acid bacteria 4. Exopolysaccharides
• By producing exopolysaccharides, both S. thermophilus and Lb. bulgaricus contribute to the viscosity and the smooth texture of yoghurt. They stabilise the yoghurt gel and decrease its tendency to synerise.
• The exopolysaccharides of the yoghurt bacteria exhibit a wide variety of chemical structures.
• The main monomers found are glucose, galactose and rhamnose, but the presence of fucose, N-acetylglucosamine and N-acetylgalactosamine has also been described.
• The molecular mass of these polymers is up to around 1500 kg/mol and the amount formed in yoghurt is limited, up to a few hundred mg/L.
• The amount and sometimes also the structure of the exopolysaccharides are
influenced by the growth conditions and the composition of the growth medium.
Thermophilic lactic acid bacteria 5. Cheese
• The thermophilic lactic acid bacteria also play an essential role in the manufacture of some cheese types.
• The starters of Swiss-type and Italian cheeses consist mainly of S. thermophilus, Lb. helveticus and Lb.
bulgaricus .
• In the ripening of Greek hard cheese types made from ewes’ and goats’ milk, the thermophilic lactic acid bacteria play a dominant role.
• Their niche in these cheese types is created by the specific high cooking temperature used in the manufacture.
• They convert lactose to lactic acid as in all dairy fermentations and this conversion is usually completed within about 24 h.
• Lactic acid plays its usual role as preservative and, for the Swiss-type cheese, is the appropriate substrate for the subsequent propionic acid fermentation, which is important for the characteristic eye formation.
• The lactobacilli in the thermophilic starters possess an elaborate proteolytic system capable of degrading milk proteins to amino acids, which are essential for their growth and that of the streptococci.
• In addition, these amino acids are the precursors for the cheese flavour as in other cheese types.
