Microbiology of starter cultures Details This article discusses the origins and role of starters in dairy fermentations, the ecology of starter bacteria, the classification of starter bacteria, the types of starter culture used and concludes with some observations on artisanal cultures. The author has provided a broader perspective on the use of starter cultures in food fermentations in the Encyclopedia of Food Microbiology. The chapter can be downloaded from Elsevier Ltd. Ecology of starter bacteria So where did modern starter cultures come from? Most starters in use to today have originated from lactic acid bacteria originally present as part of the contaminating microflora of milk. These bacteria have probably originated from vegetation in the case of lactococci (Sandine et al., 1972) or the intestinal tract in the case of Bifidobacterium spp., enterococci and Lactobacillus acidophilus. Modern starter cultures have developed from the practice of retaining small quantities of whey or cream from the successful manufacture of a fermented product on a previous day and using this as the inoculum or starter for the preceding day’s production. This practice has been called various names but the term 'back-slopping' is used widely particularly in fermented sausage manufacture. Classification of starter bacteria The bacteria used in the manufacture of fermented dairy products are generally lactic acid bacteria (LAB); however, Propionibacterium shermanii and Bifidobacterium spp. which are not lactic acid bacteria, although Bifidobacterium species do produce lactic acid, are also used. In addition, other bacteria including Brevibacterium linens, responsible for the flavour of Limburger cheese; and moulds (Penicillium species) are used in the manufacture of Camembert, Roquefort and Stilton cheeses. There are currently sixteen genera in the LAB. Species from the Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus and Tetragenococcus genera are important in food fermentations and have recently been reviewed by the author for the Encylopedia of Food Microbiology, 2nd Edition. This section will review important properties of major genera used in dairy fermentations (Enterococcus, Lactobacillus, Lactococcus, Leuconostoc and Streptococcus). Enterococcus These organisms are Gram-positive, catalase negative cocci that tend to form chains of varying length. They are normal inhabitants of the intestinal tract of man and other animals and are often used in microbiology as indicators of faecal contamination; some species of the genus are pathogens. Apart from their ability to grow at 45°C, at pH 9.6, in high concentrations of salt, in high concentrations of bile salts, their general heat tolerance and their insensitivity to a range of antimicrobial agents they are superficially similar to lactococci. The biochemical identification key developed by Manero and Blanch (1999) is particularly helpful in identifing enterococci. There are concerns about enterococci in foods partly becausesome are pathogens. However, it is their ability to exchange antibiotic resistance genes, particularly for glycopeptide antibiotics (vancomycin and teicoplanin),that perhaps raises most concern.Vancomycin is one of only a small number of antibiotics that may be effective against methicillin-resistant Staphylococcus aureus (MRSA). Prior to recent taxonomic research, the Enterococcus species used as starters were classified as faecal streptococci and Group D streptococci.The posts in the forum on this group may also be of interest to readers. Leuconostoc Leuconostoc species are important flavour producers in some fermented dairy products. There is general agreement that two species, Leuconostoc mesenteroides subsp. cremoris and Lecon. lactis are important in starter cultures. Unlike lactococci, leuconostocs grow on Rogosa agar (see Billie et al., 1992; Mullan, 2000) and are hetrofermentative producing carbon dioxide from glucose and usually fructose. While the carbon dioxide production is undesirable in Cheddar cheese gas production is desirable in some varieties e.g. Emmental. On microscopic examination, leuconostocs generally appear as Gram-positive cocci similar in size and shape (occur in pairs and in usually short chains) to lactococci. However, small rods can often be found and since leuconostocs grow on Rogosa agar, there can be a tendency to assume that these cultures are contaminated, with lactobacilli for example. Unlike lactococci, leuconostocs do not produce ammonia from arginine and produce the D isomer of lactic acid. With some exceptions leuconostocs only grow weakly in milk, and are not capable of reducing litmus before coagulation in litmus milk medium. Isolation and identification of leuconostocs in starters is time consuming and laborious (see Billie et al., 1992) and the author has found that the use of Rogosa agar to obtain initial isolates helpful. Carbohydrate fermentation and identification of the lactic acid isomer are useful elements in an identification protocol. Streptococcus Str. thermophilus is the only species of this genus found in starter cultures. This streptococcus is classified as a thermophile growing at 45°C, and higher, and is widely used in the manufacture of yoghurt and in Mozzarella, and in some other cheeses. More recently, probably since the mid-1990s-it has been used widely in the manufacture of Cheddar cheese. It is a component, along with lactococci, in some DVI/DVS cultures where it produces acid rapidly during scalding and may confer an additional measure of bacteriophage (phage) protection. Its incorporation in Cheddar-cultures also has the advantage of increasing the profitability of DVI/DVS cultures to culture-suppliers. The slide shown in plate 1 was obtained by Gram-staining a yoghurt preparation; the cocci are cells of Str. thermophilus and the rods are cells of Lb. delbrueckii subsp. bulgaricus. Like lactococci and many leuconostocs, strains of Str. thermophilus are catalase-negative; coccus shaped and occur in pairs and chains. Generally, most strains produce long chains. L-lactic acid only is produced and carbon dioxide is not produced from glucose. Some strains produce urease and have the potential to produce CO2 from urea. Since Str. thermophilus and Str. thermophilus-like organisms can grow in the regeneration section of pasteurisers high levels can occasionally occur in cheese. Urease-producing strains have the potential to cause openness in cheese (Mullan, 2000). Additionally the inability of many strains to metabolise galactose can result in cheese with significant concentrations of a fermentable carbohydrate that could be used by NSLAB for gas production. The potential involvement of Str. thermophilus should be considered during investigations of incidents of open texture or overt gas production in cheese. It is likely that the occasional problems of excessive early acidification encountered by Mozzarella manufacturers using extended production runs with pasteurised milk are due to NSLAB and in particular Str. thermophilus-like organisms that had grown to high cell densities in the regeneration section of pasteurisers. Strains differ in their ability to utilise galactose. Use of non-galactose fermenting strains will result in high levels of this reducing sugar in products. Since galactose and other reducing sugars react with amino acids in the Maillard reaction it is usual to only select galactose-utilising strains to reduce the probability of undesirable colour changes occurring in heated products. Str. salivarius, a streptococcus commonly found in saliva, has been shown by DNA: DNA hybridisation studies to be similar to Str. thermophilus (see references cited by Scheifer et al. (1991). Because of this, for some years Str. thermophilus was classified as a subspecies of Str. salivarius. However, it is now accepted that Str. thermophilus while similar, is sufficiently distinct to justify species designation. Str. thermophilus is sensitive to low levels of salts and in particular to sodium chloride concentrations of around 2%. M17 medium (Terazghi and Sandine, 1975) widely used in studies with lactococci is not an ideal medium for the growth of some strains unless modified to reduce its glycerophosphate concentration. Note that many of the Str. thermophilus-like strains isolated from pasteurisers are much less sensitive to salt and normally grow satisfactorily on M17 agar. Lactobacillus This genus consists of a large group of Gram positive, catalase negative, rod-shaped bacteria. Some species are homofermentative while others are hetrofermentative. While some species produce mainly L-lactate from glucose, others produce D-lactate. Since some strains exhibit significant racemase activity, a racemase is an isomerase enzyme, D/L lactic acid is also produced. Strains may also exhibit coccoid morphology and this can lead, as discussed previously, to confusion with leuconostocs and perhaps even lactococci. Lactobacilli are used as starters in the manufacture of yoghurt, and Mozzarella cheese. They are also used as starter adjuncts to promote faster ripening of Cheddar and similar cheeses, to reduce the incidence of bitterness and as probiotics in yoghurt type products. Note that there are several postings concerning the control of bitterness in Cheddar and Gouda cheeses in the discussion area.Lb. delbrueckii subsp. bulgaricus is widely used along with Str. thermophilus as a starter in yoghurt manufacture. This subspecies is homofermentative, produces almost 2% w/v lactic acid in milk, has an optimum temperature of 42° and grows at temperatures of 45°C and higher. It will not grow in low concentrations of salt and is sensitive to bile salts. Lb. acidophilus, which is normally present in the intestine, is generally not used as a starter; it is widely used as a probiotic. This bacterium, is homofermentative, producing high concentrations of D-lactic acid in milk, has an optimum temperature of 37°C, and is relatively tolerant of oxygen, compared with Bifidobacterium species that are frequently used in conjunction with this organism. Little growth occurs at temperatures less than 20°C and most strains show no growth at 15°C. Because Lb. acidophilus produces D-lactate there have been some concerns about its use in infant nutrition. This aspect will be discussed further in the probiotics section. Lb. casei is also a normal inhabitant of the small intestine and is resistant to bile. It is used as a probiotic although it is found in some starter cultures and is commonly one of a number of non-starter lactic acid bacteria (NSLAB) found in Cheddar cheese. L-lactate is the main isomer of lactose produced although some strains produce small concentrations of D-lactate due to weak racemase activity. Rogosa agar is widely used as a general isolation medium for lactobacilli. Further information on enumeration is given in the article on probiotic bacteria. Lb.helveticus is frequently used along with other thermophilic lactic acid bacteria in the manufacture of a range of fermented milk products including Emmental cheese, Mozzarella and yoghurt. One advantage of including this species along with Lb. delbrueckii subsp. bulgaricus is that Lb.helveticus utilises galactose and this can be useful if products free of reducing sugars are required. Since many strains have been shown to possess proline-iminopeptidase-like activity, Lb.helveticus has been used to produce modified 'Cheddar-type cheese' with some of the 'sweetness' characteristics of Swiss cheeses like Emmental. More recently, designated strains have been used as starter adjuncts to reduce bitterness in a range of cheeses, to improve flavour and/or to accelerate ripening. Bitterness is reduced due to peptidase action on starter-derived hydrophobic peptides. The species is homofermentative and produces high concentrations of D/L lactic acid in milk. Many strains grow at 45°C although lower temperatures 42-43°C generally give higher recoveries when enumerated using selective media such as Rogosa or modified MRS agars. Most strains show no or little growth at 15 °C (some atypical strains may take several weeks to grow at 15 °C or below). See the discussion area for further discussion on bitterness. Lactococcus Originally, the bacteria in this group were classified as members of the genus Streptococcus and were designated as lactic streptococci. They were differentiated from other streptococci, some of which are pathogens, by their specific reaction with Group N antiserum and by their tolerance to temperature, salt and dyes (Jones, 1978). It is now known that serotyping lactic LAB has limited value in species differentiation; strains of the same species may react with different sera and some strains may exhibit no group antigen (Schleifer and Kilpper-Balz, 1987). More information on why the lactic streptococci were reclassified is available here. Differentiation of lactococci to species level Lactococci can be differentiated to the species or biovariant level using the scheme developed for lactic streptococci-see above. Note that lactococci will not grow on Rogosa agar (Bille et al., 1992). Differential, but not selective, media are available and can be useful for quality control and strain isolation purposes. The medium, Reddys' Differential Agar, developed by Reddy et al. (1972) is still of value. This medium contains the differential ingredients lactose, calcium citrate, L-arginine and the pH indicator bromocresol purple. This indicator gives yellow and blue/purple colours under acid and alkaline conditions respectively. Lc. lactis subsp. cremoris (shown in plate 2) gives yellow colonies due to acid production from lactose. Lc. lactis subsp. lactis while producing acid also produces ammonia from arginine. The ammonia neutralises the acid and eventually produces an alkaline reaction that results in blue/purple coloured colonies. Lc. lactis subsp. lactis biovar. diacetylactis also gives a blue/purple colony. Unlike Lc. lactis subsp. lactis, however, Lc. lactis subsp. lactis biovar. diacetylactis exhibits zones of clearing around colonies because of citrate utilisation. Because some strains of Lc. lactis subsp. lactis possess only weak arginase activity streaking techniques on an improved version of this medium may be helpful in identifying these strains (Mullan and Walker, 1979). Types of starter culture Several authors have produced definitions of starters including Lawrence et al. (1976). The latter has classified the three main types of starter used commercially in Australia, New Zealand, the UK and North America as follows: a. Single-strain starters: single strains of Str. cremoris and less commonly Str. lactis. These have been used in pairs in some factories in New Zealand and in Scotland but also singly in Australia. b. Multi-strain starters: defined mixtures of three or more single strains of Str. cremoris and/or Str. lactis. Leuconostoc and Str. diacetylactis strains may also be used. Multiple-strain starters are frequently referred to as mixed-strain starters in the United States of America. c. Mixed-strain starters: mixtures of strains of Str. cremoris, Str. lactis, Str. diacetylactis and leuconostocs. The identity of the component strains is frequently unknown to the user and their composition may vary on subculture. This classification is now of limited value for several reasons. This scheme was developed before the combined use of lactococci and Str. thermophilus as starters for Cheddar and similar cheeses became common. Incidentally the so called stabilised cultures, used in the manufacture of some soft cheeses contain blends of lactococci and Str. thermophilus. In practice it now usual to define starters as either defined, meaning that the strain and species of the component strains are known or as undefined cultures. Included in this latter grouping are the artisanal cultures widely used in some European countries, particularly in Italy. Some observations on artisanal cultures Artisanal cultures are of significant scientific and technological interest. Natural whey starters, despite their unpredictable performance, are still used extensively, for example, in the manufacture of Mozzarella cheese using milk obtained from water buffaloes (Bubalus arnee) in Southern Italy. Water-buffalo whey starters are derived from the whey of a previously successful batch of cheese and are generally stored at ambient temperature for 24 h prior to use. Relatively little research has been undertaken on these natural starters but they are known to contain leuconstocs, lactobacilli, lactococci and frequently streptococciDuring a study to characterise the bacteriophage sensitivity of strains isolated from whey starters we (Aprea et al., 2005) observed large numbers of lactic acid bacteria containing inclusion bodies e.g. fig. 1. Inclusion bodies are often found in bacteria grown under certain conditions and may be composed of the biopolymers poly-ß-hydroxybutrate, polyphosphate, sulphur, lipid or polysaccharide. Fig 1:Inclusion bodies in lactic acid bacteria isolated from an artisanal whey starter. Large inclusion bodies may also be mistaken for endospores. Using specific staining techniques it is possible to distinguish and identify the inclusion bodies present. Using Neisser staining, inclusions appeared purple/black-characteristic of polyphosphate (polyP). The inclusions were subsequently confirmed as polyP by their unique yellow fluorescence (fig. 2) when stained with 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI). Because of the potential role of polyP in intracellular pH control, where it may assist in regulation of pH in a low pH environment, the presence of this polymer in starter bacteria may be an environmental adaptation to the typical high acidity storage conditions of natural starters. Fig 2: Detection and confirmation of polyphosphate inclusions in lactobacilli from whey starters. From Aprea et al., 2005.Purple/black inclusions of polyphosphate observed under Neisser stainA, Lb. paracasei 4B-10,B, Lb. fermentum 5BL6. Yellow fluorescence of polyphosphate under DAPI staining. C, Lb. paracasei 4B-10,D, Lb. fermentum 5BL6. The detrimental effects of storing starter cultures under low pH and at elevated temperatures on their subsequent growth and acid-producing potential are well documented. Since natural whey starters are subject to variable storage temperatures under high acid conditions for extended periods and generally function satisfactorily, it would appear that they have some resistance to the detrimental effects of high acidity. It is possible that the polyP metabolism of these starters may contribute, at least in part, to this resistance. Obviously further study is required to confirm this hypothesis. Interestingly, the BBC in Northern Ireland picked up on some of this work. For more information on the role of polyphosphate in environmental microorganisms read the contribution from Dr Alan Mullan of Queen's University Questor Centre. Practical use of starter cultures Cheesemakers generally use starters prepared in two main forms; bulk starter or starter concentrates. A section on bulk starter manufacture remains to be added. The article will discuss how the starter medium is sterilised, protected from contamination during cooling, the importance of head space control, how the starter vessel is inoculated aseptically and how phage-contamination can be avoided during incubation. Mention will also be made of systems for maintaining internal and external pH control and how common problems, as investigated by the author, can be solved. Search for literature cited How to cite this article Mullan, W.M.A. (2001). http://www.dairyscience.info/index.php/cheese-starters/49-cheese-starters.html . Accessed: 22 May, 2015. Revised 2004, 2005, 2006, 2007, 2008, 2011, 2013. Last revision February 2015.