Invited review: Advances in starter cultures and cultured foods.

With 2005 retail sales close to $4.8 million, cultured dairy products are driving the growth of dairy foods consumption. Starter cultures are of great industrial significance in that they play a vital role in the manufacturing, flavor, and texture development of fermented dairy foods. Furthermore, additional interest in starter bacteria has been generated because of the data accumulating on the potential health benefits of these organisms. Today, starter cultures for fermented foods are developed mainly by design rather than by the traditional screening methods and trial and error. Advances in genetics and molecular biology have provided opportunities for genomic studies of these economically significant organisms and engineering of cultures that focuses on rational improvement of the industrially useful strain. Furthermore, much research has been published on the health benefits associated with ingesting cultured dairy foods and probiotics, particularly their role in modulating immune function. The aim of this review is to describe some of the major scientific advances made in starter and non-starter lactic acid bacteria during the past 10 yr, including genomic studies on dairy starter cultures, engineering of culture attributes, advances in phage control, developments in methods to enumerate lactic acid bacteria and probiotics in dairy foods, and the potential role of cultured dairy foods in modulation of immune function.

Stability of the biodiversity of the surface consortia of Gubbeen, a red-smear cheese.

A total of 1,052 bacteria and 828 yeasts were isolated from the surface flora of 6 batches of Gubbeen cheese made in 1996-1997 and 2002-2003. Stability of the microflora was evaluated over time and also during ripening at 4, 10, and 16 d (batches 4, 5, and 6) or at 4, 16, 23, and 37 d (batches 1, 2, and 3). Bacteria were identified using pulsed-field gel electrophoresis, repetitive extragenic palindromic-PCR, and 16S rRNA gene sequencing, and yeasts were identified by Fourier transform infrared spectroscopy. The bacteria included at least 17 species, of which the most common were Staphylococcus saprophyticus (316 isolates), Corynebacterium casei (248 isolates), Brevibacterium aurantiacum (187 isolates), Corynebacterium variabile (146 isolates), Microbacterium gubbeenense (55 isolates), Staphylococcus equorum/cohnii (31 isolates), and Psychrobacter spp. (26 isolates). The most common yeasts were Debaryomyces hansenii (624 isolates), Candida catenulata (135 isolates), and Candida lusitaniae (62 isolates). In all batches of cheese except batch 2, a progression of bacteria was observed, with staphylococci dominating the early stages of ripening and coryneforms the later stages. No progression of yeast was found. Pulsed-field gel electrophoresis showed that several different strains of the 5 important species of bacteria were present, but generally only one predominated. The commercial strains used for smearing the cheese were recovered, but only in very small numbers early in ripening. Four species, B. aurantiacum, C. casei, C. variabile, and Staph. saprophyticus, were found on all batches of cheese, but their relative importance varied considerably. The results imply that significant variation occurs in the surface microflora of cheese.

Purification and characterisation of acetolactate decarboxylase from Leuconostoc lactis NCW1.

A two-step strategy involving DEAE-cellulose and POROS PI anion exchange chromatography has been developed for rapid purification of acetolactate decarboxylase (ALD) from Leuconostoc lactis NCW1. This results in 5333-fold purification with a yield of 30%. Purified ALD is a dimer of 49-kDa subunits, has a pH optimum of 6.0, a pI of 4.2 and its activity is independent of metals or branched chain amino acids. At the optimum pH, the K(m) for 2-acetolactate (ALA) was found to be 1.3 mM and the turnover number was 4000 min(-1). N-terminal sequence comparison with other ALDs showed little sequence conservation in this region. Purified ALD does not catalyse direct production of diacetyl from ALA, unlike the crude extract.

A rapid PCR based method to distinguish between Lactococcus and Enterococcus.

Phenotypic characterisation of Lactococcus and Enterococcus species remains unreliable as strains of both genera have been isolated which do not conform to the traditional criteria for separation of these genera. A bank of 131 isolates was phenotypically characterised by three methods: (a) traditional broth tests, (b) API Rapid ID 32 Strep and (c) BBL Crystal ID kits. Differences in genus designation between commercial kits were evident for 12 strains (9%), while 7 strains (5%) remained unidentified by either kit. Published 16S rRNA sequences were aligned and used to design genus-specific primers which, when used in separate PCR reactions, were capable of distinguishing all type strains of Lactococcus and Enterococcus. These primers did not react with known species of Streptococcus, Pediococcus, Lactobacillus, Leuconostoc or Tetragenococcus. Isolates which could not be identified by phenotype were assigned to either genus on the basis of the gene primers.

Enterococcal diversity in the environment of an Irish Cheddar-type cheesemaking factory.

Enterococci are natural residents of human and animal intestinal tracts and grow to high levels in a variety of artisanal cheeses. The aim of this study was to determine the diversity of enterococci in a farmhouse raw-milk cheese production unit. Putative enterococci were isolated from the faeces of all the cows and all the people associated with the cheesemaking, from the milk and cheese during manufacture and ripening and from the environment in three separate trials. Almost 1400 isolates were screened using a genus-specific primer. The results indicated that all the human, milk, curd and cheese isolates but only 33.7%, 6.7% and 4.4% of the bovine isolates from the three trials, respectively, were members of the genus Enterococcus. RAPD-PCR was used to type the enterococcal isolates. In general, only E. faecium was found in the bovine faeces while E. casseliflavus dominated the human faeces, milk and cheese followed by lower numbers of E. faecalis. Environmental sampling of the water in the milking parlour and rinses of the cows' teats, the bulk-milk storage tank and the milking machine corroborated these results as E. casseliflavus and E. faecalis were the only Enterococcus species found in these samples. The putative vancomycin-resistant enterococci (VRE), isolated in Trial 1, were shown to be Pediococcus spp. by genotypic and phenotypic analysis.

Applicability of a bacteriocin-producing Enterococcus faecium as a co-culture in Cheddar cheese manufacture.

Two strains, Enterococcus faecium RZS C5 and E. faecium DPC 1146, produce listericidal bacteriocins, so-called enterocins. E. faecium RZS C5 was studied during batch fermentation in both a complex medium (MRS) and in milk to understand the influence of environmental factors, characteristic for milk and cheese, on both growth and bacteriocin production. Fermentation conditions were chosen in view of the applicability of in situ enterocin production during Cheddar cheese production. Enterocin production by E. faecium RZS C5 in MRS started in the early logarithmic growth phase, and enterocin activity decreased during the stationary phase. The effect of pH on enterocin production and decrease of activity was as intense as the effect on bacterial growth. Higher enterocin production took place at pH 5.5 compared with pH 6.5. The use of lactose instead of glucose increased the production of enterocin, and at higher lactose concentration, production increased more and loss of activity decreased. The production in skimmed milk compared to MRS was lower and was detected mainly in the stationary phase. When casein hydrolysate was added to the milk, enterocin production was higher and started earlier, indicating the importance of an additional nitrogen source for growth of E. faecium in milk. For co-cultures of E. faecium RZS C5 with the starters used during Cheddar cheese manufacture, no enterocin activity was detected during the milk fermentation. Furthermore, the applicability of E. faecium RZS C5 and E. faecium DPC 1146 strains was tested in Cheddar cheese manufacture on pilot scale. Enterocin production took place from the beginning of the cheese manufacturing and was stable during the whole ripening phase of the cheese. This indicates that both an early and late contamination of the milk or cheese can be combated with a stable, in situ enterocin production. The use of such a co-culture is an additional safety provision beyond good manufacturing practices.

Antibiotic resistance and virulence traits of enterococci isolated from Baylough, an Irish artisanal cheese.

Eight representative Enterococcus strains from a collection of over 600 previously isolated from an Irish artisanal cheese were subjected to phenotypic and genotypic analysis of antibiotic resistance and virulence properties. Genes encoding resistance to tetracycline (tet(M) and tet(L)) and/or erythromycin (erm(B)) were detected in five strains. In addition, all strains contained two or more of the virulence genes tested (agg, gel, cyl, esp, ace, efaAfs, and efaAfm). Further investigation into the transferability and environmental dissemination of these resistance and virulence traits will allow risk assessment and safety evaluation of artisanal cheeses.

Contamination of milk by enterococci and coliforms from bovine faeces.

AIM: To determine the contribution of enterococci and coliforms from bovine faeces and teats to contamination of raw milk. METHODS: Putative enterococci (n = 301) and coliforms (n = 365) were isolated from bovine faeces (n = 20), cows' teats (n = 20), the raw milk (n = 1) and the milking environment (n = 4) on one farm. The clonal relationships of each bacterial group were investigated using Pulsed-Field Gel Electrophoresis of genomic macrorestriction fragments. Representatives of the different clusters of enterococci were identified by molecular techniques including rep-PCR, SDS protein profiling, Fluorescent Amplified Fragment Length Polymorphism (FAFLP), phenylalanyl-tRNA synthase (pheS) sequence analysis and/or 16S rDNA gene sequencing. Coliforms were identified by API 20E strips. RESULTS: The majority of the bovine faecal enterococcal isolates were identified as a potential new species of Aerococcus (100 isolates); E. faecium (28 isolates), and Aerococcus viridans (28 isolates) were also found. All coliform isolates from the bovine faeces were identified as Escherichia coli. The coliforms present in the milk were Hafnia alvei, Serratia liquefaciens, Yersinia enterocolitica and Enterobacter amnigenus. No E. coli, Enterococcus or Aerococcus from the bovine faeces were found in the milk. A single clone of H. alvei was found in the water, the milking equipment and the milk, suggesting that the water was the source of the organism in the milk. No vancomycin-resistant aerococci or enterococci were found while most of the isolates tested showed the presence of at least one virulence gene. The milk-sock retained strains that adhered to particulate faecal material. Coliforms were present at approx. 2 orders of magnitude greater than enterococci in the bovine faeces. CONCLUSIONS: The results imply that bovine faeces are not an important source of contamination of raw milk with enterococci or coliforms. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm those of two previous studies (Gelsomino et al. 2001, Int J Food Microbiol71, 177-188 and Kagkli et al. 2007, Int J Food Microbiol114, 243-251) on two other farms. The three studies show that contamination of milk by enterococci, lactobacilli and coliforms of bovine faecal origin is extremely low. The results also suggest that where raw milk is implicated in food infection, other factors in addition to faecal contamination must be involved.

Survival of surface ripening cultures during storage and monitoring their development on cheese.

AIMS: To study the survival of bacteria isolated from the surface of smear cheese and monitor their development during cheese ripening. METHODS AND RESULTS: The storage of five potential bacterial surface-ripening cheese cultures, Brevibacterium aurantiacum, Corynebacterium casei, Corynebacterium variable, Microbacterium gubbeenense and Staphylococcus saprophyticus, in maximum recovery diluent (MRD), containing 0.85% w/v or 5% w/v NaCl, at 21 or 4 degrees C for 40 days, was investigated. All five strains studied survived well with a maximum decrease of c. 2.5 log(10) CFU ml(-1) after storage for 40 days at 4 degrees C in 0.85% or 5% w/v NaCl. Survival, especially of C. variable, was less at 21 degrees C. The development of defined ripening cultures containing C. casei and Debaryomyces hansenii on two farmhouse cheeses was also evaluated. Using pulsed-field gel electrophoresis (PFGE) for the bacteria and mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) for the yeast, it was shown that the ripening cultures could be re-isolated in high numbers, 10(8) CFU cm(-2) for C. casei and 10(6) CFU cm(-2) for D. hansenii, from the cheese surface after 2.5 weeks of ripening. CONCLUSIONS: Ripening strains of surface ripening cultures can be stored in MRD containing 5% w/v salt at 4 degrees C for at least 40 days. Such cultures are recovered in high numbers from the cheese during ripening. SIGNIFICANCE AND IMPACT OF STUDY: This study has provided a low-cost and efficient way to store bacteria that could be used as ripening cultures for smear cheese. Such cultures can be recovered in high numbers from the cheese surface during ripening.

Sources of the adventitious microflora of a smear-ripened cheese.

AIMS: To determine the relationships between the major organisms from the cheese-making personnel and environment and the surface of a smear cheese. METHODS AND RESULTS: 360 yeast and 593 bacteria from the cheese surface, the dairy environment and the hands and arms of personnel were collected. Pulsed-field gel electrophoresis, repetitive sequence-based polymerase chain reaction and 16S rDNA sequencing were used for typing and identifying the bacteria, and mitochondrial DNA restriction fragment length polymorphism and Fourier-transform infrared spectroscopy for typing and identifying the yeast. The three most dominant bacteria were Corynebacterium casei, Corynebacterium variabile and Staphylococcus saprophyticus, which were divided into three, five and seven clusters, respectively, by macrorestriction analysis. The same clones from these organisms were isolated on the cheese surface, the dairy environment and the skin of the cheese personnel. Debaryomyces hansenii was the most dominant yeast. CONCLUSIONS: A 'house' microflora exists in the cheese plant. Although the original source of the micro-organisms was not identified, the brines were an important source of S. saprophyticus and D. hansenii and, additionally, the arms and hands of the workers the sources of C. casei and C. variabile. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the major contribution of the house microflora to the ripening of a smear-ripened cheese has been demonstrated.

Citrate utilization in milk by Leuconostoc cremoris and Streptococcus diacetilactis.

Citrate utilization and diacetyl, acetoin and acetaldehyde production by 2 strains each of Leuconostoc cremoris and Streptococcus diacetilactis in milk were studied. With the leuconostoc bacteria no growth and little citrate utilization occurred unless a stimulant (yeast extract) was present, when complete utilization of citrate without concomitant production of diacetyl or acetoin was obtained. The additon of Mn2+ stimulated growth resulted in diacetyl and acetoin production. Destruction of diacetyl and acetoin occurred when the citric acid level fell to c.1000 and 600 mug/g in the case of Leuc. cremoris FR8-1 and CAF1, respectively. Only strain FR8-1 produced acetaldehyde. In contrast, Str. diacetilactis produced diacetyl, acetoin and acetaldehyde concomitant with citrate utilization.

Susceptibility of cheese and yoghurt starter bacteria to antibiotics.

Eight single-strain lactic streptococci, three commercial cheese starters, and six lactic acid bacteria isolated from yoghurt were examined for their susceptibility to penicillin, cloxacillin, tetracycline-hydrochloride and streptomycin. The ranges of the antibiotics causing 50% inhibition of the bacteria were (mug/ml): penicillin, 0.009 to 0.20; cloxacillin, 0.24 to 2.50; tetracycline, 0.09 to 0.60; and streptomycin, 0.35 to 13.0. The average concentrations required to cause 50 and 100% inhibition of the cheese starters were (mug/ml): penicillin, 0.12 and 0.26; cloxacillin, 1.91 and 3.9; tetracycline-hydrochloride, 0.13 and 0.36; and streptomycin, 0.59 and 2.06. All the cocci were about equally susceptible to tetracycline, and all organisms were more resistant to cloxacillin than penicillin. The yoghurt isolates were more resistant to streptomycin and more susceptible to penicillin than the cheese starters. The 2, 3, 5-triphenyltetrazolium chloride test, using Streptococcus thermophilus BC as assay organism, does not detect low levels of streptomycin in milk. However, it is useful in detecting cloxacillin residues.

Detection of propionic acid bacteria in cheese.

Mesophilic lactic starters and thermophilic lactobacilli but not Streptococcus salivarius subsp. thermophilus grew on the sodium lactate agar (SLA) used for estimating the numbers of propionic acid bacteria (PAB) in cheese. The addition of cloxacillin (4 micrograms/ml) to SLA inhibited the starter bacteria but had no effect on the PAB. It was possible to count low numbers of PAB in the presence of high numbers of starter bacteria. A correlation coefficient of 0.9 was obtained between the level of propionic acid and the counts of PAB in cheese (n = 40). A disadvantage of the medium is that other bacteria found in cheese (mesophilic lactobacilli, enterococci, Clostridium tyrobutyricum) also grow on it; however, these bacteria are easily distinguishable from PAB on the basis of size, colour and absence of catalase.

Heat resistance of Lactobacillus spp. isolated from Cheddar cheese.

Mesophilic Lactobacillus spp. are the dominant organisms in mature Cheddar cheese. The heat resistance of broth grown cultures of Lactobacillus plantarum DPC1919 at temperatures between 50 and 57.5 degrees C, Lact. plantarum DPC2102 at temperatures between 48 and 56 degrees C and Lact. paracasei DPC2103 at temperatures between 50 and 67.5 degrees C was determined. The z-values for Lact. plantarum DPC1919, Lact. Plantarum DPC2102 and Lact. paracasei DPC2103 were 6.7 degrees C, 6.2 degrees C and 5.3 degrees C, respectively. Lactobacillus paracasei DPC2103 showed evidence of injury and recovery, especially at higher temperatures. Milk grown cultures of strains DPC2102 and DPC2103 showed greater heat resistance than broth grown cultures, tailing of the death curves and a nonlinear z-curve. Of the three strains, Lact. paracasei DPC2103 had the potential to survive pasteurization temperatures, whether grown in milk or broth.

Use of gas-liquid chromatography to determine the end products of growth of lactic Acid bacteria.

A simple gas-liquid chromatographic procedure for analyzing ethanol, acetic acid, acetoin, and racemic and meso-2,3-butylene glycol in broth media is described. Overnight broth cultures were filtered or centrifuged, and the filtrate or supernatant was treated with formic acid to aid separation of volatile fatty acids. Samples were then directly analyzed by gas-liquid chromatography on a 20% Tween 80-Chromosorb W-AW column and propionic acid as an internal standard. A complete analysis took ca. 8 min. The method can be used to distinguish homofermentative from heterofermentative lactic acid bacteria based on the level of ethanol produced and citrate-utilizing from non-citrate-utilizing lactic acid bacteria based on the levels of acetic acid produced. The method also has potential in distinguishing other bacterial fermentations. Of the 13 species of lactic acid bacteria tested, Streptococcus lactis subsp. diacetylactis was the major producer of 2,3-butylene glycol (total range, 0.3 to 3.5 mM), and, except for strain DRC1, both the racemic and meso isomers were produced in approximately equal amounts.

Diacetyl, acetoin, and acetaldehyde production by mixed-species lactic starter cultures.

Citrate utilization and acetoin, diacetyl, acetaldehyde, and lactic acid production in milk at 21 C by five different mixed-strain starters, containing Streptococcus diacetilactis (D type), Leuconostoc (B type), and S. diacetilactis and Leuconostoc (BD type), were measured. BD and D cultures utilized citrate more rapidly and produced more diacetyl, acetoin, and acetaldehyde than B types. All cultures produced much more acetoin than diacetyl, with the BD and D cultures producing four to five times larger amounts of acetoin than the B cultures. Reduction of diacetyl and acetoin toward the end of the normal incubation period was characteristic of BD and D cultures, whereas a similar reduction of acetaldehyde was characteristic of BD and especially of B cultures. Continued incubation of B cultures beyond 17 h also resulted in reduction of diacetyl and acetoin. Addition of citrate to the milk retarded diacetyl and acetoin reduction. Mn(2+) had no effect on diacetyl production by a BD culture but increased citrate utilization and, as a consequence, caused greater diacetyl destruction in one of the B cultures.

Partition of lactic streptococcal bacteriophage during the ultrafiltration concentration of milk and whey.

Milk and whey inoculated with lactic streptococcal bacteriophages 316, or 322, or both were concentrated by UF using a DDS Mini-Lab 20. The plate and frame unit was fitted with Type GR61PP polysulfone membrane with a 20,000 molecular weight cutoff. The unit was operated at an inlet pressure of .40 MPa and an outlet pressure of .23 MPa with an initial flux of 2.0 to 3.0 L/h. Samples of retentate, permeate, and membrane were analyzed for the presence of bacteriophages. Under the conditions established in this study, phage particles did not pass through the membrane but instead became trapped in the polarization concentration layer or in the membrane. Phages were recovered from the membrane by extraction in sterile buffered water with the Stomacher. The UF concentration of milk containing the host species of Streptococcus cremoris resulted in phage propagation and lysis of the host but did not result in the passage of phages through the membrane. The UF processing of milk or whey should produce a phage-free permeate.

Incidence of pathogenic bacteria in raw milk in Ireland.

Raw milk from 70 farms was sampled over 13 months for salmonellas, listerias, Escherichia coli, Staphylococcus aureus and mastitic streptococci; total bacterial counts (TBC), coliforms and somatic cells were also counted. TBC < or = 30,000/ml were obtained in 63% of samples. High count milks were found mainly during the winter months: 13% of samples had > 10(4) mastitis pathogens/ml of milk. The mean somatic cell count varied from 4.0 x 10(5) to 8.0 x 10(5)/ml throughout the year with highest counts during the late lactation period. Coliforms were present in all samples, but 65-71% of samples had < 100 coliforms/ml. Up to 60% of supplies had < or = 10 E. coli/ml. One of the 589 samples tested (0.1%) was positive for salmonellas. Yersinia enterocolitica and Y. enterocolitica-like organisms were isolated from 39% of samples with up to 68% of samples positive at some sampling periods. A total of 222 strains of yersinias were isolated; Y. enterocolitica (59%) was the most common strain followed by Y. fredriksenii (35%), Y. kristensenii (1.0%), Y. intermedia (4.5%) and Y. aldovae (0.5%). Listerias were isolated from 8.3% of samples tested; 4.9% were Listeria monocytogenes and 3.4% were L. innocua. There was a significant rise in the isolation rate between December and April from a base line of 0-5% during the spring and summer to 35-37% during the winter months while the cows were indoors. Of 66 silage samples tested from the farms involved in the survey 9% of samples were positive for listerias; 3% of these were L. monocytogenes and 6% were L. innocua.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of pH and Sugar on Acetoin Production from Citrate by Leuconostoc lactis.

The relationship between acetoin production and citrate utilization in Leuconostoc lactis NCW1 was studied. In a complex medium the organism utilized citrate at neutral pH (initial pH, 6.3) and at acid pH (initial pH, 4.5) but produced nine times more acetoin at the latter pH. In resting cells the utilization of citrate was optimum at pH 5.3. Production of acetoin as a function of citrate utilization increased as the pH decreased, and at pH 4.3 all of the citrate utilized was recovered as acetoin. Glucose (10 mM) and lactose (10 mM) markedly stimulated citrate utilization but totally inhibited acetoin production in glucose- and lactose-grown cells. Addition of glucose to cells actively metabolizing citrate caused an immediate increase in citrate uptake and a reduction in the level of acetoin. The apparent K(m) values of lactic dehydrogenase for pyruvate were 1.05, 0.25, and 0.15 mM at pH 7.5, 6.5, and 5.0, respectively. Several heterofermentation intermediates inhibited alpha-acetolactate synthetase and decarboxylase activities. The implications of these results in regulating acetoin formatin are discussed.

Inability of dairy propionibacteria to grow in milk from low inocula.

Growth of propionibacteria in complex media was independent of the initial number of cells; in contrast, growth of propionibacteria in milk and whey did not occur if the initial level of cells was < 10(6) cfu/ml. Addition of vitamins, minerals or complex nitrogen sources to the milk or whey, or incubation under anaerobic conditions had no effect on the lack of growth. Addition of freeze-dried whey, prepared from skim milk reconstituted from powder, to a complex medium prevented growth from low inocula in the complex medium, demonstrating the presence of an inhibitor or inhibitors in the whey. The inhibitor(s) was heat stable, had a low molecular mass and retained its activity for at least 4 weeks at 20 degrees C. Pregrowth of some lactic acid bacteria, used as starter cultures in Swiss-type cheese manufacture, in milk for 2 weeks at 20 degrees C removed the inhibition, which explains how propionibacteria develop in Swiss-type cheese from low numbers even though they are inhibited in milk.