Microflora Of Cheese Research Articles

Recent developments in cheese cultures with protective and probiotic functionalities

Microorganisms play essential roles in the manufacture and ripening of cheese, largely contributing to the development of organoleptic properties by their metabolism and varied enzymatic activities, and to microbiological safety through barrier effects of complex microflora and production of several low-molecular-weight antimicrobial compounds. Although extensive research has been done on bacteriocins of cheese bacteria for controlling pathogens in cheese, until now only few applications have emerged. The control of spoilage yeasts and moulds has been traditionally done by chemical additives, but the application of new antifungal protective cultures is very promising, especially for the cheese industry. It has also been recently shown that naturally established cheese microflora can efficiently prevent the growth of pathogenic or spoilage microorganisms. Cheese is also a very suitable but underused carrier for the delivery of probiotic bacteria, conferring health benefits on the host, with specific advantages compared with fermented milks and yoghurts such as high cell viability. This review addresses the latest developments in applications of protective cultures (with bacteriocin and antifungal activities) or microflora with barrier effects, and probiotic cultures for the production of high quality, safe and “healthy” cheese, as well as emphasizing some of the underlying challenges and possible solutions. Furthermore, new safety criteria for food cultures relating to the presence and transferability of antibiotic resistance genes are discussed.

Microflora of Feta cheese from four Greek manufacturers

The components of the microflora of four Feta cheeses, produced by different Greek manufacturers, were determined by culture dependent and independent techniques. Isolates from microbiological media were first grouped by Polymerase Chain Reaction–Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and then representatives of each DGGE group were sequenced for identification purposes. DNA and RNA, extracted directly from the cheese, were subjected to PCR-DGGE. Moreover, Feta cheeses were subjected to FISH analysis in order to identify viable bacterial populations. The microbial ecology, as represented by the Lactic Acid Bacteria (LAB) and yeast populations, was different for the four cheeses. The main LAB species isolated were Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus coryniformis and Lactobacillus fermentum. However, some inconsistencies were observed between the results obtained with the culture dependent and the culture independent approach. In the case of the yeasts, the results obtained by PCR-DGGE compared very well with those obtained by the conventional microbiological analysis and the main species found were Kluyveromyces lactis, Pichia fermentans and C. zeylanoides. FISH analysis highlighted viable but not culturable populations of Streptococcus thermophilus and Lactococcus spp. RAPD-PCR performed on the L. plantarum isolates revealed a cheese specific distribution and a temperature dependent clustering.

Influence of selected lab cultures on the evolution of free amino acids, free fatty acids and Fiore Sardo cheese microflora during the ripening

Fiore Sardo Protected Denomination of Origin is a traditional Sardinian (Italy) hard cheese produced exclusively from whole raw ovine milk and coagulated with lamb rennet paste. Currently, Fiore Sardo is still produced by shepherds at the farmhouse level without the addition of any starter culture and the cheese-making process is characterized by significant waste. The first objective of the present work was to investigate the autochthonous microflora present in milk and Fiore Sardo cheese in order to select lactic acid bacterial (LAB) cultures with suitable cheese-making attributes and, possibly reduce the production waste. Secondly, the ability of selected cultures to guarantee cheese healthiness and quality was tested in experimental cheese-making trials. In this study, we show that the typical lactic microflora of raw ewe's milk and Fiore Sardo cheese is mostly composed of mesophilic LAB such as Lactococcus lactis subsp. lactis, Lactobacillus plantarum and Lactobacillus casei subsp. casei. Moreover, strains belonging to the species were selected for cheese-making attributes and used in experimental cheese-making trials carried out in different farms producing Fiore Sardo. The evolution of the cheese microflora, free amino acids and free fatty acids during the ripening showed that the experimental cheeses were characterized by a balanced ratio of the chemical constituents, by a reduced number of spoilage microorganisms and, remarkably, by the absence of production waste that were significant for the control cheeses.

Antibacterial activities of the surface microflora of Kefalograviera cheese

The microflora developed on the surface of Kefalograviera, a hard cheese, during ripening in the warm room was studied. The microbial total counts and yeasts decreased ( P < 0.05) and the same was observed with the pH on ripening for 15 days. Bacilli dominated at day 4 while at 15 days Micrococcaceae and enterococci occurred frequently. A great number (38 out of 56) of the isolates exhibited narrow spectrum of antibacterial activities against Gram-positive indicator strains, while ten of the Bacillus isolates ( Bacillus pumilus) inhibited the growth of Listeria monocytogenes. The extracellular antilisterial substances were sensitive to proteinase K and heat labile (at 100 °C).

Assessment of Safety of Enterococci Isolated throughout Traditional Terrincho Cheesemaking: Virulence Factors and Antibiotic Susceptibility

Enterococci account for an important fraction of the adventitious microflora of traditional cheeses manufactured in Mediterranean countries from small ruminants' raw milk and play an important role in the development of suitable organoleptic characteristics of the final product. It has been suggested that animals used for food or animals that supply edible products are a reservoir of antibiotic-resistant enterococci. The main purpose of this research effort was thus to identify, to the species level, a total of 73 enterococci with high tolerance to acidic pH and bile salts (as prevailing environmental conditions in the first portion of the gastrointestinal tract), which were previously isolated from the milk feedstock to the final product of Terrincho cheesemaking, and to determine their profiles of antibiotic susceptibility, coupled with the occurrence of specific virulence factors (especially in those that might eventually be claimed to exhibit suitable probiotic and technological performances). Isolates, identified by both API 20 STREP and PCR methods, were found to belong to the following Enterococcus species: E. casseliflavus, E. durans, E. faecalis, E. faecium, and E. gallinarum. Susceptibility of those isolates was observed to most antibiotics tested, whereas none harbored aminoglycoside resistance genes. PCR screenings for cytolysin genes (cylLL, cylLs, cylM, cylB, and cylA), surface adhesin genes (efaAfs, efaAfm, and esp), the aggregation protein gene (agg), and the extracellular metalloendopeptidase gene (gelE) were performed. All isolates proved negative for cylLL, cylM, cylB, and agg genes. Both E. faecalis strains were positive for the cell wall–associated protein Esp and the cell wall adhesin efaAfs, whereas the cell wall adhesin efaAfm was detected in 11 of the 12 E. faecium strains. Only one strain possessed the cylLs determinant, and another possessed the cylA gene. Incidence of virulence determinants was thus very low; hence, the enterococcal adventitious microflora tested is essentially safe.

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.

Shelf-life extension and quality attributes of the whey cheese “Myzithra Kalathaki” using modified atmosphere packaging

The present work evaluated the effect of modified atmosphere packaging (MAP) on quality characteristics and shelf-life extension of the whey cheese “Myzithra Kalathaki” using microbiological, chemical and sensory analyses. Myzithra cheese was packaged in four different atmospheres: vacuum, 20% CO 2/80% N 2 (M 1), 40% CO 2/60% N 2 (M 2) and 60% CO 2/40% N 2 (M 3); identical cheese samples were packaged in air, taken as controls. All cheese samples were kept under refrigeration (4±0.5 °C) for 45 days. Of the four atmospheres, the M 2 and M 3 gas mixtures were the most effective for inhibiting growth of aerobic microflora and psychrotrophs in cheese samples until days 40 and 33 of refrigerated storage, respectively. Lactic acid bacteria (LAB) were part of the cheese microflora becoming dominant toward the end of the storage period regardless of packaging conditions. Enterobacteriaeceae were also part of the cheese microflora being effectively inhibited after day 35 of storage. Molds and yeasts were also totally inhibited by MAP (M 2 and M 3) gas mixtures throughout the entire storage period. Of the chemical quality indices determined, lipid oxidation varied below 0.005 absorbance at 532 nm for all treatments, except control samples for which absorbance values of 0.02 were recorded after 35 days of storage. Lipolysis did not vary significantly with type of packaging treatment while proteolysis values showed and increasing trend up to day 25 of storage and then decreased toward the end of the storage period. Sensory evaluation (odour and taste) showed that Myzithra cheese packaged under MAP (M 2 and M 3) retained good sensory characteristics for 30 days of storage while control samples were sensorily unacceptable after 10–12 days of storage.

Presence of halophilic and alkaliphilic lactic acid bacteria in various cheeses

We sought to confirm the presence of halophilic and alkaliphilic lactic acid bacteria (HALAB) of marine origin in cheeses and thus contribute to the understanding of the roles of LAB flora in cheese ripening. We used 7% NaCl glucose-yeast extract-peptone-fish extract broth and agar media (pH 9.5) for pour-plating and enrichment culture for 16 cheese samples produced in six European countries. HALAB were present in 9 of the 16 samples at < 20 --> 10(7) CFU g(-1). In three mould-ripened soft cheeses, HALAB counts ranged from 10(6) to 10(7) CFU g(-1) and were one order (two samples) and six orders (one sample) of magnitude greater than that of nonhaloalkaliphilic, common LAB, as enumerated on lactobacilli MRS agar. The 16S rRNA gene sequences (500 bp) of 51 of the 55 isolates examined were identical or similar to that of Marinilactibacillus psychrotolerans or Alkalibacterium olivapovliticus and related species, all of which are HALAB. HALAB of possible marine origin were present in various soft, semi-hard and semi-soft cheeses and were highly predominant in some mould-ripened cheeses. HALAB of possible marine origin are members of the microflora of various cheeses and, when dominant, may play a role in the ripening of cheeses. Microbial analysis of LAB flora in cheeses should take into consideration the presence of HALAB.

The Effect of Storage Period on the Chemical Composition and Coliform Microflora of Wara Cheese

The Effect of Storage Period on the Chemical Composition and Coliform Microflora of Wara Cheese

Identification and characterization of enterococci from bryndza cheese

To identify enterococci isolated from sheep milk cheese--bryndza, and to compare differences in the composition of enterococcal microflora affected by the season, and to evaluate the potential presence of vancomycin resistance and virulence determinants. Bacterial strains were isolated during analysis of bryndza cheese and identified on the genus and species level by phenotypic methods and with commercial biochemical sets. The identification of the species, Enterococcus faecium, Ent. durans and Ent. faecalis, was confirmed by PCR using species-specific primers for ddl genes. PCR was also used for assessment of presence of vanA and vanB genes and virulence determinants gelE, agg and cytolysin genes namely: cylL(L), cylL(S), cylM, cylB and cylA. Among 308 Enterococcus sp. strains, 177 isolates were proved to be Ent. faecium, 59 to be Ent. durans and 41 to be Ent. faecalis. Vancomycin resistance genes vanA and vanB were not detected. Agar plate testing confirmed their absence. Gene gelE, however, was found in 20 Ent. faecalis isolates, but only 13 of them showed gelatinase-positive phenotype. Seven isolates had five cytolysin genes, but none of the isolates exhibited a positive haemolytic phenotype. Four isolates possessed the agg gene. The prevalence of Ent. faecium species was highest in samples from the winter season harvest. Ent. faecium is the dominant enterococcal species in bryndza cheese and the most prevalent in the winter season product. None of the Enterococcus sp. strains was proved to have vanA or vanB genes and the vancomycin resistance. To our knowledge, this is the first report of enterococcal microflora in bryndza cheese and its evaluation for the presence of vanA and vanB genes as well as virulence determinants.

Peptides released from acid goat whey by a yeast-lactobacillus association isolated from cheese microflora

Seven lactobacilli and a variety of microflora extracted from twenty five commercial cheeses were grown on unsupplemented acid goat whey and screened for their capacity to hydrolyse whey proteins [alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg)] and to generate peptides. Fermentations were performed aerobically or anaerobically at 37 degrees C using crude or pre-heated whey (10 min at 65, 75 or 85 degrees C). Under aerobic conditions, growth of lactobacilli was poor and protein hydrolysis did not occur. Anaerobic conditions slightly increased lactobacilli growth but neither beta-lg hydrolysis nor peptide generation were observed. More than 50% of alpha-la was digested into a truncated form of alpha-la (+/- 12 kDa) in crude whey and whey pre-heated at 65 degrees C. Twenty-five microflora extracted from raw milk cheeses were screened for their proteolytic activities on acid goat whey under the conditions previously described. Eight of them were able to hydrolyse up to 50% of alpha-la mainly during aerobic growth on crude or pre-heated whey. The corresponding hydrolysates were enriched in peptides. The hydrolysate involving microflora extracted from Comté cheese after or at 18 months ripening was the only one to exhibit hydrolysis of both alpha-la and beta-lg. Microbiological analysis showed that microorganisms originating from Comté cheese and capable of growth on unsupplemented whey consisted of Candida parapsilosis and Lactobacillus paracasei. Fermentation kinetic profiles suggested that peptides were released from alpha-la hydrolysis. The co-culture of both microorganisms was required for alpha-la hydrolysis that occurred concomitantly with the pH decrease. During whey fermentation, Cand. parapsilosis excrete at least one protease responsible for alpha-la hydrolysis, and Lb. paracasei is responsible for medium acidification that is required for protease activation.

Preparation of angiotensin-I-converting enzyme inhibitory hydrolysates from unsupplemented caprine whey fermentation by various cheese microflora

Unsupplemented caprine whey was fermented by 25 cheese microflora in order to produce peptides from α-lactalbumin ( α-la) and/or β-lactoglobulin ( β-lg) hydrolysis. Fourteen hydrolysates enriched in peptides mainly released from α-la were obtained. Angiotensin-I-converting enzyme (ACE) inhibitory activity of each hydrolysate was investigated. Six of them had high ACE inhibitory activities ranging from 31% to 56%. The highest ACE inhibitory activity was obtained after whey fermentation by the microflora from 18 months ripened Comté cheese. The microflora was identified as a co-culture of Candida parapsilosis and Lactobacillus paracasei. Hydrolysate activity remained stable after pepsin, trypsin and chymotrypsin treatments simulating an in vitro gastrointestinal digestion. This hydrolysate was further fractionated by RP-HPLC. The peptide exhibiting the highest ACE inhibitory activity was characterised as WLAHK ( α-la f(104–108): Trp–Leu–Ala–His–Lys). WLAHK was resistant toward pepsin and trypsin treatments but was digested by chymotrypsin.

Characterization of the natural microflora of artisanal Mexican Fresco cheese

The objective was to characterize the microflora of Mexican artisanal Fresco cheese, as a first step towards the development of a starter culture, which would allow the making of a safe and uniform product from pasteurized milk. The numbers of several microbial groups (aerobic mesophilic flora, total and fecal coliforms, Salmonella spp., Staphylococcus spp., molds, yeasts and lactic acid bacteria) were investigated. The dominating isolates were characterized as being Lactococcus lactis ssp. lactis, Enterococcus faecium and Lactobacillus casei. The results suggest that an adequate starter in the production of Mexican Fresco cheese may be composed of strains of these species.

Discrimination of commercial Caciocavallo cheeses on the basis of the diversity of lactic microflora and primary proteolysis

A wide variety of semi-hard pasta-filata cheese fall under the common name “Caciocavallo”, including Caciocavallo Silano, a Protected Denomination of Origin (PDO) cheese produced in Southern Italy. Multivariate analysis techniques were used to investigate the specificity of Caciocavallo Silano PDO cheeses within the area of production and to compare them with cheeses of similar denomination or cheeses produced with similar technology. The composition of lactic acid bacteria (LAB) communities at the species level and the primary proteolysis of commercial cheeses were assessed. A high variability was observed in the composition of LAB communities, and no clear relationship was found with either the region of origin or the cheese denomination. However, the microflora of PDO cheeses was less heterogeneous than that of non-PDO cheeses and composed mainly of non-starter lactic acid bacteria (NSLAB) such as Lactobacillus paracasei , L. fermentum , and L. plantarum . A high diversity in the proteolytic pattern of the cheeses was also found. This diversity was associated with the trends observed in the community of LAB populating each of the cheeses. The main problems concerning the protection of the denomination are discussed.

Texture and microflora of vallagret cheese during maturation

Vallagret is a unique Jarlsberg‐like cheese that combines characteristics of Cheddar and Swiss cheeses. The objectives of this study were to elucidate the stress and strain behaviour of young (1 week) and mature (20 weeks) vallagret cheese and to enumerate the microflora of young and matured cheeses. The large deformation properties of vallagret cheese were determined using an Instron universal testing machine. Lactococci were enumerated using M17 agar. Lactobacilli and Leuconostoc mesenteroides ssp. cremoris were enumerated on Lactobacillus selective (LBS) agar while the aerobic microorganisms were enumerated using plate count agar. Fracture stress decreased significantly from 0.52 MPa in young cheese to 0.11 MPa in the matured samples and fracture strain decreased significantly from 87% in young cheese to 41% in matured cheese. Aerobic microorganisms and lactococci decreased on maturation while the numbers of lactobacilli and Leuconostoc mesenteroides ssp. cremoris increased over the 20‐week maturation period. Maturation affected the microbial counts of vallagret cheese. On maturation, vallagret cheese became softer and had lowered resistance to fracture.

Propionibacterium freudenreichii strains quantitatively affect production of volatile compounds in Swiss cheese

Cheese flavour is the result of a complex mixture of volatile compounds, originating mainly from the enzymatic degradation of curd components by cheese microflora during cheese ripening. Directing cheese flavour development requires knowledge on inter- and intra-species con- tributions to flavour development, i.e. identification of the volatile (flavour) compounds produced by each strain. The aim of this study was to identify the volatile compounds produced in Swiss cheese by Propionibacterium freudenreichii, one of the species essential for the development of the characteristic flavour of this type of cheese. The volatile profile of compounds obtained from small- scale (1/100) Swiss cheeses, with or without P. freudenreichii, were compared (three strains tested, in association with three thermophilic lactic starters, i.e. twelve cheeses, manufactured in duplicate). Neutral volatile compounds, extracted by dynamic headspace, and free fatty acids were identified using gas chromatography-mass spectrometry. The concentrations of all carboxylic acids and 14 of 58 neutral compounds were significantly higher in the presence of propionibacteria (PAB). The three PAB strains tested produced the same volatile compounds, but observed quantitative differen- ces were strain-dependent. Propionic acid and four propionate esters were detected only in the pre- sence of PAB. Moreover, cheeses with PAB contained two- to three- fold higher levels of free fatty acids derived from lipolysis and five- to fifty- fold higher levels of branched-chain compounds deri- ved from isoleucine catabolism (2-methylbutanal, 2-methylbutanol and 2-methylbutanoic acid) and from leucine catabolism (3-methylbutanoic acid). Lactic starters induced significant variations in the concentrations of some of the compounds produced by PAB, such as methylbutanoic acids and free fatty acids, which varied by 2.0 and 1.4, respectively, as a function of the lactobacilli strains. PAB strains affect the concentration of varied volatile compounds and could therefore have distinct contribu- tions to the formation of Swiss cheese flavour. Propionibacterium freudenreichii / volatile compound / Swiss cheese

Molecular characterization of semi-hard homemade cheese microflora

This decade has shown an impressive development in the application of molecular techniques based on 16S and 23S rRNA genes to study the microbial diversity in various ecosystems. Microflora of semi-hard homemade cheese was examined in this work. We developed a novel technique for DNA extraction (a bead beating based method) due to high fat content of this cheese. Rapid extraction of DNA from cheese microflora enabled a molecular identification of the LAB (Lactic Acid Bacteria) strains based on PCR amplification of 16S RNA coding sequences. The specific primers for 76S RNA gene of lactobacilli were used for amplification. The PCR reaction was performed at lower temperature, where the specificity of the annealing reaction was reduced and lactococcal sequences of 16S RNA genes were also amplified. The results of RFLP analysis revealed that the microflora of Doboj homemade cheese encompases mostly lactococci.

Effects of antifungal agent and packaging material on microflora of Kashar cheese during storage period

In this study, the effects of antimicrobial agent (natamycin) and packaging materials (PVC, Sperdex-Ref. 99017) on the microbiological properties of Kashar cheese during ripening period were investigated. Natamycin and packaging materials had no effect on the total aerobic mesophilic bacteria, yeast and lipolytic microorganism counts. However, natamycin had showed inhibitory effect on the proteolytic microorganisms by itself and combined with packaging material, Sperdex-Ref. 99017. In addition, no mould growth were detected in the Kashar cheese samples produced by combined application of natamycin and packaging materials during five month ripening period. Enterobactericeae, Coliform, Salmonella spp., and Staphylococcus aureus were not found any of the cheese samples.

Occurrence and characterization of yeasts isolated from artisanal Fiore Sardo cheese

The occurrence of yeast microflora in artisanal Fiore Sardo cheese during ripening was studied. Mean yeast counts ranged from 2.64±1 log 10 cfu ml −1 in milk to 0.65±1 log 10 cfu g −1 in 9 months cheese, with the higher counts observed in 48-h-old cheese. Strains belonging to the prevalent species Debaryomyces hansenii, Kluyveromyces lactis, Geotrichum candidum, Candida zeylanoides and Candida lambica were selected for technological and genotypic characterization. All D. hansenii strains fermented glucose and assimilated lactate, a high percentage assimilated citrate and only a few showed proteolytic and lipolytic activity. All K. lactis strains were able to both assimilate and ferment lactose, to assimilate lactate and to exhibit proteolytic activity on casein . G. candidum assimilated lactate and some strains showed proteolytic and lipolytic activity. C. zeylanoides showed lipolytic activity on tweens and the majority of strains assimilated citrate. C. lambica fermented glucose and assimilated lactate. Considering their diffusion and technological characteristics, an important role for K. lactis and G. candidum in the early stages of the ripening process and for D. hansenii after the first month of ripening can be suggested. RAPD-PCR analysis with M13 primer grouped the isolates in well-separated clusters with their type strains and confirmed the previous phenotypic identification. The high intraspecific homogeneity observed in tested strains could be explained by their isolation from a common substrate and from neighbouring geographical areas. This preliminary study allowed us to isolate autochthon yeast strains showing particular properties which can contribute to the production of typical cheese taste and flavour.

Effect of pH on microflora of Civil cheese during refrigerated storage

Microbial properties, correlation between pH and microbial properties, and the effect of pH adjustment on microflora of Civil cheese during refrigerated storage were examined. The cheeses made from skim milks with a pH of 5.35, 5.30, and 5.25 had higher counts of total aerobic bacteria, yeast and moulds, LAB and psychrotrophic organisms than those made from milks adjusted to higher pH values. A highly negative correlation between pH and total aerobic bacteria, yeast and moulds and LAB was observed in Civil cheese.