Microbial load of cheese samples (CFU/g).
The composition and production technology of the cheese are extremely diverse. There are a wide variety of microbial species on their surface, with a much smaller number inside of the product. The microbiota of the cheese may be composed of beneficial microorganisms, spoilage and foodborne pathogens. Identification and characterization of the microorganisms present in these products are important nutrition, food safety and technological aspects. During our work we evaluated the prevalence of allochthonous bacteria and microscopic fungi in traditionally processed cheeses from northeastern region of Transylvania, with classical microbiological culture methods. Based on the results the microbiota of the analysed cheeses was highly diversified. The identified bacteria with the highest prevalence from different selective media, were as follows: Escherichia coli, Enterococcus durans, Enterococcus faecalis, Shigella flexnerii, Proteus vulgaris, Stenotrophomonas maltophilia, Staphylococcus equorum subsp. equorum, Staphylococcus equorum subsp. linens, Halomonas alkaliphila, Kocuria rhizophila, Hafnia paralvei, Bacillus licheniformis and Klebsiella michiganensis.
- microbial diversity
- traditionally processed cheeses
- starter culture
- allochthonous bacteria
Cheese belongs to dairy products representing an important part of human balance diet for hundreds of years. It contains the essential macronutrients and micronutrients. Different factors as environmental conditions, geographical region, processing technologies and the local customs determine the development and release of the huge variety of cheese. One prevention method of milk spoilage over the centuries has been the production of cheese, meanwhile the nutritional benefits are conserving .
Cheese is the result of a fermentation process driven by lactic acid bacteria. Different bacteria, yeast and molds constitute the complex microbial ecosystem of this product. Throughout the ripening, the microbes release proteinases and lipases that are involved in the protein and fat hydrolysis, resulting amino acids and fatty acids. These compounds represent important flavour precursors for cheeses .
The cheese microbiota is involved in the development of the appearance texture, flavour and aroma. Both starter culture organisms and allochthonous microorganisms are part of the specific microbial ecosystem.
The quality and the processing of the milk, the environmental conditions (temperature, humidity, salt), the applied technology influence the quantity and the diversity of the specific species of microorganisms. Also, this fact is affected by the microbial conditions in the manufactures .
Cheese can be considered a privileged product with beneficial or negative impact on health, influenced by the nutritional composition, salt and bioactive compound content. The negative effect is related to the presence of pathogenic bacteria such as
2. Factors controlling microbial growth in cheeses
In various types of cheese, the growth and development of microbes are influenced by physical and chemical parameters (water activity, pH, ripening temperature, redox potential), chemical composition (salt content, presence of nitrates) and by the metabolites as organic acids and the protein like compounds released by starter cultures named bacteriocins [2, 4].
Water activity (aw) of cheese range between 0.917–0.988. Vast majority of non-starter bacteria, spoilage and pathogens are favoured by this condition. The optimum aw of starter bacteria is under these values. Osmophilic yeasts support aw less than 0.60 . The aw is related to sodium chloride (NaCl) and the produced metabolites (lactate, acetate, and propionate) content. These compounds decrease the vapour pressure of water as the result of their dissolution in the moisture of the cheese. Higher the concentration of these metabolites, the reduced the aw is. Without vacuum or sealed packaging, the moisture content is reduced, due to evaporation, causing a gradient in the aw of the cheese. This gradient commonly is proportional with the cheese size and is lower in the external part. At low aw the lag phase of bacterial growth is prolonged .
The salt content decreases the aw value of the cheese and consequently is an antimicrobial agent. For example, for the brined-cured cheeses at the beginning of the ripening process, the salt concentration is much higher on the cheese surface than in the inside the block. For this reason, salt tolerant microorganisms can grow on the surface of this type of cheeses. Surface-ripened cheeses microbiota include
Most bacteria grow best at neutral pHs. In acidic conditions (pH < 4) only several bacteria are able to grow, for example
Maturation temperature should play a role in preventing the growth of undesirable microbes as secondary flora, spoilage and pathogenic microorganisms, thus avoiding the losses .
The food hygiene is taken very seriously in the dairy industry. The quality of dairy products depends on raw milk quality and the processing conditions. Before ripening the number of microorganisms showed higher values in artisanal soft cheese from Germany named kochkäse, made from raw milk in contrast with cheese from pasteurised milk. After maturation, the amount of microorganisms was identical in the two samples. There is a close correlation between the temperature and ripening time. The ripening time is decreased at 35°C with the rising microorganism population. The maturation temperature at 15°C favour the lactic acid bacteria growth .
Redox potential (Eredox) values in cheese is approximately equal with Eredox = −250 mV, during the maturation process is reduced, due to unknown mechanisms. The redox potential is different inside and outside of the cheese that specifies the microbiota.
It was shown that the cheese surface is characterized by aerobic bacteria namely
For the prevention of bacterial gas formation by coliforms and
Environmental factors influence the growth and development of lactic acid bacteria in cheese. The environmental factors and the various amount of NaCl, glucose and lactose affect the growth and biofilm formation of bacterial strains originated from Serbian traditionally processed cheese (
3. Starter bacteria
In cheese processing lactic acid bacteria are used as starter cultures, contributing to organoleptic characteristics and preservation of the product. These strains are added in a different form, grown in milk or mediums. Also, in France or Italy the whey incubated the day before use is applied as starter culture. Starters comes from the fact that these bacteria start the lactic acid production. These belong to mesophilic and thermophilic bacteria [2, 10]. Mesophilic bacteria include lactococci (
Cheddar cheese, soft cheeses, and most Dutch cheeses are produced with mesophilic starters. Starter culture have to meet different expectations as lactic acid production, reduction of pH, to be resistant to bacteriophages, inhibition of pathogenic and spoilage bacteria, contribute to the flavour, texture formation and have health benefit. Depending on the type of cheese the starter culture contributes to the development of nature of the cheese due to lipolysis, proteolysis and autolysis of the cells [10, 11].
Beside the lactic acid bacteria
4. The characteristic cheese microbiota
Dairy products are perishable foods providing a good medium for microbial growth. Foodborne pathogens as
Superior varieties of cheeses as Emmental, Gruyère, Comté, Parmigiano Reggiano, Reblochon and Roquefort are made from raw milk. The microbial safety of these products is ensured by the heat treatment at a higher temperature > 50°C, with duration of 1 h. The challenge of this type of cheeses represents the pathogen bacteria from raw milk that can grow during the process .
The different types of cheeses are characterized by diverse microbiota. The raw milk cheeses (soft and hard, short or long maturated, starter added or nonstarter added cheeses, and ripened in brine or unbrined cheeses) own their distinct microflora.
Gram-negative bacteria and salt-tolerant bacteria belonging to different genus, including
The environmental conditions of the dairy farm have an impact on the microflora of the raw milk and therefore influence manufacturing. The main spore formers in milk are species of
Bacteria species of
Fox et al.  reported that in cheese the non-lactic acid bacteria are bacteria belonging to genera
Aged cheese microbiota includes non-starter lactic acid bacteria. The predominantly found bacteria are facultatively heterofermentative as
Non-starter lactic acid bacteria origin is the raw material and/or the processing environment. A little part of the lactobacilli are resistant to the thermal treatment, pasteurization, for example in Emmental cheese obtained from raw milk can survive the 52°C temperature.
Majority of non-starter lactic acid bacteria can be characterized as: facultative anaerobes, salt-and acid-tolerant and able to grow in cheese. The involvement of these bacteria in the production of cheese flavour is unclear .
The number of
Coryneform bacteria, especially
Swiss-type cheese as Emmental and Comte´ contain propionic acid bacteria,
Cheese can be a reservoir and carrier of probiotic bacteria for human. Due to the low redox potential, this product is a protective environment for probiotics. Their resistance to changes in pH represents an advantage in the acidic environment of gastrointestinal track. Probiotic bacteria applied in cheese production include
The level of indicator microorganisms is less than 10 CFU/g and not more than 100 CFU/g, these representing the limit values in many countries.
In those instances, where cheeses had high levels of indicator bacteria, most studies attributed these to the use of poor-quality raw milk which contained high levels of indicator flora or to unsanitary conditions, or both.
The occurrence of coliforms in dairy products reveal low grade raw milk, poor hygienic conditions under production process. Coliforms could be avoided by respecting personal hygiene and sanitation process.
Human pathogens including
At the origin of unwanted yeasts and molds appearance in cheeses could stand environmental factors, improper processing conditions or infested starter culture. Several yeast could affect negatively (sometimes positively) the cheese. These microbes can be involved in taste and flavour development, affecting the quality attributes also, causing spoilage and release unpleasant aroma compounds. Clinically relevant yeast was detected in different type of cheese as
The advanced hygiene and quality control contributed to the decrease of gas production by yeasts during cheese manufacturing .
A diverse group of yeasts represent a high proportion of surface microflora of different type of smear- and mold-ripened cheeses, including Comte´, Tilsit, Limburger, Blue, and Camembert. These microorganisms level can reach 106–107 CFU/g and are characterized by tolerance to low pH and high salt concentrations.
The most prevalent yeast species isolated from cheese were
The most common molds in cheese include
5. Microbiota and characteristics of traditionally processed cheeses
Artisanal cheeses represent a particular category of cheeses that is appreciated worldwide. Particular properties of products as the individual sensory aspects are related to local environment, cultural and historical features .
One of the challenges of artisanal cheese producers is to keep the microbial diversity of the product and exploit their beneficial effects. The traditional process, including the wooden equipment, microorganisms are the main factor in food safety issues .
In raw milk, over 400 species of lactic acid bacteria, Gram-positive and negative, catalase-positive bacteria, yeast and mould have been found. In cheese cores the diversity is not very large, typical lactic acid bacteria are the predominant microorganisms, whereas the surface of cheeses is rich in several species of bacteria, moulds and yeast.
Diversity within the cheeses results from considerable variation in distribution and growth of the same bacteria in the various cheese. Raw milk cheeses compared to the others are characterized by an intense and rich flavour. This results from the native microflora of the raw material and is not a character of cheeses made from treated milk .
The artisanal cheeses possess a heterogeneous microbiota characterized by a typical population dynamic. The unique microflora is associated with geographical indication for that region .
Perhaps natural milk cultures are used for italian traditional cheeses including Asiago, Pecorino Siciliano, Canestrato Pugliese, Castelmagno and Fossa. The origin of these cultures is the raw milk. The processing comprises a heat treatment of the raw milk for 15–30 min on 60–65°C, fast cooling and back slopping technique. Thermophilic species of that type of cultures are
Central and Southern Italian cheese is “Pecorino” with PDO status, processed traditionally. The raw milk and natural microflora contribute to the development of typical organoleptic attributes. The main representants are
In traditional cheese production crucial role have the autochthonous starter cultures, the main steps of manufacturing are more affected by it, but the final product are less. This refers only to that cheeses that are processed in the same geographical area and with settled circumstances. Numerous parameters have effect on the specific characteristics of cheese produced in different locations. This is case of PDO Pecorino Siciliano that is manufactured in different places of Sicily. There are some common parameters that must be applied during production as raw ewe’s milk, lamb rennet paste, wooden equipment and no added starter.
The inconstancy in the raw milk quality and the variable autochthonous microbiota determine the alteration of the attributes as organoleptic characteristics of artisanal cheese manufactured in different locations. With the application of lactic acid bacteria, the characteristics of PDO Pecorino Siciliano cheese production could be stabilized .
Brazilian traditional processed cheeses differ from the commercial product in their unique characteristics. These are produced with tradition, the essence of production is transferred from one generation to the next. These type of cheese are characterized by unique organoleptic properties, typical aroma, flavour, and colour due to the typical endogenous microbiota. This microbiota includes lactic acid bacteria, yeasts, fungi that source is the raw material and the production environment. Among the lactic acid bacteria isolated from artisanal cheese are mainly
The traditionally processed cheeses from raw milk are very popular and preferred by the local population, therefor is a great need for the microbiological safety of this product. For this reason, there are characterized lactic acid bacteria with multi effect traits as anti-listerial effect isolated from artisanal cheeses.
Based on Margalho et al.  research from the 220 lactic acid bacteria originated from artisanal Brasil cheese twenty-two isolates had probiotic traits and role in biopreservation. These strains are characterized by resistance to gastric acids as low pH values, bile salts, and also possess good adhesion properties, mostly to enterocyte-like cells (Caco-2). One of the lactic acid bacteria, namely
Valuable source of microorganisms with specific characteristics are the artisanal cheese due to their high biodiversity influenced by the local ecosystem.
The Russian artisanal Buryatia cottage cheese is characterized by typical taste and flavour determined by the numerous lactic acid bacteria. From the 62 isolated and assayed lactic acid bacteria of Russian cheese belonged to different 6 genera, 14 species/subspecies. The full-length 16S rRNA sequencing revealed a high diversity of bacteria including:
Northern Moroccan artisanal cheese is characteristic of rural regions. Due to the raw material, the goat’s milk, this cheese possesses unique organoleptic properties. Galiou et al.  revealed that the microbiota of 28 Northern Moroccan artisanal cheeses included
The Spanish Manchego is traditionally processed product, obtained with enzymatic coagulation. It is a maturated, uncooked and pressed cheese, characterized by high fat content. The 248 strains of predominant lactic acid bacteria obtained from manufacture and ripening process of two dairies included species as
Portuguese Serpa cheese is designated as Protected Designation of Origin (PDO) cheese that is characterized also, by particular appreciated aroma and flavour. The organoleptic attributes have resulted from the not pasteurized raw ovine milk components and the addition of extracts of
Northen French PDO Maroilles cheese is a soft type cheese with washed rind. The duration of maturation are 4–6 weeks according to the size, while the rinds are washed weakly with salt water. It is characterized by a surface microbiota
North-western Iranian Koopeh is obtained from raw sheep or cow milk in rural households, and the ripening is under the soil for four months. This cheese is semi-hard type, characterized by high fat content, by buttery and pungent flavour. It has beneficial effects as antioxidant and antimicrobial activities attributed to antioxidant peptides and aromatic herbs content (
The artisanal Mexican cheese, named Chiapas cream cheese is semi-ripened type. Organoleptic characteristics include the creamy and acidic taste. The predominant acidifying bacteria are the endogenous lactic acid bacteria adapted to the local climatic, tropical conditions. The bacterial strains originated from this type of cheese are characterized by high antioxidant capacity and proteolytic activity. It was showed that these bacteria could to produce bioactive compounds with role in functional foods, resulted from hydrolysis of proteins and also contain angiotensin-converting enzyme .
The PDO Parmigiano Reggiano traditional cheese is obtained from raw cow’s milk and added whey starters obtained with back sloping. It is a long ripened type of cheese, produced in defined geographical areas (Po river’s valley). The biochemical characteristics of cheese matrix are the result of the adventitious bacterial dynamics.
In cheese post brining process, that can take a 24 month different lactic acid bacteria were detected applying cultural and non-cultural methods:
Merchán et al.  reported that the 149 yeast from Extremadura region traditional cheese, identified by ISSR-PCR technique included
The turkish artisanal cheese Tulum is a semi-hard cheese, ripened in goat skin and characterized by a unique flavour and microbial diversity. The texture is porous thus permeable to air and water. The microorganisms identified by 16 S rRNA sequencing from this cheese during ripening included:
The MALDI-TOF MS method resulted that greek artisanal cheese non-starter lactic acid bacterial community consisted of
The PDO Robiola di Roccaverano soft Italian cheese is processed from raw goat milk with natural starters. From fresh and matured cheese samples the following microorganisms were identified
Kashkaval is an artisanal pasta filata cheese, obtained from sheep’s raw milk without added starters, but now days appeared the industrialized form of this cheese using pasteurised milk with an added commercial starter. Microbiology studies of this raw milk cheese revealed that the dominant bacteria were the mesophilic non-starter lactobacilli and enterococci. In the pasteurized milk cheese
Nicastrese goat’s cheese is a Southern Italian traditional product obtain from raw goat milk coagulated with lamb rennet, without any added starter cultures. The local aromatic Mediterranean plants impart unique sensorial characteristics to the milk. The typical flavour of this type of cheese is resulted from the macronutrient transformations during the maturation time. The autochthonous microbiota is responsible for the biochemistry of ripening leading to unique sensorial features. The following bacteria were detected from Nicastrese goat’s cheese samples:
The southern Mexican Poro de Tabasco cheese is an artisanal product obtained from raw whole cow’s milk and with natural whey starters. The unique character is attributed to salting, pressing process and the use of a paraffin coating. It is a hard fresh type of cheese with full-fat content. Dominant bacteria strains of this type of cheese include
11 artisanal Irish cheeses (soft, hard, and semi hard) microbiome analysis by high throughput DNA sequencing revealed the presence of
A traditional fried cottage cheese contained strains of
The predominant bacteria of Fresh ovine cheese were represented by
The Calabrian (Italy) artisanal cheese lactic acid bacteria possess antibacterial effect. This group of bacteria contributed to the growth reduction of
The component of the microbiota of cheeses is influenced by the manufacture conditions as the used cheese making vessels. Generally, in artisanal cheese production, the wood is a representing material, that is a natural reservoir for microorganisms. It was showed that in these circumstances the lactic acid bacteria level is high. In the maturation process the shelves made from wood also are responsible for appearance of coryneform bacteria, moulds, and yeasts .
Gram-negative bacteria strains belonging to different families were detected on the surface of maturated soft cheese. These were:
6. Occurrence of allochthonous microorganisms in traditionally processed cheese
The detection of allochthonous bacteria and microscopic fungi in raw milk cheese raise food safety issues because those products can harbour diverse spoilage and pathogenic microbes. We evaluated the microbial quality of 13 traditionally processed cheese from northeastern region of Transylvania. The processed cheese are made from unpasteurized cow and sheep milk. It is considered that the milk quality is affected by grazed grass, the special feeding strategy in that region.
The detection of the various pathogen bacteria was carried out by cultivation methods on different selective media: for the detection and enumeration of
Isolation of the most representative bacteria with high count and characteristic colony morphology was followed by identification of strains on species-level by 16 rDNA sequencing method. Genomic DNA isolation of isolated bacterial strains from the selective mediums was realized with AccuPrep® Genomic DNA Extraction Kit from Bioneer, according to the manufacturer’s protocol. A part of the bacterial 16S rDNA gene was amplified with universal primers 27f and 1492r (5’ AGAGTTTGATCMTGGCTCAG 3′, 5’ TACGGYTACCTTGTTACGACTT 3′). The PCR products were purified using
Our results of the microbiological quality of the assayed artisanal cheese showed diverse values. The highest total count of coliforms,
The count of microscopic filamentous fungi in the assayed samples obtained by us varied 102–103 CFU/g. The highest load was found in cheese 4 sample 5·104 CFU/g. The maximum value detected in the case of yeasts was 4.5 · 105 CFU/g in cheese 9 sample (Table 2). The developed filamentous fungi belonged to
The identified 27 bacterial strains isolated on various selective media according to 16S rDNA sequence analysis include:
The occurrence of
In dairy products the presence of
The plasmid-mediated colistin resistance gene in
One of the predominant bacteria from staphylococci is
In traditional fermented foods staphylococci were the dominant species. These bacteria are involved in the development of sensorial features.
Based on literature data
The other identified bacteria from cheese was
It was reported that
Jamet et al.  reported that enterococci take part of the dominant microflora of raw milk cheeses ranging from 102 to 108 CFU/g. There are contradictory aspects regarding the
The microflora of traditionally processed cheese comprise of diverse and heterogeneous group of microbes. These microorganisms have specific role in product formation providing the unique characteristics of the cheese. Also, it could appear microorganisms that are emerging pathogens. For these reason in traditional manufacture process have to pay attention to personal hygiene and sanitation process. The support of autochthonous bacteria in fermented products have to be a priority to contribute to the preservation of the organoleptic characteristics typical of that geographical region.