Enumeration of cultivable molds
Molds are the most frequent contaminants in cheese industries, which provide a suitable niche (low pH, high moisture content) for their growth [
8]. Contamination of cheese by molds can occur at various stages from dairy farms to dairy processing units thereby posing a crucial problem in the production process as well as the final quality [
9,
12,
13]. The average mold counts for various control points (floor, wall, cheese board, air and ripened cheese) in the selected 10 dairy farms were depicted in
Table 1. The counts in the floor of farm A and in walls of farm A and H were found to be undetectable. In contrast, the floor of farm F was higher than the detectable levels. The floor and wall of farm D showed higher counts of 5.9×10
2 and 1.5×10
3 cfu/cm
2, respectively. Improper cleaning and disinfection of surfaces favour the growth of molds in the wall, floor and ceiling under humid conditions [
12,
13]. Cheese boards from farms B and D showed higher counts (1.9 and 1.0×10
3 cfu/cm
2), while from farm J and A counts were lower (0.1 and 0.3×10
3 cfu/cm
2). Similarly, higher mold counts were previously reported in the Turkish white cheese vats [
13]. In cheese industries, air is the major contamination source and spoilage molds are more frequently reported due to airborne fungi since their spores easily dispersed into the air inside dairy plants [
10]. The room air in cheese ripening rooms of farms G-I was detected with higher counts of 4.8, 2.7, and 3.0×10
2 cfu/plate, respectively. The microbial load that grows on the cheese surface can be complex, influenced by the ripening and preparation conditions [
19,
22]. The counts in cheese rind samples from farms A and B were undetectable. Although molds were detectable in other farms, a higher count (3.1×10
4 cfu/cm
2) was recorded with farm G. This could be due to low pH, moisture content, high salt levels and temperature that occurs during cheese ripening process favours the growth of molds [
17]. The cheese core samples produced from all the ten farms were below detectable levels. In our study, we revealed that 8 out of 10 dairy farms did not comply with the HACCP standards. This study provides a more accurate quantitative portrait of the fungal microflora and depicts the composition of the cheese environment varies from one farm to another.
Richness of mold isolates
This part of study specified an insight into the biodiversity of fungal contaminants related to dairy products. To succeed in this goal, we employed phenotypic and genotypic tactics involving the sequencing of taxonomically related target genes to identify fungal isolates as precisely as possible. In our study, 986 fungal colonies were obtained from the samples collected in 10 dairy farms. Selection of colonies was based on differences in shape and colour. This approach increased the possibility of isolating a greater number of species. The selected isolates (
Table 2,
Figure 1a) were >150 in three farms (I, F, G), >100 in two farms (E, H), >50 in three farms and <50 in two farms (A, J).
The ITS region is preferred as the best universal barcode for fungal identification [
23]. The molecular identification tool using the ITS1-5.8S-ITS2 ribosomal DNA (rDNA) region revealed that out of 986 isolates (
Table 2), 895 isolates could be assigned to the phylum Ascomycota (886 isolates [90%], 14 genera and 37 species) and Basidiomycota (7 isolates [0.71%], 3 genera and 2 species), while remaining 89 isolates remain unclassified. In accordance, several studies on cheeses, dairy products spoilage and production environments illustrated that major molds identified belong to the phylum Ascomycota [
4,
24].
Molds in the Ascomycota phylum belong to the subdivision Pezizomycotina (12 genera; 35 species) and Saccharomycotina (2 genera; 2 species). Similar to our results, Lund et al [
25] described all the molds identified from cheese spoilage as belonging to subdivision Pezizomycotina. In this study, within the Pezizomycotina subdivision, the most represented class is the Eurotiomycetes (2 genera and 21 species) followed by the Dothideomycetes (7 genera and 8 species) the Sordariomycetes (3 genera and 6 species) and the Saccharomycetes (2 genera and 2 species). Among the Eurotiomycetes, the genera
Penicillium and
Aspergillus were most frequently associated with dairy product spoilage, cheese ripening and production environment [
3,
7,
26]. The molds in the Basidiomycota phylum belong to the subdivision Agaricomycotina in the class Agaricomycetes (2 genera and 1 species) and Ustilagomycotina in the class Exobasiomycete (1 genus).
Of the total 17 genera isolated, the genera
Penicillium,
Aspergillus (
Supplementary Figure S1) and
Cladosporium were found to be frequent with a greater number of isolates (
Table 2,
Figure 1a).
Penicillium was the only genus detected in all the 10 dairy farms, whereas
Aspergillus and
Cladosporium were prevalent in 6 farms. Out of 17 genera, 14 were acquired in only one of the farms, indicating that the later genera did not occur widely as a regular part in the cheese/dairy environments.
The most diverse and abundant isolated genus was
Penicillium (76%) with 750 isolates and 14 species found to be overly represented in all the cheese-ripening rooms, except for farm E (
Table 3,
Figure 1a). More than 100 isolates of the genus detected from farms F–J, and I, while <50 from farm A–E. The least number of isolates, 9 and 10 were obtained from farm E and A, respectively. Surprisingly, farms H and F have 129 and 164 isolates, of which 128 (99%) and 162 (98.7%), respectively were ascertained to the genus
Penicillium. This agrees with numerous earlier studies affirming that
Penicillium spp. is the most prevalent mold in the indoor environment of many cheese factories [
9,
27].
The second (9.43%) most diverse and dominant genera were
Aspergillus (93 isolates, 7 species) followed by (2.64%) by
Cladosporium (26 isolates, 4 species). The genera
Aspergillus (
Figure 1a;
Table 4) and
Cladosporium (
Figure 1a;
Table 5) were identified from 6 out of 10 dairy farms, although more frequently in farm E (76%). In contrast to
Penicillium species, most of the
Aspergillus and
Cladosporium species cannot grow under low temperatures conditions, as they are generally mesophilic [
3]. The indoor temperature of the farm E might certainly contributed to the existence of these species and repressed the dominance of
Penicillium as observed in other farms. Among 100 isolates from Farm E, 71% isolates from the cheese board, floor, wall, air and cheese rinds were identified as belonging to the genera
Aspergillus.
Aspergillus was undetectable in four farms (D, F, H, I), while the numbers were <10 in farms from A to C, G and J. The genera
Aspergillus and
Cladosporium were highly represented in farm E, while
Penicillium and
Aspergillus in A, B, and J. The genus
Penicillium alone completely dominated in farms F–I. (
Table 3–
6,
Figure 1a). It is commonly agreed that the fungal dynamics in the interior environment of a dairy farm is more influenced by the exterior atmosphere [
10,
26]. Earlier studies have revealed dominance of the genera
Penicillium,
Aspergillus, and
Cladosporium in the air of cheese ripening rooms in Portugal [
8];
Penicillium and
Cladosporium in a Greek dairy plant [
26] and
Cladosporium,
Alternaria, and
Penicillium in the indoor air of Italian dairy environments [
10]. These results clearly indicated that the genera
Penicillium,
Aspergillus, and
Cladosporium are not uncommon and constantly prevalent in cheese production farms and other dairy products.
Among the cheese ripening rooms, farm C had 9 genera mainly comprised of
Alternaria,
Aspergillus,
Cladosporium,
Fusarium, and
Penicillium. All these genera have been previously described as contaminants in cheese and their production environment across various countries [
9,
10,
13]. The occurrence of these genera in an indoor environment of a cheese production farm reveals poor hygienic processes as they were commonly attributed to the outdoor air in rural areas [
8,
10,
26].
Despite a huge number of isolates, it is possible that the mold species actually identified were only a portion of the total present. Maybe few strains can find hard to grow on a synthetic medium or exist in such low numbers that the large numbers of the other species dominated them. Nevertheless, the outline of the fungal microflora in the dairy farms affords an idea of the complexity of this ecosystem.
Diversity of mold species
The abundance of molds in cheese ripening rooms analysed at the species level resulted in identification of 39 species (
Figure 1b,
Tables 3–
6). Of which only one species of
Aureobasidium,
Debaryomyces,
Engyodontium,
Fusarium,
Mycosphaerella,
Peyronellaea,
Saccharomyces,
Trichaptum, and
Trametes were isolated. Most of the molds revealed within the cheese ripening rooms comprised of general cheese contaminants along with species not frequently isolated from cheese and other dairy products. The species of
Penicillium,
Aspergillus, and
Cladosporium described in our study are already described in earlier works [
8–
10,
27] as potentially toxigenic genera and frequent contaminants in dairy production environment.
Penicillium spp. is adapted to the cheese manufacturing process and they are well acclimated to the cheese production environment. Moreover, several studies [
3,
13,
28] reported
Penicillium species as the predominant mold in spoilage of cheeses and other dairy products. Based on a recent classification system proposed by Visagie et al [
29],
Penicillium spp. isolates fit into the section Fasciculata (
P. commune,
P. crustosum,
P. echinulatum,
P. solitum,
P. verrucosum,
P. viridicatum), Penicillium (
P. expansum,
P. griseofulvum,
P. granulatum), Chrysogena (
P. chrysogenum, P
. rubens), Brevicompacta (
P. brevicompactum), and Roquefortorum (
P. roqueforti). Most of the
Penicillium species belongs to section Fasciculata. Of the 14 different
Penicillium species, 6 (
P. cf.roqueforti,
P. crustosum,
P. griseofulvum,
P. granulatum,
P. rubens, and
P. viridicatum) were detected in only one of the farms (
Table 3,
Figure 1b).
Among thirty-nine different mold species identified in this study,
P. commune (18.6%) was especially more abundant in eight out of ten farms (except farm D and E) as contaminants from the floor, wall, cheese board, air and cheese rind (
Table 3,
Figure 1b). Likewise, several studies [
3,
9,
27] reported dominance of
P. commune on cheese environments as well as dairy products.
P. commune is one of the most common fungi in cheese spoilage and reported as the source of discolouration on cheese surfaces and production of off-flavours in cheese factories [
5,
6]. The dominance of this indoor environmental fungus could be attributed to its ability to survive under low oxygen concentration and its psychrotolerant nature [
6]. The conditions prevailing inside the indoor environment seems to favour its existence in the cheese dairy farms. The second and third abundant species
P. oxalicum (10.7%) and
P. echinulatum (5.7%), respectively were isolated from nine (except farm J) and seven farms (except farm D, E & H). The major source of
P. echinulatum isolates was found to be cheese board, since it is very well associated with wooden products and wooden shelves in the cheese production environment [
28].
P. commune and
P. echinulatum were observed in the cheese core samples of few farms, as both are highly related to the cheese flora [
30]. Other species like
P. crustosum,
P. solitum,
P. verrucosum,
P. griseofulvum,
P. chrysogenum,
P. brevicompactum, and
P. roqueforti isolates of this study were also previously reported as common contaminants in cheese production environments and dairy products [
6,
8,
15]. Most of the above-mentioned
Penicillium species acclimate to low temperatures [
15], consequently their existence in spoilage of dairy production environment is not unexpected.
The domination by
Penicillium species, which includes certain toxigenic species, raise safety concerns, owing to possible mycotoxins contamination in cheese rinds [
7,
16,
18]. Mycotoxins such as citrinin and ochratoxin A produced by
Penicillium species have been detected in cheeses. Although
P. roqueforti is well known for cheese ripening in blue cheese, a few strains have been reported as cheese contaminants and producers of mycotoxins, like PR toxin (
P. roqueforti toxin), roquefortines, sofumigaclavines, and mycophenolic acid [
16,
18,
28]. In addition, other species in our study like
P. chrysogenum,
P. expansum,
P. griseofulvum,
P. granulatum and
P. crustosum are able to produce roquefortine C.
P. verrucosum can produce a combination of citrinin and ochratoxin A, while
P. crustosum produce penitrem A and roquefortine C [
16,
28].
Next to the three most abundant
Penicillium species,
Aspergillus versicolor,
A. creber,
A. tabarinus and
A. jensenii dominated in the cheese ripening rooms (
Table 4,
Figure 1b).
A. versicolor was especially more abundant in farm E (18 out of 24 isolates) and except one isolate all were detected in the indoor air of cheese ripening rooms. As
A. versicolor has airborne conidia it is most often found to dominate in the entire cheese ripening rooms, although it hardly grows on cheeses. Frequent incidence of
A. versicolor was observed in Italian Fossa cheese and its ripening environment [
30]. Among the dairy farms,
Aspergillus species was found more dominant in farm E. Except
A. niger, all species isolated in the study were present in farm E, while the earlier was detected in farm C. Since several years,
A. versicolor and
A. niger have been often reported to cause thread mold spoilage in cheeses [
3]. Other major species like
A. versicolor,
A. creber,
A. puulaauensis,
A. jensenii presented in our study are possible sterigmatocystin producers in cheeses [
18,
31].
Our study showed that fungal contaminants belonging to the class Dothideomycetes were the second largest flora. Among them,
Cladosporium spp. are the most common airborne species which often originated from aeration structures, dust, outdoor environments or wet spots [
9,
10]. The
Cladosporium species was completely absent in farms from F–J, and <10 in numbers in farms A–D and J, although a maximum of 12 isolates was obtained in farm E (
Table 5). The air and cheese board having a higher probability of harbouring this genera.
Cladosporium spp. are well documented in spoilage of different dairy products (raw milk, butter, cream, cheese and margarine) [
4] and being present in cheese environments [
9,
10,
28]. Few species of
Cladosporium including
C. cladosporioides, which grows well in moistened wood surfaces and conditions with varying temperatures [
9], are unsafe for human health. Correspondingly, in our study we isolated
C. cladosporioides in the cheese board from two dairy farms (A and C). The two-halotolerant
Cladosporium species (
C. halotolerans and
C. sphaerospermum) (
Table 5,
Figure 1b) in our study have been associated with spoilage of butter [
4].
The majority of the species of
Penicillium,
Aspergillus, and
Cladosporium encountered in various regions of the ripening rooms also exist in the ambient air of cheese ripening rooms. The spores of filamentous fungi might be easily disseminated via airborne propagation. These results well corroborate with the findings of previous studies on dairy production environments [
8,
10,
26].
With respect to the other minor mold species (
Table 6,
Figure 1b), few species are isolated only once in a single farm. Species like
Trichoderma (3 species),
Alterneria (2 species) and
Exobasidiomycetidae were the representing ones.
Debaryomyces hansenii and
Saccharomyces cerevisiae isolated from the cheese board in a single farm each, although they are known for frequent association with the rind and core of cheese [
11,
22]. Species of
Exobasidiomycetidae, M
ycosphaerella, and
Phoma were undetermined in our study. To our knowledge, this is the first study that depicts the fungal diversity within the cheese ripening rooms from the dairy farms of the Republic of Korea. The background microflora is highly influenced by several indoor and outdoor sources that need severe attention to prevent any hazards to human health as well as economic losses.