This study assessed physical characteristics of commercially-available pasteurized and unpasteurized Gouda cheeses. According to the FDA Code of Federal Regulations (CFR) [25], the standard of identity of Gouda cheese includes a maximum moisture content of 45% and a minimum fat in solid content of 45%. The Gouda cheeses made with goat milk had higher fat in solid contents than the cheeses made with cow milk, which was expected as goat milk generally contains more fat than cow milk [32]. Four out of the 15 Gouda cheese samples had fat in solid contents which were lower than the 45% minimum specified in the CFR (43.09–44.25%), which was not expected. Also unanticipated was the fact that some of the samples taken under the rind did not have the higher salt contents as compared with the core samples. Intact Gouda cheese wheels are brined in a salt solution, resulting in greater penetration of salt to the outside areas of the cheese, and less penetration of salt to the inner core. This finding, however, may not reflect all Gouda cheeses as only 15 brands were assessed in this study.
Targeted metagenomic sequencing of the 15 brands of commercial Gouda cheese identified two common genera comprising more than 1% of the total sequencing reads among all samples: Lactococcus and Staphylococcus. In addition, unidentified members of the family Bacillaceae were also common to all cheeses. A comprehensive metagenomic study of artisanal semi-hard cheeses, of which Gouda is a member, determined that the overall population of Lactococcus was 84.5% based on sequence reads from 31 cheeses [8], which is higher than the result found in the current study (40.1–49.1%). This finding could be a reflection of the starter cultures used to make the cheese or the variation of organisms in the milk. Other studies have determined populations of Lactococcus to be 2–22% in different brands of Latin-style cheeses [9] and 49.6% in hard cheeses [8]. Lactococcus strains, such as L. lactis subspecies lactis and cremoris are commonly added as part of the starter cultures in cheese manufacture and are responsible for acidification by converting milk lactose into lactic acid. In addition to acidification, bacteria in the genus Lactococcus contribute to curd production and the conversion of amino acids into flavoring compounds.
Bacteria from the family Bacillaceae comprised 38.5–46.3% of the population of the Gouda cheeses. Many Gram-positive, heterotrophic bacterial genera are part of this family including Bacillus, Salinibacillus, Paenibacillus, Geobacillus, and Lysinibacillus. These bacteria are found in the milk production chain and are sometimes contaminants in processed cheese [33, 34]. For instance, bacteria in the family Bacillaceae comprised less than 10% of the population of Latin-style cheese [9]. The higher percent of Bacillaceae in the unpasteurized Gouda cheese in this study (46.3%) may be due to the native microbiota present in the milk used for production. Unpasteurized silo milk contains a greater proportion of Bacillaceae, especially when milk is exposed to elevated environmental temperatures [35, 36]. Organisms in this family are often harder to eliminate in the processing environment due to their ability to form biofilms and heat-resistant endospores permitting their resilience to sanitization processes.
Staphylococcus reads were found in relatively low numbers in the Gouda cheeses (2.0–13.4%). Staphylococcus species, such as Staphylococcus equorum, can be used as an additive to the starter culture for certain semi-hard cheeses such as Swiss cheese [37] and are also naturally occurring microorganisms in cheese brines. Interestingly, it has been determined that S. equorum possesses anti-listeria properties and some studies have suggested the use of this species as a protective starter culture [38]. Staphylococcus has been detected at 0.17% in semi-hard cheeses [8], and at less than 3% in Latin-style cheeses [9]. Staphylococcus has been found in high numbers (5–25%) on the surface of certain cheeses, especially early in the aging process and in cheeses made using goat milk [20, 39]. Staphylococcus species epidermidis and caprae, have also been isolated from goat milk [40].
There were a greater number of genus- or family-level identifications observed for the pasteurized goat (n = 138) and unpasteurized cow Gouda cheeses (n = 120) compared with the pasteurized cow Gouda cheeses (n = 92). This is not surprising, as unpasteurized milk has not undergone treatment to eliminate pathogens and reduce the bacterial burden. This is consistent with other studies that have shown unpasteurized cheeses contained a more diverse microbiome than pasteurized cheeses [41]. In this study, 18 genera were identified only in unpasteurized cow Gouda cheese and not in the pasteurized cow or goat Gouda cheeses. Some of the genera identified in the unpasteurized Gouda included Mycoplasma, Ochrobactrum, Nocardioides, Yaniella, and Adhaeribacter. Mycoplasma is a bacterium that can cause mastitis in dairy cattle, and Ochrobactrum has been isolated from cow teat skin [42]. Nocardioides, Yaniella, and Adhaeribacter have all previously been identified in unpasteurized milk and cheese [23, 43, 44]; Yaniella, a Gram positive coccus in the family Micrococcaceae, has been typically found in saline soils and has also been found in cheese rinds [23].
Eight bacterial genera were identified only in pasteurized cow Gouda cheese and included Anoxybacillus, Curtobacterium, and Yersinia. All three genera have previously been isolated from dairy products [45,46,47,48]. Anoxybacillus is a thermophilic spore-former frequently isolated from whole milk powder and nonfat dry milk and is sometimes used as a hygiene indicator in pasteurized diary manufacture due to its high optimum growth temperature [48]. Unpasteurized milk often contains the potential pathogen Yersinia enterocolitica and the organism can be found in curd samples when the milk is used to make cheese. However, in one study, Y. enterocolitica has been identified in one out of 265 pasteurized milk samples [47].
A total of 28 genera were identified only in the pasteurized goat Gouda cheese in this study and included Mannheimia, Leptotrichia, Balneimonas, Klebsiella, and Pseudoalteromonas. Mannheimia and Leptotrichia may have been part of the goat ecosystem which was transmitted to the milk used in the manufacture of the cheeses. The genus Mannheimia is comprised of bacteria responsible for epizootic pneumonia and mastitis in goats, sheep, and cattle [49], while Leptotrichia has been isolated from goat foot lesions [50] and are normally found in the human oral cavity [51]. Balneimonas has not previously been isolated from cheese, but has been isolated from Suanzhou (Chinese fermented cereal gruel) samples [52]. Bacteria in the genus Klebsiella, such as Klebsiella pneumoniae, are human pathogens and have also been found to cause spoilage in cheese via gas production leading to early blowing of semi-hard and hard cheeses [53]. Like many other organisms, Klebsiella lack thermoresistance, which indicates contamination of the cheeses most likely occurred during or post-manufacture. Pseudoalteromonas are mesophilic or psychrophilic marine bacteria that can survive in environments with high salinity. The genera has been identified in soft and semi-hard cow pasteurized and unpasteurized cheeses as well as in cheese rinds [8] and on the surfaces of smear-ripened cheeses [54, 55]. In one study, Pseudoalteromonas haloplanktis comprised 17% of the total mapped reads of a smear-ripened cheese as determined using 16S rDNA metagenomics sequencing [54].
Overall, the majority of all the bacterial genera identified in this study were present in all three cheese regions (under the rind, core, inside), however differences were observed in population proportions. In the samples taken under the rind, Staphylococcus and Tetragenococcus were prevalent (9.6 and 4.8% of the total sequencing reads for all cheeses, respectively). Large populations of Staphylococcus on the surfaces of cheeses have been detected previously [23, 56]. Tetragenococcus, a moderately halophilic bacterial genus, has previously been detected in unpasteurized hard cheeses and cheese rinds at 0.05 and 0.18%, respectively, but was not detected in soft or semi-hard cheeses [8]. In addition, Brachybacterium, Pseudoalteromonas, Yersinia, Klebsiella, and Weissella were only detected in the under the rind samples. Brachybacterium and Pseudoalteromonas are both halophiles, capable of growing in concentrations of salt as high as 15–18% [57, 58]. Therefore, these organisms may have contaminated the cheese during brining. Yersinia and Klebsiella contain species which are potential human pathogens and are ubiquitous in the environment and could possibly be a post-pasteurization contaminant. Yersinia can also grow at refrigeration temperatures and could survive the cheese aging and storage process. Lastly, Weissella, a facultative anaerobic lactic acid bacteria in the family Leuconostocaceae, was also only identified in the samples taken just under the rind. Although some species of Weissella are pathogenic, some species are being studied as potential pro- and prebiotic organisms. This organism has been previously identified in a wide range of habitats including milk and cheese rinds [59], Mexican Cotija cheese [60], and cheese whey [61].
Megasphaera, Caloramator, and Hymonella were only detected in the Gouda cheese cores, and Anoxybacillus and Yaniella were only detected in the inside samples. The core of a cheese represents an environment that is mainly anaerobic, explaining why the anaerobes Megasphaera and Caloramator and the facultative anaerobe Hymonella were identified in this region. Interestingly, Megasphaera is known to be a commensal organism of ruminants and has been identified in unpasteurized ewe milk cheeses [62]. Anoxybacillus and Yaniella, which were only identified in the inside, were also only present in the cow Gouda cheese samples in this study. This is only the second report of Yaniella detected in a food product [23].
In addition to assessing the microflora of Gouda cheese through milk type and spatial variability, this study also examined the differences in microflora based on cheese aging length. Unidentified members of Bacillaceae, Lactococcus, Lactobacillus, and Staphylococcus dominated the populations of the unpasteurized Gouda cheeses which were aged for 2–4, 4–6, 6–9, or 12–18 months. Bacillaceae sequencing reads decreased during aging, whereas the reverse was observed for Lactococcus. Lactic acid bacteria, including those of the indigenous microbiota and the added starter cultures, typically comprise most the population of cheese during the aging process [21]. For Swiss and Emmental cheeses, thermophilic lactic acid bacteria derived from the starter culture (such as Lactobacillus helveticus and Streptococcus thermophilus) are the dominant organisms from the start of aging up to six months [21, 63, 64]. Mesophilic lactic acid bacteria, including Lactobacillus paracasei and L. rhamnosus also become dominant during aging, especially in cheeses aged for 10–30 months [65].
Interestingly, the population of Staphylococcus was not dependent on the length of aging, but rather spatial variation. Most Staphylococcus in the aged Gouda was located in samples taken under the rind. For the Gouda sample aged 4–6 months, this genus comprised 81.1% in the samples taken under the rind. However, Staphylococcus decreased to 50.2 or 45.2% in Gouda aged for 6–9 or 12–18 months, respectively. Less than 1% of the population of the 2–4-month aged Gouda cheese samples taken under the rind was Staphylococcus. The vast differences in these results are likely due to the environmental conditions of aging, personnel handling, and the pasteurization status of the milk used. Large populations of Staphylococcus have previously been observed on cheese rinds [23, 56], possibly due to environmental contamination. Furthermore, Staphylococcus is presumed to be at a concentration of 2–3 log CFU/mL in unpasteurized milk [21].
Twenty-two and 38 genus-level identifications were observed in the unpasteurized Gouda cheese aged for 2–4 and 12–18 months, respectively. A total of 27 out of the 38 identifications in the older Gouda cheese were not found in the younger 2–4 months aged Gouda. Some of the genera identified in the Gouda cheese which was aged longer included Acidovorax, Ralstonia, Adhaeribacter, Devosia, Haemophilus, and Neisseria. Acidovorax and Ralstonia are both aerobic Gram-positive plant pathogens [66, 67]. Acidovorax has been previously identified as a contaminant of Italian Grana cheese [68], and Ralstonia has been detected in unpasteurized milk [43, 69] and can survive high salinity environments. Adhaeribacter and Devosia are both soil dwelling bacteria and have been previously identified in unpasteurized milk [44, 70]. Devosia has also been detected on cow teat skin [42]. Haemophilus and Neisseria, both genera which contain species of human pathogens, were also only detected in the Gouda cheese that was aged for 12–18 months. However, these genera have not previously been identified in dairy products.