2.1. Chemical Composition
Table 1 shows the content of water, protein fat, cholesterol, NaCl, phosphorus, and lactose in the tested cheeses. The examined cheeses were semi-hard cheeses, partially skimmed [
15]. The water content in all of the samples was in the range of 42.98–44.68%, moreover, the Os2 cheese differed significantly in water content (44.68%) from the AW and B1 cheeses (
p < 0.05). The protein content (26.41–26.93%) in the cheeses was similar, while the B1 cheese had the most significant and highest fat content (23.28%) as compared with the AW and Os2 cheeses (21.65 and 21.25%, respectively). The examined cheeses had a fairly low cholesterol content, and the lowest cholesterol content was in the Os2 cheese (47.05 mg/100 g of product). The lowest NaCl and phosphorus contents were found in the B1 cheese (1.15 and 1.19%, respectively). No lactose was found in the tested cheeses (<0.5 mg/100 g of product) or this value was below the detection level. The chemical composition of the produced cheeses was typical for traditional, regional semi-hard cheeses. The products were high in protein, whereas they were fairly low in salt, phosphorus, and cholesterol. Interestingly, no lactose was found after the production, which proves the ability of LAB strains to completely ferment lactose contained in milk (lactose content in cow’s milk is 4.6–4.9% [
16]). The resulting lactic acid significantly lowered the milk’s pH and acidity, which was important during the preservation of the product and in shaping the sensory quality.
2.2. The Fatty Acid Composition
The complete fatty acid profile is presented in the
Supplementary Materials (Table S1). An analysis of the fatty acid profile found that the main fatty acids present in the cheese samples were saturated fatty acids (SFA), which accounted for 58.40 to 59.95% (
Table 2). A statistically significant increase in the amount of SFA after storage (
p < 0.05) was found. The Os2 cheese had the lowest SFA content (
p < 0.05). At the same time, decreased (
p < 0.05) monounsaturated (MUFA; 26.70–27.48%) and polyunsaturated (PUFA; 3.68–4.48%) fatty acids during storage were observed. The content of the trans fatty acids ranged from 6.73 to 7.10%, while the B1 or Os2 treatment had a significantly lower content of trans fatty acids (
p < 0.05). The tested cheeses contained omega-3 (1.40–1.60%), omega-6 (2.15–2.85%), and omega-9 (20.35–21.18%) fatty acids, which are beneficial for human health, but their content was significantly reduced during storage of the cheeses (
p < 0.05). The tested cheeses showed a high content of conjugated linoleic acid ((CLA) C18:2 c9t11), ranging from 2.60 to 2.85% (
Supplementary Materials Table S1). The Os2 cheese had the highest content of CLA, amounting to 2.85% after 1 month and 2.80% after 2 months of storage, respectively (
p < 0.05).
Fatty acids play a positive or negative role in preventing and treating disease. Some fatty acid metabolites have anti-inflammatory and neuroprotective effects [
17]. The main fatty acids in the tested cheeses were SFA, which is also typical for dairy products. At the same time, a high content of MUFA and PUFA was found, with a low content of trans fatty acids. In general, cheeses are associated with a high concentration of long chain fatty acids. Moreover, the composition of fatty acids depends on the animal breed, season, animal diet and species, and production processes [
18].
Conjugated linoleic acid (CLA), a milk-derived fatty acid with health-promoting properties, has also been found [
19]. During maturation, CLA can be formed from linoleic acid through the action of primary or additional bacterial cultures. Then, the lipid profile changes as a result of the lipolysis reaction. It was found that some
Lactobacillus bacteria have the ability to synthesize it, however the mechanisms of this bioconversion have not been elucidated for each LAB species [
20]. CLA is produced by bacteria in the rumen and is transferred to meat and dairy products from ruminants. The daily intake of CLA from the diet is unknown, because the presence of isomers in food and the factors influencing their levels are not well understood. Nevertheless, the concentration of CLA, judged to be beneficial to health by some, is around 3–6 g/day [
21]. The high content of omega-3, -6, and -9 fatty acids also contributes to the favorable profiles of fatty acids. The milk fat, and especially the fatty acid profile, are essential for the sensory properties and the development of the correct flavor during maturation. Fat is not only a source of flavors, but also a solvent for fat-soluble flavor-forming substances [
22].
2.3. Lipid Quality Indices
The tested cheeses were characterized by a fairly low AI, ranging from 2.10 to 2.21, depending on the type of cheese and storage time, but these differences were not statistically significant among the treatments (
p > 0.05) (
Table 3). The B1 cheese was characterized by lower TI (
p < 0.05), both after storage and between the types of cheese. The cheese samples were characterized by a high DFA (41.43–42.95) and a low OFA (36.75–37.75), as well as a high H/H ratio (0.62–0.67). The HPI ranged from 0.45 to 0.48 and no significant differences were observed between the treatments and after storage (
p > 0.05). A good indicator for dairy products is the HPI, which according to research by Chen and Liu [
17], ranges from 0.16 to 0.68, depending on the type of product. The higher the HPI index, the better the impact on human health. Our tested cheeses had an HPI in the range of 0.45–0.49, with no significant differences due to product storage. The PUFA/SFA ratio is typically used to assess the effects of diet on cardiovascular health. It has been hypothesized that all PUFAs in the diet can lower low-density lipoprotein cholesterol and lower serum cholesterol, while all SFAs contribute to high serum cholesterol. Thus, the higher this index is, the more positive are the health effects [
17]. The tested cheeses were characterized by good quality indicators (milk and creamy odor, softness, moisture, and elasticity), which remained at a similar level during storage, and therefore, proves the storage stability of the products. Similar research results were obtained by Mileriene et al. [
23] who produced cheese. The AI index ranged from 1.93 to 2.77, while the TI index ranged from 1.58 to 2.52. In a Polish study, Paszczyk et al. [
24] analyzed the lipid quality indices in smoked and non-smoked cheeses and in smoked and non-smoked cheese-like products. The AI index ranged from 0.98 to 3.18, while the TI index ranged from 2.00 to 3.86 and the Hypocholesterolemic/hypercholesterolemic ratio (H/H) ranged from 0.41 to 1.00.
2.4. Microbiological Analyses
The examined cheeses were characterized by a high number of microorganisms, in particular lactic acid bacteria (~8 log CFU g
−1) as well as bacteria from the
Enterobacteriaceae family (4.42–5.70 log CFU g
−1), yeast and molds (3.79–4.85 log CFU g
−1), and coagulase-positive staphylococci (3.42–5.32 log CFU g
−1) (
Table 4). Molds and acidic bacteria are ubiquitous and could certainly develop on the surface of the cheese, especially as it is moist and ripens in moderate coolness [
25].
Cheeses with the addition of the B1 and Os2 strain were characterized by a higher number of LAB during storage, but the results were not statistically significant (
p > 0.05). The addition of B1 and Os2 culture starters increased the LAB abundance. It is known that the milk used for the research was unpasteurized, so microorganisms present in the raw material may also develop. Therefore, in the control sample with whey (AW treatment), the number of LABs was also high. The initial number of bacteria contained in the starter culture guarantees that the process would be carried out by bacteria present in the starter, which guarantees the repeatability and stability of the product. The quality of the cheeses also showed the differences between the treatments, which were probably due to the addition of the different starter cultures. According to Salazar et al. [
26], the addition of a known starter culture to unpasteurized milk makes it dominant in the developing product. In addition, Vázquez-Velázquez et al. [
27] found that the use of a starter culture helped keep the total pathogenic microbes in pasteurized cheese below standard maximum values. At the same time, a high number of lactic acid bacteria was found. The sensory judges did not find any differences between the cheese made of unpasteurized milk and that made of pasteurized milk with the addition of a starter culture.
According to Kilcawley [
28], cheeses made from raw milk are richer in microbiota, mature faster, and develop a more intense flavor and more aromatic compounds than cheeses made of pasteurized or microfiltered milk. Dairy products, especially those made from raw milk, are abundant in beneficial microorganisms (e.g., non-starter lactic acid bacteria) and in undesirable microbiota. Lactic acid bacteria are naturally present in milk, but their origin, milk quality, as well as environmental (animal diet, season, and pasture height) and hygiene conditions have a significant influence on the milk microbiota [
10].
A qualitative microbiological analysis did not reveal the presence of
Salmonella spp. and
L. monocytogenes. Psychrotrophic bacteria have an impact on the shelf life of many food products, including dairy ones, as they can multiply at refrigerated temperatures (4–10 °C). In order to maintain the high microbiological quality of products, strict control of the raw material and the course of the technological process should be carried out, and particular emphasis should be placed on the preservation of the cold chain [
29]. The cheeses were made from raw, unpasteurized cow’s milk. Most of all, acidifying microbiota is associated with milk. The use of unpasteurized raw material, on the one hand, carries a wealth of microbiota, which is so important for the sensory quality of the product, but on the other hand, carries the risk of inadequate microbiological quality [
30], which is reflected in the results of our research. As the examined cheeses were not pasteurized or preserved in any way, there was a risk of primary contamination (from the raw material) or secondary contamination (e.g., during production or packaging).
In all of the examined cheese samples, a fairly high number of coagulase-positive staphylococci (3.42–5.32 log CFU g
−1) was found.
S. aureus is ubiquitous in the environment and can be found in water, air, humans, and animals. It is also one of the leading causes of mastitis in cattle, and therefore, raw milk and raw dairy products can be contaminated with
S. aureus. This contamination is most common among unpasteurized dairy products, which is especially true of products manufactured largely by hand [
31]. Moreover, it has been observed that the number of staphylococci increases during the storage of products [
32] which was also observed in our research. This is due to the ability of
S. aureus to grow rapidly in milk and dairy products, and also due to the contamination of cheese makers’ skin. The enterotoxins which can be produced by staphylococci under appropriate conditions, are particularly dangerous to human health.
S. aureus is capable of producing enterotoxins in the temperature range of 10–46 °C (optimum 34–40 °C), pH below 5 and above 9.6, and a
w not less than 0.86, and with the NaCl concentration above 12% [
33]. By analyzing the given optimal conditions for the development of enterotoxins, it can be presumed that the cheeses produced in the present study should be free of staphylococcal toxins. However, to avoid any potent risk to consumer health we decided not to subject the produced cheeses to a sensory evaluation in terms of taste. The next stage of industrial research will be an attempt to eliminate staphylococci from unpasteurized milk, which will enable a full sensory evaluation of the products.
2.5. Physico-Chemical Analyses
Table 5 shows the values of water activity (a
w), pH, acidity, and the oxidation-reduction potential (ORP). The produced cheeses were characterized by high water activity, from the start of the production process and throughout the storage period (0.93–0.96); however, the high water activity did not guarantee microbiological stability of the product [
25]. The AW treatment had the highest water activity (0.94–0.96), but the cheeses with the addition of strains (treatment B1 and Os2) did not differ significantly (
p > 0.05). During storage, a decrease in a
w, in all cheeses, was noted, but these differences were not statistically significant (
p > 0.05). The rapid reduction in a
w can be explained by water loss by evaporation and the hydrolysis of proteins to peptides and amino acids and triglycerides to glycerol and fatty acids [
34]. The high a
w of cheeses is characteristic of traditional acid-rennet cheeses, and therefore, requires additional protection, where the preservative agent may be lactic acid bacteria in high numbers, as was the case in the present cheese production. The cheese produced on the basis of whey (AW treatment) was characterized by the lowest pH, both immediately after production and after storage (
p < 0.05). In all of the samples, a significant increase in pH was observed after 1 month of storage, and then, a decrease in pH after 2 months of storage to the value of 5.13–5.34.
Similar results were obtained in the acidity test. In the case of the AW and B1 cheeses, an increase in acidity was observed after 1 month of storage (from 103.50 and 86.00 °SH to 106.25 and 93.50 °SH, respectively), and then, a decrease in acidity after 2 months of storage (99.00 and 86.75 °SH, respectively). The acidity of the Os2 cheese decreased significantly after 1 month of storage (to 72.25 °SH,
p < 0.05), and after 2 months of storage it was 79.75 °SH. The pH values of the cheeses after production and during storage were above 5, which is the typical pH value for unpasteurized, semi-hard cow cheeses. Lowering the pH of milk through the production of lactic acid directly affects the stability of casein micelles and milk minerals [
35]. The variation in pH value depends on the buffering capacity of the cheese and the amount of protein. The acidification process, which is continued during the maturation process, is associated, apart from high lactic acid production, also with low cheese mass buffering capacity. This is a consequence of the demineralization that occurs during the coagulation and removal of the whey. The acidity of cheeses changes during storage and, similar to the pH values, it may rise at first and then fall [
35].
In all of the cheese samples, the oxidation-reduction potential increased significantly (
p < 0.05) during storage (
Table 5). Initially, the highest ORP value was characteristic for the AW treatment (338.03 mV), and the lowest Os2 cheese (258.80 mV,
p < 0.05). After 1 month of storage, a significant increase in ORP (
p < 0.05) was observed, especially in the case of the B1 and Os2 treatments (456.93 and 428.50 mV, respectively), followed by a slight decrease after 2 months of storage. The oxidation-reduction potential during storage increased significantly (
p < 0.05). This is due to the microbial and enzymatic activity of a very high number of microorganisms, in particular lactic acid bacteria [
36,
37,
38]. The high fat content of cheese may also be a predisposing factor to lipid oxidation [
39]. In the tested cheeses, no additional substance was used to inhibit the oxidation processes, and the high values of the redox potential proved their advantage over the reduction processes. This increase was lower in the samples of cow cheeses with the addition of bacterial cultures (B1 and Os2 treatment) as compared with the AW treatment. As the standardization of cheese production of this type is difficult to perform [
30], one of the solutions is the use of environmental lactic acid bacteria as starter cultures.
2.6. Measurement of Colour, Instrumental Texture Evaluation, and Sensory Evaluation
As a part of the research, it was found that the tested cheeses darkened significantly during storage (
p < 0.05). The difference in the brightness of the cheeses was 7.66 for the AW cheese, 10.17 for the B1 cheese, and 10.83 for the Os2 cheese, respectively (time 0 and 2 were compared). Cheeses made with the strain addition (B1 and Os2) significantly differed in brightness immediately after production (L* = 78.94 and 78.33, respectively,
p < 0.05) as compared with the whey cheese (AW, L* = 74.89), while after 2 months of storage no significant differences were noted in the brightness of the cheeses (
p > 0.05) (
Table 6). In terms of the a* parameter, it was found that the samples differed significantly from each other immediately after production. The color of the cheeses was greener (a* = −0.44 − 0.18) and, as the cheeses were stored, the a* parameter decreased significantly (values ranged from −0.51 to −0.57,
p < 0.05). Regarding the b* parameter, the yellow color changed during storage, i.e., after 2 months of storage, the cheeses were significantly less yellow than the fresh cheeses (b* = 19.33-20.53,
p < 0.05). Immediately after production, the most yellow cheese was the AW cheese (b* = 20.78), while after storage, the most yellow cheese was the B1 cheese (b* = 20.69).
The texture parameters of the tested acid-rennet cheeses were similar and increased during the storage of the products (
Table 6), with the exception of the adhesiveness of the products, which after production had the highest value for the Os2 cheese (1.58 mJ) and the lowest after storage (0.58 mJ), unlike other products. Among the tested products, the AW cheese was characterized by the highest value of hardness 1, elasticity, and chewiness (65.08 N, 8.57 mm, and 332.08 mJ, respectively). In addition, the value of the cohesiveness of the Os2 and B1 cheeses was similar and remained almost at the same level during the storage of the products, unlike the AW cheese, in which the cohesiveness value decreased during storage (0.44). The color and texture are important cheese quality distinguishing features, as they are important for consumers when deciding to buy a product. The color itself is one of the first quality attributes on the basis of which the consumer assesses the acceptability of a product [
7]. According to Pérez-Soto et al. [
40], the addition of bioactive compounds may affect the hardness of cheeses, but it has no effect on elasticity, firmness, and chewiness. However, it depends on the type of cheese. The color of the cheeses can be related to various internal and external factors, and the opacity of the cheeses (L*) can be affected by the degree of matrix hydration. The differences may be mainly due to the initial composition of milk and whey and the technology of cheese making [
41]. The b* color parameter is strongly correlated with the yellow color and may be related to maturation time. Springiness is a measure of the return to its original, undeformed state after the first compressive force is removed, and cohesiveness is the amount by which the cheese can be deformed before breaking. These characteristics are influenced by the composition of the milk, cheese production and procedures, microorganisms, maturation and moisture, pH, and soluble calcium. Flexibility shows whether the biochemical reactions taking place inside the cheese are not sufficient to modify the final structure, giving more or less flexibility [
34]. The texture parameters changed during the storage of the products, which correlated with the results of other physicochemical analyses. From a consumer’s point of view, a cheese that is lighter and less yellow is preferable. The tested cheeses with the addition of the B1 and Os2 strains had more consumer preferable parameters than the cheese with whey.
Figure 1 shows the results of the sensory evaluation. The color of the cheeses was towards the yellow (7.54–7.84 c.u.). After storage, a significantly more yellow color of the cheeses was found (8.12–8.32 c.u.,
p < 0.05). The cheeses produced with the addition of the B1 or Os2 strains had a significantly more intense milk and creamy odor. After storage, a significantly more intense milk odor was found (6.87–7.69 c.u.,
p < 0.05). The cheeses produced with the addition of the B1 and Os2 strains were more moist, elastic, and softer. The performed sensory analysis of the products does not give a complete picture of the sensory profile, due to the presence of
S. aureus; a taste assessment of the products was not carried out.
Acid-rennet cheeses are products whose appearance, physical, and organoleptic characteristics depend largely on the multidirectional enzymatic and metabolic activity of cultures and enzyme preparations used in their production [
42]. During maturation, the texture, structure, taste, and aroma of the cheeses changed. Changes in the structure and consistency of cheeses depend on both technological operations and the nature of fermentation processes, CO
2 formation, and the degree of protein degradation. Organoleptic characteristics are shaped by numerous end metabolites of lactic acid fermentation, proteolysis, lipolysis, and sometimes propionic fermentation. The correct proportions of compounds formed in these transformations determine the characteristic taste and smell of various types of cheese.