2.2. Acidity of Probiotic Goat’s Milk with Collagen
The application of milk from different animal species could influence the acidification rate by lactic acid bacteria, including probiotic bacteria. Therefore, some replicate better in goat’s milk than in milk from other animal species [
36]. Some authors indicated that the higher fermentation activity of lactic acid bacteria in goat’s milk is due to its specific composition and structure [
37]. The buffering capacity of goat’s milk is lower than cow’s and sheep’s milk. During the manufacture of yogurt from goat’s milk, a pH of 4.6 to 4.7 is achieved after 2 h 45 min; in cow’s milk, after 3 h 30 min; and in sheep’s milk, after 5 h 30 min. The faster pH changes in goat’s milk are mainly due to its lower casein content, while β-casein is the least phosphorylated. Moreover, the higher non-protein nitrogen content, higher vitamin concentration, higher amount of some minerals and short-chain fatty acids, and easier protein digestibility in goat’s milk may affect the faster increase in acidity [
38]. Additionally, our study observed different pH values on day 1 of storage in control samples fermented by strains:
Lacticaseibacillus casei, Lactobacillus acidophilus, Lacticaseibacillus paracasei, and
Lacticaseibacillus rhamnosus (
Figure 1,
Figure 2,
Figure 3 and
Figure 4). For all control samples, the lowest pH value was determined in LA control milk fermented by
L. acidophilus (pH = 4.33) and the highest in LP samples with
L. paracasei (pH = 4.54) on day 1 of storage. The acidity of the obtained milk is also affected by the type of collagen used. The addition of both collagen hydrolysate and bovine collagen reduced the pH value of the milk even before fermentation. However, adding hydrolysate was significantly more influenced by increasing the pH value than bovine collagen. These differences were statistically significant (
p ≤ 0.05) at 1.5% and 3.0% collagen dosage in all fermented milk samples compared to their control counterparts. It was found that the addition of 3.0% hydrolysate and bovine collagen results in an increase in pH values by 0.42 in LA3.0W samples and 0.54 in LA3.0H samples of fermented milk. Moreover, in Goto’s [
39] study, the addition of 1.5% bovine collagen resulted in a 0.14 reduction in pH values, and the addition of 3.0% collagen resulted in a 0.23 decrease in pH values.
After 21 days of storage, all samples with collagen showed higher pH values than their control counterparts. In fermented milk, after 21 days of storage, adding 1.5%, bovine collagen increased the pH value from 0.03 in LR1.5W to 0.17 in LA1.5W. However, adding 3% collagen increased the pH value to 0.17 in LR3.0W and 0.31 in LA3.0W. After 21 days of storage, the addition of 1.5% hydrolysate increased the pH value compared to controls from 0.11 in LP1.5H to 0.28 in LA1.5H. Increasing the dose resulted in higher pH values, from 0.20 in LP3.0H and LC3.0H to 0.39 in LA3.0H. Studies by Goto [
39] indicated a slow reduction in pH in milk containing 3.06% and 5.1% collagen, indicating a delayed fermentation phenomenon caused by the addition of collagen. Similar results were obtained in milk with different types of collagen, i.e., fish and pork [
39].
A study by Znamirowska et al. [
40] of cow’s milk fermented by
L. rhamnosus similarly showed higher pH values in milk with collagen hydrolysate compared to control samples during 21-day storage. A study by Dimitrellou et al. [
15] found that probiotic yogurts obtained from goat’s milk at 21 days of refrigerated storage had higher acidity (pH = 3.88) compared to those obtained from cow’s milk (pH = 3.98). Probiotic sheep’s milk with collagen hydrolysate obtained in the study by Szopa et al. [
9] was also characterized by higher pH values than the control samples.
The results in
Table 3,
Table 4,
Table 5 and
Table 6 indicate that the lactic acid content depended on the fermentation strain. On day 1 of storage, the highest lactic acid content of all control groups was found in LA milk fermented by
L. acidophilus (
Table 4). In contrast, the lowest was found in LP samples fermented by
L. paracasei (
Table 5). The lactic acid content of the samples with collagen protein hydrolysate and bovine collagen was higher on the 1st and 21st day of the study compared to the control groups of milk fermented by
L. casei, L. paracasei, and
L. rhamnosus.
On day 1 of storage of samples fermented by
L. paracasei, the addition of bovine collagen was found to increase the lactic acid content by 0.1 g L
−1 compared to the control sample while increasing the dose of bovine collagen from 1.5% to 3.0% did not significantly affect the lactic acid content of fermented milk. However, the hydrolysate addition to milk fermented by
L. paracasei increased lactic acid content by 0.08 g L
−1 but only at the 3.0% hydrolysate dose. At a dose of 1.5% hydrolysate, the lactic acid content was the same as in the control sample on day 1 of storage (
Table 5). However, the concentration of lactic acid in samples with collagen hydrolysate (LA1.5H and LA3.0H) fermented by
L. acidophilus (
Table 4) was lower than in LA control milk and milk with bovine collagen (LA1.5W and LA3.0W).
After 21 days of storage, the highest lactic acid content was determined in samples fermented by
L. casei (
Table 3) with a 3.0% addition of hydrolysate LC3.0H and samples with bovine collagen LC3.0W, where it was determined, respectively, 1.35 g L
−1 and 1.34 g L
−1. The highest concentration of lactic acid after 21 days of storage was found in goat’s milk fermented by
L. casei. In this case, adding bovine collagen at 1.5% and 3.0% increased the acid content by 0.04 g L
−1 and 0.12 g L
−1, respectively. Additionally, the addition of hydrolysate increased the lactic acid concentration in the LC1.5H sample by 0.08 g L
−1 and the LC3.0H sample by 0.13 g L
−1 after 21 days of storage. Moreover, extending the storage time to 21 days resulted in the highest increase in lactic acid concentration in samples fermented by
L. casei compared to the other fermented milk samples. A similar correlation was shown by Znamirowska et al. [
40], and according to Kavaz and Bakirci [
41], the amount of lactic acid in probiotic yogurts increases with storage time. In a study performed by Shori et al. [
42], the addition of fish collagen increased the initial titratable acidity (TA%) by about 0.2% lactic acid equivalent. In yogurts with fish collagen, there was an increase in TTA (total titratable acidity) throughout refrigerated storage compared to the control sample [
43].
2.3. Syneresis of Probiotic Goat’s Milk with Collagen
Syneresis is considered one of the most apparent defects in fermented milk, resulting from leakage of yellow-green whey on the surface of fermented milk [
16]. Syneresis occurs due to shrinkage of the protein gel, which leads to the separation of whey and curd. The process of whey separation is related to the firmness and stability of the protein network and other factors, such as the type of milk, low pH, high acidity, the type and intensity of heat treatment, storage time, and the type of additives and stabilizers used [
40,
44,
45]. Acid gel syneresis is also significantly affected by the type of starter culture used for milk fermentation due to the metabolites produced, including exopolysaccharides [
46].
In our study, on day 1 of storage, in most cases, adding a hydrolysate at 3.0% increased syneresis from 0.2% to 9.0%, depending on the type of strain used (
Table 3,
Table 4,
Table 5 and
Table 6). LA3.0H fermented milk showed a 2.42% reduction in syneresis compared to the control; however, the reported difference was not statistically significant (
Table 4). Conversely, the most significant reduction in syneresis compared to the control sample (by 2.58%) was found in milk fermented by
L. rhamnosus with the addition of 1.5% hydrolysate (
Table 6).
After 21 days of storage, all samples of milk fermented by
L. rhamnosus showed an increase in syneresis of 1.83–4.53% compared to day 1 of storage. Furthermore, significantly lower syneresis was found in samples with bovine collagen LR1.5W and LR3.0W compared to the control sample LR fermented by
L. rhamnosus both on day 1 and day 21 of storage (
Table 6). However, the milk with collagen fermented by
L. casei (
Table 3) had lower syneresis than the control milk samples on day 21 of refrigerated storage. Trends toward lower syneresis with increased storage time were also found in samples fermented by
L. paracasei (
Table 5).
Gomes et al. [
44] showed weaker curd and reduced whey leakage in beverages made from goat’s milk compared to cow’s milk. This was related to the composition and microstructure of goat’s milk, which has smaller casein micelles than cow’s milk, resulting in a protein network with smaller pores, higher density, and lower water-holding capacity. Gerhardt et al. [
47] indicated that adding collagen hydrolysate above 1.0% reduced the syneresis intensity of fermented milk, improving its stability. In our study, adding 1.5% hydrolysate reduced syneresis only in milk fermented by
L. rhamnosus, as syneresis was determined by the milk’s pH value (r = −0.6040) and lactic acid content (r = 0.6671).
One of the most commonly used additives along with fruit loading is bovine gelatin, as it has a high ability to give products increased firmness and lower syneresis [
35]. Consequently, goat’s milk yogurts with this additive have a less firm gel structure than their cow’s milk counterparts [
48,
49,
50,
51,
52].
2.4. Color of Probiotic Goat’s Milk with Collagen
Many studies reported that milk additives affect color parameters. In our study, adding hydrolysate and bovine collagen caused color darkening during 21 days of refrigerated storage (
Figure 5,
Figure 6,
Figure 7 and
Figure 8).
The most significant color darkening on day 1 of storage was found in the LP3.0W sample fermented by
L. paracasei (
Figure 7). It was also found that L* color lightness tends to decrease with increasing storage time in all samples. Additionally, in the study by Rigoto et al. [
53], the lightness and color angle values did not change for all milk samples with collagen during the storage period.
In our study, all milk samples were characterized by the proportion of green color (−a*) increasing in intensity with increasing storage time. Moreover, a significant negative correlation was shown between syneresis and the parameter b* (r = −0.6815) and color angle (r = −0.6691). The proportion of yellow color depended on the probiotic strain used and the concentration and type of collagen used. There was observed a reduction in the intensity of yellow color in milk fermented by
L. acidophilus and
L. paracasei (
Figure 6 and
Figure 7) on day 1 of storage with the addition of both collagen and hydrolysate and in milk with a 3% addition of collagen hydrolysate fermented by
L. rhamnosus (
Figure 8) compared to control milk. In milk fermented by
L. casei, the addition of collagen and hydrolysate in two doses increased the proportion of yellow color on day 1 as well as day 21 of refrigerated storage (
Figure 5). However, milk fermented by
L. rhamnosus (
Figure 8) on both day 1 and day 21 of storage showed a higher proportion of yellow color for samples with collagen. The other color components, C and h
0, were within the range: C: 7.33—10.03, h
0: 98.68—104.00.
Milk fermented by
L. acidophilus and
L. rhamnosus obtained by Szajnar et al. [
13] was also characterized by light color and the proportion of green (−a*) and yellow (+b*) color. The study of Lasik and Pikul [
54] observed that during the fermentation process in all samples, the parameter a* decreases and the parameter b* increases. This correlation is due to the acidification and coagulation process. Color changes also occurred during the non-enzymatic reaction starting with the binding of the aldehyde group of lactose to the amino group of milk proteins. The incubation temperature also affected the increase in the color parameter b*. Thus, the higher the incubation temperature, the higher the b* parameter [
54].
2.5. Texture Profile of Probiotic Goat’s Milk with Collagen
Texture characterizes the product’s physical properties, such as hardness, adhesiveness, and springiness, and is considered among the essential quality characteristics of fermented dairy products. The texture properties of fermented milk are related to the type of milk, heat treatment, type of starter cultures, dry matter content, and the type of additives used [
9,
55]. Fermented goat’s milk shows a soft gel and a high ability to spread the curd compared to cow’s milk [
55]. The rheological properties and texture of fermented goat’s milk could be improved by adding strains that produce exopolysaccharides (EPS) during incubation.
Goat’s milk gels have a lower solidity and a softer texture than cow’s milk gels. These properties are directly related to the smaller diameter of casein micelles, a lower degree of hydration, lower mineralization and casein content of milk, especially the αs
1 casein fraction, and lower non-protein nitrogen diameter in goat’s milk compared to cow’s milk [
16,
55,
56]. Smaller amounts or the lack of αs
1 casein in goat’s milk results in a softer gel with lower viscosity [
57,
58]. In order to obtain the right curd consistency in fermented goat’s milk, it is necessary to increase the solids-non-fat content through the use of whey or milk protein concentrates or isolates, as well as caseinates, stabilizers, pectins, starches, and alginates, and even the addition of lactic acid bacteria (LAB) as exopolysaccharide generators [
59,
60,
61]. Moreover, adding collagen to milk before fermentation could determine the physicochemical characteristics and hardness of the probiotic milk gel [
9,
40]. Adding proteins to goat’s milk is recommended to obtain a gel network that can be less susceptible to breaking and highly capable of immobilizing the aqueous phase in the matrix. Gel firmness increases with increasing protein levels [
57,
62]. Moreover, probiotic bacteria could determine the texture of fermented milk due to differences in the number of organic acids and exopolysaccharides produced [
57,
63,
64].
This was also confirmed in our study, where the lowest gel hardness was found in the LA control sample fermented by
L. acidophillus on both day 1 and day 21 of the experiment (
Table 4). The hardness in the remaining control samples was 0.1–0.2 N higher. Moreover, it showed a significant negative correlation between hardness and pH (r > −0.6) on days 1 and 21 of storage. However, the hardest was the LR3.0H milk gel with 3.0% hydrolysate fermented by
L. rhamnosus on day 1 of storage (
Table 6). The hardness results in
Table 3,
Table 4,
Table 5 and
Table 6 showed a tendency to increase hardness after adding collagen, especially hydrolysate, compared to control samples on days 1 and 21 of storage. However, in most cases, the differences are not significant. Additionally, the springiness of the goat’s milk gel did not change significantly after the addition of collagen. In contrast, the 1.5% addition of bovine collagen reduced the cohesiveness of fermented milk compared to the control counterparts. However, statistically significant differences were shown only for LP1.5W samples after 21 days of storage. Additionally, in the Luo et al. [
65] study, gel cohesiveness was not affected by the addition of 0.1% or 0.2% gelatin but increased significantly at higher gelatin concentrations. In a study by Pang et al. [
66], the cohesiveness of milk gel samples also decreased significantly as the gelatin concentration increased to 1%, which could be related to steric interference by gelatin addition. These authors, studying the microstructure, observed that low concentrations of gelatin did not significantly change the gel matrix of milk [
66]. An analysis of variance confirmed that the texture components (hardness, cohesiveness, and springiness) were most influenced by the type of probiotic strain used, the type of collagen, and the interaction of these two factors. In a study by Szopa et al. [
9] in sheep’s milk fermented by
L. casei, a reduction in milk cohesiveness was observed in samples with 1.5 and 3.0% collagen hydrolysate addition compared to the control sample throughout the storage period.
2.6. Viability of Probiotic Bacteria in Fermented Goat’s Milk with Collagen
Probiotic bacteria could significantly impact the quality of fermented milk and the formation of metabolites during fermentation and storage [
2]. The viability of
lactobacilli in fermented milk depends on several factors, including fermentation time and temperature, product storage conditions, acidity, dry matter and carbohydrate content, bacterial access to nutrients, the presence of oxygen, and the type, species, and strain of lactic acid bacteria used for fermentation [
67]. In Mituniewicz-Malek et al.’s [
67] study, during the whole period of 21-day refrigerated storage, the population of probiotic strains used in the production of fermented goat’s milk did not change significantly, and all experimental beverages were characterized by a normative number of viable cells (at least 6 log cfu g
−1). Minervini et al. [
68] found that in fermented goat’s milk immediately after fermentation, the number of probiotic strains reached 7–8 log cfu g
−1, while after 45 days of refrigerated storage of fermented milk, it was significantly reduced, and for
L. casei, it was 7.0 log cfu g
−1 [
68]. Yerlikaya et al. [
64] showed the highest viability for
L. acidophilus strains and the lowest for
L. casei during 30 days of storage. Kim et al. [
69] showed that collagen has a protective effect and increases the viability of probiotic strains and enhances bacterial stability, survival in the gastrointestinal tract, and heat resistance.
In our study, the acceptable survival rate of probiotic bacteria (>8 log cfu g
−1) could be attributed to the properties of probiotic strains. The best survival rate after 21 days of cold storage compared to day 1 was found in milk with 3.0% added collagen hydrolysate fermented by
L. casei (LC3.0H). The survival rate was 103.66%. This could be explained by considering the nutritional requirements of this strain, which indicate a high consumption of amino acids. Zhang et al. [
70] added various nutrients to the fermentation of milk with
L. casei, such as asparagine, cysteine, proline, glycine, glutamic acid, tyrosine, guanine, Ca-pantothenate, pyridoxine, Mn
2+, and Mg
2+, reducing the fermentation time and increasing the survival rate of the strain.
The analysis of the results in
Table 7 shows that adding collagen hydrolysate to probiotic milk had no significant effect on the viability of
L. rhamnosus and
L. paracasei on day 1 and day 21 of storage. As Morita et al. [
71] demonstrated,
L. rhamnosus can hydrolyze milk proteins to obtain the appropriate amino acids and synthesize other nutrients primarily early in the fermentation process (0–8 h).
L. rhamnosus can synthesize nutrients needed later in fermentation, but this ability is poor [
72]. Thus, the addition of collagen was also found to be insignificant.
However, milk with hydrolysate LC1.5H, LC3.0H, and LA3.0H showed significantly higher counts of live probiotic cells after 21 days than their control counterparts (
Table 7). Meng et al. [
73] analyzed the nutritional requirements of
L. acidophilus LA-5, including the consumption standards of amino acids, purines, pyrimidines, vitamins, and metal ions. The nutrients required by
L. acidophilus LA-5 were asparagine, aspartic acid, cysteine, leucine, methionine, riboflavin, guanine, uracil, and Mn
2+, and when added to milk, the fermentation time of milk fermented by
L. acidophillus LA-5 was reduced by 9 h, with a high number of viable cells that persisted during storage of the fermented milk supplemented with nutrients compared to the control. Therefore, adding an amino acid-rich hydrolysate to goat’s milk before fermentation stimulates this strain’s survival rate.
Significantly, all values (8.2–9.3 log cfu g
−1) corresponded to the recommended minimum daily intake of live probiotic cells per serving of ready-to-eat product [
74]. These results demonstrate the functional potential of final fermented milk, in which the probiotic content could be considered sufficient to provide benefits to consumers and could compensate for any possible limitations caused by passage through the gastrointestinal tract [
75].
2.7. Organoleptic Evaluation of Probiotic Goat’s Milk with Collagen
The most important problem related to applying goat’s milk to several dairy products is its goaty taste and odor [
44]. The specific odor of goat’s milk is linked to the composition of fatty acids. The content of caproic, caprylic, and capric acids in goat’s milk is slightly higher than in cow’s milk. These acids are present in large quantities in the fat of goat’s milk and are released from the membranes of the fat globules by lipases in case of improper milking and processing. During fermentation, goat’s milk changes taste and odor [
76,
77]. It was observed that in fermented milk derived from the milk of other animals (goat, sheep), the volatile compounds’ profile differs, giving them specific sensory properties. In addition to the fermentation process, many of the milk’s native compounds are metabolized using different bacterial cultures, yielding different metabolites that contribute to specific aromatic notes.
The results of the evaluation of goat’s milk fermented by four monocultures, including control and with collagen, are shown in
Figure 9,
Figure 10,
Figure 11 and
Figure 12. Among the control samples on day 1 of storage, LA (
Figure 10) milk was the sourest, and LP (
Figure 11) milk was the least, which were also the sweetest. Fermented goat’s milk with
L. acidophilus and
L. casei tested by Mituniewicz-Malek [
67] was characterized by a more noticeable sour and goaty aftertaste, minimal whey leakage, and looser texture.
In this study, the panelists found that adding collagen increased the intensity of the milky-creamy and sweet taste. These effects were especially noticeable after 21 days of storage. The intensity of off-taste and additive taste depended on the bacterial strain and storage time. The most intense off-taste and odor were found in LA milk while adding bovine collagen and hydrolysate reduced their perceptibility.
The goat’s fermented milk samples were characterized by a mildly sour odor (
Figure 9,
Figure 10,
Figure 11 and
Figure 12). Acetaldehyde is an important compound that gives milk its fermentation odor. In fermented beverages made from goat’s milk, compared to cow’s milk, it is difficult to have a high content of acetaldehyde, which is conditioned by the more than twenty-fold higher content of glycine, which has an inhibitory effect on threonine aldolase, converting threonine into acetaldehyde and glycine [
67]. According to Gao et al. [
78], milk fermented by
L. casei has a higher content of acetic acid, lactic acid, butyric acid, caproic acid, acetoin, 2-butanone, and 2-ethyl-1-hexanol, which may result in higher product acceptance. Adding whey protein products to milk increased precursors in the acetaldehyde formation pathway. Whey protein products are rich in threonine and valine AA compared to caseinates, which might be why the high concentration of acetaldehyde in yogurts is fortified with whey protein-based additives [
57].