1. Introduction
Although the application of sheep’s or goat’s milk for cheese making is well-known, few attempts have been made to systematically study the use of such milks to produce cream, butter, yoghurt and other traditional dairy products. However, products made with sheep’s or goat’s milk, due to their nutritional and antiallergenic properties, can be a beneficial alternative in terms of health benefits [
1,
2]. Hayaloglu and Karagul-Yuceer [
3] indicate that products made with sheep’s or goat’s milk have some different and interesting characteristics, mainly taste, aroma, appearance and chemical composition, compared with those made with cow’s milk. However, they also indicate that, in Turkey, over time, a significant decrease in the production of goats or sheep is evident, due to the migration of the population from rural to urban areas, low yields in production and milking, unproductive pastures, increased feed and labor prices, among other factors. The same evidence holds true for other countries, such as Portugal. Sustainable rural development requires value-added products and activities. A recent study by Tulla [
4] discusses four opportunities to valorize peripheral rural areas: (a) transformation of dairy products into competitive value-added commodities; (b) promotion of extensive livestock based on local natural grass fodder; (c) development of value-added tourist activities linked to local landscape; and (d) planning value-added cultural activities related to cultural heritage.
Sheep’s and goat’s butters are seldom reported in the literature, despite being common dairy products in some Mediterranean countries, in the Middle East and Africa. Information about its composition and characteristics is critically scarce. Only a few reports refer to the physicochemical and microbial characteristics of traditional butters made with cow’s cream or with sheep’s and goat’s cream [
5,
6,
7]. As consumers’ demands for dairy products of high nutritional and health value have increased the popularity of small ruminants’ dairy products, it is important to increase the research efforts on these valuable products. In fact, the development of new functional foods is driven by the increased public awareness regarding the health impact of food [
8,
9].
Functional foods, especially probiotics, exert a beneficial effect on the gut microbiota of the host after consumption and can prevent several health problems [
10]. Dairy products are traditionally used as vehicles for probiotics in the human diet. However, levels equal to, or greater than, 6–7 Log CFU/g have been suggested as the minimum concentration for positive effect of probiotic bacteria at the time of consumption. This aspect represents a challenge, as several factors during processing and storage affect the viability of these microorganisms [
11,
12]. A recent review comparing dairy and non-dairy fermented food products as probiotics deliver matrixes did not mention butter as a potential carrier of probiotic bacteria [
13]. However, the use of probiotic bacteria in butter can represent an excellent opportunity to increase the market potential of such product.
Cream is the main raw material to produce butter and reflects its properties in butter quality. Cream fermentation with starter cultures impacts the physicochemical and sensory properties of butter and influences its nutritional composition, texture and shelf life [
14,
15]. Usually, cream undergoes fermentation with mesophilic lactic acid bacteria, namely
Lactococcus lactis ssp.
dyacetilactis and/or
Leuconostoc citrovorum (commonly referred to as an aromatic starter) [
16]. Although the use of probiotic bacteria in cream has received little attention, some authors report their use, and their results will be compared to ours.
Buttermilk is the by-product of butter manufacture. Sweet-cream buttermilk is similar in composition to skim milk, except for its high content of phospholipids and milk fat globule membrane (MFGM) [
17]. Fat globules are stabilized by the MFGM, which is composed of phospholipids, various glycoproteins, enzymes and cholesterol [
18]. Several authors refer to the interesting nutritional and health properties of buttermilk [
19,
20,
21,
22,
23,
24,
25].
Due to its composition and proven health benefits, several papers refer to processes and products in which buttermilk is used as the main ingredient, with the addition of several ingredients such as fruits [
26] or other materials of plant origin (e.g.,
Aloe vera) [
27]. Other works refer to the modification of buttermilk composition by selective concentration of its solid components [
28], associated or not to protein hydrolysis [
29]. Moreover, in other reports, buttermilk was used as a substrate for fermentation using conventional starters [
30] or probiotic microorganisms [
31,
32,
33,
34]. The product can also be carbonated [
35].
Buttermilk is almost unknown for European consumers. Therefore, the introduction in the market of buttermilk resulting from small ruminants’ local breeds, with adequate levels of probiotic bacteria and good sensory properties, can be an interesting option for small scale producers of rural areas. Considering the importance of the subject, in the present work we compared the physicochemical (dry matter, fat, protein, ashes, fatty acids composition, color, texture and rheological properties), microbiological (lactobacilli, lactococci, yeasts and molds) and sensory characteristics of sheep’s butters produced with sweet cream and with cream ripened with aromatic cultures, kefir and probiotics. Additionally, the characteristics of the correspondent buttermilks were also assessed and compared.
3. Results and Discussion
The chemical characteristics of the butter samples are presented in
Table 1. The sweet (S), aromatic (A) and kefir (K) butter samples presented more than 80% (
w/
w) fat, while the butter produced with cream fermented by probiotic bacteria had significantly lower values, both for total solids and fat. In this case, the value obtained for fat is clearly below the legal level (80%
w/
w) indicated for salted butter by the Portuguese standard, NP-1711 [
50]. It was observed that the churning process of this type of butter was also significantly longer (45 min) compared to the other butters (15–30 min). The churning time varies from batch to batch, as it depends on the aggregation speed of destabilized fat globules to form butter grains. This process is empirically controlled by observing the characteristics and size of butter grains. The presence of higher levels of unsaturated fatty acids, which are liquid at the temperature, used during the churning process (10–14 °C) improves the aggregation of granules by sticking particles together. Therefore, as the P butter contained lower levels of unsaturated fatty acids (chiefly C18:1 c9), its churning process took longer and less moisture was expelled during the process, resulting in higher moisture in the final product.
The yields (% w/v) obtained for the different butter samples were as follows: S (43.33%), A (35.73%), K (40.95%) and P (46.95%). Adjusted yields were also calculated considering the legal limit of a maximum of 15% moisture: S (44.48%), A (36.82%), K (41.23%) and P (43.36%). In both cases, the lower yield obtained with cream fermented by aromatic and kefir cultures is clear. An increase in the total solids content of the buttermilks resulting from these two butters would explain the yield losses. However, the buttermilks’ compositions presented do not support this hypothesis. Most likely, the lower yields of the butters A and K can be explained by losses of fat in the walls of the cream churner. The higher levels of unsaturated fatty acids in the creams that originated these butters may have increased the adhesion of fat to the walls of the equipment, reducing the amount of fat recovered after churning.
The percentage fat in dry matter of all samples was similar in all cases: S (95.86 ± 0.78%), A (95.25 ± 1.08%), K (95.66 ± 1.31%), and P (95.23 ± 0.67%), indicating a similar recovery of fat in all cases. Hilali and Rischkowsky [
51], report that sheep’s milk butter contains 83.1–85.3% total solids and 81.4–83.2% fat, with a fat recovery rate of 94.1–97.5%, depending on processing conditions, especially temperature. These results agree with the data obtained in the present work.
The evolution of pH and titratable acidity of the butter samples over the 90 days is presented in
Figure 1. In all cases, the decrease in pH and the increase in titratable acidity over time is clear. The pH and acidity of the sweet butter at the first day of storage reflect the significantly higher pH and lower acidity of sweet cream when compared to those in which starters were used. The butter produced with probiotics presented the lowest pH values. It also showed high acidity values, which are like the ones obtained in the aromatic butter, after the 60th day of storage. The lower pH and higher acidity developed in the P butter indicate the good adaptation of the strains of the lactobacilli mixture used to this matrix, which is also reflected by the high counts of such microorganisms. The lactobacilli and lactococci counts in the A butter also justify its low pH and high acidity. As expected, the S butter had the highest pH values and the lowest acidity, since the initial counts of lactobacilli and lactococci are much lower when compared to those of butters produced with the addition of starter cultures. The lactic microbiota in sweet butter result from adventitious contamination and not from the intentional addition of microorganisms. The observed acidity values are higher than those reported by Ferreira et al. for sweet and fermented butters containing probiotics [
16].
The color parameters of all butters presented significant differences (
p < 0.05) regarding lightness (L*), as well as regarding parameters a* (red-green axis) and b* (blue-yellow axis) (
Table 2). Differences were observed in the same product on different days of storage and among products on the same day of storage. The lower lightness of the butter samples after the 60th day of storage can be pointed out as the greatest variation in the color parameters. Furthermore, at the end of storage, the b* values were higher, indicating an increase in the yellowness of the butter samples.
The color differences presented in
Table 3 indicate more precisely the differences observed between the samples and that resulting from storage time. Considering the ∆Eab* values (
Table 3A,B), it can be concluded that, in almost all cases, the storage time and the type of cream fermentation influenced the color of the butters, since in most comparisons these values are higher than 1, indicating that a common observer could detect differences between them [
52].
When compared to the color of sweet cow’s butter and cow′s butter containing probiotics, reported by Ferreira et al. [
16], the L* values are slightly higher, and b* values are significantly lower, indicating that sheep’s butter is lighter and cow’s butter more yellow. Since the yellow color of butter is dependent on the amount of β-carotene released from fat globules during churning, it can be concluded that sheep’s butter has lower amounts of this component.
Regarding the texture characteristics of the butters, non-significant differences (
p > 0.05) were observed, except in the case of the hardness of the aromatic butter (A), which was significantly lower when compared to all others (
Table 4). Despite its lower solids and fat contents, the highest average value of hardness was observed for the P butter, although without significant differences to the S and K butters. The S, A and K butters present lower amounts of saturated fatty acids (SFA) and higher amounts of monounsaturated fatty acids (MUFA) when compared to the P butter (
Table 5). Unsaturated fatty acids contribute to the reduction in the hardness of fat due to their lower melting points, while the saturated fatty acids increase the hardness of fats. Thus, the variations in the fatty acid profile of butters interfere with its texture and can explain the higher value observed in the P butter. The values of hardness are significantly higher than the ones of sweet cow’s butter and cow’s butter with probiotics reported in a previous work [
16].
Figure 2A shows the changes in elastic (G′) and viscous (G″) moduli as a function of frequency (Hz). It can be observed that butter samples did not follow a true gel behavior that can be observed by a power Law model (G′ = a.gb), where b (the slope in a log–log plot of G′ versus g) is equal to zero [
53]. In the present study, b presented positive values, which are characteristic of weak gels and highly concentrated dispersions (0.26 for S; 0.33 for A; 0.41 for K and 0.45 for P).
Except for sweet and kefir butters, which showed a crossover point (G′ = G″) at 0.067 Hz and 0.0738 Hz, respectively, aromatic and probiotic butters showed an elastic response (G′ > G″) within the frequency range tested (0.01–1 Hz). In the case of S and K butter, a viscous response was observed (G″ > G′) for lower frequencies. The K butter sample presented the higher elastic (G′) and viscous (G″) values, which may be associated with stronger molecular interactions [
53], while the aromatic butter presented the lowest values. Some authors have also reported that, when increasing the presence of mono and divalent salts, G′ and G″ also increase, probably because Na
+ ions can create indirect cross-links with the water molecules [
54], and Ca
2+ can be responsible for a stronger binding of carboxylate-cation-carboxylate interactions that provide greater cross-linking capacity of adjacent polymer helices [
55]. In our study, no significant differences were observed between the ash content of the butters. The proportion of these individual specific ions may be different between samples but was not investigated in the present work. Therefore, the rheological tests confirmed that the butter behaves more like a solid; that is, the deformations are elastic and recoverable [
56].
The loss tangent (tan δ), which is the ratio of G″/G′, is another characteristic for the evaluation of the viscoelastic behavior of the butters. Values of tan δ < 1 indicate predominantly elastic behavior, while tan δ > 1 values indicate predominantly viscous behavior. In this work, as reported for polymer systems [
56], it was observed that for frequencies lower than 0.1 Hz, butters behave like a dilute solution, presenting large values of tan δ (>0.3), while for higher frequencies the values of tan δ were between 0.2–0.3, revealing amorphous polymers’ behavior (data not shown).
Butters showed a shear dependent flow behavior as the complex dynamic viscosity (η*) decreased linearly with increasing frequency on a double logarithmic scale (
Figure 2B). Therefore, we can conclude that butters demonstrated a non-Newtonian shear-thinning or pseudoplastic behavior as reported by Hesarinejad et al. [
56] for
Plantago lanceolata seed mucilage. This was also confirmed by the values of the power law index (n) presented in
Figure 2B which, in all cases, was less than one.
Dias et al. tested the rheological and sensory properties of butter processed with different mixtures of cow/sheep milk cream and reported that sheep’s cream addition to cow’s cream improved the rheological properties of butters, originating lower consistency indexes and higher viscoelastic behavior, mainly due to the increased concentrations of short- and medium-chain fatty acids (SCFA; MCFA) and polyunsaturated fatty acids (PUFA). It was also reported that sheep milk cream butter was characterized by a greasy and striking aroma and a higher bitter taste [
57]. These authors report values of 3981–7943 Pa for the elastic modulus (G′) and 1995–2511 Pa for the viscous modulus (G″) of pure sheep’s butter. The power law model parameters reported were (K = 71.8 ± 1.76 Pa.s
n−1) and (n = 0.0814 ± 0.003), which are not comparable to those obtained by us, as they were obtained at 25 °C, while in the present work the rheological tests were carried out at 5 °C.
The results of the FA profiles, presented in
Table 5, show some differences between butters, specifically with regard to the following FAs: C6-C14; C17; C18:1 c12; C18:2 c9t12; C20:3; C21:0 and C22:2. The fatty acid profile of the P butter presents significantly higher concentrations of short/medium-chain fatty acids (C4-C14). These differences, with impact on the total concentration of SFA, are most probably related to the metabolic activity of the microbial culture used. Underwood et al. report the production of short-chain fatty acids by probiotic bacteria, namely,
L. rhamnosus and
L. acidophilus [
36]. Other authors also reported that the fermentation of saccharides, proteins, and peptides by intestinal bacteria originates SCFAs as end products, which appear to promote colonic homeostasis [
58].
Significant differences between butters were observed regarding ∑MUFA and ∑PUFA concentrations, with the P butter having lower levels of MUFA and slightly higher levels of PUFA. Again, these differences are, most probably, the result of the metabolic activity of the mixture of lactobacilli used in the fermentation of cream.
As presented in
Table 5, the sum of SFA was, in all cases, higher than 62%, with a significantly higher value obtained in the butter containing probiotic microorganisms (66.46%). Hilali and Rischkowsky [
51] indicate that the predominant saturated and unsaturated FAs are palmitic acid (31.7–38.3%) and oleic acid (21.6–33.7%). Özkanli et al. [
59] indicate the following ranges for pasteurized and non-pasteurized sheep’s ghee: C16:0 (29.83–31.30%); C18:0 (10.57–12.96%); C18:1 (31.08–32.67%). These results are in agreement with the ones obtained in the present work.
Figure 3 presents the results of the microbiological analyses of butters and of the correspondent buttermilks. All butter samples produced with fermented cream presented levels of lactic acid bacteria higher than 6 Log CFU/g. The aromatic and the probiotics butters presented the highest levels of lactobacilli, which tended to increase until the 30th day, but decreased until the end of storage. Considering the counts of lactobacilli, it is clear that the P butter maintained adequate numbers of probiotic microorganisms until the end of storage (>7 Log CFU/g). Even in the case of frozen probiotic butter samples, the counts of lactobacilli were higher than 6 Log CFU/g over 90 days of storage (data not shown). Erkaya et al. [
60] observed the survivability of selected probiotics during cold storage of butter produced with
Lactobacillus acidophilus ATCC 4356 and
Bifidobacterium bifidum ATCC 29521. They reported that butter with
B. bifidum maintained its probiotic characteristics until the 30th day of storage. Olszewska et al. [
61] also reported the cell viability of a
Bifidobacterium lactis strain over four weeks in butter stored under refrigeration. Ewe and Loo [
14] evaluated the properties of butter produced by cream fermented with
Lactobacillus helveticus and reported significantly higher fat content and acidity. It was also noted that butter was softer than the conventional product due to increased levels of unsaturated fatty acids. These observations are contradictory with the ones obtained in the present work.
Karaca et al. [
62] report that kefir-cultured butter presented higher amounts of
Lactococcus spp. by almost two log cycles and contained 5.24 Log CFU/g of
L. acidophilus, while the control sample did not contain
L. acidophilus.
More recently, Bellinazo et al. [
63] reported that the butter/bixin/isolated probiotic and butter/bixin/commercial probiotic formulations can be considered probiotic up to 74 and 69 days of storage, respectively, considering the concentration of ≥6 Log CFU/g.
Concerning yeast and mold counts, the aromatic and probiotic butters presented values below 4 Log CFU/g, while the sweet and the kefir butters presented values higher than 4 Log CFU/g, with a tendency to decrease over storage, in the case of the sweet butter. In kefir butter, whose starter contained the yeast Debaryomyces hansenii, the counts of yeasts and molds tended to increase until the 30th day of storage, decreasing afterwards. The same occurred with lactococci counts that reached values of >6.5 Log CFU/g at the 30th day of storage, but also decreased to ca. 5.0–5.5 Log CFU/g at the 60th and 90th days of storage.
Concerning the microbial characteristics of buttermilks, higher values of lactobacilli and lactococci were observed for the samples containing the aromatic starter and probiotics (
Figure 3D,E). In the latter, lactobacilli and lactococci counts were of the order of Log 10 CFU/mL at the beginning of storage, and levels higher than 8 Log CFU/mL were maintained during the 28 days of refrigerated storage. This observation indicates the excellent adaptation of those microorganisms to this matrix, which is an important factor regarding its use as a functional food.
The physicochemical characteristics of the buttermilks resulting from the different butters produced are presented in
Table 6. Significant differences were observed regarding almost all components. Protein levels were of the order of 4–5% (
w/
v), and fat levels were between 2 and 3% (
w/
v). Surprisingly, the buttermilk resulting from the production of probiotic butter presented the lowest level of solids, while the total solids content was the lowest in the correspondent butter. To the lower amount of solids in butter it was expectable a higher amount of solids in buttermilk, but this was not the case.
As it was expected, the pH of sweet buttermilk (>6) was higher than all others, which were around 4.5. The titratable acidity of the aromatic buttermilk and of the buttermilk containing probiotics was significantly higher than the ones of S and K samples. As it occurred in the case of butters, these observations confirm the good adaptation of the aromatic starter and of the mixture of probiotic bacteria both to butter and to buttermilk.
The evaluation of the viscosity of buttermilks (
Table 6) showed significant differences between all products, with the highest value observed corresponding to the probiotics buttermilk. Again, a possible explanation for this fact can be the higher amount of saturated fatty acids in the P butter, and hence in the correspondent buttermilk.
The viscosity of sheep’s buttermilks produced with sweet cream was significantly lower than all others. Kefir and probiotics buttermilks presented the highest values. It can be concluded that, in both cases, the type of starter cultures played a significant role regarding this parameter.
The color parameters of buttermilks also presented significant differences (
Table 6). The product resulting from the aromatic butter presented clear differences, being darker (lower L* values) and with lower yellowness (lower b* values).
Sakkas et al. compared the composition of sheep’s and cow’s sweet buttermilks and reported that the former was the most advantageous in terms of non-fat solids, protein and phosphorus contents. No significant differences were observed in the phospholipids content of ovine and bovine buttermilks. The antioxidant potential and emulsion stability of sweet ovine buttermilk were the highest [
64]. These findings indicate the nutritional interest of sweet sheep’s buttermilk, which can be further enhanced by its fermentation with probiotic bacteria.
A recent paper by Sharma et al. evaluated the production of functional buttermilk and soymilk fermented by a
Pediococcus acidilactici strain expressing the L-alanine dehydrogenase enzyme. The levels of total antioxidants, phenolics, flavonoids and especially L-alanine were significantly enhanced after LAB fermentation [
65].
Ogrodowczyk et al. tested buttermilk-based formulations fermented with several lactic acid bacteria and
Bifidobacterium strains. These authors report high growth rates for the strains tested. It was also indicated that the sensory quality of products was influenced by the profile of SCFA and free peptides. Two formulations, fermented with
L.
bulgaricus-151 and
Lactobacillus casei-LcY, were the most advantageous, with desirable sensory, immunoreactive and biochemical properties [
66].
Considering the continuous interest of researchers in the development of health-promoting products based on buttermilk, it can be inferred that this product can have potential in the European market, contributing to circular economy.
Regarding the sensory characteristics of butters (
Table S1) non-significant differences were observed between formulations, either by the preference test or by the ranking test. In general, all products were well accepted by the panelists. It is worth to note that the highest scores for aroma, taste and texture were obtained by the P and S butters at the beginning of storage. At the end of storage, all butter samples maintained good sensory characteristics.
Pandya and Ghodke [
1] found that traditional Turkish “yayik” butter made from goat’s milk had the most acceptable organoleptic characteristics when compared to “yayik” butters produced with sheep or cow milks. On the contrary, Hilali and Rischkowsky [
55] report that sheep’s milk is preferred over goat’s milk for the manufacture of ghee because of the differences in flavor. Kahyaoğlu and Çakmakç [
67] evaluated butter samples produced from cow’s, sheep’s and goat’s cream during a 90-day storage period at 4 °C, reporting that sensory analysis scores decreased in all the butter samples during the storage. The highest scores were obtained by the butter produced from cow’s milk.
As in the case of butters, non-significant differences were observed between buttermilks regarding sensory characteristics (
Table S2). In general, all products were well accepted by the panelists. It can be pointed out that both K and P buttermilks received higher scores for aroma, taste and consistency, indicating their good marketing potential.