Fermented Buffalo Milk with Conjugated Linoleic Acid-Producing Bacteria: Strain Selection and Functional Applications

Abstract

Buffalo milk is a rich source of precursor fatty acids for bioactive compounds and provides an optimal environment for bacterial growth. This study aimed to isolate and select lactic acid bacteria strains with potential to conjugated linoleic acid (CLA) production for technological application in fermented buffalo milk. Fifty-eight strains were isolated from raw milk, kefir, artisanal cheese, kombucha, and jaboticaba juice and tested for CLA biosynthesis. In milk fermentation, selected strains with linoleic acid (LA) conversion rates ranging from 65.66% to 21.86% were L. paraplantarum, L. plantarum, P. pentosaceus, and L. fermentum. The highest viability average values between 11.85 and 11.15 Log CFU/mL were observed after 8 h of fermentation for the L. plantarum, control L. plantarum, and L. fermentum treatments, while it took 10 h of fermentation for L. paraplantarum and P. pentosaceus to reach a stationary phase, with pH stabilizing at 4.60 ± 0.1 after 30 h. Despite L. paraplantarum showing the highest in vitro CLA production (0.99 mg/mL), in buffalo milk, all strains similarly produced c9t11 CLA, with no detectable t11c12 CLA. P. pentosaceus and L. fermentum showed a fatty acid profile with higher PUFA content, especially in CLA and MUFA, related to a lower degree of atherogenicity (IA) and thrombogenicity index (ThI). These findings boost understanding of dairy (raw milk, artisanal cheese, and milk kefir) and non-dairy substrates (kombucha and jaboticaba juice) as reservoirs for functional bacteria and highlight buffalo milk as a matrix for diversification of naturally enriched fermented dairy products.

1. Introduction

Fermented foods, such as yogurt, cheese, and kefir, are known for being rich in bioactive compounds with potential health benefits associated with high acceptance by the consumer market. Kefir grains consist of a symbiotic association between lactic acid bacteria, acetic acid bacteria, and yeast that coexist in a matrix of exopolysaccharides and are used to produce a fermented beverage named kefir [1,2]. Similarly, kombucha is a fermented beverage made by a symbiotic association of bacteria and yeast, with the usual substrates being green tea and/or black tea [3]. In addition to milk, other drinks can be used as a base for fermentation, such as juice from local fruits, which is typically used to attract the population’s attention [4]. Within this diversity, we can find potential probiotic microorganisms that enhance functional properties by changing nutritional characteristics, producing bioactive compounds, or maintaining the viability of probiotic cells in the product. In this context, investigating the bacterial profile of these beverages and substrates is a positive strategy for innovation in food science.

Among the bioactive compounds of interest in the dairy sector, polyunsaturated fatty acids, mainly conjugated linoleic acid (CLA), have been studied due to their beneficial effect on health [5,6,7]. Produced by microorganisms during the fermentation process and storage, CLA can be associated with several beneficial health effects, including immunomodulatory activities and anti-inflammatory, antimicrobial, and anticancer properties [8,9]. CLA has been recognized for its potential to reduce hypercholesterolemia and help control weight gain [10]. Notably, ruminant-derived dairy and meat products are among the richest natural sources of CLA [11]. CLA is primarily formed through the biohydrogenation of linoleic acid (LA) in the rumen and the desaturation of trans vaccenic acid (C18:1 t11) in the mammary gland, which converts dietary lipids into this bioactive compound [2]. Although CLA is naturally present in dairy products, its typical concentration ranges from 0.41 to 1.00 g per 100 g (4.1 to 10.0 g/kg), which is generally insufficient to meet the recommended daily intake of 1.0 to 3.0 g (approximately 0.04 g/kg body weight) required to achieve potential health benefits. [7,12].

While the recommended daily dose of CLA can be achieved through supplementation, the high cost of industrial production makes these products inaccessible to the general population. Recent studies explore the potential of microbial biosynthesis of rumenic acid (C18:2 c9, t11-CLA) as a cost-effective and accessible alternative to enrich dairy products with CLA [7,11,13]. The ability of microorganisms to synthesize CLA through multi-enzymatic pathways opens up the possibility of developing functional foods that can improve the lipid profile in dairy products [6].

Buffalo milk, the second most produced dairy matrix globally, is recognized for its high fat content and rich composition of bioactive compounds [14,15]. It is also a protective matrix for probiotic bacteria such as Bifidobacterium and Lactobacillus, increasing their resistance to gastrointestinal stress, providing a reassuring environment for the growth of beneficial bacteria [16,17]. Several studies have indicated that many bacterial strains can efficiently convert LA to CLA, with species such as L. casei, L. acidophilus, L. plantarum, and Bifidobacterium breve being among the most studied [5,18,19,20]. Higher conversion rates have been observed in buffalo milk compared to standard laboratory media like MRS broth [21], with L. casei, B. bifidum, and S. thermophilus increasing CLA levels in buffalo dairy products [22].

Developing fermented buffalo milk with health-promoting bacteria represents a promising strategy to enhance the nutritional quality of dairy products while supporting the diversification of the buffalo milk production chain. Therefore, this research aimed to isolate and select lactic acid bacteria (LAB) strains with high potential for CLA production from diverse food matrices, including milk, kefir, kefir grains, kombucha, artisanal cheeses, and jaboticaba juice. In addition, this study provides data to evaluate the fermentation process of buffalo milk by different LAB to develop fermented buffalo milk enriched with CLA and evaluate the lipid profile of the elaborated products.

2. Materials and Methods

2.1. Strains Isolation and Identification

Twenty-eight LAB strains from raw milk obtained from local farms in Bahia State, Brazil, and kefir milk produced with kefir grains obtained in the local market were isolated at the Milk and Dairy Products Inspection and Technology Laboratory (LaITLácteos), Federal University of Bahia (UFBA), through serial dilution and incubation on agar MRS medium. Additionally, 21 LAB strains were obtained from artisanal cheeses [23,24,25] provided by the Federal University of Minas Gerais (UFMG), and 9 LAB strains were isolated from kombucha and kefir grains in jaboticaba juice provided by the Faculty of Pharmacy at UFBA. Each strain was inoculated in MRS broth at 37 °C for 24 h for replication. To ensure preservation, triplicate aliquots of each strain were stored in MRS broth with glycerol at −20 °C.

Taxonomic identification of the unknown LAB samples was carried out using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis with a MALDI Biotyper (Bruker Daltonik, Billerica, MA, USA) following the protocol described by Oliveira et al. [23].

2.2. Screening for CLA Synthesis Potential

All LAB strains were evaluated for their potential to produce CLA from LA. The strains were activated in MRS broth at 37 °C for 24 h, and an aliquot of the activated culture was transferred to a micellar solution of LA from MRS broth (1.7% v/v) and incubated at 37 °C for 24 h, as adapted from [7]. Following incubation, 1 mL of the MRS broth was centrifuged (Sorvall ST16R, Thermo Scientific, Waltham, MA, USA) at 20,800× g at 4 °C for 1 min, and the supernatant was transferred to a fresh tube. Fatty acids were extracted with n-hexane, and CLA in the n-hexane extract was detected using a spectrophotometer at a characteristic absorption peak at 233 nm, and the MSR broth containing LA without bacterial strains was used as a blank, processed as described by Vieira et al. [7].

The percentage of LA conversion to CLA was calculated at the end of the incubation period according to Van et al. [21], using the following equation and considering a 1.7% v/v LA enrichment of MRS broth:

$${\displaystyle \frac{CLA}{\left(CLA+LA\right)}}\ast 100$$

2.3. Development of Fermented Buffalo Milk

Five LAB strains were selected for the development of fermented buffalo milk based on (i) in vitro CLA production capacity, (ii) genus and species identification, and (iii) food origin diversity. The selected LAB strains were Lactobacillus paraplantarum, Lactiplantibacillus plantarum, Pediococcus pentosaceus, Limosilactobacillus fermentum, and a control Lactiplantibacillus plantarum.

Raw buffalo milk from Murrah and Mediterranean breeds was obtained from the UFBA Experimental Farm located in the Municipality of Entre Rios, Bahia, Brazil, at a latitude of 11°56′31″ south and a longitude of 38°05′04″ west. It was analyzed using an ultrasonic milk analyzer to determine fat, non-fat solids, protein, lactose, and mineral salts. To produce the fermented milk, all fermentation treatments in this study were carried out using the same batch of raw buffalo milk, under identical handling and fermentation conditions, where raw buffalo milk was previously pasteurized (65 °C for 30 min) and cooled to 37 °C. Then, the selected strains were inoculated into the milk (1%, v/v), and fermentation process was monitored at intervals of 2 h from 0 to 12 h, and from 12 to 30 h, the interval was 6 h (0, 2, 4, 6, 8, 10, 12, 18, 24 and 30 h) until the pH reached 4.6 ± 0.1. Samples were taken at each point for LAB counts by plating on MRS agar and incubating at 37 °C for 48 h. The pH was measured using a previously calibrated bench pH meter. All analyses were performed in experimental duplicates (n = 2).

2.4. Lipid Extraction and Fatty Acid Analysis

Fatty acid methyl esters were identified using a commercial FAME mix to identify the FAME peaks, and all other reagents were of analytical grade (Sigma-Aldrich Chemical Co., Allentown, PA, USA).

The composition of 0.5 g of lyophilized fermented milk fatty acids was determined by the transmethylation of fat with HCl in methanol (10%), followed by gas chromatography. The FA methyl esters were separated with a column (DB-FFAP; 30 m × 0.25 mm × 0.25 μm) in a gas chromatograph equipped with a flame-ionization detector (CG-FID Clarus 680; Perkin–Elmer). The injection volume was 1 μL, and GC-FID was used to quantify CLA isomers by comparing the CLA in aliquots and CLA standards. Fatty acid methyl esters were identified by comparing the retention times obtained from the standard chromatogram (C4–C24, 189–19–AMP, Sigma-Aldrich) according to the method proposed by Souza et al. [26]. The column was held at 50 °C for 1 min after injection, and then the temperature was increased 25 °C/min to 194 °C, held for 1 min, then increased 4 °C/min to 245 °C, and held there for 2 min. C19:0 (Sigma-Aldrich Chemical Co., PA, USA) was used as an internal standard for quantification.

From the data on FA composition, the following indices of lipid quality were determined according to Vieira et al. [7] and Godinho et al. [27]

Desirable Fatty Acids (DFA):

$$\mathrm{D}\mathrm{F}\mathrm{A}=\mathrm{\Sigma }\mathrm{M}\mathrm{U}\mathrm{F}\mathrm{A}+\mathrm{\Sigma }\mathrm{P}\mathrm{U}\mathrm{F}\mathrm{A}+\mathrm{E}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{a}\mathrm{r}\mathrm{i}\mathrm{c}\left(\mathrm{C}18\text{:}0\right)$$

(1)

Index of Atherogenicity (IA):

$$IA={\displaystyle \frac{\mathrm{L}\mathrm{a}\mathrm{u}\mathrm{r}\mathrm{i}\mathrm{c}\mathrm{C}12\text{:}0+\left[4\mathrm{x}\mathrm{M}\mathrm{y}\mathrm{r}\mathrm{i}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{C}14\text{:}0\right]+\mathrm{P}\mathrm{a}\mathrm{l}\mathrm{m}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{C}16\text{:}0}{\mathrm{\Sigma }\mathrm{M}\mathrm{U}\mathrm{F}\mathrm{A}+\mathrm{\Sigma }\mathrm{P}\mathrm{U}\mathrm{F}\mathrm{A}}}$$

(2)

Index of Thrombogenicity (IT):

$$IT={\displaystyle \frac{\left[\mathrm{M}\mathrm{i}\mathrm{r}\mathrm{i}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{c}\right(\mathrm{C}14\text{:}0)+\mathrm{P}\mathrm{a}\mathrm{l}\mathrm{m}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{c}(\mathrm{C}16\text{:}0)+\mathrm{E}\mathrm{s}\mathrm{t}\mathrm{e}\mathrm{a}\mathrm{r}\mathrm{i}\mathrm{c}(\mathrm{C}18\text{:}0\left)\right]}{\left(0.5\mathrm{x}\mathrm{M}\mathrm{U}\mathrm{F}\mathrm{A}\right)+\left(0.5\mathrm{x}\mathrm{ꞷ}6\right)+\left(3\mathrm{x}\mathrm{ꞷ}3\right)+(\mathrm{ꞷ}3/\mathrm{ꞷ}6)}}$$

(3)

where ꞷ6 represents omega 6 FAs and ꞷ3 represents omega 3 FAs [24].

Hypocholesterolemic/hypercholesterolemic fatty acid ratio (hH):

$$hH{\displaystyle \frac{(\mathrm{C}18\text{:}1\mathrm{c}\mathrm{i}\mathrm{s}9+\mathrm{C}18\text{:}2\mathrm{n}6+\mathrm{C}20\text{:}4\mathrm{n}6+\mathrm{C}18\text{:}3\mathrm{n}3+\mathrm{C}20\text{:}5\mathrm{n}3+\mathrm{C}22\text{:}5\mathrm{n}3+\mathrm{C}22\text{:}6\mathrm{n}3)}{(\mathrm{C}14\text{:}0+\mathrm{C}16\text{:}0)}}$$

(4)

2.5. Statistical Analyses

The experiment was performed in duplicate, and the results were expressed as mean ± standard deviation (SD). An analysis of variance (ANOVA) was used to compare in vitro CLA production, bacterial viability, pH, and fatty acid profiles. Tukey’s test was used when significant differences between treatments were observed and declared at p-values < 0.05.

3. Results and Discussion

A total of 58 lactic acid bacteria strains were tested for their ability to produce CLA in vitro. Kefir and artisanal cheeses were the products that presented a remarkable diversity of microorganisms, highlighting the importance of these fermented dairy products as an ideal food matrix for the growth of LAB. However, jaboticaba juice proved to be a promising alternative source for obtaining species with high potential for CLA production. Lactobacillus paraplantarum isolated from jaboticaba juice demonstrated the highest CLA concentration (0.99 ± 0.35 mg/mL) and could convert LA with a conversion efficiency of 65.66%, as presented in

Table 1. Strains identified (MALDI-TOF MS) and classified in decreasing order (mean + SD) of CLA production (mg/mL) in vitro.

Species	CLA (mg/mL)	LA Conversion (%)	Food Origin
L. paraplantarum *	0.99 ± 0.35 a	65.66	Jaboticaba juice
L. plantarum *	0.76 ± 0.19 b	59.56	Kefir
L. plantarum	0.60 ± 0.05 b	53.83	Kefir grains in Jaboticaba juice
L. paraplantarum	0.56 ± 0.07 bcd	51.87	Kombucha in Jaboticaba juice
L. plantarum	0.54 ± 0.03 cd	51.24	Raw milk
L. plantarum	0.54 ± 0.06 cde	51.05	Kombucha in Jaboticaba juice
L. plantarum	0.53 ± 0.10 cde	50.79	Kombucha in Jaboticaba juice
L. plantarum	0.52 ± 0.03 cdef	49.93	Kefir grains in Jaboticaba juice
P. pentosaceus *	0.52 ± 0.11 cdef	49.93	Kefir
L. plantarum	0.50 ± 0.05 cdefg	49.15	Kefir grains
NI	0.48 ± 0.07 cdefgh	48.23	Raw milk
L. plantarum	0.48 ± 0.11 cdefgh	47.84	Kefir
L. plantarum	0.47 ± 0.02 cdefgh	47.65	Kefir grains
Lim. fermentum *	0.45 ± 0.02 cdefghi	46.34	Raw milk
P. pentosaceus	0.44 ± 0.03 cdefghi	45.91	Kefir
Leu. mesenteroides	0.38 ± 0.01 defghij	42.09	Cheese counter
L. plantarum	0.37 ± 0.12 defghijk	41.55	Kefir
Leu. pseudomesenteroides	0.36 ± 0.08 defghijk	41.32	Artesanal cheese
Lac. rhamnosus	0.36 ± 0.12 defghijkl	41.14	Artesanal cheese
NI	0.36 ± 0.06 defghijkl	41.04	Kefir
L. plantarum	0.35 ± 0.02 defghijkl	40.38	Kefir
L. plantarum	0.35 ± 0.02 defghijkl	40.29	Kefir
P. pentosaceus	0.35 ± 0.03 defghijkl	40.15	Kefir
P. pentosaceus	0.35 ± 0.06 defghijkl	40.05	Kefir
L. plantarum	0.34 ± 0.07 defghijkl	39.86	Kefir
L. plantarum	0.34 ± 0.02 defghijkl	39.47	Kefir
Lac. paracasei	0.33 ± 0.12 defghijkl	38.97	Artesanal cheese
NI	0.32 ± 0.02 efghijkl	38.37	Kefir
L. plantarum	0.31 ± 0.01 efghijkl	37.70	Kefir grains
P. pentosaceus	0.31 ± 0.03 fghijkl	37.39	Kefir
L. plantarum	0.30 ± 0.01 fghijkl	36.48	Kefir
P. pentosaceus	0.30 ± 0.03 fghijkl	36.32	Kefir
NI	0.29 ± 0.01 ghijkl	36.16	Kefir
Lim. fermentum	0.29 ± 0.09 ghijkl	35.88	Kefir
P. pentosaceus	0.29 ± 0.06 ghijkl	35.77	Kefir
P. pentosaceus	0.29 ± 0.09 ghijkl	35.66	Ripened artesanal cheese
Lactococcus lactis	0.28 ± 0.00 hijkl	35.10	Ripened artesanal cheese
P. pentosaceus	0.28 ± 0.05 hijkl	34.76	Kefir
L. plantarum	0.27 ± 0.04 hijkl	34.53	Raw milk
Lac. paracasei	0.26 ± 0.00 hijkl	33.84	Ripened artesanal cheese
Lim. fermentum	0.26 ± 0.01 hijkl	33.60	Kefir
P. pentosaceus	0.26 ± 0.04 hijkl	33.30	Kefir
L. plantarum	0.25 ± 0.05 ijkl	32.15	Kefir
Lac. casei	0.24 ± 0.01 ijkl	32.09	Ripened artesanal cheese
Lac. paracasei	0.24 ± 0.03 ijkl	31.47	Ripened artesanal cheese
Lactococcus garvieae	0.22 ± 0.00 ijkl	30.19	Artesanal cheese
L. plantarum	0.21 ± 0.03 jkl	29.33	Queijo Minas Artesanal
Levilactobacillus brevis	0.21 ± 0.02 jkl	29.13	Queijo Artesanal da Serra Geral
Lactococcus lactis	0.21 ± 0.04 jkl	29.06	Pingo
L. plantarum	0.21 ± 0.00 jkl	28.93	Queijo Artesanal da Serra Geral
L. mesenteroides	0.20 ± 0.08 jkl	28.31	Artesanal cheese
L. pseudomesenteroides	0.20 ± 0.00 jkl	28.31	Artesanal cheese
L. plantarum	0.19 ± 0.01 jkl	27.19	Queijo Minas Artesanal (Maturado)
Lac. paracasei	0.17 ± 0.04 jkl	24.70	Queijo Minas Artesanal
Lat. curvatus	0.17 ± 0.04 jkl	24.62	Artesanal cheese
L. plantarum	0.16 ± 0.02 jkl	23.70	Artesanal cheese
L. plantarum	0.16 ± 0.01 kl	23.23	Artesanal cheese
L. plantarum *	0.14 ± 0.03 l	21.86	Queijo Minas Artesanal Canastra

L. = Lactiplantibacillus; Lac. = Lacticaseibacillus; Lat. = Lactilactobacillus; Lim. = Limosilactobacillus; Leu. = Leuconostoc; P. = Pediococcus; NI = Not identified. * Strains selected to fermented beverage (T1, T2, T3, T4 and T5, respectively) production. Different letters in the column indicate significant differences between treatments (p < 0.05).

. These high CLA concentrations highlight the potential of non-dairy food matrices, such as fruit juices, for sourcing bacteria capable of producing bioactive compounds like CLA.

Among the 58 strains tested, Lactiplantibacillus plantarum was the most frequently found species, exhibiting CLA production values ranging from 0.14 ± 0.03 mg/mL to 0.76 ± 0.19 mg/mL, followed by Pediococcus pentosaceus, with CLA production ranging between 0.28 ± 0.05 mg/mL and 0.52 ± 0.11 mg/mL, and a maximum conversion capacity of 49.93%. The previous study showed that L. plantarum is widely tested for CLA production, but not all strains present potential conversion [28]. According to Coakley et al. [29], P. pentosaceus strains could survive and grow in the presence of at least 0.5 mg/mL LA in the medium, but did not produce any detectable amounts of CLA.

In other studies, Lactococcus lactis subsp. cremoris MRS47 showed the best results in MRS broth supplemented with sunflower seed oil (1.7% v/v) compared to L. casei/paracasei and Leuconostoc mesenteroides [3]. Lactobacillus acidophilus and Lactobacillus casei demonstrated CLA production ranging between 80.14 and 131.63 µg/mL when cultured in MRS broth supplemented with 0.2% LA after 24 h of incubation [30]. The LA to CLA conversion rate varied among strains, ranging from 17.0% to 35.9% in MRS broth, with higher conversion percentages observed at low LA concentrations [21]. This phenomenon may be related to the bacterial growth inhibitory effect of fatty acids, particularly LA, which increases bacterial membrane permeability due to its surfactant action, destroying the cell membrane and affecting normal metabolism in both pathogenic and Lactobacillus genera [6,31].

Although artisanal cheeses present a wide variety of LAB species, products such as milk kefir and kombucha in jaboticaba juice exhibit a profile of bacteria with greater potential for CLA production when compared to the bacteria isolated from artisanal cheeses and raw milk. These observations highlight the complexity and variability in CLA production by lactic acid bacteria, influenced by both the bacterial species and the origin of the fermented product. Since strains that efficiently converted LA to CLA in MRS broth showed even better results in buffalo milk than in the culture medium [21], this study proposed testing the possibility of naturally enhancing CLA content in buffalo dairy by selecting five strains to develop fermented buffalo milk. Strains of different species were chosen among the highest CLA producers, L. paraplantarum, L. plantarum, P. pentosaceus, and L. fermentum, with CLA conversion percentages ranging from 65.66 to 46.34%. Additionally, a strain of L. plantarum with the lowest conversion capacity (21.86%) was selected as a negative control.

The buffalo milk used in this experiment had the following composition: total solids: 13.54%; fat: 3.45%; protein: 3.69%; lactose: 5.55%; mineral salts: 0.83%. Other studies have reported higher values for total solids and fat in buffalo milk, at 15.27% to 16.81% and 5.14% to 6.58%, respectively [32,33,34], and this characteristic is appreciated for cheese manufacturing. Genetic, nutritional, and physiological factors, such as stage of lactation, animal feed system, season, parity, and age, can affect these parameters [35,36]. However, considering studies about different levels (1.5 to 6.0 g/100 g) in the fat content of buffalo milk yogurt, the physicochemical and sensory properties indicate that a more acceptable product for consumers could be produced from milk with 3 g/100 g of fat content [37]. In addition to protein and fat content, buffalo milk can also be considered a good source of minerals such as Ca, P, Mg, K, Zn, and Fe, and vitamins, including A and E [38,39], and are more bioavailable in fermented products [40].

The growth of selected strains increased significantly during fermentation, mainly in the initial hours, reaching a peak between 8 and 10 h (Figure 1).

The treatments with L. paraplantarum, L. plantarum, and L. fermentum in the logarithmic phase showed exponential growth (p < 0.05) between 0 and 8 h of fermentation. The fermented milk with P. pentosaceus also had significant growth (p < 0.05) between 0 and 4 h and from 6 to 8 h, while the L. plantarum control did not show a significant increase (p > 0.05) in the first 2 h, pointing to a marked latency phase. The highest viability was observed after 8 h of fermentation for the L. plantarum treatments (between 11.75 and 11.48 Log CFU/mL), control L. plantarum (11.15 Log CFU/mL), and L. fermentum (11.46 Log CFU/mL), while it took 10 h of fermentation for L. paraplantarum (11.85 Log CFU/mL) and P. pentosaceus (11.80 Log CFU/mL) to reach the maximum production of viable microorganisms. All treatments start with viability between 3.46 and 4.77 Log CFU/mL and reach peaks above 11.11 Log CFU/mL in 8 h, pointing to buffalo milk as a suitable food matrix for different strains. According to other studies, both the viability of LAB in fermented products and the viability post-digestive process are positively observed in buffalo dairy products due to the protective factor of fat compounds [16,17], making buffalo milk a suitable choice for fermented food matrices.

Rapid bacterial growth is an important indicator associated with the capacity to maintain high CFU counts, which may enhance CLA production during fermentation. Considering the fermentation process for up to 30 h, the treatments L. plantarum control, L. paraplantarum, and L. fermentum maintained viability without significant changes (p > 0.05) in stationary phase, with counts of 10.94 Log CFU/mL, 11.23 Log CFU/mL, and 11.49 Log CFU/mL, respectively. Meanwhile, the treatments fermented with P. pentosaceus and L. plantarum showed a reduced viability (p < 0.05), reaching 11.0 Log CFU/mL and 10.61 Log CFU/mL after 30 h of fermentation, respectively. All tested strains maintained viability above 10.61 Log CFU/mL at the end of fermentation. Different Lactobacillus strains tested for Nasrollahzadeh et al. [11] also demonstrated CLA production capabilities, with the highest production occurring after 24 h of fermentation. Most bacterial strains of Lactobacilli, Lactococci, and Bifidobacteria seem to grow and produce CLA at 37 °C to 38 °C with more than 24 h of fermentation [28,41], and it was previously observed that the Pediococcus species needs more time to adapt to a new growth environment [42].

All treatments had an initial pH close to 7.00 and showed insignificant acidification (p > 0.05) during the first 6 h of fermentation (Figure 2). The significant reduction (p < 0.05) in pH was observed after 8 h of fermentation, corresponding to the period with the highest viable cell count and intensified metabolic activity of lactic acid bacteria, indicating an increase in organic acid production [43].

After 8 h, the pH of treatments with L. paraplantarum (6.28 ± 0.02), L. plantarum (6.32 ± 0.08), P. pentosaceus (6.25 ± 0.01), L. fermentum (6.25 ± 0.01), and control L. plantarum (6.31 ± 0.04) began to show clear signs of acidification, reflected by a constant and significant reduction (p < 0.05) until the end of the fermentation process. The occurrence of pH reduction indicated a similar ability to acidify buffalo milk. After 30 h, at the end of fermentation, values were observed between 4.60 ± 0.02 and 4.65 ± 0.01 for all treatments, and were refrigerated, marking the end of the fermentation period. The acidification process can be divided into three distinct phases [44]. A slight decrease in pH is observed in the initial process, followed by a second phase with a pronounced drop in the curve, where the pH rapidly decreases. The third phase is characterized by stabilizing the pH around 4.6, with less variation. Previous studies have shown that the decrease in pH is slower in buffalo milk compared to cow or goat milk due to its greater buffering capacity, and this can be attributed to the higher total solids content, the composition of acid-base compounds, a higher casein content, and a higher concentration of inorganic phosphate [17,44]. In this study, buffalo milk, buffering capability, and unconventional bacterial strains affect pH reduction.

In cow milk, it was observed that L. plantarum and L. curvatus decreased pH levels to approximately 5.8 to 5.12 only with 24 h of fermentation [45]. Mixed cultures of the probiotic microorganism can significantly increase the acidification rate of the product as compared with a single culture, and the acidification rate of microorganisms like L. plantarum and L. rhamnosus is considerably lower than starter cultures (S. thermophilus–L. bulgaricus) commonly used in fermented products [46]. Considering that the reduction of pH is an inhibitory factor for the growth of microorganisms, the extension of the fermentation period due to the low acidification rate of the tested microorganisms may be a relevant factor that allows for greater viability of these microorganisms at the end of fermentation. The optimum conditions for growth and CLA production were described as pH levels ranging from 7.0 to 5.0, a fermentation temperature of 37 °C, and the highest CLA formation occurring between 12 and 28 h of fermentation [11,20,30,41]. Strain grown in buffalo milk showed their highest CLA production near the stationary phase of bacterial growth, as Van Nieuwenhove et al. reported [21]. So, the ability of all strains to grow and reduce pH, despite extended fermentation time, indicates their suitability for food fermentation and prolonged optimal conditions for CLA production.

Gas chromatographic analysis of free fatty acids (FFAs) provided detailed insights into the fatty acid profiles of buffalo milk fermented by different LAB strains in

Table 2. Fatty acid profile (g/100 g of fat) of buffalo milk fermented beverages (median ± SD).

Fatty Acids Profile	L. paraplantarum	L. plantarum	P. pentosaceus	L. fermentum	Ctrl L. plantarum *	p-Value
Individual fatty acids
C4:0	1.56 ± 0.29 b	1.76 ± 0.18 a	1.93 ± 0.09 a	1.04 ± 0.16 c	1.92 ± 0.11 a	<0.0001
C6:0	0.97 ± 0.09 b	1.05 ± 0.10 a	1.13 ± 0.06 a	0.80 ± 0.09 b	1.13 ± 0.07 a	0.0006
C8:0	0.46 ± 0.04 ab	0.49 ± 0.04 ab	0.53 ± 0.03 a	0.40 ± 0.04 b	0.52 ± 0.03 a	0.0024
C10:0	0.88 ± 0.08 ab	0.94 ± 0.08 ab	1.03 ± 0.06 a	0.78 ±0.07 b	0.97 ± 0.07 a	0.0038
C11:0	0.01 ± 0.01 a	0.01 ± 0.01 a	0.02 ± 0.00 a	0.01 ± 0.01 a	0.01 ± 0.01 a	0.1611
C12:0	1.30 ± 0.12 ab	1.42 ± 0.12 ab	1.53 ± 0.08 a	1.15 ± 0.10 b	1.44 ± 0.09 a	0.0036
C13:0	0.07 ± 0.01 ab	0.08 ± 0.01 a	0.09 ± 0.01 a	0.06 ± 0.01 b	0.08 ± 0.01 a	0.0002
C14:0	7.18 ± 0.37 ab	8.17 ± 1.03 ab	8.41 ± 0.28 a	6.62 ± 0.57 b	8.05 ± 0.06 a	0.0041
C14:1 n-5	0.22 ± 0.07 a	0.33 ± 0.12 a	0.27 ± 0.10 a	0.32 ± 0.02 a	0.32 ± 0.09 a	0.1510
C15:0	1.39 ± 0.13 ab	1.46 ± 0.12 ab	1.58 ± 0.09 a	1.14 ± 0.11 b	1.44 ± 0.08 a	0.0047
C15:1 n-5	0.20 ± 0.08 a	0.32 ± 0.03 a	0.25 ± 0.03 a	0.25 ± 0.02 a	0.28 ± 0.03 a	0.1010
C16:0	24.28 ± 1.69 ab	27.23 ± 4.21 ab	27.15 ± 1.51 ab	22.76 ± 2.11 b	27.67 ± 1.33 a	0.0284
C16:1 n-7	0.53 ± 0.35 b	1.21 ± 0.34 ab	1.05 ± 0.26 ab	1.10 ± 0.09 ab	1.17 ± 0.24 a	0.0480
C17:0	1.36 ± 0.33 a	1.41 ± 0.09 a	1.60 ± 0.14 a	1.10 ± 0.11 a	1.38 ± 0.14 a	0.0797
C17:1 n-7	0.28 ± 0.02 a	0.32 ± 0.05 a	0.23 ± 0.16 a	0.27 ± 0.03 a	0.32 ± 0.03 a	0.5829
C18:0	15.04 ± 2.16 a	13.48 ± 0.82 a	15.11 ± 1.52 a	10.27 ± 1.07 b	13.24 ± 1.39 a	0.0116
C18:1 n-9 c	20.73 ± 1.32 ab	24.48 ± 3.04 ab	24.66 ± 0.55 ab	20.09 ± 1.91 b	23.31 ± 0.31 a	0.0169
C18:2 n-6 t	0.13 ± 0.09 a	0.21 ± 0.03 a	0.23 ± 0.01 a	0.17 ± 0.03 a	0.21 ± 0.02 a	0.1004
C18:2 n-6 c	1.03 ± 0.14 ab	1.11 ± 0.08 ab	1.23 ± 0.08 a	0.86 ± 0.09 b	1.09 ± 0.07 a	0.0106
C18:3 n-3	0.49 ± 0.13 ab	0.47 ± 0.05 ab	0.60 ± 0.10 a	0.33 ± 0.03 b	0.47 ± 0.10 ab	0.0313
C18:2 c9 t11 CLA	0.88 ± 0.05 a	1.09 ± 0.22 a	1.21 ± 0.02 a	1.16 ± 0.19 a	1.03 ± 0.11 a	0.1170
C20:0	0.35 ± 0.04 a	0.32 ± 0.02 ab	0.37 ± 0.02 a	0.25 ± 0.03 b	0.32 ± 0.02 a	0.0087
C20:1 n-9	0.02 ± 0.03 ab	0.05 ± 0.03 ab	0.01 ± 0.01 b	0.03 ± 0.02 ab	0.05 ± 0.01 a	0.0474
C21:0	0.04 ± 0.03 a	0.00 ± 0.00 a	0.05 ± 0.04 a	0.00 ± 000 a	0.00 ± 0.00 a	0.0747
C20:3 n-6	0.00 ± 0.00 a	0.00 ± 0.00 a	0.00 ± 0.00 a	0.00 ± 0.00 a	0.00 ± 0.00 a	0.6114
C20:4 n-6	0.02 ± 0.04 a	0.00 ± 0.00 b	0.04 ± 0.02 a	0.00 ± 0.00 bb	0.00 ± 0.00 b	0.0031
C22:0	0.13 ± 0.02 a	0.11 ± 0.02 ab	0.14 ± 0.00 a	0.09 ± 0.02 ab	0.05 ± 0.04 b	0.0156
C20:5 n-3	0.02 ± 0.02 b	0.00 ± 0.00 b	0.05 ± 0.01 a	0.00 ± 0.00 b	0.00 ± 0.00 b	0.0002
C23:0	0.06 ± 0.04 ab	0.05 ± 0.04 b	0.09 ± 0.02 a	0.06 ± 0.01 ab	0.00 ± 0.00 b	0.0020
C24:0	0.07 ± 0.04 a	0.07 ± 0.05 a	0.09 ± 0.01 a	0.03 ± 0.03 a	0.02 ± 0.02 a	0.1304
C24:1 n-9	0.00 ± 0.00 b	0.00 ± 0.00 b	0.02 ± 0.01 a	0.00 ± 0.00 b	0.00 ± 0.00 b	0.0039
Sums fatty acids
SFA	55.15 ± 4.93 ab	58.07 ± 6.38 ab	60.82 ± 1.64 a	46.57 ± 4.44 b	57.23 ± 0.71 a	0.0086
MUFA	21.98 ± 1.62 a	26.71 ± 3.59 a	26.49 ± 0.93 a	22.06 ± 2.08 a	25.45 ± 0.67 a	0.1577
PUFA	2.58 ± 0.39 ab	2.89 ± 0.30 ab	3.37 ± 0.21 a	2.52 ± 0.22 b	2.80 ± 0.08 ab	0.0408
n-3	0.51 ± 0.15 ab	0.47 ± 0.06 ab	0.65 ± 0.11 a	0.33 ± 0.03 b	0.47 ± 0.10 ab	0.0205
n-6	2.07 ± 0.24 ab	2.42 ± 0.32 a	1.51 ± 0.11 b	2.19 ± 0.21 a	2.33 ± 0.02 a	0.0019
Ratios
PUFA:SFA	0.05 ± 0.00 a	0.05 ± 0.00 a	0.06 ± 0.00 a	0.05 ± 0.01 a	0.05 ± 0.00 a	0.0895
PUFA:MUFA	0.12 ± 0.01 a	0.11 ± 0.01 a	0.13 ± 0.01 a	0.011 ± 0.01 a	0.11 ± 0.01 a	0.3121
MUFA:SFA	0.40 ± 0.02 a	0.46 ± 0.02 a	0.44 ± 0.01 a	0.47 ± 0.00 a	0.44 ± 0.02 a	0.5334
n3:n6	0.24 ± 0.62 b	0.20 ± 0.05 b	0.43 ± 0.07 a	0.15 ± 0.07 b	0.20 ± 0.04 b	0.0335
DFA	39.60 ± 4.09 a	43.08 ± 4.24 a	44.96 ± 1.52 a	34.85 ± 4.00 a	41.49 ± 0.80 a	0.0381
AI	2.22 ± 0.11 a	2.07 ± 0.01 b	2.09 ± 0.03 b	2.05 ± 0.03 b	2.13 ± 0.01 ab	0.0046
ThI	3.39 ± 0.23 a	3.02 ± 0.00 b	3.09 ± 0.02 b	2.99 ± 0.03 b	3.09 ± 0.01 b	0.0008
hH	0.74 ± 0.04 a	0.78 ± 0.02 a	0.79 ± 0.03 a	0.77 ± 0.01 a	0.75 ± 0.02 a	0.0775

Treatments tested: milk fermented with L. paraplantarum; milk fermented with L. plantarum; milk fermented with P. pentosaceus; milk fermented with L. fermentum, and * milk fermented with control L. plantarum. SFA = Saturated fatty acid; MUFA = Monounsaturated fatty acid; PUFA = Polyunsaturated fatty acid; DFA = Desirable fatty acids; AI = Atherogenicity index; ThI. = Thrombogenicity index; hH = Hypocholesterolemic/hypercholesterolemic fatty acids. Different letters in the same line indicate a statistically significant difference (p < 0.05) by Tukey’s test.

. The highest c9t11-CLA production was observed in Pediococcus pentosaceus and Limosilactobacillus fermentum strains, with 1.21 and 1.16 g/100 g of fatty acids, respectively. Considering the strain control L. plantarum with low conversion of LA to CLA being statistically similar to the other treatments (p < 0.05), it is possible to infer that the amount of CLA produced by the different strains did not significantly impact the products. On the other hand, similar studies cited showed values of approximately 0.6 g/100 g of CLA in buffalo milk fermented with commercial strains [20]. In cow fermented milk, conventional strains such as Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus ensured CLA levels close to 0.39 g/100 g of fat [47,48]. Considering this data, using CLA-producing strains in buffalo milk fermentation can confer significantly higher values than commercially produced dairy products.

Interestingly, Lactobacillus paraplantarum, which showed the best in vitro results, exhibited the lowest CLA production in buffalo milk (0.88 g/100 g of fatty acids). Although L. paraplantarum has previously obtained good results in producing CLA in MRS (approximately 27.5 µg/mL), this species was not selected for further study in the production of fermented milk [45]. Another isomer of CLA of interest is t12c11-CLA, which was not detected in any treatments. The isomer c9t11 represents about 90% of CLA in ruminant products, being the most prevalent [49], and most of the bacteria tested for the potential to produce CLA have no potential to produce t12c11-CLA isomer [50]. In dairy products, natural CLA concentrations are relatively low, ranging from 0.41 to 1.0 g of CLA per 100 g of total fatty acids [12]. Studies on buffalo milk have reported CLA contents ranging between 0.21 and 0.45 g per 100 g of fat [51,52,53], but key precursors in CLA production, such as LA, can also be part of the milk fat composition and contribute to the increase in CLA content in dairy products fermented with converting strains [6]. According to Godinho et al. [27], these differences are associated mainly with animal diet; the greener and fresher the pasture, the higher the concentration of CLA precursors.

Treatments with P. pentosaceus and control L. plantarum exhibited similar levels of saturated fatty acids (SFAs), which were significantly higher (p < 0.05) compared to the other treatments. In fermented buffalo milk with both strains of L. plantarum and Pediococcus pentosaceus, the total amount of short-chain fatty acids (SCFAs), particularly butyric acid (C4:0) and caproic acid (C6:0), was significantly higher (p < 0.05) compared to fermented milk with L. paraplantarum and L. fermentum. Differences in lipoprotein lipase activity, responsible for the hydrolysis of long-chain fatty acids (LCFAs) and the formation of short- and medium-chain fatty acids, alongside the presence of bacteria, can significantly influence the fatty acid profile due to lipolytic and proteolytic activity, as well as the formation of flavor and aroma characteristics [54,55].

The higher concentrations of saturated fatty acids (SFAs) in the fermented milk were due to the presence of long-chain fatty acids such as myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0). These are the main SFAs found in foods, with stearic acid present in similar amounts across all treatments (p > 0.05), except in the beverage fermented with L. fermentum (p < 0.05). Considering the range of SFAs in buffalo milk between 64.56 and 72.0 g/100 g of total fatty acids [51,56,57], the tested strains provided products with lower values ranging from 46.57 to 61.09 g/100 g of total fatty acids, indicating good capacity to increase the unsaturated fatty acid profile. In cow-fermented milk with a probiotic CLA producer, Lactococcus lactis subsp. cremoris MRS47 observed a consistent increase in SFAs from 66.05 g/100 g of total fatty acids in cow milk to 80.77 g/100 g of total fatty acids in probiotic fermented dairy [7].

Monounsaturated fatty acid (MUFA) content was similar across all treatments (p > 0.05). In contrast, treatment with P. pentosaceus had the highest polyunsaturated fatty acid (PUFA) content (3.28 mg/mL) compared to fermented milk with L. fermentum (2.52 mg/mL) (p < 0.05). This variation may be related to differences in lipolytic activity and the ability to produce FFAs [19]. Despite the lower PUFA content, L. fermentum produced the highest amount of CLA (1.22 mg/mL), followed by P. pentosaceus with 1.20 mg/mL. In contrast, treatment with L. paraplantarum had the lowest CLA content (0.90 mg/mL). Considering that the concentration of c9t11-CLA in buffalo milk typically ranges between 0.32 and 0.45 g/100 g of fat [51,53,57], it is evident that certain Lactobacillus strains have the lipolytic ability to use LA as a substrate for CLA synthesis in fermented buffalo dairy products [20]. This process is driven by a complex multienzyme system, primarily mediated by a membrane-bound linoleate isomerase (LAI) enzyme [6].

Several strains, including Propionibacterium freudenreichii, Lactobacillus acidophilus, Lactiplantibacillus plantarum, Lactococcus lactis subsp. cremoris MRS47 and Bifidobacterium breve have been scientifically proven to have CLA-producing potential [6,7,29,50]. According to Coakley et al. [29], Pediococcus pentosaceus strains can survive and grow in the presence of at least 0.5 mg/mL of LA but do not produce detectable amounts of CLA. However, recent studies have revealed that CLA produced by probiotic P. pentosaceus GS4 (CLAGS4) exhibits strong antiproliferative and protective efficacy against colon cancer [58,59], although the exact amount of CLA produced has not been quantitatively specified.

Treatments with L. plantarum, P. pentosaceus, and L. fermentum showed a lower (p < 0.05) Atherogenicity Index (AI) and Thrombogenicity Index (ThI), indicating less potential for the development of atherosclerosis and a lower tendency for blood clot formation. This index can vary in buffalo milk throughout the year, ranging between 3.75 and 4.94 [27]. So, considering the results of fermented products ranging from 2.99 to 3.39, it is possible that these strains can reduce this negative aspect of the product, with emphasis on L. fermentum. A previous study confirms that L. fermentum used in buffalo fermented milk with 2.5% fat could reduce the markers of hyperlipidemia with reduced harmful cholesterol levels, oxidative stress, and inflammatory responses [60]. The desirable fatty acid (DFA) index was comparable across all treatments (p > 0.05), except between treatments L. fermentum and the control L. plantarum, where L. fermentum exhibited the lowest results, likely due to the reduced amount of stearic acid. Buffalo milk is considered a good source of DFA, which can be further improved through proper animal management and feeding strategies [27].

4. Conclusions

Milk kefir, artisanal cheeses, jaboticaba juice, and kombucha proved to be significant sources of a diverse range of microorganisms, with LA conversion ranging between 65.6% and 21.8%. All strains tested demonstrated good growth capacity in buffalo milk despite a lower potential for milk acidification. All selected LAB strains, particularly P. pentosaceus and L. fermentum, showed similar CLA production levels, confirming their functional dairy product development potential. These products offered balanced fatty acid profiles and reasonable quantities of available microorganisms, reaching values between 11.85 and 11.15 Log CFU per mL at the final product. Further investigations about optimization of fermentation conditions (e.g., temperature, time, and inoculum concentration), the viability of lactic acid bacteria (LAB) during storage, and the stability of CLA levels in fermented buffalo milk must be considered to maximize CLA production, enhance the fatty acid profile, and improve sensory characteristics of fermented buffalo milk products.