Boosting the functional properties of fermented camel and bovine milk: the impact of Pediococcus and Enterococcus strains on acidity, antioxidant and antibacterial capacities

The investigation aimed at assessing a comparative study on the production and characterization of ACE inhibitory, anti-diabetic, and anti-inflammatory activities, along with the production of ACE inhibitory and anti-diabetic peptides through the fermentation of buffalo and camel milk by Limosilactobacillus fermentum (KGL4) and Saccharomyces cerevisiae (WBS2A). The angiotensin-converting enzyme (ACE) inhibitory and anti-diabetic properties were evaluated at particular time intervals (12, 24, 36, and 48 h) at 37 °C, and we discovered maximum activity at 37 °C after 48 h of incubation. The maximum ACE inhibitory, lipase inhibitory activities, alpha-glucosidase inhibitory, and alpha-amylase inhibitory activities were found in the fermented camel milk (77.96 ± 2.61, 73.85 ± 1.19, 85.37 ± 2.15, and 70.86 ± 1.02), as compared to the fermented buffalo milk (FBM) (75.25 ± 1.72, 61.79 ± 2.14, 80.09 ± 0.51, and 67.29 ± 1.75). Proteolytic activity was measured with different inoculation rates (1.5%, 2.0%, and 2.5%) and incubation times (12, 24, 36, and 48 h) to optimize the growth conditions. Maximum proteolysis was found at a 2.5% inoculation rate and at a 48 h incubation period in both fermented buffalo (9.14 ± 0.06) and camel milk (9.10 ± 0.17). SDS-PAGE and 2D gel electrophoresis were conducted for protein purification. The camel and buffalo milk that had not been fermented revealed protein bands ranging from 10 to 100 kDa and 10 to 75 kDa, respectively, whereas all the fermented samples showed bands ranging from 10 to 75 kDa. There were no visible protein bands in the permeates on SDS-PAGE. When fermented buffalo and camel milk were electrophoresed in 2D gel, 15 and 20 protein spots were detected, respectively. The protein spots in the 2D gel electrophoresis ranged in size from 20 to 75 kDa. To distinguish between different peptide fractions, water-soluble extract (WSE) fractions of ultrafiltration (3 and 10 kDa retentate and permeate) of fermented camel and buffalo milk were employed in RP-HPLC (reversed-phase high-performance liquid chromatography). The impact of fermented buffalo and camel milk on inflammation induced by LPS (lipopolysaccharide) was also investigated in the RAW 264.7 cell line. Novel peptide sequences with ACE inhibitory and anti-diabetic properties were also analyzed on the anti-hypertensive database (AHTDB) and bioactive peptide (BIOPEP) database. We found the sequences SCQAQPTTMTR, EMPFPK, TTMPLW, HPHPHLSFMAIPPK, FFNDKIAK, ALPMHIR, IPAVFK, LDQWLCEK, and AVPYPQR from the fermented buffalo milk and the sequences TDVMPQWW, EKTFLLYSCPHR, SSHPYLEQLY, IDSGLYLGSNYITAIR, and FDEFLSQSCAPGSDPR from the fermented camel milk.

The comparative assessment of ACE-inhibitory and antioxidant activities of peptide fractions obtained from fermented camel and bovine milk by Lactobacillus rhamnosus PTCC 1637

Gelation of camel milk is a challenge to the industrial utilization. This study investigated the impact of low-temperature long-time (LTLT) and high-temperature short-time (HTST) pasteurization, ultra-high temperature (UHT) treatment, and high-pressure processing (HPP), at 300 and 600 MPa, on the rheological properties of the fermented milk. Both camel and bovine milk were fermented by Lactiplantibacillus plantarum and Ligilactobacillus salivarius. Both milk treated at 300 and 600 MPa showed the highest Lb. plantarum and Lb. salivarius numbers, with ∼9 log.HTST and UHT-treatment increased particle size in bovine milk but decreased it in camel milk. HPP-treatment decreased particle size in both camel and bovine milk. All fermented milk showed shear-thinning behavior, regardless of milk type, processing, culture type and storage time. For both camel and bovine milk, fermented UHT-treated milk had the highest viscosity followed by fermented HPP-treated milk. Lowest viscosities were observed for fermented LTLT-treated and HTST-treated milk. Small deformation rheology showed consistently lower tan δ values for fermented HPP-treated camel milk than for fermented heated camel milk, with the latter only giving tan δ < 1 at elevated frequencies. UHT- and HPP-treatment of milk can enhance the rheological properties of fermented camel milk compared with other heat treatments.

Chal is a traditional fermented product produced from spontaneously fermented camel milk which contains several bacterial species with potential usage in producing traditional dairy products and functional foods. The aims of this study were to isolate and identify predominant lactic acid bacteria (LAB) from Chal and investigate antioxidant activity of camel and bovine milk fermented by these isolates. Chal samples were collected from Turkman Sahra, Golestan Province, Iran. The protein hydrolysis was determined by o-phthaldialdehyde (OPA) method, and antioxidant activities of whey fractions were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals scavenging methods. Nine catalase-negative bacteria including Lactobacillus (L.) plantarum, L. paraplantarum, L. kefiri, L. gasseri, L. paracasei, Leuconostoc (Leu.) lactis, Weissella (W.) cibaria, and Enterococcus (E.) faecium were isolated and identified by conventional and molecular methods. Both camel and bovine milk were fermented by the strains for 24 h. Fermented camel milk showed significantly (P < 0.05) higher antioxidant activity than bovine milk. Camel milk fermented by Leu. lactis showed significantly (P < 0.05) higher DPPH (57.90 ± 4.59 μM) and ABTS (1484.35 ± 128.20 μM; P < 0.05) radical scavenging activity compared to samples fermented by other strains. According to sensory evaluation of fermented camel and bovine milks, camel milk fermented by Leu. lactis SM10 had the highest overall acceptance values. Our findings suggest that camel and bovine milk fermented by LAB isolated from Chal could potentially be used for producing novel functional foods.

This study assessed the fermentation performance of five lactic acid bacteria (Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lb. helveticus, Lb. casei, and Lactiplantibacillus plantarum) in camel milk (CM) and bovine milk (BM) at 42 °C for 48 h. Fluorescence microscopy revealed lower bacterial viability in fermented CM compared to BM. Acidification kinetics varied significantly between CM and BM, and proteolysis was more pronounced in fermented CM (p < 0.001), with OPA concentrations ~1.3–1.5-fold greater in CM across all strains during fermentation. Scanning electron microscopy revealed more porous, loose protein matrices in fermented CM than in BM, in line with the rheological analyses showing weaker gel networks and lower rheological strength in fermented CM. Lb. casei demonstrated superior adaptability, enhanced viability, balanced acidification, and favorable rheological properties in both milks, highlighting its potential as a possible starter or adjunct culture in fermented dairy products.

The goal of this investigation was to find antidiabetic peptides and inhibit angiotensin converting enzyme (ACE) in Lacticaseibacillus paracasei (M11) fermented dromedary camel milk (Camelus dromedaries). According to the findings, the rate of antidiabetic activity increased along with the incubation periods and reached its peak after 48 hr of fermentation. The inhibitions of α-amylase, α-glucosidase, and lipase were 80.75, 59.62, and 65.46%, respectively. The inhibitory activity of ACE was 78.33%, and the proteolytic activity was 8.90 mg/mL. M11 at 0.25 mg/mL effectively suppressed LPS-induced pro-inflammatory cytokines and their mediators such as NO, TNF-α, IL-6, and IL-1β in RAW 264.7 cells. The rate of inoculum in the optimization phase was 1.5-2.5%, and the greatest proteolytic activity was observed after 48 hr of fermentation. The investigation of the above property in the ultrafiltered fermented milk exhibited the highest antidiabetic and ACE inhibition activities in the 3kDa than 10kDa fractions. The molecular weight was determined employing SDS-PAGE, and the six-peptide sequences were identified using 2D gel electrophoresis. Due to its high proteolytic activity, the L. paracasei strain has been reported to be useful in the production of ACE-inhibitory and antidiabetic peptides. Amino acid sequences such from ɑ1, ɑ2, and β-caseins have been identified within fermented camel milk by searching on online databases, including BIOPEP (for antidiabetic peptides) and AHTPDB (for hypertension peptides) to validate the antidiabetic and ACE-inhibitory actions of several peptides. PRACTICAL APPLICATIONS: The study aims to identify antidiabetic peptides and inhibit ACE in dromedary camel milk fermented with Lacticaseibacillus paracasei M11. Maximum antidiabetic and ACE-inhibitory actions of the fermented camel milk were observed in 3kDa permeate fractions. Fermented camel milk significantly reduced the excessive TNF-α, IL-6, and IL-1β production in LPS-activated RAW 264.7 cells. RP-LC/MS was used to identify 6 bioactive peptides from dromedary fermented camel milk. This fermented camel milk could be used for the management of hypertension and diabetic related problems.

The aim of the study is to explore the biofunctional properties (antioxidative, anti-diabetic, and anti-inflammatory) with the release of bioactive peptides from fermented camel milk and Gir cow milk through yeast-lactic fermentation. Fermented camel milk and Gir cow milk exhibited higher antioxidative, antidiabetic, and anti-inflammatory activities compared to their unfermented counterparts. At 30°C, the most significant production of peptides had been discovered at 48h of incubation with 2.5% rate of inoculation of yeast-lactic culture in the fermented milks of camel and Gir cow. Additionally, both the fermented milks considerably reduced the overproduction of TNF-α, IL-6, IL-1β, and nitric oxide in RAW 267.4 cells. Confocal laser scanning microscopy revealed the visualization of protein biomolecules of camel milk and Gir cow milk pre- and post-fermentation, revealing changes in protein network structure. The structural changes that occur during fermentation were examined using Fourier-transform infraredspectroscopy by assessing changes in functional groups after fermentation. To distinguish between different peptide fractions, reversed-phase high-performance liquid chromatography was used for comparing water-soluble extracts of ultra-filtered fractions. The Peakview tool was implemented to assess the liquid chromatography-mass spectrometry (LC/MS) data. However, fermenting camel and Gir cow milk with yeast and lactic acid bacteria enhances their nutritional and therapeutic values by releasing bioactive compounds, improving antioxidative, antidiabetic, and anti-inflammatory activities, and this process supports gut health, immunity, and sustainability, offering potential for functional foods and nutraceutical innovations. PRACTICAL APPLICATION: Traditionally, camel and Gir cow milk provide health benefits beyond nutrition for the well-being of the society since long. Fermented Gir cow and camel milk contain physiologically bioactive peptides. Gir cow and camel milk fermented with Limosilactobacillus fermentum (KGL4) in combination with Saccharomyces cerevisiae (WBS2A) also provide antidiabetic and antioxidative activities. Anti-inflammatory activity of fermented Gir cow and camel milks was also observed in RAW 264.7 macrophage cell. Antidiabetic and antioxidative peptides were also identified from fermented Gir Cow and camel milks. However, functional fermented dairy products can be developed using these two potent strains in combination for providing better health benefits.

Probiotic survival, biological functionality and untargeted metabolomics of the bioaccessible compounds in fermented camel and bovine milk after in vitro digestion

Camel milk is an increasingly popular alternative to bovine milk around the world due to its reported nutritional properties and its potentially therapeutic benefits. Although Australia is home to one of the largest dromedary camel herds in the world, studies of Australian camel milk are lacking. This project aims to provide a comprehensive understanding of the physicochemical properties of Australian camel milk, with a primary emphasis on proteins.The major nutritional components, physical properties and whey protein profile of Australian camel milk samples collected over four seasons were initially analysed. The composition and physical properties of Australian camel milk varied with season, milking frequency and yield. Higher contents of fat, total solids and lactose were observed in spring samples compared with those in other seasons, while no significant (P < 0.05) differences were found for protein and ash content between the four seasons. Milk viscosity was found to be stable over the year but a smaller average particle size was observed in summer and autumn samples. Lactose content was positively correlated with milk yield in the four seasons. The milk viscosity, particle size and other components such as fat, protein and ash content were, however, normally negatively correlated with milk yield. A proteomic study showed that, in general, summer camel milk contained higher amounts of functional whey proteins, such as lactotransferrin, peptidoglycan recognition protein 1, osteopontin and lactoperoxidase.A highly sensitive, high-throughput, fluorescent method was developed for the determination of activities of enzymes with antimicrobial properties in milk, including xanthine oxidase (XO), polyamine oxidase (PAO) and lactoperoxidase (LPO). The hydrogen peroxide generation by milk XO/PAO was coupled to the horseradish peroxidase conversion of Amplex® Red (1-(3,7-dihydroxyphenoxazin-10-yl)ethanone) to its fluorescent product, resorufin. LPO activity was measured by the direct oxidation of Amplex Red by LPO, following the addition of hydrogen peroxide. These enzyme activities varied significantly between camel, bovine, goat and human milk, suggesting that different innate immune systems may be present in milk from different species. Analysis of the enzyme activities in seasonal camel milk samples revealed that summer and winter samples had the highest LPO and PAO activities, respectively, over the year. The secretion of LPO into milk varied between individual camels, while the PAO activity was negatively correlated with milk yield in all the seasons. An antimicrobial role of PAO may be important for the innate immune system in camel milk.The abundant amount of β-casein and lactotransferrin, coupled with the absence of β-lactoglobulin in camel milk, makes it a promising alternative base material for making human infant formulae. The protein digestibility of camel milk was therefore compared with that of bovine and human milk using an in vitro digestion system simulating the infant gastrointestinal tract. Camel milk formed a single clot during gastric (stomach) digestion, while no visible clot was formed in bovine and human milk. During the gastric phase of digestion, casein was the most obviously digested protein in bovine milk, while the whey proteins were most prominently digested in camel milk. The soluble milk proteins remaining in the gastric digesta were rapidly and extensively digested in the intestinal phase. A gradual hydrolysis of the proteins incorporated in the camel milk clot was observed and very small amount of the clot remained at the completion of intestinal digestion period. Different peptide profiles were observed from camel, bovine and human milk following digestion. The largest number of potentially bioactive peptides were identified in camel milk after intestinal digestion, using PeptideRanker predictions. This work provides some initial insights on the protein digestibility of camel milk under infant digestion conditions, and presages further studies on the infant consumption suitability of camel milk proteins.Finally, to increase the potential availability of camel milk, camel milk powders were produced by freeze drying or spray drying with/without pre-concentration by spray dewatering or reverse osmosis. An improved retention of lactoperoxidase activity was observed in freeze-dried camel milk powders (18-50%) when compared to spray-dried camel milk powders (9-14%). A total of 108 proteins were quantified, on a relative basis, in the camel milk powders versus unprocessed camel milk, using Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS). Smaller magnitude changes in protein profile were observed in directly freeze-dried or spray-dried camel milk powder, with the freeze-dried sample showing the closest protein profile to that of unprocessed milk, while reverse osmosis had the greatest impact in changing the milk protein profile. Among the bioactive proteins identified, lactotransferrin and oxidase/peroxidase had the most significant decreases following processing, while glycosylation-dependent cell adhesion molecule 1, peptidoglycan recognition protein 1, and osteopontin generally increased. The reverse osmosis-concentrated and freeze-dried camel milk powder exhibited the highest anti-Listeria activity among all the powders produced. The results suggest that the dehydration method should be carefully selected depending on the use and desired characteristics of the camel milk powder.

The characterization of Lactobacillus strains in camel and bovine milk during fermentation: A comparison study

Fermented camel milk offers significant nutritional benefits, enriched with probiotics that generate bioactive compounds advantageous to human health. In order to investigate the effects of camel milk with different treatments on Antioxidant Capacity and Gut Microbiota in mice, 32 ICR mice were selected and randomly divided into 4 groups, including gavage with 10 mL/kg body weight of distilled water (DW Group), camel milk (CM Group), fermented camel milk (FCM Group), and pasteurized fermented camel milk (PFCM Group) every morning, respectively. After 28 days, liver and colon samples were collected to assess liver antioxidant capacity, and metagenomic analysis was performed on alterations in microbial community structures. Results demonstrated that all camel milk treatments elevated liver total protein levels while reducing MDA and SOD activity. In addition, the PFCM group had the highest total antioxidant capacity and the lowest SOD content. In addition, the intestinal microorganisms of mice changed at the phylum, genus and species levels after being gavaged with camel milk of different treatments. A total of 4732 microorganisms were identified, of which 259, 222, 116 and 164 were unique to the DW, CM, FCM and PFCM groups, respectively. The relative abundances of Adlercreutzia caecimuris, Adlercreutzia mucosicola and Enterorhabdus sp. P55 were significantly higher in the CM, FCM and PFCM groups than in the DW group, and the relative abundances of Parvibacter caecicola, Adlercreutzia muris and Roseburia sp. 1XD42-69 were significantly higher in the CM and PFCM groups than in the DW group. In addition, the relative abundances of Faecalibaculum rodentium, Alistipes muris and Limosilactobacillus reuteri were different between the CM and FCM groups. The results of the correlation analysis between the relative abundance of microbial species and antioxidant indices showed that Adlercreutzia mucosicola, Adlercreutzia muris, Lactobacillus acidophilus, and Enterorhabdus sp. P55 were significantly correlated with the antioxidant indices of mice. Further functional annotations indicated that these microorganisms might modulate antioxidant activity via metabolic and organismal systems. In summary, camel milk and fermented camel milk can play a positive role in regulating the intestinal flora of mice, thereby regulating the antioxidant capacity of mice and alleviating the effects of oxidative stress on the body. This study provides a scientific foundation for the further exploration and utilization of camel milk.

In vitro investigation of anticancer, antihypertensive, antidiabetic, and antioxidant activities of camel milk fermented with camel milk probiotic: A comparative study with fermented bovine milk

IntroductionUlcerative colitis poses a significant threat to human health. Dysbiosis of the gut microbiota and decreased levels of short-chain fatty acids are known contributors to the pathogenesis of ulcerative colitis. While camel milk and fermented camel milk have demonstrated beneficial effects in alleviating intestinal inflammation, the differences in their preventive efficacy against ulcerative colitis and the underlying mechanisms remain unclear. Dextran sulfate sodium can induce ulcerative colitis, at least in part, through activation of the NF-κB/MAPK signaling pathway. This study aims to investigate the differential preventive effects of camel milk and fermented camel milk against dextran sulfate sodium-induced ulcerative colitis in mice and to elucidate the underlying mechanisms mediated by the gut microbiota and short-chain fatty acids.ResultsThrough the study, we found that preemptive intervention with camel milk and fermented camel milk significantly mitigated dextran sulfate sodium-triggered pathological manifestations, including body weight loss (e.g., from 97%in DSS group to 99% in FTC group, p < 0.01), elevated disease activity index (from a peak score of 2.8 to 2.0, p < 0.01), colonic shortening (from 5.8 cm to 6.2 cm, p < 0.01). Both treatments also restored intestinal barrier integrity, increasing the expression of tight junction proteins, and elevated the anti-inflammatory cytokineIL-10 (p < 0.05). Then, it can also regulate the intestinal flora imbalance in mice with ulcerative colitis. Finally, we found that both can also reduce the reduction of short-chain fatty acids in mice with ulcerative colitis (p < 0.05). Notably, the fermented camel milk exhibited superior efficacy compared to the camel milk in mitigating fermented camel milk-induced body weight loss, the decline of IL-10and E-cadherin levels, microbial diversity decreasing, the Bacteroidetes/Firmicutesratio increasing, and the relative abundance of Lachnospiraceae NK4A136 group decreasing in ulcerative colitis mice.DiscussionOur findings demonstrate that preemptive intake of camel milk and fermented camel milk effectively prevents ulcerative colitis by preserving the intestinal barrier, regulating immune responses, and restoring gut microbiota homeostasis and short chain fatty acids production, with fermented camel milk offering enhanced benefits. We hold optimistic prospects for camel milk and fermented camel milk as dietary supplements to prevent ulcerative colitis and stabilize gut microbiota homeostasis.

Camel milk (CM) is recognized for its high nutritional enrichment, and the distinction from bovine milk mainly because of its unique protein composition, proteolytic products, and anti-microbial compounds. These unique chemical properties have positive effects on the nutritional value but negative impact on the sensory attributes and consumer acceptability of fermented CM. This review summarizes the current state of knowledge on CM fermentation, emphasizing the influence of milk composition on gel and microstructure formation, texture, and overall quality of the products. While the richness of fermented CM in bioactive peptides enhances its nutritional and therapeutic values, major challenges are associated with their thin consistency and weak gel structure. Various strategies to overcome these challenges and develop unique functional fermented CM products are discussed, including the use of alternative starter cultures (e.g., Lactobacillus helveticus, Lb. casei, and Lactiplantibacillus plantarum), stabilizing additives hydrocolloids or proteins, as well as optimized heat treatments, high-pressure processing and other emerging technologies. Despite several processing and formulation procedures, no particular approach has yet offered a comprehensive solution for achieving firm and stable camel yogurt. Therefore, it is important to accept fermented CM products as being different and develop means to improve their sensory quality and consumer acceptance. Overall, this review underscores the necessity for ongoing research to optimize the quality and commercial viability of fermented CM.

In vitro investigation of anticancer and ACE-inhibiting activity, α-amylase and α-glucosidase inhibition, and antioxidant activity of camel milk fermented with camel milk probiotic: A comparative study with fermented bovine milk