Functional characteristics of peptides from whey proteins fermented with lactic acid bacteria isolated from Dongchimi

Functional characteristics of peptides from whey proteins fermented with lactic acid bacteria isolated from Dongchimi | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Functional characteristics of peptides from whey proteins fermented with lactic acid bacteria isolated from Dongchimi Jungsik Yoo, Sangjae Lee, Seoeun Song, Cheolhyun Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7899609/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Bioactive peptides (BAPs) are short amino acid sequences in food proteins that provide health benefits such as antioxidant, antihypertensive, antimicrobial, and immunomodulatory effects. Whey protein, which is rich in essential amino acids such as leucine and cysteine, is a promising source of BAPs; however, it resists enzymatic hydrolysis due to its hydrophobicity. To overcome this limitation, this study used fermentation with Lacticaseibacillus sakei DC10, a lactic acid bacteria (LAB) with strong proteolytic activity. Fermentation produced diverse peptides with antioxidant and anti-inflammatory properties and improved calcium solubility, suggesting potential bone health benefits. These peptides may reduce allergenicity and serve as multifunctional food ingredients. In addition, their calcium-binding ability makes them cost-effective alternatives to casein phosphopeptides for nutraceutical and fortified food applications. This study highlights LAB fermentation as an effective approach for generating novel whey protein-derived BAPs with enhanced bioactivity, supporting their use in functional foods and human health promotion. lactic acid bacteria whey protein concentrate fermentation antioxidant immunomodulatory effect Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Proteins can be used as substrates to produce bioactive peptides (BAPs), and can also be transformed into high value-added products such as BAPs, which is a successful management strategy [1–3]. Food proteins are currently being studied beyond their nutritional characteristics because of their positive effect on human health related to specific sequences encrypted into native proteins, known as BAPs. Such sequences are inactive when present in the parent protein, but can be released after protein hydrolysis during gastrointestinal digestion, in-vitro enzymatic hydrolysis, or microbial fermentation [4,5]. BAPs are short amino acid sequences with inactive precursor protein [6]. The potential health benefits associated with BAPs consumption have attracted the interest of many researchers [7]. The release of BAPs from their parent proteins by proteolysis is affected by several factors such as hydrolysis time, pH, temperature, and enzyme–substrate ratios [8]. Whey protein is a mixture of globular proteins isolated from whey, a liquid material created as a byproduct of cheese production [9]. Whey proteins constitute 15–20% of total milk proteins [10]. The key components of whey protein have been characterized as β-lactoglobulins and α-lactoalbumins [11]. Other constituents of whey protein include immunoglobulins, serum albumin, and lactoferrin [12]. Protein quality depends on the constituent amino acids, and whey protein has one of the highest standards. Whey proteins are also rich in essential branched-chain amino acids (BCAAs) [13]. BCAAs, including leucine, isoleucine, and valine, play crucial roles in metabolism, blood glucose homeostasis, and neural function [14]. Leucine regulates skeletal muscle protein synthesis [15]. Another essential amino acid, cysteine, is a building block of glutathione, which is a dietary antioxidant [16]. It is vital to combat oxidative stress and prevent diseases caused by redox imbalances [17]. Several studies have reported that whey proteins are valuable sources of BAPs because of their high nutritional value and the wide range of specific BAPs they release [18,19]. The enzymatic hydrolysis of food proteins is among the most promising methods for BAP production [20]. However, industrial production of BAPs remains limited by the lack of suitable large-scale technologies and the high cost of enzymes used for protein hydrolysis [21]. Whey proteins are highly hydrophobic and are resistant to enzymatic hydrolysis. Therefore, bioactive fragments may not be released from whey proteins despite the high biological potential of the protein precursor [22]. Therefore, hydrolysates produced by enzymes obtained from alternative sources, such as new strains of lactic acid bacteria (LAB) that exhibit high proteolytic activity, can be a source of several new peptides with specific bioactivities at a low production cost [23]. LAB, which have been used for lactic acid fermentation since ancient times, produce proteases that reduce the allergenic potential of milk proteins. This effect depends on the bacterial strain and regulation and optimization of proteolysis [24–26]. Food proteins and α-lactalbumin fermented by LAB can increase digestibility and hydrolyze allergenic peptides [27]. This approach can be used to develop new BAPs, and whey bioactive peptides can be targeted as compounds that exert protective effects against several diseases. The proteolytic activity of LAB is exerted in a strain-dependent manner, resulting in a variety of proteolytic activities [28]. Moreover, due to the specificity of the enzyme to the substrate, the peptide composition of hydrolysates, and therefore peptide activities, change [29]. This suggests that LAB can generate a considerable variety of BAPs and highlights the need to correctly select the strains that will be applied for BAP production [28]. In this regard, the purpose of this study was to develop bioactive peptides derived from fermented whey protein with superior physiological properties, calcium solubilization ability, and effects on immunity. This research is significant for the potential application of calcium-binding peptides as ingredients in functional foods. 2. Materials and Methods 2.1 LAB Screening and Identification LAB were isolated from homemade Dongchimi collected from different regions of Korea. Dongchimi samples (10 mL) were mixed with 40 mL of phosphate-buffered saline (PBS) (pH 7.4) and homogenized by vortexing. Diluted samples (1 mL) were serially diluted with 0.85% sterile saline, and 100 µL of each dilution was then spread onto De Man–Rogosa–Sharpe (MRS; BD Difco, USA) agar containing 0.02% sodium azide, and incubated at 37°C for 48 h. LAB colonies were selected from the plates and inoculated into MRS broth for stock preparation. For long-term storage, all cultures were maintained as frozen stocks at -72°C in MRS broth containing 20% glycerol. Screening of selected isolates was performed based on the colony shape on plates, Gram staining, and observation of strain morphology using a microscope. The selected strains were identified by 16S rRNA gene sequencing. The 16s rRNA sequences were analyzed using the GenBank database (Macrogen, Korea), and identification was performed based on 16S rRNA sequence homology. 2.2 Tolerance to Artificial Gastric Juice and Bile Tolerance to artificial gastric juice and artificial bile acid was measured according to the method developed in a previous study [30]. To investigate the survival of LAB strains under acidic conditions, each strain was harvested by centrifugation at 7,000 ×g for 10 min, washed twice with PBS, then inoculated (1%) into MRS broth acidified to pH 1.5, 2.0, or 3.0 (adjusted using HCl) containing 1,000 units of pepsin (Sigma–Aldrich, St. Louis, MO, USA) or into non-acidified MRS broth. The cultures were incubated at 37°C for 2 h. Following gastric juice treatment, strains were subjected to bile tolerance testing by centrifugation at 7,000 ×g for 10 min, washing twice with PBS (pH 7.4), and incubation at 37°C for 24 h in artificial bile acid consisting of MRS broth supplemented with 0.3% oxgall (Difco, Detroit, MI, USA). Finally, viable cell counts were determined by serial 10-fold dilutions in 0.85% saline, and plating 1-mL aliquots evenly on BCP agar plates which were then aerobically incubated at 37°C for 48 h before the number of colony-forming units (CFU) were estimated. 2.3 Proteolytic Activity Proteolytic activity was assessed by an agar well diffusion method according to the previously described method [31]. For the agar well-diffusion test, selected LAB strains were screened for proteolytic activity by agar-well diffusion test on skim milk containing 2.0% (w/v) agar (Difco, Detroit, MI, USA). The supernatant (100 µL) obtained by centrifuging bacterial cultures grown in MRS broth at 5000 ×g for 10 min were loaded into 9 mm diameter wells of skim milk agar plates. Proteolysis resulted in the formation of a clear zone around the wells. Protease activity was determined by estimating the diameter of clear zone area for 72 h at 37°C. 2.4 Whey Protein Concentrate (WPC) Solution Preparation WPC (Meegle, Germany) was reconstituted with distilled water to a final concentration of 10% (w/v), and glucose (Sigma–Aldrich Co., USA) was added at a concentration of 5% (w/v) for growth of LAB. The solution was adjusted to the optimum active pH for LAB using 1 N NaOH (Sigma–Aldrich Co., USA), sterilized for 30 min at 80°C, and then stored at 4 ℃ for ≤ 1 week [32]. 2.5 Fermentation Conditions LAB were cultured in MRS broth at 37°C for 18 h. The cultures were centrifuged at 8,000 ×g for 15 min at 4°C and the supernatant was discarded. The resulting cell pellets were resuspended in 10 mL of 1 X PBS to prepare the bacterial suspension. This suspension was inoculated into the WPC solution at a final concentration of 1% (v/v) and fermented at 37°C with shaking at 150 rpm in a water bath for 20 h. The fermented solution was subsequently heat-treated at 95°C for 10 min to obtain the final fermentation product. 2.6 Degree of Hydrolysis (DH) of WPC Solution The DH of the WPC solution was measured in fermented WPC samples (every 4 h for 20 h) using the 2,4,6-trinitrobenzenesulfonic acid (TNBS) method [33]. The TNBS reagent consisted of 0.1% (v/v) TNBS in distilled water. All samples and standard solutions were prepared in 1% (w/v) sodium dodecyl sulfate (SDS) solution. Samples (2 mL) were dissolved in distilled water to 10% (v/v) in a test tube, and 4 mL of 0.72 N trichloroacetic acid (TCA) was added. Samples were then incubated at 25°C for 20 min. Filtration through a 0.45-µm sterile polyvinylidene fluoride (PVDF) syringe membrane filter (SIGL, Germany) to takes 0.2 mL of samples were added to test tubes containing 2 mL of 0.2125 M sodium phosphate buffer (pH 8.2). The TNBS reagent were added 2 mL each test tube and then incubated at 50°C for 60 min to exclude light. After incubation, the reaction was stopped by addition of 4 mL of 0.1 N HCl to each test tube. The samples were then allowed to cool at room temperature for 30 min before absorbance values were measured at 420 nm using a UV-Vis spectrophotometer (X-ma 1200, Human Corp., Korea). DH is defined as the proportion of the total number of peptide bonds cleaved during hydrolysis [33], and was calculated as follows: DH, % = h/htot x 100, (1) where h is the number of hydrolyzed peptide bonds, and htot is the total number of peptide bonds present [34]. Leucine was used as a standard (at concentrations ranging from 0–2 mM) to determine the free amino group content of the samples. Each sample was analyzed in triplicate. 2.7 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Fermented WPC solution was analyzed by SDS-PAGE according to the method of [35] using a 12% polyacrylamide separation gel under reducing conditions with a Mini-PROTEAN III system (Bio-Rad, USA). The operating conditions were set to 20 V for 20 min, followed by 100 V for 2 h. After staining with Coomassie blue R-250 (water:methanol:acetic acid [45:45:10, v/v/v]) and water:methanol:acetic acid (45:45:10 v/v/v) solutions, SDS-PAGE analysis was performed using Precision Plus protein dual xtra standards (Bio-Rad, U.S.A). 2.8 Fast Protein Liquid Chromatography (FPLC) To obtain the peptide fractions from the WPC solution fermented with DC10, gel filtration was performed using a preparative chromatography system (Waters, U.S.A). A Waters preparative pump and Preparative liquid chromatography W600 were used with a Waters Dual λ Absorbance detector W2487 and Waters W717 Autosampler. In the first purification step, Fermented WPC solution was dissolved in 50 mM sodium phosphate buffer with 0.15 M NaCl (Sigma–Aldrich Co., USA) (pH 7.0). It was then filtered through a PVDF 0.45-µm sterile syringe membrane filter (SIGL, Germany). Next, the filtrate was loaded onto a Hiprep 16/60 Sephacryl S-100 HR column (GE Healthcare Life Sciences, U.S.A) and eluted at a 0.5-mL/min flow rate. The detection wavelength was set at 280 nm. All fractions obtained during gel filtration with the Hiprep 16/60 Sephacryl S-100 HR column (GE Healthcare Life Sciences, U.S.A) in a Preparative chromatography system were collected and stored at -20°C until analysis. Table 1 Analysis condition of fast protein liquid chromatography (FPLC). Instrument Condition Column Hiprep 16/60 Sephacryl S-100 HR column (GE Healthcare Life Sciences, U.S.A) Mobile phase 50 mM Sodium phosphate buffer (pH 7.0) Detector (Detection) Waters Dual Wave length Absorbance W2487 detector Flow rate 1 mL/min Injection volume 3 mL 2.9 Calcium Solubilization Ability In the calcium binding activity experiment, the method described previously [36,37] was slightly modified to measure the effect of calcium phosphate formation on precipitation in solution. Calcium chloride (10 Mm; Sigma–Aldrich Co., USA) and sodium phosphate buffer (20 mM) were prepared. Then, 0.5 mL of 10 mM calcium chloride and the casein hydrolysate fraction (0.5 mL) were mixed, and 1.0 mL of a 20 mM sodium phosphate buffer was added. The mixed solution was incubated at 37°C for 2 h and centrifuged at 2,000 ×g for 30 min at 25°C to remove insoluble calcium phosphate salts. Calcium solubility was measured using a calcium colorimetric kit (Gene tex, Inc., USA), and the supernatant was collected after centrifugation. The whole solution and supernatant samples were dispensed into each 10-µL 96-well plate, followed by mixing of chromogenic reagent to 90 µL and calcium assay buffer to 60 µL. After incubation for 5 min in a dark room at room temperature, absorbance was measured at 570 nm. The calcium concentration was calculated according to Eq. (2), and the calcium solubility was calculated according to Eq. (3). The protein concentration of all fractions from fermented WPC solution samples was 200 µg/mL. Calcium concentration (mg/mL) = Sa/Sv (2) *Sa = Sample amount from the standard curve (3) *Sv = Sample volume (4) Calcium solubility (%) = (calcium concentration in supernatant)/(calcium concentration in whole solution) × 100 (5) 2.10 Antioxidant Activity 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Assay ABTS radical scavenging activity was assessed by first preparing a 7 mM ABTS solution in ethanol (Samchun Chemical, Seoul, Korea) using ABTS powder (Sigma–Aldrich Co., USA). To generate the ABTS•⁺ radical solution, the ABTS stock was mixed with 2.45 mM potassium persulfate (Sigma–Aldrich Co., USA) in a 1:1 (v/v) ratio and incubated at an ambient temperature in the dark for 12–16 h. The resulting ABTS•⁺ solution was diluted with ethanol to achieve an absorbance of 0.70 ± 0.02 at 734 nm, measured using a Multiskan EX355 microplate reader (Thermo Fisher Scientific, Waltham, USA). For the assay, 190 µL of the diluted ABTS•⁺ solution was added to 10 µL of each sample fraction in a 96-well plate (SPL Life Sciences, Pocheon, Korea). After 3 absorbance was measured at 734 nm, and antioxidant activity was expressed as the Trolox equivalent antioxidant capacity in mM/L [38]. 2,2-diphenyl-1-picrylhydrazyl (DPPH) Assay DPPH free radical-scavenging activity was determined with slight modifications [39]. A DPPH solution (0.2 mM) was prepared by dissolving 2,2-diphenyl-1-picrylhydrazyl and 2,4,6-tripyridyl-s-triazine (Sigma–Aldrich Co., USA) in ethanol. This solution was mixed with the fermented WPC samples in equal volumes (1:1, v/v), followed by incubation in the dark at room temperature for 30 min. After the reaction, absorbance was recorded at 570 nm, and the antioxidant capacity was quantified as ascorbic acid equivalents (mM), using ascorbic acid (Sigma–Aldrich Co., USA) as the standard [38]. Ferric-Reducing Antioxidant Power (FRAP) Assay A FRAP assay was performed. The FRAP solution was prepared as follows: 300 mM acetate buffer was prepared by mixing sodium acetate trihydrate (Sigma–Aldrich Co., USA) with acetic acid (J.T Baker Co., USA). A 10 mM TPTZ solution was prepared by dissolving 2,4,6-tri(2-pyridyl)-1,3,5-triazine (Sigma–Aldrich Co., USA) in 40 mM HCl (DAEJUNG Co., Korea). Additionally, a 20 mM Iron(III) chloride hexahydrate solution was prepared by dissolving Iron(III) chloride hexahydrate 97% A.C.S reagent (Sigma–Aldrich Co., USA) in ethanol (Samchun Chemical Co., Korea). The three solutions were mixed at a 10:1:1 (v/v) ratio. For the assay, 1.5 mL of the FRAP solution, preheated to 37 ℃, was combined with 50 µL of fermented WPC fraction samples or standard solutions. The mixture was vortexed and allowed to react in the dark at room temperature. Absorbance was measured at 593 nm, and the results were expressed as ascorbic acid equivalent capacity in mM [40,41]. The protein concentration of all fermented WPC fraction samples was adjusted to 200 µg/mL. 2.11 Cell Culture RAW 264.7 cells were maintained in Dulbecco’s modification of Eagle’s medium supplemented with 10% heat-inactivated FBS at 37 ℃ in a 5% CO2 atmosphere. The medium was replaced every 2 d. To assess cell protection, RAW 264.7 cells were seeded into 96-well plates at a density of 5 × 103 cells/mL (100 µL per well) for cell viability assays and into 24-well plates at a density of 1.5 × 104 cells/mL (500 µL per well) for the nitric oxide (NO) assay and enzyme linked immunosolvent assay (ELISA). 2.12 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay Cell viability was assessed using the MTT assay following protocols outlined in previous studies [42,43]. RAW 264.7 cells were seeded into 96-well plates and allowed to adhere for 24 h. After removing the culture medium, the cells were treated with various concentrations of the fractions diluted in serum-free medium and incubated for 24 h. MTT solution (5 mg/mL) was then added to each well, and the cells were incubated for 3 h at 37 ℃ in a 5% CO₂ atmosphere. After incubation, the supernatant was carefully discarded and the formazan crystals produced by metabolically active cells were solubilized using dimethyl sulfoxide. Absorbance was recorded at 540 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA) to evaluate cell viability. The viability percentage was determined by comparing the absorbance values of the treated groups to those of the untreated control group, which was considered to have 100% viability. 2.13 NO Assay NO concentrations were determined by the nitrite concentration using the Griess reaction, based on the method outlined in a previous study [44]. RAW 264.7 cells were incubated in a serum-free medium with different concentrations of the fraction samples for 2–3 h after removing the existing medium. Subsequently, lipopolysaccharide (LPS) (1 ng/mL) was added to an equal volume of serum-free medium, and the cells were incubated for 20 h to induce stimulation. Following this, 0.1 mL of the reaction mixture was collected and placed in a 96-well microplate, and Griess reagent was added. The mixture was allowed to react in the dark at room temperature for 15 min, and absorbance was measured at 540 nm. NO scavenging activity was calculated as a percentage by comparing the absorbance of the samples at 540 nm to that of the blank sample. 2.14 Measurement of Cytokine Production (ELISA) RAW 264.7 cells were seeded in 96-well cell culture plates for 24 h. After removing the medium, RAW 264.7 cells were treated with various concentrations of the fraction samples in serum-free medium for 2–3 h. LPS at a final concentration of 1 ng/mL was then treated and stimulated in the same volume in serum-free medium for 20 h. Cell-free supernatants were collected and the cytokine content was measured using a Mouse IL-1, IL-6 and TNF-α ELISA kit (Komabiotech, Korea) using ELISA. The optical density of the microplates was measured at 450 nm. 2.15 Amino Acid Profiling Amino acid profiling was performed according to the Waters Amino Acid Analysis AccQ Tag Manual. A Waters AccQ-Tag∙Fluor reagent kit was used for derivatization of the sample and standard. An amino acid standard (Sigma–Aldrich Co., USA) was used. The sample amount ranged from 0.02–0.08 µg (20–1,000 pmol). The other system conditions are listed in Table 2 . Table 2 Chromatographic conditions of FPLC. Instrument Condition Column Waters AccQ-Tag (Waters, U.S.A) Mobile phase A: 10% ( v/v ) Waters AccQ-Tag eluent A in water B: 60% ( v/v ) Acetonitrile in water (pH 5.02) Detector (Detection) Waters 474 scanning fluorescence detector Flow rate 1 mL/min Injection volume 10 µL 2.16 Peptide Identification by Mass Spectrometry The fraction samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Ultimate 3000 HPLC system (Dionex, Sunnyvale, CA, USA) and a Micro-TOF III mass spectrometer (255748, Bruker Daltonics, Germany) at Proteinworks Co. (Daejeon, Korea). The analytical conditions are listed in Table 3 . Table 3 Chromatographic conditions for liquid chromatography-mass spectrometry (LC-MS). Instrument Condition Column Porshell 120 EC-C18 (2.1 X 100 nm, 2.7 µm, Agilent) Injection volume 5 µL Flow rate 0.2 mL/min Column temperature 30 ℃ Mobile phase / Time (min) Solvent composition A (%) B (%) 0 95 5 5 95 5 28 70 30 33 5 95 40 5 95 41 95 5 46 95 5 Note: Mobile phase A consisted of water containing 0.2% (v/v) fluoroacetic acid (FA), and mobile phase B consisted of acetonitrile containing 0.2% (v/v) FA. 2.17 Statistical Analysis A one-way analysis of variance was performed to assess statistical differences among the experimental groups. The analysis revealed significant group effects ( p < 0.05), indicating that the mean values differed among the treatments. Microsoft Excel was used for data visualization and group comparisons to facilitate a clear interpretation of the variance patterns. 3. Results 3.1 LAB Screening and Identification Morphological characteristics of the isolated LAB strains were examined under microscopy. Various microorganisms were observed, and all strains were confirmed to be Gram-positive. Ten strains were identified using 16S-rRNA sequencing. Based on 16S-rRNA sequence analysis, the lactic acid-producing strains were classified into eight strains of Leuconostoc mesenteroides , one strain of Pediococcus pentosaceus , and one strain of Lactiactobacillus sakei . The 16S-rRNA gene sequences are listed in Table 4 . The isolated strains were named Leuconostoc mesenteroides DC1, 2, 3, 4, 5, 7, 8, and 9, Pediococcus pentosaceus DC6, and Lactilactobacillus sakei DC10. Table 4 Identification of strains isolated from Dongchimi with 16S-rRNA sequence analysis. Strain 16s-rRNA sequence species ID (%) DC1 Leuconostoc mesenteroides DS3_KCTC13016BP 16S ribosomal RNA gene, partial sequence 99 DC2 Leuconostoc mesenteroides strain DS3_KCTC13016BP 16S ribosomal RNA gene, partial sequence 99 DC3 Leuconostoc mesenteroides DNA, complete genome, strain: LK-151 99 DC4 Leuconostoc mesenteroides strain SCWL 03 16S ribosomal RNA gene, partial sequence 99 DC5 Leuconostoc mesenteroides strain DS3_KCTC13016BP 16S ribosomal RNA gene, partial sequence 99 DC6 Pediococcus pentosaceus strain DAEM1 16S ribosomal RNA gene, partial sequence 99 DC7 Leuconostoc mesenteroides strain DS3_KCTC13016BP 16S ribosomal RNA gene, partial sequence 99 DC8 Leuconostoc mesenteroides strain DS3_KCTC13016BP 16S ribosomal RNA gene, partial sequence 99 DC9 Leuconostoc mesenteroides strain DS3_KCTC13016BP 16S ribosomal RNA gene, partial sequence 99 DC10 Lactilactobacillus sakei strain TUB/2013/10(3–72) 16S ribosomal RNA gene, partial sequence 99 3.2 Tolerance to Artificial Gastric and Bile Juice The LAB strains demonstrated strong survival ability in artificial gastric juice during 2 h of incubation at pH 3.0, 2.0, and 1.5 at 37°C, which simulates human gastric transit time and pH (approx. 120 min; pH 1.5–3.0) [45,46]. The observed survival rates (Table 5 ) are consistent with previous findings, indicating that tolerance varied among strains. Similar tolerance has been reported in strains from Leuconostoc , Lactobacillus , and Pediococcus genera [47]. Following gastric acid exposure, LAB strains were further tested for bile tolerance by incubation in MRS broth containing 0.3% oxgall for 24 h at 37°C. The strains exhibited high survival rates under bile salt conditions (Table 5 ). These results are consistent with previous reports on bile salt resistance in LAB isolated from food sources. Collectively, these findings highlight the robustness of acid and bile tolerance which is essential for effective probiotic applications [48–50]. Table 5 Tolerance to artificial gastric and bile juice. CFU – colony-forming units. Strains Log (CFU/mL) Control pH 3.0 pH 2.0 pH 1.5 DC1 8.93 ± 0.16 6.06 ± 0.2 4.52 ± 0.21 4.52 ± 0.21 DC2 8.69 ± 0.12 6.08 ± 0.11 4.91 ± 0.12 4.91 ± 0.12 DC3 8.97 ± 0.19 6.85 ± 0.13 5.57 ± 0.18 5.57 ± 0.18 DC4 8.87 ± 0.11 6.46 ± 0.13 4.64 ± 0.18 4.64 ± 0.18 DC5 8.39 ± 0.14 6.75 ± 0.36 4.51 ± 0.24 4.51 ± 0.24 DC6 8.85 ± 0.11 6.34 ± 0.19 4.46 ± 0.16 4.46 ± 0.16 DC7 8.85 ± 0.18 6.51 ± 0.18 4.59 ± 0.14 4.59 ± 0.14 DC8 8.19 ± 0.11 7.66 ± 0.17 5.73 ± 0.11 5.73 ± 0.11 DC9 8.38 ± 0.13 6.82 ± 0.16 5.88 ± 0.13 5.88 ± 0.13 DC10 8.53 ± 0.14 6.73 ± 0.15 6.01 ± 0.12 6.01 ± 0.12 LA-5 8.76 ± 0.14 6.97 ± 0.15 5.93 ± 0.15 5.93 ± 0.15 Note: Leuconostoc mesenteroides DC1, DC2, DC3, DC4, DC5, DC7, DC8, and DC9; Pediococcus pentosaceus DC6; Lactilactobacillus sakei DC10; Lactilactobacillus acidophilus LA-5 (Chr. Hansen, Denmark) were used. CFU refer to the colony-forming units. All data are mean values ± standard error (n = 3). 3.3 Proteolytic Activity Bacterial proteolytic activity was assessed based on the ability to produce clear zones on skim milk agar [51,52]. The agar well diffusion assay revealed that the 10 LAB strains initially screened for proteolytic activity could hydrolyze milk proteins. A clear zone around the wells was present for these 10 strains, indicating their proteolytic activity. Of the 10 tested strains, L. sakei DC10 was the most efficient for proteolytic activity on skim milk agar (clear zone diameter > 23 mm; Table 6 ). Table 6 Proteolytic activity of lactic acid bacteria (LAB) strains. Strains Diameter of clear zone (mm) 24h 48h 72h DC1 13 ± 0.03 16 ± 0.13 22 ± 0.11 DC2 12 ± 0.09 13 ± 0.07 18 ± 0.04 DC3 12 ± 0.07 14 ± 0.09 19 ± 0.07 DC4 13 ± 0.07 15 ± 0.15 20 ± 0.07 DC5 13 ± 0.1 14 ± 0.21 21 ± 0.03 DC6 11 ± 0.03 15 ± 0.13 19 ± 0.13 DC7 14 ± 0.13 16 ± 0.2 22 ± 0.09 DC8 13 ± 0.1 16 ± 0.12 18 ± 0.12 DC9 14 ± 0.13 17 ± 0.18 22 ± 0.13 DC10 13 ± 0.11 18 ± 0.17 23 ± 0.18 LA-5 13 ± 0.13 16 ± 0.13 22 ± 0.11 Note: Leuconostoc mesenteroides DC1, DC2, DC3, DC4, DC5, DC7, DC8, and DC9; Pediococcus pentosaceus DC6; Lactilactobacillus sakei DC10; Lactilactobacillus acidophilus LA-5 (Chr. Hansen, Denmark) were used. All data are mean values ± standard error of the mean (n = 3). 3.4 DH of WPC solution LAB possess a complex system of proteinases and peptidases that enable them to use milk protein as a source of amino acids and nitrogen. Hydrolysis during whey protein fermentation was monitored by analyzing the amount of primary amino groups released and the soluble protein content. In the present study, we inoculated LAB into a whey protein solution for fermentation. The degree of whey protein hydrolysis by LAB is shown in Fig. 1 . During fermentation, whey proteins were hydrolyzed by LAB, resulting in an increased number of free amino groups and peptides (Fig. 1 ). The DH of WPC solutions fermented by LAB rapidly increased from 0–4 h. The DH progressed slowly after 16 h (DH %), indicating that whey protein hydrolysis reached its highest proteolytic rate at 16 h. The extent of proteolysis was time dependent. These results indicated that the LAB strain showed proteolytic activity in the whey protein solution (Fig. 1 ). 3.5 SDS-PAGE Figure 2 shows the SDS-PAGE analysis of the whey proteins using L. sakei DC10. Protein bands with molecular weights ranging from 13–250 kDa were identified. The main components of whey protein such as lacttoferrin, bovine serum albumin (BSA), immunoglobulin, β-lactoglobulin and α-lactalbumin etc., were found in the band from 14–100 kDa. Components with higher molecular weights were degraded during fermentation. After 16 h of fermentation, lactoferrin, BSA, and immunoglobulin were degraded. 3.6 FPLC The fermented WPCs were identified using SDS-PAGE and separated on a preparative scale. L. sakei DC10 was selected based on the DH and SDS-PAGE results. Fractions of the WPCs fermented with DC10 were isolated by molecular weight using a Hiprep 16/60 Sephacryl S-100 HR column in a preparative liquid chromatography system (Fig. 3 ). 3.7 Calcium Solubilization Capacity Casein phosphopeptide (CPP) contains phosphoserine and binds calcium to promote its calcium-solubilizing ability. In this study, calcium solubility was measured to determine whether each fraction promoted a calcium-solubilizing ability similar to that of CPP. The experimental results showed that CPP had a high calcium solubility of 93.67%. The phosphoserine contained in CPP dissolves calcium and increases its solubility [53]. All of the fractions from fermented WPC solution with L. sakei DC10 had lower calcium solubilization capacity than CPP but showed higher calcium solubilization capacity than unhydrolyzed whey protein, of which fraction 3 had lower than CPP but approximately 85% calcium solubilization capacity (Fig. 4 ). This may have indicated the potential calcium solubilization capacity. 3.8 Antioxidant Activity ABTS Assay The Trolox equivalent antioxidant capacity assay measures the ability of a compound to eliminate or scavenge radicals compared to Trolox (Vitamin E) as an antioxidant reference [54]. The ABTS assay results are shown in Fig. 5 . Except for F1 and F4, the other fractions showed higher ABTS radical scavenging activities than non-hydrolyzed whey protein. Fraction 3 had the highest value. DPPH Assay DPPH is a stable free radical that has been widely accepted as a tool for estimating the free radical-scavenging activities of antioxidants [55]. The antioxidant activity of each fraction was evaluated by measuring its DPPH scavenging activity. DPPH scavenging activity values were statistically analyzed based on the standard curve of the Trolox solution. Fractions 2 and 3 showed a DPPH radical scavenging ability similar to that of CPP (Fig. 5 ). FRAP Assay The decrease in absorbance is proportional to the antioxidant content [56]. The FRAP assay is a direct method for measuring antioxidant ability. This is based on the ability of antioxidants to reduce Fe3 + to Fe2 + in the presence of TPTZ, forming an intense blue Fe2+–TPTZ complex with absorption at 593 nm, which is associated with their electron-donating ability to break the free radical chain reaction [55]. According to the results (Fig. 5 ), fraction 3 exhibited a higher iron reduction capacity than CPP. 3.9 MTT Assay Cytotoxicity tests based on the MTT assay are widely used for in-vitro toxicology experiments. The experimental results confirmed that increasing the concentration improved cell viability. In addition, all fractions showed > 80% cell viability (Fig. 6 ). Therefore, we confirmed that the macrophage fractions were not cytotoxic. 3.10 NO Assay NO plays important roles in blood coagulation, blood pressure regulation, and immune function in cancer cells. However, it is oxidized to reactive oxygen species and converted into active NO. NO produces oxidants that cause cytotoxicity. NO production in cells exposed to inflammatory mediators is increased by tissue damage and inflammatory diseases. According to the results of this study, NO production was inhibited as the concentration of the fractions increased (Fig. 7 ). The MTT experiments showed that the inhibition of NO production was not caused by cytotoxicity. 3.11 Measurement of Cytokine Production (ELISA) Direct or indirect interactions between immune cells are needed to maintain immune balance. Cytokines can induce the proliferation, differentiation, and changes in the function and activity of various immune cells. Disease is mostly associated with inflammation, and inflammatory cells secrete inflammatory cytokines that induce inflammation [57]. Expression levels of IL-1α, IL-6 and TNF-α were measured in the present study. According to the results of cytokine measurements using ELISA, production of three cytokines (IL-1α, IL-6, and TNF-α) was significantly lower than LPS(+) group. Based on these results, anti-inflammatory activity was observed in the hydrolysate fractions. In addition, the low expression level of TNF-α from macrophage effect by samples prevented the activation of pro-inflammatory cytokines which was produced by the expression of TNF-α (Fig. 8 ). It is believed to have reduced the inflammatory response. 3.12 Amino Acid Profiling Based on the above experimental results, fraction 3 was selected as the fraction with superior functional properties and chosen for identification of its amino acid composition and peptide sequencing. By confirming the amino acid sequences of the selected fractions using the AccQ-Tag system, 17 amino acid species and several species of unknown amino acids were identified (Fig. 9 ; Table 7 ). Fraction 3 had the highest leucine content and lowest cysteine content. Table 7 Amino acids concentrations of fraction 3. Amino acid WPC (%) Fraction 3 (%) Asp 7.55 2.78 Thr 8.49 4.83 Ser 6.53 3.31 Glu 17.8 1.89 Pro 8.27 2.88 Gly 3.87 0.43 Ala 9.07 8.16 Val 4.9 17.49 Cys 0.68 0.13 Met 1.66 0.34 Ile 4.93 13.58 Leu 10.36 21.1 His 1.73 1.91 Phe 2.81 8.45 Lys 8.29 5.86 His 1.42 1.91 Arg 1.64 4.95 Total molar ratio 100 100 3.13 Peptide Identification by Mass Spectrometry Fraction 3 was subjected to LC-MS and LC-MS/MS for peptide separation and sequence identification, respectively. MS analysis confirmed the presence of many different peptides; however, four were fully sequenced, and additional purification of fraction 3 was likely required. Four peptides were identified from fraction 3 (Table 8 ) with molecular masses of 905.49, 658.36, 893.96 and 883.46 Da. Molecular mass and tandem mass spectrometry results for the peptides are shown in Figs. 10 and 11, respectively. Table 8 Peptide sequencing results of fraction 3. Peak name m/z 1) RT (min) 2) Sequencing 1 905.49 16.1 TVQVTSTAV 2 658.36 22.3 DKTEIPTINTIA 3 893.96 22.3 DKTEIPTINTIASGEPT 4 883.46 26.3 SLVYPFPGPIHNSLPQ 4. Discussion The fermentation of WPC by LAB is an effective alternative for producing functional dairy foods with enhanced nutritional and functional properties owing to the high biological value of whey proteins [58]. In the present study, 10 LAB strains isolated from Dongchimi were screened, and three proteolytic strains, Leuconostoc mesenteroides DC3, L. mesenteroides DC8, and Latilactobacillus sakei DC10, were selected based on physiological and functional assays. The DH of WPC increased in a similar pattern for all selected LAB strains, showing no further significant changes after 16 h of fermentation. SDS-PAGE analysis confirmed the degradation patterns of the major whey protein components. WPC fermented with DC10 exhibited proteolytic patterns similar to those observed with other effective LAB strains [59]. The peptide fractions of the fermented whey were analyzed for molecular weight using a HiPrep 16/60 Sephacryl S-100 HR column. The peptides in fraction 3 showed the highest calcium solubilization capacity. Peptides generally bind calcium ions by forming a chelate through electron pairs on the α-amino group and carboxyl group, with side chains of certain amino acids further stabilizing the complex [60]. In particular, leucine, aspartic acid, and glutamic acid play key roles in such structural interactions, and amino acid analysis of fraction 3 revealed high levels of BCAAs (leucine, isoleucine, and valine) [61]. In addition to direct binding, BCAAs can enhance intestinal calcium absorption by upregulating Vitamin D receptor expression, thereby promoting Vitamin D-dependent pathways [62]. BCAAs activate both transcellular and paracellular transport mechanisms in the intestinal epithelium and regulate calcium and phosphorus transporter expression in the kidney to maintain blood calcium homeostasis [63]. Fraction 3 showed antioxidant activity comparable to that of CPP. LC/MS analysis identified four peptide sequences in fraction 3, among which TVQVTSTAV and SLVYPFPGPIHNSLPQ are well-known antioxidative peptides [64]. In the anti-inflammatory assays, fraction 3 significantly inhibited NO production and suppressed pro-inflammatory cytokines (IL-1α, IL-6, and TNF-α) while improving cell viability, consistent with previous reports showing enhanced bioactivities of hydrolyzed whey proteins compared to that of their non-hydrolyzed forms [65]. In other studies, TVQVTSTAV and DKTEIPTINTIA were reported as peptides with notable antimicrobial activities and SLVYPFPGPIHNSLPQ exhibited immunomodulatory effects [64]. These results suggested that fraction 3 obtained in the present study may have antioxidant, anti-inflammatory, antibacterial, and immunomodulatory properties. In this study, we confirmed that fermentation of WPC using specific LAB produced bioactive peptide fractions with diverse physiological functions, including calcium solubility and antioxidant, anti-inflammatory, and antibacterial activities. Furthermore, this study demonstrated the potential of dairy proteins in the design of diverse functional and specific bioactive ingredients. Based on these biological activities, future research should focus on elucidating its mechanism of action, conducting clinical trials to evaluate its stability and bioavailability, and demonstrating its potential application as a functional food. Declarations Funding This research was financially supported by the Ministry of Agriculture, Food and Rural Affairs through the project “Next-generation Food Processing of High Value-added Food Technology Development” (Project No. RS-2024-00407186). Author Contribution Conceptualization: Kim CH.; Data curation: Yoo JS and Lee SJ.; Formal analysis: Yoo JS, Lee SJ and Song SE.; Methodology: Yoo JS, Lee SJ and Song SE.; Software: Yoo JS, Lee SJ and Song SE.; Validation: Kim CH.; Investigation: Yoo JS, Lee SJ and Song SE.; Writing - original draft: Yoo JS.; Writing - review & editing: Lee SJ and Kim CH. All authors have read and agreed to the published version of the manuscript. Acknowledgement This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the High Value-added Food Technology Development Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA) (RS-2024-00407186). 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16:24:03","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":160002,"visible":true,"origin":"","legend":"","description":"","filename":"ea1ba39643b54decbb4f69f38a9b49e81structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/925093b98e8f7d4ddde2a723.xml"},{"id":95108937,"identity":"a89dfad7-283f-497b-8b19-3bf6ae9bc770","added_by":"auto","created_at":"2025-11-04 11:32:19","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":179308,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/10b8f447cc89cfc592e1dbc4.html"},{"id":95108902,"identity":"7a7cffde-8d82-475d-ace4-2c8027057efc","added_by":"auto","created_at":"2025-11-04 11:32:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80872,"visible":true,"origin":"","legend":"\u003cp\u003eDegreeof hydrolysis (DH %) of fermented whey protein concentrate (WPC) with LAB strains. Note: \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e DC3 and DC8, and \u003cem\u003eLatilactobacillus sakei\u003c/em\u003e DC10 were used in this study. All data are mean values ± standard deviation (n = 3).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/23bfb6954300c29d665523ee.png"},{"id":95108904,"identity":"ad51021f-62f8-47e7-af61-b41c6ad4117a","added_by":"auto","created_at":"2025-11-04 11:32:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":309589,"visible":true,"origin":"","legend":"\u003cp\u003eSodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of fermented WPC with DC10. Note: (a) raw bovine milk. (b) Lane 1 contains Precision Plus Protein Dual Xtra standards (Bio-Rad, U.S.A.), which provide 12 pre-stained recombinant protein bands ranging from 2–250 kD, including 9 blue-stained and 3 pink reference bands. Lanes 2, 3, 4, 5, and 6 represent hydrolysates obtained after 0, 4, 8, 12, and 16 h of hydrolysis, respectively. DC10 refers to \u003cem\u003eLatilactobacillus sakei\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/6ee90f9df29acbba96525c97.png"},{"id":95108903,"identity":"8bd1b5f8-c377-41c4-8392-da22030ede8e","added_by":"auto","created_at":"2025-11-04 11:32:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":80219,"visible":true,"origin":"","legend":"\u003cp\u003eSeparation of the WPC fermented with DC10 using an FPLC system with a Hiprep 16/60 Sepacryl S-100R column. Note: \u003cem\u003eLatilactobacillus sakei\u003c/em\u003eDC10 was used in this study. Absorbance was measured at 280 nm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/6d4676f35ad5f0219e0e97af.png"},{"id":95223865,"identity":"11d37e11-f83b-462b-9fd6-43d7287fb82e","added_by":"auto","created_at":"2025-11-05 16:22:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51028,"visible":true,"origin":"","legend":"\u003cp\u003eCalciumsolubilization capacity of fractions from fermented WPC-80 with DC10. Note: The control sample consisted of WPC-80 solution after pasteurization. CPP refers to casein phosphopeptide. F# indicates the fraction number obtained from the FPLC system. All samples were adjusted to a protein concentration of 200 µg/mL. All data are as mean values ± standard deviation (n = 3). DC10 represents Latilactobacillus sakei. Superscript letters (a–d) indicate significant differences at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/734e47561b0fdf59d33a71a1.png"},{"id":95224726,"identity":"0c4f8b2a-a033-4a52-b4b5-3f4e5e301b4b","added_by":"auto","created_at":"2025-11-05 16:24:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":104482,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidantactivities of fractions from fermented WPC-80 with DC10. Note: The control sample consisted of WPC-80 solution after pasteurization. CPP refers to casein phosphopeptide. F# indicates the fraction number obtained from the FPLC system. DPPH refers to 2,2-diphenyl-1-picrylhydrazyl. ABTS refers to 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). All samples were adjusted to a protein concentration of 200 µg/mL. All data are mean values ± standard deviation (n = 3). DC10 represents Latilactobacillus sakei. Superscript letters (a–d) indicate significant differences at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/9776ca4b2419f7835e23991a.png"},{"id":95224463,"identity":"6e4e730c-7b7e-4c84-80da-db79c16518ad","added_by":"auto","created_at":"2025-11-05 16:23:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":232895,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of fractions from fermented WPC-80 with DC10 on cell viability in RAW 264.7 cells. Note: The control refers to untreatmentWPC-80 solution. CPP indicates casein phosphopeptide. F# denotes the fraction number obtained from the FPLC system. Samples were prepared at protein concentrations of 2.0, 1.0, 0.5, and 0.25 mg/mL. All data are mean values ± standard deviation (n = 3). Superscript letters (a–d) indicate significant differences at p \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/e76bd02c9b44aab193359be8.png"},{"id":95224480,"identity":"9354c0fd-7154-441e-a0c8-49ab089ef027","added_by":"auto","created_at":"2025-11-05 16:23:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":58528,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of fractions from fermented WPC-80 with DC10 on inhibition of NO production in LPS-activated RAW 264.7 cells. Note: CPP indicates casein phosphopeptide. F# denotes the fraction number obtained from the FPLC system. Samples were prepared at protein concentrations of 1.0 mg/mL. All data are mean values ± standard deviation (n = 3). Superscript letters (a–d) indicate significant differences at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/9c762891426e14c13d901213.png"},{"id":95224022,"identity":"95bb28eb-6ee6-4e89-8014-f6099eadc383","added_by":"auto","created_at":"2025-11-05 16:23:13","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":185000,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of fractions from fermented WPC-80 with DC10 on inhibition of cytokine production in LPS-activated RAW 264.7 cells. Note: CPP refers to casein phosphopeptide. F# denotes the fraction number obtained from the FPLC system. Samples were prepared at a protein concentration of 1.0 mg/mL. All data are mean values ± standard deviation (n = 3). Superscript letters (a–d) indicate significant differences at \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/5788784d039e206c38a1502f.png"},{"id":95108912,"identity":"7dd30b97-6181-4184-90af-0c98a13a0639","added_by":"auto","created_at":"2025-11-04 11:32:18","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":112847,"visible":true,"origin":"","legend":"\u003cp\u003eHigh-performance liquid chromatography chromatogram of amino acids of fraction 3.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/9b636abe10fe14f39d734082.png"},{"id":95108914,"identity":"8e5e7791-6f1b-40d6-970a-a615f32b503f","added_by":"auto","created_at":"2025-11-04 11:32:18","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":20863,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 3.\u003c/strong\u003e Liquidchromatography-mass spectrometry base peak chromatogram of fraction 3.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/97b51b73a678d40e87a1f2dc.png"},{"id":95108921,"identity":"43660f00-b8a5-4600-8a92-2d4ed0d0615f","added_by":"auto","created_at":"2025-11-04 11:32:18","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":281860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 4.\u003c/strong\u003e Mass spectrometric de-novo sequencing\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/3ad90ac15b2c67fa050e3007.png"},{"id":95230512,"identity":"a7ad36e0-f1a5-42b4-b5ac-baafa84d16bc","added_by":"auto","created_at":"2025-11-05 16:37:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2737446,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7899609/v1/12cc77de-a186-4fed-a574-401d3093ef9d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functional characteristics of peptides from whey proteins fermented with lactic acid bacteria isolated from Dongchimi","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eProteins can be used as substrates to produce bioactive peptides (BAPs), and can also be transformed into high value-added products such as BAPs, which is a successful management strategy [1\u0026ndash;3]. Food proteins are currently being studied beyond their nutritional characteristics because of their positive effect on human health related to specific sequences encrypted into native proteins, known as BAPs. Such sequences are inactive when present in the parent protein, but can be released after protein hydrolysis during gastrointestinal digestion, in-vitro enzymatic hydrolysis, or microbial fermentation [4,5]. BAPs are short amino acid sequences with inactive precursor protein [6].\u003c/p\u003e\u003cp\u003eThe potential health benefits associated with BAPs consumption have attracted the interest of many researchers [7]. The release of BAPs from their parent proteins by proteolysis is affected by several factors such as hydrolysis time, pH, temperature, and enzyme\u0026ndash;substrate ratios [8].\u003c/p\u003e\u003cp\u003eWhey protein is a mixture of globular proteins isolated from whey, a liquid material created as a byproduct of cheese production [9]. Whey proteins constitute 15\u0026ndash;20% of total milk proteins [10]. The key components of whey protein have been characterized as β-lactoglobulins and α-lactoalbumins [11]. Other constituents of whey protein include immunoglobulins, serum albumin, and lactoferrin [12]. Protein quality depends on the constituent amino acids, and whey protein has one of the highest standards. Whey proteins are also rich in essential branched-chain amino acids (BCAAs) [13]. BCAAs, including leucine, isoleucine, and valine, play crucial roles in metabolism, blood glucose homeostasis, and neural function [14]. Leucine regulates skeletal muscle protein synthesis [15]. Another essential amino acid, cysteine, is a building block of glutathione, which is a dietary antioxidant [16]. It is vital to combat oxidative stress and prevent diseases caused by redox imbalances [17]. Several studies have reported that whey proteins are valuable sources of BAPs because of their high nutritional value and the wide range of specific BAPs they release [18,19].\u003c/p\u003e\u003cp\u003eThe enzymatic hydrolysis of food proteins is among the most promising methods for BAP production [20]. However, industrial production of BAPs remains limited by the lack of suitable large-scale technologies and the high cost of enzymes used for protein hydrolysis [21]. Whey proteins are highly hydrophobic and are resistant to enzymatic hydrolysis. Therefore, bioactive fragments may not be released from whey proteins despite the high biological potential of the protein precursor [22]. Therefore, hydrolysates produced by enzymes obtained from alternative sources, such as new strains of lactic acid bacteria (LAB) that exhibit high proteolytic activity, can be a source of several new peptides with specific bioactivities at a low production cost [23].\u003c/p\u003e\u003cp\u003eLAB, which have been used for lactic acid fermentation since ancient times, produce proteases that reduce the allergenic potential of milk proteins. This effect depends on the bacterial strain and regulation and optimization of proteolysis [24\u0026ndash;26]. Food proteins and α-lactalbumin fermented by LAB can increase digestibility and hydrolyze allergenic peptides [27]. This approach can be used to develop new BAPs, and whey bioactive peptides can be targeted as compounds that exert protective effects against several diseases.\u003c/p\u003e\u003cp\u003eThe proteolytic activity of LAB is exerted in a strain-dependent manner, resulting in a variety of proteolytic activities [28]. Moreover, due to the specificity of the enzyme to the substrate, the peptide composition of hydrolysates, and therefore peptide activities, change [29]. This suggests that LAB can generate a considerable variety of BAPs and highlights the need to correctly select the strains that will be applied for BAP production [28].\u003c/p\u003e\u003cp\u003eIn this regard, the purpose of this study was to develop bioactive peptides derived from fermented whey protein with superior physiological properties, calcium solubilization ability, and effects on immunity. This research is significant for the potential application of calcium-binding peptides as ingredients in functional foods.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 LAB Screening and Identification\u003c/h2\u003e\u003cp\u003eLAB were isolated from homemade Dongchimi collected from different regions of Korea. Dongchimi samples (10 mL) were mixed with 40 mL of phosphate-buffered saline (PBS) (pH 7.4) and homogenized by vortexing. Diluted samples (1 mL) were serially diluted with 0.85% sterile saline, and 100 \u0026micro;L of each dilution was then spread onto De Man\u0026ndash;Rogosa\u0026ndash;Sharpe (MRS; BD Difco, USA) agar containing 0.02% sodium azide, and incubated at 37\u0026deg;C for 48 h. LAB colonies were selected from the plates and inoculated into MRS broth for stock preparation. For long-term storage, all cultures were maintained as frozen stocks at -72\u0026deg;C in MRS broth containing 20% glycerol. Screening of selected isolates was performed based on the colony shape on plates, Gram staining, and observation of strain morphology using a microscope. The selected strains were identified by 16S rRNA gene sequencing. The 16s rRNA sequences were analyzed using the GenBank database (Macrogen, Korea), and identification was performed based on 16S rRNA sequence homology.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Tolerance to Artificial Gastric Juice and Bile\u003c/h2\u003e\u003cp\u003eTolerance to artificial gastric juice and artificial bile acid was measured according to the method developed in a previous study [30]. To investigate the survival of LAB strains under acidic conditions, each strain was harvested by centrifugation at 7,000 \u0026times;g for 10 min, washed twice with PBS, then inoculated (1%) into MRS broth acidified to pH 1.5, 2.0, or 3.0 (adjusted using HCl) containing 1,000 units of pepsin (Sigma\u0026ndash;Aldrich, St. Louis, MO, USA) or into non-acidified MRS broth. The cultures were incubated at 37\u0026deg;C for 2 h. Following gastric juice treatment, strains were subjected to bile tolerance testing by centrifugation at 7,000 \u0026times;g for 10 min, washing twice with PBS (pH 7.4), and incubation at 37\u0026deg;C for 24 h in artificial bile acid consisting of MRS broth supplemented with 0.3% oxgall (Difco, Detroit, MI, USA). Finally, viable cell counts were determined by serial 10-fold dilutions in 0.85% saline, and plating 1-mL aliquots evenly on BCP agar plates which were then aerobically incubated at 37\u0026deg;C for 48 h before the number of colony-forming units (CFU) were estimated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Proteolytic Activity\u003c/h2\u003e\u003cp\u003eProteolytic activity was assessed by an agar well diffusion method according to the previously described method [31]. For the agar well-diffusion test, selected LAB strains were screened for proteolytic activity by agar-well diffusion test on skim milk containing 2.0% (w/v) agar (Difco, Detroit, MI, USA). The supernatant (100 \u0026micro;L) obtained by centrifuging bacterial cultures grown in MRS broth at 5000 \u0026times;g for 10 min were loaded into 9 mm diameter wells of skim milk agar plates. Proteolysis resulted in the formation of a clear zone around the wells. Protease activity was determined by estimating the diameter of clear zone area for 72 h at 37\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Whey Protein Concentrate (WPC) Solution Preparation\u003c/h2\u003e\u003cp\u003eWPC (Meegle, Germany) was reconstituted with distilled water to a final concentration of 10% (w/v), and glucose (Sigma\u0026ndash;Aldrich Co., USA) was added at a concentration of 5% (w/v) for growth of LAB. The solution was adjusted to the optimum active pH for LAB using 1 N NaOH (Sigma\u0026ndash;Aldrich Co., USA), sterilized for 30 min at 80\u0026deg;C, and then stored at 4 ℃ for \u0026le;\u0026thinsp;1 week [32].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Fermentation Conditions\u003c/h2\u003e\u003cp\u003eLAB were cultured in MRS broth at 37\u0026deg;C for 18 h. The cultures were centrifuged at 8,000 \u0026times;g for 15 min at 4\u0026deg;C and the supernatant was discarded. The resulting cell pellets were resuspended in 10 mL of 1 X PBS to prepare the bacterial suspension. This suspension was inoculated into the WPC solution at a final concentration of 1% (v/v) and fermented at 37\u0026deg;C with shaking at 150 rpm in a water bath for 20 h. The fermented solution was subsequently heat-treated at 95\u0026deg;C for 10 min to obtain the final fermentation product.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Degree of Hydrolysis (DH) of WPC Solution\u003c/h2\u003e\u003cp\u003eThe DH of the WPC solution was measured in fermented WPC samples (every 4 h for 20 h) using the 2,4,6-trinitrobenzenesulfonic acid (TNBS) method [33]. The TNBS reagent consisted of 0.1% (v/v) TNBS in distilled water. All samples and standard solutions were prepared in 1% (w/v) sodium dodecyl sulfate (SDS) solution. Samples (2 mL) were dissolved in distilled water to 10% (v/v) in a test tube, and 4 mL of 0.72 N trichloroacetic acid (TCA) was added. Samples were then incubated at 25\u0026deg;C for 20 min. Filtration through a 0.45-\u0026micro;m sterile polyvinylidene fluoride (PVDF) syringe membrane filter (SIGL, Germany) to takes 0.2 mL of samples were added to test tubes containing 2 mL of 0.2125 M sodium phosphate buffer (pH 8.2). The TNBS reagent were added 2 mL each test tube and then incubated at 50\u0026deg;C for 60 min to exclude light. After incubation, the reaction was stopped by addition of 4 mL of 0.1 N HCl to each test tube. The samples were then allowed to cool at room temperature for 30 min before absorbance values were measured at 420 nm using a UV-Vis spectrophotometer (X-ma 1200, Human Corp., Korea). DH is defined as the proportion of the total number of peptide bonds cleaved during hydrolysis [33], and was calculated as follows:\u003c/p\u003e\u003cp\u003eDH, % = h/htot x 100, (1)\u003c/p\u003e\u003cp\u003ewhere h is the number of hydrolyzed peptide bonds, and htot is the total number of peptide bonds present [34]. Leucine was used as a standard (at concentrations ranging from 0\u0026ndash;2 mM) to determine the free amino group content of the samples. Each sample was analyzed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)\u003c/h2\u003e\u003cp\u003eFermented WPC solution was analyzed by SDS-PAGE according to the method of [35] using a 12% polyacrylamide separation gel under reducing conditions with a Mini-PROTEAN III system (Bio-Rad, USA). The operating conditions were set to 20 V for 20 min, followed by 100 V for 2 h. After staining with Coomassie blue R-250 (water:methanol:acetic acid [45:45:10, v/v/v]) and water:methanol:acetic acid (45:45:10 v/v/v) solutions, SDS-PAGE analysis was performed using Precision Plus protein dual xtra standards (Bio-Rad, U.S.A).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Fast Protein Liquid Chromatography (FPLC)\u003c/h2\u003e\u003cp\u003eTo obtain the peptide fractions from the WPC solution fermented with DC10, gel filtration was performed using a preparative chromatography system (Waters, U.S.A). A Waters preparative pump and Preparative liquid chromatography W600 were used with a Waters Dual λ Absorbance detector W2487 and Waters W717 Autosampler. In the first purification step, Fermented WPC solution was dissolved in 50 mM sodium phosphate buffer with 0.15 M NaCl (Sigma\u0026ndash;Aldrich Co., USA) (pH 7.0). It was then filtered through a PVDF 0.45-\u0026micro;m sterile syringe membrane filter (SIGL, Germany). Next, the filtrate was loaded onto a Hiprep 16/60 Sephacryl S-100 HR column (GE Healthcare Life Sciences, U.S.A) and eluted at a 0.5-mL/min flow rate. The detection wavelength was set at 280 nm. All fractions obtained during gel filtration with the Hiprep 16/60 Sephacryl S-100 HR column (GE Healthcare Life Sciences, U.S.A) in a Preparative chromatography system were collected and stored at -20\u0026deg;C until analysis.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAnalysis condition of fast protein liquid chromatography (FPLC).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInstrument\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCondition\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColumn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHiprep 16/60 Sephacryl S-100 HR column \u003c/p\u003e\u003cp\u003e(GE Healthcare Life Sciences, U.S.A)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMobile phase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50 mM Sodium phosphate buffer (pH 7.0)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDetector (Detection)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWaters Dual Wave length Absorbance W2487 detector\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFlow rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 mL/min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection volume\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3 mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Calcium Solubilization Ability\u003c/h2\u003e\u003cp\u003eIn the calcium binding activity experiment, the method described previously [36,37] was slightly modified to measure the effect of calcium phosphate formation on precipitation in solution. Calcium chloride (10 Mm; Sigma\u0026ndash;Aldrich Co., USA) and sodium phosphate buffer (20 mM) were prepared. Then, 0.5 mL of 10 mM calcium chloride and the casein hydrolysate fraction (0.5 mL) were mixed, and 1.0 mL of a 20 mM sodium phosphate buffer was added. The mixed solution was incubated at 37\u0026deg;C for 2 h and centrifuged at 2,000 \u0026times;g for 30 min at 25\u0026deg;C to remove insoluble calcium phosphate salts. Calcium solubility was measured using a calcium colorimetric kit (Gene tex, Inc., USA), and the supernatant was collected after centrifugation. The whole solution and supernatant samples were dispensed into each 10-\u0026micro;L 96-well plate, followed by mixing of chromogenic reagent to 90 \u0026micro;L and calcium assay buffer to 60 \u0026micro;L. After incubation for 5 min in a dark room at room temperature, absorbance was measured at 570 nm. The calcium concentration was calculated according to Eq.\u0026nbsp;(2), and the calcium solubility was calculated according to Eq.\u0026nbsp;(3). The protein concentration of all fractions from fermented WPC solution samples was 200 \u0026micro;g/mL.\u003c/p\u003e\u003cp\u003eCalcium concentration (mg/mL)\u0026thinsp;=\u0026thinsp;Sa/Sv (2)\u003c/p\u003e\u003cp\u003e*Sa\u0026thinsp;=\u0026thinsp;Sample amount from the standard curve (3)\u003c/p\u003e\u003cp\u003e*Sv\u0026thinsp;=\u0026thinsp;Sample volume (4)\u003c/p\u003e\u003cp\u003eCalcium solubility (%) = (calcium concentration in supernatant)/(calcium concentration in whole solution) \u0026times; 100 (5)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Antioxidant Activity\u003c/h2\u003e\u003c/div\u003e\n\u003ch3\u003e2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Assay\u003c/h3\u003e\n\u003cp\u003eABTS radical scavenging activity was assessed by first preparing a 7 mM ABTS solution in ethanol (Samchun Chemical, Seoul, Korea) using ABTS powder (Sigma\u0026ndash;Aldrich Co., USA). To generate the ABTS\u0026bull;⁺ radical solution, the ABTS stock was mixed with 2.45 mM potassium persulfate (Sigma\u0026ndash;Aldrich Co., USA) in a 1:1 (v/v) ratio and incubated at an ambient temperature in the dark for 12\u0026ndash;16 h. The resulting ABTS\u0026bull;⁺ solution was diluted with ethanol to achieve an absorbance of 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 at 734 nm, measured using a Multiskan EX355 microplate reader (Thermo Fisher Scientific, Waltham, USA). For the assay, 190 \u0026micro;L of the diluted ABTS\u0026bull;⁺ solution was added to 10 \u0026micro;L of each sample fraction in a 96-well plate (SPL Life Sciences, Pocheon, Korea). After 3 absorbance was measured at 734 nm, and antioxidant activity was expressed as the Trolox equivalent antioxidant capacity in mM/L [38].\u003c/p\u003e\n\u003ch3\u003e2,2-diphenyl-1-picrylhydrazyl (DPPH) Assay\u003c/h3\u003e\n\u003cp\u003eDPPH free radical-scavenging activity was determined with slight modifications [39]. A DPPH solution (0.2 mM) was prepared by dissolving 2,2-diphenyl-1-picrylhydrazyl and 2,4,6-tripyridyl-s-triazine (Sigma\u0026ndash;Aldrich Co., USA) in ethanol. This solution was mixed with the fermented WPC samples in equal volumes (1:1, v/v), followed by incubation in the dark at room temperature for 30 min. After the reaction, absorbance was recorded at 570 nm, and the antioxidant capacity was quantified as ascorbic acid equivalents (mM), using ascorbic acid (Sigma\u0026ndash;Aldrich Co., USA) as the standard [38].\u003c/p\u003e\u003cp\u003e\u003cb\u003eFerric-Reducing Antioxidant Power (FRAP) Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA FRAP assay was performed. The FRAP solution was prepared as follows: 300 mM acetate buffer was prepared by mixing sodium acetate trihydrate (Sigma\u0026ndash;Aldrich Co., USA) with acetic acid (J.T Baker Co., USA). A 10 mM TPTZ solution was prepared by dissolving 2,4,6-tri(2-pyridyl)-1,3,5-triazine (Sigma\u0026ndash;Aldrich Co., USA) in 40 mM HCl (DAEJUNG Co., Korea). Additionally, a 20 mM Iron(III) chloride hexahydrate solution was prepared by dissolving Iron(III) chloride hexahydrate 97% A.C.S reagent (Sigma\u0026ndash;Aldrich Co., USA) in ethanol (Samchun Chemical Co., Korea). The three solutions were mixed at a 10:1:1 (v/v) ratio. For the assay, 1.5 mL of the FRAP solution, preheated to 37 ℃, was combined with 50 \u0026micro;L of fermented WPC fraction samples or standard solutions. The mixture was vortexed and allowed to react in the dark at room temperature. Absorbance was measured at 593 nm, and the results were expressed as ascorbic acid equivalent capacity in mM [40,41]. The protein concentration of all fermented WPC fraction samples was adjusted to 200 \u0026micro;g/mL.\u003c/p\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Cell Culture\u003c/h2\u003e\u003cp\u003eRAW 264.7 cells were maintained in Dulbecco\u0026rsquo;s modification of Eagle\u0026rsquo;s medium supplemented with 10% heat-inactivated FBS at 37 ℃ in a 5% CO2 atmosphere. The medium was replaced every 2 d. To assess cell protection, RAW 264.7 cells were seeded into 96-well plates at a density of 5 \u0026times; 103 cells/mL (100 \u0026micro;L per well) for cell viability assays and into 24-well plates at a density of 1.5 \u0026times; 104 cells/mL (500 \u0026micro;L per well) for the nitric oxide (NO) assay and enzyme linked immunosolvent assay (ELISA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.12 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay\u003c/h2\u003e\u003cp\u003eCell viability was assessed using the MTT assay following protocols outlined in previous studies [42,43]. RAW 264.7 cells were seeded into 96-well plates and allowed to adhere for 24 h. After removing the culture medium, the cells were treated with various concentrations of the fractions diluted in serum-free medium and incubated for 24 h. MTT solution (5 mg/mL) was then added to each well, and the cells were incubated for 3 h at 37 ℃ in a 5% CO₂ atmosphere. After incubation, the supernatant was carefully discarded and the formazan crystals produced by metabolically active cells were solubilized using dimethyl sulfoxide. Absorbance was recorded at 540 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA) to evaluate cell viability. The viability percentage was determined by comparing the absorbance values of the treated groups to those of the untreated control group, which was considered to have 100% viability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.13 NO Assay\u003c/h2\u003e\u003cp\u003eNO concentrations were determined by the nitrite concentration using the Griess reaction, based on the method outlined in a previous study [44]. RAW 264.7 cells were incubated in a serum-free medium with different concentrations of the fraction samples for 2\u0026ndash;3 h after removing the existing medium. Subsequently, lipopolysaccharide (LPS) (1 ng/mL) was added to an equal volume of serum-free medium, and the cells were incubated for 20 h to induce stimulation. Following this, 0.1 mL of the reaction mixture was collected and placed in a 96-well microplate, and Griess reagent was added. The mixture was allowed to react in the dark at room temperature for 15 min, and absorbance was measured at 540 nm. NO scavenging activity was calculated as a percentage by comparing the absorbance of the samples at 540 nm to that of the blank sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Measurement of Cytokine Production (ELISA)\u003c/h2\u003e\u003cp\u003eRAW 264.7 cells were seeded in 96-well cell culture plates for 24 h. After removing the medium, RAW 264.7 cells were treated with various concentrations of the fraction samples in serum-free medium for 2\u0026ndash;3 h. LPS at a final concentration of 1 ng/mL was then treated and stimulated in the same volume in serum-free medium for 20 h. Cell-free supernatants were collected and the cytokine content was measured using a Mouse IL-1, IL-6 and TNF-α ELISA kit (Komabiotech, Korea) using ELISA. The optical density of the microplates was measured at 450 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.15 Amino Acid Profiling\u003c/h2\u003e\u003cp\u003eAmino acid profiling was performed according to the Waters Amino Acid Analysis AccQ Tag Manual. A Waters AccQ-Tag∙Fluor reagent kit was used for derivatization of the sample and standard. An amino acid standard (Sigma\u0026ndash;Aldrich Co., USA) was used. The sample amount ranged from 0.02\u0026ndash;0.08 \u0026micro;g (20\u0026ndash;1,000 pmol). The other system conditions are listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eChromatographic conditions of FPLC.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInstrument\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCondition\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColumn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWaters AccQ-Tag (Waters, U.S.A)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMobile phase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA: 10% \u003cem\u003e(\u003c/em\u003ev/v\u003cem\u003e)\u003c/em\u003e Waters AccQ-Tag eluent A in water\u003c/p\u003e\u003cp\u003eB: 60% \u003cem\u003e(\u003c/em\u003ev/v\u003cem\u003e)\u003c/em\u003e Acetonitrile in water (pH 5.02)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDetector (Detection)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWaters 474 scanning fluorescence detector\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFlow rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 mL/min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection volume\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2.16 Peptide Identification by Mass Spectrometry\u003c/h2\u003e\u003cp\u003eThe fraction samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Ultimate 3000 HPLC system (Dionex, Sunnyvale, CA, USA) and a Micro-TOF III mass spectrometer (255748, Bruker Daltonics, Germany) at Proteinworks Co. (Daejeon, Korea). The analytical conditions are listed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eChromatographic conditions for liquid chromatography-mass spectrometry (LC-MS).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInstrument\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eCondition\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColumn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003ePorshell 120 EC-C18 (2.1 X 100 nm, 2.7 \u0026micro;m, Agilent)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInjection volume\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e5 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFlow rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.2 mL/min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColumn temperature\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e30 ℃\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMobile phase / Time (min)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eSolvent composition\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eB (%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: Mobile phase A consisted of water containing 0.2% (v/v) fluoroacetic acid (FA), and mobile phase B consisted of acetonitrile containing 0.2% (v/v) FA.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e2.17 Statistical Analysis\u003c/h2\u003e\u003cp\u003eA one-way analysis of variance was performed to assess statistical differences among the experimental groups. The analysis revealed significant group effects (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the mean values differed among the treatments. Microsoft Excel was used for data visualization and group comparisons to facilitate a clear interpretation of the variance patterns.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.1 LAB Screening and Identification\u003c/h2\u003e\u003cp\u003eMorphological characteristics of the isolated LAB strains were examined under microscopy. Various microorganisms were observed, and all strains were confirmed to be Gram-positive. Ten strains were identified using 16S-rRNA sequencing. Based on 16S-rRNA sequence analysis, the lactic acid-producing strains were classified into eight strains of \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e, one strain of \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e, and one strain of \u003cem\u003eLactiactobacillus sakei\u003c/em\u003e. The 16S-rRNA gene sequences are listed in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The isolated strains were named \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e DC1, 2, 3, 4, 5, 7, 8, and 9, \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e DC6, and \u003cem\u003eLactilactobacillus sakei\u003c/em\u003e DC10.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIdentification of strains isolated from Dongchimi with 16S-rRNA sequence analysis.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16s-rRNA sequence species\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eID (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e DS3_KCTC13016BP 16S ribosomal RNA gene, partial sequence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e strain DS3_KCTC13016BP 16S ribosomal RNA gene, partial sequence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e DNA, complete genome, strain: LK-151\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e strain SCWL 03 16S ribosomal RNA gene, partial sequence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e strain DS3_KCTC13016BP 16S ribosomal RNA gene, partial sequence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e strain DAEM1 16S ribosomal RNA gene, partial sequence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e strain DS3_KCTC13016BP 16S ribosomal RNA gene, partial sequence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e strain DS3_KCTC13016BP 16S ribosomal RNA gene, partial sequence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e strain DS3_KCTC13016BP 16S ribosomal RNA gene, partial sequence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLactilactobacillus sakei\u003c/em\u003e strain TUB/2013/10(3\u0026ndash;72) 16S ribosomal RNA gene, partial sequence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Tolerance to Artificial Gastric and Bile Juice\u003c/h2\u003e\u003cp\u003eThe LAB strains demonstrated strong survival ability in artificial gastric juice during 2 h of incubation at pH 3.0, 2.0, and 1.5 at 37\u0026deg;C, which simulates human gastric transit time and pH (approx. 120 min; pH 1.5\u0026ndash;3.0) [45,46]. The observed survival rates (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) are consistent with previous findings, indicating that tolerance varied among strains. Similar tolerance has been reported in strains from \u003cem\u003eLeuconostoc\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, and \u003cem\u003ePediococcus\u003c/em\u003e genera [47]. Following gastric acid exposure, LAB strains were further tested for bile tolerance by incubation in MRS broth containing 0.3% oxgall for 24 h at 37\u0026deg;C. The strains exhibited high survival rates under bile salt conditions (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results are consistent with previous reports on bile salt resistance in LAB isolated from food sources. Collectively, these findings highlight the robustness of acid and bile tolerance which is essential for effective probiotic applications [48\u0026ndash;50].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTolerance to artificial gastric and bile juice. CFU \u0026ndash; colony-forming units.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eStrains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eLog (CFU/mL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003epH 3.0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003epH 2.0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003epH 1.5\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e4.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e4.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e5.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e5.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e4.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e4.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e4.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e4.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e5.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e5.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e5.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e5.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e6.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e6.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLA-5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e5.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e5.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e DC1, DC2, DC3, DC4, DC5, DC7, DC8, and DC9; \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e DC6; \u003cem\u003eLactilactobacillus sakei\u003c/em\u003e DC10; \u003cem\u003eLactilactobacillus acidophilus\u003c/em\u003e LA-5 (Chr. Hansen, Denmark) were used. CFU refer to the colony-forming units. All data are mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (n\u0026thinsp;=\u0026thinsp;3).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Proteolytic Activity\u003c/h2\u003e\u003cp\u003eBacterial proteolytic activity was assessed based on the ability to produce clear zones on skim milk agar [51,52]. The agar well diffusion assay revealed that the 10 LAB strains initially screened for proteolytic activity could hydrolyze milk proteins. A clear zone around the wells was present for these 10 strains, indicating their proteolytic activity. Of the 10 tested strains, \u003cem\u003eL. sakei\u003c/em\u003e DC10 was the most efficient for proteolytic activity on skim milk agar (clear zone diameter\u0026thinsp;\u0026gt;\u0026thinsp;23 mm; Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eProteolytic activity of lactic acid bacteria (LAB) strains.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eStrains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eDiameter of clear zone (mm)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e72h\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLA-5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e DC1, DC2, DC3, DC4, DC5, DC7, DC8, and DC9; \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e DC6; \u003cem\u003eLactilactobacillus sakei\u003c/em\u003e DC10; \u003cem\u003eLactilactobacillus acidophilus\u003c/em\u003e LA-5 (Chr. Hansen, Denmark) were used. All data are mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (n\u0026thinsp;=\u0026thinsp;3).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.4 DH of WPC solution\u003c/h2\u003e\u003cp\u003eLAB possess a complex system of proteinases and peptidases that enable them to use milk protein as a source of amino acids and nitrogen. Hydrolysis during whey protein fermentation was monitored by analyzing the amount of primary amino groups released and the soluble protein content. In the present study, we inoculated LAB into a whey protein solution for fermentation. The degree of whey protein hydrolysis by LAB is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. During fermentation, whey proteins were hydrolyzed by LAB, resulting in an increased number of free amino groups and peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The DH of WPC solutions fermented by LAB rapidly increased from 0\u0026ndash;4 h. The DH progressed slowly after 16 h (DH %), indicating that whey protein hydrolysis reached its highest proteolytic rate at 16 h. The extent of proteolysis was time dependent. These results indicated that the LAB strain showed proteolytic activity in the whey protein solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.5 SDS-PAGE\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the SDS-PAGE analysis of the whey proteins using L. sakei DC10. Protein bands with molecular weights ranging from 13\u0026ndash;250 kDa were identified. The main components of whey protein such as lacttoferrin, bovine serum albumin (BSA), immunoglobulin, β-lactoglobulin and α-lactalbumin etc., were found in the band from 14\u0026ndash;100 kDa. Components with higher molecular weights were degraded during fermentation. After 16 h of fermentation, lactoferrin, BSA, and immunoglobulin were degraded.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.6 FPLC\u003c/h2\u003e\u003cp\u003eThe fermented WPCs were identified using SDS-PAGE and separated on a preparative scale. L. sakei DC10 was selected based on the DH and SDS-PAGE results. Fractions of the WPCs fermented with DC10 were isolated by molecular weight using a Hiprep 16/60 Sephacryl S-100 HR column in a preparative liquid chromatography system (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Calcium Solubilization Capacity\u003c/h2\u003e\u003cp\u003eCasein phosphopeptide (CPP) contains phosphoserine and binds calcium to promote its calcium-solubilizing ability. In this study, calcium solubility was measured to determine whether each fraction promoted a calcium-solubilizing ability similar to that of CPP. The experimental results showed that CPP had a high calcium solubility of 93.67%. The phosphoserine contained in CPP dissolves calcium and increases its solubility [53]. All of the fractions from fermented WPC solution with L. sakei DC10 had lower calcium solubilization capacity than CPP but showed higher calcium solubilization capacity than unhydrolyzed whey protein, of which fraction 3 had lower than CPP but approximately 85% calcium solubilization capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This may have indicated the potential calcium solubilization capacity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Antioxidant Activity\u003c/h2\u003e\u003cp\u003e\u003cb\u003eABTS Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Trolox equivalent antioxidant capacity assay measures the ability of a compound to eliminate or scavenge radicals compared to Trolox (Vitamin E) as an antioxidant reference [54]. The ABTS assay results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Except for F1 and F4, the other fractions showed higher ABTS radical scavenging activities than non-hydrolyzed whey protein. Fraction 3 had the highest value.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDPPH Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDPPH is a stable free radical that has been widely accepted as a tool for estimating the free radical-scavenging activities of antioxidants [55]. The antioxidant activity of each fraction was evaluated by measuring its DPPH scavenging activity. DPPH scavenging activity values were statistically analyzed based on the standard curve of the Trolox solution. Fractions 2 and 3 showed a DPPH radical scavenging ability similar to that of CPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFRAP Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe decrease in absorbance is proportional to the antioxidant content [56]. The FRAP assay is a direct method for measuring antioxidant ability. This is based on the ability of antioxidants to reduce Fe3\u0026thinsp;+\u0026thinsp;to Fe2\u0026thinsp;+\u0026thinsp;in the presence of TPTZ, forming an intense blue Fe2+\u0026ndash;TPTZ complex with absorption at 593 nm, which is associated with their electron-donating ability to break the free radical chain reaction [55]. According to the results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e), fraction 3 exhibited a higher iron reduction capacity than CPP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e3.9 MTT Assay\u003c/h2\u003e\u003cp\u003eCytotoxicity tests based on the MTT assay are widely used for in-vitro toxicology experiments. The experimental results confirmed that increasing the concentration improved cell viability. In addition, all fractions showed\u0026thinsp;\u0026gt;\u0026thinsp;80% cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Therefore, we confirmed that the macrophage fractions were not cytotoxic.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003e3.10 NO Assay\u003c/h2\u003e\u003cp\u003eNO plays important roles in blood coagulation, blood pressure regulation, and immune function in cancer cells. However, it is oxidized to reactive oxygen species and converted into active NO. NO produces oxidants that cause cytotoxicity. NO production in cells exposed to inflammatory mediators is increased by tissue damage and inflammatory diseases. According to the results of this study, NO production was inhibited as the concentration of the fractions increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The MTT experiments showed that the inhibition of NO production was not caused by cytotoxicity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\u003ch2\u003e3.11 Measurement of Cytokine Production (ELISA)\u003c/h2\u003e\u003cp\u003eDirect or indirect interactions between immune cells are needed to maintain immune balance. Cytokines can induce the proliferation, differentiation, and changes in the function and activity of various immune cells. Disease is mostly associated with inflammation, and inflammatory cells secrete inflammatory cytokines that induce inflammation [57]. Expression levels of IL-1α, IL-6 and TNF-α were measured in the present study. According to the results of cytokine measurements using ELISA, production of three cytokines (IL-1α, IL-6, and TNF-α) was significantly lower than LPS(+) group. Based on these results, anti-inflammatory activity was observed in the hydrolysate fractions. In addition, the low expression level of TNF-α from macrophage effect by samples prevented the activation of pro-inflammatory cytokines which was produced by the expression of TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003e). It is believed to have reduced the inflammatory response.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\u003ch2\u003e3.12 Amino Acid Profiling\u003c/h2\u003e\u003cp\u003eBased on the above experimental results, fraction 3 was selected as the fraction with superior functional properties and chosen for identification of its amino acid composition and peptide sequencing. By confirming the amino acid sequences of the selected fractions using the AccQ-Tag system, 17 amino acid species and several species of unknown amino acids were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Fraction 3 had the highest leucine content and lowest cysteine content.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAmino acids concentrations of fraction 3.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmino acid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWPC (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFraction 3 (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAsp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThr\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.83\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlu\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePro\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.88\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGly\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAla\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCys\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMet\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeu\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLys\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eArg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal molar ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec35\" class=\"Section2\"\u003e\u003ch2\u003e3.13 Peptide Identification by Mass Spectrometry\u003c/h2\u003e\u003cp\u003eFraction 3 was subjected to LC-MS and LC-MS/MS for peptide separation and sequence identification, respectively. MS analysis confirmed the presence of many different peptides; however, four were fully sequenced, and additional purification of fraction 3 was likely required. Four peptides were identified from fraction 3 (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) with molecular masses of 905.49, 658.36, 893.96 and 883.46 Da. Molecular mass and tandem mass spectrometry results for the peptides are shown in Figs.\u0026nbsp;10 and 11, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePeptide sequencing results of fraction 3.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePeak name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e\u003csup\u003e1)\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRT (min) \u003csup\u003e2)\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSequencing\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e905.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTVQVTSTAV\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e658.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e22.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDKTEIPTINTIA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e893.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e22.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDKTEIPTINTIASGEPT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e883.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e26.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSLVYPFPGPIHNSLPQ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe fermentation of WPC by LAB is an effective alternative for producing functional dairy foods with enhanced nutritional and functional properties owing to the high biological value of whey proteins [58]. In the present study, 10 LAB strains isolated from Dongchimi were screened, and three proteolytic strains, Leuconostoc mesenteroides DC3, L. mesenteroides DC8, and Latilactobacillus sakei DC10, were selected based on physiological and functional assays.\u003c/p\u003e\u003cp\u003eThe DH of WPC increased in a similar pattern for all selected LAB strains, showing no further significant changes after 16 h of fermentation. SDS-PAGE analysis confirmed the degradation patterns of the major whey protein components.\u003c/p\u003e\u003cp\u003eWPC fermented with DC10 exhibited proteolytic patterns similar to those observed with other effective LAB strains [59].\u003c/p\u003e\u003cp\u003eThe peptide fractions of the fermented whey were analyzed for molecular weight using a HiPrep 16/60 Sephacryl S-100 HR column. The peptides in fraction 3 showed the highest calcium solubilization capacity. Peptides generally bind calcium ions by forming a chelate through electron pairs on the α-amino group and carboxyl group, with side chains of certain amino acids further stabilizing the complex [60]. In particular, leucine, aspartic acid, and glutamic acid play key roles in such structural interactions, and amino acid analysis of fraction 3 revealed high levels of BCAAs (leucine, isoleucine, and valine) [61].\u003c/p\u003e\u003cp\u003eIn addition to direct binding, BCAAs can enhance intestinal calcium absorption by upregulating Vitamin D receptor expression, thereby promoting Vitamin D-dependent pathways [62]. BCAAs activate both transcellular and paracellular transport mechanisms in the intestinal epithelium and regulate calcium and phosphorus transporter expression in the kidney to maintain blood calcium homeostasis [63].\u003c/p\u003e\u003cp\u003eFraction 3 showed antioxidant activity comparable to that of CPP. LC/MS analysis identified four peptide sequences in fraction 3, among which TVQVTSTAV and SLVYPFPGPIHNSLPQ are well-known antioxidative peptides [64].\u003c/p\u003e\u003cp\u003eIn the anti-inflammatory assays, fraction 3 significantly inhibited NO production and suppressed pro-inflammatory cytokines (IL-1α, IL-6, and TNF-α) while improving cell viability, consistent with previous reports showing enhanced bioactivities of hydrolyzed whey proteins compared to that of their non-hydrolyzed forms [65].\u003c/p\u003e\u003cp\u003eIn other studies, TVQVTSTAV and DKTEIPTINTIA were reported as peptides with notable antimicrobial activities and SLVYPFPGPIHNSLPQ exhibited immunomodulatory effects [64]. These results suggested that fraction 3 obtained in the present study may have antioxidant, anti-inflammatory, antibacterial, and immunomodulatory properties.\u003c/p\u003e\u003cp\u003eIn this study, we confirmed that fermentation of WPC using specific LAB produced bioactive peptide fractions with diverse physiological functions, including calcium solubility and antioxidant, anti-inflammatory, and antibacterial activities. Furthermore, this study demonstrated the potential of dairy proteins in the design of diverse functional and specific bioactive ingredients.\u003c/p\u003e\u003cp\u003eBased on these biological activities, future research should focus on elucidating its mechanism of action, conducting clinical trials to evaluate its stability and bioavailability, and demonstrating its potential application as a functional food.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was financially supported by the Ministry of Agriculture, Food and Rural Affairs through the project \u0026ldquo;Next-generation Food Processing of High Value-added Food Technology Development\u0026rdquo; (Project No. RS-2024-00407186).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Kim CH.; Data curation: Yoo JS and Lee SJ.; Formal analysis: Yoo JS, Lee SJ and Song SE.; Methodology: Yoo JS, Lee SJ and Song SE.; Software: Yoo JS, Lee SJ and Song SE.; Validation: Kim CH.; Investigation: Yoo JS, Lee SJ and Song SE.; Writing - original draft: Yoo JS.; Writing - review \u0026amp; editing: Lee SJ and Kim CH. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the High Value-added Food Technology Development Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA) (RS-2024-00407186).\u003c/p\u003e\n\u003cp\u003eInstitutional rview board statement\u003c/p\u003e\n\u003cp\u003eThis study did not involve human participants or animals; therefore, ethical approval was not required.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSmithers GW. Whey-ing up the options\u0026ndash;Yesterday, today and tomorrow. Int Dairy J. 2015;48:2\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYadav JSS, Yan S, Pilli S, Kumar L, Tyagi RD, Surampalli RY. Cheese whey: A potential resource to transform into bioprotein, functional/nutritional proteins and bioactive peptides. Biotechnol Adv. 2015;33(6):756\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChourasia R, Chiring Phukon L, Abedin MM, Padhi S, Singh SP, Rai AK. Bioactive peptides in fermented foods and their application: a critical review. Syst Microbiol Biomanuf. 2023;3(1):88\u0026ndash;109.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKorhonen H, Pihlanto A. Bioactive peptides: production and functionality. Int Dairy J. 2006;16(9):945\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKorhonen H. Milk-derived bioactive peptides: From science to applications. J Funct Foods. 2009;1(2):177\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDziuba B, Dziuba M. Milk proteins-derived bioactive peptides in dairy products: Molecular, biological and methodological aspects. Acta Sci Pol Technol Aliment. 2014;13(1):5\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi-Chan EC. Bioactive peptides and protein hydrolysates: research trends and challenges for application as nutraceuticals and functional food ingredients. Curr Opin Food Sci. 2015;1:28\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUdenigwe CC, Aluko RE. Food protein-derived bioactive peptides: production, processing, and potential health benefits. J Food Sci. 2012;77(1):R11-24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMadureira AR, Pereira CI, Gomes AM, Pintado ME, Malcata FX. Bovine whey proteins\u0026ndash;Overview on their main biological properties. Food Res Int. 2007;40(10):1197\u0026ndash;211.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSindayikengera S, Xia WS. Nutritional evaluation of caseins and whey proteins and their hydrolysates from Protamex. 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J Food Sci Technol. 2021;58(10):4225\u0026ndash;4238.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-science-of-animal-resources","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Science of Animal Resources](https://link.springer.com/journal/44463)","snPcode":"44463","submissionUrl":"https://submission.springernature.com/new-submission/44463/3?","title":"Food Science of Animal Resources","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"lactic acid bacteria, whey protein concentrate, fermentation, antioxidant, immunomodulatory effect","lastPublishedDoi":"10.21203/rs.3.rs-7899609/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7899609/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBioactive peptides (BAPs) are short amino acid sequences in food proteins that provide health benefits such as antioxidant, antihypertensive, antimicrobial, and immunomodulatory effects. Whey protein, which is rich in essential amino acids such as leucine and cysteine, is a promising source of BAPs; however, it resists enzymatic hydrolysis due to its hydrophobicity. To overcome this limitation, this study used fermentation with \u003cem\u003eLacticaseibacillus sakei\u003c/em\u003e DC10, a lactic acid bacteria (LAB) with strong proteolytic activity. Fermentation produced diverse peptides with antioxidant and anti-inflammatory properties and improved calcium solubility, suggesting potential bone health benefits. These peptides may reduce allergenicity and serve as multifunctional food ingredients. In addition, their calcium-binding ability makes them cost-effective alternatives to casein phosphopeptides for nutraceutical and fortified food applications. This study highlights LAB fermentation as an effective approach for generating novel whey protein-derived BAPs with enhanced bioactivity, supporting their use in functional foods and human health promotion.\u003c/p\u003e","manuscriptTitle":"Functional characteristics of peptides from whey proteins fermented with lactic acid bacteria isolated from Dongchimi","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-04 11:32:13","doi":"10.21203/rs.3.rs-7899609/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-10T12:40:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-09T04:52:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-21T10:43:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"203098958442777267114677660823719656992","date":"2025-11-10T05:35:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110726297218941154245708148370416975321","date":"2025-10-23T03:00:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-22T05:34:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-22T05:20:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-22T01:39:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Science of Animal Resources","date":"2025-10-19T15:33:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-science-of-animal-resources","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Science of Animal Resources](https://link.springer.com/journal/44463)","snPcode":"44463","submissionUrl":"https://submission.springernature.com/new-submission/44463/3?","title":"Food Science of Animal Resources","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bc562245-b73a-43f6-aa9c-4592ec90c952","owner":[],"postedDate":"November 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-30T05:23:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-04 11:32:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7899609","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7899609","identity":"rs-7899609","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}