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

Abstract

Bioactive peptides (BAP) are short amino acid sequences in food proteins that provide health benefits, including 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 owing to its hydrophobicity. To overcome this limitation, we performed fermentation with Latilactobacillus sakei DC10, a lactic acid bacterium (LAB) with strong proteolytic activity. Fermentation produces diverse peptides with antioxidant and anti-inflammatory properties and improves calcium solubility, suggesting potential bone health benefits. Thus, these peptides may reduce allergenicity and serve as multifunctional food ingredients. Additionally, 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 promoting human health.

Introduction

Proteins can be used as substrates to produce bioactive peptides (BAPs) and can also be transformed into high value-added products (Smithers, 2015; Yadav et al., 2015; Chourasia et al., 2023). Protein obtained from food is currently being researched beyond its nutritional characteristics because of its positive effects on human health, particularly owing to specific sequences encrypted within 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 (Korhonen & Pihlanto, 2006; Korhonen, 2009). BAPs are short amino acid sequences that contain inactive precursor protein (Dziuba & Dziuba, 2014). The potential health benefits associated with BAPs consumption have attracted the interest of many researchers (Li-Chan, 2015). The release of BAPs from their parent proteins by proteolysis is affected by a variety of factors, including hydrolysis time, pH, temperature, and enzyme–substrate ratios (Udenigwe & Aluko, 2012).

Whey protein is a mixture of globular proteins isolated from whey, a liquid material created as a byproduct of cheese production (Madureira et al., 2007). Whey proteins constitute 15–20% of the total milk protein (Sindayikengera & Xia, 2006). The key components of whey protein are β-lactoglobulins and α-lactalbumins (Marshall, 2004). Other constituents include immunoglobulins, serum albumin, and lactoferrin (Law & Leaver, 2000). Protein quality depends on the constituent amino acids, and whey protein has one of the highest quality standards. Whey proteins are also rich in essential branched-chain amino acids (BCAA) (Devries & Phillips, 2015). BCAAs, including leucine, isoleucine, and valine, play crucial roles in metabolism, blood glucose homeostasis, and neural function (Joy et al., 2013). Leucine regulates skeletal muscle protein synthesis (Rieu et al., 2007). Another essential amino acid, cysteine, is a building block of glutathione, which is a dietary antioxidant (Micke et al., 2002). It is vital to combat oxidative stress and prevent diseases caused by redox imbalances (Trachootham et al., 2008). Several studies have reported that whey proteins are valuable sources of BAPs owing to their high nutritional value and the wide range of specific BAPs they release (Ferreira et al., 2007; Théolier et al., 2013).

The enzymatic hydrolysis of food proteins is one of the most promising methods for BAP production (Daliri et al., 2017). Although commercial enzymes are economically advantageous for protein hydrolysis and peptide production, it is difficult to obtain peptides with specific amino acid sequences using conventional enzymes (Wu et al., 2013). Whey proteins are highly hydrophobic and resistant to enzymatic hydrolysis. Therefore, bioactive fragments may not be released from whey protein despite the high biological potential of the protein precursor (Minkiewicz et al., 2011). Hydrolysates produced by enzymes obtained from alternative sources, such as new strains of lactic acid bacteria (LAB) that exhibit high proteolytic activity, can be sources of several new peptides with specific bioactivities at low production costs (Rizzello et al., 2017).

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 (]Kleber et al., 2006, El-Ghaish et al., 2011, Pescuma et al., 2008). Food proteins and α-lactalbumin fermented by LAB can increase digestibility and hydrolyze allergenic peptides (Bertrand-Harb et al., 2003). This approach could be used to develop new BAPs, and whey bioactive peptides could 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 (Raveschot et al., 2018). Moreover, differences in enzyme substrate specificity lead to changes in the peptide composition of hydrolysates and, consequently, their biological activities (Jensen & Ardö, 2010). This suggests that LAB can generate a considerable variety of BAPs, highlighting the need to select appropriate strains for use in BAP production (Raveschot et al., 2018).

Dongchimi, a traditional Korean radish-based kimchi, harbors diverse lactic acid bacteria (LAB) with well-documented probiotic potential and good adaptability to dairy environments. Dongchimi-derived LAB strains were selected as ideal candidates because they exhibit both probiotic properties and technological compatibility with dairy applications. Previous studies reported that LAB isolated from general kimchi can impart undesirable sensory characteristics when used in fermented milk or dairy systems (Baick & Kim, 2015). Therefore, in this study, pre-screened Dongchimi-derived LAB strains were employed to ensure both the generation of highly functional bioactive peptides during whey protein fermentation and the feasibility of their incorporation into commercial dairy products.

This study aimed to develop bioactive peptides derived from fermented whey proteins with superior physiological properties, calcium solubilization abilities, and effects on immunity. The objective was to highlight potential applications of calcium-binding peptides as ingredients in functional foods.

Materials and methods

LAB screening and identification

LAB were isolated from homemade Dongchimi collected from different regions of South 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 microscopic observation of strain morphology. The selected strains were identified using 16 S rRNA gene sequencing. The 16 s rRNA sequences were analyzed using the GenBank database (Macrogen, Korea), and 16 S rRNA sequence homology was used for identification.

Tolerance to artificial gastric juice and bile

Tolerance to artificial gastric juice and bile acids was measured according to the method developed in a previous study (Kobayashi et al., 1974). To investigate the survival of the LAB strains under acidic conditions, each strain was harvested by centrifugation at 7,000 × g for 10 min, washed twice with PBS, and 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 incubating 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 1-mL aliquots were plated evenly on BCP agar plates, which were then aerobically incubated at 37 °C for 48 h before the number of colony-forming units (CFU) was estimated.

Proteolytic activity

Proteolytic activity was assessed using an agar well diffusion method, as previously described (Riffel & Brandelli, 2006). For the agar well diffusion test, selected LAB strains were screened for proteolytic activity on skim milk containing 2.0% (w/v) agar (Difco, Detroit, MI, USA). Bacterial cultures grown in MRS broth centrifuged at 5,000 × g for 10 min, and the resulting supernatant (100 µL) was loaded into 9-mm diameter wells of skim milk agar plates. Proteolysis resulted in the formation of clear zones around the wells. Protease activity was determined by estimating the diameter of the clear zone area for 72 h at 37 °C.

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 the 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 (Fialho et al., 1999).

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 1X 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.

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 (Adler-Nissen, 1986). The TNBS reagent comprised 0.1% (v/v) TNBS in distilled water. All samples and standard solutions were prepared in a 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. After filtration through a 0.45-µm sterile polyvinylidene fluoride (PVDF) syringe membrane filter (SIGL, Germany), 0.2 mL of samples were added to test tubes containing 2 mL of 0.2125 M sodium phosphate buffer (pH 8.2). TNBS reagent (2 mL) was added to each test tube, which were then incubated at 50 °C for 60 min away from light. After incubation, the reaction was stopped by adding 4 mL of 0.1 N HCl to each test tube. The samples were allowed to cool to room temperature for 30 min before the absorbance values were measured at 420 nm using a UV-Vis spectrophotometer (Human Corporation, Seoul, Korea). DH was defined as the proportion of the total number of peptide bonds cleaved during hydrolysis (Adler-Nissen, 1986) and was calculated as follows:

1

where h is the number of hydrolyzed peptide bonds, and h_tot is the total number of peptide bonds (Nielsen et al., 2001). Leucine was used as a standard (0–2 mM) to determine the free amino group content in the samples. Each sample was analyzed in triplicate.

SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)

The fermented WPC solution was analyzed by SDS-PAGE according to a previously described method (Laemmli, 1970) using a 12% polyacrylamide separation gel under reducing conditions with a Mini-PROTEAN III system (Bio-Rad, USA). The operating conditions were set at 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).

Fast protein liquid chromatography (FPLC)

To obtain peptide fractions from the WPC solution fermented with DC10, gel filtration was performed using a preparative chromatography system (Waters, USA). 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, the Fermented WPC solution was dissolved in 50 mM sodium phosphate buffer with 0.15 M NaCl (Sigma–Aldrich Co., USA) at pH 7. It was then filtered through a PVDF 0.45-µm sterile syringe membrane filter (SIGL, Germany). The filtrate was loaded onto a HiPrep 16/60 Sephacryl S-100 h column (GE Healthcare Life Sciences, USA) 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 a HiPrep 16/60 Sephacryl S-100 h column (GE Healthcare Life Sciences, USA) in a preparative chromatography system were collected and stored at − 20 °C until analysis (Table 1).

Table 1.

Analysis conditions of fast protein liquid chromatography (FPLC)

Instrument	Condition
Column	HiPrep 16/60 Sephacryl S-100 h column (GE Healthcare Life Sciences, USA)
Mobile phase	50 mM Sodium phosphate buffer (pH 7.0)
Detector (Detection)	Waters Dual Wavelength Absorbance W2487 detector
Flow rate	1 mL/min
Injection volume	3 mL

Calcium solubilization ability

For the calcium binding activity experiment, a previously described method (Hou et al., 2018; Jung & Kim, 2007) was slightly modified to measure the effect of calcium phosphate formation on precipitation in solution. Calcium chloride (10 mM; Sigma–Aldrich) and sodium phosphate buffers (20 mM) were prepared. Then, 10 mM calcium chloride (0.5 mL) and the 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 the dark at room temperature, the absorbance was measured at 570 nm. A standard curve was prepared using calcium standards at concentrations of 40–200 µg/mL. The protein concentration of all fractions from fermented WPC solution sample was 200 µg/mL. Calcium concentration was calculated according to Eq. (2) and calcium solubility was calculated according to Eq. (5):

2

3

4

5

Antioxidant activity

2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay

The ABTS radical scavenging activity was assessed by 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 reacting at 37 ℃ for 10 min, the absorbance was measured at 734 nm. Antioxidant activity was expressed as the Trolox equivalent antioxidant capacity in mM/L (Cano et al., 2023). The protein concentration of all fermented WPC fraction samples was adjusted to 200 µg/mL.

2,2-diphenyl-1-picrylhydrazyl (DPPH) assay

DPPH free radical-scavenging activity was determined with some modifications (Tomassi et al., 2025). 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 equal volumes of fraction samples (1:1, v/v) and incubated in the dark at room temperature for 30 min. After the reaction, absorbance was measured at 517 nm. The antioxidant activity was expressed as the Trolox equivalent antioxidant capacity in mM/L as the standard (Cano et al., 2023). The protein concentration of all fermented WPC fraction samples was adjusted to 200 µg/mL.

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) with acetic acid (J. T. Baker). 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 in 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 dark at 37 ℃ for 10 min. The absorbance was measured at 593 nm, and the results were expressed as the ascorbic acid equivalent capacity in mM (Tang et al., 2017; Rumpf et al., 2023). The protein concentration of all fermented WPC fraction samples was adjusted to 200 µg/mL.

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).

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 (Sornsenee et al., 2025; Lee et al., 2025). RAW 264.7 cells were seeded into 96-well plates and allowed to adhere for 24 h. After removing the culture medium, cells were treated with various protein concentrations (0.25–2.25 mg/ml) of the peptide fractions or CPP diluted in serum-free medium and incubated for 24 h. MTT solution (0.5 mg/mL) was then added to each well, followed by incubation 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. The absorbance was recorded at 540 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA) to evaluate cell viability. Percentage viability was determined by comparing the absorbance values of the treated groups with those of the untreated control group, which was considered to have 100% viability.

NO assay

NO concentrations were determined from the nitrite concentration using the Griess reaction based on the method outlined in a previous study (Kang et al., 2025). RAW 264.7 cells were pretreated with the fraction samples (1 mg/mL protein concentration) in serum-free medium for 2–3 h after removing the existing medium. Subsequently, lipopolysaccharide (LPS) (100 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 the absorbance was measured at 540 nm. NO production was calculated as a percentage by comparing the absorbance of the samples at 540 nm with that of the LPS-treated control.

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 pre-treated with fraction samples (1 mg/mL protein concentration) in serum-free medium for 2–3 h. Subsequently, LPS was added at a final concentration of 100 ng/mL in serum-free medium and the cells were incubated for 20 h at 37℃ in a 5% CO₂ atmosphere. 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). The optical density of the microplates was measured at 450 nm.

Amino acid profiling

Amino acid profiling was performed according to 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) was used. The sample amounts ranged from 0.02 to 0.08 µg (20–1,000 pmol). The other system conditions are listed in Table 2.

Table 2.

HPLC conditions for amino acid analysis

Instrument	Condition
Column	Waters AccQ-Tag (Waters, USA)
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

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 × 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

Statistical analysis

A one-way analysis of variance (ANOVA) was performed followed by Tukey’s HSD test for multiple comparisons among treatment groups, with independent Student’s t-tests used for pairwise comparisons where appropriate (n = 3 replicates per group). Statistical significance was accepted at p < 0.05. Data were analyzed using Microsoft Excel for descriptive statistics (means ± SD or SE) and Python (statsmodels) for inferential analyses.

Results

LAB screening and identification

The morphological characteristics of the isolated LAB strains were examined using a microscope. Various microorganisms were observed, and all strains were confirmed to be Gram-positive. Ten strains were identified using 16 S-rRNA sequencing. Based on the 16 S-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 Latilactobacillus sakei. The 16 S-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 Latilactobacillus sakei DC10.

Table 4.

Identification of strains isolated from Dongchimi with 16 S-rRNA sequence analysis

Strain	16s-rRNA sequence species	ID (%)
DC1	Leuconostoc mesenteroides DS3_KCTC13016BP 16 S ribosomal RNA gene, partial sequence	99
DC2	Leuconostoc mesenteroides strain DS3_KCTC13016BP 16 S ribosomal RNA gene, partial sequence	99
DC3	Leuconostoc mesenteroides DNA, complete genome, strain: LK-151	99
DC4	Leuconostoc mesenteroides strain SCWL 03 16 S ribosomal RNA gene, partial sequence	99
DC5	Leuconostoc mesenteroides strain DS3_KCTC13016BP 16 S ribosomal RNA gene, partial sequence	99
DC6	Pediococcus pentosaceus strain DAEM1 16 S ribosomal RNA gene, partial sequence	99
DC7	Leuconostoc mesenteroides strain DS3_KCTC13016BP 16 S ribosomal RNA gene, partial sequence	99
DC8	Leuconostoc mesenteroides strain DS3_KCTC13016BP 16 S ribosomal RNA gene, partial sequence	99
DC9	Leuconostoc mesenteroides strain DS3_KCTC13016BP 16 S ribosomal RNA gene, partial sequence	99
DC10	Latilactobacillus sakei strain TUB/2013/10(3–72) 16 S ribosomal RNA gene, partial sequence	99

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) (Gilliland, 1979; Havenaar et al., 1992). The observed survival rates (Table 5) were consistent with previous findings, indicating that tolerance varied among the strains. Similar tolerance has been previously reported in strains of the genera Leuconostoc, Lactobacillus, and Pediococcus (Mansouri et al., 2025). 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. These strains exhibited high survival rates in the presence of bile salts (Table 5). These results are consistent with previous studies 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 (Bove et al., 2012; Jensen et al., 2012; Turchi et al., 2013).

Table 5.

Tolerance to artificial gastric and bile juice

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; Latilactobacillus sakei DC10; Latilactobacillus acidophilus LA-5 (Chr. Hansen, Denmark) was used for analysis. All data represent the mean ± SE (n = 3). CFU, colony-forming units

Proteolytic activity

The bacterial proteolytic activity was assessed by the formation of clear zones on skim milk agar (Pailin et al., 2001; Němečková et al., 2009). An agar well diffusion assay revealed that the 10 LAB strains initially screened for proteolytic activity could hydrolyze milk proteins. A clear zone was observed around the wells for these ten strains, indicating their proteolytic activity. Among the ten tested strains, L. sakei DC10 exhibited the strongest 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)
	24 h	48 h	72 h
DC1	13 ± 0.03a	16 ± 0.13a	22 ± 0.11a
DC2	12 ± 0.09b	13 ± 0.07c	18 ± 0.04c
DC3	12 ± 0.07b	14 ± 0.09d	19 ± 0.07d
DC4	13 ± 0.07a	15 ± 0.15e	20 ± 0.07e
DC5	13 ± 0.1a	14 ± 0.21d	21 ± 0.03f
DC6	11 ± 0.03c	15 ± 0.13e	19 ± 0.13d
DC7	14 ± 0.13d	16 ± 0.2a	22 ± 0.09a
DC8	13 ± 0.1a	16 ± 0.12a	18 ± 0.12c
DC9	14 ± 0.13d	17 ± 0.18f	22 ± 0.13a
DC10	13 ± 0.11a	18 ± 0.17b	23 ± 0.18b
LA-5	13 ± 0.13a	16 ± 0.13a	22 ± 0.11a

Note: Leuconostoc mesenteroides DC1, DC2, DC3, DC4, DC5, DC7, DC8, and DC9; Pediococcus pentosaceus DC6; Latilactobacillus sakei DC10; Latilactobacillus. acidophilus LA-5 (Chr. Hansen, Denmark) was used for analysis. Values are expressed as mean ± SD (n = 3). Means within the same column followed by different superscript letters are significantly different (Tukey’s HSD test, p < 0.05)

DH of WPC solution

LAB possess a complex system of proteinases and peptidases that enables 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 soluble protein content. In this 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 are 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 to 4 h. DH progressed slowly after 16 h (DH %), indicating that the whey protein hydrolysis reached its highest proteolytic rate at 16 h. The extent of proteolysis was time-dependent. These results indicate that the LAB strain showed proteolytic activity in the whey protein solution (Fig. 1).

Fig. 1.

Degree of hydrolysis (DH, %) of fermented whey protein concentrate (WPC) with LAB strains. Note: Leuconostoc mesenteroides DC3 and DC8, and Latilactobacillus sakei DC10 were used in this study. All data represent the mean ± SD (n = 3)

SDS-PAGE

Figure 2 shows the SDS-PAGE analysis of whey proteins using L. sakei DC10. Protein bands with molecular weights ranging from 13 kDa to 250 kDa were identified. The main components of whey protein, including lactoferrin, bovine serum albumin (BSA), immunoglobulin, β-lactoglobulin and α-lactalbumin, were observed at bands from 14 to 100 kDa. Components with higher molecular weights were degraded during fermentation. After 16 h of fermentation, the lactoferrin, BSA, and immunoglobulins were degraded.

Fig. 2.

Sodium dodecyl 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, USA), which provide 12 pre-stained recombinant protein bands ranging from 2–250 kD, including nine blue-stained and three 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 Latilactobacillus sakei

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 h column in a preparative liquid chromatography system (Fig. 3).

Fig. 3.

Separation of the WPC fermented with DC10 using an FPLC system with a HiPrep 16/60 Sephacryl S-100R column. Note: Latilactobacillus sakei DC10 was used in this study. Absorbance was measured at 280 nm

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 calcium-solubilizing ability similar to that of CPP. The experimental results showed that CPP had high calcium solubility (93.67%). The phosphoserine contained in CPP dissolves calcium and increases its solubility (Kim et al., 2021). All fractions from the fermented WPC solution with L. sakei DC10 had lower calcium solubilization capacity than CPP but showed higher calcium solubilization capacity than unhydrolyzed whey protein. Among them, Fraction 3 had lower calcium solubilization CPP but approximately 85% calcium solubilization capacity (Fig. 4). This highlights the potential calcium solubilization capacity.

Fig. 4.

Calcium solubilization 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. DC10 represents Latilactobacillus sakei. All samples were adjusted to a protein concentration of 200 µg/mL. Data are expressed as the mean ± SD (n = 3). Statistical analyses were performed using one-way ANOVA followed by Tukey’s HSD test. Different superscript letters (a–d) indicate significant differences at p < 0.05

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 (Leung et al., 2009). The results of the ABTS assay are shown in Fig. 5. Except for F1 and F4, the other fractions showed higher ABTS radical scavenging activities than non-fermented whey protein. Fraction 3 had the highest value.

Fig. 5.

Antioxidant activities 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). DC10 represents Latilactobacillus sakei. All samples were adjusted to a protein concentration of 200 µg/mL. All data represent the mean ± SD (n = 3). Statistical analyses were performed using one-way ANOVA followed by Tukey’s HSD test. Superscript letters (a–d) indicate significant differences at p < 0.05

DPPH assay

DPPH is a stable free radical that is widely accepted as a tool for estimating the free radical-scavenging activity of antioxidants (Lin & Chang, 2000). The antioxidant activity of each fraction was evaluated by measuring the DPPH scavenging activity. DPPH scavenging activity values were statistically analyzed based on a standard curve of the Trolox solution. Fractions 2 and 3 showed DPPH radical scavenging ability similar to that of CPP (Fig. 5).

FRAP assay

The decrease in absorbance was proportional to the antioxidant content (Benzie & Strain, 1996). The FRAP assay is a direct method for measuring the 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 (Lin & Chang, 2000). The results indicated that Fraction 3 exhibited a higher iron reduction capacity than CPP (Fig. 5).

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 of fractions and CPP improved cell viability. In addition, all fractions showed > 80% cell viability (Fig. 6). This confirmed that the macrophage fractions were not cytotoxic.

Fig. 6.

Effect of fractions from fermented WPC-80 with DC10 on cell viability in RAW 264.7 cells. Note: The control refers to the untreated WPC-80 solution. CPP indicates casein phosphopeptide. F# denotes the fraction number obtained from the FPLC system. Samples were prepared at protein concentrations of 0.25, 0.5, 1.0, and 2.0 mg/mL. All data represent the mean ± SD (n = 3). Statistical analyses were performed using one-way ANOVA followed by Tukey’s HSD test. Superscript letters (a–d) indicate significant differences at p < 0.05

Nitric oxide (NO) assay

Nitric oxide (NO) plays important roles in blood coagulation, blood pressure regulation, and immune function in cancer cells. However, it is oxidized to reactive oxygen species (ROS) and converted into active NO, which produces cytotoxic oxidants. NO production in cells exposed to inflammatory mediators is increased in response to 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.

Fig. 7.

Effect 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 represent the mean ± SD (n = 3). Statistical analyses were performed using one-way ANOVA followed by Tukey’s HSD test. Superscript letters (a–d) indicate significant differences at p < 0.05

Measurement of cytokine production (ELISA)

Direct or indirect interactions between immune cells are required to maintain immune balance. Cytokines can induce proliferation, differentiation, and changes in the function and activity of various immune cells. Diseases are mostly associated with inflammation, and inflammatory cells secrete inflammatory cytokines that induce inflammation (Barland et al., 2004). The 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, the production of three cytokines (IL-1α, IL-6, and TNF-α) was significantly lower than that of the LPS(+) group. Based on these results, anti-inflammatory activity was observed in the hydrolysates. 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 reduce the inflammatory response.

Fig. 8.

Effect 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 represent the mean ± SD (n = 3). Statistical analyses were performed using one-way ANOVA followed by Tukey’s HSD test. Superscript letters (a–d) indicate significant differences at p < 0.05

Amino acid profiling

Fraction 3 was selected for amino acid composition analysis and peptide sequencing because of its superior functional properties, including the highest calcium solubilization, strongest antioxidant activity, and significant anti-inflammatory effects without cytotoxicity, as demonstrated in previous experiments. By confirming the amino acid sequences of selected fractions using the AccQ-Tag system, 17 amino acid species and several unknown amino acid species were identified (Fig. 9; Table 7). Fraction 3 had the highest leucine and lowest cysteine content.

Fig. 9.

High-performance liquid chromatography chromatogram of amino acids of Fraction 3

Table 7.

Amino acids concentrations of fraction 3

Amino acid	WPC (%)	Fraction 3 (%)
Asp	7.55 ± 0.04a	2.78 ± 0.06b
Thr	8.49 ± 0.01a	4.83 ± 0.05b
Ser	6.53 ± 0.09a	3.31 ± 0.04b
Glu	17.8 ± 0.3a	1.89 ± 0.05b
Pro	8.27 ± 0.47a	2.88 ± 0.05b
Gly	3.87 ± 0.29a	0.43 ± 0.05b
Ala	9.07 ± 0.08a	8.16 ± 0.05b
Val	4.9 ± 0.3a	17.49 ± 0.05b
Cys	0.68 ± 0.03a	0.13 ± 0.03b
Met	1.66 ± 0.22a	0.34 ± 0.05b
Ile	4.93 ± 0.09a	13.58 ± 0.04b
Leu	10.36 ± 0.52a	21.1 ± 0.21b
His	1.73 ± 0.37a	1.91 ± 0.2a
Phe	2.81 ± 0.84a	8.45 ± 0.16b
Lys	8.29 ± 0.25a	5.86 ± 0.12b
Tyr	1.42 ± 0.19a	1.91 ± 0.19a
Arg	1.64 ± 0.8a	4.95 ± 0.8b
Total molar ratio	100	100

Note: WPC refers to non-fermented whey protein concentrate and Fraction 3 refers to the fraction derived from whey protein concentrate fermented with Latilactobacillus sakei DC10. All samples were adjusted to a protein concentration of 1.0 mg/mL and measured in triplicate. Data represent the mean ± SD. Statistical significance between WPC and Fraction 3 for each amino acid was evaluated using an independent Student’s t-test. Different superscript letters indicate significant differences (n = 3; significance set at p < 0.05)

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, only four were fully sequenced, and additional purification of Fraction 3 was required. Four peptides were identified from Fraction 3 (Table 8) with molecular masses of 905.49, 658.36, 893.96, and 883.46 Da. The 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/za	RT (min)b	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

^aMeans m/z value of (M + H)⁺

^bMeans retention time

Fig. 10.

Liquid chromatography-mass spectrometry base peak chromatogram of Fraction 3

Fig. 11.

Mass spectrometric de novo sequencing

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 (Smithers, 2008). In this 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 the results of subsequent physiological and functional assays.

The DH of the 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 (Bu et al., 2010).

The peptide fractions of fermented whey were analyzed for molecular weight using a HiPrep 16/60 Sephacryl S-100 h column. The peptides in Fraction 3 exhibited 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 (Liu et al., 2021). In particular, leucine, aspartic acid, and glutamic acid play key roles in such structural interactions. Amino acid analysis of Fraction 3 revealed high levels of BCAAs (leucine, isoleucine, and valine), which may further enhance calcium metabolism by modulating intestinal absorption and vitamin D receptor expression (Daliri et al., 2018; Tang & Skibsted, 2016; Habibi et al., 2022).

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 (Skrzypczak et al., 2017).

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 (Mansinhbhai et al., 2021).

In other studies, TVQVTSTAV and DKTEIPTINTIA were reported to be peptides with notable antimicrobial activities, whereas SLVYPFPGPIHNSLPQ exhibited immunomodulatory effects (Skrzypczak et al., 2017). These results suggested that Fraction 3 obtained in the present study may have antioxidant, anti-inflammatory, antibacterial, and immunomodulatory properties.

Collectively, these results highlight the novelty of Fraction 3 from WPC fermented with L. sakei DC10, which uniquely integrates calcium-binding, antioxidant, and immunomodulatory properties within a single peptide fraction. This multifunctional profile highlights the potential of LAB-fermented WPC as a source of bioactive peptides for use in functional food formulations. Thus, future studies should investigate the underlying molecular mechanisms, assess in vivo bioavailability, and explore clinical applications to substantiate its physiological benefits.

Author contributions

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.

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).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.