This work was supported by the Yunling Scholar Program (Grant No. XDYC-YLXZ-2023-0032), the National Natural Science Foundation of China (Grant No. 32060572) and the Yun-Ling Industrial Technology Leading Talent program (Grant No. 2014-1782). We sincerely thank Li Guanyuan (College of Food Science and Technology, Yunnan Agricultural University) for their help in this study.
Binglangjiang buffalo fermented milk (BBFM) is rich in bioactive peptides and exhibits significant immunomodulatory activity, although the specific components and effects are not well understood. In this study, we investigated the anti-inflammatory activity peptides from BBFM using peptidomics and proteomics. A total of 769 peptides were identified using LC-MS/MS. Among these, a novel peptide (GPGAPADPGRPTG (GG13)), was screened out using LPS-stimulated RAW264.7 cells. The molecular weight of peptide GG13 was 1149.56 Da, and it exhibited high in-vitro safety and thermal stability. Furthermore, ELISA and Western blot analysis showed that peptide GG13 significantly inhibited the secretion of pro-inflammatory cytokines NO and TNF-α, as well as the expression of proteins iNOS and TNF-α in LPS-stimulated RAW264.7 cells, and proteomics analysis showed that peptide GG13 significantly down-regulated the protein expression of STAT1, NOS2, COX2, and CD40. The study provides a basis for further explore the development of health fermented milk products.
Inflammation is the body's immune response to external stimuli, infections, and tissue damage. During instances of inflammation, different inflammatory agents, cytokines, and reactive oxygen species (ROS) become activated. Among these are nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), along with various other immune cells. Excessive production of these elements may result in tissue harm and impaired immune system performance, contributing to various health conditions like type 2 diabetes, obesity, bowel inflammation, and dementia. Therefore, managing inflammation is crucial for health. Previous studies have focused on regulating the secretion pathway of inflammatory factors to control the activity of pro-inflammatory cytokines by macrophages. Researchers have turned their focus to investigating bioactive peptides from food as potential anti-inflammatory agents, due to worries surrounding the long-term impact of anti-inflammatory medications. Bioactive peptides derived from milk are produced through various processes, including hydrolysis of proteins or peptides, microbial fermentation, and digestion in the gastrointestinal tract. Research has demonstrated that dairy products can contribute to lowering blood pressure and providing antioxidative, anticancer, and immunomodulatory effects. Therefore, bioactive peptides derived from milk, which appear safe and with no adverse effects, have garnered significant interest in the creation of functional foods and dietary supplements, and serving as a means to combat chronic inflammation.
Fermented milk is obtained as a result of the fermentation of milk by characteristic bacteria, being widely popular for its attractive flavor, texture, and health benefits, especially among people with special nutritional needs. During milk fermentation, lactic acid bacteria (LAB) secrete enzymes like peptidyl peptidase, aminopeptidase, and carboxypeptidase in order to break down milk proteins into peptides that possess diverse biological characteristics, including antibacterial, anti-inflammatory, angiotensin converting enzyme (ACE) inhibition, and immune modulation activities. For example, fermenting goat milk with the Lactobacillus plantarum KGL4 strain has been demonstrated to reduce the levels of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β in LPS-stimulated RAW 264.7 cells, thereby potentially managing the inflammatory response effectively. Furthermore, camel milk fermented with the Lactobacillus plantarum KGL3A strain could inhibit pro-inflammatory mediators and nitric oxide in LPS-stimulated RAW264.7 macrophages, hence displaying anti-inflammatory properties. Consequently, bioactive peptides generated from the fermentation of cow milk, goat milk, and camel milk fermentation by LAB have demonstrated promising anti-inflammatory effects. A new species of buffalo known as the Binglangjiang buffalo was found in Tengchong City, Yunnan Province, China, which yields a milk containing more protein and fat than cow's milk. In previous studies conducted by our research group, bioactive peptides were isolated from casein and whey protein of Binglangjiang buffalo milk which showed notable antibacterial, hypoglycemic, and anti-inflammatory activity. However, studies on the identification of anti-inflammatory peptides from Binglangjiang buffalo milk and their activity using LAB fermentation are still lacking.
In the present study, peptidomics combined with LPS-stimulation of RAW264.7 cells was used to screen peptides with anti-inflammatory activity from BBFM. Furthermore, the structural properties, stability, and in vitro safety of anti-inflammatory peptides were investigated. Finally, the effects of these peptides on the secretion of pro-inflammatory factors (i.e., NO, TNF-α and iNOS) in LPS-stimulated RAW264.7 cells were analyzed using ELISA and Western blotting, and the potential anti-inflammatory pathways involved were also elucidated using proteomics.
Fresh Binglangjiang buffalo milk was obtained in Tengchong City, Yunnan Province, China. Reagents like fetal bovine serum (FBS), high glucose medium (DMEM), phosphate-buffered saline (PBS) with a pH of 7.2, penicillin, and streptomycin were obtained from the Biological Industries (Beit Haemek, Israel). The chemicals 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) and lipopolysaccharide (LPS) were obtained from Solarbio (Beijing, China). Nitric oxide (NO) Griess reagent was
Proteins from Binglangjiang buffalo milk were hydrolyzed into bioactive activity peptides through LAB fermentation, and amino acid sequences of peptides with MW less than 3000 Da were identified using LC-MS/MS (Supplementary Fig. S1). A total of 769 peptides were discovered in Binglangjiang buffalo fermented milk (Supplementary Table S1) and classified according to molecular weight. Among these, 22, 101, 609, and 37 peptides were identified with MW greater than 3000 Da, 2000–3000 Da, 1000–2000 Da, and less than 1000 Da,
Milk bioactive peptides are mainly derived from casein and whey protein, and can be obtained through proteolysis, microbial fermentation, and digestion in the gastrointestinal tract. These peptides have shown to regulate inflammation and alleviate various disease symptoms in humans. Milk fermented by LAB not only enhances the sensory, nutritional, and health properties of the food, but also results in the production of bioactive peptides which are considered safer and
Fermented milk is rich in bioactive peptides. Herein, a total of 769 peptides were identified from BBFM. An anti-inflammatory activity peptide with potent safety and heat resistance, GG 13, was screened from the BBFM using peptidomics and proteomics. Peptide GG13 could effectively inhibit the expression of pro-inflammatory cytokines and the overproduction of NO in LPS-stimulation RAW264.7 cells by down regulating key protein expression of STAT1, NOS2, COX2, and CD40 in the JAK-STAT and
The experimental protocol involving animal experiment was in accordant with the national law of China. In the course of carrying out the study, appropriate protocols were utilized and all procedures were carried out in accordance with the relevant laws and institutional guidelines and have been approved by the appropriate institutional committees.
Wentao Zheng: Writing – original draft, Methodology, Formal analysis, Data curation, Conceptualization. Yufang Li: Validation, Formal analysis, Data curation. Yuzhu Wang: Investigation, Data curation. Jinze He: Investigation, Data curation. Qiong Zhao: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision. Aixiang Huang: Writing – review & editing, Writing – original draft, Visualization, Project administration, Funding acquisition.
We declare that we do not have any personal, financial, commercial or associative interest that represents a conflict of interest in connection with the work submitted.
J.K. Ashokbhai et al. Antioxidative, antimicrobial and anti-inflammatory activities and release of ultra-filtered antioxidative and antimicrobial peptides during fermentation of sheep milk: In-vitro, in-silico and molecular interaction studies
