Activities of proteases in deep eutectic solvents and removal of protein from chitin by subtilisin A in betaine/glycerol.

This research intended to remove residual protein from chitin with proteases in deep eutectic solvents (DESs). The activities of some proteases in several DESs, including choline chloride/p-toluenesulfonic acid, betaine/glycerol (Bet/G), choline chloride/malic acid, choline chloride/lactic acid, and choline chloride/urea, which are capable of dissolving chitin, were tested, and only in Bet/G some proteases were found to be active, with subtilisin A, ficin, and bromelain showing higher activity than other proteases. However, the latter two proteases caused degradation of chitin molecules. Further investigation revealed that subtilisin A in Bet/G did not exhibit "pH memory", which is a universal characteristic displayed by enzymes dispersed in organic phases, and the catalytic characteristics of subtilisin A in Bet/G differed significantly from those in aqueous phase. The conditions for protein removal from chitin by subtilisin A in Bet/G were determined: Chitin dissolved in Bet/G with 0.5 % subtilisin A (442.0 U/mg, based on the mass of chitin) was hydrolyzed at 45 °C for 30 min. The residual protein content in chitin decreased from 5.75 % ± 0.10 % to 1.01 % ± 0.12 %, improving protein removal by 57.20 % compared with protein removal obtained by Bet/G alone. The crystallinity and deacetylation degrees of chitin remained unchanged after the treatment.

The antioxidant peptides from walnut protein hydrolysates and their protective activity against alcoholic injury.

In this study, walnut protein was hydrolyzed, separated by ultrafiltration, purified by RP-HPLC, identified by LC-MS/MS, and screened by molecular docking to finally obtain three novel antioxidant peptides HGEPGQQQR (1189.584 Da), VAPFPEVFGK (1089.586 Da) and HNVADPQR (949.473 Da). These three peptides exhibited excellent cellular antioxidant activity (CAA) with EC50 values of 0.0120 mg mL-1, 0.0068 mg mL-1, and 0.0069 mg mL-1, respectively, which were superior to that of the positive control GSH (EC50: 0.0122 mg mL-1). In the ethanol injury model, three antioxidant peptides enhanced the survival of cells treated with ethanol from 47.36% to 62.69%, 57.06% and 71.64%, respectively. Molecular docking results showed that the three antioxidant peptides could effectively bind to Keap1, CYP2E1 and TLR4 proteins. These results suggested that walnut-derived antioxidant peptides could be potential antioxidants and hepatoprotective agents for application in functional foods.

Effect of the phosphorylation structure in casein phosphopeptides on the proliferation, differentiation, and mineralization of osteoblasts and its mechanism.

Our previous studies have shown that highly phosphorylated casein phosphopeptides (residues 1-25) P5 could efficiently bind calcium and promote intestinal calcium absorption, and enhanced bone development in rats. The purpose of this study was to investigate the effect of the phosphorylation structure in P5 on the proliferation, differentiation, and mineralization of osteoblasts (MC3T3-E1) and its mechanism. P5 was obtained by high-performance liquid chromatography (HPLC) and non-phosphorylated peptide P5-0 was obtained by chemical synthesis. Compared with the control group, the proliferation rate of MC3T3-E1 cells treated by P5 was 1.10 times that of P5-0 at 200 µg mL-1. P5 caused the cell cycle retention of MC3T3-E1 cells in the G2/M phase, while P5-0 had no significant difference in the G2/M phase. MC3T3-E1 cells incubated with P5 showed stronger alkaline phosphatase (ALP) activity than with P5-0, suggesting a tendency to promote cellular differentiation. Compared to the P5-0 treatment group, the P5 treatment group at concentrations of 10 µg mL-1 showed significant differences in the mineralization rates (p < 0.05). P5 significantly upregulated the expressions of Runx2, ALP, ColIα1, and OCN compared with the control group (p < 0.05). In addition, in silico molecular docking showed that the binding force of the P5-EGFR complex was stronger than that of the P5-0-EGFR complex, which was significantly related to the phosphorylation structure in P5 and might be an important reason for osteoblast proliferation. In conclusion, the phosphorylation structure and amino acid composition in P5 stimulated the osteogenic activity of MC3T3-E1 cells, and could be expected to be a functional food for the prevention of osteoporosis.

Four novel sleep-promoting peptides screened and identified from bovine casein hydrolysates using a patch-clamp model in vitro and Caenorhabditis elegans in vivo.

Bovine casein hydrolysates (CHs) have demonstrated sleep-promoting activities. However, only few peptides were identified from CHs with sleep-promoting effects. In this work, an in vitro model based on the electrophysiology of brain neurons was established for the evaluation of sleep-promoting effects. Based on this model, four novel peptides were systematically separated from CH. Compared with the control group, the action potential (AP) inhibitory rate of four peptides increased by 38.63%, 340.93%, 233.28%, and 900%, respectively, and the membrane potential (MP) change rate of four peptides increased by 319.78%, 503.09%, 381.22%, and 547.10%, respectively. These results suggested that four peptides have sleep-promoting activities. Furthermore, Caenorhabditis elegans (C. elegans) sleep behavior results indicated that all the four peptides could significantly increase the total sleep duration, the motionless sleep duration of C. elegans, implying that these four peptides can significantly improve sleep. The LC-MS/MS results showed that the primary structures of these novel peptides were HQGLPQEVLNENLLR (αs1-CN, f8-22), YKVPQLEIVPNSAEER (αs1-CN, f104-119), HPIKHQGLPQEVLNENLLR (αs1-CN, f4-22), and VPQLEIVPNSAEER (αs1-CN, f106-119). Overall, this study revealed that the four novel sleep-promoting peptides identified were strong candidates as potential functional ingredients in the development of sleep-promoting products.

Purification and activity evaluation of novel anti-inflammatory peptides from pearl oyster (Pinctada martensii) hydrolysates.

Pearl oyster meat, a by-product of pearl production, is rich in protein, but has a low utilization rate. Our previous study showed that pearl oyster meat hydrolysates have potential anti-inflammatory activity. In this study, highly active peptides from pearl oyster meat hydrolysates were purified, identified, and extracted, and their anti-inflammatory activity was further investigated. A total of 206 peptides were identified, and three novel anti-inflammatory peptides, TWP (402.1903 Da), TAMY (484.1992 Da) and FPGA (390.1903 Da), were screened by molecular docking. The molecular docking results showed that TWP, TAMY and FPGA can bind to key regions in the cyclooxygenase-2 (COX-2) active site. Furthermore, the three anti-inflammatory peptides can effectively regulate the release of inflammatory mediators from RAW264.7 macrophages by reducing the levels of nitric oxide (NO) and pro-inflammatory cytokines (such as TNF-α, IL-6 and IL-1ß), and increasing the production of anti-inflammatory cytokine IL-10, showing great anti-inflammatory activity. This study provides a new theoretical reference for the development of functional foods or nutritional supplements with natural anti-inflammatory effects.

The nutritional and functional properties of 1-oleoyl-2-palmitoyl-3-linoleoylglycerol-rich oil: promoting early-life growth and intestinal health with alterations in the intestinal microbiota of Micropterus salmoides.

1-Oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL), a key structural lipid in the breast milk fat, plays a critical role in providing nutrients and energy for infants. OPL is more abundant in Chinese breast milk fat and might be better for Chinese infants' growth. However, few studies have investigated the effect of OPL on the growth and intestinal health of the organism in early life. OPL-rich oil with 45.77% OPL was prepared by immobilized lipase-catalyzed synthesis and purification. The effects of OPL on the nutritional properties and the regulation of intestinal microbiota in early life were further investigated in vivo (Micropterus salmoides). Dietary OPL-rich oil significantly increased the juvenile fish weight gain rate, protein content, and muscular polyunsaturated fatty acids, which in turn markedly altered the muscle texture in springiness and cohesiveness. Dietary OPL-rich oil could also protect intestinal tissues by significantly increasing fish intestinal fold height, mucosal thickness, and intestinal wall thickness. Furthermore, dietary OPL-rich oil regulated intestinal microbiota. Particularly, OPL significantly increased the probiotics (Cetobacterium_sp014250685, Streptomyces_mutabilis, Saccharopolyspora_spinosa, and Nocardiopsis_kunsanensis) and reduced the potential pathogens (Staphylococcus_nepalensis, Salmonella_enterica, the Candidatus_berkiella). The structured OPL significantly promoted fish growth and improved nutritional composition due to its higher bioavailability relative to tripalmitate (PPP). Moreover, OPL significantly improved the growth, cholesterol metabolism, and intestinal health than the mixed oil (MO), which was attributed to the higher palmitic acid content in the sn-2 position. Overall, the structure of triacylglycerols and its distribution of fatty acids affected early growth and intestinal health, and OPL was more effective in the improvement of juvenile growth and intestinal health.

Purification, identification and hypolipidemic activities of three novel hypolipidemic peptides from tea protein.

In this study, hypolipidemic peptides were obtained from tea protein by enzymatic hydrolysis, ultrafiltration and high-performance liquid chromatography. Subsequently, the hypolipidemic peptides were identified by mass spectrometry and screened through molecular docking technology, and the hypolipidemic activities and mechanisms of the active peptides were explored. The results showed that the hydrolysate of hypolipidemic peptides obtained by pepsin hydrolysis for 3 h had good bile salt binding ability. After purification, identification and molecular docking screening, three novel hypolipidemic peptides FLF, IYF and QIF were obtained. FLF, IYF and QIF can interact with the receptor proteins 1LPB and 1F6W through hydrogen bonds, π-π bonds, hydrophobic interactions and van der Waals forces, thus exerting hypolipidemic activities. Activity studies showed that, compared with the positive controls, FLF, IYF and QIF had excellent sodium taurocholate binding abilities, pancreatic lipase inhibitory activities and cholesterol esterase inhibitory activities. Moreover, FLF, IYF and QIF can effectively inhibit lipogenic differentiation of 3T3-L1 preadipocytes, reduce intracellular lipid and low-density lipoprotein content and increase high-density lipoprotein content. These results indicated that the three novel hypolipidemic peptides screened in this study had excellent hypolipidemic activities and were expected to be used as natural-derived hypolipidemic active ingredients for the development and application in functional foods.

Purification, Identification, and Mechanistic Investigation of Novel Selenium-Enriched Antioxidant Peptides from Moringa oleifera Seeds.

In this study, five novel Se-enriched antioxidant peptides (FLSeML, LSeMAAL, LASeMMVL, SeMLLAA, and LSeMAL) were purified and identified from Se-enriched Moringa oleifera (M. oleifera) seed protein hydrolysate. The five peptides showed excellent cellular antioxidant activity, with respective EC50 values of 0.291, 0.383, 0.662, 0.1, and 0.123 µg/mL. The five peptides (0.025 mg/mL) increased the cell viability from 78.72 to 90.71, 89.16, 93.92, 83.68, and 98.29%, respectively, effectively reducing reactive oxygen species accumulation and significantly increasing superoxide dismutase and catalase activities in damaged cells. Molecular docking results revealed that the five novel Se-enriched peptides interacted with the key amino acid of Keap1, thus directly blocking the interaction of Keap1-Nrf2 and activating the antioxidant stress response to enhance the ability of scavenging free radicals in vitro. In conclusion, Se-enriched M. oleifera seed peptides exhibited significant antioxidant activity and can be expected to find widespread use as a highly active natural functional food additive and ingredient.

Two Novel Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from Rice (Oryza sativa L.) Bran Protein.

To realize the high-value utilization of rice byproducts, the rice bran protein hydrolysate was separated and purified by ultrafiltration and reversed-phase high-performance liquid chromatography (RP-HPLC), then the sequences of peptides were identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and their molecular docking analysis and activities in vitro and in the cell were carried out. Two novel peptides FDGSPVGY (840.3654 Da) and VFDGVLRPGQ (1086.582 Da) were obtained with IC50 values of 0.079 mg/mL (94.05 µM) and 0.093 mg/mL (85.59 µM) on angiotensin I-converting enzyme (ACE) inhibitory activity in vitro, respectively. Molecular docking results showed that two peptides interacted with ACE receptor protein through hydrogen bonding, hydrophobic interactions, etc. Through the EA.hy926 cells, it was found that FDGSPVGY and VFDGVLRPGQ could promote the release of nitric oxide (NO) and reduce the content of ET-1 to achieve the effect of antihypertension. In conclusion, the peptides from rice bran protein exhibited significant antihypertension activity and may be expected to realize the high-value utilization of rice byproducts.

Rapid screening of novel tyrosinase inhibitory peptides from a pearl shell meat hydrolysate by molecular docking and the anti-melanin mechanism.

Pearls are an edible and medicinal resource with whitening activity and nutritional value in China. In the previous study, we found that the pearl shell meat hydrolysate showed dual activities of antioxidation and tyrosinase inhibition, which were similar to the activities of pearls. In this research, a pearl shell meat hydrolysate was isolated, identified and screened by molecular docking, and three peptides FLF, SPSSS and WLL with high tyrosinase inhibitory activities were obtained. The results indicated that FLF, SPSSS and WLL could effectively inhibit tyrosinase activities and the inhibition rates (1.0 mg mL-1) were 54.32%, 65.26% and 57.50%, respectively. The results of a zebrafish whitening experiment showed that the tyrosinase activities of zebrafish treated with FLF, SPSSS and WLL decreased by 75.41%, 62.87% and 64.99% (p < 0.05), respectively, and the melanin content decreased by 37.34%, 38.52% and 40.39% (p < 0.05), respectively. In a B16F10 cell whitening experiment, compared with a control group, FLF, SPSSS and WLL also showed a significant whitening effect, the tyrosinase activities decreased by 84.08%, 79.08% and 77.45% (p < 0.05), respectively, and the melanin content decreased by 42.23%, 34.37% and 34.02% (p < 0.05), respectively. Moreover, the active peptides could act on three signal pathways including Wnt/ß-catenin, MAPK and MC1R/α-MSH and significantly downregulated the expressions of the signaling factors WNT4, MITF, ß-catenin, ERK, JNK, TRP1 and TRP2 (p < 0.05). The results demonstrated that the whitening active peptides were edible natural antioxidants, tyrosinase inhibitors and skin anti-melanin agents, which could be added to functional foods as food ingredients.

Novel Antioxidant Peptides from Pearl Shell Meat Hydrolysate and Their Antioxidant Activity Mechanism.

Free radicals are associated with aging and many diseases. Antioxidant peptides with good antioxidant activity and absorbability are one of the hotspots in antioxidant researches. In our study, pearl shell (Pinctada martensii) meat hydrolysate was purified, and after identification by proteomics, six novel antioxidant peptides SPSSS, SGTAV, TGVAS, GGSIT, NSVAA, and GGSLT were screened by bioinformatics analysis. The antioxidant peptides exhibited good cellular antioxidant activity (CAA) and the CAA of SGTAV (EC50: 0.009 mg/mL) and SPSSS (EC50: 0.027 mg/mL) were better than that of positive control GSH (EC50: 0.030 mg/mL). In the AAPH-induced oxidative damage models, the antioxidant peptides significantly increased the viability of HepG2 cells, and the cell viability of SGTAV, SPSSS, and NAVAA were significantly restored from 79.41% to 107.43% and from 101.09% and 100.09%, respectively. In terms of antioxidant mechanism by molecular docking, SGTAV, SPSSS, and NAVAA could tightly bind to free radicals (DPPH and ABTS), antioxidant enzymes (CAT and SOD), and antioxidant channel protein (Keap1), suggesting that the antioxidant peptides had multiple antioxidant activities and had structure-activity linkages. This study suggests that the antioxidant peptides above are expected to become new natural materials for functional food industries, which contribute to the high-value applications of pearl shell meat.

Identification and functional analysis of three novel osteogenic peptides isolated from tilapia scale collagen hydrolysate.

On the basis of our previous study that 133 peptides were identified from the tilapia scale peptide-calcium chelate, the potential osteogenic peptide monomers through the calcium-binding properties of peptides and molecular docking were screened, and the osteogenic activity and active mechanism of the peptides were further researched in this study. Three highly osteogenic peptides GPAGPHGPVG (844.4191 Da), APDPFRMY (995.4534 Da), and TPERYY (827.3813 Da) were screened. Molecular docking showed that the three osteogenic peptides had the same interaction sites in the epidermal growth factor receptor (EGFR), namely ARG 285, GLN 8, GLY 317, THR 406, and HIS 409. Compared to the blank control group, within 50 µg/mL of GPAGPHGPVG, APDPFRMY, and TPERYY increased the proliferation of MC3T3-E1 cells by changing the cell proportion in the S and G2/M phases, and the alkaline phosphatase (ALP) activity of MC3T3-E1 cells treated with 50 µg/mL of the three active peptides increased by 25%, 37%, and 56%, respectively. The three active peptides at 10 µg/mL concentration significantly promoted the mineralization of osteoblasts, and the mineralized calcium nodules increased by 166%, 161%, and 111%, respectively. TPERYY significantly increased the expression of osteogenic genes (osteocalcin (OCN), type I collagen (Col I α) and transcription factor (OSX)). Moreover, TPERYY significantly increased the mRNA and protein expression of ß-catenin to 1.39 and 2.6 times of the blank control group, respectively, while decreased the mRNA and protein expression of glycogen synthesis kinase (GSK3ß) to 1.6 and 2.3 times of the blank control group, respectively. This study provides a theoretical basis for using GPAGPHGPVG, APDPFRMY, and TPERYY peptides as functional foods to prevent osteoporosis.

A comprehensive review on preparation, structure-activities relationship, and calcium bioavailability of casein phosphopeptides.

Calcium is one of the important elements for human health. Calcium deficiencies can lead to numerous diseases. Calcium chelating peptides have shown potential application in the management of calcium deficiencies. Casein phosphopeptides (CPP) are phosphoseryl-containing fragments of casein by enzymatic hydrolysis or fermentation during manufacture of milk products as well as during intestinal digestion. An increasing number of CPP with the ability to facilitate and enhance the bioavailability of calcium are being discovered and identified. In this review, 249 reported CPP derived from four types of bovine casein (αs1, αs2, ß and κ) were collected, and the amino acid sequence and phosphoserine group information were sorted out. This review outlines the current enzyme hydrolysis, detection methods, purification, structure-activity relationship and mechanism of intestinal calcium absorption in vitro and in vivo as well as application of CPP.

Preparation, characterization, and osteogenic activity mechanism of casein phosphopeptide-calcium chelate.

Casein phosphopeptides (CPPs) are good at calcium-binding and intestinal calcium absorption, but there are few studies on the osteogenic activity of CPPs. In this study, the preparation of casein phosphopeptide calcium chelate (CPP-Ca) was optimized on the basis of previous studies, and its peptide-calcium chelating activity was characterized. Subsequently, the effects of CPP-Ca on the proliferation, differentiation, and mineralization of MC3T3-E1 cells were studied, and the differentiation mechanism of CPP-Ca on MC3T3-E1 cells was further elucidated by RNA sequencing (RNA-seq). The results showed that the calcium chelation rate of CPPs was 23.37%, and the calcium content of CPP-Ca reached 2.64 × 105 mg/kg. The test results of Ultraviolet-Visible absorption spectroscopy (UV) and Fourier transform infrared spectroscopy (FTIR) indicated that carboxyl oxygen and amino nitrogen atoms of CPPs might be chelated with calcium during the chelation. Compared with the control group, the proliferation of MC3T3-E1 cells treated with 250 µg/mL of CPP-Ca increased by 21.65%, 26.43%, and 28.43% at 24, 48, and 72 h, respectively, and the alkaline phosphatase (ALP) activity and mineralized calcium nodules of MC3T3-E1 cells were notably increased by 55% and 72%. RNA-seq results showed that 321 differentially expressed genes (DEGs) were found in MC3T3-E1 cells treated with CPP-Ca, including 121 upregulated and 200 downregulated genes. Gene ontology (GO) revealed that the DEGs mainly played important roles in the regulation of cellular components. The enrichment of the Kyoto Encyclopedia of Genes and Genomes Database (KEGG) pathway indicated that the AMPK, PI3K-Akt, MAPK, and Wnt signaling pathways were involved in the differentiation of MC3T3-E1 cells. The results of a quantitative real-time PCR (qRT-PCR) showed that compared with the blank control group, the mRNA expressions of Apolipoprotein D (APOD), Osteoglycin (OGN), and Insulin-like growth factor (IGF1) were significantly increased by 2.6, 2.0 and 3.0 times, respectively, while the mRNA levels of NOTUM, WIF1, and LRP4 notably decreased to 2.3, 2.1, and 4.2 times, respectively, which were consistent both in GO functional and KEGG enrichment pathway analysis. This study provided a theoretical basis for CPP-Ca as a nutritional additive in the treatment and prevention of osteoporosis.

Purification, identification, and antioxidative mechanism of three novel selenium-enriched oyster antioxidant peptides.

Natural organic selenium (Se) has multiple physiological health benefits and has become a hotspot of research in recent years. In this study, the Se-enriched antioxidant peptides were purified from Se-enriched oyster hydrolysate. Three novel Se-enriched antioxidant peptides LLVSeMY (685.2953 Da), MMDSeML (687.1875 Da) and VSeMDSeML (703.1599 Da) were identified from fraction F6-4, which all exhibited strong cellular antioxidant activity (CAA) with EC50 values of 0.739, 0.423, and 0.395 µg/mL, respectively. These three Se-enriched antioxidant peptides (0.025 mg/mL) could significantly enhanced cell viability to 84.60 ± 3.32% âˆ¼ 86.18 ± 1.36% compared with the AAPH injury group (75.99 ± 0.79%), and the cytoprotective effects were even better than that of GSH (80.47 ± 2.78%). Moreover, these three Se-enriched peptides also significantly protected HepG2 cells from AAPH-induced oxidative injury by inhibiting ROS production and enhancing the activities of antioxidant enzymes. The molecular docking results showed that these three Se-enriched peptides can form stable hydrogen and hydrophobic bonds with key amino acid residues of Keap1 protein, thereby potentially regulating the Keap1-Nrf2 pathway. In conclusion, the three novel Se-enriched oyster antioxidant peptides are expected to be used in medicine or functional food, providing a new theoretical basis for the high-value utilization of natural organic Se.

High-Performance Thermal Interface Materials with Magnetic Aligned Carbon Fibers.

Thermal interface materials with high thermal conductivity and low hardness are crucial to the heat dissipation of high-power electronics. In this study, a high magnetic field was used to align the milled carbon fibers (CFs, 150 µm) in silicone rubber matrix to fabricate thermal interface materials with an ordered and discontinuous structure. The relationship among the magnetic field density, the alignment degree of CFs, and the properties of the resulting composites was explored by experimental study and theoretical analysis. The results showed higher alignment degree and enhanced thermal conductivity of composites under increased magnetic flux density within a certain curing time. When the magnetic flux density increased to 9 T, the CFs showed perfect alignment and the composite showed a high thermal conductivity of 11.76 W/(m·K) with only 20 vol% CF loading, owing to the ordered structure. Meanwhile, due to the low filler loading and discontinuous structure, a low hardness of 60~70 (shore 00) was also realized. Their thermal management performance was further confirmed in a test system, revealing promising applications for magnetic aligned CF-rubber composites in thermal interface materials.

Antioxidant Activity and Mechanism of Resveratrol and Polydatin Isolated from Mulberry (Morus alba L.).

Natural stilbenes have unique physiological effects, such as anti-senile dementia, anti-cancer, anti-bacterial, lowering blood lipid, and other important biological functions, which have attracted great attention from scholars in recent years. In this study, two stilbene compounds, resveratrol (RES) and polydatin (PD), were isolated from Mulberry (Morus alba L.), and their antioxidant activity and mechanism were investigated. The results showed that the contents of RES and PD in mulberry roots were 32.45 and 3.15 µg/g, respectively, significantly higher than those in mulberry fruits (0.48 and 0.0020 µg/g) and mulberry branches (5.70 and 0.33 µg/g). Both RES and PD showed high antioxidant potential by DPPH, ABTS free-scavenging methods, and ORAC assay, and provided protection against oxidative damage in HepG2 cells by increased catalase (CAT) activity, superoxide dismutase (SOD) activity, and Glutathione (GSH) content, and decreasing generation of reactive oxygen species (ROS), lactate dehydrogenase (LDH) level, and malondialdehyde (MDA) content. Therefore, RES and PD treatment could be effective for attenuating AAPH-induced oxidative stress in HepG2 cells. This study will promote the development and application of stilbene compounds. Furthermore, the RES and PD could be used as antioxidant supplements in functional foods, cosmetics, or pharmaceuticals, contributing to health improvement.

Effects of Antibacterial Peptide F1 on Bacterial Liposome Membrane Integrity.

Previous studies from our lab have shown that the antimicrobial peptide F1 obtained from the milk fermentation by Lactobacillus paracasei FX-6 derived from Tibetan kefir was different from common antimicrobial peptides; specifically, F1 simultaneously inhibited the growth of Gram-negative and Gram-positive bacteria. Here, we present follow-on work demonstrating that after the antimicrobial peptide F1 acts on either Escherichia coli ATCC 25922 (E. coli) or Staphylococcus aureus ATCC 63589 (S. aureus), their respective bacterial membranes were severely deformed. This deformation allowed leakage of potassium and magnesium ions from the bacterial membrane. The interaction between the antimicrobial peptide F1 and the bacterial membrane was further explored by artificially simulating the bacterial phospholipid membranes and then extracting them. The study results indicated that after the antimicrobial peptide F1 interacted with the bacterial membranes caused significant calcein leakage that had been simulated by different liposomes. Furthermore, transmission electron microscopy observations revealed that the phospholipid membrane structure was destroyed and the liposomes presented aggregation and precipitation. Quartz Crystal Microbalance with Dissipation (QCM-D) results showed that the antimicrobial peptide F1 significantly reduced the quality of liposome membrane and increased their viscoelasticity. Based on the study's findings, the phospholipid membrane particle size was significantly increased, indicating that the antimicrobial peptide F1 had a direct effect on the phospholipid membrane. Conclusively, the antimicrobial peptide F1 destroyed the membrane structure of both Gram-negative and Gram-positive bacteria by destroying the shared components of their respective phospholipid membranes which resulted in leakage of cell contents and subsequently cell death.

Promoting the Calcium-Uptake Bioactivity of Casein Phosphopeptides in vitro and in vivo.

Casein phosphopeptides have been studied widely for their ability to chelate calcium. However, systematic studies on the effects of casein phosphopeptides (CPP) on calcium absorption in vitro and in vivo are scarce. The purities of two commercially available products, CPP1 and CPP2, are 18.37 and 25.12%, respectively. Here, the in vitro calcium binding capacity of CPP2 was 142.56 ± 7.39 mg/g, which was higher than that of CPP1 (107.15 ± 6.27 mg/g). The calcium transport results in a Caco-2 monolayer model indicated that, relative to controls, CPP1 and CPP2 increased calcium transport by 21.78 and 53.68%, respectively. Subsequent animal experiments showed that the CPP2-Ca-H group (1% Ca, 0.4% CPP2) had significant increases in the femur index, serum Ca2+ and serum osteocalcin levels, and femoral Ca content. The CPP2-Ca-H animal also had decreased serum alkaline phosphatase levels, parathyroid hormone content, and urinary pyridinoline content. Overall, our results demonstrated that CPP2 had stronger effects on promoting calcium uptake than CPP1.

Comprehensive Utilization of Immature Honey Pomelo Fruit for the Production of Value-Added Compounds Using Novel Continuous Phase Transition Extraction Technology.

The immature honey pomelo fruit (IPF) is a huge agro-industrial by-product generated during pomelo planting. Although IPF is rich in nutrients, more than 95% of IPF is discarded annually, which causes resource waste and a serious environmental problem. Here, we report a novel continuous phase transition extraction technology (CPTE) to improve the comprehensive utilization of IPF by sequentially generating high value products and solve pollution problems related to their disposal. First, essential oil was successively extracted by CPTE at a yield of 1.12 ± 0.36%, in which 43 species were identified. Second, naringin extraction parameters were optimized using the response surface methodology (RSM), resulting in a maximum extraction rate of 99.47 ± 0.15%. Finally, pectin was extracted at a yield of 20.23 ± 0.66%, which is similar to the contents of commercial pectin. In conclusion, this study suggested that IPF was an excellent potential substrate for the production of value-added components by CPTE.