Novel antibacterial lactoferrin peptides generated by rennet digestion and autofocusing technique

Bovine lactoferrin was hydrolysed with a range of proteolytic enzymes including calf rennet, fungal rennin, and porcine pepsin. Lactoferrin hydrolysates were assessed for their antibacterial activities against Escherichia coli and Bacillus subtilis. At pH 3, calf rennet lactoferrin hydrolysate before (LFH) showed the highest antimicrobial activity, then pepsin LFH, while fungal rennin LFH was the least active. The calf rennet and pepsin LFH were fractionated using autofocusing and chromatographic techniques. The activity-guided fractionation of calf rennet LFH identified a potent antimicrobial peptide of 11-residues, lactoferricin B (Lf-cin B), and three other novel antibacterial peptides. The 11-residues Lf-cin B was the most potent antibacterial peptide and was isolated from both rennet and pepsin LFH. Pepsin LFH had a main antimicrobial peptide of 25-residues, which was not detected in calf rennet LFH. It could be concluded that calf rennet LFH had stronger antibacterial properties than porcine pepsin LFH. Besides, autofocusing could be used for scaling up the isolation of the potent rennet LFH peptides that would have a widespread commercial use as a natural food preservative substituting porcine pepsin digest, especially in Islamic communities.     

Antilisterial activity of dromedary lactoferrin peptic hydrolysates

The aim of this study was to explore the antibacterial peptides derived from dromedary lactoferrin (LFc). The LFc was purified from colostrum using a batch procedure with a cation exchange chromatography support and was hydrolyzed with pepsin to generate peptic digest. This peptic digest was fractionated by cation exchange chromatography, and the antilisterial activity of LFc, peptic digest, and obtained fractions was investigated using the bioscreen method. The growth of Listeria innocua ATCC 33090 and LRGIA 01 strains was not inhibited by LFc and its hydrolysates. Two fractions of dromedary lactoferrin peptic hydrolysate were active against both strains. A tandem mass spectroscopy analysis revealed that the 2 active fractions comprised at least 227 different peptides. Among these peptides, 9 found in the first fraction had at least 50% similarity with 10 known antimicrobial peptides (following sequence alignments with the antimicrobial peptide database from the University of Nebraska Medical Center, Omaha). Whereas 9 of these peptides presented homology with honeybee, frog, or amphibian peptides, the 10th peptide, F₁₅₂SASCVPCVDGKEYPNLCQLCAGTGENKCACSSQEPYFGY₁₉₂ (specifically found in 1 separated fraction), exibited 54% homology with a synthetic antibacterial peptide (AP00481) derived from human lactoferrin named kaliocin-1. Similarly, the second fraction contained 1 peptide similar to lactoferrampin B, an antibacterial peptide derived from bovine milk. This result suggests that peptic hydrolysis of LFc releases more active antimicrobial peptides than their protein source and thus provides an opportunity for their potential use to improve food safety by inhibiting undesirable and spoilage bacteria.     

黄鲫(Setipinna taty)蛋白酶解液的抑菌活性及稳定性研究

以黄鲫为原料测定其氨基酸组成,比较风味蛋白酶、胰蛋白酶、胃蛋白酶、碱性蛋白酶和木瓜蛋白酶水解黄鲫蛋白所得酶解液对大肠杆菌的抑菌作用,并对抑菌效果最强的蛋白酶酶解液进行抑菌稳定性研究。结果表明：黄鲫蛋白必需氨基酸含量丰富,其中胃蛋白酶的水解液对大肠杆菌抑菌作用强,相对分子质量主要分布在3000～1000;黄鲫胃蛋白酶酶解液对热稳定,酸性低pH值可增强其抑菌效果,可耐受胰蛋白酶和β-内酰胺酶处理,该酶解液有作为天然抗菌剂应用的前景。     

黄鲫胃蛋白酶酶解液体外抗氧化、抑菌作用研究

采用胃蛋白酶水解黄鲫,测定酶解液的蛋白提取率、可溶性肽含量、水解度及氨基酸组成,并对酶解液体外抗氧化和抑菌作用进行研究。结果表明：黄鲫胃蛋白酶酶解液的蛋白提取率、可溶性肽提取率、水解度分别为 (110.28 ± 4.81)%、(81.78 ± 0.04)% 和 (18.12 ± 0.39)%,黄鲫胃蛋白酶酶解液的人体必需氨基酸相对含量可达37.91%。黄鲫胃蛋白酶酶解液具有很强的抗氧化能力,清除羟自由基和DPPH 自由基的ED50 分别为4.55μg/mL 和4.46μg/mL,还原力随着黄鲫胃蛋白酶酶解液质量浓度的增加而增大,在3.4 μg/mL 和13.6μg/mL 低、中质量浓度时螯合金属离子能力与EDTA 接近。体外抑菌实验显示：黄鲫胃蛋白酶酶解液具有广谱抑菌性,对大肠杆菌的抑菌效力分别与 13.21～15.44mcg/mL 的氨苄青霉素和 325.00～340.00U/mL 的硫酸链霉素相当 (P ＞ 0.05)。     

Peptides from the N-terminal end of bovine lactoferrin induce apoptosis in human leukemic (HL-60) cells

To determine the effects of the multifunctional iron-binding glycoprotein, lactoferrin (LF) and related compounds on the growth of leukemic cells, human myeloid leukemic cells (HL-60) were exposed to bovine lactoferrin (bLF) and proteolytic hydrolysates of bLF. Pepsin hydrolysates of bLF showed a greater growth suppressive effect than tryptic hydrolysates or mature bLF. Four peptides with proliferation inhibition activity were purified from pepsin hydrolysates by ion-exchange chromatography, reverse-phase HPLC, and gel-filtration. All peptides were from the N-terminal end, in a region where lactoferricin B (Lfcin B), an antibacterial peptide, is located. Among the four peptides, peptide 1 (pep1) was found to exhibit highest activity and corresponded to residues 17 to 38 of bLF, with a molecular weight of 2753.88. The IC50 value of this peptide was 6.3 micrograms/ml. Three other peptides were less active and corresponded to sequences 1 to 16 and 45 to 48, linked by disulfide-bridge (pep2, molecular mass of 2430.13), 1 to 15 and 45 to 46 linked by disulfide bridge (pep3, molecular mass of 2017,92) and from residues 1 to 13 (pep4, molecular mass of 1558.73). Cell proliferation inhibition activity of the peptides was thought to be due to induction of apoptosis, which was evaluated by DNA ladder formation, DNA fragmentation, enhanced expression of phosphatidyl serine, and morphological changes. The IC50 values of the three peptides were confirmed using synthetic peptides and were consistent with those of purified peptides.     

沙棘籽渣酶解产物的体内外抑菌作用

以沙棘籽渣为原料,采用水提法提取沙棘籽渣蛋白,再用ProteAX复合蛋白酶和胃蛋白酶两种酶酶解,利用膜过滤对其分离纯化,得到具有抑菌活性的沙棘籽渣蛋白酶解物。体外实验结果表明：ProteAX复合蛋白酶水解多肽对金黄色葡萄球菌、沙门氏菌、绿脓杆菌均有抑制作用,胃蛋白酶水解多肽对大肠杆菌、金黄色葡萄球菌、沙门氏菌有抑制作用。动物实验表明：胃蛋白酶水解多肽灌胃的小鼠粪便中,沙门氏菌的菌落总数与阳性对照组相比显著减少（P＜0.05）;而灌胃ProteAX复合蛋白酶水解多肽的小鼠粪便中,金黄色葡萄球菌的菌落总数比阳性对照组显著降低（P＜0.05）。     

Functionalities and antioxidant properties of protein hydrolysates from the muscle of ornate threadfin bream treated with pepsin from skipjack tuna

Functional properties and antioxidant activities of protein hydrolysates prepared from ornate threadfin bream (Nemipterus hexodon) muscle, using skipjack tuna pepsin, with different degrees of hydrolysis (DH: 10%, 20% and 30%), were evaluated. Emulsifying and foaming properties of hydrolysates were governed by their DH and concentrations used. Hydrolysates with 20% DH had the highest scavenging activities for ABTS and DPPH radicals. However, chelating activity of hydrolysates for ferrous ion increased as DH increased. Size exclusion chromatography of the hydrolysate with 20% DH using Sephadex G-25 revealed that antioxidative peptides with molecular weight of approximately 1.3 kDa exhibited the highest ABTS radical-scavenging activity. In vitro simulated gastrointestinal digestion indicated that ABTS radical-scavenging activity of the antioxidative peptides was not affected by pepsin hydrolysis, whilst further digestion by pancreatin enhanced the activity. Therefore, protein hydrolysate from the muscle of ornate threadfin bream produced by skipjack tuna pepsin can be used as a promising source of functional peptides with antioxidant properties.     

In vitro antioxidant activities of hydrolysates obtained from Iranian wild almond (Amygdalus scoparia) protein by several enzymes

A protein extract from wild almond was hydrolysed using five different enzymes (pepsin, trypsin, chymotrypsin, alcalase and flavourzyme). The hydrolysates were then assayed for their antioxidant activities. The highest extent of proteolysis was obtained with alcalase (0.35; determined as the change in the absorbance at 340 nm, ΔA₃₄₀) and the lowest was with pepsin (ΔA₃₄₀ = 0.12). Radical scavenging activities obtained by 2, 2′‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulphonic acid) and ferric‐reducing abilities of the hydrolysates demonstrated that the hydrolysate from alcalase had significantly (P < 0.05) greater antioxidant activity. Analysis of the molecular weight distributions showed that peptides produced by alcalase were smaller than those produced by chymotrypsin, trypsin and flavourzyme. Based on the current study, the hydrolysates produced by alcalase can be suggested as potential antioxidant agents in food industry and for use in functional foods.     

Antimicrobial properties of milkfat globule membrane fractions

Milkfat globule membranes (MFGMs) were prepared from bovine cream according to standard procedures. These membranes and peptide hydrolysates, which were generated by proteolysis with immobilized digestive enzymes, were screened for antibacterial activity against Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica Typhimurium, Pseudomonas fluorescens, Bacillus cereus, Lactobacillus acidophilus, and Lactobacillus gasseri. Assays were first performed on beef heart infusion (BHI) plates spotted with test protein-peptide fractions and then seeded with lawns of indicator cells to monitor the zone of growth inhibition. Under these experimental conditions, MFGMs were most active against Salmonella Typhimurium and P. fluorescens. However, antibacterial activity was not seen after plating on Luria-Bertani (LB) medium. We determined that the antimicrobial effects observed on BHI plates were due to the generation of H2O2 by xanthine oxidase, a major protein constituent of the MFGMs, as a result of purine catalysis. This substrate is present in BHI but lacking in LB medium. Evaluation of purified xanthine oxidase alone resulted in analogous data trends. The growth of probiotic Lactobacillus strains were affected only marginally when grown on lactobacilli deMan Rogosa Sharpe plates, suggesting the decreased sensitivity of these bacteria to H2O2. In this study, several MFGM hydrolysates exhibited variable antibacterial activity against test food pathogens on agar plates prepared with M9 minimal media, and this variation was not attributable to xanthine oxidase enzymatic activity. The probiotic microorganisms were mostly resilient to these antibacterial fractions. Bovine MFGM fractions may represent an excellent resource material from which to generate native, naturally occurring biodefensive proteins and/or peptides.     

High pressure increases bactericidal activity and spectrum of lactoferrin, lactoferricin and nisin

We have studied the inactivation of a panel of eight test bacteria (two Escherichia coli strains, Salmonella enteritidis, Salmonella typhimurium, Shigella sonnei, Shigella flexneri, Pseudomonas fluorescens and Staphylococcus aureus) by high pressure in the presence of bovine lactoferrin (500 microg/ml), pepsin hydrolysate of lactoferrin (500 microg/ml), lactoferricin (20 microg/ml) and nisin (100 IU/ml). None of these compounds, at the indicated dosage, were bactericidal when applied at atmospheric pressure, except nisin, which caused a low level of inactivation of the bacteria. Under high pressure, lactoferrin, lactoferrin hydrolysate and lactoferricin displayed bactericidal activity against some of the test bacteria, however, the former had a narrower bactericidal spectrum than the two latter compounds. The bactericidal efficiency and spectrum of nisin were also enhanced under high pressure. The sensitisation of the test bacteria to these antimicrobials under pressure was transient, since no bactericidal activity was observed when bacteria were pressure treated before exposure to the compounds. We propose a mechanism of pressure-promoted uptake of these antimicrobial proteins and peptides in gram-negative bacteria to explain this sensitisation.