Identification of antibacterial peptides from ovine αs2-casein

The aim of this work was to isolate and identify antibacterial peptides present in a pepsin digest of ovine α_s2-casein. A protein digest with antibacterial properties was first separated by ion exchange chromatography. The fractions most active against Escherichia coli ATCC 25922 were fractionated by semi-preparative RP-HPLC, and the identification of the active peptides was carried out by on-line and off-line RP-HPLC-ESI-MS/MS. Following this strategy, 10 different peptides were identified, all corresponding to the C-terminal region of the ovine α_s2-casein. Four of them were chemically synthesized and showed antibacterial activity against several Gram-positive and Gram-negative bacteria. Among the synthesized peptides, ovine α_s2-casein f(165–181) exhibited the highest antibacterial potency against all bacteria tested. The antimicrobial activity was compared with that of other previously described peptides like lactoferricin and fragment f(183–207) of bovine α_s2-casein.     

Isolation and characterisation of antibacterial peptides derived from the f(164–207) region of bovine αS2-casein

A chymosin digest of sodium caseinate, which showed antibacterial activity against Listeria innocua, was fractionated using reverse phase high performance liquid chromatography and the purified antibacterial peptides were characterised by mass spectrometry, N-terminal amino acid sequencing and comparison to peptide masses of theoretical enzymic digests of milk proteins. Five antibacterial peptides, Cr1, Cr3, Cr4, Cr5 and Cr7 corresponding to amino acid residues 181–207, 180–207, 175–207, 164–207 and 172–207 of bovine α_S2-casein, respectively, were isolated. The minimal inhibitory concentration of peptides Cr1, Cr4 and Cr5 was determined against a range of Gram-positive and Gram-negative bacteria and showed similar activities to those of the bacteriocin peptide, nisin and the antibacterial peptide, lactoferricin B against certain Gram-positive bacteria. A partially purified chymosin digest of sodium caseinate (Cr_MIX) was prepared and observed to be heat stable for up to 15 min on exposure to 121 °C. Although Cr_MIX showed bactericidal activity against Salmonella Typhimurium in 0.1% (w/v) peptone medium, no antibacterial activity was observed when tested in skim milk at the same concentration.     

Enhanced antimicrobial activity of nisin in combination with allyl isothiocyanate against Listeria monocytogenes,Staphylococcus aureus,Salmonella Typhimurium and Shigella boydii

This study was designed to evaluate the synergistic antimicrobial effect of nisin and allyl isothiocyanate (AITC) against Listeria monocytogenes, Staphylococcus aureus, Salmonella Typhimurium and Shigella boydii. The synergistic interactions between nisin and AITC were observed against all foodborne pathogens, showing the fractional inhibitory concentrations <1. The populations of L. monocytogenes and S. aureus at the combined treatment of nisin and AITC were decreased to below 1 log CFU mL^?1 after 10‐h incubation at 37 °C. The changes in fatty acid profiles of all strains were substantially influenced by nisin alone and the combined treatment of nisin and AITC. A good agreement was observed among cell viability, membrane permeability and depolarisation activity in response to nisin and AITC. The results suggest that nisin and AITC as synergistic inhibitors could be an effective approach to achieve satisfactory antimicrobial activity against a wide range of foodborne pathogens.     

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.     

Short communication: Bovine α-casein is a ferritin-binding protein and inhibitory factor of milk ferritin immunoassay

Commercial bovine milk α-casein, but not β- and κ-caseins, bound to bovine spleen ferritin, as determined by an immunoassay for ferritin. In contrast, α-casein did not bind to apoferritin. The binding of α-casein to bovine spleen ferritin was strongly inhibited by increasing ionic strength by the addition of 0.5 M (NH₄)₂SO₄. The addition of α-casein to a known amount of bovine spleen ferritin resulted in significantly lower recovery (78-80%) of added ferritin, although β- and κ-caseins showed little inhibitory effect in the ferritin immunoassay. These results indicate that bovine α-casein is a specific ferritin-binding protein that may inhibit milk ferritin immunoassay.     

Generation of the antimicrobial peptide caseicin A from casein by hydrolysis with thermolysin enzymes

The generation of the antimicrobial peptide caseicin A (IKHQGLPQE) from a casein substrate using proteolytic enzymes was assessed in silico. This bioinformatic analysis predicted that thermolysin (EC 3.4.24.27) and thermolysin-like enzymes (EC 3.4.24.28) are likely candidates to liberate the bioactive peptide from α_S1-casein. Commercially available sources of the thermolysin enzyme from industrial suppliers were subsequently shown to liberate the 1049 Da caseicin A peptide, under various conditions of hydrolysis, at both lab and pilot scale. The antimicrobial ability of the hydrolysates to reduce pathogen numbers spiked in infant formula trials was subsequently confirmed. For example, numbers of Cronobacter sakazakii were reduced 1000 fold after 3 h of incubation at 37 °C. In conclusion, this study demonstrates the potential to improve the safety of infant milk formula using milk-derived bioactive peptides.     

常用防腐剂对牛乳铁蛋白抗菌活性的影响

乳铁蛋白的抗菌活性常受到其他共存成分的影响。本文研究了常用防腐剂对乳铁蛋白抗菌活性的影响。在研究过程中,以大肠杆菌为实验菌株,进行了实验研究。研究表明,在牛肉膏蛋白胨培养基中,pH6.0,菌浓为106CFU/ml的条件下,乳铁蛋白对大肠杆菌的最小抑菌质量浓度为3mg/ml;苯甲酸钠、山梨酸钾、乳酸链球菌素、对羟基苯甲酸等分别与乳铁蛋白共同作用时,对乳铁蛋白的抗菌活性都有明显的影响。其中,山梨酸钾和苯甲酸钠减弱了乳铁蛋白的抗菌活性,乳酸链球菌素和对羟基苯甲酸丁酯加强了乳铁蛋白的抗菌活性。     

Antibacterial activities of peptides from the water-soluble extracts of Italian cheese varieties

Water-soluble extracts of 9 Italian cheese varieties that differed mainly for type of cheese milk, starter, technology, and time of ripening were fractionated by reversed-phase fast protein liquid chromatography, and the antimicrobial activity of each fraction was first assayed toward Lactobacillus sakei A15 by well-diffusion assay. Active fractions were further analyzed by HPLC coupled to electrospray ionization-ion trap mass spectrometry, and peptide sequences were identified by comparison with a proteomic database. Parmigiano Reggiano, Fossa, and Gorgonzola water-soluble extracts did not show antibacterial peptides. Fractions of Pecorino Romano, Canestrato Pugliese, Crescenza, and Caprino del Piemonte contained a mixture of peptides with a high degree of homology. Pasta filata cheeses (Caciocavallo and Mozzarella) also had antibacterial peptides. Peptides showed high levels of homology with N-terminal, C-terminal, or whole fragments of well known antimicrobial or multifunctional peptides reported in the literature: α_S1-casokinin (e.g., sheep α_S1-casein (CN) f22-30 of Pecorino Romano and cow α_S1-CN f24-33 of Canestrato Pugliese); isracidin (e.g., sheep α_S1-CN f10-21 of Pecorino Romano); kappacin and casoplatelin (e.g., cow κ-CN f106-115 of Canestrato Pugliese and Crescenza); and β-casomorphin-11 (e.g., goat β-CN f60-68 of Caprino del Piemonte). As shown by the broth microdilution technique, most of the water-soluble fractions had a large spectrum of inhibition (minimal inhibitory concentration of 20 to 200 μg/mL) toward gram-positive and gram-negative bacterial species, including potentially pathogenic bacteria of clinical interest. Cheeses manufactured from different types of cheese milk (cow, sheep, and goat) have the potential to generate similar peptides with antimicrobial activity.     

Short communication: Activity of nisin,lipid bilayer fragments and cationic nisin-lipid nanoparticles against multidrug-resistant Staphylococcus spp. isolated from bovine mastitis

Staphylococci are the main etiological agents of bovine mastitis. Bacteriocins and nanoparticles have emerged as promising alternatives for the future development of antimicrobial agents. This study evaluated the activity of the bacteriocin nisin and bicelles of the synthetic cationic lipid dioctadecyldimethylammonium bromide, alone and in combination, against multidrug-resistant Staphylococcus spp. strains isolated from bovine mastitis. In summary, cationic nisin/dioctadecyldimethylammonium bromide nanoparticles are shown to be a promising alternative for the control of mastitis caused by multidrug-resistant Staphylococcus spp.     

Effect of heat treatment on the antibacterial activity of bovine lactoferrin against three foodborne pathogens

The effect of different heat treatments on the antimicrobial activity of bovine lactoferrin against Escherichia coli O157:H7, Salmonella enteritidis and Listeria monocytogenes has been studied. We have observed that the heat treatments lower than 85°C for 10 min did not affect the antibacterial activity of the protein. Hydrolysates of bovine lactoferrin were found to be more active than the native protein against the three pathogens. Moreover, the antibacterial effect of bovine lactoferrin was also assayed in milk and whey, and although we found a reduction in the number of viable cells, this reduction was lower than in culture media.     

Identification and molecular mechanism of action of antibacterial peptides from Flavourzyme-hydrolyzed yak casein against Staphylococcus aureus

Antibacterial peptides can be released from yak milk casein. To date, the amino acid sequences and mechanism of action of yak casein–derived antibacterial peptides remain unknown. The current study identified antibacterial peptides from yak casein and their molecular mechanism of action. Our results showed that yak α-casein, β-casein, and κ-casein could be effectively hydrolyzed by Flavourzyme (Solarbio Science and Technology Co. Ltd.), and the 2-h hydrolysate showed the highest antibacterial rate of 43.07 ± 2.59% against Staphylococcus aureus. The 1,000 to 3,000 Da fraction accounted for 23.61% of the 2-h hydrolysate and had an antibacterial rate of 62.64 ± 4.40%. Three novel peptides with antibacterial activity were identified from this fraction, and the β-casein–derived peptide APKHKEMPFPKYP showed the strongest antibacterial effect (half-maximal inhibitory concentration = 0.397 mg/mL). Molecular docking predicted that APKHKEMPFPKYP interacted with 2 important enzymes of Staph. aureus, dihydrofolate reductase and DNA gyrase, through hydrophobic, hydrogen bonding, salt bridge, and π-π stacking interactions. Our findings suggest that the yak casein–derived peptides may serve as a potential source of natural preservatives to inhibit Staph. aureus.     

Synergistic effect of nisin and garlic shoot juice against Listeria monocytogenes in milk

The aim of this research was to determine the synergistic effect of nisin and garlic shoot juice (GSJ) against Listeria monocytogenes ATCC 19118 found in whole (3.5%), low (1%) and skim (no fat content) milk. Garlic shoot juice (GSJ) at concentrations of 2.5%, 5% and 10% revealed strong and similar patterns of antilisterial effect against L. monocytogenes ATCC 19118 in all categories of milk. Nisin only at concentrations of 62.5, 125, 250 and 500 IU/ml displayed a strong antilisterial effect as compared to the control group. Also, the synergistic combinations of GSJ (2.5%, 5%) and nisin (62.5, 125, 250 and 500 IU/ml) had a remarkable antilisterial activity in all categories of whole, low and skim milk after 14 days. Results of this study indicated the synergistic effect of GSJ and nisin as a potential antilisterial agent for the food industry.     

Short communication: Antimicrobial effect of lactoferrin and its amidated and pepsin-digested derivatives against Salmonella Enteritidis and Pseudomonas fluorescens

The antimicrobial effect of bovine lactoferrin (LF) and its amidated and pepsin-digested derivatives, at concentrations varying from 0.25 to 20 mg/mL, against 3 Salmonella Enteritidis strains and 3 Pseudomonas fluorescens strains was investigated. Lactoferrin showed its maximum antimicrobial effect at 10 mg/mL against the 3 Salmonella strains, with reductions ranging from 1.3 to 2.0 log units, and the 3 Pseudomonas strains, with reductions ranging from 1.8 to 5.4 log units. In the case of amidated LF, the maximum effect on the 3 Salmonella strains was recorded at 0.25 mg/mL, with reductions in the range of 0.8 to 1.2 log units, whereas it was recorded at 1 mg/mL for the 3 Pseudomonas strains, with reductions in the range of 4.4 to 6.0 log units. Pepsin-digested LF showed its maximum antimicrobial effect at 1 mg/mL against the 3 Salmonella strains, with reductions ranging from 2.6 to 3.4 log units, and at 20 mg/mL against the 3 Pseudomonas strains, with reductions ranging from 4.5 to 5.4 log units. It is worth noting the pronounced effect (reductions exceeding 2.5 log units) of a low (1 mg/mL) concentration of pepsin-digested LF, which is naturally formed in the gastrointestinal tract, on Salmonella and Pseudomonas strains. A highly significant inverse correlation was found between capsule polysaccharide levels of bacterial strains and their lethality in the presence of different concentrations of amidated lactoferrin.     

Isolation and characterisation of a novel antibacterial peptide from bovine αS1-casein

Bovine casein was hydrolysed with a range of proteolytic enzymes including pepsin, trypsin, α-chymotrypsin and β-chymotrypsin, and assessed for antibacterial activity. The pepsin digest of bovine casein, which showed antibacterial activity, was fractionated using reverse phase high performance liquid chromatography and the antibacterial peptides isolated were characterised using electrospray ionisation mass spectrometry. Two antibacterial peptides were identified, a novel peptide (Cp1) which corresponded to residues 99–109 of bovine α_S1-casein and a previously reported peptide (Cp2) which corresponded to residues 183–207 of bovine α_S2-casein. The minimum inhibitory concentration (MIC) of Cp1 and Cp2 were determined against a range of bacterial cultures. Cp1 exhibited an MIC of 125 μg mL⁻¹ against all Gram-positive bacteria tested, and MIC ranging between 125 and >1000 μg mL⁻¹ against the Gram-negative bacteria tested. Cp2 was generally far more potent against the Gram-positive bacteria, exhibiting an MIC of 21 μg mL⁻¹, compared to MICs ranging from 332 to >664 μg mL⁻¹ against most of the Gram-negative bacteria tested.     

Plasmin digestion of photooxidized milk proteins

Plasmin-mediated hydrolysis of 6 different milk protein preparations [α_S-casein (α_S1 + α_S2), β-casein, κ-casein, α-lactalbumin, β-lactoglobulin, and lactoferrin] was found to be very dependent on photooxidation of the said proteins. Changes in plasmin proteolysis were investigated in a peptide-mapping study applying liquid chromatography-mass spectrometry. The changes were seen in the formation of peptides formed by plasmin-mediated hydrolysis after photooxidation, which was initiated with the naturally occurring photosensitizer riboflavin in all the milk protein preparations studied. The changes in the plasmin-mediated hydrolysis of photooxidized proteins are discussed in relation to changes introduced in the protein structure upon photooxidation. Plasmin-mediated hydrolysis of α_S-casein, consisting of a mixture of α_S1- and α_S2-casein and a preparation of β-casein, was most highly affected by photooxidation, which is in agreement with the fact that those 2 proteins have been found to be most labile toward photooxidation. Changes in the formation of potential angiotensin-I-converting enzyme-inhibitory peptides as well as peptides proposed to have antibactericidal activities by plasmin were observed by oxidation of milk proteins before plasmin-mediated hydrolysis.     

Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese

The objective of this study was to evaluate if Mexican Fresco cheese manufactured with specific lactic acid bacteria (LAB) presented angiotensin I-converting enzyme inhibitory (ACEI) activity. Water-soluble extracts (3 kDa) obtained from Mexican Fresco cheese prepared with specific LAB (Lactococcus, Lactobacillus, Enterococcus, and mixtures: Lactococcus-Lactobacillus and Lactococcus-Enterococcus) were evaluated for ACEI activity. Specific peptide fractions with high ACEI were analyzed using reverse phase-HPLC coupled to mass spectrometry for determination of amino acid sequence. Cheese containing Enterococcus faecium or a Lactococcus lactis ssp. lactis-Enterococcus faecium mixture showed the largest number of fractions with ACEI activity and the lowest half-maximal inhibitory concentration (IC₅₀; <10 μg/mL). Various ACEI peptides derived from β-casein [(f(193-205), f(193-207), and f(193-209)] and α_S1-casein [f(1-15), f(1-22), f(14-23), and f(24-34)] were found. The Mexican Fresco cheese manufactured with specific LAB strains produced peptides with potential antihypertensive activity.     

Variation in caseinolytic properties of six cheese related Lactobacillus helveticus strains

A large degree of strain variation was observed in caseinolytic properties of six cheese related Lactobacillus helveticus strains. Activity on intact α_s1- and β-casein was observed only after growth in milk and not in MRS. Totally 27 peptides from α_s1- and 22 from β-casein were identified from MS/MS fragmentation patterns. All six strains released peptides from the amino end of α_s1-casein, and the bonds Ile₆-Lys₇ and Gln₉-Gly₁₀ were identified as primary cleavage sites. Strain variation in the activity on intact β-casein was observed and five of the six strains released peptides from the C-terminal region. The strains had very different activities and some strains had only trace activities. L. helveticus CNRZ 32 had the highest activity towards α_s1-casein while L. helveticus LHC2 had the highest activity towards β-casein, and these two strains also produced unique peptides from both α_s1- and β-casein.     

Analysis and comparison of key proteins in Maiwa yak and bovine milk using high-performance liquid chromatography mass spectrometry

Yak milk is an essential and predominant food resource for Tibetan people for subsistence purposes and to combat altitude-induced challenges. Due to its unique qualities, yak milk has recently been gaining broader attention from consumers across China as well in other parts of the world. One of the key characteristics of yak milk is the protein content, which is about 40 to 60% higher than that of native bovine milk. In this work, a sensitive and reproducible high-throughput analytical method was developed employing both ultra high-performance liquid chromatography Orbitrap (Thermo Fisher Scientific) high-resolution accurate mass spectroscopy (UHPLC-HRAM-MS) and UHPLC coupled with triple quadrupole tandem MS (UHPLC-QqQ-MS) to simultaneously analyze 8 milk proteins. A total of 15 Maiwa yak milk samples and 15 bovine milk samples were qualitatively and quantitatively analyzed using targeted proteomics and compared for α-lactalbumin, β-lactoglobulin, α_S1-casein, α_S2-casein, β-casein, κ-casein, lactoferrin, and osteopontin. Peptides of β-lactoglobulin were used to specifically distinguish yak and bovine milk. The results showed that this novel detection method could quantitatively detect these major and minor milk proteins with >0.99 linear correlation coefficient and a recovery rate between 90 and 120%, with relative standard deviations typically less than 10%. The data revealed that yak milk not only had higher overall milk protein content than bovine milk but higher lactoferrin and osteopontin contents as well. The lactoferrin content of yak milk was about 30% higher than that of bovine milk, and the osteopontin content of yak milk was nearly twice that of bovine milk. The application of this method demonstrates that UHPLC-HRAM-MS and UHPLC-QqQ-MS are suitable for high-throughput qualitative and quantitative analysis of major and minor proteins of yak and bovine milk.     

The molecular characterisation and antimicrobial activity of amidated bovine lactoferrin

Chemical modification of bovine lactoferrin (LF) by amidation increased the net positive charge of the protein and markedly enhanced its activity against most Gram-positive and Gram-negative bacteria tested, but not against Saccharomyces cerevisiae or Penicillium candidum. Bovine lactoferrin was amidated with 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide in the presence of ammonium ions. Mass spectrometric analysis of the modified protein confirmed that amidation converted aspartyl or glutamyl residues to asparaginyl or glutaminyl residues. The changes in net charge and electrophoretic mobility of amidated LF were confirmed by electrophoretic and chromatographic methods. Isoionic point titration was used to determine changes to net charges and ion exchange chromatography results confirmed that the increased positive charge played an important role in the antimicrobial action of the amidated protein. Thus, chemical amidation of LF introduced substantial structural changes in the LF molecule, reflected in amino acid composition and increased net positive charge, and enhanced biological activity.     

Effect of nanovesicle-encapsulated nisin on growth of Listeria monocytogenes in milk

Commercial nisin was encapsulated in nanovesicles (mean diameter 140 nm) prepared from partially purified soy lecithin. Nisin-loaded liposomes and unencapsulated (free) nisin were initially tested in BHI medium and skim milk inoculated with Listeria monocytogenes and incubated for 48 h at 30 °C. At such abuse temperature conditions, free nisin showed better inhibitory than the liposomal counterparts. Subsequently, the effect of encapsulated or free nisin was evaluated in combination with refrigeration (7 ± 1 °C) in both whole (3.25% fat) and skim (0% fat) milk for up to 14 day. A decrease of 3–4 log cycles in L. monocytogenes counts was observed for free and encapsulated nisin at 0.5 mg/ml concentration. Liposome encapsulation of antimicrobial peptides may be important to overcome stability issues and interaction with food components. The utilization of nanovesicle-encapsulated nisin in combination with low temperatures appeared to be effective to control L. monocytogenes in milk, emphasizing the importance of hurdle technology to assure food safety.