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Antimicrobial activity of buttermilk and lactoferrin peptide extracts on poultry pathogens

  • Catherine Jean (a1) (a2), Martine Boulianne (a2), Michel Britten (a1) and Gilles Robitaille (a1)

Antibiotics are commonly used in poultry feed as growth promoters. This practice is questioned given the arising importance of antibiotic resistance. Antimicrobial peptides can be used as food additives for a potent alternative to synthetic or semi-synthetic antibiotics. The objective of this study was to develop a peptide production method based on membrane adsorption chromatography in order to produce extracts with antimicrobial activity against avian pathogens (Salmonella enterica var. Enteritidis, Salmonella enterica var. Typhimurium, and two Escherichia coli strains, O78:H80 and TK3 O1:K1) as well as Staphylococcus aureus. To achieve this, buttermilk powder and purified lactoferrin were digested with pepsin. The peptide extracts (<10 kDa) were fractionated depending on their charges through high-capacity cation-exchange and anion-exchange adsorptive membranes. The yields of cationic peptide extracts were 6·3 and 15·4% from buttermilk and lactoferrin total peptide extracts, respectively. Antimicrobial activity was assessed using the microdilution technique on microplates. Our results indicate that the buttermilk cationic peptide extracts were bactericidal at less than 5 mg/ml against the selected avian strains, with losses of 1·7 log CFU/ml (Salm. Typhimurium) to 3 log CFU/ml (E. coli O78:H80); viability decreased by 1·5 log CFU/ml for Staph. aureus, a Gram-positive bacterium. Anionic and non-adsorbed peptide extracts were inactive at 5 mg/ml. These results demonstrate that membrane adsorption chromatography is an effective way to prepare a cationic peptide extract from buttermilk that is active against avian pathogens.

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Agyei, D & Danquah, MK 2011 Industrial-scale manufacturing of pharmaceutical-grade bioactive peptides. Biotechnology Advances 29 272277
Arnould, VMR, Soyeurt, H, Gengler, N, Colinet, FG, Georges, MV, Bertozzi, C, Portetelle, D & Renaville, R 2009 Genetic analysis of lactoferrin content in bovine milk. Journal of Dairy Science 92 21512158
Bellamy, W, Takase, M, Wakabayashi, H, Kawase, K & Tomita, M 1992a Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin. Journal of Applied Bacteriology 73 472479
Bellamy, W, Takase, M, Yamauchi, K, Wakabayashi, H, Kawase, K & Tomita, M 1992b Identification of the bactericidal domain of lactoferrin. Biochimica et Biophysica Acta (BBA) – Protein Structure and Molecular Enzymology 1121 130136
Branen, J & Davidson, PM 2000 Activity of hydrolysed lactoferrin against foodborne pathogenic bacteria in growth media: the effect of EDTA. Letters in Applied Microbiology 30 233237
Brenes, A & Roura, E 2010 Essential oils in poultry nutrition: main effects and modes of action. Animal Feed Science and Technology 158 114
Brewster, JD 2003 A simple micro-growth assay for enumerating bacteria. Journal of Microbiological Methods 53 7786
Brogden, KA 2005 Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Reviews Microbiology 3 238250
Chan, JCK & Li-Chan, ECY 2007 Production of lactoferricin and other cationic peptides from food grade bovine lactoferrin with various iron saturation levels. Journal of Agricultural and Food Chemistry 55 493501
Church, FC, Swaisgood, HE, Porter, DH & Catignani, GL 1983 Spectrophotometric assay using o-phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins. Journal of Dairy Science 66 12191227
Clare, DA, Zheng, Z, Hassan, HM, Swaisgood, HE & Catignani, GL 2008 Antimicrobial properties of milkfat globule membrane fractions. Journal of Food Protection 71 126133
Dahiya, JP, Wilkie, DC, Van Kessel, AG & Drew, MD 2006 Potential strategies for controlling necrotic enteritis in broiler chickens in post-antibiotic era. Animal Feed Science and Technology 129 6088
Dewettinck, K, Rombaut, R, Thienpont, N, Le, TT, Messens, K & Van Camp, J 2008 Nutritional and technological aspects of milk fat globule membrane material: Netherlands association for the advancement of dairy science 1908–2008. International Dairy Journal 18 436457
Dionysius, DA & Milne, JM 1997 Antibacterial peptides of bovine lactoferrin: purification and characterization. Journal of Dairy Science 80 667674
Ellison, RT & Giehl, TJ 1991 Killing of gram-negative bacteria by lactoferrin and lysozyme. Journal of Clinical Investigation 88 10801091
Foschiatti, M, Cescutti, P, Tossi, A & Rizzo, R 2009 Inhibition of cathelicidin activity by bacterial exopolysaccharides. Molecular Microbiology 72 11371146
Hancock, REW & Chapple, DS 1999 Peptide antibiotics. Antimicrobial Agents and Chemotherapy 43 13171323
Hernández-Ledesma, B, García-Nebot, MJ, Fernández-Tomé, S, Amigo, L & Recio, I 2014 Dairy protein hydrolysates: peptides for health benefits. International Dairy Journal 38 82100
Joerger, RD 2003 Alternatives to antibiotics: bacteriocins, antimicrobial peptides and bacteriophages. Poultry Science 82 640647
Joo, HS & Otto, M 2015 Mechanisms of resistance to antimicrobial peptides in staphylococci. Biochimica et Biophysica Acta – Biomembranes 1848 30553061
Kaur, IP, Kuhad, A, Garg, A & Chopra, K 2009 Probiotics: delineation of prophylactic and therapeutic benefits. Journal of Medicinal Food 12 219235
Kreub, M & Kulozik, U 2009 Separation of glycosylated caseinomacropeptide at pilot scale using membrane adsorption in direct-capture mode. Journal of Chromatography A 1216 87718777
Kuwata, H, Yamauchi, K, Teraguchi, S, Ushida, Y, Shimokawa, Y, Toida, T & Hayasawa, H 2001 Functional fragments of ingested lactoferrin are resistant to proteolytic degradation in the gastrointestinal tract of adult rats. Journal of Nutrition 131 21212127
Lahov, E & Regelson, W 1996 Antibacterial and immunostimulating casein-derived substances from milk: casecidin, isracidin peptides. Food and Chemical Toxicology 34 131145
Lin, J, Hunkapiller, AA, Layton, AC, Chang, YJ & Robbins, KR 2013 Response of intestinal microbiota to antibiotic growth promoters in Chickens. Foodborne Pathogens and Disease 10 331337
Lynch, JM & Barbano, DM 1999 Kjeldahl nitrogen analysis as a reference method for protein determination in dairy products. Journal of AOAC International 82 13891392
Marshall, BM & Levy, SB 2011 Food animals and antimicrobials: impacts on human health. Clinical Microbiology Reviews 24 718733
Murdock, CA & Matthews, KR 2002 Antibacterial activity of pepsin-digested lactoferrin on foodborne pathogens in buffered broth systems and ultra-high temperature milk with EDTA. Journal of Applied Microbiology 93 850856
Pellegrini, A, Thomas, U, Bramaz, N, Hunziker, P & Von Fellenberg, R 1999 Isolation and identification of three bactericidal domains in the bovine α-lactalbumin molecule. Biochimica et Biophysica Acta – General Subjects 1426 439448
Pellegrini, A, Dettling, C, Thomas, U & Hunziker, P 2001 Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin. Biochimica et Biophysica Acta – General Subjects 1526 131140
Plate, K, Beutel, S, Buchholz, H, Demmer, W, Fischer-Frühholz, S, Reif, O, Ulber, R & Scheper, T 2006 Isolation of bovine lactoferrin, lactoperoxidase and enzymatically prepared lactoferricin from proteolytic digestion of bovine lactoferrin using adsorptive membrane chromatography. Journal of Chromatography A 1117 8186
Robitaille, G 2013 Growth-promoting effects of caseinomacropeptide from cow and goat milk on probiotics. Journal of Dairy Research 80 5863
Shin, K, Yamauchi, K, Teraguchi, S, Hayasawa, H, Tomita, M, Otsuka, Y & Yamazaki, S 1998 Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157:H7. Letters in Applied Microbiology 26 407411
Sibel Akalin, A 2014 Dairy-derived antimicrobial peptides: action mechanisms, pharmaceutical uses and production proposals. Trends in Food Science and Technology 36 7995
Stern, NJ, Svetoch, EA, Eruslanov, BV, Kovalev, YN, Volodina, LI, Perelygin, VV, Mitsevich, EV, Mitsevich, IP & Levchuk, VP 2005 Paenibacillus polymyxa purified bacteriocin to control Campylobacter jejuni in chickens. Journal of Food Protection 68 14501453
Teeters, MA, Root, TW & Lightfoot, EN 2002 Performance and scale-up of adsorptive membrane chromatography. Journal of Chromatography A 944 129139
Théolier, J, Fliss, I, Jean, J & Hammami, R 2014 Antimicrobial peptides of dairy proteins: from fundamental to applications. Food Reviews International 30 134154
Troost, FJ, Steijns, J, Saris, WHM & Brummer, RJM 2001 Gastric digestion of bovine lactoferrin in vivo in adults. Journal of Nutrition 131 21012104
Voswinkel, L & Kulozik, U 2014 Fractionation of all major and minor whey proteins with radial flow membrane adsorption chromatography at lab and pilot scale. International Dairy Journal 39 209214
Wong, CL, Sieo, CC, Tan, WS, Abdullah, N, Hair-Bejo, M, Abu, J & Ho, YW 2014 Evaluation of a lytic bacteriophage, Φ st1, for biocontrol of Salmonella enterica serovar Typhimurium in chickens. International Journal of Food Microbiology 172 92101
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Journal of Dairy Research
  • ISSN: 0022-0299
  • EISSN: 1469-7629
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