Preservation of red meat with natural antimicrobial peptides produced by lactic acid bacteria

Kohrs, Gertruida Ansia (2004-12)

Thesis (MScVoedselwet)--University of Stellenbosch, 2004.

Thesis

ENGLISH ABSTRACT: Red meat has a limited shelf-life at refrigerated temperatures, where spoilage is mainly due to the proliferation of bacteria, yeast and moulds, acquired during the dressing process. In addition, almost a fifth of food-borne disease outbreaks, caused by microorganisms such as Escherichia coli 0157:H7, Listeria monocytogenes and Staphylococcus aureus are associated with red meat. To improve the microbiological quality of red meat, systems such as HACCP, GHP and GMP are currently practiced; however, these practices are not able to extend the shelf-life of these products. At present suitable food-grade preservatives are recommended, but the use of some of these preservatives is increasingly being questioned with regard to their impact on human health. Additionally, food service customers demand high quality products that have a relatively long shelf-life, but still prefer the appearance of minimally processed food. All these factors challenge the food manufacturing industry to consider more natural means of preservation. Antimicrobial metabolites of food grade bacteria, especially lactic acid bacteria, are attracting increasing attention as food preservatives. Bacteriocins are antimicrobial peptides (3 to 10 kDa) with variable activity spectra, mode of action, molecular weight, genetic origin and biochemical properties that are bacteriostatic or bactericidal to bacteria closely related and bacteria confined within the same ecological niche. Micro-organisms were isolated from beef, lamb and pork, obtained from four commercial retailers. The number of viable cells three days after the sell-by date at 4ºC ranged from 80 cfu.g-1 to 1.4 × 108 cfu.g-1. Fifty-three percent were Gram-negative bacteria, 35% Gram-positive and 12% yeast. The microbial population of the meat was greatly influenced by the origin, i.e. the retailer. Bacteriocins produced by Enterococcus faecalis BFE 1071, Lactobacillus curvatus DF 38, Lb. plantarum 423, Lb. casei LHS, Lb. salivarius 241 and Pediococcus pentosaceus ATCC 43201 were screened for activity against bacteria isolated from the different meat samples. Sixteen to 21% of the isolates, identified as members of Klebsiella, Shigella, Staphylococcus, Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Streptococcus and Bacillus were sensitive to the bacteriocins. Curvacin DF 38, plantaricin 423 and caseicin LHS (2.35 to 3.4 kDa) had the broadest activity range and inhibited species of Lactobacillus, Pediococcus, Enterococcus, Listeria, Bacillus, Clostridium and Propionibacterium. The bacteriocins remained stable at 121ºC for 20 min, in buffers with a pH ranging from 2 to 10 and in NaCl concentrations of between 0.1 and 10% (m/v). Like most peptides, they were sensitive to proteolytic enzymes. Curvacin DF 38 is sensitive to amylase, suggesting that the bacteriocin might be glycosylated. To assess the efficiency of curvacin DF 38, plantaricin 423 and caseicin LHS as meat preservatives, they were partially purified by ammonium sulphate precipitation and separation in a Sep Pak C18 cartridge. The shelf-life of pork may be extended by up to two days. Meat samples treated with bacteriocins were darker than the control (untreated) sample. Descriptive sensory evaluation by a seven-member panel indicated that there were significant differences (P ≤ 0.05) regarding the aroma, sustained juiciness, first bite and metallic taste attributes of the control and the 4 day-treated samples. The control and 2 day-treated samples and the 2 day- and 4 day treated samples did not differ significantly regarding these attributes. There were no significant differences regarding the initial juiciness, residue and pork flavour attributes. Concluded from the results obtained in this study, bacteriocins produced by Lb. curvatus DF 38, Lb. plantarum 423 and Lb. casei LHS effectively extended the shelf-life of pork loins by up to 2 d at refrigerated temperatures with no drastic changes on sensory characteristics. In edition, the stability of these bacteriocins broadens their application as preservatives in many foods.

AFRIKAANSE OPSOMMING: Die rakleeftyd van rooivleis by yskastemperature is beperk, waar bederf hoofsaaklik deur die vermenigvuldiging van bakterieë, giste en swamme veroorsaak word. Die meeste van hierdie kontaminante is afkomstig van die slagtingsproses. Byna ’n vyfde van alle uitbrake van voedselvergiftigings wat deur organismes soos Escherichia coli 0157:H7, Listeria monocytogenes en Staphylococcus aureus veroorsaak word, word met rooivleis geassosieër. Die praktyke HACCP, GMP en GHP word tans toegepas om die mikrobiologies kwaliteit van vleis te handhaaf, maar is egter nie voldoende om die rakleeftyd van rooivleis the verleng nie. Die preserveermiddels wat huidiglik aanbeveel word vir dié doel, word toenemend bevraagteken aangaande die invloed daarvan op die menslike gesondheid. Hierby is daar ’n aanvraag na hoë kwaliteit, ongeprosesseerde produkte met ’n verlengde rakleeftyd. Gevolglik word die voedsel vervaardigings industries aangemoedig om meer natuurlike vorms van preservering the oorweeg. Die aandag word tans op die anti-mikrobiese metaboliete van voedselgraad microbes, veral melksuurbakterieë, gevestig. Bakteriosiene is anti-mikrobiese peptiede (3 tot 10 kDa) met verskeie aktiwiteitsspektra, werkswyse, molekulêre massa, genetiese oorsprong en biochemiese eienskappe. Bakteriosiene is meestal bakterie-dodend of - staties teen taksonomies naby geleë organismes en organismes vanuit dieselfde ekologiese nis. Mikroörganismes is geïsoleer vanuit bees-, skaap- en varkvleis, verkry vanaf vier supermarkte. Die aantal lewensvatbare selle per gram (cfu.g-1) het drie dae na die “verkoop”-datum by 4ºC vanaf 80 cfu.g-1 tot 1.4 × 108 cfu.g-1 gevarieër. Drie en vyftig persent van die isolate is as Gram-negatief, 35% as Gram-positief en 12% as giste geïdentifiseer. Die sensitiwiteit van hierdie isolate teen bakteriosiene wat deur Enterococcus faecalis BFE 1071, Lactobacillus curvatus DF 38, Lb. plantarum 423, Lb. casei LHS, Lb. salivarius 241 en Pediococcus pentosaceus ATCC 43201 geproduseer is, is vervolgens getoets. Tussen 16% en 21% van die isolate was sensitief teen die bacteriosiene en is onder andere as Klebsiella, Shigella, Staphylococcus, Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Streptococcus en Bacillus geïdentifiseer. Die bakteriosiene met die wydste aktiwiteitsspektrum, naamlik, curvacin DF 38, plantaricin 423 en caseicin LHS is verder ondersoek. Hierdie antimikrobiese peptiede (2.35 tot 3.4 kDa) toon aktiwiteit teen spesies van Lactobacillus, Pediococcus, Enterococcus, Listeria, Bacillus, Clostridium and Propionibacterium. Die bakteriosiene is stabiel by 121ºC vir 20 min, in buffers met ‘n pH-reeks van tussen 2 en 10 en soutkonsentrasies vanaf 0.1% tot 10%. Soos die geval by meeste peptiede is hierdie bakteriosiene sensitief vir proteolitiese ensieme. Curvacin DF 38 is ook sensitief vir amylase, wat daarop dui dat hierdie bakteriosien moontlik geglikosileer is. Die effektiwiteit van curvacin DF 38, plantaricin 423 en caseicin LHS as preserveermiddel in voedselsisteme is getoets deur dit te suiwer (ammonium sulfaat presipitasie en Sep Pak C18 kolom) en op vark lendestukke aan te wend. Mikrobiese analise het bewys dat die rakleeftyd van vark met sowat 2 dae verleng kan word. Volgens die vleiskleurevaluering was die bakteriosien behandelde vark donkerder as die kontrole. Die aroma-, sappigheid-, tekstuur- en metaalgeur-eienskappe van die kontrole en die 4-dag behandelde monster het volgens ‘n opgeleide sensoriese paneel betekenisvol verskil (P ≤ 0.05). Die kontrole en die 2-dag behandelde en die 2-dag behandelde en die 4-dag behandelde monsters het nie betekenisvol verskil nie. Daar was geen betekenisvolle verskil aangaande die aanvanklike sappigheid-, residu- en varkgeur-eienskappe nie. Hierdie sensoriese eienskappe is belangrik ten opsigte van die verbruiker se aanvaarding van die produk. Vervolgens kan uit hierdie resultate afgelei word dat die bakteriosiene wat deur Lb. curvatus DF 38, Lb. plantarum 423 en Lb. casei LHS geproduseer word voldoende is om die rakleeftyd van vark lendestuk by 4ºC met 2 dae te verleng met min of geen effek op die sensoriese persepsie van die vleis. Hierdie bakteriosiene is ook stabiel onder verskeie kondisies wat die toepassing as preserveermiddel aansienlik verbreed.

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