食品保鲜技术Chapter-4-antimicrobial-enzymes

1、Chapter 4 Antimicrobial Enzymes,Outline,4.1 Introduction 4.2 Lysozymes 4.3 Lactoperoxidase (LP) 4.4 Glucose Oxidase 4.5 Safety & Regulations,4.1 Introduction,Enzymes are used in food processing Rennet in cheese making amylases in bread baking pectinase in fruit juice production Purpose promote speci

2、fic transformations,4.1 Introduction,Few types of enzymes are food preservatives Most studied enzymatic preservatives Hen egg white lysozyme Glucose oxidase Lactoperoxidase,4.2 Lysozymes,Peptideglycan N-acetylmuramoylhydrolase (E.C. 3.2.2.17) Present in eggs, mammalian tears, insects and fish Hen eg

3、g white is the major source for commercial extraction,4.2.1 Antimicrobial Mechanism,Catalyse cleavage of glycosidic bonds(糖苷键) in peptideglycan (肽聚糖) Natural substrate is peptideglycan Major component of the bacterial cell wall Catalyze hydrolysis of the -1,4 glycosidic bonds of the peptidoglycan,4.

4、2.1 Antimicrobial Mechanism (cont.),Peptidoglycan is required for bacterial growth Forms a closed network around the entire cell Defines cellular shape and absorbs cellular turgor pressure Losozyme results in a punctured cell wall Lysis of the bacterial cell membrane Cell death or growth inhibition,

5、4.2.2 Antimicrobial Effects,Sensitivity of bacterial cells to lysozyme activity is determined by their cell-wall structure Accessibility Thickness Composition,4.2.2 Antimicrobial Effects,Especially active against G+ Clostridia, listeria, staphylococci, bacilli, and LAB Structure difference between G

6、+ & G- G- Peptidoglycan is less accessible Sandwiched between the cytoplasmic membrane and an outer membrane G+ Peptidoglycan is more exposed,4.2.3 Improve the Efficiency of Lysozyme,1. Activity enhancement Antimicrobial spectrum is narrow Few, specific, G+ Combine with other substrates EDTA Microfl

7、ora: shrimp L. monocytogenes : freshly prepared vegetable products Glycine, lysine, salt (NaCl),1. Activity Enhancement (cont.),Combine with other enzymes Lipase Combine with physio-chemical stressing of the cells decreased pH and temperature Combine with bacteriocin & other food preservatives Combi

8、ne with ultrasonic treatment,2. Spectrum Extension to G-,Combine with EDTA Destabilize the outer lipopolysaccharide (脂多糖) layer and perturb the membrane Partial thermal inactivation Autolysin (自溶) enzymes activated and induce cell lysis Enhanced interaction with the bacterial membranes Increased mem

9、brane permeabilisation Chemical modification Attachment of fatty acids Facilitate the penetration through the outer membrane,4.2.4 Application in Food,Food Cheese, whole milk, wines, beverages, fresh vegetables, fish, tofu, seafoods, meat products Component of antimicrobial packaging,4.3 Lactoperoxi

10、dase (LP),Enzyme that occurs in raw milk, colostrums, saliva and other biological secretions Lactoperoxidase system (LPS) Composed of LP, thiocyanate (SCN-), H2O2 Active only in the presence of all these three components Antibacterials are generated,4.3.1 Antibacterial Mechanism,LP catalyse oxidatio

11、n of SCN- to thiocyanogen (SCN)2 (SCN)2 hydrolyses spontaneously to hypothiocyanite (OSCN-) Antibacterial effect of (OSCN-) Oxidase protein sulfhydryl (-SH) groups to disulphides (S-S) Result in inactivation of vital bacterial enzyme system Lead to inhibition of bacterial metabolic functions and con

12、sequently cell death,Schematic Reaction of LPS,2SCN-+H2O2+2H+ (SCN)2+2H2O,(SCN)2+H2O OSCN- +SCN-+2H+,SCN-+H2O2 OSCN- +H2O,Net reaction:,Lactoperoxidase:,LP,4.3.2 Source of LPS compounds,Naturally occuring SCN- H2O2 (generated by LAB) Co-addition or individual addition,4.3.3 Spectrum of Antimicrobial

13、 Effects,Bacteria G- inhibited or killed G+ inhibited but not killed Reason for the difference Cell wall structure Fungi Viruses,4.3.4 Improve the Efficiency of the LPS,Combine with nisin and glucose oxidase L. monocytogenes Combine with nisin and LAB L. monocytogenes (skim & raw milk) Addition of m

14、onolaurin E. coli O 157:H7, Staphylococcus aureus Combine with hydrostatic pressure treatment E. coli, Listeria innocua,4.3.5 Application,Most widely recommended application Preservation of raw milk Guideline for the use of the LP system Published by International Dairy Federation 0.25 mM SCN- and H

15、2O2 Milk quality: 72C,15 s80C,15 s Bovine LP completely inactivated at 80C Residual LP is 70% at 72C Other applications infant formula, ice cream, cream, and cheeses,4.4 Glucose Oxidase,Produced by fungi, Penicillium and Aspergillus Aspergillus niger strain NRRL 3 The common source for the commercia

16、l production of glucose oxidase,4.4.1 Reaction Catalysed by GO,Catalyses the oxidation of -D-glucose by O2 producing -D-gluconolactone (葡糖酸内酯)and H2O2 D-gluconalactone hydrolyses spontaneously to D-gluconic acid Net reaction: O2+C6H12O6 H2O2+C6H12O7,4.4.2 Antibacterial Activity,Production of H2O2 De

17、pendent on the presence of catalase Impurity in commercial glucose oxidase Decrease in pH caused by the gluconate generation More important than H2O2,4.4.3 Application,Suppress significantly the growth of Pseudomonas fragi Common fish spoilage organism Fish extract 2.0 Enzyme Units/mL +16mg/mL glucose lowered the pH to 4.0 Shrimp Psedumonas flurorescens inhibited Liquid eggs 5 Enzyme units/mL GO + 5 mg/mL glucose Sammonella, Micrococcus and Bacillus,4.5 Safety & Regulations,United States several enzyme preparations have GRAS status EU Differentiate the aids Aids: processing, f