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Lactic acid bacteria spoilage What are LABs? • • • • • • • • • • • • • Lactococcus sp. Lactobacillus sp. Leuconostoc sp. Pediococcus sp. Oenococcus sp. Streptococcus sp. Enterococcus sp. Sporolactobacillus sp. Carnobacterium sp. Aerococcus sp. Tetragenococcus sp. Vagococcus sp. Weisella sp. • Sugars lactic acid • Fermentative Physiologic features • Lactic acid main end product • Limited capacity to synthesize amino acids, vitamins (esp. Lactobacillus, Lactococcus) – Lb. johnsonii can’t make any amino acids, for example • Strictly fermentative metabolism – No cytochromes, etc. – Energy generated by substrate-level phosphorylation • Many prefer lack of oxygen – Many lack catalase, superoxide dismutase, other enzymes for dealing with oxygen radicals. • More acid resistant (in general) than aerobic spoilage bacteria (especially lactobacilli) Spoilage or beneficial? Process Beneficial (controlled) Detrimental (uncontrolled) Conversion of sugars to lactic acid Acid flavor in fermented meats and dairy products Sourness of fresh meats, pasteurized milk Malty flavor (3methylbutanal) Malt powder Pasteurized milk Ropiness (exopolysaccharides) Yogurt Meats, milk Acetaldehyde (product of threonine catabolism) Yogurt (green apple flavor) Milk Diacetyl Buttermilk (butter flavor) Fresh milk Homofermentative lactose utilization Lactococcus lactis lactose phospho-b-galactosidase ATP ADP EmbdenMeyerhoff pathway glucose glucose-6-P fructose-6-P fructose-1,6-diP ATP ADP galactose-6-P tagatose-6-P tagatose-1,6-diP 4 triose-P tagatose pathway 4 triose-P 4 ADP 4 ATP 4 pyruvic acid 4 lactic acid Lactose 2 ATP + 4 lactic acid Heterofermentative lactose utilization Some lactobacilli lactose b-galactosidase glucose ATP ADP galactose 2 glucose-6-P ATP ADP 2 glucose-6-P 2 CO2 2 xyulose-5-P 2 triose-P 2 acetyl phosphate 2 pyruvic acid acetaldehyde 2 lactic acid ethanol ADP ATP acetate Spoilage or beneficial? Process Beneficial (controlled) Detrimental (uncontrolled) Conversion of sugars to lactic acid Acid flavor in fermented meats and dairy products Sourness of fresh meats, pasteurized milk Malty flavor (3methylbutanal) Malt powder Pasteurized milk Ropiness (exopolysaccharides) Yogurt Meats, milk Acetaldehyde (product of threonine catabolism) Yogurt (green apple flavor) Milk Diacetyl Buttermilk (butter flavor) Fresh milk Acetaldehyde • From L-threonine • Threonine aldolase • In yogurt, mainly L. delbrueckii subsp. bulgaricus threonine acetaldehyde glycine ethanol chemistry.about.com Second pathway to acetaldehyde Fig 4.3 from Microbiology and technology of fermented foods (R. Hutkins) Branched chain amino acid catabolism Lactococcus lactis subsp. lactis biovar maltigenes http://femsre.oxfordjournals.org/content/29/3/591 Diacetyl production • From citrate • Movie popcorn; common in buttermilks (L. lactis subsp. lactis biovar. diacetylactis) • Functions often plasmidencoded citrate (C6) Acetate (C2) oxaloacetate (C4) CO2 pyruvate (C3) CO2 a-acetolactate (C5) CO2 diacetyl (C4) non-enzymatic http://www.biokemi.org/biozoom /issues/522/articles/2368 acetoin (C4) Ropiness • 50-5000 monomers • Glucose and galactose most common – Also rhamnose, mannose, fucose, arabinose, xylose, N-acetylglucosamine, Nacetylgalactosamine • Homopolysaccharides – Dextrans, mutans, fructans • Heteropolysaccharides • Extracellular polysaccharides (EPS) – Capsular polysaccharides (CPS) – Ropy polysaccharides http://www.healthyfacilitiesinstitute.com/p_281-Biofilm Snapshot of EPS diversity (yogurt LAB) Organism strain Lb. delbrueckii LY03, rr, Lfi5 subsp. bulgaricus Streptococcus thermophilus Exopolysaccharide composition Sugar ratios in subunits Glucose, galactose, rhamnose Gal:Glu:Rha; 5:1:1 291 Glucose, galactose Glu:Gal; 3:2 SY89, SY102, SFi39, Glucose, galactose Gal:Glu; 1:1 IMD01, CNCMI Glucose, galactose Gal:Glu; 3:1 SFi 2 Galactose, rhamnose, glucose Gal:Rha:Glu; 3:2:1 MR-IC Galactose, rhamnose, fucose Gal:Rha:Fuc; 5:2:1 OR 901 Galactopyranose, rhamnopyranose GalP:RhaP; 5:2 Importance of EPS production for yogurt production • Increases viscosity • Reduce syneresis • Save money: – Decrease milk solids added prior to heating – Increase water content • “Clean” label