lactic acid
TRANSCRIPT
Benar atau Salah
• Mikroorganisme mengeluarkan enzim ekstra seluler untuk memecah karbohidrat
• Monomer dari karbohidrat bisa langsung masuk kedalam sel
• Ada 3 jalur pemecahan glukosa menjadi asam piruvat
• Bakteri asam laktat homofermentatif digunakan dalam pengawetan makanan
Jelaskan
• Kelompok bakteri asam laktat• Homofermentatif• Heterofermentatif
Poly lactic acid (PLA) for plastics production
Polyhydroxyalkanoates (PHA’s)
Overview – Lactic Acid Bacteria• Bacteria Basics• Introduction• Taxonomy
– Lactobacillus– Oenococcus– Pediococcus
• Nutritional Requirements• Metabolism
– Glucose– Arginine– Malate– Mannitol and Erythritol– Diacetyl and Other Odor/Flavor Compounds
Introduction – Lactic Acid Bacteria• Lactic acid bacteria comprise an ecologically
diverse group of microorganisms united by the formation of lactic acid as the primary metabolite of sugar metabolism
• Utilize sugars by either homo- or hetero-fermentative pathways, as well as L-malic acid, a major acid present in grape must
• Whereas growth of some bacteria in certain wines is desirable, growth of other species can lead to spoilage.
Nutrition/Metabolism – Lactic Acid Bacteria
• All lactic acid bacteria are capable of producing diacetyl or 2,3 Butanedione
• Utilize malic and citric acid• Generally less citric acid in wine but taken up
by cell at much slower rate• O. oeni can produce higher alcohols as by
products of fermentation
Nutrition/Metabolism – Lactic Acid Bacteria
• Lactic acid bacteria have very limited biosynthetic capabilities and are therefore described as nutritionally fastidious.
• Do not eat 5 carbon sugars• Early work noted that all strains of wine lactic
acid bacteria required nicotinic acid, riboflavin, pantothenic acid and either thiamine or pyridoxine
• May result in acetate and VA development under oxidative conditions
Nutrition/Metabolism – Lactic Acid Bacteria
• Citric acid is a major factor in diacetyl production
• Deacidification is the most major winemaking effect
Taxonomy – Lactic Acid Bacteria
• Lactobacillus• Oenococcus• Pediococcus
Lactobacillus• Gram +• Short Rods• Homo or hetero fermentative• All ferment frucose• Most produce lactic acid from
D/L Glucose• Produce sour taste
Oenococcus• Gram +• Heterofermentative• Can be difficult to distinguish from
Lactobacillus under a microscope• Produce lactic acid from malic acid• Makes mannitol from fructose• Makes lactic acid from glucose• Diacetyl character is most balanced
compared to other lactics
Pediococcus
• Gram +• Form spherical cells in tetrads• Aerobic to microaerophillic• Homofermentative• Can produce acetate, diacetyl• Complex nutrient requirements• Can produce “ropiness” sensory
character but is rare
Bacteria Basics
• Homofermentative vs. Heterofermentative• Gram + vs. Gram –• Oxygen usage
Homo vs. Hetero Fermentative
• Homofermentative bacteria only produce lactic acid from glucose
• Heterofermentative produce acetic acid, CO2 and lactic acid from glucose
Sugar Transport in L.A.B.ce
llm
embr
ane
OUT
IN
ConcentrationGradient (S1 & S2) PEP
Sugar
Sugar
H+
H+
Symport
Permease
Sugar
Sugar-PO4
PEP-PTSAntiportSugar1
Sugar1
Sugar2
Sugar2
Permease
PEP Pyruvate
EIIBCS
EIIAS
HPr
EI
ATP
H+
H+
ADP
F1F0 ATPase
PMF(pH + )
Energyfrom:
PEP-PTS
Sugar Transport Systems are Sugar-Specific ce
llm
embr
ane
OUT
IN
Lactose
Lactose
H+
H+
LactosePermease
Glucose
Glucose
H+
H+
GlucosePermease
Galactose
Galactose
H+
H+
GalactosePermease
Also sugar specific…… Antiport permease PEP-PTS – EIIA and EIIBC
Example: Lactose Transport
Type of Transport System for a Sugar is Species Specific
PEP-PTS:LactococcusLactobacillus casei
Antiport (lactose/galactose):S. thermophilusLactobacillus delbrueckii subsp. bulgaricus
SymportLactobacillus helveticusLactobacillus acidophilusLeuconostocPediococcus
Lactose
Lactose
H+
H+
Lactose
Lactose
Galactose
Galactose
Lactose
Lactose-PO4
Homolactic Fermentation of Glucose (Embden-Meyerhof Pathway)
Glucose
Fructose-1,6-diphosphate
ATPADP
(2) 3-Phosphoglycerate(2) ATP
(2) ADP(2) 1,3-Diphosphoglycerate
(2) H2PO4- (2) NAD+
(2) NADH + (2) H+
(2) 2-Phosphoglycerate
(2) Phosphoenolpyruvate (PEP)
(2) H2O
(2) Pyruvate(2) ATP
(2) ADP
(2) Glyceraldehyde-3-phosphateDihydroxyacetone-phosphate
FDP aldolase
Products:2 ATP2 Lactate
Key enzymes:FDP aldolaseLactate dehydrogenase
(2) Lactate(2) NAD+
(2) NADH + (2) H+
Lactate dehydrogenase (LDH)
cofa
ctor
rege
nera
tion
O-P-OO
O
phosphate groupGlucose-6-phosphate
ATPADP O-P-O
O
O
Fructose-6-phosphate
Pyruvate = key intermediate
(Glycolysis + LDH)
Two roles for PEP
Transport (PEP-PTS) or ATP generation
Mixed Acid Fermentation: Alternative endproducts for pyruvate
Glucose
(2) ATP(2) ADP
(2) H2PO4- (2) NAD+
(2) NADH + (2) H+
(2) Pyruvate(2) ATP
(2) ADP
(2) Glyceraldehyde-3-phosphateDihydroxyacetone-phosphate
FDP aldolase
2 ATP2 ADP
(2) Lactate
(2) NAD+ (2) NADH + (2) H+
(LDH)Homolactic
cofa
ctor
rege
nera
tion
ATPADP
Acetate
Acetyl-phosphate
(2) Acetyl-CoA
Acetaldehyde
Ethanol
NAD+
NADH + H+
(2) Formate(2) CoA
NAD+
NADH + H+
CoAH2PO4
-
CoA
Products:3 ATP 2 Formate1 Ethanol1 Acetate
Key enzymes:FDP aldolasePyruvate formate lyase (PFL)
Homolactic vs. Mixed Acid Fermentation
Homolactic fermentation prevails when glucose is abundant.
Mixed acid fermentation prevails when sugars are limited – “semi-starvation”.
Substrate availability and the nature of the substrate determine which pathway is used…..
Mixed acid fermentation prevails during growth on galactose as the primary sugar source.
Homolactic fermentation prevails under aerobic conditions because the pyruvate formate lyase (PFL) enzyme is oxygen sensitive.
Glucose
Glucose-6-phosphate
ATPADP
1,3-Diphosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate (PEP)
Pyruvate
Lactate
ATPADP
H2PO4- NAD+
NADH + H+
(2) H2O
ATPADP
NAD+
NADH + H+
LDH
Heterolactic Fermentation of Glucose (Pentose Phosphate Pathway)
6-phospho-gluconate
NAD+
NADH + H+
Xylulose-5-phosphate
Ribulose-5-phosphate
NAD+
NADH + H+CO2
Products:2 ATP1 CO2
1 Lactate1 Acetate
Key enzymes:PhosphoketolaseLactate dehydrogenaseNADH oxidase
Glyceraldehyde-3-phosphate
H2PO4-
Acetyl-phosphatePhosphoketolase
ATPADP
Acetate
O2
NAD+
NADH + H+
NAD+
NADH + H+H2O2
2 H2O
NADH oxidase
NADH oxidase
--- Aerobic conditions
Glucose
Glucose-6-phosphate
6-phospho-gluconate
Ribulose-5-phosphate
Glyceraldehyde-3-phosphate
1,3-Diphosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate (PEP)
Pyruvate
Lactate
ATPADP
ATPADP
H2PO4- NAD+
NADH + H+
(2) H2O
ATPADP
NAD+
NADH + H+
LDH
Heterolactic Fermentation of Glucose --- Anaerobic conditions
NAD+
NADH + H+
Xylulose-5-phosphateH2PO4
-
NAD+
NADH + H+CO2
Acetyl-phosphate
Acetyl-CoA
Acetaldehyde
Ethanol
NAD+
NADH + H+
H2PO4-
CoA
NAD+
NADH + H+
CoA
Products:1 ATP1 CO2
1 Lactate1 Ethanol
Key enzymes:PhosphoketolaseLactate dehydrogenase
Phosphoketolase
Hexoses other than glucoseFructose, mannose and galactose enter the major pathways at the level of glucose-6-phosphate or fructose-6-phosphate after isomerization and phosphorylation steps
Glucose
Glucose-6-phosphate
Fructose-6-phosphate
HOMOLACTIC & MIXED ACID
Glucose
Glucose-6-phosphate
6-phospho-gluconate
HETEROLACTIC
Galactose
Galactose-1-P
Glucose-1-P
Fructose
(when galactose is transported by permease – Leloir – next slide)
Galactose metabolism pathway depends on transport system used
GalactoseH+
H+
GalactosePermease
Galactose
Galactose
Galactose-6-PO4
PEP
PyruvateEIEI
LeloirPathway
Galactose-1-PO4
Glucose-1-PO4
Glucose-6-PO4
ATPADP
Homolactic, *mixed acid, or heterolactic pathway
Tagatose-1,6-diPO4
ATPADP
(2) Glyceraldehyde- 3-phosphate
Dihydroxyacetone-phosphate
TagatosePathway
Tagatose-6-PO4
Homolactic or *mixed acid pathway
* Mixed acid fermentation dominates if galactose is the most abundant sugar available
Disaccharides: broken into monosaccharides before metabolized
sucrose maltoselactose
galactose glucose
glucose fructose glucoseglucose
Lactose Breakdown: depends on transport system
+
+
PO4 PO4
-galactosidase
Phospho--galactosidase
Homolactic, mixed acid, or heterolactic
pathway
Homolactic, mixed acid, or heterolactic
pathway
Tagatose pathway
Leloir pathway
Some L.A.B. cannot metabolize galactose
Lactose
Lactose
Galactose
Galactose
Permeasecell
mem
bran
e
OUT
IN
Example: S. thermophilus and Lb. delbrueckii subsp. bulgaricus
Galactose is exported via antiport system
Glucose
Homolactic, mixed acid or heterolactic
pathway
Summary of Lactose Metabolism in L.A.B.
How many ATPs from one lactose?
LactococcusLb. casei
*S. thermophilus, *Lb. delbrueckii, Lb. Helveticus, Lb. lactis
* S. thermophilus, and Lb. delbrueckii do not metabolize the galactose part of lactose. They export galactose from the cell.
LeuconostocGroup III Lactobacillus
(Figure from Fox et al. 1990. Critical Reviews in Food Science and Nutrition. 29:237-253.)
Tagatose Pathway Homolactic Pathway
Leloir Pathway
Heterolactic Pathway
-galactosidasephospho--galactosidase
CO2
Pentoses
Pentose
Pentose
Pentose-PO4
Xylulose-PO4
orRibulose-PO4
Heterolactic fermentationpathway
ATPADP
isomerization
Pentoses cannot enter the homolactic or mixed acid pathways
Glucose
Glucose-6-phosphate
ATPADP
1,3-Diphosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate (PEP)
Pyruvate
Lactate
ATPADP
H2PO4- NAD+
NADH + H+
(2) H2O
ATPADP
NAD+
NADH + H+
LDH
Heterolactic Fermentation: Pentose-PO4 entry
6-phospho-gluconate
NAD+
NADH + H+
Xylulose-5-phosphate
Ribulose-5-phosphate
NAD+
NADH + H+CO2
Products:2 ATP1 Lactate1 Acetate
Glyceraldehyde-3-phosphate
H2PO4-
Acetyl-phosphatePhosphoketolase
ATPADP
Acetate
O2
NAD+
NADH + H+
NAD+
NADH + H+H2O2
2 H2O
NADH oxidase
NADH oxidase
L.A.B. groups based on fermentation pathways
• Obligately homofermentative
• Obligately heterofermentative
• Facultatively heterofermentative
Obligately homofermentative
Group I Lactobacillus species and a few other species
• Hexoses are fermented by homolactic fermentation pathway (glycolysis + LDH)
• Can do mixed acid fermentation of hexoses under certain conditions
• Do not ferment pentoses
• Have FDP aldolase enzyme
• Do not have phosphoketolase enzyme
Homolactic Fermentation of Glucose (Glycolysis + LDH)
Glucose
Fructose-1,6-diphosphate
ATPADP
(2) 3-Phosphoglycerate(2) ATP
(2) ADP(2) 1,3-Diphosphoglycerate
(2) H2PO4- (2) NAD+
(2) NADH + (2) H+
(2) 2-Phosphoglycerate
(2) Phosphoenolpyruvate (PEP)
(2) H2O
(2) Pyruvate(2) ATP
(2) ADP
(2) Glyceraldehyde-3-phosphateDihydroxyacetone-phosphate
FDP aldolase
Products:2 ATP2 Lactate
Key enzymes:FDP aldolaseLactate dehydrogenase
(2) Lactate(2) NAD+
(2) NADH + (2) H+
Lactate dehydrogenase (LDH)
cofa
ctor
rege
nera
tion
O-P-OO
O
phosphate groupGlucose-6-phosphate
ATPADP O-P-O
O
O
Fructose-6-phosphate
Glucose
Glucose-6-phosphate
ATPADP
1,3-Diphosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate (PEP)
Pyruvate
Lactate
ATPADP
H2PO4- NAD+
NADH + H+
(2) H2O
ATPADP
NAD+
NADH + H+
LDH
Heterolactic Fermentation of Glucose --- Aerobic conditions
6-phospho-gluconate
NAD+
NADH + H+
Xylulose-5-phosphate
Ribulose-5-phosphate
NAD+
NADH + H+CO2
Products:2 ATP1 CO2
1 Lactate1 Acetate
Key enzymes:PhosphoketolaseLactate dehydrogenaseNADH oxidase
Glyceraldehyde-3-phosphate
H2PO4-
Acetyl-phosphatePhosphoketolase
ATPADP
Acetate
O2
NAD+
NADH + H+
NAD+
NADH + H+H2O2
2 H2O
NADH oxidase
NADH oxidase
Obligately heterofermentative
Group III Lactobacillus species, Leuconostoc, Oenococcus
• Hexoses are fermented by heterolactic fermentation pathway (phosphoketolase pathway)
• Pentoses are fermented by heterolactic fermentation pathway (phosphoketolase pathway)
• Have phosphoketolase enzyme
• Do not have FDP aldolase enzyme
Facultatively heterofermentative
Group II Lactobacillus species, Lactococcus, Pediococcus, Streptococcus thermophilus• Hexoses are fermented by homolactic fermentation pathway (glycolysis + LDH)
• Can do mixed acid fermentation of hexoses under certain conditions
• Pentoses are fermented by heterolactic fermentation pathway (phosphoketolase pathway)
• Have both FDP aldolase and phosphoketolase enzymes
Lb. rhamnosus
Lactobacillus
Homolactic and facultatively heterolactic – no CO2 from glucose, FDP aldolase presentObligately heterolactic – CO2 from glucose, phosphoketolase present
Lb. sanfranciscoLb. kefir
Citrate Transport
CitrateH+
CitrateH+
CitratePermease
Citrate permease is pH dependent – only functions pH 5 – 6. Optimum = pH 5.2
CH2 - COOH
CH2 - COOH
HO - C - COOH
~1.5 mg/ml citrate in milk
Citrate Metabolism
NAD+
NADH + H+
NAD+
NADH + H+
Acetate
CO2
Leuc. mesenteroides subsp. cremorisLc. lactis subsp. lactis biovar. diacetylactis
citrate lyase
CH2 - COOH
CH2 - COOH
HO - C - COOH
Pathway does not generate ATP, but regenerates NAD+.
citrate
CH3 – C – C – CH3
O O
Exopolysaccharide types and applications
• Capsular: yogurts (thickener), cheeses (increase moisture)
• Ropy: some fermented milks such as viili and långfil
(Perry et al. 1997. Journal of Dairy Science. 80:799-805.)
L.A.B.: Carbohydrate Metabolism
Transport of sugars across cell membrane
Catabolism of sugars for energy
Synthesis of polysaccharides
Homolactic fermentationMixed acid fermentation Heterolactic fermentation
• Pathways for glucose fermentation
• Pathways for other hexoses
• Pathways for pentoses
• Pathway for citrate
• Disaccharides
Fermentasi karbohidrat
• Polisakarida dipecah menjadi gula sederhana sebelum difermentasi
• Tahap pertama fermentasi, glukosa diubah menjadi asam piruvat
• Tahap kedua fermentasi, asam piruvat diubah menjadi produk akhir yang lebih spesifik
4 jalur pemecahan glukosa menjadi asam piruvat
• Jalur Embden-meyerhoff-Parnas (EMP) atau glikolisis. Ditemukan pada fungi, bakteri (mayoritas), hewan danmanusia
• Glukosa + 2ADP + 2 NAD+ + Pi 2 Piruvat + 2 ATP + 2 NADH + H+
• Jalur Entner Doudoroff (ED), hanya ditemukan pada beberapa bakteri
• Glukosa + NADP+ + NAD+ + ADP + Pi 2 Piruvat + NADP + H+ + NADH + ATP
• Jalur Heksosamonofosfat (HMP) menghasilkan pentosa yang diperlukan untuk sisntesis asam nukleat, beberapa asam amino aromatik dan vitamin, dilakukan beberapa organisme
• Glukosa + 12 NADP+ + ATP 6CO2 + 12 NADPH + 12 H+ + ADP + Pi
• Jalur Fosfoketolase (FK) ditemukan pada bakteri yang tergolong laktobasili heterofermentatif
• Glukosa + NAD+ + ADP + Pi Piruvat + etanol + CO2 + NADH + H+ + ATP