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

The Citric Acid Factor

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