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 (PHAs)

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 heterofermentative 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.

Symport
OUT H

Antiport
Sugar1 Sugar2

PEP-PTS
Sugar

Sugar

F1F0 ATPase

Permease

Permease

EIIBCS
EIIAS

IN

ATP ADP

+ H
Sugar Sugar1 Sugar2

HPr

+ H

EI

Sugar-PO4

PEP Energy from: PMF (pH + ) Concentration Gradient (S1 & S2)

Pyruvate

PEP

PEP-PTS

Sugar Transport Systems are Sugar-Specific

+ H
Lactose

OUT

Glucose

Galactose

Lactose Permease

Glucose Permease

Galactose Permease

IN H

+
Lactose

+
Glucose

+
Galactose

Also sugar specific Antiport permease PEP-PTS EIIA and EIIBC

Type of Transport System for a Sugar is Species Specific

Example: Lactose Transport
PEP-PTS: Lactococcus Lactobacillus casei
Lactose-PO4 Lactose Galactose Lactose

Antiport (lactose/galactose): S. thermophilus Lactobacillus delbrueckii subsp. bulgaricus

Lactose

Galactose

Symport Lactobacillus helveticus Lactobacillus acidophilus Leuconostoc Pediococcus

Lactose H+

H+ Lactose

Homolactic Fermentation of Glucose (Embden-Meyerhof Pathway)

(Glycolysis + LDH)

Glucose
Glucose-6-phosphate Fructose-6-phosphate

ATP ADP

O O O-P-O O-P-O O O phosphate group

Fructose-1,6-diphosphate

ATP ADP

FDP aldolase
Dihydroxyacetone-phosphate (2) Glyceraldehyde-3-phosphate (2) H2PO4(2) 1,3-Diphosphoglycerate (2) NAD+ (2) NADH + (2) H+

(2) 3-Phosphoglycerate
Products: 2 ATP 2 Lactate Key enzymes: FDP aldolase Lactate dehydrogenase (2) 2-Phosphoglycerate

(2) H2O (2) Phosphoenolpyruvate (PEP) (2) Pyruvate (2) ADP (2) ATP (2) NADH + (2) H+ (2) NAD+

Lactate dehydrogenase (LDH)

Pyruvate = key intermediate

(2) Lactate

cofactor regeneration

(2) ADP (2) ATP

Two roles for PEP

Transport (PEP-PTS) or ATP generation

Mixed Acid Fermentation: Alternative endproducts for pyruvate Glucose

2 ATP 2 ADP

FDP aldolase
Dihydroxyacetone-phosphate (2) Glyceraldehyde-3-phosphate (2) H2PO4(2) NAD+ (2) NADH + (2) H+ (2) ADP (2) ATP (2) ADP (2) ATP

Homolactic
(LDH)

(2) Lactate
(2) NAD+
(2) NADH + (2) H+ H2PO4Products: 3 ATP 2 Formate 1 Ethanol 1 Acetate Key enzymes: FDP aldolase Pyruvate formate lyase (PFL)

(2) Pyruvate

(2) CoA

(2) Formate
CoA NADH + H+ Acetaldehyde NAD+ NADH + H+ NAD+

(2) Acetyl-CoA CoA

Acetyl-phosphate ADP ATP

Acetate

Ethanol

cofactor regeneration

Homolactic vs. Mixed Acid Fermentation

Substrate availability and the nature of the substrate determine which pathway is used..

Homolactic fermentation prevails when glucose is abundant. Mixed acid fermentation prevails when sugars are limited semi-starvation.

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.

Heterolactic Fermentation of Glucose (Pentose Phosphate Pathway)

Glucose
Glucose-6-phosphate 6-phospho-gluconate

--- Aerobic conditions

ATP ADP NAD+ NADH + H+ NAD+ NADH + H+

NADH +

H+

O2
NADH oxidase
H2O2

NAD+
NADH + H+ NAD+

CO2

NADH oxidase
2 H2 O

Ribulose-5-phosphate Xylulose-5-phosphate H2PO4-

Phosphoketolase
Glyceraldehyde-3-phosphate H2PO4
-

Acetyl-phosphate NAD+ NADH + H+ ADP ATP ADP ATP

1,3-Diphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate

Acetate
Products: 2 ATP 1 CO2 1 Lactate 1 Acetate Key enzymes: Phosphoketolase Lactate dehydrogenase NADH oxidase

(2) H2O Phosphoenolpyruvate (PEP) Pyruvate ADP ATP NADH + H+ NAD+

LDH

Lactate

Heterolactic Fermentation of Glucose --- Anaerobic conditions

Glucose
Glucose-6-phosphate 6-phospho-gluconate

ATP ADP NAD+ NADH + H+ NAD+ NADH + H+

CO2

Ribulose-5-phosphate Xylulose-5-phosphate H2PO4-

Phosphoketolase
Glyceraldehyde-3-phosphate H2PO41,3-Diphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate

Acetyl-phosphate NAD+ NADH + H+ ADP ATP CoA Acetyl-CoA CoA NADH + H+ NAD+ H2PO4-

Acetaldehyde
(2) H2O

NADH + H+ NAD+

Phosphoenolpyruvate (PEP) Pyruvate

Ethanol

ADP ATP NADH + H+ NAD+

Products: 1 ATP 1 CO2 1 Lactate 1 Ethanol Key enzymes: Phosphoketolase Lactate dehydrogenase

LDH

Lactate

Hexoses other than glucose

Fructose, mannose and galactose enter the major pathways at the level of glucose-6-phosphate or fructose-6-phosphate after isomerization and phosphorylation steps

HOMOLACTIC & MIXED ACID Glucose

Galactose Galactose-1-P Glucose-1-P

HETEROLACTIC Glucose

Glucose-6-phosphate
Fructose Fructose-6-phosphate

Glucose-6-phosphate
6-phospho-gluconate

(when galactose is transported by permease Leloir next slide)

Galactose metabolism pathway depends on transport system used

H+ Galactose Galactose

Leloir Pathway

Galactose Permease H+ Galactose

ATP ADP

PEP Pyruvate EI EI

Tagatose Pathway
Galactose-6-PO4 Tagatose-6-PO4 Tagatose-1,6-diPO4

Galactose-1-PO4 Glucose-1-PO4 Glucose-6-PO4

ATP ADP

Dihydroxyacetonephosphate

(2) Glyceraldehyde3-phosphate

Homolactic, *mixed acid, or heterolactic pathway

* Mixed acid fermentation dominates if galactose is the most abundant sugar available

Homolactic or *mixed acid pathway

Disaccharides: broken into monosaccharides before metabolized

lactose

sucrose

maltose

glucose

fructose

glucose

glucose

galactose

glucose

Lactose Breakdown: depends on transport system

+

-galactosidase
Leloir pathway Homolactic, mixed acid, or heterolactic pathway

PO4

PO4

+ Phospho--galactosidase
Tagatose pathway

Homolactic, mixed acid, or heterolactic pathway

Some L.A.B. cannot metabolize galactose

Example: S. thermophilus and Lb. delbrueckii subsp. bulgaricus Galactose is exported via antiport system Lactose OUT Galactose

Permease

IN Glucose
Homolactic, mixed acid or heterolactic pathway

Lactose Galactose

Summary of Lactose Metabolism in L.A.B.

phospho-galactosidase

Lactococcus Lb. casei

*S. thermophilus, *Lb. delbrueckii, Lb. Helveticus, Lb. lactis

Leuconostoc Group III Lactobacillus

-galactosidase

How many ATPs from one lactose?

CO2

* S. thermophilus, and Lb. delbrueckii do not metabolize the galactose part of lactose. They export galactose from the cell.

(Figure from Fox et al. 1990. Critical Reviews in Food Science and Nutrition. 29:237-253.)

Tagatose Pathway

Homolactic Pathway

Leloir Pathway

Heterolactic Pathway

Pentoses
Pentose

Pentose Pentose-PO4
isomerization

ATP ADP

Xylulose-PO4 or Ribulose-PO4

Heterolactic fermentation pathway

Pentoses cannot enter the homolactic or mixed acid pathways

Heterolactic Fermentation: Pentose-PO4 entry

Glucose
Glucose-6-phosphate 6-phospho-gluconate

CO2
Ribulose-5-phosphate Xylulose-5-phosphate H2PO4-

ATP ADP NAD+ NADH + H+ NAD+ NADH + H+

NADH + H+ NAD+ NADH + H+ NAD+

O2
NADH oxidase
H2O2

NADH oxidase
2 H2 O

Phosphoketolase
Glyceraldehyde-3-phosphate H2PO4
-

Acetyl-phosphate NAD+ NADH + H+ ADP ATP ADP ATP

1,3-Diphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate

Acetate
Products: 2 ATP 1 Lactate 1 Acetate

(2) H2O Phosphoenolpyruvate (PEP) Pyruvate ADP ATP NADH + H+ NAD+

LDH

Lactate

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

Glucose-6-phosphate Fructose-6-phosphate Fructose-1,6-diphosphate ATP ADP ATP ADP

O O O-P-O O-P-O O O phosphate group

FDP aldolase
Dihydroxyacetone-phosphate (2) Glyceraldehyde-3-phosphate (2) H2PO4(2) 1,3-Diphosphoglycerate (2) NAD+ (2) NADH + (2) H+

(2) 3-Phosphoglycerate
Products: 2 ATP 2 Lactate Key enzymes: FDP aldolase Lactate dehydrogenase (2) 2-Phosphoglycerate

(2) H2O (2) Phosphoenolpyruvate (PEP) (2) Pyruvate (2) ADP (2) ATP (2) NADH + (2) H+ (2) NAD+

Lactate dehydrogenase (LDH)

(2) Lactate

cofactor regeneration

(2) ADP (2) ATP

Heterolactic Fermentation of Glucose --- Aerobic conditions

Glucose
Glucose-6-phosphate 6-phospho-gluconate

ATP ADP NAD+ NADH + H+ NAD+ NADH + H+

NADH +

H+

O2
NADH oxidase
H2O2

NAD+
NADH + H+ NAD+

CO2

NADH oxidase
2 H2 O

Ribulose-5-phosphate Xylulose-5-phosphate H2PO4-

Phosphoketolase
Glyceraldehyde-3-phosphate H2PO4
-

Acetyl-phosphate NAD+ NADH + H+ ADP ATP ADP ATP

1,3-Diphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate

Acetate
Products: 2 ATP 1 CO2 1 Lactate 1 Acetate Key enzymes: Phosphoketolase Lactate dehydrogenase NADH oxidase

(2) H2O Phosphoenolpyruvate (PEP) Pyruvate ADP ATP NADH + H+ NAD+

LDH

Lactate

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

Lactobacillus

Lb. rhamnosus

Lb. sanfrancisco Lb. kefir

Homolactic and facultatively heterolactic no CO2 from glucose, FDP aldolase present Obligately heterolactic CO2 from glucose, phosphoketolase present

Citrate Transport
H+ Citrate
Citrate Permease

~1.5 mg/ml citrate in milk

H+ Citrate

CH2 - COOH
HO - C - COOH CH2 - COOH

Citrate permease is pH dependent only functions pH 5 6. Optimum = pH 5.2

The Citric Acid Factor

Citrate Metabolism
CH2 - COOH

HO - C - COOH
CH2 - COOH

citrate lyase

Acetate CO2

citrate

O O CH3 C C CH3

Pathway does not generate ATP, but regenerates NAD+.

NADH + H+ NAD+

NADH + H+ NAD+

Leuc. mesenteroides subsp. cremoris Lc. lactis subsp. lactis biovar. diacetylactis

Exopolysaccharide types and applications

Capsular: yogurts (thickener), cheeses (increase moisture)

(Perry et al. 1997. Journal of Dairy Science. 80:799-805.)

Ropy: some fermented milks such as viili and lngfil

L.A.B.: Carbohydrate Metabolism

Transport of sugars across cell membrane

Catabolism of sugars for energy

Pathways for glucose fermentation Homolactic fermentation Mixed acid fermentation Heterolactic fermentation Pathways for other hexoses Disaccharides

Synthesis of polysaccharides

Pathways for pentoses

Pathway for citrate

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