ITP 321 Lecture Note 7 `

Fermentation of Ingredients and

F d Additives
Food Additi

Lilis Nuraida and Ratih Dewanti-Hariyadi

Department of Food Science and Technology
Bogor Agricultural University
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Ingredients from
microorganisms
microorganism s
• Yeast-derived flavouring agents
• Biogum:
• Xanthan
• Pullulan
• etc
• Vitamins
• Oils
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 Yeast-Derived Products
Contains guanosine 5’-
• Yeast Extract monophosphate (GMP),
• Yeast Autolysates ’
ionosine 5’-
monophosphate (IMP),
glutamic acid

 Application:
– Flavoring agent in soup, sauces,
gravies,
i stew, snackk food
f d etc.
– Main component of savoury spreads:
Vegemite and Marmite

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Yeast Extract Components

• Amino acids
• Peptides
• Nucleotides
• Proteins
• Carbohydrates
• Vitamins
Vit i
• Flavor compounds

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 Product definition

• Yeast Extract:
Yeast extract comprises the water soluble
components of the yeast cell, the composition
of which is primarily amino-acids, peptides,
carbohydrates and salts. Yeast extract is
produced through the hydrolyses of peptide
b d by
bonds b the
th naturally
t ll occurring
i enzymes
present in edible yeast or by the addition of
food grade enzymes
The Food Chemical Codex

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Product definition

• Yeast Autolysates/Autolysed yeast:

Autolyzed yeast is the concentrated, not
extracted, partially soluble digest obtained
from food-grade yeast. Solubilization is
accomplished by enzyme hydrolysis or
autolysis of yeast cells. Autolyzed yeast
contains both soluble and insoluble
components derived from the whole yeast
cells.
The Food Chemical Codex

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 Composition
Yeast Extract Yeast Autolysate

• Protein content: 50-75% • Protein content: 50-69%

• Total carbohydrate: 4- • Total carbohydrate
13% content: 15-25%
• Lipid content: very little • Lipid content: 3-10%

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Preparation of Yeast Extract

Requires disruption of cell walls:
• Autolysis by endogenous enzymes*)
• Plasmolysis: modified autolyses in the
presence of accelerator, i.e. salt or
organic solvent
• Mechanical desruption
• Hydrolysis by acid or by exogenous
enzymes
*)practiced in industrial scale

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 Production Process of Yeast
Extract Absent in
production of
Yeast cream Clarification yeast
autolysate

Plasmolyses Concentration

Liquid: 50-65% dry matters

Autolyses
y Paste: 70-80%
70 80% dry matters

Pasteurisation Packed in Spray, roller

pails, drums drying

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Source of yeast cream

• Conventional:
• Baker’s Yeast
• Brewer’s Yeast
• Alternative:
• Candida utilis
• Kluyveromyces marxianus
Enriched yeast extract in amino acid cystein produced
through the application of GE-yeast (genetically engineered
yeast) to over express and over produce the yeast cystein-
rich protein metallothionein (Stam et al., 2000)

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 Condition for Yeast Extract Production

Steps Condition

Autolyses and 55 oC for 24 h, pH 5.5

Plasmolyses
Pasteurisation 1st : 70oC for 15 h
2nd: 70-75oC for 2-5 h
(after clarification)
Concentration Temperature <55oC

Adapted from Biocatalyst Ltd

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Common problems encountered in yeast

extract production
Problem Solution*)

Enzyme not working Ensure no direct heat

Low yield autolysis
Over heat
Clarification problems caused by
insoluble glucans
Burnt flavor
Treatment with protease followed by nuclease resulted in the highest 5’-GMP
(Chae et al., 2001)
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Check pH and temperature of slurry at
each stage
Extend autolyses time
Add protease
Add protease
Add glucanase
Keep final temperature <55oC during
final evaporation
*)Adapted from Biocatalyst Ltd

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 Other yeast-derived products

• Colorants
• Pigmentedd yeasts suchh as Rhodotorula,
h d l
Phodospondium, Cryptococcus, Sporidiobolus,
Sporobolomyces, GE-Saccharomyces, Candida
• Yeast Polysaccharides:
• Food application and nutraceutical potential
• Potential species S.
S cerevisiae
cerevisiae, Pichia holstii
holstii,
Hansenula sp, Candidia, Rhodotorula
• Yeast Enzymes

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Microbial Polymers
Xanthan gum Emulsion stabilization and suspension agent in foods

from X. Foam stabilization in foods

camprestis Crystallization inhibition in foods
Viscosity control in oil drilling mud and inkjet printing
Bacterial Moisture retention in wound dressings
cellulose High strength acoustic diaphragms in sound reproductio
Hyalluronic acid Hydrating agents in cosmetics and pharmaceuticals
Replacement for synovial fluid and vitreos humor in biomedicine
Emulsan Emulsifier and vaccine adjuvant
Curdlan Gelling agent in foods
Gellan Gelling agent in foods
Pullulan Food coatings

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 Advantage of Using Microorganisms
for Gum Production

• Produce consistent gum

• Independence of plant resources
• Independence of external factors
• Possibility of invention of new gum with unique
properties : certain rheological properties for food
additives
• Consistent quality of gum
• Exploration is being carried out continuously

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Polysaccharide Producing Microbes

and their Polysaccahrides
Microorganisms Polysaccharides

Azotobacter vinelandii, Pseudomonas sp. Alginate

Saccharomyces cerevisiae Baker yeast Glucan
Acetobacter sp. Cellulosa
Alcaligenes faecalis Curdlan
Leuconostoc mesentroides, L. dextranicum Dextran
Pseudomonas elodea Gellan
Aureobasidium (Pullularia) pullulans Pullulan
Xanthomonas sp. Xantan
Alcaligenes spp. Welan
Sclerotium spp. Skleroglucan
Schizophyllum communis Skhizopilan

*Lapasin dan Sabrina (1995)

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 Sucrose
invertase
Glucose Fructose
glucokinase phosphoglucose Fructokinase
isomerase
Glucose 6-Phosphate Fructose 6-Phosphate
phosphomannose
p p siomerase
Mannose 6-Phosphate
phosphomanno mutase
Mannose 1-Phosphate
GDP-mannose pyrophosphorylase
GDP-Mannose
GDP-mannose dehydrogenase
GDP-Mannuronic Acid
polymerase
Plymannuronic acid
polymannuronic acid 5-epimerase

acetyltransferase
Alginic Acid
Alginic acid biosynthetic pathway in Azotobacter vinelandii
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Xanthan gum
Colonies of Xanthomonas campestris
produced copious extracellular slime - a
complex polysaccharide composed of
more than one type of sugar (a
heteropolymer)
It is termed xanthan
It is used as a gelling and stabilising
agent in salad dressings, ice creams,
toothpastes, cosmetics, water-based
paints etc.,
paints, etc and also as a drilling lubricant
in oil wells.
Xanthomonas campestris pv. campestris is an agricultural and industrial
important bacterium.
In agriculture it is a pathogen that causes black rot on a number of crops.

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 Xanthan Gum

• Xanthan gum is a high molecular

weight exopolysaccharides with:
• Cellulose backbone
• Trisaccharides side chain

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Xanthan Gum

• Characteristics :
• High viscosity
• Stable properties in extreme conditions
• Pseudoplastic behavior
• Applied as:
as:
• Stabilizing
Stabilizing// Viscosifying/
Viscosifying/Emulsifyin
Emulsifying/
g/
Thickening or Suspending agents
agents

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 Growth of X. campestris

• X. campestris is able to use glucose, sucrose,

and starch as carbon source but not lactose.
• This is due to the low level of enzyme 
galactosidase.
• This enzyme converts lactose to galactose and
glucose.

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Pullulans

• Aureobasidium pullulans produces the

exopolysaccharide pullulan.
• Linear mixed linkage a-D-glucan consisting
mainly of maltotriose units interconnected via
-(1-6) linkage
• This polymer has unique film-forming and
adhesive properties that make it useful for
producing a film-wrap for foods

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 Production of Pullulans

• Substrate: sugar and starch

• Industrial waste: potato starch waste,
whey, mollases, brewery waste
• A. pullulans also produced other
exopolysaccharides other than pullulans

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FACTORS INFLUENCING BIOGUM

PRODUCTION

● Nitrogen and Carbon Source

▪ Requires high C/N ratio
▪ Arthrobacter viscosus: Xylosa 3%, N 0.033%
▪ Xanthomonas: glukosa 1-5%, N 0.048%

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 FACTORS INFLUENCING BIOGUM
PRODUCTION
● pH
▪ Gum production is inhibited at low pH, needs buffering
agents
▪ Xanthomonas: optimum pH 6-7.5
▪ Propionibacterium: pH 6
▪ pH optimum for biogum production may not be similar to
that for biomass production
▪ Aeromonas almonicida requires excessive phosphate

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FACTORS INFLUENCING BIOGUM

PRODUCTION
● Mineral
▪ Some requires K, P, Mg, Mn dan Ca
▪ In some instance, mineral inhibits EPS production
(Enterobacter agglomerans, Erwinia)

● Incubation time
▪ Production at the end of logarithmic phase
▪ Maximum when N is low
▪ At the end of fermentation, EPS may decrease due to
enzymatic activity that cleaves EPS

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 FACTORS INFLUENCING BIOGUM
PRODUCTION
● Incubation temperature
▪ EPS is generally formed at suboptimal temperature
when growth is stressed
▪ Xanthomonas: optimum at 28oC
▪ P. acidipropioni: 25oC

● Harvesting methods
▪ Agglutination by organic solvents (ethanol, aceton,
isopropanol)

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Production
d i off Vitamins
i i b by
Microorganisms
Microorganism s

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 Vitamin B12 Production by Various
Microorganisms
Strain Carbon Source
Micromonospora sp. Glucose
Nocardia rugosa Glucose-cane molasses
Propionibacterium reudenreichii Glucose
Propionibacterium shermanii Glucose
Propionibacterium vannielli Glucose
Pseudomonas denitrificans Beet molasses
Streptomyes olivaceus Glucose-lactose
Mixed methanogenic bacteria Methanol
B t i
Bacterium FM-O2T
FM O2T M th
Methanol
l
Methanobacillus omellanskii Methanol
Protoaminobacter ruber Methanol
Corynebacterium and n-Parafins
Rhodopseudomonas
Nocardia gardneri Hexadecane
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SYNTHESIS OF VITAMIN B12

Cobinamid + ATP Cobinamid-P + ADP

Cobinamid-P + GTP GDP-Cobinamid + PPi

GDP-Cobinamid
GDP Cobinamid + α
α-ribazole
ribazole 5’-P
5 P Cobalamine 5
5’-P
P + GMP

Cobalamine 5’-P Cobalamine + Pi

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 Synthesis of L-Ascorbic Acid

• Ascorbic acid ((vitamin C)) is currentlyy

synthesized by a very expensive
process which includes:
• A microbial fermentation step
• A number of chemical steps
• convert glucose to ascorbic acid

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Commercial Synthesis of Ascorbic Acid

2-KLG : 2-keto-gluconic acid

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 Synthesis of L-Ascorbic Acid

 Glucose  2,5-diketo-D-gluconic acid (2,5-DKG).

microrganisms capable of 2,5-DKG
2 5 DKG synthesis:
 Acetobacter, Gluconobacter, Erwinia
 Enzyme 2,5-DKG reductase will convert 2,5-DKG
 2-KLG (2-keto-L-gluconic acid).
microrganisms having this enzyme include:
 Corynebacteria,
C b t i Brevibacterium,
B ib t i Arthrobacter
A th b t
 Last step in the process involves the conversion of:
2-keto-L-gluconic acid (2-KLG)  L-ascorbic acid.

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Microbial Synthesis of LAA

• Glucose 2 5-
2,5
5-DKG
Erwinia herbicola

• 2,5
2,5--DKG KLG
Corynebacterium sp

• Genetically enginered E. herbicola (recombinant having

DKG reductase from Corynebacterium)
Glucose KLG

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 Production of
2-KLG

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Production of microbial oils

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 Production of Gamma-
Gamma-linolenic
acid (GLA)

• Produced by Mucorales
• Gamma
Gamma--linolenic acid is the only 18:3
structure in Mucorales and produced in
higher amount than by other molds
• Commecial process:
• UK: ja anic s (Mucor
UK oil of javanicus M co javanicus
ja anic s)
• Japan: Mortierella isabellina.

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Production of GLA

• Carbon source: glucose

g
• 15 to 18% carbon source is converted to
oil
• Production of oil depend on growth rate and
substrate conc
concentration
• Oil production induced by high C/N ratio,
i.e. 80:1

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 Preservatives produced by
l
lactic
i acid
id bacteria:
b i
BACTERIOCIN

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Bacteriocins

• Small proteins produced by bacteria

• h
harm only
l microbes
i b that th t closely
l l resemble
bl
the bacteria that manufactured them
• attack potentially fatal food-
food-poisoning germs
(ie. L. monocytogenes and C. botulinum):
• Disturb membrane
membrane stability

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 Bacteriocins: Highly Selective

 Act against one target pathogen and

probably will not sicken beneficial bacteria
or humans
 Bacteriocins considered for protection of
food against Listeria are useless against
S l
Salmonella
ll andd E.
E colili
E.c

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Bacteriocin, Examples

 Nisin
 Pediocin
 Lacticin 3147
 Enterocins A and B

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 Nisin
 Secreted by some Lactococcus lactis ssp.
Lactis strains
 Interacts with phospholipids in the
cytoplasmic membrane of undesirable
bacteria
 This action disrupts membrane function,
preventing bacterial growth
 P
Prevents outgrowthh off spores b
by inhibiting
i hibi i
the swelling process of germination

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Pediocin

 Produced by Pediococcus acidilactici, a

l ti acid
lactic id bacteria
b t i (LAB)
 Together with fermentation-
fermentation-derived organic
acids produced by LAB, pediocin attac
attack
ks
gram--positive bacteria such as Lactobacillus
gram
and Listeria monocytogenes

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 Lacticin 3147

 Produced byy Lactococcus lactis subsp.p Lactis

 Inhibits growth in all gram positive bacteria
including L . monocytogenes, C. botulinum,
& Staphylococcus aureus
 Inserts into cell membrane of sensitive cells,
leadingg to the loss of potassium
p ions and
the collapse of the membrane potential
resulting in cell death

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Enterocins A and B

 Produced by Enterococcus faecium CTC492

 Antilisterial
 Used in
– fermented sausages
– cooked ham
– pate
– minced pork
– deboned chicken breasts

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