CUBT 402 : FOOD BIOTECHNOLOGY

Ms B.
Madzora
FOOD BIOTECHNOLOGY
INTRODUCTION
Food additives and processing aids from use of genetically modified
organisms

Enzymes Amylase, isomerase High fructose corn

syrup
  Rennet Cheese making
  Protease Meat tenderising
  Pullulanase “Lite beer”
Amino acids Methionine, lysine, Nutritional
tryptophan supplements
  Aspartic acid, Phenylalanine Ingredients in
sweetener production
Flavours of Vanillin Flavouring and
pigments colouring agents
Low-calorie Aspartame, thaumatin, Non-nutritive
products monellin sweeteners
  Modified fatty acids Food additives
triglycerides Cooking oil
Microbial Xanthan gum Stabilizers, thickeners
polysaccharid and gelling agents
es
FOOD BIOTECHNOLOGY
INTRODUCTION
Production of Enzymes

Aspergillus niger Phytase

Chymosin
Lipase
Aspergillus oryzae Esterase-lipase
  Aspartic proteinase
Glucose oxidase
Laccase
Lipase

Bacillus licheniformis α amylase

  Pullulanase
Bacillus subtilis α acetolactate decarboxylase
  α amylase
Maltogenic amlyase
Pullulanase

Escherichia coli K-12 Chymosin

Fusarium venenatum Xylanase
 
Kluyveromyces marxianus var. lactis Chymosin
 
Pseudomonas fluorescens α amylase
Trichoderma reesei Pectin lyase
FOOD FERMENTATION TECHNOLOGY
FUNGI
FOOD FERMENTATION TECHNOLOGY

FUNGI

• Production of beverages (juices, drinks, wine, beers etc)

• Production of enzymes
• Production of metabolites
• Used industrially and for GMO processes
• Bioprocesses
FOOD FERMENTATION TECHNOLOGY

Production of acids:
-Citric acid
-Gluconic acid
-Lactic acid
-Succinic acid
-Malic acid
-Fumaric acid
-Itaconic acid

Fungi and bacteria

• Cell factories
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS

Commonly used fungi in organic acid production are:

Aspergillus sp.
Rhizopus sp.
Saccharomyces sp.
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
FUMARIC ACID
Applications
-Food additive
-Polymer industry
-Production of succinic and malic acid

Fumaric acid
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
FUMARIC ACID
-Rhizopus sp e.g R. oryzae
-Microbial strain, morphology, neutralizing agents
-Submerged fermentation: carbon e.g. glucose , nitrogen
e.g. urea
-Accumulation of fumarate:
-Glycolysis (pyruvate) and citrate cycle and what is known
as the reductive carboxylation pathway
-L-malic to fumarate high activity in low N2
-Cytosolic fumarase activity increased in low N2
-Nutrients: C:N molar ratio of 200:1 critical in fumaric acid
production , limitation in nitrogen required
Trace metals: Zn, Mg,Fe
-CaCO3 used as a neutralising agent, can also be a source
of CO2
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
FUMARIC ACID

Roa Engel et al., 2008

FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
CITRIC ACID
-Acidulant in food, confectionary, and beverage

-Pharmaceutical e.g. soluble aspirin preparation

-Industrial : complexes with metals such as iron and copper

to be stabilizer of oil and fats
-Detergents and cleaning products
-A. niger also the yeast Yarrowia Lipolytica

Citric acid
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
CITRIC ACID
Pathway in production of citric acid by A. niger
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
CITRIC ACID
Production of citric acid by A. niger
KEY ASPECTS
Fermentation
-Beet, sugar cane mollases, glucose syrup
Requirements: Carbon, low level nitrogen, low level
phospate, zinc, iron, magnesium and manganese high
oxygen(air), low pH (2 or3)
-Manganese regulates some key enzymes
-Potassium of Sodium ferrocyanide added for regulating
trace metals
-Limitations in nitrogen and phosphate favour citric acid
production
-Media is inoculated with spores of fungi/Mycellium
-Mycellium can be immobilised in alginate beads,
polyurethane
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
CITRIC ACID
Production of citric acid by A. niger
KEY ASPECTS
Fermentation: Down stream
-Fermentation liquor is separated from biomass by filtration
or centrifugation
Recovery: Solvent extraction, membrane filtration, resins
Recovery processes for food-grade citric acid (USA):
lime/sulfuric acid precipitation and liquid extraction
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
CITRIC ACID
Production of citric acid by A. niger
Lime/sulfuric acid precipitation
• Fermentation liquor is then treated with calcium hydroxide
to precipitate calcium citrate, which is filtered from the
slurry, washed free of impurities, and dissolved with
sulfuric acid.
• The insoluble calcium sulfate generated is separated from
the citric acid solution and the solution is deionized and
concentrated for crystallization to form either anhydrous
or monohydrated citric acid.
• Disadvantage of this process is the calcium sulfate by-
product, which constitutes a significant disposal problem.
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
CITRIC ACID
Production of citric acid by A. niger
Liquid extraction
• Counter-current liquid extraction, which employs a
mixture of tri-laurylamine, n-octanol and decane to
extract citric acid from the fermentation broth, followed
by extraction of the citric acid back into water at higher
temperature (Baniel et al., 1981).
• Purification steps include solvent washes, passage
through activated carbon, concentration by evaporation,
crystallization, and drying.

RESIN:
Citric acid is recovered from aqueous solution via anion-
exchange with a tertiary amine resin followed by thermal
desorption.
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
Malic acid

Applications
-Used in confectionery

It give tartness
Natural occurs in fruits such as grapes, apples etc

Malic acid
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
Production of Malic acid
-An acidulant in carbonated soft drinks, jams, candies, fruit
and vegetable juices
-Immobilised microbial cells (Brevibacterium flavum) using
conversion of fumarate (sodium fumarate) to L-malic acid
-Conversion is achieved by enzyme fumarase from the
microorganism
-Carrageena beads used as supports for immobilisation
-Monthly production of 30 tonnes of L-malic acid from 1 000
litres column fed at flow rate of 450 litre/hour of 1M sodium
fumarate
-Fumarase (enzyme) isolated from microbial cells can be
immobilised on cellulose triacetate and used for this
conversion
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
Gluconic acid
Applications
-Food additive
-Pharmaceutical(treating burns)
-Cleaning products (chelates with ions)

gluconic acid
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
Production of gluconic acid
Cells
-A. niger
-Gluconic acid produced from glucose by enzymes from the
microorganism

Enzymes
-Produced extracellularly by glucose oxidase from glucose
-An enzymatic process which is efficient and requires no
purification
FOOD FERMENTATION TECHNOLOGY

PRODUCTION OF ORGANIC ACIDS

Itaconic acid
Applications
-Building block in polymer industry
Styrene-butadiene-acrylonitrile and acrylate latexes used in
paperand architectural coating industry
-Additive to improve adhesion properties
-Detergents, shampoos and pharmaceuticals

COOH
CH2 = C – CH2 - COOH
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
Itaconic acid
Production
-Aspergillus terreus
-Carbon sources (glucose and sucrose)
-Glycolysis (pyruvate) and tricarboxylic acid cycles
(oxolaacetate to itaconic acid)
-Glucose converted to pyruvate which produces Itaconic acid
-Adequate oxygen supply
-Media should have the following present: Fe, Mn, Mg, Cu,
Zn, P and N
-Submerged fermentation (pretreated molasses inoculated
with spores the fungi)
-Immobilization also used fungi entrapped in polyvinyl
alcohol beads
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
OTHER ACIDS

Lactic acid- Rhizopus sp. e.g Rhizopus oryzae, Lactic acid

bacteria

Conditions
Carbon sources: Sucrose from sugar cane/sugar beet juice;
Lactose from cheese whey, glucose, etc
Nitrogen sources: corn steep liquor, yeast extract, malt
extract

Factors: pH<6 for Rhizopus sp.

FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
OTHER ACIDS

Lactic acid
Downstream processing
-Neutralise the acid with CaCO3 or Ca(OH)2
-Temperature increased (80-1000C), pH lowered to 10-11
-Microorganism is affected, proteins coagulated, calcium
lactate solubilizes
-Removal of biomass by filtering liquid
-Addition of sulphuric acid to obtain lactic acid
-Calcium sulphate removed
-Concentrating of lactic acid
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ORGANIC ACIDS
OTHER ACIDS

Succinic acid- Aspergillus sp.

Research on that
FOOD FERMENTATION TECHNOLOGY
ENZYMES
FOOD PROCESSING AIDS
HOSTS OF EXPRESSION

Sources: Fungi (yeasts and moulds),

-Yeasts preferred to bacteria as they secrete
proteins in active form, they also do not
produce toxins
-Filamentous fungi they secrete a wide
variety of metabolites
-Bacterial are also used they are easy to
transform (for genetic purposes)
FOOD FERMENTATION TECHNOLOGY
ENZYMES

Groups
Proteases
Lipases
Amylases
Pectinases
Cellulases
Arabinases
Lactases
Xylanases
Lignases
Lipoxygenases
FOOD FERMENTATION TECHNOLOGY
ENZYMES
Classification Reactions E.C.
Number
Oxidoreductase Catalyse 1
s oxidation/reduction reaction
Transferases Transfer a functional group 2
Hydrolases Catalyse hydrolysis of 3
bonds
Lyases Cleave various bonds by 4
means other than
hydrolysis and oxidation
Isomerases Catalyze isomerization 5
changes within a single
molecule
Ligases Join two molecules with 6
covalent bonds
FOOD FERMENTATION TECHNOLOGY

ENZYMES
Commodity Possible enzymes
Dairy products Chymosin, Lactases, Lipases,
Proteases
Fish hydrolysates Proteases
Meat Proteases
tenderization
Gelatin production Proteases
Vegetable/Fruit Pectinases, amylases,
processing Polyglacturonases
Starch processing α and β amylase,α-D-glucosidase,
cyclomaltodetrin-D-glycotransferase,
glucoamylase, glucoisomerase,
pullulanase

Food processing enzymes from micro-oganisms targeted for genetic

engineering
Joshi and Pandey, 2004
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ENZYMES
PROTEASES
-Interest of the type of enzymes produced and
characteristics is dependent on the target area(market)
-Enzymes produced on large scale by microbial
fermentation include: proteases (rennins, bovine enzymes)
-Proteases used in detergent industry: high activity,
stability in alkaline conditions, unaffecetd by oxidising and
chelating agents, available in bulk at low prices
-Characteristics required of these proteases e.g. peptide
bond specificity

Trehan K., 1990

FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ENZYMES
Proteases
-Thermophilic –thermolysin ( a zinc metalloprotease from B.
thermoproteolyticus)
-Thermophilic proteases are isolated from thermophilic
microorganisms
-Optimum growth of thermophiles 55-650C some extreme
thermophiles above 650C
Genetic manipulation
-B. stearothermophilus neutral protease was expressed and
cloned into a plasmid
-Recombinant plasmid was expressed in B.
stearothermophilus 15-fold production of protease was
observed
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ENZYMES
Proteases
-Proteolytic enzymes hydrolize peptide bonds in proteins
and peptides.
-Essential for digestion of proteins to amino acids.
-In G.I. tract the proteolytic enzyme pepsin (stomach ) and
small intestine enzymes trypsin, chymotrypsin,
amnopeptidases convert protein to amino acids.They can
exit as zymogens (inactive requiring cleaving for activation
e.g. propepsin)
-Proteolytic enzymes can be specific e.g. Chymotrypsin
hydrolyses peptide bonds hen tyrosine, phenylalanine and
tryptophan residues provide carbonyl groups of the peptide
bond.
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ENZYMES
Proteases
FOOD FERMENTATION TECHNOLOGY
ENZYME IN FOOD PROCESSING
-Fruit juice types (clear, nectar and turbid juices)
-Cell wall polysaccharide degrading enzymes
Pectinases essential in juice clarification, liquefaction and
other e.g. pectin methyl esterases used in jam
manufacturing
Cellulases liquefaction increase yield
-Glucoamylase and amylase crucial in production of high
fructose corn syrup
-Enzyme crucial in fermentation degrading starch e.g. Koji
fermentation , removal of A.N.F e.g. phytate
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ENZYMES
Commercial applications of proteases
-Detergent preparations
-Meat and fish hydrolysates
-Gelatin hydrolysis
-Tenderization
-Clarifying wine and beer
-Digestive aids, treatment of burns, ulcers etc

Trehan K., 1990

FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ENZYMES
High Temperature proteases

Protease Type Source/Organism

Thermolysin (a Bacillus
metaloprotease form thermoproteolyticus
Zn-ligand)
Thermitase Thermoactinomyces
vulgaris
S Protease Streptomyces rectus

Aqualysin II Thermus aquaticus YTI

Trehan K., 1990

FOOD FERMENTATION TECHNOLOGY

PRODUCTION OF ENZYMES
FUNGAL SPECIES:
Enzyme production from Aspergillus species:
-Highly studied species with A. niger having the largest fungal
source of enzymes.
-SSF usual preferred when enzymes are isolated from fungi. The
metabolic differences between SSF and SmF have a direct impact on
the productivity of the fungus.

Subramaniyam, R. and Vimala, R;2012

FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ENZYMES
FUNGAL SPECIES:
Enzyme production from Aspergillus niger:
- Generally regarded as safe (GRAS) status
-High level production of secondary metabolites
-Ability to grow on inexpensive substrates
-Ability to produce and secrete recombinant proteins

Disadvantages
-The fungal cell wall of the fungi hampers DNA transformation thus DNA
transformation in Aspergillus is extremely inefficient compared to yeast or E.
coli
-Plasmids not retained for a long time
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ENZYMES
BACTERIAL SPECIES:

-Use of SMF and SSF in producing these enzymes

Subramaniyam, R. and Vimala, R;2012

FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ENZYMES
OTHER MICROORGANISMS USED TO PRODUCE
COMMERCIAL ENZYMES

Saccharomyces sp.
Rhizopus sp.
Mucor sp. Mucor Miehei
Candida sp.
FOOD FERMENTATION TECHNOLOGY
PRODUCTION OF ENZYMES
EXAMPLES OF ENZYMES PRODUCED BY GENETIC
ENGINEERING:

Enzyme Application/Indus Host Donor

Activity try Organisms Organism
Amylase(fungal Baking, brewing, A. oryzae, A. Aspergillus spp.
) starch niger
Amylase Starch Bacillus Bacillus spp.
(bacterial) amylolique
faciens
Cellulase Baking,brewing, Trichoderma Trochoderma,
detergents, textiles reesei Aspergillus
Pectate lyase Fruits and Bacillus Bacillus spp.
vegetables, textiles lichenformis
Protease Detergents Bacillus subtilis Bacillus spp.
(alkaline)
Protease(acidic Dairy (milk A. niger, A. Calf Stomach
) clotting) oryzae Mucor
Xylanase Pulp and paper A. niger, Bacillus Actinomedia, T.
(hemicellulase) subtilis reesei
J.E. Smith, 2009
FOOD FERMENTATION TECHNOLOGY

PRODUCTION OF ENZYMES
STRATEGY USED:
Cloning strategy for enzymes
-Growth of microorganism with useful enzymes
-Purification of mRNA- Production of cDNA (identification of
clones with oligoprobes)
-OR purifying enzyme, synthesis of oligonucleotides then
production of cDNA clones
-Transforming host organism e.g A. niger
-Industrial production of enzyme
THERAPEUTIC ENZYMES
Enzyme EC number Reaction Use

Asparaginase 3.5.1.1 L-Asparagine H2O  L-aspartate + NH3 Leukaemia

Collagenase 3.4.24.3 Collagen hydrolysis Skin ulcers

Glutaminase 3.5.1.2 L-Glutamine H2O  L-glutamate + NH3 Leukaemia

Hyaluronidasea 3.2.1.35 Hyaluronate hydrolysis Heart attack

Lysozyme 3.2.1.17 Bacterial cell wall hydrolysis Antibiotic

Rhodanase b 2.8.1.1 S2O32- + CN-  SO32- + SCN- Cyanide poisoning

Ribonuclease 3.1.26.4 RNA hydrolysis Antiviral 

b-Lactamase 3.5.2.6 Penicillin  penicilloate Penicillin allergy

Streptokinasec 3.4.22.10 Plasminogen  plasmin Blood clots

Trypsin 3.4.21.4 Protein hydrolysis Inflammation

Uricase d 1.7.3.3 Urate + O2   allantoin Gout

Urokinase e 3.4.21.31 Plasminogen  plasmin Blood clots

FOOD FERMENTATION TECHNOLOGY

FOOD ADDITIVES

Food additives are substances added to food to preserve

flavor or enhance its taste and appearance

There are some interesting food additives produced via

biotechnological means (in this case we shall consider
microbial and/or enzymatic)

These compounds include:

Aspartame
Xanthan gum
Monellin
Vanillin
Thaumatin
There are many more others not mentioned such as
glutamate.
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
Thaumatin
-Sweet tasting protein from the fruit of Thaumatococcus
daniellii (West Africa) locally known in Ghana as katemfe.
-22kDa with 207 amino acids
-Aqueous solution of thaumatin have a sweetness that is
5,500 times that of sucrose
-Solubility in ethanol allows the protein to be used in flavour
essences or oils
-It is found in five different forms thaumatin I,II, III and b
and c
-These forms are isolated by ion-exchange chromatography
-Isoelectric point of all forms is pH 12, soluble in water,
stable at 1000C for a few hours
-Onset of sweetness is slow, licorice after taste limiting its
use in some products
-Claims of protein engineering to alter this issue of after
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
Thaumatin

-Low calorie substitute for sugar- production of low calorie

products (limitation is after taste)
-Trade name: Talin , sold by a company Tate and Lyle, U.K.
-Companies such as Ingene, USA using thaumatin genes
expressed in yeasts
Unilver,U.K thaumatin genes expressed in E.coli
FOOD FERMENTATION TECHNOLOGY
PRODUCTION FOOD ADDITIVES
Thaumatin
Genetic manipulation
-mRNA was obtained from species of T. daniellii
-mRNA was converted to cDNA and then cloned into S.
cerevisae, Kluyveromyces lactis
-Thaumatin II was fused to a promoter e.g lac UV5 using E.
coli as a host
-E.coli-showed low yields of the protein
-In the yeast successful expression was associated with
plant signal sequence
-An invertase signal sequence was used which showed
0.4mg/litre of production
-A stable multicopy system was also considered

K. Trehan,2008 ; Joshi and Pandey, 2004

FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
Xanthan gum
A polysaccharide
Consists of glucose units, mannose units , glucuronic acid
unit in repeated unit of pentasaccharide

Garcia-Ochoa et al.,2000
Garcia-Ochoa et al.,2000
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
Xanthan gum
Applications are numerous
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
Xanthan gum
Microorganism:Xanthomonas campestris
-Has different strains , the L- strain (large) is used as it
produces high yield of xanthan
-The microorganism is grown on solid surfaces or liquid media
for use in large bioreactors
-The growth media use is the YM media
-The growth of microorganisms is also influenced by : type of
bioreactor used, the mode of operation (batch or continuous),
the medium composition, and the culture conditions
(temperature, pH, dissolved oxygen concentration).
-Culturing conditions: 25-300C
-Requirements: Macronutrients : C and N
Carbon source: glucose and sucrose Nitrogen source:
glutamate
Micronutrients : potassium, iron, and calcium salts
Carbon and phosphorus limiting for improved xanthan
production.
FOOD FERMENTATION TECHNOLOGY

FOOD ADDITIVES
Xanthan gum

Garcia-Ochoa et al.,2000
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
Xanthan gum
-Fermentation broth consists of xanthan, bacterial cells, and
other chemicals.
-To recover the xanthan, the cells are usually removed first,
either by filtration or centrifugation
-Purification :precipitation using water-miscible non-solvents
(isopropanol, ethanol, acetone) addition of certain salts, and
pH adjustments
-However the Food and Drug Association (FDA) for food
grade xanthan gum use of isopropanol for precipitation is
advised
-Xanthan is then mechanically dewatered and dried.
-The product is then milled and packed into containers with
a low
permeability to water.
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
Xanthan gum
-Pyruvate and acetate play a role in influencing the
interaction between molecules of xanthan, and between
xanthan and other polymers. These components increase as
xanthan increases.(The components also affected by
temperature optimisations during fermentation)
-Xanthan is affected by pH changes in terms of losind
groups like acetyl groups at pH 9 and below pH 3
-Shows pseudoplastic rheology, emulsion stabilization and
temperature stability,

OTHER components similar to xanthan gum are

locust bean gum and guar gum
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
Monellin
-Natural sweet plant protein from serendipity berry (West
Africa)
-Dioscoreophyllum cumminsii
-3000 times sweeter than sugar
-Consists of two subunits chain A (45 amino acids-β strands)
and chain B (50 amino acids forming two β strands
separated by an alpha helix)
-Bioengineering in microorganisms (E. coli, yeasts S.
cerevisae and Candida utilis) and transgenic plants has
been considered
 Genes encoding the two chains of monellin have been
cloned into a modified vector under promoters such as T7
promoters.
 The expression vector containing the genes of the two
chains has been expressed in E. coli
-Monellin loses its taste above 500C and in acidic conditions
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
Aspartame
-Low calorie sweetener
-180-200 times sweeter than sucrose
-It has no after taste like some sweeteners
Used in diet drinks and food
-Metabolised by body to methanol and the two component
amino acids(its precursors)
-Individuals with phenylketonuria cannot metabolise
phenylalanine so are at risk
-Produced using enzymes (thermolysin a Metalloprotease
from Bacillus Thermolyticus)
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
Aspartame
-Produced from precursors phenylalanine and aspartic acid
Phenylalanine is esterified to produce methyl phenylalanine
methyl ester and the other is carbobenzoxylated to
benzyloxycarbonyl-L-aspartates
-Amino acids phenylalanine and aspartic acid can be
produced via fermentation using microorganisms such as
E.coli they are used as a precursors in production of
aspartame
-The precursors are enzymatically coupled at neutral pH
-Benzyloxy carbonyl group is removed living aspartame
-Alpha aspartame (natural) and beta aspartame (bitter) are
produced
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
Aspartame
-For microbial production recombinant DNA technology is
used. In recombinant DNA technology the gene
(dodecanucleotide) was chemically synthesized cloned into
a plasmid and expressed
-The gene was synthesized as repeating unit of the
aspartame dipeptide
-A controllable tryptophan promoter is used
Aspartame associated with some health concerns such as
allergic reactions e.g. hives, swelling

The company Showa, Japan is using microbes to produce aspartame

(technology not mentioned)
Toyo-Soda, Japan uses microbes e.g. Pseudomonas sp. and enzymes
in producing aspartame
Bioeurope, France uses enzymes to produce aspartame
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
Vanillin
-Flavouring and colouring agent
-The bean or pod of the tropical orchid Vanilla planifolia, and
also in Vanilla tahitiensis and Vanilla pompona
-Chemical synthesis and biotechnological means
-Controlled oxidation of lignin from wood waste
-Biotechnological production by biotransformations of
caffeic acid, veratraldehyde and mainly ferulic acid
-Recombinant DNA technology also considered
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
Vanillin
-Caffeic acid :using enzymes to mimic biosynthetic
pathways from the original plant
-Ferulic acid: Use of microorganisms that can utilise ferulic
acid and convert it to vanillin e.g. Pseudomonas fluorescens
-Recombinant technology: Microorganisms which have been
used in the synthesis of vanillin
 E.coli: Genes from Pseudomonas fluorescens cloned into
E.coli to enable it to utilise ferulic acid rich substrates.
Mutation made to vanillin dehydrogenase (VDH) that
prevents vanillin-to-vanillic acid oxidation so that the by
product would not be produced.
 A. niger and Pycnoporus cinnabarinus- investigated in
bioconversion of agroproducts to vanillin such as in
utilising maize, sugar beet pulp
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
FOOD ADDITIVES OF CONCERN:
• Monosodium glutamate (MSG)-Flavour enhancer
found
in soups, salad dressings, chips, and restaurant foods .
-Gives immune response by stimulating nervous system.
Can cause nerve damage. Also cause high blood pressure
and irregular heart rate.
Possibly associated with Chinese restaurant syndrome-
symptoms such as headaches, chest tightness, sweating ,
nausea.
L-glutamate (an amino acid) can be produced by chemical
synthesis or from bacterial (Corynebacterium) fermentation.

Tartrazine-Synthetic yellow azo dye found in sweets,

drinks and biscuits etc . Allergic reactions e.g. rash ,
asthma, possible carcinogen. It is chemically synthesized.
FOOD FERMENTATION TECHNOLOGY
FOOD ADDITIVES
LOOK AT THE BENEFITS AND PRODUCTION OF FOOD
ADDITIVES:
Xylitol
Xylose

FOOD ADDITIVES OF CONCERN:

There are many other food additives which have raised

concern including dyes (sunset yellow-headaches, gastric
effect etc), butylated hydroxytoluene (BHT), tartrazine,
carmoisine (banned in the US, causes hyperactivity)
• ,erthrocin