Download Enzymes in Food Technology

Enzymes in Food Technology
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Asparaginase-an enzyme for acrylamide reduction in food products
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In 2002, it was discovered that relatively high concentrations of acrylamide are
found in common carbonhydrate rich foods prepared by baking or frying.
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Acrylamide is classified as probably cancerogenic to humans and ist occurence in
food products has therefore caused intensive debate concerning the potential
health risk through dietary exposure.
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Acrylamid content of several typical food categories
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Schematic overview of acrylamide formation from a reducing sugar and the
amino acid asparagine
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Asparaginase catalyze the hydrolysis of the amide group of the side chain of
asparagine to produce aspartic acid and ammonium.
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Asparaginases participate in basic amino acid catabolism by shuttling asparagine to
aspartic acid which, after subsequent conversion to oxalacetate, can enter the
citric acid cycle.
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Implementing an asparaginase treatment broadly into a range of food products is
not a simple undertaking, since for each product the food matrix or components
may influence enzyme action and reactivity.
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Schematic overview of acrylamid formation from a reducing sugar and the
amino acid asparagine
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Asparaginase
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Asparaginase in cereal food Products
• Asparaginase can be successfully applied for acrylamide reduction in a
range of cereal-basedrecipes without changing the taste and appearance
of the final product.
• Asparaginase has been shown to work in both hard and short doughs, and
at temperatures between 10°C and 40°C.
• Acrylamide reduction of 50-90% was seen for the different recipes and
process parameters.
• For cereal food applications, enzyme performance is dependant upon
processing conditions, for example temperature, pH and resting time, and
most importantly water availability.
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Enzymes in dairy product manufacture
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Milk contains proteins, specifically caseins, that maintain its liquid form.
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Proteases are enzymes that are added to milk during cheese production, to
hydrolyze caseins, specifically kappa casein, which stabilizes micelle formation
preventing coagulation.
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Rennet and rennin are general terms for any enzyme used to coagulate milk.
Technically rennet is also the term for the lining of a calf's fourth stomach.
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Casein
• Casein (from Latin caseus, "cheese") is the name for a family of related
phosphoprotein proteins (αS1, αS2, β, κ).
• These proteins are commonly found in mammalian milk, making up 80%
of the proteins in cow milk and between 60% and 65% of the proteins in
human milk.
• Casein has a wide variety of uses, from being a major component of
cheese, to use as a food additive, to a binder for safety matches.
• As a food source, casein supplies essential amino acids; carbohydrates;
and two inorganic elements, calcium and phosphorus.
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Κ-Casein
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Enzymes in dairy product manufacture
• The most common enzyme isolated from rennet is chymosin.
• Chymosin can also be obtained from several other animal, microbial
or vegetable sources, but indigenous microbial chymosin (from fungi
or bacteria) is ineffective for making cheddar and other hard cheeses.
• Limited supplies of calf rennet have prompted genetic engineering of
microbial chymosin by cloning calf prochymosin genes into bacteria.
• Bioengineered chymosin may be involved in production of up to 70%
of cheese products.
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Enzymes used in dairy technology
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Milk proteins
• Milk consists of water, fat, protein, phosphate, lactose, citric acid and
inorganics such as calcium phosphate. The protein component of milk can
be divided into two groups, the casein fraction and the whey proteins.
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Casein
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alpha(s1)-casein: (molecular weight 23,000; 199 residues, 17 proline residues) Two
hydrophobic regions, containing all the proline residues, separated by a polar
region, which contains all but one of eight phosphate groups. It can be
precipitated at very low levels of calcium.
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alpha(s2)-casein: (molecular weight 25,000; 207 residues, 10 prolines)
Concentrated negative charges near N-terminus and positive charges near Cterminus. It can also be precipitated at very low levels of calcium.
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Casein
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ß -casein: (molecular weight 24,000; 209 residues, 35 prolines) Highly charged Nterminal region and a hydrophobic C-terminal region. Very amphiphilic protein acts
like a detergent molecule. Self association is temperature dependant; will form a
large polymer at 20° C but not at 4° C. Less sensitive to calcium precipitation.
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kappa-casein: (molecular weight 19,000; 169 residues, 20 prolines) Very resistant
to calcium precipitation, stabilizing other caseins. Rennet cleavage at the Phe105Met106 bond eliminates the stabilizing ability, leaving a hydrophobic portion, parakappa-casein, and a hydrophilic portion called kappa-casein glycomacropeptide
(GMP), or more accurately, caseinomacropeptide (CMP).
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Caseins
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Found in milk in the form of a Micelle (a dense protein granule).
The phosphate groups covalently bound to the casein molecules are involved in
binding Calcium (Ca) via ionic bonds.
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After caseins are phosphorylated, Ca++ binds to the phosphate to initiate
polymerization of the micelle particles. This structure is key to micelle formation.
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The casein micelle functions as a source of nutrients for the neonate: supplying
amino acids, calcium and phosphate.
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Casein
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Micelles are ~140 nanometers in diameter. They are composed of alpha- (as-),
beta-, and kappa-caseins. α-Caseins are in multiphosphorylated forms (αs2, αs3,
αs4, αs5, and αs6).
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ß-Casein is a major casein in cow milk, but is the minor casein in human milk. κCasein (a glycoprotein) is distributed throughout the casein micelle and acts to
stabilize the micelle.
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Gamma-caseins (part of the proteose peptone fraction of milk) are C-terminal
fragments of ß-casein, which are released by plasmin digestion, mostly while the
milk is in the gland.
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Rennet
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Rennet is usually a natural complex of enzymes produced in any mammalian
stomach to digest the mother's milk, and is often used in the production of
cheese.
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Rennet contains many enzymes, including a proteolytic enzyme (protease) that
coagulates the milk, causing it to separate into solids (curds) and liquid (whey).
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The active enzyme in rennet is called chymosin or rennin but there are also other
important enzymes in it, e.g., pepsin and lipase.
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Rennets
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Because of the above imperfections of microbial rennets, some producers sought
further replacements of natural rennet. With the development of genetic
engineering, it became possible to use cow genes to modify some bacteria, fungi
or yeasts to make them produce chymosin.
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Chymosin produced by genetically modified organisms was the first artificially
produced enzyme to be registered and allowed by the FDA. In 1999, about 60% of
U.S. hard cheese was made with genetically engineered chymosin and it has up to
80% of the global market share for rennet.
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By 2008, approximately 80 - 90% of commercially made cheeses in the United
States were made utilizing GMO-based rennet.
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Rennets
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By 2008, approximately 80 - 90% of commercially made cheeses in the United
States were made utilizing GMO-based rennet. One example of a commercially
available genetically engineered rennet is Chymax, created by Pfizer. Today the
most widely used genetically engineered rennet is produced by the fungus
Aspergillus niger.
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Chymosin reaction
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The chhymosin content of the rennin causes a specific and rapid cleavage of the
kappa-casein component of the casein micelles.
This protein stabilises the micelles and after cleavage, the casein proteins
precipitate under the influence of the calcium ions.
Chymosin specifically recognises the sequence His 98 to Lys 111 and cleaves the
peptide bond between Phe 105 and Met 106 in the kappa-casein chain.
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Lactose intolerance
• Lactose intolerance is the inability to metabolize lactose, because of a lack
of the required enzyme lactase in the digestive system.
• It is estimated that 75% of adults worldwide show some decrease in
lactase activity during adulthood.
• The frequency of decreased lactase activity ranges from as little as 5% in
northern Europe, up to 71% for Sicily, to more than 90% in some African
and Asian countries.
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Lactose intolerance
• Disaccharides cannot be absorbed through the wall of the small
intestine into the bloodstream, so in the absence of lactase,lactose
present in ingested dairy products remains uncleaved and passes intact
into thecolon.
• The operons of enteric bacteria quickly switch over to lactose
metabolism, and the resulting in-vivo fermentation produces copious
amounts of gas (a mixture of hydrogen, carbon dioxid, and methane).
• This, in turn, may cause a range of abdominal symptoms, including
stomach cramps, nausea, bloating, acid reflux and flatulence. In
addition, as with other unabsorbed sugars (such as sorbitol, mannitol,
and xylitol), the presence of lactose and its fermentation products
raises the osmotic pressure of the colon contents.
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Lactose intolerance
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The normal mammalian condition is for the young of a species to experience
reduced lactase production at the end of the weaning period (a species-specific
length of time). In humans, in non-dairy consuming societies, lactase production
usually drops about 90% during the first four years of life, although the exact drop
over time varies widely.
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However, certain human populations have a mutation on chromosome 2 which
eliminates the shutdown in lactase production, making it possible for members of
these populations to continue consumption of fresh milk and other dairy products
throughout their lives without difficulty.
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This appears to be an evolutionarily recent adaptation to dairy consumption, and
has occurred independently in both northern Europe and east Africa in
populations with a historically pastoral lifestyle.
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Lactose intolerance by group
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Lactase
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Lactase is a glycoside hydrolase enzyme that cuts lactose into it's constituent
sugars, galactose and glucose.
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Without sufficient production of lactase enzyme in the small intestine, humans
become lactose intolerant, resulting in discomfort (cramps, gas and diarrhea) in
the digestive tract upon ingestion of milk products.
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Lactase is used commercially to prepare lactose-free products, particularly milk,
for such individuals. It is also used in preparation of ice cream, to make a creamier
and sweeter-tasting product. Lactase is usually prepared from Kluyveromyces sp.
of yeast and Aspergillus sp. of fungi.
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Lactase properties
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The properties of the enzyme depend on its source. Temperature and pH optima
differ from source to source and with the type of particular commercial
preparation. Immobilization of the enzymes, method of immobilization, and type
of carrier can also influence these optima values.
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In general, fungal lactase have pH optima in the acidic range 2.5–4.5, and yeast
and bacterial lactases in the neutral region 6–7 and 6.5–7.5, respectively. The
variation in pH optima of lactases makes them suitable for specific applications, for
example fungal lactases are used for acid whey hydrolysis, while yeast and
bacterial lactases are suitable for milk (pH 6.6) and sweet whey (pH 6.1) hydrolysis.
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Lactase properties
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Product inhibition, e.g. inhibition by galactose, is another property which also
depends on the source of lactase. The enzyme from A. niger is more strongly
inhibited by galactose than that from A. oryzae. This inhibition can be overcome by
hydrolysing lactose at low concentrations by using immobilized enzyme systems or
by recovering the enzyme using ultrafiltration after batch hydrolysis.
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Lactase from Bacillus species are superior with respect to thermostability, pH
operation range, product inhibition, and sensitivity against high-substrate
concentration.
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Thermostable enzymes, able to retain their activity at 60°C or above for prolonged
periods, have two distinct advantages viz. they give higher conversion rate or
shorter residence time for a given conversion rate, and the process is less prone to
microbial contamination due to higher operating temperature.
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Using lactase
• The enzymatic hydrolysis of lactose can be achieved either by free
enzymes, usually in batch fermentation process, or by immobilized
enzymes or even by immobilized whole cells producing intracellular
enzyme.
• Although numerous hydrolysis systems have been investigated, only few of
them have been scaled up with success and even fewer have been applied
at an industrial or semi-industrial level. Several acid hydrolysis systems
have been developed to industry-scale level.
• Large-scale systems which use free enzyme process have been developed
for processing of UHT-milk and processing of whey, using K. lactis lactase
(Maxilact, Lactozyme).
• Several commercial immobilized systems have been developed for
commercial exploitation.
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Using lactase
• Several commercial immobilized systems have been developed for
commercial exploitation.
• Snamprogretti process of industrial-scale milk processing technology in
Italy is one such working systems. They make use of fibre-entrapped
yeast lactase in a batch process, and the milk used is previously sterilized
by UHT.
• For pilot plants, there are three other processes designed and developed
to handle milk; (i) by Gist-Brocades, Rohm GmbH (Germany), and (ii) by
Sumitomo, Japan. These are continuous processes with short residence
times. Processing of whey UF-permeate is accomplished by the system
developed by Corning Glass, Connecticut, Lehigh, Valio and Amerace corp.
The process by Corning Glass is being applied at commercial scale in the
bakers yeast production using hydrolysed whey23.
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Catalase
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The enzyme Catalase has found limited use in one particular area of cheese
production.
Hydrogen peroxide is a potent oxidizer and toxic to cells. It is used instead of
pasteurization, when making certain cheeses such as Swiss, in order to preserve
natural milk enzymes that are beneficial to the end product and flavour
development of the cheese
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These enzymes would be destroyed by the high heat of pasteurization. However,
residues of hydrogen peroxide in the milk will inhibit the bacterial cultures that are
required for the actual cheese production, so all traces of it must be removed.
Catalase enzymes are typically obtained from bovine livers or microbial sources,
and are added to convert the hydrogen peroxide to water and molecular oxygen.
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Lipases
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Lipases are used to break down milk fats and give characteristic flavours to
cheeses. Stronger flavoured cheeses, for example, the italian cheese, Romano, are
prepared using lipases.
The flavour comes from the free fatty acids produced when milk fats are
hydrolyzed.
Animal lipases are obtained from kid, calf and lamb, while microbial lipase is
derived by fermentation with the fungal species Mucor meihei.
Although microbial lipases are available for cheese-making, they are less specific in
what fats they hydrolyze, while the animal enzymes are more partial to short and
medium-length fats.
Hydrolysis of the shorter fats is preferred because it results in the desirable taste
of many cheeses. Hydrolysis of the longer chain fatty acids can result in either
soapiness, or no flavour at
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Cheese ripening enzymes
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Cheese ripening or alternatively cheese maturation is a process in cheesemaking.
It is responsible for the distinct flavour of cheese, and through the modification of
"ripening agents", determines the features that define many different varieties of
cheeses, such as taste, texture, and body.
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The process is "characterized by a series of complex physical, chemical and
microbiological changes” that incorporate the agents of: “bacteria and enzymes of
the milk, lactic culture, rennet, lipases, added moulds or yeasts, and
environmental contaminants.”
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Enzymes in bread making
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Wheat flour constituents:
Flour contains mostly starch. However,othercomponents in the flour also clearly
affect ist properties.
The main components are starch (70-75%), protein (9-14%), lipids (1-3%), non
starch cabohydrates (1-2%), ash (around0,5%), lipids (1-2,5%) and moisture (1314%).
Starch:
Starch is the most abundant component and the most relevant reserve constituent
of cereals.
Starch consists primarilyof D-glucopyranose polymers linked together by α 1,4 and
α 1,6 glycosidic bonds.
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Schematic representations of amylopektin
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Different charakterisitics of amylose and amylopectin
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Gluten proteins
Gluten proteins play a key role in determining the unique baking quality of
wheat by conferring water absorption capacity, cohesivity, viscosity and
elasticity on dough.
Gluten proteins can be divided into two main fractions according to their
solubility in aqueous alcohols: the soluble gliadins and the insoluble
glutenins. Both fractions consist of numerous, partially closely related
protein components characterized by high glutamine and proline contents.
Gliadins are mainly monomeric proteins with molecular weights (MWs)
around 28,000–55,000 and can be classified according to their different
primary structures into the α,β,γ- and ω-type.
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Gluten proteins
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Disulphide bonds are either absent or present as intrachain crosslinks. The
glutenin fraction comprises aggregated proteins linked by interchain disulphide
bonds; they have a varying size ranging from about 500,000 to more than 10
million.
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After reduction of disulphide bonds, the resulting glutenin subunits show a
solubility in aqueous alcohols similar to gliadins. Based on primary structure,
glutenin subunits have been divided into the high-molecular-weight (HMW)
subunits (MW = 67,000–88,000) and low-molecular-weight (LMW) subunits (MW=
32,000–35,000). Each gluten protein type consists or two or three different
structural domains; one of them contains unique repetitive sequences rich in
glutamine and proline.
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Non-starch polysaccharides (NSP)
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NSP originate from the cell wall of the aleurone and the endosperm of the wheat
kernel. NSP represent different polysacchaides.
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One group of polysaccharides is built up by pentose sugars and some hexose
sugars.
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These are called pentosans and main components are pentose sugars arabinose
and xylose.
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Desipte their low content in flour (2-3% at normal extraction rates), pentosans are
very important in determining dough properties, gluten quality and final bread
quality.
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Plant xylan structure with bound ferulic acid
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Lipids
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Wheat flour lipids form a highly heterogenous group of molecules with different
chemical structures and compositions.
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The lipids can be divided into free lipids and bound lipids.
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Both fractions contain polar and non-polar components.
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Polar lipids can be divided into glycolipids and phospholipids.
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Both lipids are mainly bound to starch and to lesser extent to proteins.
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Enzymes in bread making
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Bakery products have undergone radical improvements in quality over the past 10
years in terms of flavour, texture and shelf life.
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The usage of enzymes is the biggest contributor to these improvements.
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Among the enzymes used in food applications, those used in bakery industry
constitute nearly one-third of the market.
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The baking industry predominantly makes use of five types of enzymes.
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Attack sites and breakdown products for various starch degrading enzymes
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Anti-staling enzymes
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About 85 million ton of wheat flour is used every year tobake bread.
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By adding specific agents, such as emulsifiers or enzymes, bread stays fresh longer.
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It is assumed that 10-15% of bread is thrown away because it not longer fulfils the
consumer demands (quality, crumb, softness, tast..)
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Starch retrogradation is seen as the main factor responsible for the observed
changes.
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Retrogradation
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Retrogradation is a reaction that takes place in gelatinized starch when the
amylose and amylopektin chains realign themselves, causing the liquid to gel.
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When native starch is heated and dissolves in water, the crystalline structure of
amylose and amylopectin molecules are lost and they hydrate to form a viscous
solution.
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If the viscous solution is cooled or left at lower temperature for long enough
period, the linear molecules, amylose, and linear parts of amylopectin molecules
retrograde and rearrange themselves again to a more crysatalline structure. The
linear chains place themselves parallel and form hydrogen bridges.
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In viscous solutions the viscosity increases to form a gel. At temperatures between
–8 and +8 °C the aging process is enhanced drastically.
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Retrogradation can expel water from the polymer network. This is a process
known as syneresis. A small amount of water can be seen on top of the gel.
Retrogradation is directly related to the staling or aging of bread.
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Effect of various starch degrading enzymes on crumb softness as a function of
time
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The effects of various amylases on staling
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Enzymes in Fruit Juice Production and Fruit Processing
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The first application of enzymes in the fruit juice industry was the use of
pectinases for apple juice clarification in the 1930s.
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The fast clarification of juice after breakdown of pectin by pectinases and the
decrease in juice viscosity resulted in a shorter process and greatly improved the
quality of industrial apple juice.
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Biochemistry of fruit cell walls:
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Three major independent domains are distinguished: the xyloglucan network, the
pectin matrix, and the structural proteins.
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The cellulose xyloglucan network is embedded in the pectin matrix.
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Pectin is the major structural polysaccharide component of fruit lamellas and cell
walls.
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Enzyme in fruit juice production
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Enzymes are an integral component of modern fruit juice manufacturing and
are highly suitable for optimising processes. Their main purposes are:
• increase extraction of juice from raw material
• increase processing efficiency (pressing, solid settling or removal)
• generate a final product that is clear and visually attractive
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Cell-Wall Degrading Enzymes
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Many microorgansms produce enzymes that degrade fruit cell walls.
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Commercial pectinases for the fruit juice industry come from selected strains of
Aspergillus sp.
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Enzymes are produced during fungal growth, purified and concentrated.
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Hemicellulases
Hemicellulases are enzymes that hydrolyze arabinogalactans, galactans,
xyloglucans, and xylans.
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Amylases In fruit juice industry, fungal acid amylase and amyloglucosidase are
used to process fruits which contain starch. Aspergillus niger produces acid
amylase and amyloglucosidase.
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Cell wall degrading Enzymes
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Fruit cell wall compositionin g/kg fresh matter
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Production of apple concentrate
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Pectin
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Pectin forms a familiy of complex polysaccharids that contain 1,4 linked α-D
galacturonic acid. To date, they have been clasified into three groups:
homogalacturonan, rhamnogalacturonans and substituted galacturonans.
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Apple Juice depectinization
• Pectin is the main cause of juice turbidity.
• One liter of juice with 13% dry matter can contain 2-5 g of pectin after
pressing., depending on the ripeness of the fruit.
• Pectinlyase or pectin methylesterase plus polygalacturase and arabanase
are the most important enzymes.
• Aspergills niger is the main microorganism used for large scale production
of pectinases for the fruit industry.
• Wild strains of this fungus secrete large amounts of different enzymes to
break down the substrate on which they grow into nutrients for their own
metabolism.
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Pectin degrading enzymes
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Production of blackcurrant concentrate
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Brewing
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The basic ingredients of beer are water; a starch source, such as malted barley,
able to be fermented (converted into alcohol); a brewers yeast to produce the
fermentation; and a flavouring such as hops.
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A mixture of starch sources may be used, with a secondary starch source, such as
maize (corn), rice or sugar, often being termed an adjunct, especially when used as
a lower-cost substitute for malted barley.
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Less widely used starch sources include millet, sorghum and cassava root in Africa,
potato in Brazil, and agave in Mexico, among others. The amount of each starch
source in a beer recipe is collectively called the grain bill.
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Types of brewing yeasts
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Two types of brewing yeasts, originally classified on flocculation behavior
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Top-fermenting
Ale yeast: Predominant brewing yeast prior to the mid-1800s. Displaced by lager
yeast, Strains are genetically more diverse - several origins, Warm fermentation
temperatures: 65 to 72°F.
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Weiss yeast: Bavarian origins - closely related, Produces beer that has spicy, clove,
vanilla, and nutmeg flavor notes. PAD1 gene phenylacrylic acid decarboxylase,
Decarboxylation of ferulic acid forms 4-vinyl-guaiacol, which gives the
characteristic clove flavor, Warm fermentation temperatures: 65 to 72°F
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Bottom-fermenting
Lager yeast: Bavarian origin,1400s in Munich - cool fermentations (selective
pressure), Taken to Pilsen and Copenhagen in 1840s, became very popular displaced ale yeast, Strains are closely related - common origins, Cool fermentation
temperatures: 42 to 52°F, Beers are more delicate, clean, drinkable, and less
aromatic.
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Schematic of the brewing process
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Schematic of the brewing process
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Milling: reducing the size of the dry malt
Mashing: adding water to the malt; controlled rise of temperature to allow
enzymes to degrade proteins and carbohydrates
Brewing: boiling the mash
Filtration: separate spent grain and most
Cooling of the most
Fermentation: convert sugars into alkohol and carbon dioxid
Filtration: separate yeast from bright beer
Maturation: settling of insolubles, reduction of vicinal diketones
Packaging: in casks, kegs, bottles, cans
Pasteurization: improving microbiological stability
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Brewing process
• All beers are brewed using a process based on a simple formula. Key to the
process is malted grain— mainly barley, though other cereals, such as
wheat or rice, may be added.
• Malt is made by allowing a grain to germinate, after which it is then dried
in a kiln and sometimes roasted.
• The germination process creates a number of enzymes, notably α-amylase
and β-amylase, which convert the starch in the grain into sugar.
• Depending on the amount of roasting, the malt will take on a dark colour
and strongly influence the colour and flavour of the beer.
• The malt is crushed to break apart the grain kernels, expose the cotyledon
which contains the majority of the carbohydrates and sugars, increase
their surface area, and separate the smaller pieces from the husks..
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Brewing process
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There are several steps in the brewing process, which include malting, milling,
mashing, lautering ,boiling, fermenting, conditioning, filtering, and packaging.
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Malting is the process where the barley grain is made ready for brewing. Malting is
broken down into three steps, which help to release the starches in the barley.
First, during steeping, the grain is added to a vat with water and allowed to soak
for approximately 40 hours.
During germination, the grain is spread out on the floor of the germination room
for around 5 days. The goal of germination is to allow the starches in the barley
grain to breakdown into shorter lengths.
When this step is complete, the grain is referred to as green malt. The final part of
malting is kilning.
Here, the green malt goes through a very high temperature drying in a kiln. The
temperature change is gradual so as not to disturb or damage the enzymes in the
grain.
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Brewing process
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The next step in the brewing process is milling. This is when the grains that are
going to be used in a batch of beer are cracked. Milling the grains makes it easier
for them to absorb the water that they are mixed with and which extracts sugars
from the malt. Milling can also influence the general characteristics of a beer.
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Mashing is the next step in the process. This process converts the starches
released during the malting stage, into sugars that can be fermented. The milled
grain is dropped into hot water in a large vessel known as a mash tun. In this
vessel, the grain and water are mixed together to create a cereal mash. The
leftover sugar rich water is then strained through the bottom of the mash in a
process known as lautering. Prior to lautering, the mash temperature may be
raised to about 75 °C (165-170 °F) (known as a mashout) to deactivate enzymes.
Additional water may be sprinkled on the grains to extract additional sugars (a
process known as sparging.
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Brewing process
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At this point the liquid is known as wort. The wort is moved into a large tank
known as a "copper" or kettle where it is boiled with hops and sometimes other
ingredients such as herbs or sugars.
This stage is where many chemical and technical reactions take place, and where
important decisions about the flavour, colour, and aroma of the beer are made.
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The boiling process serves to terminate enzymatic processes, precipitate proteins,
isomerize hop resins, and concentrate and sterilize the wort. Hops add flavour,
aroma and bitterness to the beer.
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After the whirlpool, the wort then begins the process of cooling. This is when the
wort is transferred rapidly from the whirlpool or brew kettle to a heat excanger to
be cooled. The heat exchanger consists of tubing inside a tub of cold water. It is
very important to quickly cool the wort to a level where yeast can be added safely.
Yeast is unable to grow in high temperatures.
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Brewing process
• After the wort goes through the heat exchanger, the cooled wort goes into
a fermentation tank. A type of yeast is selected and added, or "pitched",
to the fermentation tank. When the yeast is added to the wort, the
fermenting process begins, where the sugars turn into alcohol, carbon
dioxid and other components.
• The second to last stage in the brewing process is called racking. This is
when the brewer racks the beer into a new tank, called a conditioning
tank.
• Conditioning of the beer is the process in which the beer ages, the flavour
becomes smoother, and flavours that are unwanted dissipate.
• After one to three weeks, the fresh (or "green") beer is run off into
conditioning tanks. After conditioning for a week to several months, the
beer enters the finishing stage. Here, beers that require filtration are
filtered, and given their natural polish and colour. Filtration also helps to
stabilize the flavour of the beer. After the beer is filtered, it undergoes
carbonation, and is then moved to a holding tank until bottling.
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Warm-/cold fermenting
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Warm fermenting
Yeasts such as Saccharomyces cerevisiae are generally fermented at warm
temperatures between 15–20 °C (59–68 °F), occasionally as high as 24 °C (75 °F),
while the yeast used by Brasserie Dupont for saison ferments even higher at 29 °C
(84 °F) to 35 °C (95 °F).
Warm fermented beers are generally ready to drink within three weeks after the
beginning of fermentation, although some brewers will condition them for several
months.
Cool fermenting
Lager is beer that has been cool fermented, at around 10 °C (50 °F), compared to
typical warm fermentation temperatures of 18 °C (64 °F). It is then stored for 30
days or longer close to the freezing point, and during this storage sulfur
components developed during fermentation dissipate.
67
Warm-/cold fermenting
•
Though it is the cool fermenting that defines lager, the main technical difference
with lager yeast is its ability to processraffinose (a trisaccharide composed of the
sugars galactose,fructose, and glucose) which means that all sugars are fermented.
•
Top-fermenting yeast only cleaves and ferments the fructose portion of raffinose,
leaving melibiose, which it cannot further cleave into two monosaccharides due to
its lack of melibiase and the beer is sweeter with a lower conversion of sugar into
alcohol.
68
Yeast (Saccharomyces) DNA

Laboratory Yeast
– Linear nuclear DNA
– Located on 16 chromosomes
– Mitochondrial DNA (circular)

Industrial yeast strains
– Genetically complex
 Typically more than 1 copy of each chromosome
– Polyploid (n x 16 chromosomes)
– Aneuploid (n x ± 16 chromosomes)
– Ploidy procures a robust quality to yeast
– 16 chromosomes average (230kb to 2.2Mb)
69
Differences between Ale and Lager Yeast
Ale
– Top fermenting
– Ferment at 18-22°C
– Melibiose negative
Lager
– Bottom fermenting
– Ferment at 7-15°C
– Melibiose positive
Genetically very different!
70
Diversification of Yeasts
• Yeast strains selected over many years for desirable
fermentation characteristics
• Ale strains are diverse
– Genetically and phenotypically
• Lager strains more closely related
– Originate from 1 or 2 sources (Tuborg/Carlsberg)
– Display strain-strain differences
• The significance of diversity is product variation
71
The Importance of Yeast Genetics to Fermentation
• Genetic make-up of yeast can influence
– Fermentation rate
– Alcohol production
• Strain dependent flavors
– Metabolism
– Specific genes (e.g. POF+ yeast)
• Mutations
– Nuclear mutants
– Respiratory deficient (petite) mutants
• Variety of defects
R.D. Mutant on
WLN agar
72
Random Amplified Polymorphic DNA (RAPD-PCR)
Denature DNA
• Non specific PCR
• Smaller primers
• Attach at multiple sites
• Produce a PCR ‘fingerprint’
1
2
4
DNA Template
Attach primers
3
5
6
Elongate
PCR Reaction
Product A
Product B
73
RAPD for Detection of Cross-Contamination
1
2
3
4
5
6
7
8
1)
Ale 1
2)
Ale 2
3)
95% #1 / 5% #2
4)
90% #1 / 10% #2
5)
80% #1 / 20% #2
6)
70% #1 / 30% #2
7)
60% #1 / 40% #2
8)
50% #1 / 50% #2
RAPD-PCR of mixtures of
Saccharomyces strains
74
Yeast Genetic Stability
• Desirable characteristic for all yeast used in
fermentation
– Mutations arising during a fermentation typically have a
negative effect on product quality
– Can form part of selection criteria for new strains
• Important in brewing fermentations
– Selective pressures caused by serial repitching can lead to
genetic drift
•
•
•
•
•
Product inconsistencies over time
Changes to flocculation
Altered sugar uptake rates
Loss of ability to ferment maltotriose
Inappropriate flavor production (diacetyl)
75
Fingerprinting Methods
• Pulse-Field Gel Electrophoresis (PFGE)
– Karyotyping (Chromosome profiling)
• Restriction Fragment Length Polymorphism
(RFLP)
– Detection of sequences within the genome which are
known to change frequently
– Indication of wider genetic changes
76
Karyotyping by Pulse-Field Gel Electrophoresis (PFGE)
• Separation of whole chromosomes
• Number and size varies according to each strain
of yeast
– Strain differentiation
– Determine chromosomal stability
• Total genomic DNA is loaded into agarose
• Electric field is applied in alternative directions
to move the chromosomes through the agarose
gel
77
Genetic Stability Analysis by Karyotyping
DNA Ladder
DNA Ladder
10 colonies of a brewing yeast
strain showing genetic variability
in chromosome profiles
10 colonies of a brewing yeast
strain showing identical
chromosome profiles
Variable
Regions
78
Restriction Fragment Length Polymorphisms (RFLP)
• Extract and digest genomic DNA with restriction
enzymes
• Use a hybridization probe to target repeated
sequences within the DNA fragments
– Specific for retrotransposons (TY elements)
• Detect DNA using antibodies to the probe linked to
alkaline phosphatase
– Causes color change in the presence of TY sequences
79
Optimization of Serial Repitching
Generation of yeast

Some yeast strains are more
susceptible to genetic change
– Strange fermentation patterns
noticed in ‘older’ yeast
– Altered RFLP profile was observed
G0
G1
G2
G10
G20
RFLP Analysis
80
Fig. 9-11
Pyruvate
CO2
NAD+
CoA
NADH
+ H+
Acetyl CoA
CoA
CoA
Citric Acid
Cycle
Overview
(Krebs
Cycle)
Citric
acid
cycle
2 CO2
FADH2
3 NAD+
3 NADH
FAD
+ 3 H+
ADP + P
i
ATP
81
Acetyl CoA
CoA—SH
NADH
+H+
H2O
1
NAD+
8
Oxaloacetate
2
Malate
Citrate
Isocitrate
NAD+
H2O
Citric
acid
cycle
7
Fumarate
NADH
+ H+
3
CO2
CoA—SH
6
-Ketoglutarate
4
CoA—SH
5
FADH2
NAD+
FAD
Succinate
Pi
GTP GDP
ADP
ATP
Succinyl
CoA
NADH
+ H+
CO2
Purification of Citric Acid
A typical method used for purification of citric acid from a
fermentation broth involves two major purification techniques:
precipitation and filtration. The following schematic displays a
generic citric acid purification scheme. The scheme will be
discussed in detail in the next few slides.
React citric acid
Prec
with calcium
carbonate
Purified Citric
Acid
Filter
precipitate
React precipitate
with sulfuric
•ffff
acid
Filter
precipitate
83
Purification of Citric Acid
The citric acid broth from the production fermenter is highly
contaminated by leftover biomass, salts, sucrose, and water. First,
the citric acid must be reacted with calcium carbonate to
neutralize the broth and form the insoluble precipitate calcium
citrate. Calcium citrate contains about 74% citric acid. The
stoichiometric equation is as follows:
CaCO3 + Citric Acid → CO2+ Calcium Citrate
Calcium Carbonate, CaCO3
Contaminated Citric Acid
CSTR
Calcium Citrate as
a precipitate plus
contaminants
84
Purification of Citric Acid
The calcium citrate is then washed, heated, and filtered to remove
any number of the contaminants. Depending on the specific
design of the purification scheme, filters can be placed before the
first reaction with calcium carbonate, in series between the two
precipitation reactions or in any other combination that works.
Also, it is important to choose the best kind of filter for what is
being removed. For simplicity, the filters here will remove larger
contaminants first (sucrose and salts) and the smaller
contaminants later.
Calcium Citrate as
a precipitate plus
contaminants
Filter
Ex. Plate filter, Rotary
presses, rack-and-frame
presses
Calcium Citrate,
biomass, water
85
Purification of Citric Acid
To crack the calcium citrate precipitate, sulfuric acid is needed.
The temperature of this reaction should stay below 60ºC. The
reaction will produce free citric acid and a new precipitate,
calcium sulfate, which will need to be removed later. The
stoichiometric coefficients for this reaction are all one.
Sulfuric
Acid, SO4
Calcium Citrate,
biomass, water
CSTR
Calcium sulfate as a
precipitate, free citric
acid, biomass, water
Kirk, 15
86
Purification of Citric Acid
In this filter, the calcium sulfate is washed away from the citric
acid and the leftover biomass is removed. Again, the
contaminants that were present in the fermentation broth can be
removed by additional filtration means, such as microfiltration or
ultrafiltration.
Calcium sulfate as a
precipitate, free citric
acid,
Kirk
15 biomass, water
Filter
Ex. Plate filter, Rotary
presses, rack-and-frame
presses
Citric acid,
water
87
Yoghurt production
•
•
•
The starter culture for most yoghurt production is asymbiotic blend of
Streptococcus salivarius subsp. Thermophilus and Lactobacillus delbruckii suspbulgaricus.
Although they can grow independently, the rate ofacid production is much higher
when used together—
These microorganisms are ultimately responsible for the formation of typical
yogurt flavour and texture.
88