Transcript ACETIC ACID

Enzymatic and
Colorimetric Analyses
1
Analytical methods to determine
important parameters in the following
sectors:




Food & Beverage
Environmental
Clinical
Pharmaceutical
Now internationally recognised as official
methods because:
 They are simple to use
 They are fast (especially with automatic
machines)
 They have low analysis costs
 The kits are long-life (if liquid)
 Based on a Spectrophotometric reading of
absorbance correlated with the
concentration
 Colorimetric at end point or differential
 Enzymatic at end point after kinetics or self
blank
The principle parameters analysed are of
particular importance as regards:
 Product stability
 Product genuineness
 Product transformation
 Product toxicity
Steroglass kit list
• Acetic acid
• D-Gluconic acid
• D-Lactic and L-Lactic
acid
• D-Malic and L- Malic
acid
• Pyruvic acid
• Tartaric acid
• Acetaldehyde
• CO2
• Anthocyanin
• Alpha Amino Nitrogen
• Ammoniacal Nitrogen
• Calcium
• Catechins
•
•
•
•
•
•
•
•
•
•
•
•
•
Chlorides
Colour
Glycerol
Glucose-Fructose
Sucrose
Iron
Magnesium
Polyphenols
Potassium
Copper
Free SO2
Total SO2
L-Ascorbic
Kit List – the most popular and why....
ACETIC ACID:
This is linked to product perishability and
must be analysed before bottlingpackaging (alcoholic drinks).
Kit List – the most popular and why....
MALIC ACID:
It is one of the organic acids responsible for
secondary fermentation (e.g. malolactic
fermentation in wine).
Found naturally in some samples (e.g. apple
juice)
It must be analysed in the original product
before bottling.
Kit List – the most popular and why....
CITRIC ACID:
This is an antioxidant which increases
titrable acidity and, as a result, protects
against bacterial attacks.
It also establishes product acidity or fruit
ripeness.
Kit List – the most popular and why....
GLUCONIC ACID:
This indicator develops when moulds are
present in the product (and therefore a
high possibility of degradation). It
indicates a raw material or even the
finished product is “rotten”.
Kit List – the most popular and why....
READILY ASSIMILABLE NITROGEN (R.A.N.):
SUM OF AMMONIACAL NITROGEN AND
ALPHA AMINO NITROGEN
It is fundamental because it “feeds” the
yeasts and can ferment....
To avoid counterfeits (Amino/Ammoniacal
ratio)
Kit List – the most popular and why....
R.A.N.:
It replaces the FORMALIN n°!!!!
Method used until now using FORMALIN
which proved to be carcinogenic!
Kit List – the most popular and why....
It replaces the FORMALIN n°!!!!
The Formalin no indicates when Nitrogen
has combined with the aldehyde group of
formaldehyde. It does not specify whether
this is ammoniacal or alpha amino
nitrogen.
The yeasts prefer to eat the more readily
assimilable ammoniacal nitrogen.
Kit List – the most popular and why....
It replaces the FORMALIN n°!!!!
The Formalin no is analysed for Titration
using concentrated Formaldehyde raised
to pH 8.1 and titrated with Soda!
Kit List – the most popular and why....
SUGARS:
GLUCOSE-FRUCTOSE-SUCROSE
These are linked to the alcoholic strength of
a drink (if it ferments) or to the sweetness
and energy power of a product.
ENZYMATIC ACTIVITY
 The activity of an enzyme is measured as the
speed of the reaction it catalyses.
 The speed of reaction is measured in terms of
quantity of transformed substrate, or of the
product which develops within the time unit.
 The speed of the chemical reactions is subjected
to well-defined chemical-physical laws which
enable equations for different speeds to be
obtained according to the type of reaction. These
equations associate the speed of the reaction with
the concentration of the reactants.
How enzymes work
the speed of a reaction, measured
as a quantity of product obtained
in a set time, depends on the activating
energy
To raise the temperature:
increase the number of molecules
I can lower the activating with a sufficient quantity of energy
energy by adding a catalyst to overcome the energy barrier
Diagram of the variations in
energy of a reaction
S
P
The enzymes modify only the
speed of the reaction and not
the equilibria
lowers activation energy
Weak, non-covalent
interactions between S
and E release free energy
produces specific enzymes
E+S
FREE ACTIVATION
•
•
•
•
The enzymes act as a catalyst
to lower free activation
energy.
As a result the presence of
the
concentration of the species
during
the transition state will be
greater- The speed of
reaction is proportionate to
the concentration of the
species during the transition
state.
Therefore, the enzymes act
as a catalyst and increase the
speed of the chemical
E+P
ENERGY

Transition
state
Free Activation
Energy of the
forward reaction
(not cathalized)
energia libera
•
ES
The free activation energy Δ G
is the quantity of energy
required to bring all the
molecules of 1 mole of a
substance to a given
temperature at the transition
state
Free Ativation
Energy of the
reverse reaction
(not cathalized)
Free Activation
Energy or the
reverse reaction
(cathalized)
start
Free Energy
completed
changed during
reaction
Free Activation
Energy of the
forward reaction
(cathalized)
end
direzione della reazione
UNIT OF MEASUREMENT
 An enzyme can be measured in terms of concentration in the same
way as any other protein.
 From an analytical viewpoint, however, it is interesting to measure the




catalytic activity (or enzymatic activity).
Enzymatic activity is expressed in International Units (I.U.)
A unit of enzymatic activity is defined as the quantity of enzyme
capable of catalysing
the transformation of a micromole of substrate per minute under
standard
conditions.
•Gli enzimi sono suddivisi in sei classi principali a seconda della natura
generale delle reazioni catalizzate:
CLASSIFICATION OF ENZYMES






oxido-reductase
transferase
hydrolase
lyase
isomerase
ligase
Each class consists of sub-classes which, in turn consist of sub-subclasses.
An enzyme usually has:
- A common name
- A systematic name
- A classification number
ACTIVE SITE
a specific region of the enzymatic molecules where the substrate binds
and reacts to form the product. There are two models to interpret the
active-substrate site interaction.
Lock and key model (Fischer 1894)
(Over-simplified model)
The enzyme is a lock, into which only
the specific key can turn; in fact, only
a particular molecule of substrate has
the shape which will allow it to enter
the active site slit of the enzyme to enable
a reaction to take place.
Induced fit model (Koshland 1954)
The shape of the enzyme is suitable to
enable the reaction to take place only
after the latter has bound with the
substrate. This phenomenon brings
the specific functional groups to the
correct position required to catalyse
the reaction.
Enzymatic Relative Activity
TEMPERATURE EFFECT
20
40
60
T (°C)
• Enzymes have different thermal stability
• The descending part of the curve is due to thermal
denaturation
Effect of pH on the reactions
catalysed by the enzymes
The change in pH alters the degree of ionisation of
the various ionisable groups present in an enzymatic
molecule; if the substrate is also an ionic compound,
its ionisation will vary with the pH.
Both the ionic nature of the substrate
and that of the enzyme modify the
way in which they bind together
(Trend of the speed of the enzymatic
reaction according to the pH)
An optimum pH and a range of pH
values within which the enzyme
demonstrates maximum efficiency
How to obtain kinetic data
 Spectrophotometric methods
 Fluorimetric methods
 Potenziometric methods (pH)
 Manometric methods
 Radio-isotopic methods
 Continuous methods
 Discontinuous methods
 Joint methods
JOINT METHODS: A to 340 nm
WINE
Processing and
ANALYTICAL checks
Composition of the cluster
Rachis 2 – 6%
Skin
1–3%
Grape seeds 4 – 6 %
Pulp 75 – 90 %
Pedicel trace
Contribution to the wine
 Rachis:
 Skin:
 Pulp:
polyphenols (pigments), minerals
polyphenols
water 70-90%
sugars
organic acids
What to check for grape
ripeness
 Sugar content
 Titratable acidity
 Polyphenolic maturity
Vinification
Stripping
Pressing
Must = check analysis
fermentable sugars
R.A.N.
Titrable acidity
Gluconic Acid
Enzymatic treatment (?)
Wine
 Wine is produced from sugar solutions obtained by crushing
the bunch of grapes left to ferment with unicellular yeasts
from the Saccharomyces genus present in the grape skin or
from selected cultures. The characteristic organoleptic
qualities (colour, flavour, bouquet) are differentiated by the
conditions of fermentation. These become more pronounced
during the subsequent stages in the process. Under anaerobic
conditions, yeast transforms 100 grams of sugar into 51.1 of
spirit . This is the ideal yield. In reality, part of the sugar
available is used by the yeast to multiply. Furthermore, the
must yeasts produce not only alcohol and carbon dioxide
during fermentation, but also secondary products (glycerol,
acetic acid, succinic acid) which contribute towards giving the
finished product its characteristic bouquet.
Fermentation
 Spontaneous autochthonous yeasts
 Induced with selected yeasts
 Competition between Yeasts – Bacteria
Useful analyses during fermentation
SUGARS Glucose Fructose
Total Polyphenols
Anthocyanins Tannic acids
Alcoholic content
Acetaldehyde
Catechins
RAN
Volatile Acidity (Acetic
Acid)
Polyphenols /
Anthocyanins
COLOUR 420-520
Pyruvic Acid
TYPES OF FERMENTATION
Lactic Fermentation
Alcoholic Fermentation
Acetic Fermentation
FERMENTATION
 From a strictly chemical viewpoint, fermentation is an
anaerobic oxidation process by numerous organisms depending
on glucides to produce energy. As this type of metabolism is so
important in the preparation of numerous foods, the term
fermentation has been widely used to indicate any
transformation catalysed by a micro-organism. Sometimes the
term is still used in this sense. The word fermentation comes
from the Latin fervere (simmer), a term used to describe the
appearance of the must as the wine is prepared. Louis Pasteur
gave a better explanation saying that fermentation was due to as
yet not clearly identified bodies described as ferments, contained
in yeasts. Subsequent studies discovered the protein content of
the ferments and identified them as containing enzymes which
enable the fermentative processes. Later, the main paths of
energy metabolism in general and of fermentation in particular
were clarified.
ALCOHOLIC FERMENTATION
 Alcoholic fermentation is a form of energy
metabolism which occurs in some yeasts in the
absence of oxygen.
 This type of fermentation is due to the action of yeasts
belonging to the Saccharomyces genus, of which the
most well-known is S. cerevisiae, found on the grape
skin and in beer yeast, which transform glucose into
spirit or ethanol and carbon dioxide.
Diagram of fermentation
 The alcohol produced acts as an antiseptic
against the other micro-organisms present,
which die when the concentration of alcohol
increases.
 C6H12O6 — 2 CO2 + CH3 CH2OH ethilic
alcohol
 Alcoholic fermentation is used to produce
wine, beer, champagne, alcoholic drinks in
general and in bread making.
Production of alcohol
C12H22O11+ H2O = C6H12O6 + C6H12O6
C6H12O6 -> 2CH3CH2OH + CO2 + energia
Other types of fermentation:
Malolactic
Pyruvic
Acetic
Types of fermentation
 Lactic fermentation
 Lactic fermentation is due to the action of bacteria
of the Lactobacillus genus (bulgaricus) and the
Streptococcus genus (thermophilus), which use the
glucose obtained from the degradation of the
lactose which they transform into lactic acid.
C6H12O6 → 2 CH3 CHOH COOH lactic acid
Acetic fermentation
 Unlike the other types of fermentation, acetic
fermentation requires the presence of oxygen from the
atmosphere to be able to take place. Acetic bacteria of
the genus Acetobacter are responsible and are exploited
to produce vinegar as they are able to transform the
glucose, and also spirit into acetic acid and water.
C6H12O6 + 2 O2 → 2 CO2 + CH3 COOH +
2H2O acetic acid
Devatting - Filtration
 Addition of sulphur (gas or solid)
 Combination demand
 Acetaldehyde
 Total SO2
 Free SO2
 Combined SO2
Physical treatments
 Acidification
 Disacidification
 Demetallisation ( Cu, Fe, Ca )
 Stabilisation: Tartaric
Protein
Bottling
Alcoholic contentTotal
Polyphenols
Glucose Fructose
Alcoholic contentTotal
Polyphenols
Catechins
Titratable Acidity
Anthocyanins
Organic Acids
Colour
pH
Dissolved oxygen
Free and Total SO2
Microbiology
STEROGLASS KITS:
Advantages
 Ready to use (Liquid)
 Easy to use
 Stable and reproducible (automatic)
 Separate Standards (and multiparametric)
 Long expiry dates (minimum 2 years)
 "Unbeatable" prices
 Analytical Application Support
 Continual Research and Development
Official Methods
 Titration, Gravimetric analysis, HPLC and
Atomic absorption, etc. are all internationally
recognised analytical techniques. Each country
acknowledges the official methods according
to its own directives. Enzymatic methods are
often used as an alternative/ to complete the
official methods, especially because they are
practical, rapid and economical.
Official Methods – e.g. Wine
OIV and Official Gazette. The following enzymatic
kits are officially recognised:
 Glucose and Fructose
 Lactic Acid
 Citric Acid
 L-Malic and D-Malic Acid
Main Examples of Application
WINE
FRUIT JUICES
ACETIC ACID
ACETIC ACID
L-MALIC ACID
L-MALIC ACID
ACETALDEHYDE
ACETALDEHYDE
ASCORBIC ACID
ASCORBIC ACID
CITRIC ACID
CITRIC ACID
GLUCONIC ACID
GLUCONIC ACID
LACTIC ACID
LACTIC ACID
RAN
AMMONIACAL NITROGEN
GLUCOSE AND FRUCTOSE
GLUCOSE AND FRUCTOSE
SUCROSE
SUCROSE
Main Examples of Application
WINEGAR
ACETIC ACID
LACTIC ACID
SAUCES
ACETIC ACID
Main Examples of Application
BEER
CHEESES
ACETIC ACID
ACETIC ACID
ASCORBIC ACID
ACETALDEHYDE
GLUCONIC ACID
ASCORBIC ACID
LACTIC ACID
CITRIC ACID
RAN
GLUCOSE AND FRUCTOSE
SUCROSE
Main Examples of Application
YOGURT
MAYONNAISE
ACETIC ACID
ACETIC ACID
ACETALDEHYDE
CITRIC ACID
LACTIC ACID
Main Examples of Application
FRUIT AND VEGETABLE
PRODUCTS
MALIC ACID
HOMOGENISED BABY
FOODS
SUCROSE
BREAD AND BAKERY
PRODUCTS
ACETALDEHYDE
COFFEE
ACETALDEHYDE
Main Examples of Application
COCOA
ACETALDEHYDE
TOBACCO
SUCROSE
TEA
ACETALDEHYDE
POTATOES
ASCORBIC ACID
SUCROSE
Main Examples of Application
FLOUR
MEAT BY-PRODUCTS
ASCORBIC ACID
ASCORBIC ACID
RAN
GLUCONIC ACID
LACTIC ACID
RAN
SUCROSE
OIL, MARGARINE
CITRIC ACID
COSMETICS
CITRIC ACID
LACTIC ACID
Main Examples of Application
DETERGENTS
CITRIC ACID
LIQUEURS
GLUCOSE AND
FRUCTOSE
JAMS
SUCROSE
GLUCOSE AND
FRUCTOSE
HONEY
GLUCOSE AND
FRUCTOSE
Main competitors
 R-Biopharm (formerly Boheringer,
formerly Difchamb)
 Megazyme
THANKS FOR YOUR ATTENTION
57