ENZYMES

1
ENZYMES LYVEN PRODUCTSPRESENTATION
2
ENZYMES PROTEINS

• Biochemicals Molecules composed of different
  amino-acid and other radicals
• Produce from a microorganism or cell
• Thermolabile Proteins able to work in or out the
  production cell.
• Active site is the operational part of the enzyme

3
ENZYMESPROTEINS
Active site
4
Side Activity
Other proteins
Primary activity
Side activity
Chromatography of a xylanase extraction
5
Micro-organisms

• Two kinds of micro-organisms can produce enzyme
  Fungal or Bacterial way
• Different side activities

Fungal/Yeast Bacterial
pH Acid-Neutral Neutral-Alkaline
Temperature Low Temperature High Temperature
6
Enzymes Enzymatic Catalysis Agent

• CatalysisIncrease in the velocity of a chemical
  reaction or process produced by the presence of a
  substance that is not consumed in the net
  chemical reaction or process

SSubstrate PProduct EEnzyme
7
Enzymatic Action Scheme
E
G
G
G
G
E
Maltose
Glucose
G
Enzyme
E
8
Enzymes Specific Catalysts

• Each enzyme catalyses one and only one reaction
• Nowaday, near 3 000 enzymes were discovered but
  only 25 to 30 enzymes are industrialy produced

9
Enzymatic Action

• Oxidoreductases (EC 1) oxidationreduction
  reactions, which entail the transfer of electrons
  from a substrate to another substrat
• Transferases (EC 2) move a chemical group from
  one compound to another compound
• Hydrolases (EC 3) Hydrolysis of a chemical bond
• Lyases (EC 4) formation of a double bond
• Isomerases (EC 5) catalyzes substrate to an
  isomeric form
• Ligases (EC 6) catalyzes a reaction that joins
  two substrates

10
Lock and Key Model
One and only one molecule could match with one
enzyme
Enzymes changes shape slightly as substrate binds
Products
Substrate
Active Site
Enzyme/Substrate complex
Substrate entering active site of enzyme
Products leaving active site of enzyme
11
EFFECTOR
INHIBITOR
I
I
E
E
S
S
E Enzymes I Inhibitor S Substrate
e.g Cu, Hg, Fe inhibit amylase
12
EFFECTOR
S
S
ACTIVATOR
A
E
AE
E Enzymes A Activator S Substrate
e.g Calcium activates amylase
13
Enzymatic catalysis principle

• Any reaction catalysed by an enzyme can occur
  naturally but with a more important energy cost.

A, B Substrate E Enzyme AB Product
Source wikipedia
14
Factors acting on the enzymatic activity

• Substrate concentration
• Water Enzymes work only in aqueous phase
• pH There is always an optimum pH
• Temperature There is an optimum and over 70C,
  main enzymes are distorted
• Duration

15
pH influences
e.g Amylase activity
16
Temperature influences
e.g Amylase activity
17
Parameters Variation
-
-


TC
TC
Optimum
Optimum
-
-
Dose
Dose

-

-


p.H
p.H
Optimum
Time
Time
-
-


DOSECOST
18
Enzymes deactivation

• By thermic treatment (e.g Baking)
• By settling/filtration (e.g Fruit Juice)
• By increasing pH with Sodium carbonate or
  decreasing pH with acid (e.g Textile)

19
EnzymeProcessing Aids

• Normally enzymes are not present in final
  products, they were destroyed or degraded during
  the process
• (Specifications from FAO, WHO, JECFA, FCC and
  French law 19/10/2006)

20
Enzymes in Food-Industry

• FRUIT JUICE
• BAKING
• BREWING
• WINE
• FEED

21
ENZYMES ACTIONON FRUIT
22
Fruit Structure

• Vegetable raw material is composed of
  polysaccharides more or less complex. The main
  are
• Cellulose
• Hémi-cellulose
• Lignine
• Pectins

23
The Vegetal Cell Wall of a Fruit
Source Dean MADDEN, 2000
24
Chemical Constitution of Vegetal Cell Wall
OTHER CELL
CYTOPLASM
Vegetal Cell
Cellulose
Hémicellulose
PECTINS
Source Bayou Cortes
Lignine
25
PECTINS

• Fruit Juice cloudiness at the end of the pressing
  step is caused by pectins

26
Pectin

• A carbohydrate consisting of one primary chain of
  galacturonic acid and branched out with several
  sugar molecules including rhamnose, galactose,
  arabinose

Méthyl Galacturonic acid
Galacturonic acid
Galacturonic acid Methyl ac. Gal. Rhamnose Other
oses
A detail of pectin


Calcium ions
Source Jussieu University
27
Pectinases

• Three basical enzymatic activities are present in
  commercial preparation
• PG PolyGalacturonase
• PL PectineLyase
• PME Pectine Methyl Esterase

28
How work Pectinases
Source Dean MADDEN, 2000
29
WINE

• FRUIT JUICE

30
ENZYMES IN WINE
31
Grape Berry Structure
Red Grape
White Grape
Berry Stem (Pedicel)
Brush
Skin
Anthocyanes
Flavones
Seed
Tannins, Oil
Vascular Bundle
Flesh
Glucose, Fructose
32
Red Wine Making Process
PECLYVE V Extraction
Maceration Alcoholic Fermentation
DESTEMMER
Grapes
CRUSHER
Stem
PRESSING
 Vin de Goutte 
MALOLACTIC FERMENTATION
SKIN
Blending
 Vin de Presse 
PECLYVE V CLARIFICATION
33
Red Wine Making Process flash détente
PECLYVE V EXTRACTION

DESTEMMER
Grape Berries
CRUSHER
Stem
PRESSING
PECLYVE V CLARIFICATION
FERMENTATION
CLARIFICATION
SKIN
34
PECLYVE V EXTRACTION in Red Wine

• Allows to extract faster the color from the red
  grape berry
• Avoid to extract too much tannins
• Give less astringent wine

35
Action of Peclyve V extr
Color extraction with Peclyve V extraction
Color extraction without Peclyve V extraction

Tannins Extraction
Days
36
DECANTATION STEP WITH PECLYVE V CLARIFICATION
-
-


-


-


-
-
-
-

PECLYVE V Clarification
CLOUDY JUICE
CLEAR JUICE
MUST
ENZYMATIC ACTION
protein
pectin
37
White Wine Making Process
PECLYVE V EXTRACTION PECLYVE VP
MACERATION
DESTEMMER
Grape Berries
CRUSHER
PRESSING
Stem
LYVAROME A5
ALCOHOLIC FERMENTATION
CLARIFICATION
PECLYVE V Clarification
38
PECLYVE V EXTR in White Wine

• Allows to extract faster the flavour from the
  white grape berry
• Avoid to extract too much tannins
• Give less astringent wine

39
Flavour in Wine

• Flavour in the wine comes from terpenols which
  are derived from terpen.
• Sometimes, terpens must be transformed in
  terpenols by enzymes help.

Yeast
Enzymes
T
T
TL
G
G Glucose T Terpen TL Terpenol
40
LYVAROME A5

• At the last quarter of the fermentation, adding
  LYVAROME A5 will transform Terpen in Terpenol,
  giving more flavour to the wine

LYVAROME A5
GÉRANYL DIPHOSPHATE
GERANIOL
NEROL
NERYLE DIPHOSPHATE
CH2OH
OPP
OH
LINALYL DIPHOSPHATE
LINALOL
41
(No Transcript)
42
Action of Peclyve V extraction
Flavour extraction with Peclyve V extr
Flavour extraction without Peclyve V extr

Tannins Extraction
Days
43
PECLYVE VP

• Sometimes, Pectinases enzymes with
  Cinnamoyl-Esterase (CIE) could lead to release
  vinyl-phenols.
• Vinyl-phenol could give a bad profil aroma to the
  white wine
• Peclyve VP with a very low level of CIE activity
  avoid this problem

44
Optimum pH
45
Thermostability
46
Washing Efficiency
47
ENZYMES FOR FRUIT JUICE
48
Pectinases in Fruit Juice Process

• The commercial preparations of pectinase are
  adapted to the process and to the types of fruit
• Different preparations exist for pulp treatment
  and for Juice
• All pectinases from LYVEN are from Aspergillus
  niger and are GMO free

49
FRUIT JUICE PROCESS
PECLYVE PR
MASHING
PRESSING
MACERATION STEP
CLEAR JUICE
DECANTATION STEP
CONCENTRATION STEP
PECLYVE CP PECLYVE UF AMYLYVE TC SUPER
PACKING
50
PECLYVE PR

• PECLYVE PR is well balanced in PG, PL PE to
  increase the yield during pressing

Pulp Treatment Fresh Apple Frozen Apple
Without enzyme 80-82 70-74
With PECLYVE PR 90-92 85-90
51
AMYLYVE TC SUPER

• When the fruit is harvested early in the season,
  some residual starch could remain in the fruit
  causing cloudiness at the pasteurisation and
  filtration step. AMYLYVE TC Super allows to
  degrade this residual starch.

52
DECANTATION STEP WITH PECLYVE CP
-
-


-


-


-
-
-
-

PECLYVE CP
CLOUDY JUICE
CLEAR JUICE
MUD
ENZYMATIC ACTION
protein
pectin
53
Pectin Test
To check presence of pectin, a test with alcohol
could be make.
54
OTHER FRUITS
FRUIT PECTIN() REMARK
APPLE 0.5-1.6
PEAR 0.5-0.8 ARABAN
LEMON 3.0-4.0
GRAPEFRUIT/ ORANGE 1.6-4.5 CLOUDY JUICE
STRAWBERRY 0.2-0.5
CRANBERRY 0.7-1.0 ACID
55
PEAR

• Due to araban, a haze could appear. PEARLYVE is
  specially formulated to avoid that.

56
CITRUS

• To reduce viscosity, improving the concentration,
  
• PECLYVE CITRUS JUICE is the best product.
• To clarify the juice (e.g lemon)
• PECLYVE CITRUS CLAR is highly recommended

57
CITRUS

• To increase yield and extract the residual juice
  of the pulp, PECLYVE CITRUS WASH is the ideal
  product.
• For extraction of essential oil
• PECLYVE CITRUS OIL
• To remove peel from citrus without damage for the
  general aspect of the fruit
• PECLYVE CITRUS PEEL is perfectly adapted.

58
FRUIT FIRMING

• Thanks to its high content in Pectin Methyl
  Esterase, PECLYVE PME PLUS allows an improvement
  of fruit consistency (e.gfruit inclusion in
  yoghourt, cider)

59
TROPICAL FRUITS

• With a high level in hemicellulase, PECLYVE
  PINEAPPLE allows to reduce viscosity of the juice
• To make a total clarification of cloudy juice
  from tropical fruit, PECLYVE TROPICA is well
  adapted.

60
PECLYVE OLIVA

• During maceration step,
• PECLYVE OLIVA allows to reduce the emulsifying
  phase.

Oil
Emulsion with enzymes
Emulsion without enzymes
61
RED FRUITS VEGETABLE

• Red fruit mash is particularly acid and PECLYVE
  FR is well adapted for acid phase.
• Vegetable have a high content in cellulose and
  pectins and it is why our PECLYVE LI is
  specially formulated for vegetables

62
WARNING

• The dosage of all these enzymes must be very
  precise. If too much enzymes is added, it will
  lead to a liquefaction and the filtration will
  not be able anymore.
• Due to its experience Lyven is ready to help you
  to find the best dosage to use.

63
ENZYMES IN BAKING
64
BAKING PRINCIPLES

• BREAD Food made from dough of flour or meal and
  usually raised with yeast or baking powder and
  then baked
• 3 elements are imperatives
• WATER Allows to give a network of gluten
• YEAST Allows to give CO2 and flavour molecules
  producing from flour components
• CO2 is trapped in gluten network and gives bread
  volume

65
FLOUR COMPOSITION
COMPONENT
STARCH 60-72
DIFFERENT OSES 1-2
PROTEINS 8-12
LIPIDS 1,2-1,4
MINERALS 0,5-0,6
HEMICELLULOSE 2-3
WATER 13-16
66
BREAD PROCESS
MOULDING STAGE
PROVING STAGE
67
Enzymes in Baking

• AMYLASE
• HEMICELLULASE
• GLUCOSE-OXYDASE
• PROTEASE
• PHOSPHOLIPASE LIPASE

68
AMYLASES

• Starch is composed of amylose and amylopectine
• Amylase Enzyme that liquefy starches and convert
  them to maltose (sugar) and dextrins.

AMYLOSE
amylase
69
LYVEN Amylases

• Panlyve Amy Fungal Alpha-Amylase
• Increase bread volume by faciltating yeast
  activity
• Increase colorness of the crust
• Panlyve AR Bacterial Alpha-Amylase
• Retard the staling of baked goods
• Avoid starch retrogradation

70
Correlation between Falling Number and Amylase
dose

• Examples
• If the falling number is 350 seconds, 2300-2900
  FAU per 100 kg of flour should be added.
• If after malt addition, the falling number is 300
  seconds, 900-1700 FAU per 100 kg of flour should
  be added.

FAU (Fungal Amylase Unit) Amylase activity Unit
71
HEMICELLULASE

• Hemicellulase (xylanase) Enzyme that breaks down
  hemicellulose. Hemicellulose catagorizes a
  variety of complex polysaccharides chain (mainly
  pentosans).
• Within hydrolising pentosans, xylanases give more
  free water and increase the dough workability and
  bread volume

72
LYVEN Xylanase

• Panlyve HCB increases bread volume and gives
  better color and structure to the crumb.

73
GLUCOSE-OXIDASE

• Glucose-Oxidase converts glucose to gluconic
  acid and hydrogen peroxyd and will lead to
  molecule oxidation.

GLUCOSE
GLUCOSE H202
HS
Di-sulfure bonds created
Gluten proteins
S
S
SH
74
LYVEN Glucose-Oxidase

• Panlyve GO Fungal Glucose Oxidase
• Well adapted for weak flour
• Increase tenacity and decrease elasticity of the
  dough
• Partials ascorbic acid replacer
• Well adapted to deep-frozen raw dough

Panlyve GO should be avoided in dough with butter
(rancid flavour)
75
PROTEASE

• Protease Hydrolysis of the protein chains of the
  gluten and the formation of fragments of smaller
  size

76
LYVEN Protease

• Panlyve BI Bacterial protease
• Replacer of chemical reducers (metabisulfite)
• Specially adapted for use in the manufacture of
  Biscuits and crackers

77
LIPASE PHOSPHOLIPASE

• Enzyme that catalyzes the hydrolysis of
  triglycerides to yield glycerol and fatty acids

triglycerides
78
LYVEN Lipase Phospholipase

• Panlyve TDR Enzymatic complex with lipase
  phospholipase
• Replacer of Chemical emulsifier as Datem or SSL.
• Increasing bread volume
• Giving a thiner crumb

79
SPECIAL FORMULATION

• Panlyve BR
• Alpha-AmylaseHemicellulaseGlucose oxidase
• Bromate replacer
• Improvement of machenability and strength of the
  dough
• Improvement of crispiness of the crust
• Panlyve NPB
• Alpha-AmylaseProtease
• Dough relaxer (actives on gluten)
• Gives expanding dough
• Well adapted for strong flour

80
SPECIAL FORMULATION

• Panlyve AG
• Fungal amylaseamyloglucosidase
• Gives directly glucose and maltose
• Increases the yeast activity
• Increase the colour of the bread
• Panlyve ASN
• Alpha-AmylaseHemicellulase
• Improves softness and fineness of the crumb
• Extends the shelf-life of the bread

81
SPECIAL FORMULATION

• Panlyve B2G
• Alpha-amylaseshemicellulases
• Specially adapted for retarder proven processes
• Specially adapted for deep freezing of unbaked
  doughs

82
SPECIAL FORMULATION

• Panlyve B2S
• Alpha-amylase specific hemicellulases
• Specially recommended for good quality flours
• Improves crumb softness and crust aspect
• Panlyve XT
• Alpha-Amylaseendo-xylanase
• Well adapted on rye flour and short fermentation
  process
• Convenient for low hydrated dough (biscuit)

83

• Enzymes dosage should be adapted to the quality
  of the flour.
• Due to his good knowledge in Flour and Baking,
  LYVEN is ready to help you in the choice of
  enzymes and dosages.

84
ENZYMES IN BEER
85
BEER

• It is a fermented beverage obtained from malted
  cereals (barley, wheat, corn) water, yeast and
  hops.
• For several reasons, malted cereals could be
  replaced by cereals like wheat, oats
• Enzymes will attack the cereal in order to help
  yeast during the fermentation

86
Brewing Flowchart
Brewhouse
Adjuncts Cooker
Mash Tub
Lauter Tub
Copper
Malt
Fermentation Filtration
Beer Tank
Fermentation
Fermentation
FILTRATION
Yeast
87
AMYLASES

• Starch is composed of amylose and amylopectine
• Amylase Enzyme that liquefy starches and convert
  them to maltose (sugar) and dextrins.

AMYLOSE
amylase
88
Lyven Amylases in Brewing

• Amylyve UHT Bacterial Alpha-Amylase
• Heat Stable enzymes
• Liquefaction of the starch at high temperature
• Amylyve A30 Bacterial Alpha-Amylase
• Liquefaction of starch at intermediate
  temperature
• Amylyve AN2300LFungal Alpha-Amylase
• Maltose and maltotriose forming effect
• Amylyve AG400L Fungal Glucamylase
• Transforms dextrins into glucose

89
Brewing Flowchart
Amylyve UHT
Amylyve A30 Amylyve AG 400L
Brewhouse
Adjuncts Cooker
Mash Tub
Lauter Tub
Copper
Malt
Amylyve AG 400L Amylyve AN 2300L
Fermentation Filtration
Beer Tank
Fermentation
Fermentation
FILTRATION
Yeast
90
GLUCANASE

• An enzyme that act on beta glucans, a type of gum
  found in the endosperm of unmalted barley,
  oatmeal, and wheat.

Beta-glucanase
Beta-glucans
Dextrins
91
LYVEN Glucanase in Brewing

• GLUCALYVE PB 750 Fungal Beta Glucanase
• Heat stable
• Reduces viscosity of the wort
• BREWLYVE AXC 1500L Fungal xylanase
• Acts on the pentosane
• Completes action of Glucalyve PB750
• FILTERLYVE 1500L Fungal Beta Glucanase
• Improve filterability of beer
• Efficient at the pH of beer

92
Brewing flowchart
Glucalyve PB 750 Brewlyve AXC 1500L
Brewhouse
Adjuncts Cooker
Mash Tub
Lauter Tub
Copper
Malt
Fermentation Filtration
Filterlyve 1500L
Beer Tank
Fermentation
Fermentation
FILTRATION
Yeast
93
PROTEASE

• An enzyme that hydrolyzes (breaks down a bond and
  adds water) proteins, especially to peptides

94
LYVEN Protease in Brewing

• Brewlyve NP900 Bacterial Protease
• Improves the content of peptides and amino-acid
  in the wort
• Gives more FAN (Free Amino Acid) to the yeast
• Lypaine 6500 Vegetal Protease
• Prevent hazing of beer kept in cold storage

95
Brewing Flowchart
Brewlyve NP 900
Brewhouse
Adjuncts Cooker
Mash Tub
Lauter Tub
Copper
Malt
Fermentation Filtration
Lypaine 6500
Beer Tank
Fermentation
Fermentation
FILTRATION
Yeast
96
ENZYMES IN ANIMAL FEED
97
Enzymes using in Animal Feed

• Enzymes in animal feed will allow to higher
  absorption of nutritional particles and to a
  better digestibility of feedingstuff, leading to
  an increasing growth of the animals
• Three enzymes are mainly used in Animal Feed
• XYLANASE
• GLUCANASE
• PHYTASE

98
Animal Feed Definitions

• Feed Conversion Ratio
• FCRQuantity of feed used/ Quantity of meat
  produced
• Apparent Metabolisable Energy
• AMEGeneral Energy (GE) of feed-Energy Contained
  in Excretion (ECE)

AME
TE
ECE
99
Non-Starch Polysaccharides

• Non-Starch Polysaccharides (NSP) are present in
  cereal based diets
• Traps of valuable nutrients in the cells of
  vegetable feedstuffs
• Increase the viscosity of the digesta in the
  intestine.

100
Non-Starch Polysaccharides (NSP)
AME/GE
rice
maize
sorgho
wheat
barley
rye
NSP
101
FEEDLYVE AXC
- contains xylanase as the main enzymatic
activity, which degrade pentosan contained in
cereal like wheat and rye fungal xylanase
from Trichoderma longibrachiatum xylanase
concentration is described in units of AXC -
contains main side activities (B glucanase,
pectinases,cellulases...)

• Feedlyve AXC is available
• - in 2 different forms liquid and powder
  (Feedlyve AXC L and Feedlyve AXC P)
• in different concentration
• Feedlyve AXC 200 contains at least 200 u AXC / g
• Feedlyve AXC 400 contains at least 400 u AXC / g
• Feedlyve AXC 1500 contains at least 1500 u AXC /
  g
• Feedlyve AXC 6000 contains at least 6000 u AXC /
  g
• Feedlyve AXC 12000 contains at least 12000 u AXC
  / g

102
AME with FEEDLYVE AXC
Apparent Metabolisable Energy Roslin Institute,
Scotland

• Results

AME levels, in MJ / kg
4.04
103
FCR with FEEDLYVE AXC in broilers
104
FEEDLYVE AGL
- contains endo glucanase as the main enzymatic
activity which degrade glucans contained in
cereal like barley fungal B glucanase from
Aspergillus Niger B glucanase concentration
is described in units of AGL - contains main
side activities (xylanase, pectinases, ..)

• Feedlyve AGL is available
• - in 2 different forms liquid and powder
  (Feedlyve AGL L et Feedlyve AGL P)
• in different concentration
• Feedlyve AGL 200 contains at least 200 u AGL / g
• Feedlyve AGL 400 contains at least 400 u AGL / g
• Feedlyve AGL 1500 contains at least 1500 u AGL /
  g
• Feedlyve AGL 6000 contains at least 6000 u AGL /
  g
• Feedlyve AGL 12000 contains at least 12000 u AGL
  / g

105
AME with FEEDLYVE AGL
106
FCR with FEEDLYVE AGL in Broilers
107
PHYTATES

• Oilseed (soya) and Cereals (wheat)contain 90
  of their phosphorus locked up in phytate which is
  almost indigestible to monogastric animals. The
  unabsorbed phytate passes through the
  gastrointestinal tract, elevating the amount of
  phosphorus in the manure, leading to
  environmental problem.

Furthermore, Other nutrients are linked with
phytates unavalaible for the intestinal
absorption.
minerals
Phytates
108
FEEDLYVE PHY 5000

• FEEDLYVE PHY 5000 is a phytase which will release
  the phophorus and the minerals allowing the
  absorption by the intestinal cells.

Ca
OH
STARCH
P
O-
FEEDLYVE PHY 5000
O
FEEDLYVE PHY 5000
109
FEEDLYVE PHY 5000 for broilers
110
Legislation in Feed Enzymes

• European authorisation
• xylanase 
• definitive authorisation in chicken (3 trials
  already done)
• definitive authorisation in turkey (3rd trial
  running in spring)
• B glucanase 
• definitive authorisation in chicken (3 trials
  already done)
• definitive autorisation in turkey (2nd trial
  running in spring, 3rd trial in late summer)
• For other country than european countries, used
  should be adapted to the legislation.
• LYVEN plans
• new segments (mainly pig)
• to think for new enzymes activities (alpha
  galactosidase, cellulases, proteases, amylases
  for ex)

111
Enzymes shelf-life

• All the Lyven enzymes get only Best Before Date.
  It means that after this date the activitycould
  be lower than the specification. .
• This Best Before Date last for 1 year for the
  liquids and 2 years for the powders

112
THANK YOU FOR YOUR ATTENTION