PROMUTASE™ 200

Melon extract

Raw material for animal feed

Natural complex
of antioxidants
 PROMUTASE™ 200
Summary

Technical data & Technical data & Specifications

Specifications
Description
Active ingredients
Nutritional facts
Manufacturing process Specifications
& Analysis
Packaging and storage
Uses

Coating characteristics
& bioavailability
Manufacturing process & Analysis
Manufacturing process
Oxidants/antioxidants
Analysis
balance Physical data

In vitro antiradical Coating characteristics & bioavailability

properties Description of the coating
Efficiency of coating protection
Bioavailability of PROMUTASE™ 200
Impact on stress
markers
Oxidants/antioxidants balance
Introduction
Impact on dairy cow
Oxidants
mixed-milk cells
Antioxidants
Oxidative stress

in vitro antiradical properties

Test description
Results

Impact on stress markers

Description
Results

Impact on dairy cow mixed-milk cells

Description
Results
P/3142/GB/01/September 2005 - 0
 PROMUTASE™ 200
Technical data & Specifications

PROMUTASE™ 200 was developed by a partnership between

Description
BIONOV and SAINT-HERBOT. The result of this partnership
allowed to launch an application in animal nutrition.

PROMUTASE™ 200 is composed by a melon extract coated

Active ingredients
with a vegetable fat matter and adsorbed on a mineral carrier
which makes easy the blending with animal feed.

The melon juice extract is obtain from a specific patented

Nutritional facts
variety of Cantaloup melon selected for its high content in
antioxidants.

Specifications
Description
The product is a natural and efficient source of antioxidants
and particularly antioxidant enzymes as superoxide
Packaging and storage dismutase (SOD) and catalase.

Active ingredients
Uses

Antioxidants Value (%)

SOD (1) (Cu/Zn; Mn) Min. 2 600 000 IU/ kg
Catalase 400 000 IU/ g
(1) SOD activity is measured according to the Oberley and Spitz method 1985 and/or
Cytochrome C method 1969 based on the activity of SOD to inhibit the reduction of O ⎯
2

generated by a xanthine/xanthine oxidase system ( ∆ DO initial = 0,025 ). One unit of

560

SOD gives 50 % inhibition of the reduction of NBT.

Minerals Value (%)

Phosphorus 0.40
Magnesium 0.28
Calcium 13.83
Sodium 0.14

Nutritional analysis
Proteins 9.5 %
Lipids 16.6 %
Carbohydrates (Dubois) 34.9 %
Ashes 39.0 %
Specifications
Characteristics Standards
Appearance Powder
Colour Pale yellow
Odour Characteristic
Dry matter 96.1 %
Conservator None
Total germs Max. 10 000 CFU / g
Yeast & Mould Max. 100 CFU / g
Enterobacteria Absence / g
Escherichia coli Absence / g
Salmonella Absence / 10 g
Staphylococcus aureus Absence / g

Packaging and storage

Packaging 1 kg plastic jar or 20 kg plastic drum
Selflife 18 months
Storage Store in a dark, cool and dry place

Uses
All animal species:
- Cattle, goat, sheep ;
- Pig, poultry, rabbit ;
- Breeding fish, shrimp ;
- Pets and sport animals such as : dogs, cats, horses …

P/3142/GB/01/September 2005 - 1
 PROMUTASE™ 200
Manufacturing process & Analysis

Manufacturing process
Manufacturing process
The raw materials used to manufacture the microgranules of the
melon juice concentrate are
- a specific variety of cantaloupe melon registered into the
Analysis European variety catalogue and exclusively reserved for
BIONOV developments thanks to exclusive contracts with
producers;
- an vegetable fat matter authorised in animal feed ;
Physical - a mineral matter authorised in animal feed
characteristics
The melon juice concentrate is obtained by an industrial process
developed by BIONOV and requiring usual filtration and
separation techniques. This patented process (FR 2 716 884, US
5 616 323) only uses melon pulp without any solvent or additive.

The vegetable fat matter coated melon juice concentrate is

adsorbed on mineral and vegetable carriers authorized in animal
feed.

Extraction and concentration of melon juice

Coating with a fat matter authorized in animal feed

Adsorption on mineral and vegetable carriers

authorized in animal feed

Dilution on food carrier

authorized in animal feed

PROMUTASE™ 200
Analyses
The analysis of PROMUTASE™ 200 is based on the dosage of SOD activity made after removal
of the coating.
Dosage of SOD activity follows a very specific method published by Beauchamp & Fridovich in
1971 and modified by Oberley & Spitz in1985. This method consists in the evaluation of SOD
activity through its capacity to inhibit a superoxide anion flux generated by the
xanthine/xanthine oxidase system. Superoxide radicals produced by this system reduce
Nitroblue Tetrazolium (NBT) into formazan blue stable at 560 nm.
An enzymatic unit of SOD corresponds to the quantity of vegetable extract liable to a 50%
inhibition of NBT reduction.

Physical characteristics
Loss on drying <5%
Solubility none
Temperature max. stable at T< 41 °C out of press
Density 0.798
Particle size 81.73 % between 250 and 100 µm

P/3142/GB/01/September 2005 - 2
 PROMUTASE™ 200
Coating characteristics & bioavailability

The melon juice concentrate is coated to protect the actives

Description of the
coating of PROMUTASE™ 200. This double protection prevents their
destruction by the acidity of the digestive tract and secure its
resistance to high temperatures.
Efficiency of coating
protection
Description of the coating
The hydrogenated vegetable fat matter (palm oil, 100% fatty
Bioavailability of acid), the mineral and the food carrier are authorized in
PROMUTASE™ 200 animal feed and don’t contain GMO.

It is the coating which best protects SOD in the melon juice

from gastric acidity and high temperatures and which makes
easy its incorporation in animal feed.

Efficiency of coating protection

Comparison between the melon juice (powder) and melon
juice concentrate coated with different content of vegetable
fat matter shows that the coating protects the SOD activity
from the gastric acidity (pH 1, trypsin and pepstatin).

Efficacy of the coating

Melon juice (powder)

Coating melon juice concentrate
100
(% of initial SOD activity)

80
SOD activity into
the gastric juice

60

40

20

0
0 15 30 45 60
Time in the gastric juice
(min)
Bioavailability of PROMUTASE™ 200
Recent results obtained by test on piglets (INRA study, Rennes, 2004) have permitted to show
the bioavailability of antioxidant enzymes and particularly SOD in PROMUTASE™ 200.

Piglets feed was supplemented with PROMUTASE™ 200 at 5g/T equivalent to 0.3 UI SOD/kg
BW/day and 20 g/T equivalent to 1.2 UI SOD/kg BW/day for 12 days.

Bioavailability of PROMUTASE™ 200

379
400
Plasma SOD activity

350 335
(IU/ml)

300
269

250

200
Control 5 g/T 20 g/T

The results of the study show that PROMUTASE™ 200 permits to increase the SOD plasma
activity between 25 to 40%.

P/3142/GB/01/September 2005 – 3
 PROMUTASE™ 200
Oxidant/antioxidant balance

Introduction
Introduction
Under normal conditions, animal body constantly produces
oxygen reactive derivatives, oxidant molecules, rapidly
destroyed or scavenged by antioxidant defence systems.
Oxidants This happens generally for all biochemical reactions that
bring molecular oxygen.
In the first time oxygen free radicals, also called primary
free radicals (ºO , H O , OHº) are produced. Defence systems
2
¯
2 2

Antioxidants in charge of eliminate these primary free radicals are make

up of antioxidant enzymes (SOD, catalase, peroxidases),
called primary antioxidants.
SOD
Oxidative stress

O 2 º
O 2
¯
HO2 2 HO + O
2 2

OHº Catalase
Peroxidase

Free radicals non-eliminated by these enzymes led to the

formation of secondary free radicals (ROO°), which are
able to cause chain oxidative reactions. Their toxicity is
important for cell structures that can cause several
pathologies. To scavenge these secondary free radicals, the
body throws in a second defence line constituted by
exogenous molecules brought by food (vitamin E, C, A,
polyphenols), also called free radical scavengers or
secondary antioxidants.

SOD
O 2
º
O 2
¯
H2O2 H2O + O2
Fe3+
Fe2+ Catalase
Peroxidase

OHº ROOº

Vitamin E, C, A
Polyphenols
Then primary antioxidants prevent the formation of oxygen free radicals while secondary
antioxidants participate to the slowing down of chain oxidative reactions limiting cell
damages.

Under certain conditions, the production of oxidant molecules highly increases, leading to a
disequilibrium between formation and destruction of these molecules by antioxidants ending
at a stage called oxidative stress.
During this oxidative stress, more or less important damages can be inflicted on the body
either directly by deterioration, destruction, disorganisation of structures, either indirectly by
perturbation of its normal functions.
All inflammatory process, stress, intensive efforts, ageing are concerned by these
phenomenon.

To limit this phenomenon of oxidative stress and help cells and the body eliminating free
radicals, it is then interesting to reinforce the pool of primary antioxidant such as SOD by an
exogenous intake.

SOD

O2 º
O2¯ H2O2 H2O + O2
Fe
3+

Fe
Catalase
2+

Peroxidase
OHº ROOº

Vit E, C, A, Polyphenols

In this way, SOD brought by PROMUTASE™ 200, through oral route is a concept
innovative and efficient in the reduction of the oxidative risk.

Oxidants
They are called reactive oxygen species (ROS) or, more commonly, free radicals (FR). Their
constant production constitute a normal physiological phenomenon, permanently under
antioxidant control. Abnormal increase of their production and the insufficient control by
antioxidants constitute the oxidative stress.

Free radicals are molecules with an odd number of electron on the external orbital of one or
several of its atoms. This particularity makes them instable, because electrons naturally tend
towards a two by two organization on the last orbital. With an unpaired electrons, these free
radicals are eager for the “missing electron” to form a more stable new molecule. Oxidant
molecules, or free radicals, are then very reactive molecules. This reactivity is a useful
property for numerous physiological reactions but can also led to pathological disequilibrium.

P/3142/GB/01/September 2005 – 4a
Two main classes of free radicals are described : primary free radicals ( O , H O , OH°), °
2
¯
2 2

directly formed from oxygen and secondary or organic free radicals (ROO°) generate by
the action of primary free radicals.

Primary free radicals

• Superoxide anion (ºO2¯) is the first and the most abundant of free radicals formed in cells
and tissues.
It is formed by the addition of one electron on molecular oxygen :

º
O + e- 2
º
O 2
¯

It is an unstable molecule at acid pH (4.7) where it spontaneously dismutes into hydrogen

peroxide and oxygen.
In other pH conditions, this dismutation appears thanks to the enzymatic action of superoxide
dismutase (SOD).

SOD

º
O 2
¯
+ ºO 2
¯
+ 2H +
HO + O
2 2 2

• Hydrogen peroxide (H2O2) formed by superoxide anion dismutation is a low reactive

molecule whose cell can withstand relatively important concentrations, used by certain cells
specialized in antimicrobial fight.
In presence of catalase or peroxidases, hydrogen peroxide is destroyed.

Catalase or Peroxidase

HO
2 2 H0 + O
2 2

• Hydroxyl radical (OH°) is formed by a second dismutation way of superoxide anion in the
presence of iron. Hydroxyl radical (OH°) is the most powerful single electron oxidant known. It
is very toxic. Superoxide anion ( O ) turns ferric iron (Fe ) into ferrous iron (Fe ) whose reacts
°
2
¯ 3+ 2+

then with hydrogen peroxide (H O ) and releases hydroxyl radical (OH°).

2 2

º
O2-

Fe3+ Fe2+ + O 2

H 2O 2 OHº + OH¯ + O + Fe3+ 2

Hydroxyl radical (OH°) is then able to oxidize all substrates in its area and represents a
potential source of important molecular, cellular, tissue lesions. More over, it crosses cell
membranes and is able to act far away from its formation site. It initiates a cascade of
oxidative reactions producing the main part of secondary free radicals.

Nevertheless, the first dismutation way of superoxide anion to hydrogen peroxide thanks to
superoxide dismutase (SOD) leaves few superoxide anion available for the dismutation by
the hydroxyl radical way.

• Singulet oxygen is a single oxygen atom. It is formed by the action of the light on oxygen.
It plays a minor role in the display of toxic effects of oxygen reactive species.

Secondary free radicals

They are not spontaneously formed. They are formed from a primary free radical.

• Organic peroxides form the main part of secondary free radicals. They are formed by an
oxidative reaction chain started by hydroxyl radical (OH°) and concerning the constituent of
the cell.

For example, a peroxide radical (ROO°) is formed by the action of the hydroxyl radical on an
unsatured fatty acid of the cell membrane. This reaction is propagated from one to one
leading to an alteration of the cell membrane permeability. It is the lipid peroxidation that, if
it is not controlled, drag towards a disintegration of the cell membrane.
In the same way, protein amino acids can suffer a carbonylation (=C=O) making theses
proteins sensitive to proteases and DNA bases can suffer an oxidation might leading to
mutations or stop in the DNA replication.

Besides these direct effects, organic peroxides indirectly participate in the inflammatory
process and cell lesions.
These is for example phospholipases activation releasing arachidonic acid which is the
most abundant precursor of eicosanoids bringing, after oxidation by COX1 and COX2 ways,
prostanoids (prostaglandins, prostacyclins, thromboxans) and by LOX way, leucotriens. This is
also the activation of inflammatory cytokine synthesis and apoptosis, the main form of
programmed cell death.

Thus organic peroxides are synthesis intermediate of numerous chemical mediators of

acute or chronic inflammations either from exogenous (infections, UV irradiations, X-rays,
ischemic attacks) or endogenous causes.

P/3142/GB/01/September 2005 – 4b
 Free radicals and inflammatory process

ROO°
Organic peroxides

Phospholipase activation

Arachidonic Acid Release

which is, after oxidation, the most abundant precursor of
Eicosanoids

Cyclooxygenase way Lipoxygenase way

(COX) (LOX)

COX 1 COX 2

Leucotriens Hepoxylins

Prostanoids Cystenyl leucotriens Lipoxins

Prostaglandins Thromboxans

Prostacyclins

• Microbiocide molecule formation during inflammatory process in the specialised cells

(neutrophil polynuclears, monocytes, macrophages) is also one of the important indirect
effects of free radicals. At the time of their activation during bacteria invasion, oxygen O2 is
transformed in superoxide anion °O by NADPH oxidase. Thanks to SOD, this superoxide anion
2
¯

is dismutated into hydrogen peroxide, which is the first bactericide. It could be transformed
into hypochlorite HOCl, second bactericide, thanks to myeloperoxidase (MPO) released by
white corpuscle azureophile granules at the time of phagocytosis. This hypochloride is able to
react with a primary amine RNH2 to form a chloramine RNHCl, third bactericide and
antiprotease inhibitor. This last property permits the lyses by the proteases of the phagocyted
bacteria proteins that reinforce the antimicrobial fight of already formed microbiocides.

Antimicrobial fight in specialized cells

polynuclears, monocytes et macrophages

NADPH oxidase SOD Cl- RNH2

O2 º
O2- H 2O 2 HOCl RNHCl Antiprotease
inhibition
d’ i
SOD is then a key enzyme in the antibacterial defence process because of favouring
dismutation way of superoxide anion to hydrogen peroxide to the detriment of the
dismutation way to hydroxyl radical.

• Peroxinitrite (ONOO-) is a powerful oxidant generated on the occasion of high production

of nitric oxide (NO°) and which is able to split up into other oxidants including hydroxyl radical
(OH°).
Many cells are able to produce nitric oxide (NO°) from arginin and oxygen thanks to nitric
oxide synthetase (NOS). It is a physiologic production which the role is essential in vascular
tonus, water absorption in the digestive tract, inhibition of blood platelet aggregation… At the
time of certain microbe infections releasing cytokines and endotoxins, there is a massive
production of nitric oxide (NO°) which reacts with superoxide anion ( O ) to form peroxinitrite
°
2
¯

(ONOO-), a powerful oxidant extremely toxic for the cells.

Catalysing the dismutation of superoxide anion, SOD is thus the key enzyme in the body
protection against extremely harmful effects of peroxinitrite.

Primary Secondary
free radical free radical Cell
inflammation
- Phospholipase
activation
STRESS
Protein - Cytokine
carbonylation activation

Lipid Apoptosis
OH° ROO°
O 2
º
O 2
¯
peroxidation

DNA base Cell

oxidation destruction

Primary antioxidants Secondary antioxidants

(SOD, Catalase…) (Vit E, Se , Vit C…)

Induction of superoxide anion dismutation by SOD enzyme towards hydrogen

peroxide is a key process in the antioxidant chain implemented by the body.
This dismutation way to hydrogen peroxide prevents important production of
secondary free radicals and, consequently, permits a protection of cell constituents,
a preservation of normal biological activity of the cell and, indirectly, an optimal
functioning of the body.

P/3142/GB/01/September 2005 – 4c
Antioxidants
They are molecules able to regulate or neutralize reactive oxygen species. The result of
oxidant/antioxidant equilibrium is a preservation of the physiological levels of free radicals.
Antioxidants act at 2 levels :
- They prevent the formation of primary free radicals : primary antioxidants ;
- They neutralize secondary free radicals : secondary antioxidants.

Primary antioxidants : antioxidant enzymes

alive organisms set against the primary free radical production, a chain of very efficiently
enzymatic protections. This defence line is successively constituted by three families of
antioxidant enzymes :
- superoxide dismutases (SOD),
- catalase,
- glutathion peroxidases.

These antioxidant enzymes are very efficient, constantly produced by the body and act at very
low doses.
Chronologically there are SOD whose first intervene inducing by catalysis the dismutation of
superoxide radicalºO . Then, with a more or less intensive action, catalase or glutathione
2
¯

peroxidases intervene depending of the formation site of hydrogen peroxide.

SOD

O 2 º
O
2
¯
HO2 2 HO + O
2 2

OH º Catalase
Peroxidase
• Superoxide dismutases (SOD)
These enzymes are proteins containing several metal atoms. There are metalloproteins.
Their real biological function was discovered in 1969 by par Mc Cord and Fridovich whose
demonstrated for the first time their activity in the dismutation of superoxide anion.
In human and animal bodies, 3 forms of SOD are distincts :
- SOD (1) with Cu/Zn called cytosolic
- SOD (2) with Mn called mitochondrial
- SOD (3) with Cu/Zn called extra-cellular

SOD (1) is produced into the cytosol. Very stable into the cell, it efficiently protects it form
secondary free radicals by catalysing the dismutation of superoxide anion through the
peroxide way.
It escapes from the cytosol during the phagocytosis by specialized cells and also after cell
necrosis. It is thus release in a huge quantity in the inflammatory centres where it is very
active.
SOD (2) is produced into the mitochondria. It induces the dismutation of superoxide anion
generated in a high quantity into the mitochondria, protecting it against damages of
secondary free radicals. The increase of superoxide anion production into the mitochondria
(intensive food consumption, cell aggression, ageing) leads to an oxidant/antioxidant
disequilibrium which endanger it. It releases then free radicals into the cytosol through
membrane canals. Then SOD (1) takes over. This process is known under the name « free
radical theory of ageing » and lets see the way SOD might protect the body from premature
ageing.
SOD (3) is out of the cells. It is present in a low quantity in the blood and in a high quantity in
the tissues. Its presence protects cells against numerous sources of extracell free radicals.

• Catalase and peroxidases eliminate hydrogen peroxide. The involvement of each of these
2 enzymes is determined by the site of hydrogen peroxide formation. Catalase has a speed of
reaction very rapid that not need energy supply by the cell. Peroxidases need energy and
cofactors (ascorbate for ascorbate peroxidase, selenium for glutathion peroxidase).

SOD is the first link of the primary antioxidant defence system. It IS Then the key
enzyme of the antiradical defence system, favouring superoxide dismutation way to
hydrogen peroxide to the detriment of the dismutation way to hydroxyl radical.

Secondary antioxidants : free radical scavengers

To neutralize secondary free radicals, the body throws in a complementary second defence
line : the free radical scavengers.
All substance able to scavenge unpaired electron of free radical without giving itself a radical
product could be defined as a free radical scavenger. A first class of compounds ensure the
direct scavenge of free radicals as glutathion, ubiquinol, vitamin A, vitamin C, bilirubin, uric
acid, lipoic acid… The synthesis potential of these compounds is very low in the foetus and
the new-born whose radical defences is then dependent on exogenous supply.
A second class of compounds reacts with the secondary free radicals (tissue components
attacked by primary free radicals) by neutralizing them molecule to molecule. It is typically the
case of vitamin E that contributes to eliminate lipid peroxidation. Neutralisation is molecule to
molecule and despite a partial regeneration of vitamin E (thanks to vitamin C), this protection
could be insufficient that brings to an alteration of fat quality and animal meat with a lower
conservation. Exogenous supply of vitamin E is efficient till a certain level of assimilation
beyond which is destroyed or eliminated in the faeces. Moreover, it has to be noted that to
obtain a level of vitamin E having an optimal protection in the bovine muscle for example, it
needs a minimum 2 to 4 month distribution. In fact, a part of these secondary antioxidants is
lost and has to be constantly renewed by new supplies. In many situations their efficiency
reaches limits, leaving free way to chain oxidations and leading to what is called oxidative
stress.
.

P/3142/GB/01/September 2005 – 4d
Oxidative stress
Oxidative stress is the result of a disequilibrium between free radical production and
destruction process.
In normal physiological conditions, the animal body continuously produces free radicals. This
basic production is necessary for the good functioning of the body.

It is the intensity, the duration and the site of their production which will determine the effects
of free radicals :

- a moderate and transitional increase of their production corresponds to a defence

mechanism of the cell permitting it the destruction of cancer cells or pathogenous
microorganisms, for example.

- a moderate but recurrent or chronic increase of free radicals disturbs the balance between
their production and destruction in continuous way. This leads to disequilibrium of cellular
and organic functioning. It is then called oxidative stress. It is for example what takes place
during :
• chronic inflammations (arthritis, tendonitis, milk cells, nidation, …)
• stress
sport animals before and after competition (horses, dogs, …)
breeding starting periods (chick and fish stock, nurseries, …)
transition period (dropping, laying, weaning, pond change, transport, …)
vaccination
intensive feeding (effective productions, …)

• prolonged and intensive efforts (running, work, endurance, walking, …)

• ageing

- an important increase sufficient to alter irreversibly vital cell processes inducing apoptosis
(cell death by implosion, without release of cell content) : this is a major mechanism of the
immune system organisation particularly at the level of T and B lymphocytes. An excessive
activation of apoptosis triggers off neurodegenerative, neurological and cardiac diseases,
whereas its insufficiency leads the development of malignant tumours.

- a massive production of free radicals leads to a cell necrosis (cell death by explosion with
release of all the cell content). It is very rapidly what takes place during acute inflammations of
microbe, viral, physical, chemical or ionic origin.
Oxidative stress leads to a weakening of antioxidant defences : decrease of tissue levels of
free radical scavenger compounds (ubiquinol, glutathion, vitamin C, carotenoids, vitamins A,
vitamins E), decrease of the activity of antioxidant enzymes SOD, catalase, peroxidases. That
is directly linked to animal vitality, performances and disease resistance.

Prevention of oxidative stress in animals go through a reinforcement of their

primary antioxidant pool.

PROMUTASE™ 200, vegetable source of SOD brought in animal feed is an innovative

concept efficient in the reduction of oxidative stress.

P/3142/GB/01/September 2005 – 4e
 PROMUTASE™ 200
In vitro antiradical properties

Test principle
Test principle
Determination of the radical scavenging effect is based on
the inhibition or the decrease in the speed of the reduction
of an uncoloured oxidised molecule which is coloured when
it is reduced, cytochrome C for example, by adding to the
Test description
reactive medium the radical scavenging molecule to study.
The ROS is generated, for example by the action of xanthine
oxidase on xanthine (production of superoxide anion). In the
absence of a molecule able to capture it, the ROS leads to
Results
the reduction of the cytochrome C. The appearance of
reduced molecule is monitored using a spectrophotometer
(at 550 nm for cytochrome C) in the presence of radical
scavenging molecules.

xanthine oxidase Oxidised cytochrome C

(colourless)

xanthine --------------> O ----------------> Reduced cytochrome C

°
2
¯

(superoxide anion) (coloured)

appearance measurement
(OD/min)

RADICAL SCAVENGING MOLECULE ⇒ REACTION / INHIBITION

appearance measurement
(OD/min)

Test description
3 concentrations of freeze-dried melon juice (active
ingredient of PROMUTASE™ 200) are put in presence of
superoxide anion : 0.1, 0.3 and 0.6 mg/ml respectively
corresponding to 7, 21 and 42 IU/mg of SOD activity. Results
permit the measurement of the capacity of freeze-dried
melon juice to inhibit reduced cytochrome C apparition.
Results
in vitro test in presence of superoxide anion
0.16
Absorbance (550 nm)

°O ¯ in vitro
2
0.14
Freeze-dried melon juice 0,1 mg/ml
Freeze-dried melon juice 0,3 mg/ml
0.12 Freeze-dried melon juice 0,6 mg/ml

0.1
0 5 10 15 20 25 30
Time (min)

The results show a protective effect in vitro of freeze-dried melon juice (active ingredient of
PROMUTASE™ 200 against superoxide anion.

P/3142/GB/01/September 2005 – 5
 PROMUTASE™ 200
Impact on stress markers

At the cell level, stress (environment change, transport, bacteria

Description
or viral infections, …) are responsible for inflammatory reactions
due to the increase of oxygen reactive species (ORS)
SOD is highly involved in the fight against ORS and thus in the
prevention of inflammatory reactions.
Results
In order to show the impact of PROMUTASE™ 200 on stress
markers, BIONOV and SAINT-HERBOT in partnership with INRA
UMRVP (Saint-Gilles, France), carried out an in vivo study on
piglets during the weaning period.

Description
Animals & breeding
48 piglets whose never eat solid food have been weaned at 21
days old.

2 days with empty stomach (water supply only) were

immediately imposed after the weaning in order to exaggerate
digestive tract alteration.

Piglets of different litters were mixed for 24 hours in order to

exaggerate stress.

At last, piglets were breed in individual cage in room which were

voluntarily kept in a mediocre hygiene in order to increase the
risk of sanitary problems.

Food and feed

2 age type of feed based on cereals and vegetable proteins
nd

(wheat, barley, soy ground) without food additive antibiotics was

formulated and supplied in granules.

3 different foods were formulated with this feed :

T : control food without PROMUTASE™ 200
EV1: food with 5g of PROMUTASE™ 200 per tonne
(0.3 IU SOD/kg BW/d)
EV2 : food with 20 g of PROMUTASE™ 200 per tonne
(1.2 IU SOD/kg BW/d)

Piglets were feed in the following conditions : quantities

supplied intra triplets daily lined up on the lowest individual
intra-triplet consumption (triplet-feeding)
.
Experimental protocol
Two repetition were conducted on 48 piglets, that means 24 piglets per repetition.

Piglets were divided up into intra-litter quadruplets with the same body weight at the weaning (1
or 2 quadruplets per litters depending on the number and the homogeneity of piglets). Each
quadruplet is then formed by :
- One piglet killed at 2 days (with a 2-day empty stomach)
- Three piglets killed at 7 or 14 days

Weight equilibrium between groups J+2, J+7 et J+14 was globally checked

After killing, oxidative stress markers, HSP (heat shock proteins) and NOS (NO synthase), were
measured in gastrointestinal tissues.

Results
Impact of PROMUTASE™ 200 on stress markers of piglets
during weaning period
Control 5 g/T 20 g/T
50
45
unit / g of tissue

40
35
30
25
20
15
10
5
0
HSP 27 HSP 90 NOS
(stomach) (stomach) (colon)

PROMUTASE™ 200 MARKEDLY REDUCE – around 50%- the tissular concentration of HSP and NOS.
These molecules are produced by the cell in response to stressful conditions.

PROMUTASE™ 200 allows the reduction of the weaning stress effects.

P/3142/GB/01/September 2005 – 6
 PROMUTASE™ 200
Impact on dairy cow mixed milk cells

Description
Description
Measurement of the level of cells contained in milk reflects the
health state of cow udder. It is one of the quality criteria of the
milk of a bovine breeding. When milk cell level is under 250 000
cells/ml, the udder is considered as in a good health. During
Results
udder inflammation, milk cell level increases. Above 400 000
cells/ml, milk is unfit for human consumption.
PROMUTASE™ 200 impact on udder inflammation was tested in
a breeding of dairy cows with a permanent high level of cells in
the mixed milk of the milk tank.

Feed of 35 cows of this herd was supplemented with 50mg of

PROMUTASE™ 200 per day and per cow for 18 months. URCILL,
the official French milk control organism, carried out every
month the countage of mixed-milk cells.

Results
Impact of PROMUTASE™ 200 on milk inflammatory cells
650
600
Before PROMUTASE 200
550
cells / ml of milk

During PROMUTASE 200

500
450
400
350
300
250
200
3

150
10

100
50
oct-02
dec-02
jan-01

oct-01

aug-02
dec-01
feb-02
apr-02
june-02

feb-03
apr-03

oct-03
dec-03
nov-00

mar-01
may-01
july -01
sept-01

june-03
-03
-
-
-
-
-
-
-
-

-
-

-
-
-
-
-
-
-

-
aug-

Follow period of dairy cows (months)

URCILL’s countage shows a decrease of milk inflammatory cells

– around 60% - as soon as the distribution of PROMUTASE™ 200.
Nota

The analytical specifications warranted are only those mentioned on the certificate of analysis supplied with each
delivery of the product.

Except as set forth above, SEPPIC* makes no warranties, whether express, implied or statutory, as to the product
which is the subject of this document . Without limiting the generality of the foregoing, SEPPIC* makes no warranty
of merchantability of the product or of the fitness of the product for any particular purpose. Buyer assumes all risk
and liability resulting from the use or sale of the product, whether singly or in combination with other goods. The
information set forth herein is furnished free of charge and is based on technical data that SEPPIC* believes to be
reliable. It is intended for use by persons having technical skill and at their own discretion and risk. Since conditions
of use are outside SEPPIC*'s control, SEPPIC* makes no warranties, express or implied, and assumes no liability in
connection with any use of this information. Nothing herein is to be taken as a license to operate under or a
recommendation to infringe any patents.

PROMUTASE™ 200 is a commercial trade name of SAINT-HERBOT (France).

* SEPPIC being : and, depending on the country :

SEPPIC S.A. SEPPIC UK Ltd SEPPIC ITALIA Srl

75, quai d’Orsay
75321 Paris cedex 07
FRANCE
Tel. : +33 (0) 1 40 62 55 55
Fax : +33 (0) 1 40 62 52 53
50 Salisbury Road Via Quarenghi 27
PO Box 338 - Hounslow 20151 Milano
TW4 6SH - ENGLAND ITALY
Tel. : +44 208 577 8800 Tel. : +39 02 38009110
Fax : +44 208 570 2106 Fax : +39 02 38009140

GIVAUDAN LAVIROTTE S.A. SEPPIC Inc. SEPPIC Chine

56, rue Paul Cazeneuve
BP 8344 – 69008 Lyon
FRANCE
Tel. : +33 (0) 4 78 61 55 00
Fax : +33(0) 4 72 05 60 89
30, Two Bridges Road, suite 210
Fairfield, New Jersey 07004-1530
USA
Tel. : +1 973 882 5597
Fax : +1 973 882 5178
Room 510 Jin Tai Building
58 South Mao Ming Road
Shanghai 200020 CHINA
Tel. : +86 (21) 64 66 01 49
Fax : +86 (21) 64 66 11 09

SEPPIC GmbH SEPPIC Belgium NV

ABC Tower Köln Nieuwe Weg 1

Ettore - Bugatti - Str.6-14 Haven 1053
51149 Köln-Porz B – 2070 Zwijndrecht
GERMANY BELGIUM
Tel. : +49 (0) 220 3890 3100 Tel. : +32 3 250 3911
Fax : +49 (0) 220 3890 3199 Fax : +32 3 250 3912

www.seppic.com

Subsidiary of the AIR LIQUIDE group

© SEPPIC 2005 P/3047/F/01/Juillet 2005

P/3142/GB/01/September 2005 – 7