Yeast in aquaculture by GFMT

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Yeast products are getting more and more popular in aquaculture. However many products (as a whole or as fractions) are on the aquaculture market at the moment and differentiating between one from another can be difficult. This small article aims at shading some lights on the subject and explains that all yeast products are not equal.

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									                  November | December 2012
                                Yeast in aquaculture




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The International magazine for the aquaculture feed industry
         FEATURE




Yeast in aquaculture
by Philippe Tacon PhD, Lesaffre Feed Additives, France




Y
            east products are getting more         shrimp and marine fish larval feeds or included      Nutritional properties of yeast:
            and more popular in aquaculture.       as a protein source in aquafeeds.                        Typical dry yeast composition is 93-97
            However many products (as a                The reasons for this extensive use is its        percent dry matter and can contain from 40%
            whole or as fractions) are on the      excellent nutritional contents, its easy supply      to 60 percent crude protein nitrogen, 35-45
aquaculture market at the moment and dif-          in dried form or under liquid form when              percent carbohydrates, and 5-9 percent lipids.
ferentiating between one from another can          bakery yeast plants or breweries are near            A quite important fraction of the nitrogen is
be difficult. This small article aims at shading   aquafeed plants, and nowadays a competitive          under the form on nucleic acids (up to 12%)
some lights on the subject and explains that       price in regards to other protein sources such       that can lead to produce significant level of
all yeast products are not equal.                  as fish or soybean meal. Further applications        uric acid if consumed at high concentra-
                                                   are being developed for yeast as functional          tion, like meat. The Amino acid profile of
   Yeast is a unicellular organism belonging to    feed additives as probiotic live yeast, yeast        yeast is close to soybean meal and therefore
the kingdom of Fungi. More than a thousand         fractions (yeast cell walls, yeast extracts) or as   well adapted to animal nutrition; it is rich in
species have been found in two major phyla:        a source for more purified products such as          Glutamic acid and Lysine (up to 8%). Yeast
Basidiomycota and Ascomycota in which              beta-glucans and nucleotides. The production         is naturally rich in B vitamins such as biotin,
belong species able to duplicate by budding        process of yeast can allow the possibility to        thiamine and folic acid. It also produces niacin
such as Saccharomyces cerevisiae.                  incorporate trace minerals and then produce          but contrary to some belief does not produce
   Due to their unique properties to grow          highly bioavailable organic trace minerals, also     B12 Vitamin. Ergosterol which is a significant
under aerobic conditions and produce gas           known as selenium and chromium yeast.                fraction of yeast cell wall, also is also a precur-
and ethanol under anaerobic conditions, some           The pink yeast Phaffia rhodozyma, is natu-       sor of Vitamin D2 by using UV treatments.
yeast (mostly S. cerevisiae) have been used        rally rich in astaxanthin and has been used for
for the manufacture of fermented foods such        some time as natural source of the pigment           Baker’s yeast
as bread , beer and wine for a long time.          in salmonids. Although now it tends to be                 Even if their name remains Saccharomyces
Yeasts are also used as single sell protein        replaced by bacterial products which have a          cerevisiae (cerevisiae for beer), most of the
source in animal nutrition and in aquaculture      higher concentration and whose cell wall is          strains of Baker’s yeast have been selected
under various forms and species (Torulaspora,      more easily degraded. We will only refer in          for their high fermentative power, particularly
Torulopsis, Kluyveromyces, Saccharomyce et         the following article on products coming from        useful for bakers.Strains are specific to the
caetera). It can be found for example in           S. cerevisiae origin.                                type of bread and the region where it is sold,
                                                                                                        in order to respond to different bread making
                                                                                                          conditions (French bread, white bread, flat
                                                                                                          bread, croissant, etc.) and resist to different
                                                                                                          process conditions (osmotic pressure from
                                                                                                          high sugared bread, freezing, acidity of sour
                                                                                                          dough,…).
                                                                                                              Baker’s yeast comes as a pure and pri-
                                                                                                          mary culture grown on sugar substrate such
                                                                                                          as molasses. The production is performed
                                                                                                          under very strict conditions in order to main-
                                                                                                          tain the genetic purity, consistency, specificity
                                                                                                          and efficacy of the strains. (Figure 1). It can
                                                                                                          be sold under different forms and packaging
                                                                                                          (instant dried yeast, active dry yeast, com-
                                                                                                          pressed, cream).
                                                                                                              The primary grown culture controlled
                                                                                                          process makes also a very consistent base for
                                                                                                          the production of yeast extracts, autolysed
                                                                                                          yeast, yeast cell walls and their derivate:
                                                                                                          nucleotides and beta-glucans. Yeast cell
                                                                                                          walls produced from Baker’s yeast usually
     Figure 1: Yeast manufacturing process (primary grown culture)
                                                                                                          have a high content of mannans. They are

                                            14 | InternatIonal AquAFeed | november-December 2012
                                                                                                                                    FEATURE
                                                                                                                                   FEATURE

                                                          encounters as
      important for vaccinator safety but on growth parameters in tilapia a
         Table 1: Effect of live yeast Actisaf also the
                                    fish.                 contract vaccina-
      safety and welfare of the(n=3, P<0.05, measures with different letters are
         under stress conditions.
         significantly different)
           The fish are then delivered onto a stainless tion team, is poor
      steel table where the fish then(%) vaccinated anaesthesia of PER
                    Treatment Survival are        SGR         FCR            the
      in a very specific area, with only a 3 mm toler- fish and also poor
      ance. The team must achieve a 96 percent grading, prior to vac-
          CON 40% -10 fry          75.0ab       3.33a        3.11e        0.83ab
      accuracy target and considering most vaccina- cination of the fish.
          CON 40% -20 fry           64.8a       3.47a        3.26e
      tors handle between 15-20,000 fish each this Poor anaesthesia of    0.78ab
            Act some 10 fry
      is quite 40% - achievement!  87.5bc       5.80d       1.43abc       1.89cd
                                                          the fish can lead to
           The 40% are then returned to a recovery high mortalities. If
            Act fish - 20 fry       92.6c        5.43c       1.01a         2.64d
      tankAct 27% - 10come round from the anaes- fish are under anaes-
             and should fry        91.7bc      5.46cd        1.62bc        2.26c
      thetic within about two to three minutes. thetised, the whole
            Act 27% - 20 fry       96.29c        5.24c      1.17ab         3.17e
      There is always some level of mortality after process becomes
      this high risk, stressful process but usually it is stressful and danger-
        recognised as good 100 binders. Fractions drum them (imag-
      just a few fish, around toxin for every 100,000 ous for dried. It can also
                                                                                           Figure 2: Schema of a process to produce yeast
        coming from High yeast have a light beige been grown major
      fish vaccinated.baker’s mortalities immediately ine having a as a more               extracts and yeast cell walls
        colour.                                            controlled product and
      after vaccination are usually attributed to poor operation          whilst
                                                           specific strains and
      anaesthesia rather than aquaculture application only partly sedated!). find
            The most popular the injection.
        of The consequenceshatcheries where it is a On way to human care as a food supplement tively high and and its amino acid content is
            Baker’s yeast is in of poor vaccination        its the other hand,
        major feed source forapparent and rotifer (see ifand holistic therapeutic, also known as natural similar to baker’s yeast. Numerous works
      usually only become         artemia months after         they are over
      vaccination and can last et al 1990).                brewer’s yeast.
        for example Couteau up until harvest where anaesthetised, they                                     have shown the efficacy of Brewer’s yeast to
                                                                Brewer’s yeast for aquafeed applications replace partially or totally the proteins found
      the financially consequences become appar- risk not recover-
        Brewer’s Yeast
      ent. The main problems are:                         ing sold by trading companies as a commodity in fish and vegetable meal in fish and shrimp.
                                                           is quickly enough,
           Incorrect needle depth resulting in either resultingon the protein content, or by local Shrimp feeds formulators typically incorporate
            Brewer’s yeast can be identified either based in piles of
        as the ferment used (needle too short) or fish in the in need to dispatch their slurry. The brewer’s yeast in their formula at the rate of
      intra-muscular injection in brewery industries breweries recovery
      internal primary damage, including granuloma tank causing the supply of these products can two to four percent.
        (Yeast organ production) or the by-product quality and suffoca-
      (needle too long) which results in the fish not tion inconsistent and depends greatly on the
        of these industries which is the form mainly be or even worse,                                        Brewer’s yeast can be used to produce
        used in properly due to damage tocase, gut.
      growing                                              source of supply.
                  aquaculture. In the latter the yeast not recovering at all.                              yeast fractions, however due to the nature of
           Fish is being immune to fermentation
        biomassnot harvested from the the disease             The nutritional content is similar as the brewer’s yeast and the specificity of the pro-
                                                                The other risk
                                                                                            Innovative and proven
        vats at the end of beer fermentation. vaccine) factorin baker’s yeast, but contains more trace duction processes, the quality is less consistent
      because of incorrect dosage (or no        It can be one here is to the
      being under liquid form (18-20% of dry matter) vaccinators. If as selenium and chromium. than in baker’s yeast. Products coming from
        sold delivered.                                    minerals such the
                                                                                                yeast products
        but preferentiallymaininactive yeastSalmovac1 mineral oils used in of brewer yeast is rela- brewery yeast tend to have a distinctive bitter
                  of the                                   The protein content
                              as problems spray or 29/10/2012 13:57
           Two ridgewaybio_quarterpage_print.pdf

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                                                   november-December 2012 | InternatIonal AquAFeed | 15
                                                  november-December 2012 | InternatIonal AquAFeed | 11
         FEATURE

                                                                             enzymes (notably pro- transfer from lab conditions to farm using
                                                                             teolysis) or playing on commercial feeds.
                                                                             the osmotic pressure            All the work published so far was made
                                                                             to rupture the cell wall with yeast either top dressed on feeds or
                                                                             (plasmolysis). Different incorporated in pressed (uncooked) feeds.
                                                                             grades of autolysed Nevertheless some direct effects to the gut
                                                                             yeast can be obtained maturation have been found in sea bass with a
                                                                             depending on the level species extracted from the rainbow trout gut
                                                                             of autolysis (from partial Debaryomyces hansenii (see the works from
                                                                             to total). The final prod- Tovar-Ramirez and also the reviews by Chi et
                                                                             uct is a mixture of cel- al 2006 and Gatesoupe 2007). Marine yeasts
                                                                             lular content and yeast and yeasts isolated from fish seem a very
        Figure 4: Cumulative mortality after immersion                       cell wall. Furthermore logical choice to use in species of aquaculture
        with L. anguiilarum (blue line is control, orange line
        is Pronady at 0.5g/kg. n=3, Pronady significantly
                                                                             the autolysis process interest. However, such material is often dif-
        decreases mortality at 120h. P<0.01)                                 degrades protein and ficult to grow under industrial conditions and
                                                                             forms peptides (dipep- did not lead to the development of an actual
                                                                             tides to tetra peptides) product yet. The products on the market are
smell and taste and a darker colour than the          and oligonucleic acids which are readily digest- therefore often from S. cerevisiae origin. It has
ones coming from baker’s yeast.                       ible by the animal. Again here depending on to be noted that up to now, no yeast prod-
                                                      the original yeast material used, autolysed and ucts have been registered in EU as a probiotic
Ethanol Yeast                                         inactive yeast quality can be very different.     in aquaculture.
    Ethanol Yeast are harvested after hav-                                                                  As an example of S. cerevisiae effects,
ing performed alcoholic fermentation and Live Yeast as probiotics                                       (Lara Flores et al 2003, 2010) Table 2 shows
distillation for the conventional production              Live yeast helps regulate the gut microbio- some works done in tilapia fry fed for 3 weeks
of Bioethanol from sugarcane, beet sugar ta. Its effects have been shown, first in human with feeds supplemented with Actisaf (also
or grains syrup. In the first case, the yeast where it can reduce diarrhoea, especially knwn as Biosaf) at 1 kg/T in two diets (40%
biomass is harvested and then dried with with children. Specific strains have then been and 27% proteins) and at 2 crowded condi-
the recycled energy used to heat the vegetal developed and produced industrially such as tions (1 fry per L or 1 fry per 2L).
material. The majority of ethanol yeast comes S. cerevisiae boulardii or S. cerevisiae Sc 47                All the yeast treatments also increased the
from Brazil.                                          (Actisaf) for the animal nutrition market. It is alkaline Phosphatase activity, and we can see a
    Production prices and selling prices are a common practice now to supplement feeds better improvement of feed conversion ratio
very low, however the quality, such as the to increase milk production in dairy cows (FCR) and survival under stressful conditions
protein content is very inconsistent. The sup- or help piglets
ply depends on the activity of the bioethanol survival.
plants and can also be inconsistent. Another              Live yeast
concern is the sanitary safety of these prod- are             charac-
ucts. Antibiotics are sometimes added to the terized                 by
process in order to prevent bacteria compet- their               living
ing with the yeast for nutrients andavoiding cells              count,
yield decrease. It is therefore possible that expressed by
some antibiotic residues and possibly other colony forming
toxins might be left in the final dried product. unit (cfu per
    Autolysed yeast – Inactive Dried Yeast            gram), typically
    Inactive and Autolysed yeast come from ten                 billions
primary grown cultures or Brewer’s yeast. cfu/g. Dosages
They are major products within the food are made in
industry as flavour enhancers and in pet food the feeds as                      Figure 3: Number of pellets remaining in the feeding tray one
                                                                                hour after feeding (n=4, YE are significantly different than
as feed attractants. They are used in aquacul- dilutions to get
                                                                                control at P<0.05).
ture feeds as a source of protein and nitrogen. an efficient cfu
Brewer’s yeast, and its ethanol equivalent, is count per g of
mostly favoured as it is cheaper than baker’s feed, a 1000 fold dilution giving a 10e107 per (low protein percentage and crowded condi-
yeast. They are also easier supplied as yeast g of feed for example. Viability of the yeast is tions). There is also a better protein efficiency
suppliers prefer to sell the more controlled mandatory for its effect and cfus should be ratio (PER) and digestive enzyme activity
and tailored Baker’s yeast on food markets.           checked before and after pelleting using plate when Actisaf is used.
    Inactive yeast is a yeast that has been counts.                                                         Live yeast can be used directly on farm,
deactivated by high temperature drying (often             Despite the increasing use of yeast as where it has been showed (empirically) to
spray drying). The cells come as a whole a probiotic in terrestrial animals, there improve water quality in shrimp and fish
and the cell wall is not ruptured making the are only a few numbers of works studying ponds. It is either used alone or mixed with
access to intracellular material (amino acids, its effect in fish as a gut functions stabi- bacteria. Farms producing mash feed onsite
vitamins…) difficult. A way to access these liser. The major reason is that live yeast also add yeast in order to degrade cellulolytic
materials is to partially hydrolyse the yeast does not resist the severe conditions of material to ensure a better digestion.
cell wall to let the cellular content be partially the manufacturing processes of aquafeeds
released from the cell. This can be facilitated (high temperatures, steam, long condi- Yeast Culture or fermented yeast.
by activating the internal autolytic enzymes tioning times, see Aguirre-Guzzman et al                       Yeast culture is a particular product in
of the live yeast (autolysis), adding external 2002). The studies are then difficult to which yeast is allowed to ferment. Yeast bio-

                                             16 | InternatIonal AquAFeed | november-December 2012
                                                                                                                     FEATURE




     Figure 5: Yeast rich in organic selenium manufacturing process



mass, substrate and fermented extracellular   in functional feeds, and hatcheries, as a      aquafeed in shrimp. In a trial performed in
metabolites are then dried.                   source of nucleotides complementing the        Thailand with white shrimp L. vannamei.
                                              de novo synthesis of cells in multiplication   Feed containing YE at 2 kg/T of feed was
Yeast Extracts.                               and helping boost immunity and anti-stress     presented in feeding trays at the corner of
   Yeast extracts (YE) come from the          mechanisms.                                    hapas and the remaining feed was counted
further hydrolysis and purification of           Autolysed yeast and inactive yeast are      after one hour. We can see a faster feed-
autolysed yeast. Insoluble yeast cell walls   commonly mistakenly sold on the label          ing when YE are included. (Tacon and
are separated from the cellular content       yeast extract in aquaculture. A good way       Suyawanish 2011).
by centrifugation. YE are very soluble,       to differentiate them is to look at the
rich in peptides (up to 65%-70% of the        carbohydrate levels. Autolysed yeast has       Yeast Cell walls
product), free amino-acids like glutamic      around 20-22% (mostly from the remaining          Yeast Cell Walls (YCW) represent
acid and vitamins. They also contain a high   YCW) whereas YE contain only three to          the shell of the yeast cell and are roughly
level of nucleic acid which can be further    six percent of carbohydrates.                  40-50 percent of the mass of the cell.
purified to increase the level of tasty 5’       The small peptides and free amino acids     YCW are composed mainly of fibrous
nucleotides. They are used in aquaculture     in YE can also prove a potent attractant for   polysaccharides glucans with beta 1,3 and




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                                         november-December 2012 | InternatIonal AquAFeed | 17
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                                                                  FEATURE
                        FEATURE

 a             beta 1,6 links, (50% and 8% respectively ), tration (2 kg/T) only for a short period, or              as both alpha and beta forms)or mannans.
 s are         mannans under the form of Mannoproteins a low concentration continuously (0.5 g/Kg).                  Not all the YCW are equal. Efficiency should
               (40%) and chitin (2%) (see Lippke and                     An example of sea-bass juveniles fed with   be checked as a prerequisite to use, or
   PER         Ovalle 1998). Further purification can lead Pronady (a YCW of the Lesaffre group) at                  change, YCW.
               to the production of either purified beta- 0.5 g/kg of feed for 8 weeks can be seen                       At LFA we have conducted a survey
  .83ab        glucans (50% and up) and mannoprotein in Figure 4, showing a significant protection                   of four YCW (2 bakery and 2 brewery
               (often used in wine making for clarifica- against L. Anguillarum without any growth                   yeasts) produced in 4 of our own factories
  .78ab
               tion). The presence of these compounds difference with the control. However a mini-                   in the same L. Anguillarum challenge in sea
  .89cd        often leads to the mislabelling of YCW as mal amount of YCW seems needed to be                        bass supplemented at 0.5 g/kg of feed for
2.64d          MOS or Beta-glucans.                                 ingested before challenge in order to provide    8 weeks. Only 2 responded significantly (1
 2.26c             These two carbohydrate types are very an efficient immunostimulation and so there                 bakery, 1 brewery), the remaining 2 had even
 3.17e         interesting for the aquaculture market, beta- might be a gap period when the product is               negative results at 4 weeks (lower survival
               glucans are direct stimulators of the immune not efficient. (data from Dr. Morgane Henry,             than control). This result shows first that not
               systems in shrimp and fish, upon the stimula- Hellenic Center for marine Research , 2011)             all is understood in the way these products
   can also
               tion of specific Schemacellsa(granulocytes or
                      Figure 2: blood of process to produce yeast        YCW products, depending on the quality      work and that one particular YCW cannot
 s a more
                      extracts  Mannans are involved in the of the autolysed yeast separation, contain
               macrophages). and yeast cell walls                                                                    be replaced by another.
 duct and
               binding to pathogenic bacteria (especially also significant percentages of proteins and
   and find
               those with pili having mannose receptors) lipids. It should be noted that the lower the               Selenium Yeast
                                                   and and                                  is
man care as a food supplement tively high intestine. its amino acid contentthe higher of level of carbo-
               and eliminate them from the                   It level of proteins,                                       Yeast can be induced to be a source of
                                                                      Numerous then
  rapeutic, also known as naturalthatsimilar to baker’s yeast. hydrates, and worksthe better immunostimu-
               is also suspected       the mannanes act as                                                           organic selenium, mainly under the form of
                                      have shown beneficial lation from the to
               prebiotics promoting the growth of the efficacy of Brewer’s yeast YCW is. Various quality of          seleniomethionine, which is then stored in
                                                                     the proteins the animal production market
east for aquafeed applications replace partially or totally YCW are on found
               bacteria.                                                                                             proteins. During the growth of baker’s yeast,
                   YCW have been in fish to vegetable meal in major differences
 ng companies as a commodity shown and be effective and fish and shrimp. can be found between                        selenium is added to the medium and is
               to improve local Shrimp bacterial chal- products depending
   protein content, or bythe resistance to feeds formulators typically incorporate on the strain, the sub-           replacing sulphur in methionine. The excess
               lenges in numerous brewer’s species. Beta strate used rate of
eed to dispatch their slurry. The aquaculture yeast in their formula at theto produce the yeast, and event           of selenium is then eliminated by careful
               glucans have to be two to four percent.
    supply of these products can use carefully in aquacul- the drying process.                                       washing steps (see Figure 5) to ensure that
                                          Brewer’s negative
 t and depends greatly on experiments report yeast can be used to produce
               ture as some the                                          Mannans represent as most 25-27 per-        the selenium left is 97-99 percent organic.
 ly.                                   used for prolonged cent of YCW in of
               effects in fish when yeast fractions, however due to the naturegood quality YCW from pri-             Selenium yeast should be then checked for
onal content is similar high the brewer’s yeast and the specificity of the pro- but can be found as low
               periods at as concentrations.. This can be mary grown yeasts                                          the highest percentage of selenomethio-
               avoided by trace duction the source of as is percent in crude preparation coming
  yeast, but contains morecareful choosing processes, the quality 9 less consistent                                  nine and the consistency between batches.
                                      either baker’s yeast.         from coming from
   as selenium andand using them than in at high concen- Productsindustry by-products. Glucans or poly-
               YCW chromium.                                                                                         Seleniomethionine is the main carbon-asso-
ontent of brewer yeast is rela- brewery yeast tend to have a distinctive bitterfrom 18 To 40 percent.
                                                                    glucose can range                                ciated form of selenium in the animal’s body
                                                                                                 YCW Protein         and then allow making organic selenium
                                                                                                 level remains       which are readily available when oxidative

                        ming
                                                                                                 the most con-       stress reactions occur.

                  Upco r          2013
                                                                                                 venient indica-         The main application would be in aquacul-
                       fo                                                                        tor of quality,     ture as fish meal is a main supply of selenium
               E vents         World Aquaculture                                                 the best prod-      and the development of diets with less fish
                                                                                                 ucts being those    meal will require compensation of selenium
                       The international triennial
                       conference & exposition of                                                having lower        in aquafeed formulae. Such an application
                       World Aquaculture Society                                                 nitrogen con-       could be useful in preventing the oxidation


3
                                                                                                 tent. The vari-     of poly unsaturated fatty acids (PUFA) in
                                                                                                 ability between     fish flesh. Chromium yeast is seldom used in
                       February 21 - 25, 2013
                                                                                                 batches can also    aquaculture diets.
                       Nashville, Tennessee                    For all info contact us on
sia                                                                                              be very high.
                       USA                                                 www.was.com
                                                                   worldaqua@aol.com             Texture should      Conclusion
                                                                                                 be checked first.        Yeast products are getting more fre-
                                                        Aquaculture Europe
                                                                                                 Good       YCW      quently used in aquaculture. Some appli-
                                                                                                 often have a        cations are promising as the use as an
                                                         August 9 - 12, 2013
                                                                                                 smooth,      fine   alternative source of proteins or as a sanitary
                                                         Trondheim, Norway
                                                                                                 texture,     low    and welfare enhancer. However many prod-
                                                          Organised by European Aquaculture
                                                                                    Society      granulometry        ucts ranging from crude ethanol yeast by-
                                                            For all info contact us on           and a light beige   products to more purified beta-glucans are
                                                                   www.easonline.org             colour. There is    available on the market. Therefore potential
W                        Making Sense of Science                                                 also the tenden-    users must accurately select them in func-
  at                                                                                             cy to believe       tion of their targeted application. It is also
                        Asia Pacific Aquaculture                                                 that all YCW        as important to select a reliable source of
                        December 10-13th,                                                        are the same        the products to ensure a consistency of the
                                                                                                 and that dif-       supply.
                        2013
                                                                                                 ferentiation of
                        Ho Chi Minh City, Vietnam
                                                                                                 products must
                                                               For all info contact us on        be done to the        More InforMatIon:
                                                                           www.was.com
                                                                   worldaqua@aol.com
                                                                                                 highest level of      Website: www.lesaffre.com
                                                                                                 glucans (some-
                                                                                                 times measured

                                                           18 | InternatIonal AquAFeed | november-December 2012
 012 | InternatIonal AquAFeed | 15
The Future of Feeds
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                                                  Instant Algae in our
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                      VO L U M E 1 5 I S S U E 6 2 0 1 2
                                                                                          •	 See	the	full	issue
                                                An overview
                          of the UK fish vaccination industry                             •	   Visit	the	International	Aquafeed	website
                                   Why check selenomethionine
                                       levels in selenium yeast?

                               Extrusion technology for the
                          production of micro-aquatic feeds
                                                                                          •	   Contact	the	International	Aquafeed	Team
                                          and shrimp feeds

                                                           EXPERT TOPIC
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IAF12.06.indd 1                                                        07/11/2012 17:39




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