November | December 2012 Yeast in aquaculture

International Aquafeed is published five times a year by Perendale Publishers Ltd of the United Kingdom. All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. Copyright 2012 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058

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FEATURE

Yeast in aquaculture
by Philippe Tacon PhD, Lesaffre Feed Additives, France
east 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. Yeast is a unicellular organism belonging to the kingdom of Fungi. More than a thousand species have been found in two major phyla: Basidiomycota and Ascomycota in which belong species able to duplicate by budding such as Saccharomyces cerevisiae. Due to their unique properties to grow under aerobic conditions and produce gas and ethanol under anaerobic conditions, some yeast (mostly S. cerevisiae) have been used for the manufacture of fermented foods such as bread , beer and wine for a long time. Yeasts are also used as single sell protein source in animal nutrition and in aquaculture under various forms and species (Torulaspora, Torulopsis, Kluyveromyces, Saccharomyce et caetera). It can be found for example in

shrimp and marine fish larval feeds or included as a protein source in aquafeeds. The reasons for this extensive use is its excellent nutritional contents, its easy supply in dried form or under liquid form when bakery yeast plants or breweries are near aquafeed plants, and nowadays a competitive price in regards to other protein sources such as fish or soybean meal. Further applications are being developed for yeast as functional feed additives as probiotic live yeast, yeast fractions (yeast cell walls, yeast extracts) or as a source for more purified products such as beta-glucans and nucleotides. The production process of yeast can allow the possibility to incorporate trace minerals and then produce highly bioavailable organic trace minerals, also known as selenium and chromium yeast. The pink yeast Phaffia rhodozyma, is naturally rich in astaxanthin and has been used for some time as natural source of the pigment in salmonids. Although now it tends to be replaced by bacterial products which have a higher concentration and whose cell wall is more easily degraded. We will only refer in the following article on products coming from S. cerevisiae origin.

Nutritional properties of yeast:

Typical dry yeast composition is 93-97 percent dry matter and can contain from 40% to 60 percent crude protein nitrogen, 35-45 percent carbohydrates, and 5-9 percent lipids. A quite important fraction of the nitrogen is under the form on nucleic acids (up to 12%) that can lead to produce significant level of uric acid if consumed at high concentration, like meat. The Amino acid profile of yeast is close to soybean meal and therefore well adapted to animal nutrition; it is rich in Glutamic acid and Lysine (up to 8%). Yeast is naturally rich in B vitamins such as biotin, thiamine and folic acid. It also produces niacin but contrary to some belief does not produce B12 Vitamin. Ergosterol which is a significant fraction of yeast cell wall, also is also a precursor of Vitamin D2 by using UV treatments.

Bakers yeast
Even if their name remains Saccharomyces cerevisiae (cerevisiae for beer), most of the strains of Bakers yeast have been selected for their high fermentative power, particularly useful for bakers.Strains are specific to the 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,). Bakers yeast comes as a pure and primary culture grown on sugar substrate such as molasses. The production is performed under very strict conditions in order to maintain 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, compressed, 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 Bakers yeast usually have a high content of mannans. They are

Figure 1: Yeast manufacturing process (primary grown culture)

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 contract vaccinasafety and welfare of the(n=3, P<0.05, measures with different letters are fish. under stress conditions. significantly different) The fish are then delivered onto a stainless tion team, is poor the steel table where the fish then(%) vaccinated anaesthesia of PER Treatment Survival are SGR FCR 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 vacCON 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 0.78ab tors handle between 15-20,000 fish each this Poor anaesthesia of Act some 10 fry 87.5bc 5.80d 1.43abc 1.89cd the fish can lead to is quite 40% - achievement! The 40% are then returned to a recovery high mortalities. If fish - 20 fry Act 92.6c 5.43c 1.01a 2.64d tankAct 27% - 10come round from the anaes- fish are under anaesand 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 dangerrecognised as good 100 binders. Fractions drum them (imagjust 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.bakers mortalities immediately ine having a as a more extracts and yeast cell walls controlled product and colour. whilst after vaccination are usually attributed to poor operation specific strains and The most popular the injection. anaesthesia rather than aquaculture application only partly sedated!). find its the other hand, 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 Bakers yeast is in of poor vaccination major feed source forapparent and rotifer (see ifand holistic therapeutic, also known as natural similar to bakers yeast. Numerous works artemia months after they are over usually only become have shown the efficacy of Brewers yeast to brewers yeast. for example Couteau up until harvest where anaesthetised, they vaccination and can last et al 1990). Brewers yeast for aquafeed applications replace partially or totally the proteins found the financially consequences become appar- risk not recoveris quickly enough, Brewers Yeast ing sold by trading companies as a commodity in fish and vegetable meal in fish and shrimp. ent. The main problems are: Brewers yeast can be identified either based in piles of Incorrect needle depth resulting in either resultingon the protein content, or by local Shrimp feeds formulators typically incorporate as the ferment used (needle too short) or fish in the in need to dispatch their slurry. The brewers yeast in their formula at the rate of intra-muscular injection in brewery industries breweries recovery (Yeast organ production) or the by-product quality and suffocainternal primary damage, including granuloma tank causing the supply of these products can two to four percent. Brewers yeast can be used to produce of these industries which is the form mainly be or even worse, (needle too long) which results in the fish not tion inconsistent and depends greatly on the yeast fractions, however due to the nature of source of supply. used in properly due to damage tocase, gut. aquaculture. In the latter the yeast not recovering at all. growing The other risk biomassnot harvested from the the disease The nutritional content is similar as the brewers yeast and the specificity of the proFish is being immune to fermentation vats at the end of beer fermentation. vaccine) factorin bakers yeast, but contains more trace duction processes, the quality is less consistent It can be one here is to the because of incorrect dosage (or no minerals such the sold delivered. being under liquid form (18-20% of dry matter) vaccinators. If as selenium and chromium. than in bakers yeast. Products coming from The protein content but preferentiallymaininactive yeastSalmovac1 mineral oils used in of brewer yeast is rela- brewery yeast tend to have a distinctive bitter as problems spray or 29/10/2012 13:57 Two ridgewaybio_quarterpage_print.pdf of the

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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 the autolysis process interest. However, such material is often difis Pronady at 0.5g/kg. n=3, Pronady significantly degrades protein and ficult to grow under industrial conditions and decreases mortality at 120h. P<0.01) forms peptides (dipep- did not lead to the development of an actual tides to tetra peptides) product yet. The products on the market are and oligonucleic acids which are readily digest- therefore often from S. cerevisiae origin. It has smell and taste and a darker colour than the ible by the animal. Again here depending on to be noted that up to now, no yeast prodones coming from bakers yeast. the original yeast material used, autolysed and ucts have been registered in EU as a probiotic inactive yeast quality can be very different. in aquaculture. Ethanol Yeast As an example of S. cerevisiae effects, Ethanol Yeast are harvested after hav(Lara Flores et al 2003, 2010) Table 2 shows ing performed alcoholic fermentation and Live Yeast as probiotics 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 condithe recycled energy used to heat the vegetal developed and produced industrially such as tions (1 fry per L or 1 fry per 2L). All the yeast treatments also increased the material. The majority of ethanol yeast comes S. cerevisiae boulardii or S. cerevisiae Sc 47 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 characucts. 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 Brewers yeast. cfu/g. Dosages They are major products within the food are made in Figure 3: Number of pellets remaining in the feeding tray one industry as flavour enhancers and in pet food the feeds as 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 Brewers yeast, and its ethanol equivalent, is count per g of mostly favoured as it is cheaper than bakers feed, a 1000 fold dilution giving a 10e107 per (low protein percentage and crowded condiyeast. 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 Bakers yeast on food markets. checked before and after pelleting using plate when Actisaf is used. Live yeast can be used directly on farm, Inactive yeast is a yeast that has been counts. 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 bio16 | InternatIonal AquAFeed | november-December 2012

FEATURE

Figure 5: Yeast rich in organic selenium manufacturing process

mass, substrate and fermented extracellular metabolites are then dried.

Yeast Extracts.
Yeast extracts (YE) come from the further hydrolysis and purification of autolysed yeast. Insoluble yeast cell walls are separated from the cellular content by centrifugation. YE are very soluble, rich in peptides (up to 65%-70% of the product), free amino-acids like glutamic acid and vitamins. They also contain a high level of nucleic acid which can be further purified to increase the level of tasty 5 nucleotides. They are used in aquaculture

in functional feeds, and hatcheries, as a source of nucleotides complementing the de novo synthesis of cells in multiplication and helping boost immunity and anti-stress mechanisms. Autolysed yeast and inactive yeast are commonly mistakenly sold on the label yeast extract in aquaculture. A good way to differentiate them is to look at the carbohydrate levels. Autolysed yeast has around 20-22% (mostly from the remaining YCW) whereas YE contain only three to six percent of carbohydrates. The small peptides and free amino acids in YE can also prove a potent attractant for

aquafeed in shrimp. In a trial performed in Thailand with white shrimp L. vannamei. Feed containing YE at 2 kg/T of feed was presented in feeding trays at the corner of hapas and the remaining feed was counted after one hour. We can see a faster feeding when YE are included. (Tacon and Suyawanish 2011).

Yeast Cell walls

Yeast Cell Walls (YCW) represent the shell of the yeast cell and are roughly 40-50 percent of the mass of the cell. YCW are composed mainly of fibrous polysaccharides glucans with beta 1,3 and

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FEATURE

FEATURE
as both alpha and beta forms)or mannans. Not all the YCW are equal. Efficiency should be checked as a prerequisite to use, or change, YCW. At LFA we have conducted a survey of four YCW (2 bakery and 2 brewery yeasts) produced in 4 of our own factories in the same L. Anguillarum challenge in sea bass supplemented at 0.5 g/kg of feed for 8 weeks. Only 2 responded significantly (1 bakery, 1 brewery), the remaining 2 had even negative results at 4 weeks (lower survival than control). This result shows first that not all is understood in the way these products work and that one particular YCW cannot be replaced by another.

beta 1,6 links, (50% and 8% respectively ), tration (2 kg/T) only for a short period, or mannans under the form of Mannoproteins a low concentration continuously (0.5 g/Kg). An example of sea-bass juveniles fed with (40%) and chitin (2%) (see Lippke and PER Ovalle 1998). Further purification can lead Pronady (a YCW of the Lesaffre group) at to the production of either purified beta- 0.5 g/kg of feed for 8 weeks can be seen glucans (50% and up) and mannoprotein in Figure 4, showing a significant protection .83ab (often used in wine making for clarifica- against L. Anguillarum without any growth .78ab tion). The presence of these compounds difference with the control. However a mini.89cd often leads to the mislabelling of YCW as mal amount of YCW seems needed to be 2.64d ingested before challenge in order to provide MOS or Beta-glucans. These two carbohydrate types are very an efficient immunostimulation and so there 2.26c interesting for the aquaculture market, beta- might be a gap period when the product is 3.17e glucans are direct stimulators of the immune not efficient. (data from Dr. Morgane Henry, systems in shrimp and fish, upon the stimula- Hellenic Center for marine Research , 2011) can also YCW products, depending on the quality tion of specific Schemacellsa(granulocytes or Figure 2: blood of process to produce yeast s a more macrophages). and yeast cell walls Mannans are involved in the of the autolysed yeast separation, contain extracts 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 and and is man care as a food supplement tively high intestine. its amino acid contentthe higher of level of carboand eliminate them from the It level of proteins, Numerous then rapeutic, also known as naturalthatsimilar to bakers yeast. hydrates, and worksthe better immunostimuis also suspected the mannanes act as have shown beneficial lation from the to prebiotics promoting the growth of the efficacy of Brewers yeast YCW is. Various quality of the proteins the animal production market east for aquafeed applications replace partially or totally YCW are on found bacteria. ng companies as a commodity shown and be effective and fish and shrimp. can be found between YCW have been in fish to vegetable meal in major differences protein content, or bythe resistance to feeds formulators typically incorporate on the strain, the subto improve local Shrimp bacterial chal- products depending eed to dispatch their slurry. The aquaculture yeast in their formula at theto produce the yeast, and event lenges in numerous brewers species. Beta strate used rate of supply of these products can use carefully in aquacul- the drying process. glucans have to be two to four percent. Brewers negative t and depends greatly on experiments report yeast can be used to produce Mannans represent as most 25-27 perture as some the ly. effects in fish when yeast fractions, however due to the naturegood quality YCW from priused for prolonged cent of YCW in of onal content is similar high the brewers yeast and the specificity of the pro- but can be found as low periods at as concentrations.. This can be mary grown yeasts yeast, but contains morecareful choosing processes, the quality 9 less consistent avoided by trace duction the source of as is percent in crude preparation coming as selenium andand using them than in at high concen- Productsindustry by-products. Glucans or polyfrom coming from YCW chromium. either bakers yeast. ontent of brewer yeast is rela- brewery yeast tend to have a distinctive bitterfrom 18 To 40 percent. glucose can range YCW Protein level remains the most convenient indica2013 tor of quality, the best prodWorld Aquaculture ucts being those The international triennial having lower conference & exposition of nitrogen conWorld Aquaculture Society tent. The variability between February 21 - 25, 2013 batches can also Nashville, Tennessee For all info contact us on be very high. sia www.was.com USA Texture should worldaqua@aol.com be checked first. Aquaculture Europe Good YCW often have a August 9 - 12, 2013 smooth, fine Trondheim, Norway texture, low Organised by European Aquaculture granulometry Society and a light beige For all info contact us on www.easonline.org colour. There is Making Sense of Science W also the tendenat cy to believe Asia Pacific Aquaculture that all YCW are the same December 10-13th, and that dif2013 ferentiation of Ho Chi Minh City, Vietnam products must For all info contact us on be done to the www.was.com highest level of worldaqua@aol.com glucans (sometimes measured

a s are

Selenium Yeast
Yeast can be induced to be a source of organic selenium, mainly under the form of seleniomethionine, which is then stored in proteins. During the growth of bakers yeast, selenium is added to the medium and is replacing sulphur in methionine. The excess of selenium is then eliminated by careful washing steps (see Figure 5) to ensure that the selenium left is 97-99 percent organic. Selenium yeast should be then checked for the highest percentage of selenomethionine and the consistency between batches. Seleniomethionine is the main carbon-associated form of selenium in the animals body and then allow making organic selenium which are readily available when oxidative stress reactions occur. The main application would be in aquaculture as fish meal is a main supply of selenium and the development of diets with less fish meal will require compensation of selenium in aquafeed formulae. Such an application could be useful in preventing the oxidation of poly unsaturated fatty acids (PUFA) in fish flesh. Chromium yeast is seldom used in aquaculture diets.

ming Upco r fo vents E

Conclusion
Yeast products are getting more frequently used in aquaculture. Some applications are promising as the use as an alternative source of proteins or as a sanitary and welfare enhancer. However many products ranging from crude ethanol yeast byproducts to more purified beta-glucans are available on the market. Therefore potential users must accurately select them in function of their targeted application. It is also as important to select a reliable source of the products to ensure a consistency of the supply.

More InforMatIon:
Website: www.lesaffre.com

012 | InternatIonal AquAFeed | 15

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