November | December 2012

Yeast in aquaculture

The International magazine for the aquaculture feed industry

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

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 dif-ferentiating 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 natu-rally 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 concentra-tion, 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 precur-sor of Vitamin D2 by using UV treatments.

Baker’s yeastEven if their name remains Saccharomyces

cerevisiae (cerevisiae for beer), most of the strains of Baker’s 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,…).

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 have a high content of mannans. They are

Yeast in aquacultureby Philippe Tacon PhD, Lesaffre Feed Additives, France

14 | InternatIonal AquAFeed | november-December 2012

FEATURE

november-December 2012 | InternatIonal AquAFeed | 15

Figure 1: Yeast manufacturing process (primary grown culture)

IAF12.06.indd 14 07/11/2012 17:40

recognised as good toxin binders. Fractions coming from baker’s yeast have a light beige colour.

The most popular aquaculture application of Baker’s yeast is in hatcheries where it is a major feed source for artemia and rotifer (see for example Couteau et al 1990).

Brewer’s YeastBrewer’s yeast can be identified either

as the ferment used in brewery industries (Yeast primary production) or the by-product of these industries which is the form mainly used in aquaculture. In the latter case, yeast biomass is harvested from the fermentation vats at the end of beer fermentation. It can be sold under liquid form (18-20% of dry matter) but preferentially as inactive yeast spray or

drum dried. It can also been grown as a more controlled product and specific strains and find its way to human care as a food supplement and holistic therapeutic, also known as natural brewer’s yeast.

Brewer’s yeast for aquafeed applications is sold by trading companies as a commodity based on the protein content, or by local breweries in need to dispatch their slurry. The quality and the supply of these products can be inconsistent and depends greatly on the source of supply.

The nutritional content is similar as the one in baker’s yeast, but contains more trace minerals such as selenium and chromium. The protein content of brewer yeast is rela-

tively high and and its amino acid content is similar to baker’s yeast. Numerous works have shown the efficacy of Brewer’s yeast to replace partially or totally the proteins found in fish and vegetable meal in fish and shrimp. Shrimp feeds formulators typically incorporate brewer’s yeast in their formula at the rate of two to four percent.

Brewer’s yeast can be used to produce yeast fractions, however due to the nature of brewer’s yeast and the specificity of the pro-duction processes, the quality is less consistent than in baker’s yeast. Products coming from brewery yeast tend to have a distinctive bitter

Table 1: Effect of live yeast Actisaf on growth parameters in tilapia under stress conditions. (n=3, P<0.05, measures with different letters are significantly different)

Treatment Survival (%) SGR FCR PER

CON 40% -10 fry 75.0ab 3.33a 3.11e 0.83ab

CON 40% -20 fry 64.8a 3.47a 3.26e 0.78ab

Act 40% - 10 fry 87.5bc 5.80d 1.43abc 1.89cd

Act 40% - 20 fry 92.6c 5.43c 1.01a 2.64d

Act 27% - 10 fry 91.7bc 5.46cd 1.62bc 2.26c

Act 27% - 20 fry 96.29c 5.24c 1.17ab 3.17e

14 | InternatIonal AquAFeed | november-December 2012 november-December 2012 | InternatIonal AquAFeed | 15

FEATURE

Figure 2: Schema of a process to produce yeast extracts and yeast cell walls

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important for vaccinator safety but also the safety and welfare of the fish.

The fish are then delivered onto a stainless steel table where the fish then are vaccinated in a very specific area, with only a 3 mm toler-ance. The team must achieve a 96 percent accuracy target and considering most vaccina-tors handle between 15-20,000 fish each this is quite some achievement!

The fish are then returned to a recovery tank and should come round from the anaes-thetic within about two to three minutes. There is always some level of mortality after this high risk, stressful process but usually it is just a few fish, around 100 for every 100,000 fish vaccinated. High mortalities immediately after vaccination are usually attributed to poor anaesthesia rather than the injection.

The consequences of poor vaccination usually only become apparent months after vaccination and can last up until harvest where the financially consequences become appar-ent. The main problems are:

Incorrect needle depth resulting in either intra-muscular injection (needle too short) or internal organ damage, including granuloma (needle too long) which results in the fish not growing properly due to damage to the gut.

Fish not being immune to the disease because of incorrect dosage (or no vaccine) being delivered.

Two of the main problems Salmovac

encounters as a contract vaccina-tion team, is poor anaesthesia of the fish and also poor grading, prior to vac-cination of the fish. Poor anaesthesia of the fish can lead to high mortalities. If fish are under anaes-thetised, the whole process becomes stressful and danger-ous for them (imag-ine having a major operation whilst only partly sedated!). On the other hand, if they are over anaesthetised, they risk not recover-ing quickly enough, resulting in piles of fish in the recovery tank causing suffoca-tion or even worse, not recovering at all.

The other risk factor here is to the vaccinators. If the mineral oils used in

10 | InternatIonal AquAFeed | november-December 2012 november-December 2012 | InternatIonal AquAFeed | 11

FEATURE

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Consistent products supported bythe Lesaffre group experience andits unique know-how in biotechnologyand nutrition;

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Dedicated range of products:live yeast, yeast cell wall, yeast extractand enriched yeast.

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IAF12.06.indd 11 07/11/2012 17:40

smell and taste and a darker colour than the ones coming from baker’s yeast.

Ethanol Yeast Ethanol Yeast are harvested after hav-

ing performed alcoholic fermentation and distillation for the conventional production of Bioethanol from sugarcane, beet sugar or grains syrup. In the first case, the yeast biomass is harvested and then dried with the recycled energy used to heat the vegetal material. The majority of ethanol yeast comes from Brazil.

Production prices and selling prices are very low, however the quality, such as the protein content is very inconsistent. The sup-ply depends on the activity of the bioethanol plants and can also be inconsistent. Another concern is the sanitary safety of these prod-ucts. Antibiotics are sometimes added to the process in order to prevent bacteria compet-ing with the yeast for nutrients andavoiding yield decrease. It is therefore possible that some antibiotic residues and possibly other toxins might be left in the final dried product.

Autolysed yeast – Inactive Dried Yeast Inactive and Autolysed yeast come from

primary grown cultures or Brewer’s yeast. They are major products within the food industry as flavour enhancers and in pet food as feed attractants. They are used in aquacul-ture feeds as a source of protein and nitrogen. Brewer’s yeast, and its ethanol equivalent, is mostly favoured as it is cheaper than baker’s yeast. They are also easier supplied as yeast suppliers prefer to sell the more controlled and tailored Baker’s yeast on food markets.

Inactive yeast is a yeast that has been deactivated by high temperature drying (often spray drying). The cells come as a whole and the cell wall is not ruptured making the access to intracellular material (amino acids, vitamins…) difficult. A way to access these materials is to partially hydrolyse the yeast cell wall to let the cellular content be partially released from the cell. This can be facilitated by activating the internal autolytic enzymes of the live yeast (autolysis), adding external

enzymes (notably pro-teolysis) or playing on the osmotic pressure to rupture the cell wall (plasmolysis). Different grades of autolysed yeast can be obtained depending on the level of autolysis (from partial to total). The final prod-uct is a mixture of cel-lular content and yeast cell wall. Furthermore the autolysis process degrades protein and forms peptides (dipep-tides to tetra peptides)

and oligonucleic acids which are readily digest-ible by the animal. Again here depending on the original yeast material used, autolysed and inactive yeast quality can be very different.

Live Yeast as probiotics Live yeast helps regulate the gut microbio-

ta. Its effects have been shown, first in human where it can reduce diarrhoea, especially with children. Specific strains have then been developed and produced industrially such as S. cerevisiae boulardii or S. cerevisiae Sc 47 (Actisaf) for the animal nutrition market. It is a common practice now to supplement feeds to increase milk production in dairy cows or help piglets survival.

Live yeast are charac-terized by their living cells count, expressed by colony forming unit (cfu per gram), typically ten billions cfu/g. Dosages are made in the feeds as dilutions to get an efficient cfu count per g of feed, a 1000 fold dilution giving a 10e107 per g of feed for example. Viability of the yeast is mandatory for its effect and cfus should be checked before and after pelleting using plate counts.

Despite the increasing use of yeast as a probiotic in terrestrial animals, there are only a few numbers of works studying its effect in fish as a gut functions stabi-liser. The major reason is that live yeast does not resist the severe conditions of the manufacturing processes of aquafeeds (high temperatures, steam, long condi-tioning times, see Aguirre-Guzzman et al 2002). The studies are then difficult to

transfer from lab conditions to farm using commercial feeds.

All the work published so far was made with yeast either top dressed on feeds or incorporated in pressed (uncooked) feeds. Nevertheless some direct effects to the gut maturation have been found in sea bass with a species extracted from the rainbow trout gut Debaryomyces hansenii (see the works from Tovar-Ramirez and also the reviews by Chi et al 2006 and Gatesoupe 2007). Marine yeasts and yeasts isolated from fish seem a very logical choice to use in species of aquaculture interest. However, such material is often dif-ficult to grow under industrial conditions and did not lead to the development of an actual product yet. The products on the market are therefore often from S. cerevisiae origin. It has to be noted that up to now, no yeast prod-ucts have been registered in EU as a probiotic in aquaculture.

As an example of S. cerevisiae effects, (Lara Flores et al 2003, 2010) Table 2 shows some works done in tilapia fry fed for 3 weeks with feeds supplemented with Actisaf (also knwn as Biosaf) at 1 kg/T in two diets (40% and 27% proteins) and at 2 crowded condi-tions (1 fry per L or 1 fry per 2L).

All the yeast treatments also increased the alkaline Phosphatase activity, and we can see a better improvement of feed conversion ratio (FCR) and survival under stressful conditions

(low protein percentage and crowded condi-tions). There is also a better protein efficiency ratio (PER) and digestive enzyme activity when Actisaf is used.

Live yeast can be used directly on farm, where it has been showed (empirically) to improve water quality in shrimp and fish ponds. It is either used alone or mixed with bacteria. Farms producing mash feed onsite also add yeast in order to degrade cellulolytic material to ensure a better digestion.

Yeast Culture or fermented yeast. Yeast culture is a particular product in

which yeast is allowed to ferment. Yeast bio-

16 | InternatIonal AquAFeed | november-December 2012

FEATURE

november-December 2012 | InternatIonal AquAFeed | 17

Figure 4: Cumulative mortality after immersion with L. anguiilarum (blue line is control, orange line is Pronady at 0.5g/kg. n=3, Pronady significantly decreases mortality at 120h. P<0.01)

Figure 3: Number of pellets remaining in the feeding tray one hour after feeding (n=4, YE are significantly different than control at P<0.05).

IAF12.06.indd 16 07/11/2012 17:40

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 feed-ing when YE are included. (Tacon and Suyawanish 2011).

Yeast Cell wallsYeast 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

16 | InternatIonal AquAFeed | november-December 2012 november-December 2012 | InternatIonal AquAFeed | 17

FEATURE

Figure 5: Yeast rich in organic selenium manufacturing process

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beta 1,6 links, (50% and 8% respectively ), mannans under the form of Mannoproteins (40%) and chitin (2%) (see Lippke and Ovalle 1998). Further purification can lead to the production of either purified beta-glucans (50% and up) and mannoprotein (often used in wine making for clarifica-tion). The presence of these compounds often leads to the mislabelling of YCW as MOS or Beta-glucans.

These two carbohydrate types are very interesting for the aquaculture market, beta-glucans are direct stimulators of the immune systems in shrimp and fish, upon the stimula-tion of specific blood cells (granulocytes or macrophages). Mannans are involved in the binding to pathogenic bacteria (especially those with pili having mannose receptors) and eliminate them from the intestine. It is also suspected that the mannanes act as prebiotics promoting the growth of beneficial bacteria.

YCW have been shown to be effective to improve the resistance to bacterial chal-lenges in numerous aquaculture species. Beta glucans have to be use carefully in aquacul-ture as some experiments report negative effects in fish when used for prolonged periods at high concentrations.. This can be avoided by careful choosing the source of YCW and using them either at high concen-

tration (2 kg/T) only for a short period, or a low concentration continuously (0.5 g/Kg).

An example of sea-bass juveniles fed with Pronady (a YCW of the Lesaffre group) at 0.5 g/kg of feed for 8 weeks can be seen in Figure 4, showing a significant protection against L. Anguillarum without any growth difference with the control. However a mini-mal amount of YCW seems needed to be ingested before challenge in order to provide an efficient immunostimulation and so there might be a gap period when the product is not efficient. (data from Dr. Morgane Henry, Hellenic Center for marine Research , 2011)

YCW products, depending on the quality of the autolysed yeast separation, contain also significant percentages of proteins and lipids. It should be noted that the lower the level of proteins, the higher of level of carbo-hydrates, and then the better immunostimu-lation from the YCW is. Various quality of YCW are on the animal production market and major differences can be found between products depending on the strain, the sub-strate used to produce the yeast, and event the drying process.

Mannans represent as most 25-27 per-cent of YCW in good quality YCW from pri-mary grown yeasts but can be found as low as 9 percent in crude preparation coming from industry by-products. Glucans or poly-glucose can range from 18 To 40 percent.

YCW Protein level remains the most con-venient indica-tor of quality, the best prod-ucts being those having lower nitrogen con-tent. The vari-ability between batches can also be very high. Texture should be checked first. Good YCW often have a smooth, fine texture, low granulometry and a light beige colour. There is also the tenden-cy to believe that all YCW are the same and that dif-ferentiation of products must be done to the highest level of glucans (some-times measured

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.

Selenium YeastYeast 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 baker’s 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 selenomethio-nine and the consistency between batches. Seleniomethionine is the main carbon-asso-ciated form of selenium in the animal’s body and then allow making organic selenium which are readily available when oxidative stress reactions occur.

The main application would be in aquacul-ture 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.

Conclusion Yeast products are getting more fre-

quently used in aquaculture. Some appli-cations are promising as the use as an alternative source of proteins or as a sanitary and welfare enhancer. However many prod-ucts ranging from crude ethanol yeast by-products to more purified beta-glucans are available on the market. Therefore potential users must accurately select them in func-tion 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

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IAF12.06.indd 18 07/11/2012 17:40

recognised as good toxin binders. Fractions coming from baker’s yeast have a light beige colour.

The most popular aquaculture application of Baker’s yeast is in hatcheries where it is a major feed source for artemia and rotifer (see for example Couteau et al 1990).

Brewer’s YeastBrewer’s yeast can be identified either

as the ferment used in brewery industries (Yeast primary production) or the by-product of these industries which is the form mainly used in aquaculture. In the latter case, yeast biomass is harvested from the fermentation vats at the end of beer fermentation. It can be sold under liquid form (18-20% of dry matter) but preferentially as inactive yeast spray or

drum dried. It can also been grown as a more controlled product and specific strains and find its way to human care as a food supplement and holistic therapeutic, also known as natural brewer’s yeast.

Brewer’s yeast for aquafeed applications is sold by trading companies as a commodity based on the protein content, or by local breweries in need to dispatch their slurry. The quality and the supply of these products can be inconsistent and depends greatly on the source of supply.

The nutritional content is similar as the one in baker’s yeast, but contains more trace minerals such as selenium and chromium. The protein content of brewer yeast is rela-

tively high and and its amino acid content is similar to baker’s yeast. Numerous works have shown the efficacy of Brewer’s yeast to replace partially or totally the proteins found in fish and vegetable meal in fish and shrimp. Shrimp feeds formulators typically incorporate brewer’s yeast in their formula at the rate of two to four percent.

Brewer’s yeast can be used to produce yeast fractions, however due to the nature of brewer’s yeast and the specificity of the pro-duction processes, the quality is less consistent than in baker’s yeast. Products coming from brewery yeast tend to have a distinctive bitter

Table 1: Effect of live yeast Actisaf on growth parameters in tilapia under stress conditions. (n=3, P<0.05, measures with different letters are significantly different)

Treatment Survival (%) SGR FCR PER

CON 40% -10 fry 75.0ab 3.33a 3.11e 0.83ab

CON 40% -20 fry 64.8a 3.47a 3.26e 0.78ab

Act 40% - 10 fry 87.5bc 5.80d 1.43abc 1.89cd

Act 40% - 20 fry 92.6c 5.43c 1.01a 2.64d

Act 27% - 10 fry 91.7bc 5.46cd 1.62bc 2.26c

Act 27% - 20 fry 96.29c 5.24c 1.17ab 3.17e

14 | InternatIonal AquAFeed | november-December 2012 november-December 2012 | InternatIonal AquAFeed | 15

FEATURE

Figure 2: Schema of a process to produce yeast extracts and yeast cell walls

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Special themes

IAF12.06.indd 15 07/11/2012 17:40

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VOLUME 15 I S SUE 6 2 012

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An overview of the UK fish vaccination industry

Why check selenomethionine levels in selenium yeast?

Extrusion technology for the production of micro-aquatic feeds

and shrimp feeds

EXPERT TOPIC– Salmon

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