Digital Re-print January | February 2013

Yeast in aquaculture
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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 Bakers yeast the manufacture of fermented foods such as Even if their name remains Saccharomyces bread , beer and wine for a long time. Yeasts cerevisiae (cerevisiae for beer), most of the are also used as single sell protein source in Nutritional properties of yeast strains of Bakers yeast have been selected animal nutrition and in aquaculture under Typical dry yeast composition is 93-97 for their high fermentative power, particuvarious forms and larly useful for bakers. Strains species (Torulaspora, are specific to the type of Torulopsis, bread and the region where Kluyveromyces, it is sold, in order to respond Saccharomyces and to different bread making caetera). They can conditions (French bread, be found for example white bread, flat bread, in shrimp and marine croissant, etc.) and resist to fish larval feeds or different process conditions included as a protein (osmotic pressure from high source in aquafeeds. sugared bread, freezing, acidThe reasons for ity of sour dough,). this extensive use is Bakers yeast comes as its excellent nutria pure and primary culture tional contents, its grown on sugar substrate easy supply in dried such as molasses. The proform or under liqduction is performed under uid form when bakvery strict conditions in order ery yeast plants or to maintain the genetic puribreweries are near ty, consistency, specificity and aquafeed plants, and efficacy of the strains. (Figure Figure 1: Yeast manufacturing process (primary grown culture) nowadays a competi1). It can be sold under differ26 | January - february 2013 Grain

tive 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.

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.

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FEATURE
Table 1: Effect of live yeast Actisaf on growth and survival parameters in tilapia under stress conditions. (n=3, P<0.05, measures with different letters are significantly different) Treatment CON 40% -10 fry CON 40% -20 fry Act 40% - 10 fry Act 40% - 20 fry Act 27% - 10 fry Act 27% - 20 fry Survival (%) 75.0ab 64.8a 87.5bc 92.6c 91.7bc 96.29c SGR 3.33a 3.47a 5.80d 5.43c 5.46cd 5.24c

ent 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 recognised as good toxin binders. Fractions coming from bakers yeast have a light beige colour. The most popular aquaculture application of Bakers yeast is in hatcheries where it is a major feed source for artemia and rotifer (see for example Couteau et al 1990).

Brewers yeast
Brewers 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 brewers yeast. Brewers 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 bakers yeast, but contains more trace minerals such as selenium and chromium. The protein content of brewer yeast is relatively high and and its amino acid content is similar to bakers yeast. Numerous works have shown the efficacy of Brewers yeast to replace partially or totally the proteins found in fish and vegetable meal in fish and shrimp. Shrimp feeds formulators typically incorporate brewers yeast in their formula at the rate of two to four percent.
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summit is another best-practice of the value VIV 3.11e 0.83ab adds to its events. Developed in 3.26e 0.78ab close co-operation with loyal advisors 1.43abc 1.89cd to VIV, this is a premium-quality 1.01a 2.64d conference based on my personal 1.62bc 2.26c initiative, he says. Figure 2: Schema of a process to produce yeast 1.17ab 3.17e VIV Asia presents three special extracts and yeast cell walls A very special seminar features: CropTech-FeedTech Perendale Publishers will be takAsia, focusing cost-effective feed ing part in one of the CropTechFeedTech Asia seminars on of Brewers yeast canproduction, MeatTech, highlighting concern is the sanitary safety March be used to produce Another the latest the nature produce 13, Antibiotics Digital engineering yeast fractions, however due totechnologies tothese products. 2013. Called are sometimes of brewers yeast and safe products that can be added to the process in order to this unique the specificity of the used eas- in feed manufacturing, prevent by quality is less bacteria competing is for those working in production processes,ily the the consumers, and the VIV seminar with the yeast for nutri-the Animal Health Summit ents area yield technology is thereconsistent than in bakers yeast. Products Asia. and avoiding of milldecrease. It and aims at The summit is the fore possible that some antibiotic residues coming from brewery yeast tend to have a first confer- providing background information on ence in Asia a address and possibly other toxins might that have been distinctive bitter smell and taste andto darker the rapidly intelligent solutions be left in the colour than the ones growing concerns about the use of product. coming from bakers final dried introduced to address processing antibiotics in animal protein produc- chain dilemmas. yeast. tion, both at CEO and Autolysed yeast Inactive Dried technical Whats unique for our industry level. On a personal note, Berculo about this event is its format, says Ethanol yeast Yeast is particularly excited about the VIV and Autolysed publisher of Grain and Ethanol yeasts are harvested after having Inactive Roger Gilbert, yeast come from Animal Health distil- primary grown cultures Technology yeast. performed alcoholic fermentation andSummit Asia. The Feed Milling or Brewers magazine lation 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 supply depends on the activity of the bioethanol plants and can also be inconsistent.
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FEATURE They are major products within the food industry as flavour enhancers and in pet food as feed attractants. They are used in aquaculture feeds as a source of protein and nitrogen. Brewers yeast, and its ethanol equivalent, is mostly favoured as it is cheaper than bakers yeast. They are also easier supplied as yeast suppliers prefer to sell the more controlled and tailored Bakers 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 proteolysis) 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 product is a mixture of cellular content and yeast cell wall. Furthermore the autolysis process degrades protein and forms peptides (dipeptides to tetra peptides) and oligonucleic acids which are readily digestible by the animal. Again here depending on the original yeast material used, autolysed and inactive yeast quality can be very different. 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 difficult 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 products 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 conditions (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 conditions). There is also a better

Live yeast as probiotics

Live yeast helps regulate the gut microbiota. 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 characterized 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 10.107 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

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). 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.

a probiotic in terrestrial animals, there are only a few numbers of works studying its effect in fish as a gut functions stabiliser. The major reason is that live yeast does not resist the severe conditions of the manufacturing processes of aquafeeds (high temperatures, steam, long conditioning 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
28 | January - february 2013

Yeast culture or fermented yeast

Yeast culture is a particular product in which yeast is allowed to ferment. Yeast biomass, 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
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FEATURE

Figure 5: Yeast rich in organic selenium manufacturing process 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 under the label yeast extract in aquaculture. A good way to differentiate them is to look at the carbohydrate levels. Autolysed yeast has a level 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 to be 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
30 | January - february 2013

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 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 clarification). 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, betaglucans are direct stimulators of the immune systems in shrimp and fish, upon the stimulation 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 challenges in numerous aquaculture species. Beta glucans have to be use carefully in aquaculture 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 concentration (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 minimal 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
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FEATURE 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 carbohydrates, and then the better immunostimulation 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 substrate used to produce the yeast, and event the drying process. Mannans represent as most 25-27 percent of YCW in good quality YCW from primary 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 convenient indicator of quality, the best products being those having lower nitrogen content. The variability 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 tendency to believe that all YCW are the same and that differentiation of products must be done to the highest level of glucans (sometimes 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. 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.

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 by-products 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 and the quality. More
InforMatIon:

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-

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