July | August 2012 Unlocking the hidden potential of plant proteins using solid state fermentation enzymes

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

The International magazine for the aquaculture feed industry

FEATURE

Unlocking the hidden potential of plant proteins using solid state fermentation technology
by John Sweetman1, Ioannis Nengas2 and Serge Corneillie3 n the terrestrial animal feed industry the use of exogenous enzymes is a relatively common practice and today the global feed enzyme market is worth more than US$550 million. This saves the global feed market an estimated US$3-5 billion per year. To date the use of enzymes in aquaculture feeds has been limited, but interest is growing due to the increasing use of plant based protein ingredients and their by-products. The incorporation of Synergen™ (Alltech Inc., USA), a natural solid state fermentation complex that improves profitability by maximizing nutrient release, has enabled the fishmeal content of diets for several commercial carnivorous fish species to be reduced by up to 65 percent. These diets have also maintained the same or achieved even better growth performance when compared to the high fishmeal diets. This article highlights the recent work carried out with Gilthead sea bream (Sparus aurata), red sea bream (Pagrus major), amberjack (Serioli dumerili), pangasius catfish and Nile tilapia.

I

Modern aquafeed challenges: maximising nutrient availability
The economic success and sustainability of aquaculture depends on minimising production cost to maintain profitability, and feed generally comprises some 50 to 60 percent of the total cost in intensive culture. Traditionally fishmeal has been the preferred ingredient due to its high protein content, favourable amino acid and mineral profiles, oil and other benefits. Its replacement with plant based protein ingredients has been brought about by the increasing cost of fishmeal, its limited availability and the requirement to ensure that this expanding industry remains both financially profitable and environmentally sustainable. Plant derived feed ingredients however have several limitations. Non starch polysaccharides (NSPs) are a complex group of polysaccharides which act as energy storage carbohydrates in grains and seed. While they may provide a cheap source of dietary energy

NSPs cannot be broken down by the digestive Alltech’s Bioscience Centers in Dunboyne, enzymes of many fish species especially the Ireland; Bangkok, Thailand; and Kentucky, carnivorous ones. USA, have resulted in a $40 million state-ofA wide range of anti-nutritional factors the-art production facility in Serdan, Mexico. (ANFs) such as protease inhibitors, nonThe SSF process involves the careful digestible carbohydrates, lectins, saponins and selection of specific strains of naturally occurphytates may also be present in a number of ring fungi which have the ability to ferment plant derived materials such as legume seeds, a wide range of agricultural products such as soybean meal, rapeseed meal etc. These can DDGS, corncob, palm kernel, wheat bran, impede digestion in fish often decreasing rapeseed oil cake and soy bean. The selected intestinal viscosity and bacterial loads which fungi are first propagated in a liquid media to in turn affect the animals’ performance. These produce a large volume of inoculum which ANFs have therefore to be removed before is mixed with pre-sterilised selected solid they can be incorporated in commercial feeds. substrate media to produce a mixture known The use of highly digestible and processed soybean meal such as low antigen SPC (soy protable 1: Diet composition for Gilthead sea bream tein concentrates) or proc1 2 3 essed corn gluten to replace Diet fish meal is possible but these Control natural Pre-treated complex with natural highly processed ingredients complex are expensive and the economic savings can therefore be relatively low. Fish meal 25 25 25 Another solution for counSoybean meal 40 40 40 terbalancing the digestibility Wheat meal 16.7 16.65 16.65 problems of such ingredients is Corn gluten 5 5 5 to use enzymes that improve Fish oil 13 13 13 substrate digestibility resulting in higher nutrient availability Vit-Min 0.3 0.3 0.3 thus improving growth, food Synergen 0.05 0.05 conversion rates and therefore economic efficiency. table 2: effect of diet on the performance of Gilthead sea bream Today a better underafter 12 weeks. standing of how enzymes Pre-treated natural work in animal feeds and the Control with natural complex increasing evidence of their complex ability to improve feed quality, shown in the last 10 years, Initial weight (g) 31,11±0,65 30,77±1,37 31,65±0,80 has allowed a more flexible approach by feed formulators. final weight (g) 84,04±5,76 95,57±3,57 100,75±1,90

Solid State Fermentation Technology
Alltech has pioneered the production of SSF technologies for the animal feed industry. Eight years of collaborative research between

FCr SGr

1,45±0,13a 0,83±0,05a

1,18±0,01b 0,94±0,07b

1,13±0,02b 0,97±0,04b

Values are means of three replicates expressed with the standard deviation between tanks. Values with common superscripts demonstrate no significant differences among groups (p<0.05) Specific growth rate (SGR) = (ln final weight - ln initial weight)*100/days Feed conversion ratio (FCR) = feed consumed g / weight increase g

32 | InternAtIonAl AquAFeed | July-August 2012

FEATURE

Figure 1. Feed conversion ratio (a) and Specific growth rate (b) of the Gilthead sea bream fed different diets table 3: experimental diet composition for red sea bream. 1 2 3 4 5 6

Ingredients anchovy meal Soybean meal Corn gluten meal Fish oil Soybean oil Ca(H2Po4)2 taurine Phytase (IU/g) natural complex others*

FM50 50 0 5 5 5 1 34

FM20 20 18 23 5 7.4 1 25.6

Ft 20 18 23 5 7.4 1 0.2 25.4

FtP 20 18 23 5 7.4 1 0.2 1000 25.2

Fte0.05 Fte 0.1 20 18 23 5 7.4 1 0.2 0.05 25.35 20 18 23 5 7.4 1 0.2 0.1 25.3

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Consistent products supported by the Lesaffre group experience and its unique know-how in biotechnology and nutrition; Selected strains and controlled production; Designed to solve nutritional and sanitary issues; Dedicated range of products: live yeast, yeast cell wall, yeast extract and enriched yeast.

others: Starch, vitamin premix and wheat flour

as ‘Koji’. Under strict aseptic conditions the Koji is then evenly distributed onto trays and introduced into environmentally controlled SSF culture chambers for up to five days. During this time the fungus grows rapidly, breaking down the fibrous and non-fibrous portions of the chosen substrate. Doing so dramatically changes the nutritional profile of the material and results in the generation of products that can be used to reformulate diets. On day five, the Koji is extracted and the by-product is dried. The product from the Alltech SSF process, Synergen™, allows for a more flexible approach to feed formulation through the inclusion of by-products or by reducing nutrient constraints in the diet. It has also been shown, through animal performance, to remain

effective over a wide range of feed processing conditions.

Carnivorous fish: Gilthead sea bream
At the Institute of Aquaculture of the Hellenic Centre for Marine research an experiment was performed in which approximately 31 g juvenile Gilthead sea bream were fed 3 different diets (Table

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July-August 2012 | InternAtIonAl AquAFeed | 33

FEATURE that requires additional facilities and treatments therefore it Parameter FM50 FM20 Ft FtP Fte0.05 Fte 0.1 would need a change in the production Final body weight (g) 71.5c 49.8a 55.2ab 59.7ab 74.5c 67. 8bc line. Since Weight gain(g) 57.7b 35.9a 40.4ab 59.7ab 59.9b 53.7b the results SGr (%/day) 1.96c 1.52a 1.57ab 1.70ab 1.94c 1.87c obtained from FCr 1.12a 1.30b 1.27b 1.25b 1.16ac 1.21ac the ‘natural complex’ Feed intake (g/day) 64.7a 46.6b 51.2ab 56.6ab 69.6a 64.9a diet have no a,b Means differ P<0.05. significant dif1) in triplicate at a water temperature of 18 ± ference from the ‘pre-treated with natural 2 °C. The sea bream were fed with a control complex’ diet, this indicates that the natural diet (1) with a moderate 25 percent fishmeal complex can be effectively added directly to inclusion and two other diets in which a the ingredient mix. The ‘natural complex’ diet portion of the wheat meal was replaced by gave an improvement of 18 percent in FCR Synergen, incorporated at 0.05%. In diet 2, and a 13 percent improvement in SGR when ‘Natural complex’, Synergen was incorpo- compared to the control diet. Currently industrial trials and commercial rated into the whole ingredient mix prior to extrusion while in diet 3, ‘Pre-treated with application are underway. Based solely on the improvement in FCR, a return on investment of 1: 43 can be expected when Synergen is added in this manner for this species with additional increased profit benefits to be added from the growth rate improvements.
table 4: effect of dietary treatment on the performance of red sea bream after 12 weeks. Figure 2: Growth of red sea bream fed different diets

only obtained by the low fishmeal diets containing Synergen. The lowest growth and highest FCR were obtained with diet 2 (low fishmeal). Adding taurine alone or taurine / phytase to the low fishmeal diet improved the performance but this performance was still much lower than the high fishmeal diet or the Synergen treated groups. Adding higher amounts of the natural complex (1 kg of Synergen/tonne) did not further improve the results. These results show clearly that highly carnivorous fish (RSB) can be fed with low fishmeal levels and that ordinary plant proteins can be used if appropriate ingredients are added. Field trials in Japan with red sea bream have confirmed these results. In commercial cages, red sea bream performed better when Synergen was incorporated in the diet with an increased SGR (0.70% when compared to 0.55% in the control group) and a final weight gain of 175 g which was greater than the 138 g achieved by the control groups. The FCR was 21 percent lower in the fish fed the diet incorporating Synergen and the feed efficiency of the Synergen incorporated diet was also improved. (86.6 versus 68%). Amberjack (Serioli dumerili) also showed similar growth results growing from 2.2 kg to 2.6 kg (without Synergen) or 3.1 kg (with Synergen).

Carnivorous fish: Red sea bream

Pangasius and tilapia
It is strongly believed that omnivorous and herbivorous fish can digest plant proteins better than carnivorous fish and therefore do not need additional ingredients in their diets. However the inclusion of Synergen in diets for fish species such as tra and basa catfish and tilapia has resulted in significantly improved growth and lowered significantly the FCR in both low and high fishmeal diets. In recent trials, by Hung and Kim (2007), a comparison was made between a 15 percent fishmeal diet and a five percent fish meal diet (65% reduction) with added Synergen (200 or 500 g per tonne feed) to both diets. They demonstrated that irrespective of the diet used (low or high fish meal) the addition of the natural complex resulted in dramatic improvements in performance parameters. Growth reached approximately double that of the diet without the enzyme present. Tra catfish grew from 14 g to 36 g (without Synergen) or 70 g (with Synergen) while Basa catfish grew from 6 g to 57 g (without Synergen) or 82 g (with Synergen). Similar results have been recorded for Nile Tilapia in which a zero percent fishmeal diet was used as the control diet where growth from 3 g to 21 g occurred without Synergen and to 35 g (with Synergen). This indicates that supplementing the low

Improved growth rates have also been achieved by Satoh et al. (2011) with juvenile red sea bream. In this case red sea bream, of approximately 13.5 g were fed six different diets (Table 3). A high fishmeal diet with 50 percent anchovy meal was fed as a control and five other diets all with low fishmeal incluFigure 3: Growth of Amberjack with and without sion (20%) but to which Synergen different components were added (taurine, taurine and natural complex’, the plant ingredients were phytase enzyme, taurine and Synergen). In hydrolyzed with the Synergen for four hours these diets the fishmeal was replaced with ordinary corn gluten and soybean meal, at 40 °C prior to extrusion. The diet whose ingredients were pre- which are cheaper and industrially available treated by hydrolysing them with Synergen ingredients. Diet 2 was low fishmeal, diet 3 was low before the extrusion gave slightly better but not significantly different results than the fishmeal and taurine, diet 4 low fishmeal and diet which incorporated Synergen as a sup- taurine and phytase enzyme, diet 5 was low plemented ingredient, mixed directly with fish meal and taurine and 500 g Synergen/ the rest of the ingredients. Both these diets tonne feed and diet 6 low fishmeal and performed significantly better than the control taurine and 1 kg of Synergen/tonne feed. The best performance was obtained with diet with improved growth, SGR and FCR the positive high fishmeal diet (best growth (Table 2 and Figure 1). Pre-treatment of raw materials in com- and lowest FCR). Comparable growth and mercial feed production units is a procedure performance to the high fishmeal diet was
34 | InternAtIonAl AquAFeed | July-August 2012

metals were analysed for five farms over two croppings (10 months). The study helped

three international standards for tilapia farming, i.e. BAP, GlobalGAP, and ASC, was held in Haikou

Sustainable Fisheries Partnership Website: www.sustainablefish.org FEATURE

and high fish meal diets with the natural complex has enhanced the availability of dietary nutrients and compensated for the poorer quality of the raw ingredient characteristics in the low fishmeal diet so improving overall dietary performance.

References
Hung LT and TNH Kim. 2007. Reducing fish meal utilization in Pangasius Catfish feeds through application of enzymes. Presented at Asian Pacific Aquaculture 2007, Hanoi, Vietnam, 5-8 August 2007. Satoh S, Hanini I, Sarker MSA, Haga Y, Ohkuma T and H Nakayama 2011. Effect of Taurine, Phytase and enzyme complex supplementation to low fish meal diet on growth of juvenile red sea bream Pagrus major. Presented at World Aquaculture 2011, Natal, Brazil, 6-10 June 2011.

Conclusion
The application of this SSF technology in this manner opens the door more flexible feed formulation and allows the incorporation of lower cost vegetable protein substitutes such as simple soybean meal and corn gluten. Improved nutrient availability impacts directly growth and performance so increasing dietary efficiency both in terms of cost and environmental impact. The savings in fishmeal usage addresses consumer concerns and sustainability issue in the industry. There is still much to learn about these complex interactions but the indicators show the potential of this technology. ■

1Alltech Aqua, 28200 Lixouri, Kefalonia, Greece 2Institute of Aquaculture, Hellenic Centre of Marine Research, Agios Kosmas, Elliniko, 16610 Athens, Greece 3Alltech Japan, Shiba-Koen 2-3-27, Minato, Tokyo 1050011, Japan

July-August 2012 | InternAtIonAl AquAFeed | 27

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July-August 2012 | InternAtIonAl AquAFeed | 35

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