Unlocking the hidden potential of plant proteins using solid state fermentation enzymes

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July | August 2012Unlocking the hidden potential of plantproteins using solid state fermentation

enzymes

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 formor by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058
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In the terrestrial animal feed industrythe use of exogenous enzymes is arelatively common practice and todaythe global feed enzyme market is worth

more than US$550 million. This saves theglobal feed market an estimated US$3-5billion per year. To date the use of enzymes

in aquaculture feeds has been limited, butinterest is growing due to the increasing useof plant based protein ingredients and theirby-products.

The incorporation of Synergen (AlltechInc., USA), a natural solid state fermentation complex that improves profitability bymaximizing nutrient release, has enabled thefishmeal content of diets for several com-mercial carnivorous fish species to be reducedby up to 65 percent. These diets have also

maintained the same or achieved even bettergrowth performance when compared to thehigh fishmeal diets.

This article highlights the recent workcarried out with Gilthead sea bream (Sparusaurata ), red sea bream (Pagrus major ), amberjack (Serioli dumerili), pangasius catfish andNile tilapia.

Modern aquafeed challenges:maximising nutrient availability

The economic success and sustainability ofaquaculture depends on minimising production cost to maintain profitability, and feedgenerally comprises some 50 to 60 percent of the total cost in intensive culture. Traditionallyfishmeal has been the preferred ingredientdue to its high protein content, favourableamino acid and mineral profiles, oil and otherbenefits. Its replacement with plant based protein ingredients has been brought about by the increasing cost of fishmeal, its limited avail-ability and the requirement to ensure that thisexpanding industry remains both financiallyprofitable and environmentally sustainable.

Plant derived feed ingredients howeverhave several limitations. Non starch polysac-charides (NSPs) are a complex group ofpolysaccharides which act as energy storagecarbohydrates in grains and seed. While theymay provide a cheap source of dietary energy

NSPs cannot be broken down by the digestiveenzymes of many fish species especially thecarnivorous ones.

A wide range of anti-nutritional factors(ANFs) such as protease inhibitors, non-digestible carbohydrates, lectins, saponins andphytates may also be present in a number of

plant derived materials such as legume seeds,soybean meal, rapeseed meal etc. These canimpede digestion in fish often decreasingintestinal viscosity and bacterial loads whichin turn affect the animals performance. TheseANFs have therefore to be removed before they can be incorporated in commercial feeds.

The use of highly digestible andprocessed soybean meal suchas low antigen SPC (soy protein concentrates) or proc-essed corn gluten to replace

fish meal is possible but thesehighly processed ingredientsare expensive and the eco-nomic savings can thereforebe relatively low.

Another solution for coun- terbalancing the digestibilityproblems of such ingredients is to use enzymes that improvesubstrate digestibility resultingin higher nutrient availability thus improving growth, foodconversion rates and there-fore economic efficiency.

Today a better under-standing of how enzymeswork in animal feeds and theincreasing evidence of theirability to improve feed quality,shown in the last 10 years,has allowed a more flexibleapproach by feed formulators.

Solid StateFermentation

TechnologyAlltech has pioneered the production of SSF tech-nologies for the animal feedindustry. Eight years of col-laborative research between

Alltechs Bioscience Centers in Dunboyne,Ireland; Bangkok, Thailand; and Kentucky,USA, have resulted in a $40 million state-of- the-art production facility in Serdan, Mexico.

The SSF process involves the carefulselection of specific strains of naturally occur-ring fungi which have the ability to ferment

a wide range of agricultural products such asDDGS, corncob, palm kernel, wheat bran,rapeseed oil cake and soy bean. The selectedfungi are first propagated in a liquid media toproduce a large volume of inoculum whichis mixed with pre-sterilised selected solidsubstrate media to produce a mixture known

Unlocking the hiddenpotential of plantproteins using solid statefermentation technologyby John Sweetman 1, Ioannis Nengas 2 and Serge Corneillie 3

table 1: Diet compositio for Gilthead sea bream

Diet 1 2 3

Co trol naturalcomplex Pre-treated with aturalcomplex

Fish meal 25 25 25Soybea meal 40 40 40 Wheat meal 16.7 16.65 16.65Cor glute 5 5 5

Fish il 13 13 13 Vit-Mi 0.3 0.3 0.3

Sy erge 0.05 0.05

table 2: ffect of diet o the performa ce of Gilthead sea breamafter 12 weeks.

Co trol naturalcomplex

Pre-treated with atural

complex

I itial weight (g) 31,110,65 30,771,37 31,650,80

fi al weight (g) 84,045,76 95,573,57 100,751,90FC 1,450,13a 1,180,01b 1,130,02bSG 0,830,05a 0,940,07b 0,970,04b

Values are means of three replicates expressed with the standard deviation between tanks. Values with common superscriptsdemonstrate no significant differences among groups (p


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as Koji. Under strict aseptic conditions theKoji is then evenly distributed onto trays andintroduced into environmentally controlledSSF culture chambers for up to five days.During this time the fungus grows rapidly,breaking down the fibrous and non-fibrousportions of the chosen substrate. Doing sodramatically changes the nutritional profileof the material and results in the generationof products that can be used to reformulatediets. On day five, the Koji is extracted and the by-product is dried.

The product from the Alltech SSF proc-ess, Synergen, allows for a more flexibleapproach to feed formulation through theinclusion of by-products or by reducing nutri-ent constraints in the diet. It has also beenshown, through animal performance, to remain

effective over awide range of feedprocessing conditions.

Carnivorousfish: Giltheadsea bream

At the Instituteof Aquaculture of the Hellenic Centrefor Marine research

an experiment wasperformed in whichapproximately 31 g juvenile Gilthead seabream were fed 3different diets (Table

table 3: xperimen al die composi ion for red sea bream.

1 2 3 4 5 6

Ingredien s FM50 FM20 Ft FtP Ft 0.05 Ft 0.1

nchovy meal 50 20 20 20 20 20

Soybean meal 0 18 18 18 18 18

Corn glu en meal 5 23 23 23 23 23

Fish oil 5 5 5 5 5 5

Soybean oil 5 7.4 7.4 7.4 7.4 7.4

Ca(H2P 4)2 1 1 1 1 1 1

taurine - - 0.2 0.2 0.2 0.2

Phy ase - - - - - -

(IU/g) - - - 1000 - -

a ural complex - - - - 0.05 0.1

hers* 34 25.6 25.4 25.2 25.35 25.3

hers: S arch, vi amin premix and whea flour

Figure 1. Feed conversion ratio (a) and Specific growth rate (b) of the Gilthead sea bream fed different diets

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July-August 2012 | In ernA IonA AquAFeed | 33

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1) in triplicate at a water temperature of 18 2 C. The sea bream were fed with a controldiet (1) with a moderate 25 percent fishmealinclusion and two other diets in which aportion of the wheat meal was replaced bySynergen, incorporated at 0.05%. In diet 2,Natural complex, Synergen was incorpo-rated into the whole ingredient mix prior to

extrusion while in diet 3, Pre-treated with

natural complex, the plant ingredients werehydrolyzed with the Synergen for four hoursat 40 C prior to extrusion.

The diet whose ingredients were pre- treated by hydrolysing them with Synergenbefore the extrusion gave slightly better butnot significantly different results than thediet which incorporated Synergen as a sup-plemented ingredient, mixed directly with

the rest of the ingredients. Both these dietsperformed significantly better than the controldiet with improved growth, SGR and FCR(Table 2 and Figure 1).

Pre-treatment of raw materials in com-mercial feed production units is a procedure

that requiresadditionalfacilities and therefore itwould need achange in theproductionline. Since the result sobtained from the natura lcomplexdiet have nosignificant dif-

ference from the pre-treated with naturalcomplex diet, this indicates that the naturalcomplex can be effectively added directly to the ingredient mix. The natural complex dietgave an improvement of 18 percent in FCRand a 13 percent improvement in SGR whencompared to the control diet.

Currently industrial trials and commercial

application are underway. Based solely on the improvement in FCR, areturn on investment of 1:43 can be expected whenSynergen is added in thismanner for this species withadditional increased profitbenefits to be added from the growth rate improve-ments.

Carnivorous fish:

Red sea breamImproved growth rateshave also been achievedby Satoh et al. (2011) with juvenile red sea bream. In this case red sea bream,of approximately 13.5 gwere fed six different diets(Table 3). A high fish-meal diet with 50 percentanchovy meal was fed as acontrol and five other dietsall with low fishmeal inclu-sion (20%) but to which

different components wereadded (taurine, taurine and

phytase enzyme, taurine and Synergen). In these diets the fishmeal was replaced withordinary corn gluten and soybean meal,which are cheaper and industrially availableingredients.

Diet 2 was low fishmeal, diet 3 was lowfishmeal and taurine, diet 4 low fishmeal and taurine and phytase enzyme, diet 5 was lowfish meal and taurine and 500 g Synergen/

tonne feed and diet 6 low fishmeal and taurine and 1 kg of Synergen/tonne feed.

The best performance was obtained with the posit ive high fishmeal diet (best growthand lowest FCR). Comparable growth andperformance to the high fishmeal diet was

only obtained by the low fishmeal diets con- taining Synergen. The lowes t growth andhighest FCR were obtained with diet 2 (lowfishmeal). Adding taurine alone or taurine /phytase to the low fishmeal diet improved the performance but this performance wasstill much lower than the high fishmeal dietor the Synergen treated groups.

Adding higher amounts of the naturalcomplex (1 kg of Synergen/tonne) did notfurther improve the results. These resultsshow clearly that highly carnivorous fish(RSB) can be fed with low fishmeal levelsand that ordinary plant proteins can be usedif appropriate ingredients are added.

Field trials in Japan with red sea breamhave confirmed these results. In commercialcages, red sea bream performed betterwhen Synergen was incorporated in the dietwith an increased SGR (0.70% when com-pared to 0.55% in the control group) and afinal weight gain of 175 g which was greater

than the 138 g achieved by the controlgroups. The FCR was 21 percent lower in the fish fed the diet incorporating Synergenand the feed efficiency of the Synergenincorporated diet was also improved. (86.6versus 68%). Amberjack (Serioli dumerili )also showed similar growth results growingfrom 2.2 kg to 2.6 kg (without Synergen) or3.1 kg (with Synergen).

Pangasius and tilapiaI t is strongly believed that omnivo-

rous and herbivorous fish can digest plantproteins better than carnivorous fish and therefore do not need addit ional ingredi-ents in their diets. However the inclusion ofSynergen in diets for fish species such as traand basa catfish and tilapia has resulted insignificantly improved growth and loweredsignificantly the FCR in both low and highfishmeal diets.

In recent trials, by Hung and Kim (2007),a comparison was made between a 15percent fishmeal diet and a five percentfish meal diet (65% reduction) with addedSynergen (200 or 500 g per tonne feed) to both diets . They demonstrated thatirrespective of the diet used (low or highfish meal) the addition of the natural com-plex resulted in dramatic improvements inperformance parameters. Growth reachedapproximately double that of the diet with-out the enzyme present. Tra catfish grewfrom 14 g to 36 g (without Synergen) or 70g (with Synergen) while Basa catfish grewfrom 6 g to 57 g (without Synergen) or 82g (with Synergen).

Similar results have been recorded for NileTilapia in which a zero percent fishmeal dietwas used as the control diet where growthfrom 3 g to 21 g occurred without Synergenand to 35 g (with Synergen).

This indicates that supplementing the low

table 4: ffec of die ary rea men on he performance of red sea bream af er 12 weeks.

trea men sParame er FM50 FM20 Ft FtP Ft 0.05 Ft 0.1

Final body weigh (g) 71.5c 49.8a 55.2ab 59.7ab 74.5c 67. 8bc Weigh gain(g) 57.7b 35.9a 40.4ab 59.7ab 59.9b 53.7b

SG (%/day) 1.96c 1.52a 1.57ab 1.70ab 1.94c 1.87c

FC 1.12a 1.30b 1.27b 1.25b 1.16ac 1.21acFeed in ake (g/day) 64.7a 46.6b 51.2ab 56.6ab 69.6a 64.9a

a,b Means differ P


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and high fish meal diets with the natural com-plex has enhanced the availability of dietarynutrients and compensated for the poorerquality of the raw ingredient characteristicsin the low fishmeal diet so improving overalldietary performance.

ConclusionThe application of this SSF technology in

this manner opens the door more flexiblefeed formulation and allows the incorpo-rat ion of lower cost vegetable proteinsubstitutes such as simple soybean meal andcorn gluten. Improved nutrient availabilityimpacts directly growth and performance soincreasing dietary efficiency both in terms ofcost and environmental impact. The savingsin fishmeal usage addresses consumer con-cerns and sustainability issue in the industry.There is still much to learn about thesecomplex interactions but the indicators

show the potential of this technology.

References

Hung LT and TNHKim. 2007. Reducingfish meal utilization inPangasius Catfish feeds through application ofenzymes. Presentedat Asian PacificAquaculture 2007,

Hanoi, Vietnam, 5-8August 2007.

Satoh S, Hanini I, SarkerMSA, Haga Y, Ohkuma Tand H Nakayama 2011.Effect of Taurine, Phytaseand enzyme complexsupplementation tolow fish meal diet ongrowth of juvenile redsea bream Pagrus major .Presented at WorldAquaculture 2011, Natal,

Brazil, 6-10 June 2011.

1 Alltech Aqua, 28200 Lixouri, Kefalonia, Greece2Institute of Aquaculture, Hellenic Centre of MarineResearch, Agios Kosmas, Elliniko, 16610 Athens, Greece

3 Alltech Japan, Shiba-Koen 2-3-27, Minato, Tokyo 105-0011, Japan

July-August 2012 | In ernA IonA AquAFeed | 35

FEATURE

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Unlocking the hidden potential of plant proteins using solid state fermentation enzymes

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