Journal of Engineering Science and Technology Special Issue on SOMCHE 2014 & RSCE 2014 Conference, January (2015) 30 - 40 © School of Engineering, Taylor’s University
30
POTENTIALITY OF SELECTED SEAWEED FOR THE PRODUCTION OF NUTRITIOUS FISH FEED USING SOLID
STATE FERMENTATION
N. N. ILIAS, P. JAMAL*, I. JASWIR, S. SULAIMAN, Z.. ZAINUDIN, A. S. AZMI
Biotechnology Engineering Department, Kulliyyah of Engineering, International Islamic
University Malaysia, Jalan Gombak, 53100, Kuala Lumpur, Malaysia
*Corresponding author: [email protected]
Abstract
The rising cost of imported fish feed ingredients have called for an intensive
research towards the production of high nutritional fish feed using cheap and
natural nutrient sources. In this study, seaweed and palm kernel cake were used
as alternative protein ingredients in fish feed and the enrichment of its
nutritional value was achieved by using solid state fermentation. Following the
fermentation process, the nutrient compositions of the three types of seaweeds:
Caulerpa lentillifera (Chlorophyta), Eucheuma cottonii (Rhodophyta) and
Sargassum fulvellum (Phaeophyta) were compared. Solid state fermentation
was conducted using fungus (Phanerochaete chrysosporium) and yeast
(Candida utilis) to improve the bioprotein production. The comparison between the nutrient compositions before and after the fermentation showed that
fermentation process can increase the nutrient value of the bioprotein. Results
of the analysis showed that Sargassum fulvellumis the best type of seaweed
with the highest total protein, 52.28 mg/g, high percentage improvement of ash
after fermentation, 61.31% and contains high amount of carbohydrate, 445.89
mg/g which are very important in fish diet. Hence, Sargassum fulvellum can be used as an alternative protein ingredient in the fish feed since it was rich in
nutritional compounds compared to the other types of seaweed tested.
Keywords: Seaweed, Solid state fermentation, Phanerochaete chrysosporium,
Candida utilis, Fish feed.
Potentiality of Selected Seaweed for the Production of Nutritious Fish Feed . . . . 31
Journal of Engineering Science and Technology Special Issue 3 1/2015
1. Introduction
Several studies are directed to an efficient and cost-effective supplementary fish
feed because of the importance of fish as a protein source for human and
animal’s diet. Aqua feed is the major determinant that influences the successful
growth and intensification of aquaculture production. According to Pereira et al.
[1], fish feeding constitutes over 50% of operating cost in intensive aquaculture,
where protein is the most expensive dietary source. Fish meal is one of the
major ingredients in making fish feed, considering its high importance as a
source of protein. However, it is an expensive feed ingredient and the supplies
often vary unpredictably due to the overfishing or large-scale transient oceanic
changes [2].
The use of terrestrial plants like leguminous plants and oil seed meals as an
alternative source in fish feed has been limited due to the presence of anti-
nutritional compounds, nutritional profile that differs from fish requirement as
well as to the palatability problem [3]. Nowadays, various types of seaweeds
have become the centre of attention due to the possibility of these aquatic plants
as an alternative source of protein for cultured fish. This is also because of the
high content of protein and high production rate [4]. Seaweeds are macroscopic
marine algae. There are about 6000 species of seaweeds that have been
identified and grouped into three different classes which are green
(Chlorophyceae), red (Rhodophyceae) and brown (Phaeophyceae) algae [5].
Various types of seaweeds had been used in Asia since ancient time as a source
of human food, fertiliser, animal feed, herbicides as well as fungicides [6]. In
Malaysia, seaweed has become an economically important natural source with
resource mobilisation of about RM 21.16 billion in the year of 2002. Total
export of dried seaweed from Sabah in 2002 was 1750 MT by weight and
RM14 million by value [7].
Palm kernel cake (PKC) is one of the by-products that was obtained from
mechanically pressed nuts of the palm fruit [8]. The cost of imported fish
ingredients like soybean meal, corn flour and fish meal affects the local fish
farmers where the profits obtained are not sufficient enough to cope with the
Nomenclatures
FeSO4.7H2O Iron (II) sulphate heptahydrate
KCl Potassium chloride
KH2PO4 Potassium dihydrogen phosphate
MgSO4.7H2O Magnesium sulfate heptahydrate
NH4H2PO4 Ammonium dihydrogen phosphate
Abbreviations
AOAC The Association Of Analytical Communities
CU Candida utilis
MARDI Malaysian Agricultural Research and Development Institute
MT Metric tonnes
PC Phanerochaete chrysosporium
PKC Palm Kernel Cake
YEPG Yeast Peptone Glucose media
32 N. N. Ilias et al.
Journal of Engineering Science and Technology Special Issue 3 1/2015
cost required in maintaining aquaculture enterprise. Therefore, the use of
locally available feed ingredients like PKC, which can be easily obtained at a
low cost, is very beneficial and profitable in fish feed production. Several
studies have already been carried out by using PKC as a substrate in solid state
fermentation for various purposes such as in the production of lipase [9] and
xynalase [10].
Solid state fermentation (SSF) is defined by Couto and Sanroman [11] as ‘a
fermentation process conducted on non-soluble material that has a role as both
physical support as well as a source of nutrients in the absence or near absence
of water’. Jecu [12] has reported that the highest possibility for SSF method to
be successful is when fungi were used. This is due to the ability of fungi to
grow in nature on solid substrates like seeds, piece of wood, roots, stems
and some animals’ dried parts. The usage of co-culture system (fungi and
yeast as the fermentative microorganisms) was reported to be beneficial as it
can enhance the protein synthesis through their synergistic effect on solid
matrix as well as improving the protein content and the digestibility of the
substrate [13,14].
Nutrition plays an important role in efficient aquaculture production as it gives
high influences not only to the production costs, but also to the fish health, growth
and production of waste. Specific nutritional requirement of a particular fish
should be known before the formulation of any fish feed in order to produce
nutritious and cost-effective diets.The use of various seaweeds and other aquatic
plants are getting more attention as possible alternative protein sources for
cultured fish due to the high production rate and protein content [4]. In order to
further improve the nutritional value of seaweed, this experiment was conducted
with the aim of evaluating the nutritional compounds of few seaweeds and
selecting one of them with the potentiality of producing nutritious fish feed using
solid state fermentation process involving Phanerochaete chrysosporium and
Candida utilis.
2. Materials and Methods
2.1. Preparation of raw materials (substrates)
Three types of seaweeds (Caulerpa lentillifera, Eucheuma cottonii and
Sargassum fulvellum) were used as the substrate in the production of high
nutritional fish feed. These seaweeds were collected from the near-shore waters
of Port Dickson, Negeri Sembilan. It was thoroughly washed and was dried
in an air forced oven at 60 °C for 24 hours. Then, the dried seaweed was
grounded to the size of 2 mm and was kept in an airtight container at 4 °C
for further use. The other substrate that was used in this study is Palm kernel
cake (PKC). PKC was obtained from a local palm oil plantation (Sime Darby,
Pulau Carrey, Banting, Selangor). It was dried at 60 °C for 72 hours in order to
reduce the moisture content to approximately 5% and was grounded to the size
of 2 mm [13].
Potentiality of Selected Seaweed for the Production of Nutritious Fish Feed . . . . 33
Journal of Engineering Science and Technology Special Issue 3 1/2015
2.2. Microorganisms
Two different types of microorganisms were used in this study. They were
Phanerochaete chrysosporium (PC) (PC 2094) and Candida utilis (CU) (FTCC
8100). PC strains were sourced from the Laboratory stock of Bioenvironmental
Laboratory, IIUM while CU cultures were bought from MARDI, Serdang. CU
stock cultures were maintained on nutrient solution containing YEPG media:
yeast extract 0.3%, peptone 0.5%, glucose 1%, malt extract 0.3% and distilled
water. The ingredients were thoroughly mixed, and was then kept in culture
tubes and sterilised at 121 °C for 15 minutes. The sterilised slants were
inoculated with these microorganisms. They were then be incubated at 32 °C to
obtain luxuriant growth. PC was subcultured fortnightly for subsequent use.
2.3. Inoculums preparation
Spores of PC were then harvested from 7-days old plates and were washed with
25 mL of sterile distilled water. The filtered spore suspension was collected in
250 mL Erlenmeyer flask and maintained at 7.5×109 spores/mL. For CU, 1-2
loopfull of cell suspension was added into the YEPG media in 250 ml conical
flasks that was then incubated at 30 °C and 150 rpm for 24 hours. Then, the
inoculums were stored at 4 °C for further use.
2.4. Solid state fermentation
About 4 g of seaweed was added into a 250 mL flask containing 2 g of
accurately weighed palm kernel cake (PKC) in order to obtain the biomass. The
fermentation medium (pH 4) containing the following pre-optimised mineral
concentration (g/L): 47.9 sucrose, 5 KH2PO4, 6.1 MgSO4.7H2O, 5 KCl, 4.4
NH4H2PO4, 3 peptone and 3 FeSO4.7H2O were then added into the flask [13].
The substrate moisture was adjusted to 70% using distilled water and growth
media and was then autoclaved at 121°C for 15 minutes. After that, the
specified amount of inoculums was added and the mixture was incubated at 32
°C in an incubator for 6 days. After the fermentation, the biomass was
harvested. Flasks containing the fermentation media and substrate (without
undergoing fermentation process) were also prepared to act as a control for
comparison purposes.
2.5. Culture harvesting
Biomass (residue after the six days of fermentation process) was harvested by
drying in hot air oven at 60 °C for 48 hours and cooled in desiccators until weight
equilibrates. The biomass was then grinded for further analysis.
2.6. Proximate analysis
Proximate compositions of bioprotein after the fermentation were evaluated using
several types of analysis. Ash content was determined using the method of AOAC
Official Method 923.03 [15]. Crude protein was determined using the Lowry
34 N. N. Ilias et al.
Journal of Engineering Science and Technology Special Issue 3 1/2015
method (Folin-Phenol Reagent) Lowry et al. [16] while total carbohydrate was
identified using the method used by Dubois et al. [17] and Krishnaveni et al. [18]
with slight modifications. The total reducing sugars was analysed by using the
method used by Miller [19].
3. Results and Discussion
3.1. Analysis of total protein content
One of the major growth promoting factors in fish feed is protein. Protein
including enzymes, hormones and immunoglobins are needed by normal bodily
functions and the deficiency of this nutrient can affect the protein synthesis and
lead to the reduction in weight and other symptoms to the fish [20]. Study done
by Al Mahmud et al. [21] showed that the protein requirement of fish was
influenced by several factors, including fish size, water temperature, water
quality, rearing environment, the genetic compositions and also feeding rates of
the fish. In present study, Fig. 1 shows the comparison between the total protein
content (mg/g of substrates) of the three types of seaweed namely S. fulvellum, C.
lentillifera and E. cottonii. From the graph, the protein content after the
fermentation using only seaweed as a substrate showed that C. lentillifera from
the group of green seaweed has the highest amount of protein content which was
(31.714 mg/g) compared to the other two types of seaweed that contains about
similar amount of protein content (14.751 mg/g). This result was identical with
the study reported by Matanjun et al. [22] which showed that green seaweed has
the protein content (10.41%) higher than red (9.76%) and brown seaweed
(5.40%). Figure 1 also shows the comparison between the methods of addition of
seaweed whether it was added during the fermentation along with PKC or after
the fermentation process. For all three types of seaweed, it was observed that
when the seaweed was added during the fermentation together with PKC as
substrate, the amount of protein content was higher compared to the addition at
the end of the fermentation process. This shows that the role of seaweed as
substrate is very important in order to make sure it works well with the PKC to
produce nutritive bioprotein. This finding has been supported by a study done by
Al Azad et al. [23] which has reported that the use of seaweed as substrate during
the fermentation process is a promising way in preparing a feed supplement for
finfish larvae.
Other than that, the addition of PKC as the substrate along with the seaweed
showed better results compared to the usage of the three types of seaweed only as
substrate during the fermentation. This is due to the high nutritional value of PKC
that contains an abundant amount of nutrients like protein (14.5-19.6%),
carbohydrate (34.9-52.5%) and fibre (13.0-20.0%) [24]. The comparison between
the protein content before (control) and after the fermentation was also compared
where the maximum amount of the protein content after the fermentation was
obtained from S. fulvellum followed by C. lentillifera and E. cottonii. It was also
observed that the total protein contents got increased from 37.53 mg/g to 52.28
mg/g and 35.16 mg/g to 36.06 mg/g for S. fulvellum and E. cottonii respectively.
Our results were in agreement with the study done by Igbabul et al. [25], where
the fermentation process has improved the protein content of the fermented
Potentiality of Selected Seaweed for the Production of Nutritious Fish Feed . . . . 35
Journal of Engineering Science and Technology Special Issue 3 1/2015
Mahogany Bean (Afzelia africana) flour. This is due to the extensive hydrolysis
of the protein molecules to simpler components like peptides and amino acids
caused by the increase in microbial biomass during the fermentation process.
Similar observations were reported by Azoulay et al. [26] which showed that the
fermentation process has improved the protein content of cassava from 8.75%-
20.6%. On the other hand, the protein content of green seaweed, C. lentillifera
was reduced after the fermentation. A similar result was reported by Osman [27]
which stated that the protein content was not significantly changed after the
fermentation process and a gradual and insignificant decrease in the amount of
protein was observed after the 20 hours of fermentation.
Fig. 1. The amount of protein content (mg/g of substrates)
for three types of seaweeds (Mean ±±±±SD; n=3).
3.2. Analysis of total ash content
Ash content is the indication of the amount of mineral elements present in the
bioprotein produced after the fermentation process [28]. According to Craig and
Helfrich [29], the minimum amount of ash content required for fish is about 8.5%.
Hence, the ash content found in the three types of seaweeds (used in this study)
can be considered as high compared to the minimum requirement for fish with the
maximum percentage of ash was observed in E. cottonii (15.1%) followed by C.
lentillifera (14.97%) and S. fulvellum (11.27%) before the fermentation as
depicted in Fig. 2. The same results were also reported by Al Azad et al. [23]
where the ash content of C. lentillifera was higher than Sargassum sp.. However,
the results obtained from this study were slightly different from the study done by
Matanjun et al. [22] in which they found that the least ash content was shown by
C. lentillifera instead of S. polycystum. This might be due to the difference in the
seasonal and environmental growth conditions of the place where the seaweeds
had been collected. Although the ash content after the fermentation using S.
fulvellum was found to be the least among the other two seaweeds, the percentage
improvement from control (before the fermentation process) to the enriched
bioprotein was higher (61.31%) compared to C. lentillifera (51.17%) and E.
cottonii (57.02%), as shown in Table 1. This shows that fermentation process
really gives significant improvement for S. fulvellum.
36 N. N. Ilias et al.
Journal of Engineering Science and Technology Special Issue 3 1/2015
Table 1.Percentage improvement in ash content (%) from
control to the enriched biomass (after fermentation).
Fig. 2. The amount of ash content (%) for the three types of seaweed (Mean ±±±± SD; n=3).
3.3. Analysis of carbohydrate and total reducing sugars
Soluble carbohydrate in fish feed is important not only as a source of energy, but
also because of its ability to increase the pellet integrity and stability, which is a
crucial characteristic for fish feed [20]. In this study, the maximum amount of
carbohydrate was observed after the fermentation of using green seaweed (C.
lentillifera). Contradict results were observed from the study done by Murugaiyan
et al. [30] which found that Sargassum sp. has the maximum carbohydrate content
(25.5±1.37%) followed by the other types of brown seaweed which is
Stoechospermum marginatum (15.8±0.8%). Green seaweed, Caulerpa taxifolia
and Caulerpa racemosa on the other hand showed a minimum carbohydrate
content which are (9.7±0.47%) and (8.5±0.84%) respectively. Nutrition status of
the cells has contributed to the variations of lipids and carbohydrate contents in
the seaweed. It was also observed from Table 2 that the amount of carbohydrate is
decreasing at the end of the fermentation process compared to the control (before
the fermentation process) and S. fulvellum was found to have the highest
carbohydrate content in the final bioprotein produced. This is due to the utilisation
of glucose by the microorganisms to support their growth [27].
Due to high carbohydrate content of S. fulvellum after the fermentation,
additional analysis about the total reducing sugars for S. fulvellum was conducted.
The results proved that the excellent growth of microorganisms has caused the
increment of the amount of total reducing sugars at the end of the fermentation
(day 6) from 56.5 mg/g to 345 mg/g due to the breaking down of the cellulose
Type of Seaweed Percentage Improvement of Ash(%)
Sargassum fulvellum 61.31
Caulerpa lentillifera 51.17
Eucheuma cottonii 57.02
Potentiality of Selected Seaweed for the Production of Nutritious Fish Feed . . . . 37
Journal of Engineering Science and Technology Special Issue 3 1/2015
since it has been metabolised by the microorganisms during the fermentation
process. This was clearly proven in Figs. 3(a) to (c) which show that the growth
of the P. chrysosporium and C. utilis were observed to be well-supported by S.
fulvellum compared to the other two types of seaweeds.
Table 2. Amount of carbohydrate content (mg/g of substrates)
for the three types of seaweed (Mean ±±±± SD; n=3).
Sargassum fulvellum Caulerpa lentillifera Eucheuma cottonii
Control 549.420 ± 15.21 623.044 ± 120.42 565.986 ± 17.68
PKC+Seaweed
(Fermentation) 445.886 ± 1.77 419.658 ± 17.68 258.145 ± 30.05
(a) (b) (c)
Fig. 3. Solid state fermentation of (a) Sargassum fulvellum
(b) Eucheuma cottonii and (c) Caulerpa lentillifera.
4. Conclusions
From all the results of analysis, S. fulvellum was found to be the best type of
seaweed in which the nutritional compounds were significantly improved after the
fermentation compared to C. lentillifera and E. cottonii. The protein content,
percentage improvement of ash and total carbohydrate of S. fulvellum were found
to be 52.28 mg/g, 61.31% and 445.89 mg/g of substrates respectively. The
amount of reducing sugars was also increased which shows that the substrate was
metabolised by the microorganisms during the fermentation process. It can also
be concluded that the solid state fermentation process has the ability to improve
the nutritional value of the seaweed, which is very important in the diet of fish
and can lead to the production of high nutritional fish feed. The nutritious fish
feed will then produce high quality and healthy fish that is also one of the
important sources of protein for human and animals consumption. Further study
can be done in narrowing the scope of the nutritional analysis by analysing the
amino acid content and fibre compositions of the seaweed which is also important
for fish.
38 N. N. Ilias et al.
Journal of Engineering Science and Technology Special Issue 3 1/2015
Acknowledgements
Research was supported by a research grant FRGS13-025-0266 approved by the
Ministry of Higher Education (MOHE), Malaysia. The authors would also like
to thank the Research Management Centre and Department of Biotechnology
Engineering, IIUM, for supporting and providing the laboratories facilities for
this project.
References
1. Pereira, R.; Valente, L.M.P.; Sousa-Pinto, I.; and Rema, P. (2012). Apparent
nutrient digestibility of seaweeds by rainbow trout (Oncorhynchus mykiss)
and Nile tilapia (Oreochromis niloticus). Algal Research, 1(1), 77-82.
2. Guroy, B.K.; Cirik, S; Guroy, D.; Sanver, F.; and Tekinay, A.A. (2007).
Effects of Ulva rigida and Cystoseira barbata meals as a feed additive on
growth performance, feed utilization, and body composition of Nile Tilapia,
Oreochromis niloticus. Turkish Journal of Veterinary Animal Science, 31(2),
91-97.
3. Francis, G.; Makkar, H.P.S.; and Becker, K. (2011). Antinutritional factors
present in plant-derived alternate fish feed ingredients and their effects in
fish. Aquaculture, 199 (3-4), 197-227.
4. Nakagawa, H.; and Montgomery, W.L. (2007). Algae. In: Dietary
supplements for the health and quality of cultured fish. Edited by Nakagawa,
H.; Sato, S.; and. Gatlin III. D. CABI North American Office Cambridge,
MA 02139 USA, 133-168.
5. Devi, G.K.; Manivannan, K.; Thirumaran, G.; Rajathi, F.A.A.; and
Anantharaman, P. (2011). In vitro antioxidant activities of selected seaweeds
from Southeast coast of India. Asian Pacific Journal of Tropical Medicine,
4(3), 205-211.
6. Benjama, O.; and Masniyom, P. (2011). Nutritional composition and
physicochemical properties of two green seaweeds (Ulva pertusa and U.
intestinalis) from the Pattani Bay in Southern Thailand. Songklanakarin
Journal of Science and Technology, 33(5), 575-583.
7. Ahemad, S.; Ismail, A.; and Mohammad Raduan, A. (2006). The seaweed
industry in Sabah, East Malaysia. Jati, 11, 97-107.
8. Chong, C.H.; Zulkifli, I.; and Blair, R. (2008). Effects of dietary inclusion
of palm kernel cake and palm oil, and enzyme supplementation on
performance of laying hens. Asian-Australasian Journal of Animal
Sciences, 21(7), 1053-1058.
9. Imandi, S.B.; Karanam, S.K.; and Garapati, H.R. (2010). Optimization of
media constituents for the production of lipase in solid state fermentation by
Yarrowia lipolytica from palm Kernel cake (Elaeis guineensis). Advances in
Bioscience and Biotechnology, 1(2), 115-121.
10. Kheng, P.P. and Ibrahim, C.O. (2005). Xylanase production by a local
fungal isolate, Aspergillus niger USM AI 1 via solid state fermentation using
palm kernel cake (PKC) as substrate. Songklanakarin Journal of Science
and Technology, 27(2), 325-336.
Potentiality of Selected Seaweed for the Production of Nutritious Fish Feed . . . . 39
Journal of Engineering Science and Technology Special Issue 3 1/2015
11. Couto, S.R.; and Sanroman, M.A. (2006). Application of solid-state
fermentation to food industry- A review. Journal of Food Engineering,
76(3), 291-302.
12. Jecu, L. (2000). Solid-state fermentation of agricultural wastes for
endoglucanase production. Industrial Crops and Products, 11(1), 1-5.
13. Saheed, O.K. (2012). Bio-protein Production by Solid State Bioconversion
using Banana Peel as Carbon Source. Master Thesis. International Islamic
University Malaysia, Malaysia.
14. Ke, L.; Wu, Q.; and Zhang, D. (2011). Bioconversion of rape straw into a
nutritionally enriched substrate by Ganoderma lucidum and yeast. African
Journal of Biotechnology, 10(29), 5648-5653.
15. Association of Official Analytical Chemists (AOAC) (2000). Official
Methods of Analysis, Method 923.03, Ash of flour: direct method (17th Ed.).
Arlington, VA.
16. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; and Randall, R.J. (1951). Protein
measurement with the Folin phenol reagent. Journal of Biological
Chemistry, 193(1), 265-275.
17. Dubois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers. P.A.; and Smith, F.
(1956). Colorimetric method for determination of sugars and related
substances. Analytical Chmistry,28(3), 350-356.
18. Krishnaveni, S.; Theymoli, B.; and Sadasivam, S. (1984). Sugar distribution
in sweet stalk sorghum. Food Chemistry, 15(3), 229-232.
19. Miller, G.L. (1959). Use of dinitrosalicylic acid reagent for determination of
reducing sugar. Analytical Chemistry, 31(3), 426-428.
20. Gatlin, D.M. (2010). Principles of fish nutrition. Retrieved April 26, 2014,
from https://www.extension.org/mediawiki/files/3/3b/Priciples_of_Nutrition.pdf.
21. Mahmud, N.A.; Robiul Hasan, M.D.; Belal Hossain, M.; and Minar, M.H.
(2012). Proximate composition of fish feed ingredients available in
Lakshmipur region, Bangladesh. American-Eurasian Journal of Agricultural
and Environmental Sciences, 12 (5), 556-560.
22. Matanjun, P.; Suhaila, M.; Noordin, M.M.; and Kharidah, M. (2009).
Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa
lentillifera and Sargassum polycystum. Journal of Applied Phycology, 21(1),
75-80.
23. Azad, S.A.; and Teo, Z.X. (2012). Suitability of seaweed meal incorporated
with Rhodovulum sp. bacterium as feed supplement for finfish larvae.
Borneo Science, 30, 57-63.
24. Jørgensen, H.; Sanadi, A.R.; Felby, C.; Lange, N.E.K.; Fischer, M; and
Ernst, S. (2010). Production of ethanol and feed by high dry matter
hydrolysis and fermentation of palm kernel press cake. Applied Biochemistry
and Biotechnoogy.,161(1-8),318-322.
25. Igbabul, B.; Hiikya, O.; and Julius, A. (2014). Effect of fermentation on the
proximate composition and functional properties of mahogany bean (Afzelia
africana) Flour. Current Research in Nutrition and Food Science, 2(1), 1-7.
26. Azoulay, E.; Jouanneau, F.; Bertrand, J.C.; Raphael, A.; Janssens, J.; and
Lebeault, J.M. (1980). Fermentation methods for protein enrichment of
40 N. N. Ilias et al.
Journal of Engineering Science and Technology Special Issue 3 1/2015
cassava and corn with Candida tropicalis. Applied and Environmental
Microbiology¸ 39(1), 41-47.
27. Osman, M.A. (2011). Effect of traditional fermentation process on the
nutrient and antinutrient contents of pearl millet during preparation of
Lohoh. Journal of the Saudi Society of Agricultural Sciences, 10(1), 1-6.
28. Ratana-arporn, P.; and Chirapart, A. (2006). Nutritional evaluation of
tropical green seaweeds Caulerpa lentillifera and Ulva reticulate. Kasetsart
Journal (Natural Science), 40, 75-83.
29. Craig, S.; and Helfrich, L. A. (2009). Understanding fish nutrition, feeds,
and deeding, Retrieved January 14, 2014, from https://pubs.ext.vt.edu/420/
420-256/420-256_pdf.
30. Murugaiyan, K.; Narasimman, S.; and Anatharaman, P. (2012). Proximate
composition of marine macro algae from Seeniappa Dharka, Gulf of Mannar
region, Tamil Nadu. International Journal of Research in Marine Sciences,
1(1), 1-3.
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