Analysis of Proximate Composition and Aflatoxins of Some Poultry Feeds
J. John Prabakaran
In the present study raw samples like bamboo rice, pani varagu, thiri varagu, kampu, saamai and koran thinai used for poultry feeds production were collected from Kolli hills region of Central Tamil Nadu in India. When they were analyzed for proximate composition, high moisture content (11.70%), high protein content (11.34%), high crude fiber (10.16%), crude fat (4.69%) and high total ash content (5.12%) were reported in koran thinai, bamboo rice, thiri varagu, kampu and pani varagu, respectively. Aflatoxins were detected in kampu (220 ppb of AFB1 and 45 ppb AFB2) and in saamai (15 ppb AFB1) only and absent in other samples. Generally aflatoxins production increased in autoclaved samples compared to non autoclaved samples. Significantly kampu showed high level of AFB1 (900 ppb) followed by pani varagu (630 ppb). From this study it is clear that the aflatoxins presence in the raw food samples must be checked properly as it will cause economic loss of crops to be used as source. So, proper storage and harvesting methods should be adopted to prevent aflatoxins contamination.
Aflatoxins are a group of polyketide-derived furanocoumarins which are carcinogenic
among the known mycotoxins. They are mainly of four major groups, AFB1, AFB2,
AFG1 and AFG2 of which, AFB1 is a potent carcinogen. They are hepatotoxic secondary
metabolites produced by some strains of Aspergillus flavus, A. parasiticus,
A. nomius and A. tamarii (Dvorack, 1999).
They cause health problem to live stock and human beings by contaminating agricultural
commodities (Benneth et al., 2003; Bhatnagar
et al., 1993; Payne, 1998). High amount of
aflatoxin is produced in agricultural crops like groundnut, cotton seed, wheat,
rice, barley, coconut, corn, dried peas, oat, sweet potato, millet and cassava
(Goldblatt, 1969). Due to acute aflatoxin poisoning 1000
people were affected out of this 100 people were died in India in 1974 (Krishnamachari
et al., 1975; Tandon and Tandon, 1988).
Aflatoxins are mainly produced in poultry feeds by many fungi such as Aspergillus
species and Fusarium species. Nearly 20% Aspergillus species
exhibit toxigenic activity in poultry feeds. Due to high stability they can
be easily destroyed by any methods. In humans and animals they may be implicated
in high incidence of hepato-cellular and lung carcinoma (Massey
et al., 1995). The development of aflatoxins depends on the infection
and growth of fungi in grains. Moisture acts as an important factor for the
growth of fungi. The minimum moisture level for aflatoxin production at 30°C
by A. flavus is equal to the moisture content of a product in equilibrium
with 83% relative humidity or higher, depending on the nature of the substrate
and the duration of storage. For starchy cereal seeds such as maize and wheat,
the limiting moisture level for growth of A. flavus is about 18.5% whereas
in oil seeds such as peanuts it is 8 or 9% (Christen and Kaufmann,
Aflatoxins are of industrial importance due to the economic loss resulting
from the contaminated crops especially in poultry industry (Pillet,
1998). The AFB1 is always present in varying degrees in poultry which are
probably the most sensitive food animals to its toxic effects and small amounts
of AFB1 cause reductions in growth rate, feed efficiency, hatchability and increased
susceptibility to disease (Coulombe, 1993).
In the present investigation some important poultry feeds produced frequently in Kollihills region of Namakkal district,Tamil Nadu,India were analyzed for aflatoxins. This area was selected as it is characterized with high moisture. In Namakkal area there are many poultry farms which obtain poultry feeds from Kolli hills. The main objectives were to determine moisture, crude protein, fat, fiber and total ash that is proximate analysis of poultry feeds, to detect AFB1 and AFB2 by thin layer chromatography and to find out the effect of autoclaving on the production of AFB1 and AFB2.
MATERIALS AND METHODS
Collection of Samples
Samples like bamboo rice, pani varagu, thiri varagu, kampu, saamai and koran
thinai were collected from Kolli hills region during winter season (November-December,
2008) and analyzed for proximate composition and aflatoxins in the Biotechnology
Research Centre of KSR College at Tiruchengode, Namakkal Dt, Tamilnadu, India.
In Kolli hills, plants which act as the sources of these poultry feeds are cultivated.
Kolli hills is a small mountain range located in Central Tamil Nadu in India.
The mountains are about 1000 to 1300 m in height and cover an area of approximately
280 km2. The Kolli hills is a part of the Eastern Ghats, which is
a mountain range that runs mostly parallel to the East coast of South India
reached by road easily from Namakkal city (50 km) (Fig. 1).
The proximate composition of raw poultry feeds such as moisture, crude protein,
crude fibre, crude fat and total ash content were analyzed using the procedures
described by (Sundaram et al., 2001).
Determination of Moisture
Five grams of poultry feed was weighed in a petridish, placed in a hot air
oven at 105-110°C for a minimum of 6 h, cooled in a dessicator. The process
of heating and cooling was repeated till a constant weight was obtained. The
moisture was removed as vapour.
|| Kolli hills region of Tamilnadu in India selected for poultry
feed samples collection
Determination of Crude Protein
Crude protein was determined using MicroKjeldajhls distillation assembly
(Hexatec, Maharashatra, India). Two grams sample was digested in H2SO4
using CuSO4 5H2O as catalyst and by adding Na2SO4
(anhydrous) or K2SO4 to elevate the boiling point converting
organically bound N to ammonium sulphate which when heated with excess alkali
(40% NaOH), NH3 is liberated which is distilled into a known excess
of standard acid (0.1 N H2SO4). The unrelated (unneutralised)
acid is back titrated with standard alkali (0.1 N NaOH). From the titre values
the nitrogen content and crude protein were calculated multiplying by 6.25.
Determination of Crude Fat
The crude fat (a combination of simple fat, fatty acid, esters, compound
fat, neutral fat, sterols, waxes, vitamins (A, D2, E, K), carotene, chlorophyll,
etc) soluble in ether was estimated by extracting in ether which was continuously
volatilized at 60-80°C condensed and allowed to pass through the thimble
containing the sample in a Soxhlets apparatus (Borosil, Mumbai, India).
Crude fibre consisting of cellulose, hemicellulose and lignin etc was estimated
by successive digestion of two grams of sample with dilute acid (0.255 N) and
alkali (0.313 N).The entire residue transferred into a silica crucible and kept
in a hot air oven at 105°C, cooled in a dessicator, weighed and finally
concordant values were obtained.
Determination of Total Ash
To find out the total content of mineral matter or total ash i.e., non-
combustible portion of the feed, 2 g of sample was weighed accurately in a silica
crucible. The sample was ignited on a burner till smoke ceases. The crucible
was placed in a muffle furnace and heated to 600°C and kept for 2 h. At
this temperature all organic matter was burnt leaving behind minerals. The crucible
was removed from the furnace carefully and cooled in a dessicator at room temperature
and weighed again.
Aflatoxins were analyzed by thin layer chromatography technique according
to the method of Romer (1975).
In addition to raw feeds analysis, the effect of autoclaving on aflatoxin
production was studied. For this 20 g of finely powdered sample was placed in
each 500 mL cortical flask and enough water (about 20 mL) was added to just
moisten the powder. The flasks were divided into two sets. Set 1 was autoclaved
at 15 pounds pressure for 15 min and set 2 was not autoclaved. Two loopful of
5 to 6 days old culture of A. flavus was streaked and grown on Sabourauds
dextorse agar. The inoculated flasks were incubated at 26±1°C for
8 days. At the end of incubation period flasks were sprayed with 95% alcohol,
dried overnight at 80°C (Nagaragan et al., 1973)
and used for aflatoxin analysis.
Detection and Estimation
Raw 10 g of dried poultry feed samples were beat with 40 mL of distilled
water for 2 min. Then 60 mL of acetone was added and again beat for 2 min and
then the contents were slightly heated up. The contents were filtered and to
the 30 mL of filterate approximateky 0.6 g of cupric carbonate in beaker (A)
was added. Thirty four milliliter of 0.2 M NaOH and 6 mL of FeCl3 (0.41
M) was taken in beaker (B) and swirled. Then both the contents were mixed and
filtered through whatman No.1 filter paper. Forty milliliters of the filterate
was taken in 250 mL separating funnel. Then, 0.03% of 40 mL sulphuric acid and
10 mL of chloro form was added and mixed. The chloroform layer was collected
in a 200 mL beaker and again 20 mL of chloroform was added again, mixed thoroughly,
allowed to settle and the chloroform layer in the same flask was collected.
Another separating funnel having 40 mL of 0.02 M KOH and 1% KCl mixture to this
collected 20 mL of chloroform extract was added and mixed it slowly and the
bottom layer was collected through anhydrous sodium sulphate bed drop by drop
to remove any traces of moisture. The chloroform extract was kept in an oven
set at 50°C till it becomes dry. Then aflatoxin film was dried, rediluted
with 0.2 mL chloroform and spotted on the TLC plate taking exactly 5, 10, 20
and 40 μL besides the standard spots of 5 and 10 μL. A colour change
from blue colour fluorescence band to pale yellow colour confirms the presence
of aflatoxin. The results were interpreted by visually comparing with standards
purchased from Hi-media Co., Mumbai, India and the concentration was ascertained
at 364 nm in a UV viewing cabinet.
All the determinations that is for proximate composition and aflatoxins analysis were carried out in triplicates and the date obtained were expressed as Mean±SD.
RESULTS AND DISCUSSION
All the samples were analyzed for moisture, crude protein, crude fiber, crude fat and total ash content. High amount of moisture (11.70%) was observed in koran thinai whereas low amount (9.38%) in bamboo rice. In case of crude protein high amount (11.34%) was resulted in bamboo rice, low amount (6.55%) in thiri varagu. High amount (10.16%) of crude fibre was observed in thiri varagu whereas bamboo rice showed low (0.79%) fiber content. Kampu showed high amount (4.69%) of crude fat whereas bamboo rice showed low amount (1.01%). The total ash content of pani varagu was high (5.12%), while bamboo rice showed low amount (1.63%) (Table 1).
Then aflatoxins were quantified for all the samples using TLC. Out of six samples only two samples were positive for aflatoxin presence. Kambu had 220 ppb of AFB1 and 45 ppb of AFB2 whereas saamai had only 15 ppb of AFB1 (Table 2).
Samples were inoculated with two loopful of standard strain of Aspergillus flavus to produce aflatoxin and the effect of autoclaving on the production of aflatoxin was studied. The results clearly show that the autoclaved samples showed high level of AFB1 and AFB2 than non autoclaved samples. In autoclaved and non autoclaved samples, kambu showed high levels of aflatoxins B1 (900 and 600 pbb, respectively), while Saamai showed high levels of B2 (330 and 210 ppb, respectively). The data shows on average there was three fold increase in toxin production in autoclaved samples compared to non autoclaved samples (Table 3).
|| Proximate analysis of poultry feeds
|The results are presented as Mean±SD
|| Quantification of aflatoxins in raw poultry feeds by TLC
|The results are presented as Mean±SD. -: Negative
|| Aflatoxin quantification in A.flavus inoculated poultry
feeds by TLC
|The results are presented as Mean±SD
The proximate composition influences the contamination of aflatoxins in raw
poultry food samples. Owen et al. (2008) have
reported proximate composition of heat treated poultry litter and obtained the
values for crude protein (20%),crude fiber (10.4%) and ash (18.5%).The total
ash content of pani varagu was high (5.12%) but bamboo rice showed low amount
(1.63%).The variations in the values may be related to the anatomical structure
of the plant sources. Similar results for ash content have been reported for
legumes (Elegbede, 1998).
No aflatoxins were detected in four of the samples in our studies except kampu
and saamai. It may be correlated to their high fat content compared to other
samples. Similarly aflatoxin was not detected in soyabean meal or broken rice
analyzed by Purwoko et al. (1991). Even though
they were absent in raw samples after milling aflatoxin may be produced during
storage (Mazen et al., 1990). Present results
confirm the importance of ingredients before incorporating them into mixed feeds
as reported by Thirumala-Devi et al. (2002) in
some important Indian poultry feeds. Escobar and Regueiro
(2002) have reported aflatoxin B1 with the biggest percentage in sorghum
and peanut, 83.3 and 40.4%, respectively in the analyzed food stuffs.
Aspergillus flavus isolates produce only AFB1 and AFB2,
while Aspergillus parasiticus and Aspergillus nominus produce
AFB1, AFB2, AFG1 and AFG2. The chemical
composition of the feeds determines the growth of the different fungal species.
Also seasonal and geographical factors and the conditions required for the cultivation
of the crops influence the aflatoxin contamination. (Jewers
et al., 1986). In Kambu the AFB1 level is less than 250 ppb. Poultry
diets containing above this level, when fed to poultry predispose them to attack
by bacteria and viruses. Aflatoxin is a hepatotoxin causing an excessive build
up of biliary ducts (Adav and Godinwar, 1997). The maximum
tolerated aflatoxin in the food samples should be 20 μg kg-1
(Smith and Moss, 1985).
Various methods have been tried to decontaminate aflatoxin contaminated commodities
such as groundnut, cotton seed, palm kernel cake/meal and maize. These include
physical methods (sorting, irradiation techniques, heating), chemical methods
(acids, bases, oxidizing agents), biological methods (microbiological) and solvent
extraction (Coker, 1986). Ammonia gas appears to be
the most promising approach as it is capable of reducing the aflatoxin level
in situ by more than 95% and is applicable to a variety of contaminated commodities
using batch and continuous processing methods (Coker et
The result revealed that aflatoxin production was increased in autoclaved samples
compared to non autoclaved. Similar results have been reported with As pergillus
tamarii which produces B group of aflatoxin and cyclopiazonic acid (Tetsuhisa
et al., 1996). It may be related to the denaturation of any compound
which may be inhibitory to aflatoxin synthesis in normal conditions and the
necessary compound may be available as reported in soyabeans.Phytic acid inhibits
aflatoxin formation by combining with zinc. During autoclaving zinc was liberated
from phytic acid, zinc was known to have a pronounced simulatory effect on aflatoxin
production. The destruction of phytic acid by heat results in the availability
of zinc for aflatoxin synthesis. Here also aflatoxin production increased in
autoclaved samples. Soybeans produce aflatoxin but extent of production depends
on the variety of soybean and the toxigenic potential of the isolates used (Nagaragan
et al., 1973). Aspergillus flavus strain NRRL 2999 produces aflatoxins
on the solid substrate rice. Optimal yields more than 1mg of AFB1 per gram of
starting material obtained in 5 days at 28°C have also been reported by
Mateles and Adye (1965). So, our poultry feed raw samples,
kambu and saamai also act as good substrate for the growth of aflatoxin producing
fungi. So proper harvesting and storage method have to be implemented to prevent
As the cost of feeding the animals and birds comes to nearly 70 to 75% of the production cost of the products, assessing the quality of raw materials to regular testing is very important. To inhibit the aflatoxin production the concerned gene should be inactivated in fungi. Some bacterial isolates which have detoxification effect on aflatoxin are also economically important. Bamboo rice, pani varagu, thiri varagu, kampu, saamai and koran thinai have been mostly used as poultry feeds in our local area as these are easily available and cost effective. So, much attention in the processing procedure should be given.
The authors are grateful to Executive Director and Principal of KS.R. College of Arts and Science, Tiruchengode for giving the facilities to carry out the work and Mr. R. Rajendiran, Cromopark Biotech Lab, Namakkal for his help in the sample analysis.
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