**2. Material and methods**

The total of 30 (thirty) samples (500g each) of fish flour from Brazil were collected from pub‐ lic markets at the Amazon region. The samples were sailed in a bulk.

The methods of analysis were:

flour for some Amazon communities, because they are geographically far from the power energy supplies to keep poultry food. On the other hand, some environmental conditions from the Amazon region, such as high temperature (>30°C) and RH >80% associated to the poor safety conditions of the process can favor the contamination, especially by fungi that can be toxigenic, such as the mycotoxin producers [12]. The aflatoxin is one of those meta‐ bolic produced by some fungi strains with carcinogenic action to human beings and their

They have been reported, not only in nuts and vegetable products, but also in animal feed and meat products. Some aflatoxigenic moulds have been isolated from salted fish samples such as *Candida* spp., *Rhodotorulla* spp. and *Aspergillus* spp. [14]. Concerning the possibility of aflatoxigenic moulds in animal feed and to prevent contamination in the Amazon region consumers diet, a work was carried out in order to evaluate the presence of aflatoxin in fish flour samples from the Amazon Region a work was carried out concerning the evaluation of water activity (*aw*), moisture content (*mc*), aflatoxigenic fungi strains and total aflatoxin.

The total of 30 (thirty) samples (500g each) of fish flour from Brazil were collected from pub‐

lic markets at the Amazon region. The samples were sailed in a bulk.

level in food supply must be studied [13].

200 Aflatoxins - Recent Advances and Future Prospects

**Figure 3.** Flowchart of general Fish Flour Process

**2. Material and methods**


*Sample preparation:* the samples were visually inspected in order to identify the presence of bones. The samples were finely ground in a mill (particle size <100 µm) and homogenized*;*

*Chemicals*: aflatoxin standards and trifluoroacetic acid (TFA) were purchased by Sigma-Al‐ drich while acetonitrile, methanol (HPLC grade) and n-hexane were purchased by Nuclear;

*Instrumentation*: The HPLC operating conditions were as follows: Colum type and size: C18 Supelco; 25cm x 4.6 mm id; 5 micron particle size; Temperature: room temperature 25C; Mobile phase: deionized water: acetonitrile:methanol:water (8:27:65) and the flow rate was fixed at 1.0 ml min-1; membrane filter and degassed in an ultrasonic bath for 25 min prior to use;

*Standards preparation*: the aflatoxin B1, B2, G1 and G2 standards (1.0 mg of each aflatoxin) in capped amber bottles) were used to the working solutions were prepared according to the AOAC [19] procedure by injecting 1 ml of acetonitrile into each vial to dissolve the aflatoxins.

*Extraction and clean-up*: 20 g of sample was extracted with 80 mL acetonitrile:water (9:1) mix‐ ture for 30 min by shaking under high speed and then filtered using a N°. 04 Whatman filter paper. A 1 mL portion of the filtrate was loaded on a multifunctional column and passed through at a flow rate of 2 mL/min. Then 1mL of acetonitrile:water (9:1) was applied to the column for 5 times. The filtrates were combined and evaporated to dryness under nitrogen and the residue was used for the derivatisation.

*Derivatization:* a 100 µl of the TFA solution and 300 µl of n-hexane were added to the residue from the sample extracted or to the aflatoxin work standards, vortexed for 30 s and kept in the dark for 15 minutes in room temperature. Nine hundred microlitres of acetonitrile:water (9:1) was added to the vial and vortexed for 30 s. The mixture was left to stand to allow the two layers to be separated. Twenty microlitres of the derivatized product (bottom layer) was injected into the HPLC column.

**3.** *Water activity* (*aw*): was determined in triplicate in an Aqualab series 3TE instrument (Decagon, USA) at 25±0.1°C;

The presence of aflatoxigenic fungi strains can be explained by the environment contam‐ ination, since the product was disposable in room temperatures with no regards of hy‐ gienic standards. The Brazilian regulation does not require the fungi analysis in fish or fish products [21]. In the process of fish flour temperatures of > 60-80° C for 60 min are applied with the binomial time-temperature acting on the microbiological control. The fish flour is rich in protein and nutrients to be spoiled by aflatoxigenic fungi strains. Adding NaCl 2%, during the process seems to not affect efficiently as a preservative fac‐ tor to avoid fungi strains. In cured fish, for example, slight inhibition of mycelial growth and/or sporulation was recorded when isolates were cultured in basal medium contain‐ ing 5% sodium chloride. On the other hand, the extent of inhibition increased with in‐ creasing salt concentrations, at 25% level, all the species had their growth completely

Aflatoxin in Fish Flour from the Amazon Region

http://dx.doi.org/10.5772/51948

203

The samples presented the following results with mean (range) described in Table 03. The *(a) aw*: 0.65 (0.64-0.70); *(b) mc*: 15.5 (10.0-20.8) % and *(c)* total aflatoxin (B1+B2+G1+G2): 10.5 (1.5-18.0) µg/kg. The aflatoxin was found in 20% of the samples under the LOQ. All the posi‐ tive samples were under the limit of the Brazilian regulation for animal feed of 50 µg/kg [23]. The 05 (five) positive samples for aflatoxin belong to the group of samples with *A. fla‐ vus* isolated in the fungi test, and identified as aflatoxin producers. This fact, confirms the association between the presence of aflatoxigenic strains and the aflatoxin production in fish

> **Aw Mc % Total Aflatoxin µg/kga Mean (range) Mean (range) Positive samples Mean (range)**

30 0.65 (0.64-0.70) 15.5 (10.0-20.8) 05 (20%) 10.5 (1.5-18.0)

The aflatoxin production in the fish flour could be affected by the levels of *aw* and *mc*. Those parameters have shown to allow the toxigenic fungi strains into the aflatoxin pro‐ duction, as showed in other dry food, such as nuts [24]. In previous work [25], the *aw* levels ranged from 0.1-0.90 and the microbiological stability of piracuí was showed at *aw* < 0.6 if *mc* will be below 10g%. The levels of our findings of *mc* were higher than 10%, so these levels must be concerned, because in *aw* below 0.6, there was reported shortly halophilic bacteria growth. Our results, concerning the *mc* levels were below 18.6%, re‐

inhibited [22].

flour samples.

**Number of Samples**

Total aflatoxin= B1+B2+G1+G2

ported by Santos & Freitas [26].

**Table 3.** *Aw, Mc* and Total Aflatoxin in fish flour from the Amazon region

a

**3.2. Aw, Mc and total aflatoxin**

*(d)Moisture Content (mc):* the *mc* levels were determined by the gravimetric method [19];
