**3. Results and discussion**

## **3.1. Aflatoxigenic moulds**

All the samples (100%) presented fungi growth. The *Aspergillus spp.* was identified in 85% of the samples and 15 isolated were obtained and tested concerning aflatoxin production as showed in Table 02. According to other authors, *Aspergillus* spp. was the most frequent strain reported in feed and fish products. Hassan et al. [14] also found levels of the presence of *Aspergillus* spp. (66.6%). The *Penicillium* spp. was found in 43% of the samples and 90% of the isolated tested for aflatoxin production was negative.


**Table 2.** Fungi and aflatoxin production in fish four

In our work, from the *Aspergillus* spp strains identified as *A. flavus,* 85% presented aflatoxin production as showed in Figure 4. Alinezhad et al. [20] reported *A. flavus* (60.66%) isolated from feed ingredients as well as pellet feed. Among 37 *A. flavus* isolates, 19 (51.35%) were able to produce AFB1 in the range of 10.2 to 612.8 µg/g fungal dry weight. The aflatoxigenic behavior with fluorescence was showed in Figure 4.

**Figure 4.** Aflatoxigenic behavior with fluorescence production from *A. flavus* from fish flour

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 inhibited [22].

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

**3.** *Water activity* (*aw*): was determined in triplicate in an Aqualab series 3TE instrument

All the samples (100%) presented fungi growth. The *Aspergillus spp.* was identified in 85% of the samples and 15 isolated were obtained and tested concerning aflatoxin production as showed in Table 02. According to other authors, *Aspergillus* spp. was the most frequent strain reported in feed and fish products. Hassan et al. [14] also found levels of the presence of *Aspergillus* spp. (66.6%). The *Penicillium* spp. was found in 43% of the samples and 90% of

> **Number of isolated testeda**

*Aspergillus spp.* 85 15 85% 15% *Penicillium spp.* 43 10 10% 90%

In our work, from the *Aspergillus* spp strains identified as *A. flavus,* 85% presented aflatoxin production as showed in Figure 4. Alinezhad et al. [20] reported *A. flavus* (60.66%) isolated from feed ingredients as well as pellet feed. Among 37 *A. flavus* isolates, 19 (51.35%) were able to produce AFB1 in the range of 10.2 to 612.8 µg/g fungal dry weight. The aflatoxigenic

**Toxigenic Strains Positive Negative**

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

(Decagon, USA) at 25±0.1°C;

202 Aflatoxins - Recent Advances and Future Prospects

**3. Results and discussion**

the isolated tested for aflatoxin production was negative.

**samples (%)**

**Fungi Strains Incidence in the**

**Table 2.** Fungi and aflatoxin production in fish four

behavior with fluorescence was showed in Figure 4.

**Figure 4.** Aflatoxigenic behavior with fluorescence production from *A. flavus* from fish flour

Strains identified as *Aspergillus flavus*

**3.1. Aflatoxigenic moulds**

a

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 flour samples.


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

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‐ ported by Santos & Freitas [26].
