**1. Introduction**

The Amazon region is well-known for biodiversity and nutritious food. The sustainable use of wildlife is considered strategically as an action for the conservation of the natural tropical environments and maintaining biodiversity [1]. The fish trade market, for example, compris‐ es different countries and consumers with different intention of use, and requires a process chain of frozen fish as support. On the other hand, the Fish waste management has been of the problems with the greatest inpect on teh environment. Most of the waste is discarded or used in other fish products. Treated fish waste has found many applications among which the most important are animal feed, biodiesel/biogas, dietic products (chitosan), natural pig‐ ments (after extraction), food-packaging applications (chitosan), cosmetics (collagen), en‐ zyme isolation, soil fertilizer and moisture maintenance in foods (hydrolysates)[2]. The muscle of some fish species with low fat, for example, can be useful in the flour production. In Brazil, the fish industries waste provides environmental pollution due to the inadequate disposable ways, in most of the cases. About 50% of the biomass produced by the industries is discarded along the process. Thus, there is an increasing interest for other ways of profita‐ bility of those wastes, since a high amount of fish protein has been lost [3]. With the increas‐ ing world population, it became necessary to search for alternative foods, to increase the demand and supply. These sources of food should be nutritious, have good sensory charac‐ teristics and be low cost, to achieve much of the population [4]. The alternative that has grown tremendously in the market is to concentrate the protein of raw materials. The pro‐ tein concentrate which has a high nutritional value and has a low cost of raw material used, aims to provide a product with the human element constructor, no fat, avoiding the intake of saturated fats cause high cholesterol, obesity and other consequences negative health [3]. Thus, a more directed waste recovery of slaughtered animals can be used in the form of di‐ rect consumption by humans, or indirectly by means of the feeding [4]. This protein concen‐

properly cited.

© 2013 Kluczkovski and Kluczkovski Junior; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is © 2013 Kluczkovski and Kluczkovski Junior; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

trate product could be obtained by other sources of animals such as fish [5] and soy or nuts [6, 7]. The fish flour is an alternative protein source for the natives of the Amazon region and came from the Indian culture. Beyond that, the fish flour is used as animal feed in some regions. The flour of the acari-bodó (*Liposarcus pardalis*) fish, for example, is called "piracui" and it is considered the "classic" fish flour. In the Tupi language, "piracuí" means fish dry fire, pounded in a mortar, grind him to powder, sifted, put into and kept in a smokehouse. The idea was born of conserving food for all the Indian tribes of the Amazon. At the time of low water (drought) had plenty of food (hunting and fishing). And at the time of the great waters (floods), food was difficult because the fish moved around. Thus was born the idea of storing food: game meat (boiling them with herbs that will retain for several days). The native long dominate technique resulting in dehydration of fish product known as piracuí. Only one type of fish derived from fish muscle, dried and shredded, which represents a ma‐ jor source of protein, average of 70% protein of optimum digestibility in the diet of some population especially the poor [8].The production involves an artisanal drying process, with the raw material of the fish waste or the whole meat from the fish. The flour is regularly sailed in a bulk in local markets of the Amazon region at the Amazon environmental condi‐ tions with temperature above 25°C and relative humidity (RH) above 70%. In most of the markets it is sailed between other products. The illustration of the fish flour presentation is presented in Figure 1 (a and b).

(a) Fish Flour (Piracuí) sold in the market (b) Fish Flour (Piracuí)

duction region has different procedures of process. It can be visible in the product the pres‐

Samples of piracui from *Liposacus pardalis*; b Protein concentrate from nine different fish species (range); c

The production of piracuí takes some stages and the flowchart is described in Figure 3. The fish or fish waste are washed and, the fish is eviscerated. They are cooked in an oven (100°C) and Sodium Chloride 2% is added. Then, a stage of drying is applied with temperatures of 60 to 80°C for 50 to 60 min. The material is cooled in room temperature and packaged in

The low water activity (*aw*) and moisture content (*mc*) levels in the product can increase the stability and shelf life, because the flour does not require refrigeration or low temperatures of storage, and can be kept in the environmental conditions. This is an advantage of the fish

**Table 1.** Proximate composition of fish and fish products samples according different authors.

**Proximate Composition of fish products Peixoto Castro[9] a Murueta [10] b Romanelli&Schmidt [11]c**

Piracuí done by grilled fish; f

Samples of

Piracuí done by cooked fish; g

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ence of bones and collagen fibers such as showed in Figure 2.

Caloriesd 356.8e 350.5f 3888.4-5015.9 - Moisture g 7.3 11.8 69.9-82.5 2.4-3.8 Proteing 76.4 75.5 57.4-77.8 36.8-63.4 Lipidsg 4.7 4.7 0.6-16.5 22.2-52.5 Ashg 9.4 6.5 8.1-20.2 2.3-12.4

Viscera Flour from *Caiman yacare* ; d Expressed in kcal; e

**Figure 2.** Small bones in Fish Flour (Piracuí)

polyethylene bags and stored at room temperature.

a

expressed in g%.

#### **Figure 1.** Fish Flour (Piracuí) presentation

It is consumed as ingredient in the local cuisine or as a protein source. Some authors report‐ ed the final product proximate composition of flour and protein concentrate as described in Table 01.

The protein levels around 78% from piracuí seems to be higher than other protein concen‐ trate obtained from other fish ranging from 57.4 to 77.8g% [10]. The fish flour is sailed in common markets and there is no color or granulometry standard, since each artisanal pro‐ duction region has different procedures of process. It can be visible in the product the pres‐ ence of bones and collagen fibers such as showed in Figure 2.


a Samples of piracui from *Liposacus pardalis*; b Protein concentrate from nine different fish species (range); c Samples of Viscera Flour from *Caiman yacare* ; d Expressed in kcal; e Piracuí done by grilled fish; f Piracuí done by cooked fish; g expressed in g%.

**Table 1.** Proximate composition of fish and fish products samples according different authors.

**Figure 2.** Small bones in Fish Flour (Piracuí)

trate product could be obtained by other sources of animals such as fish [5] and soy or nuts [6, 7]. The fish flour is an alternative protein source for the natives of the Amazon region and came from the Indian culture. Beyond that, the fish flour is used as animal feed in some regions. The flour of the acari-bodó (*Liposarcus pardalis*) fish, for example, is called "piracui" and it is considered the "classic" fish flour. In the Tupi language, "piracuí" means fish dry fire, pounded in a mortar, grind him to powder, sifted, put into and kept in a smokehouse. The idea was born of conserving food for all the Indian tribes of the Amazon. At the time of low water (drought) had plenty of food (hunting and fishing). And at the time of the great waters (floods), food was difficult because the fish moved around. Thus was born the idea of storing food: game meat (boiling them with herbs that will retain for several days). The native long dominate technique resulting in dehydration of fish product known as piracuí. Only one type of fish derived from fish muscle, dried and shredded, which represents a ma‐ jor source of protein, average of 70% protein of optimum digestibility in the diet of some population especially the poor [8].The production involves an artisanal drying process, with the raw material of the fish waste or the whole meat from the fish. The flour is regularly sailed in a bulk in local markets of the Amazon region at the Amazon environmental condi‐ tions with temperature above 25°C and relative humidity (RH) above 70%. In most of the markets it is sailed between other products. The illustration of the fish flour presentation is

(a) Fish Flour (Piracuí) sold in the market (b) Fish Flour (Piracuí)

It is consumed as ingredient in the local cuisine or as a protein source. Some authors report‐ ed the final product proximate composition of flour and protein concentrate as described in

The protein levels around 78% from piracuí seems to be higher than other protein concen‐ trate obtained from other fish ranging from 57.4 to 77.8g% [10]. The fish flour is sailed in common markets and there is no color or granulometry standard, since each artisanal pro‐

presented in Figure 1 (a and b).

198 Aflatoxins - Recent Advances and Future Prospects

**Figure 1.** Fish Flour (Piracuí) presentation

Table 01.

The production of piracuí takes some stages and the flowchart is described in Figure 3. The fish or fish waste are washed and, the fish is eviscerated. They are cooked in an oven (100°C) and Sodium Chloride 2% is added. Then, a stage of drying is applied with temperatures of 60 to 80°C for 50 to 60 min. The material is cooled in room temperature and packaged in polyethylene bags and stored at room temperature.

The low water activity (*aw*) and moisture content (*mc*) levels in the product can increase the stability and shelf life, because the flour does not require refrigeration or low temperatures of storage, and can be kept in the environmental conditions. This is an advantage of the fish 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 level in food supply must be studied [13].

The methods of analysis were:

min prior to use;

the aflatoxins.

and the residue was used for the derivatisation.

injected into the HPLC column.

**a.** *Aflatoxigenic moulds:* the samples were prepared and examined according to the techni‐ que recommended by APHA [15]. The identification of isolated mould and yeast genera was carried out according to Pitt & Hocking [16]. For the evaluation of aflatoxigenic strains, we used the method of incubation of strains from coconut agar (5-7 d, 26-28° C) [17]. To the *Aspergillus* spp. strain, only that identified as *A. flavus* were tested. After in‐ cubation the colonies were observed in UV light. The fluorescence indicative of the

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**b.** Total aflatoxin (B1+B2+G1+G2): the samples were analyzed by HPLC [18]. The Limit of Quantification (LOQ) was 0.95 µg/kg. Five points were used to build an analytical curve, in order to obtain the correlation coefficient (R) values for LOD and LOQ. Each point corresponded to a mean of five injections of each extract. The recoveries for each

*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

*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

*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

*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

aflatoxin (B1, B2, G1 and G2) were: 91.0; 75.0; 95.0 and 92.0%, respectively.

presence of aflatoxins was observed at the reverse of the plate.

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

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.
