Influence of a homeopathic product on performance and on quality flour and cookie (Grissini) of Nile tilapia

Animals can also benefit from homeopathic remedies by having their immunological system and organic responses to stress reduction stimulated, as well as their balance reestablished. In this context, the objective of this study was to assess the performance of Nile tilapias ( Oreochromis niloticus ) treated with food containing a homeopathic product (Homeopatila 100®), as well as the physical, chemical, technological and sensory quality of flour and cookie (grissini) based on the fish co-product. An eighty-four days assessment was conducted on two types of diet for fishes with sexual inversion: control (CT), added with 40 ml of hydroalcoholic solution (alcohol 30° GL) per kilo of food; and homeopathic treatment (HT) added with 40 ml of homeopathic product Homeopatila 100® per kilo of food. The flours (NTF) were obtained using the head and neck-ends of Nile tilapias with subsequent physical, chemical and technological analyses. In order to assess the applicability of the flours from both treatments, we formulated the grissini (cookies) added with 10% NTF substituting wheat flour; physical, chemical, technological and sensory characteristics were assessed. Considering the studied period, the fish treated with food containing the homeopathic product Homeopatila 100® presented final total weight significantly higher (p < 0.05) than fish in the control group. The homeopathic product had no significant influence (p < 0.05) on the physical and chemical of the NTF, as well as the results of the assessments on cookies (grissini) produced with the addition of 10% NTF, including the sensory test.


INTRODUCTION
Aquaculture provides about one-third of fish-based products worldwide with a major role of Nile tilapia (Oreochromis niloticus) production. The production of this type of fish has expanded since the last decade because  of the technological advances associated with more intensive growth practices, especially in China, Philippines, Thailand, Indonesia and Egypt (Josupeit, 2010;Yarnpakdee et al., 2014).
Nile tilapias are the most cultivated fresh-water fish in Brazil; according to the Ministry of Fishing and Aquaculture, in 2011, the production exceeded 253 thousand tons (Brazil, 2011). The increase in the fish production also increases the quantity of generated subproducts that may be used to develop processed foods due to excellent sensory and nutritional characteristics. In this context, the development of new products, based on fish, residues represent a viable technological alternative since fish are historically associated with healthy food (Brazil, 2011).
Homeopathy is a form of complementary medicine that uses high dilutions of substances derived from plants, minerals or animals based on the principle of similarity (Khuda-Bukhsh and Pathak, 2008). Bell and Koithan (2012) proposed an innovative model for the action of homeopathic remedies on living organisms; the research indicates that homeopathic remedies are constituted by nanoparticles and this model provides a basis to guide researches on the function of nanomaterial in living systems, the action mechanism of homeopathic remedies and their use in nanomedicine. Animals can also benefit from homeopathic products by having their immunological system and organic responses to stress reduction stimulated, as well as their balance reestablished. In addition to contributing with prophylaxis by reducing stress, these products may also reduce the application of chemotherapy and antibiotics, preventing environmental risks for animals and consumers (Siena et al., 2010).
The use of homeopathic products in Nile tilapias diet (40 ml kg -1 of food) benefited fish' survival, muscle fiber hypertrophy, reduced the hepatic lipid inclusion, hepatosomatic index and total lipids content in the muscle tissue (Valentim-Zabott et al., 2008;Júnior et al., 2012;Andretto et al., 2014). However, there is only one study approaching the development and evaluation of food products (nuggets) based on fish treated with homeopathic medicines (Lima et al., 2015). In this study the aim was to evaluate the performance of Nile tilapia treated with foods containing a homeopathic product (Homeopatila 100®), as well as physical, chemical, technological and sensory quality of flour and biscuit (grissini) produced as a co-product of this fish.

Experimental protocol and sampling
The experiment was carried out at the experimental station for fishery of the State University of Maringá (UEM) -CODAPAR, district of Floriano, city of Maringá, state of Paraná. A hundred and sixty Nile tilapia males with sexual inversion were weighed, measured and distributed in eight fiber glass tank with capacity of 600 L, maintained at the density of 20 fish per box. The design was completely randomized with two treatments -control (CT) added with 40 ml hydro-alcoholic solution (30° GL) (vehicle as the homeopathic product) per kilo of food; homeopathic (HT) added with 40 ml Homeopatila 100® per kilo of food; each with four replicates (C1, C2, C3 and C4; H1, H2, H3 and H4, respectively). Table 1 presents the composition of the studied homeopathic product. The eighty-four day experiment was approved by the Animal Experimentation Ethics Committee of UEM (protocol 019/2013). The fish were treated with food containing 32% crude protein; the quantity provided was ad libitum in two daily portions (9 am and 4 pm). On a weekly basis, the homeopathic product Homeopatila 100® was incorporated into the food sprinkled with a manual sprayer; subsequently, the food was homogenized and dried at room temperature with periodical removal for 24 h. The food was stored in a dry, well-ventilated location without the presence of sunlight, chemical products or equipment emitting a magnetic field until reaching no alcohol odor and clusters. Equal inclusion process was carried out for the control treatment using hydroalcoholic solution.
At the final stage of the experiment, all fish were captured by nets, stunned with water and ice at 0°C and sacrificed through section of the spinal cord and separated per treatment. The whole fish were weighed and measured; heads were removed, washed, weighed and vacuum-packed in polyethylene bags. The neck-ends were extracted from the carcasses using High Tech® deboner (model HT C100), weighed and vacuum-packed in polyethylene bags. The heads and the neck-ends were stored at -18°C. The apparent feed conversion (AFC) was determined by the following equation: AFC = (Weight of meal provided during the period) × (Final weight -Initial weight) -1

Obtaining the Nile tilapia flour (NTF)
Four flours were obtained per treatment (one per replicates). The preparation of NTF followed the methodology by Franco et al. (2013), with modifications. Initially, heads and neck-ends were subjected to one-hour cooking process in a pressure pot; the residues were pressed in manual press for foods and grinded in a stainless steel mill (worm thread) Skymsen® (model PSEE-98MHD). Subsequently, the mass obtained was Quimis® kiln (model Q317B), allowed to remain at 60°C for 24 h. The obtained product was then grinded in cutting mill SOLAB® (model CE-430/Mini) and sieved at 60 mesh. The flours were weighed, vacuum-packed in polyethylene bags and stored at -18°C until the analyses and grissini production.

Characterizing Nile tilapia flour (NTF)
Moisture content, crude protein, ashes and total lipids were determined according to the methodology proposed by the Association of Official Analytical Chemists (AOAC, 2005). Minerals, calcium, iron and phosphorus were determined according to the analytical methods of the National Laboratory of Animal Reference (LANARA, 1981) and Institute Adolfo Lutz (IAL, 2008). Water activity (Aw) was assessed at 25°C in Aqualab® (model 4TE) equipment for the determination of water activity, according to the manufacturer's instructions. For color measurement, we used Minolta® colorimeter (model CR 400) with illuminant D65 and angle of view of 10°. The values of L (luminosity), a* (intensity of red color) and b* (intensity of yellow color) were expressed according to the color system by the Commission Internationale de L'Eclairage (CIELAB) (Minolta, 1998). The determination of the NTF yield was carried out gravimetrically through the relationship between the weight of heads and neck-ends in nature, and the weight of the extracted flours expressed in percentage. All of the analyses were carried in 3 replicates.

Cookie (grissini) formulation
In order to assess the applicability of the flours from both treatments, we formulated grissini added with 10% NTF substituting wheat flour. For the grissini production, the NTF from the four repetitions of TC (C1, C2, C3, and C4) were mixed and homogenized; equal process was employed for the HT with repetitions H1, H2, H3, and H4. The following ingredients and proportions were applied to produce the grissini: fresh yeast 2.3%; refined sugar 3%; distilled water 25%; salt 2.1%, oil 6%, wheat flour 56% and NTF (CT or HT) 5.6%. The cookies were baked in a Fisher® conventional oven at 200°C for 10 min; immediately after being cooked, the cookies were cooled at room temperature (25°C) and stored in polyethylene bags of high density and hermetically sealed glasses identified per treatment until the analyses.

Cookie (grissini) characterization
The moisture content, crude protein, ashes, total lipids, Aw, color and minerals (calcium, iron, phosphorus) were determined according to the description for NTF. Total carbohydrates were calculated by difference (100 g -total grams of moisture, crude protein, total lipids, and ashes). The content of soluble and insoluble dietary fibers was determined through the enzymaticgravimetric method 985.29 by the AOAC (Prosky et al., 1985); total dietary fiber was calculated using the sum of these two fractions. All of the analyses were carried out in triplicate. The characteristics of the cookies were assessed according to method 10 -50D (AACC, 2000); the dough was determined using pre-and post-heat weight expressed in grams. The width, thickness and length of the cookies were determined using millimeter scale ruler with pre-and post-heat values expressed in centimeters. The specific volume was calculated from the relationship between the apparent volume (determined using millet displacement method) and the post-heat weight of the cookies (Pizzinatto et al., 1993). The yield was determined through the difference between pre-and post-heat weight of the cookies. The results represented an arithmetic mean of 10 determinations derived from samples of a single test. The tensile strength was determined using Stable Micro Systems Texture Analyser TA.XT2 texture meter, with probe 3-Point bending Rig (HDP/3PB) and platform HDP/90. Results were expressed in Newton (N) representing an arithmetic mean of 15 determinations of tensile strength for samples derived from a single test randomly selected. The following parameters were employed: pretest velocity = 1.0 mm s -1 ; test velocity = 3.0 mm s -1 ; post-test velocity = 10.0 Fuzinatto et al. 677 mm s -1 ; distance 5 mm, with measure of compression force (Clerici et al., 2013).

Sensory evaluation
The sensory evaluation was approved by the Human Research Ethics Committee of UEM (623.527/2014). For the grissini acceptance test for both treatments, we applied a nine-point hedonic scale (1 = dislike extremely to 9 = like extremely) assessing the attributes of color, aroma, texture, taste and overall evaluation with the collaboration of 120 non-trained tasters -56 female, 64 male, ages ranging from 15 and 57 years old, all employees and students at the Federal Technological University of Paraná (Dutcosky, 2013). After the sensory evaluation, we calculated the Acceptance Index (AI) for the attribute of overall evaluation by using the following expression: Where A = average score for the attribute, and B = maximum score for the attribute (Lawless and Heymann, 2010).

Statistical and multivariate analyses
Fish performance results, the physical, chemical and technological analyses of the NTFs and grissini as well as the sensory evaluation were submitted to the t-Student means difference test (p<0.05). The multivariate analysis indicated the individual values for each replicate in the four NTF repetitions (n=24) for both treatments -CT and HT; the grissini had one repetition (n=6) and analyses in triplicate for both cases divided into data matrices. The first matrix contained the data of the NTF characterizations, and two matrices for the results obtained for the grissini. The data were preprocessed through self-scaling; in this case, data are centered on the mean and each individual value is divided by the standard deviation. The procedure is to guarantee that all of the variables have equal importance, that is, equal weight. Posteriorly, we applied the principal component analysis (PCA) using NIPALS algorithm with the scores (samples) and loadings (variables) decomposed in a dimensional graph. Statistical software, version 8.0 (StatSoft, 2007) was applied using 5% significance level (p<0.05) for the rejection of the null hypothesis and selection of the principal component. Table 2 presents the mean values of fish performance during the experimental period. No significant difference was observed between the treatments in relation to initial total weight, initial total length, final total length, head and neck-end weight, and apparent feed conversion (AFC); however, significant difference between the treatments (p < 0.05) was observed in relation to final total weight. The weight gain after 84 days for the tilapias treated with homeopathy (HT) was 207.16 g, while those in the control treatment (CT) presented 192.05 g. According to Rocha et al. (2002), weight gain is a very important factor when growing fish and determinant for the economic exploration of the area. Intensive growth systems impose animals with conditions that are opposite to the one found in their natural environment, such as high density and constant handling, factors that damage their well-being and have a negative influence on production. Stressed fish generally have their development compromised and possible hematological alterations followed by hyperglycemia and hemoglobin increase caused by the higher release of cortisol, which induces hepatic glycogenesis boost (Mazeaud et al., 1977;Carneiro, 2001). Siena et al. (2010) treated Nile tilapia (O. niloticus) with food containing four levels of the homeopathic product Homeopatila 100® in hydroalcoholic solution per kilo of food (0.20, 40 and 60 ml) concluding that the fish treated with 40 ml kg -1 of food presented higher survival rate and lower hepatosomatic index than the control group. Braccini et al. (2013) demonstrated adding Homeopatila 100® (40 ml kg -1 of food) to young Nile tilapias' diet reduced the hepatosomatic index, increased the amount of hepatocytes and the percentage of intracellular glycogen compared with fish in the control group.

Fish performance
The formulation of the studied homeopathic product has four compounds (iodum, sulphur, Natrum muriaticum and Streprococcinum); according to Vijnovsky (2003), they have different indications for the treatment of behavior disorders. Therefore, the product was developed in order to reduce production stress by acting on the vital energy and maintaining the organic homeostasis of the fish.

Characterizing Nile tilapia flour (NTF)
According to the t-Student test (p<0.05), no minimum significant difference was observed between the NTF treatments in relation to the physical, chemical analyses ( Table 3). The results of ashes and crude protein indicate that the NTF present high content of minerals in addition to being excellent sources of animal protein. Franco et al. (2013) studied the flour of Nile tilapia (O. niloticus) carcass and found contents of moisture, ashes and crude protein similar to those indicated in this study -2.15, 38.03, and 45.32%, respectively. The chemical composition of fish-based products may vary according to the source of acquisition of heads and neck-ends, the food provided to the fish and the applied filleting method. Another factor that may have influence on the proximate composition of fish-based flour is the methodology employed in the production. Monteiro et al. (2012) developed a flour based on the head and carcass of Nile tilapia and verified contents of 4.01% moisture, 56.15% crude protein, 30.14% total lipids, and 3.89% ashes.
The residues of Nile tilapia (O. niloticus), especially head and bones, present high levels of calcium, iron and phosphorus (Oetterer, 2002). Calcium was the most abundant mineral present in the flours developed in this study followed by phosphorus and iron (Table 3). Petenuci et al. (2008) also found higher amount of calcium followed by phosphorus and iron when developing a flour based on Nile tilapia neck-end -2715.9 mg 100 g -1 , 1132.7 mg 100 g -1 and 1.3 mg 100 g -1 .
The intensity of yellow color (b*) in the NTF for the different treatments in this study was higher than the Brazilian catfish flour (Brachyplatystoma vaillantii) studied by Oliveira et al. (2015), while the luminosity parameter (L) and the intensity of red color (a*) presented lower values. The color of a certain food is influenced mostly by the employed raw materials and possible chemical and biochemical alterations occurring during processing and storage (Ribeiro et al., 2007).

Principal component analysis
The principal component analysis (PCA) was carried out to enable a wider exploration of the results, considering the weight of all of the obtained experimental measures. The data obtained from the four repetitions of NTF of the CT (C1, C2, C3 and C4) and the HT (H1, H2, H3 and H4) enabled the achievement of the significant principal components (p<0.05): PC1 and PC2, explaining 20.65 and 17.10%, respectively, of the results variability. We observe the formation of two distinguished groups (Figure Table 3. Physical and chemical composition and quality of Nile tilapia flour (NTF) and grissini cookie for the different treatments.  1): the group formed by the blue ellipse containing the CT repetitions and the one formed by the red ellipse containing the HT repetitions. The HT repetitions obtained higher influence of the following loadings: water activity (0.8001), total lipids (0.7521) and moisture (0.6186). The loadings of crude protein (0.8464) and phosphorus (0.6619) (PC2) presented higher weight when distinguishing the CT repetitions (PC1); a possible explanation would be higher values of water activity, total lipids and moisture for the HT, and crude protein and phosphorus for the CT (Table 3).

Characterizing the (cookie) grissini
The results of the physical, chemical and technological analyses of the grissini for the different treatments are presented in Table 4. When statistically analyzed (t-Student p<0.05), the results demonstrated no significant difference between the treatments. Grissini are classified as cookies prepared with wheat flour, fat, water, salt and other food substances in the shape of thin, short cylinders (Brazil, 1978). Following the pattern in the NTF, calcium was the most abundant mineral found in the grissini followed by phosphorus and iron. The NTF has a dark color, consequently, the cookies presented more intense color than cookies produced exclusively with wheat flour (Sharmas and Gujral, 2014). Preheat weight, width, thickness and length of the grissini for both treatments presented no statistical differences (p<0.05), which indicated a high level of homogeneity during the processing. This allows the assumption that any differences possible to be observed after the cooking would derive from the NTF behavior in the dough. We observed that the width, thickness and length of the grissini in both treatments increased after the heat, while weight decreased; similar results were found by Fasolin et al. (2007) in cookies produced with banana flour, and  Hedonic Scale: (9) like extremely, (8) like very much, (7) like moderately, (6) like slightly, (5) neither like nor dislike, (4) dislike slightly, (3) dislike moderately, (2) dislike very much, (1) dislike extremely. AI = Acceptance Index. Moraes et al. (2010) in cookies with variations in the contents of sugar and lipids.

Sensory evaluation
No minimum significant difference (p<0.05) was observed among the means of the evaluation of attributes referent to the acceptance of the products ( Table 4). All of the evaluations were within categories 'like slightly' and 'like regularly'. The attribute color received the most satisfactory score for both treatments; color is a major parameter to be considered when adding different types of flour into the cookies formulation since it may influence its appearance, and consequently its acceptance. Lima et al. (2015) carried out a sensory evaluation on nuggets containing fillet and mechanically separated meat of Nile tilapia treated with homeopathic product Homeopatila 100® and also found no significant differences (p<0.05) compared to the control treatment, for all attributes.  Rebouças et al. (2012) produced salted cookie added with protein concentrate of Nile tilapia and obtained means for the sensory evaluation close to 5 ('indifferent') for all of the attributes. Rocha (2011) carried out a sensory evaluation on salted cookie containing Nile tilapia flour and obtained 56.66% acceptance with scores within 'like regularly' and 'like extremely'. Neiva et al. (2011) produced low commercial value cookies based on fish meat mechanically separated (Menticirrhus americanus and Umbrina corides), and verified that the samples presented good scores, varying from 6 ('like slightly') to 9 ('like extremely') for all of the evaluated attributes.
According to Dutcosky (2013), for a product to be considered well-received in consumers market in terms of sensory properties, it is required an Acceptance Index (AI) of at least 70%. As observed in Table 4, the grissini IA for both treatments was satisfactory.

Principal component analysis
The grissini developed with NTF from both the CT and the HT were grouped in two PCA dimensional graphs (Figures 2 and 3). Figure 2 presents the data on proximate composition (moisture, ashes, crude protein and total lipids), minerals as well as the sensory attributes, explaining a data variance of 73.78%. It was possible due to the selection of the PC1 with 47.12%, and PC2, responsible for 26.85% of the results variability presenting 5% significance level. According to Figure 2, the grissini containing NTF from the HT (red ellipse) obtained higher contribution in all of the evaluated organoleptic properties -color (-0.9534), odor (-0.9263), texture (-0.9512), taste (-0.9312) and overall impression (-0.9147) through PC1. The loadings ashes (0.9150), insoluble dietary fiber (IDF) (0.6681) and total dietary fiber (TDF) (0.6495) in PC2 also had an influence on the characterization of the HT grissini. The multivariate analysis enabled the identification of the differences between the treatments indicating better sensory preference for the grissini containing NTF from the HT (Figure 2), even though such results did not present minimum significant difference (p<0>05) through t-Student test (Table 4).
according to the statistical multivariate analysis, the use of NTF in both treatments had no influence on the grissini processing.

Conclusion
During the studied period, the Nile tilapias treated with food containing the homeopathic product Homeopatila 100® presented final total weight significantly higher (p<0.05) than the fish in the control group. The homeopathic product had no significant influence (p<0.05) on the physical and chemical properties of the NTF, as well as the results of the assessment on the cookies (grissini) produced with addition of 10% NTF, including the sensory test. The multivariate analysis enabled an improved grouping and distinction of the samples through the respective physical, chemical compositions and technological and sensory characteristics. The obtained results suggest that the homeopathic product Homeopatila 100® used in Nile diet (40 ml kg -1 of food) is able to improve weight gain without compromising the physical, chemical, technological and sensory quality of the fish flour and cookie (grissini).