Evaluation of the biological quality of defatted pequi ( Caryocar brasiliense Cambess ) seed fl our protein supplemented with lysine to rats ( Rattus norvegicus )

Objective In the biome of the Brazilian Cerrado, there are a lot of fruit tree species that stand out for their sensory quality and for presenting potentialities in the market of pulp and almond. Among these species, the pequi deserves


I N T R O D U C T I O N
Knowledge of the chemical composition of native species helps to understand nutrition and biodiversity, especially in terms of food production and processing for human consumption [1].In the http://dx.doi.org/10.1590/1678-9865201932e180129 Brazilian cerrado, the second largest biome in Brazil, there are several native fruit species that present potential for use in human nutrition, and in this context pequi fruit (Caryocar brasiliense Cambess) has an edible seed that is not consumed, even by the local population [2].
The pequi is a typical fruit of the Brazilian cerrado, constituted by a pericarp of greenish coloration; an external mesocarp (pulp with grayish brown); an internal mesocarp, with yellowish color and representing the edible pulp of the fruit.The endocarp, which is prickly, protects the edible seed, which is coated by a thin, brown integument [3][4][5][6][7].The pulp of in natura pequi fruit has a high lipid content (19.60% to 33.40%) and is also a good source of dietary fiber (10.02% to 11.20%) [3,[8][9][10].Therefore, this fruit exerts a great influence on both eating habits and income of the inhabitants of the Brazilian cerrado.It is an indispensable product in the diet of the populations that live in the areas of occurrence of the species because it provides part of the energy and nutritional needs, mainly for low-income families [11].
In addition to the pulp, the pequi fruit has a seed that is rich in nutrients and which can be better utilized as a food source.Typically, edible seeds are rich in lipids, sources of protein and also contain vitamins and minerals in considerable amounts.These can also be a good source of fibers [12].The pequi seed is white and has a characteristic flavor.As well as the pulp, it is also edible, being used in natura or in several preparations like sweets, farofas (toasted cassava flour mixture), paçocas (a candy made out of ground peanuts, sugar and salt) and snacks [5,13,14].It has a high content of lipids, which allows the extraction of a clear, good quality oil that has been used in the cosmetics industry for the production of beauty creams and soaps as well as in the production of regular soap [15,16].
A striking feature of pequi seed is its high protein content, around 25% [9].However, few data on the amino acid profile and nutritional benefits of this seed are available, therefore, it is very important to determine the protein quality of the seed [2].
It is known that proteins of plant origin do not always present an adequate qualitative and quantitative profile of amino acids, being considered incomplete proteins because they have deficiencies in one or more essential amino acids.Deficiencies in the amino acid profile of proteins can be corrected through the following methods: addition of small amounts of another type of protein that is rich in the first limiting amino acid; through complementation, in which the combination of specific foods is made and the mixture of the proteins of these foods causes that the deficiencies of one are compensated by the excesses of the same amino acids of others; and through the addition of the first amino acid limiting, which was the option used in the present study [17,18].
Considering that pequi seed is rich in proteins, and that proteins play important roles in the body, chemical and biological methods were used to determine the protein quality of the pequi seed [19][20][21].Chemical methods are used to evaluate protein quality because they determine the composition of amino acids and enable certain correlations, such as the determination of the limiting amino acid through the Chemical Score (CS) and the calculation of the amino acid chemical score corrected by protein digestibility (PDCAAS, Protein Digestibility-Corrected Amino Acid Score) [22][23][24].The biological methods used to determine the nutritional value of a protein are based on the response of an organism to the intake of the studied protein.The most commonly used biological indices are the Protein Efficiency Ratio (PER), the Net Protein Ratio (NPR) [25][26][27][28], the True Digestibility (TD) [29,30], the Net Protein Utilization (NPU) [31][32][33] and the Apparent Nitrogen Balance (BNap).
Based on this information and with the purpose of valuing and stimulating the consumption of regional foods, the objective of this study was to evaluate the biological quality of the protein of defatted pequi seed flour supplemented with lysine, through the analysis of nutritional parameters taken from tests with young animals.

Samples of pequi seeds
Pequi seeds were obtained in Japonvar, a city located in the north of the state of Minas Gerais, Brazil.The seeds were ground until obtaining a homogeneous flour that was used in the analysis of the centesimal, mineral and amino acid composition.Afterwards, the flour was defatted by direct extraction with petroleum ether in Soxhlet [34], dried in a ventilated oven at 45°C for 72 hours and kept under refrigeration until determination of the composition and preparation of the diets.

Centesimal composition
The determination of the centesimal composition of the pequi seed and of Defatted Pequi Seed Flour (DPSF) was carried out at the Laboratory of Bromatology of the School of Nutrition, Universidade Federal de Ouro Preto (UFOP, Federal University of Ouro Preto).The determination of proteins, lipids, ashes and moisture were performed in triplicate, in accordance with the Adolfo Lutz Institute [34].Total dietary fiber was determined by enzymatic-gravimetric method, in accordance with the Association of Official Analytical Chemists [35].The amount of carbohydrates in the sample was obtained by difference from the other components, i.e., the value 100 is subtracted from the sum of the values obtained for proteins, lipids, moisture, ash and fiber.

Amino acid profile
The composition of amino acids was determined at the Centro de Ciência e Qualidade de Alimentos (CCQA, Center for Food Science and Quality) of the Instituto de Tecnologia de Alimentos (ITAL, Institute of Food Technology), in the city of Campinas, Brazil [36][37][38].

Mineral composition
To quantify the levels of Sodium (Na), Potassium (K) and Phosphorus (P), 200mg of the sample was transferred to digestion tubes with 4mL P.A. nitric acid (65% purity grade) and slowly heated (20ºC every 30 minutes) in a digestion block with exhaust system, until reaching 130°C.After this process, the sample remained at the same temperature for about 4 hours, until it became light yellow.Subsequently, with the sample now at room temperature, 1mL of P.A. perchloric acid (70% purity grade) was added and heating was resumed as described above.The sample was transferred to a volumetric flask (50mL) and volume was completed.The determination of sodium and potassium was performed in the flame photometer, using Na/K 100 parts per million standard.For phosphorus quantification, a standard curve with phosphorus solution (0.01mg/mL) was prepared, and the absorbance of the standard curve and the sample was taken at the wavelength of 680 nanometers in the spectrophotometer.To determine the other minerals, the sample was incinerated and the http://dx.doi.org/10.1590/1678-9865201932e180129ashes obtained were sent to the Laboratory of Atomic Absorption of the Universidade Federal de Minas Gerais (UFMG, Federal University of Minas Gerais).The analyzes were carried out in an atomic absorption spectrophotometer coupled to a graphite furnace [34,35].

Composition of experimental diets
The isocaloric and isoprotein diets (10% protein -100g protein/1000g diet) were prepared at the beginning of the experiment and followed the specific preparation recommendations from the Association of Official Analytical Chemist (AOAC) (39.133) [39] for biological evaluation of protein quality.According to this recommendation the diets must have (for every 100 grams): enough for 10% of proteins; 8% lipids; 1% blend of vitamins; 5% blend of minerals; 1% cellulose (fibers); 5% water and starch, enough to complete 100%.Prior to the preparation of the diets, the centesimal composition of the defatted pequi seed flour was determined so that the nutrients could be discounted when the ingredients of the diet were added.A protein-free diet was also prepared to be used in the determination of NPR, NPU, TD and BNap.The calculation for determining the amount of lysine to be added in the diet was done using as reference the daily amino acid requirements of children from two to five years of age, according to the Food and Agriculture Organization (FAO) standard [40].The compositions of the experimental diets are shown in Table 1.

Experimental design
Seventy-six recently weaned male Fischer rats from 21 to 23 days old were used, weighing approximately 45 grams, from the Laboratory of Experimental Nutrition of the School of Nutrition of UFOP.The animals were housed individually in metal cages arranged in an environment which allowed for air circulation, with temperature, luminosity and humidity control.Filtered water and food were available at all times.To verify the proposed objectives, two experiments were performed, since the indices for calculating the protein quality have different characteristics, such as the followup time.In addition, when a group of animals that do not receive protein through the diet is included, a shorter period of experimentation is recommended due to the stress caused to the animals, and it is necessary to quantify nitrogen in the carcass for the calculation of the Net Protein Utilization.
In experiment 1, which lasted 14 days, the rats were divided into four groups with 10 animals each.The control group received the standard diet with 10% protein from casein, the protein-free diet group received a protein-free diet.A diet with 10% of protein derived from the pequi seed was offered to the Defatted Pequi Seed Flour (DPSF) group, and the Lysine-supplemented Defatted Pequi Seed Flour (DPSFL) group received the same diet as the DPSF group with the addition of lysine, which is the first limiting amino acid of the pequi seed.In experiment 2, the animals were divided into three groups with 12 animals each.The diets offered to each group presented the same composition of the diets of experiment 1, except for the protein-free diet that was not included in this experimental design.The animals were kept under the same conditions as in the first experiment and were followed-up for 28 days.
The animals were weighed weekly for monitoring body mass and calculating weight gain (or loss).Food consumption and fecal collection were evaluated for seven consecutive days from the second week of each experiment.In order to control ingestion and excretion, paper trays were placed under each of the cages, being replaced daily and the separation and weighing of the feces and food leftovers.Concomitantly with this step, the amount of food offered for each animal was also controlled.
At the end of the experiments, the animals were fasted for 12 hours, anesthetized using a vaporizer calibrated with isoflurane (3% to 5%) and euthanized by incision of the blood vessels adjacent the brachial plexus to full bleeding.The experiment was approved by the Committee on Ethics in the Use of Animals of the Federal University of Ouro Preto (CEUA/UFOP), protocol No.2015/43.

Protein quality evaluation indices
Methods for evaluating biological protein quality were determined using the formulas described below.The Chemical Score (CS) is given by the ratio: CS=(mg of the essential aac per g of the test protein/mg of the essential aac per g of the reference protein)x100.The PDCAAS is calculated by multiplying the lowest chemical score of the essential amino acid by the True Digestibility (TD):PDCAAS=CSxTD.PER and NPR are calculated, respectively: PER=(weight gain (g)/protein consumed (g)) and NPR=(weight gain in the test group (g)-weight loss in the protein-free group (g)/protein consumed by the test group (g)).The corrected PER=[(PER of the test group/PER of the control group)×2.5]was also determined.For the calculation of Apparent Nitrogen Balance (BNap), True Digestibility (TD) and Net Protein Utilization (NPU), it was necessary to quantify the Ingested Nitrogen (NI), Fecal Nitrogen (NF) and Carcass Nitrogen (NC), and the following formulas were used: BNap = NI(g)-[NF (test group)(g)-NF(protein-free group)(g)]; TD={NI(g)-[NF(test group)(g)-NF(protein-http://dx.doi.org/10.1590/1678-9865201932e180129free group)(g)]/NI(test group)(g)}x100 and NPU={[NC(test group)g)-NC(protein-free group)(g)]/NI(test group)(g)}x100.

Statistical analysis
Data were submitted to the Kolmogorov-Smirnov normality test.Data with parametric distribution were treated by the One-Way ANOVA test, Tukey post-hoc test and presented as Mean±Standard Deviation (M±SD).Data with non-parametric distribution were evaluated through the Kruskal-Wallis test followed by Dunn post-hoc test and were expressed as median and interquartile range.The differences were considered significant when p≤0.05.The GraphPad Prisma software, v5.00 compatible with the Windows operating system (San Diego, California, United States) was used for the analysis.

R E S U L T S A N D D I S C U S S I O N
The results of the centesimal composition of the pequi seed and the defatted pequi seed flour used in this study are shown in Table 2.The concentration of lipids found in the pequi seed was 55.4% and the protein content was 25.3%.In the defatted flour, the lipids represented only 3.0% of the sample and the proteins, 65.5%.Significant amounts of total dietary fiber were found both in the seed and the flour, representing 8.2% and 15.6%, respectively.
In the pequi seed, high levels of lipids are observed, which according to Lima et al. [9] is the major component of this seed, accounting for 51.5% of its composition.In the present study, we found a percentage of lipids of 55.4%, a value close to that reported by Lima.Other studies corroborate with this finding, Ramos & Souza [7] found a mean lipid content of 48.5% in the pequi seed of the species Caryocar coriaceum and Sousa et al. [41] reported a proportion of lipids of 50.0% for the pequi seed of the Caryocar brasiliense species.Regarding the protein content of the pequi seed, the result found in this study showed a protein content of 25.3%, similar to those found by Lima et al. [9] and Sousa et al. [41], who found 25.3% and 29.6% respectively.Damiani et al. [5] found a protein content of 13.4% for the raw almond, differing from our study.Ramos & Souza [7] worked with pequi seeds of the Caryocar coriaceum species, and also found a protein concentration similar to that of this study, 27.1%.The average value of the amount of fibers found in this study was 8.2g/100g, close to that reported in Sousa et al. [41], 10.4g/100g and higher than that described by Lima et al. [9], which was 2.2g/100g.These results reinforce the nutritional importance of the pequi seed and the need for further studies to stimulate the consumption and use of this seed as an extra option in the almond market.
In the analysis of the mineral content of the pequi seed (Table 2), it was observed that the sodium content found in this study was 24.80mg per 100 grams of the seed, which corroborates with the study of Freitas & Naves [42], which reports that nuts and edible seeds have reduced sodium concentration.In addition, the potassium, phosphorus, magnesium, copper and chromium contents found in the pequi seed meet the Recommended Daily Intake (IDR) minimum recommendation of 15%, therefore pequi seeds can be considered a source of these minerals [43][44][45].For some microminerals (chromium, barium, aluminum, cobalt, cadmium and lead) no data were found in the literature concerning the pequi seed.
The amino acid composition is an important chemical property of proteins, and as such represents one of the determinants of the seed's nutritional value [46,47].Based on the essential amino acid requirement model for preschoolers from 2 to 5 years old [40], the chemical lysine score found in our study corresponds to only 39% of the requirement, and is therefore considered the first limiting amino acid of the pequi seed (Table 3).It should be noted that the seed was a good source of valine, methionine and cystine, isoleucine, histidine, phenylalanine and tyrosine, with values higher than those recommended by the FAO in 1985 [40].Lysine was also considered the first limiting amino acid of the pequi seed in the study of Sousa et al. [41] and several nuts, such as the Brazil nut (Bertholetia excelssa), cashew nut (Anacardium occidentale), hazelnut (Corylus hazelnut), pine nuts (Pinus pinea) and walnuts (Juglans regia), according to Venkatachalan & Sathe [12].Due to these findings, the study of lysine supplementation in defatted pequi seed flour was proposed.

Design 1
The digestibility is the first factor that reflects the efficiency of the protein utilization of the diet, therefore, it can be considered a conditioner of its quality [48].The true digestibility of the casein diet of our study (94.3%) presented in table 4 was higher and statistically different from the other diets, whose values were 86.9% for the DPSF diet and 89.4% for the DPSFL diet.Sousa et al. [41] evaluated  the digestibility of the pequi seed and found a value of 88.1%, a result that corroborates the finding in this study.Some studies evaluated the digestibility of other typical seeds from the Brazilian cerrado and found values of true digestibility of 83.5% for bocaiúva seeds (Acronomia aculeata), 79.4% for baru seeds (Dipteryx alata), and 88.0% for Cashew nuts (Anacardium othonianum) [1,41,49].The DPSF and DPSFL diets presented good digestibility, that is, an effective absorption of the amino acids.A digestibility value around 80.0% to 90.0%, which is considered a good percentage of amino acid uptake, is observed in some plant products and is what has been found for the pequi seed in the present study [30,[50][51][52].
In relation to the NPR, it was verified that the addition of lysine did not result in statistical difference between the DPSF and DPSFL groups.The food and protein intake of the groups that received the pequi seed did not differ significantly and the same could be observed for the weight gain.The control group presented statistically higher averages of protein intake, weight gain and NPR, however, it was observed that the pequi seed proteins were able to guarantee the maintenance of the animals' weight.Sousa et al. [41] found an NPR value of 2.19±0.29 for the pequi seed, higher than the one found in this study (1.65±0.33)(Table 4).
It was also determined the apparent nitrogen balance, since it was not possible to collect the urine from the animals for quantification of nitrogen.BNap analysis revealed that the use of pequi seed protein was enough to promote a positive nitrogen balance, which is expected for growing animals.Thus, the ingested nitrogen was superior to the nitrogen excreted in the faeces, evidencing retention of nitrogen.
The NPU results of the groups fed with the DPSF and DPSFL diets showed that the quality of the pequi seed protein was lower than casein to promote protein synthesis, since NPU measures how much of the protein ingested is retained in the organism [53].As observed through digestibility, amino acids may be well absorbed, but may not be involved in protein synthesis, due to a deficiency in some essential amino acids of the pequi seed [54].The addition of lysine did not significantly alter endogenous protein retention, and in addition, animals in the DPSF and DPSFL groups consumed significantly lower amounts of nitrogen (protein), which may explain the low NPU values.No data were found in the literature regarding the NPU of the pequi seed.

Design 2
The values obtained in the second design for the PER and the corrected PER differed statistically among the diets evaluated (Table 5).The PER of the group fed with the casein diet showed to be significantly superior to the other groups, which may be justified by the higher food and protein intake and, consequently, higher weight gain by this group.Because it is an animal protein, casein has all the necessary essential amino acids in adequate amounts for the growth and maintenance of the organism [55].
When comparing pequi seed-based diets, the lysine-supplemented diet had a PER value approximately 50% higher than the PER of the DPSF diet.Observing the PER value of both diets, we can see an effect of supplementation with the first limiting amino acid.Other studies

C O N C L U S I O N
The findings contained in this study regarding the centesimal composition of the pequi seed showed that it has significant contents of lipids, proteins, fibers and minerals such as potassium, phosphorus and magnesium, thus representing a food of good nutritional value.The low consumption of food from the diet by the animals that received the pequi seed directly reflected in the results obtained from the biological tests in this study.For the NPR and NPU no differences were observed between the DPSF and DPSFL groups, possibly due to the short experimental period that prevented animals from having a beneficial adaptation.However, for the PER, which among the methods used in this study, is the most demanding one in terms of protein quality, it was observed a positive effect of lysine supplementation, with an increase of approximately 50% in the value of this index, compared to the non-supplemented group.However, when compared with the control group, lysine supplementation was not satisfactory, which may be related to the fact that pequi protein is deficient in other essential amino acids, such as threonine, leucine and tryptophan.As a perspective for the promotion of the pequi seed, future studies are suggested to be performed with the supplementation including other limiting amino acids in order to achieve a PER closer to the control group.

: 1
Lysine: the first limiting amino acid; 2 FAO/WHO (1985): Established theoretical standard of essential amino acids for children between 2 and 5 years of age.FAO: Food and Agriculture Organization; WHO: World Health Organization; PDCAAS: Protein Digestibility-Corrected Amino Acid Score.
C O N T R I B U T O R S MMA FAGUNDES, AMF VIANA and MMF CARVALHO Participated in the development and performance of the experimental design; data collection, analysis and discussion, and writing and reviewing the article.ME SILVA was responsible for the development of the project, interpretation of data, and final review of the article.

Table 2 .
Centesimal composition, caloric value and mineral composition of the pequi seed and defatted pequi seed flour.

Table 4 .
Food and protein intake, weight gain, nitrogen determination and protein quality indices of animals on protein-free, control, DPSF and DPSFL diets.Ouro Preto (MG), Brazil, 2017.