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Production Process and Optimization of Solid Bioethanol from Empty Fruit Bunches of Palm Oil Using Response Surface Methodology

This study aimed to observe the potential of solid bioethanol as an alternative fuel with high caloric value. The solid bioethanol was produced from liquid bioethanol, which was obtained from the synthesis of oil palm empty fruit bunches (PEFBs) through the delignification process by using organosolv pretreatment and enzymatic hydrolysis. Enzymatic hydrolysis was conducted using enzyme (60 FPUg−1 of cellulose) at a variety of temperatures (35 ◦C, 70 ◦C, and 90 ◦C) and reaction times (2, 6, 12, 18, and 24 h) in order to obtain a high sugar yield. The highest sugars were yielded at the temperature of 90 ◦C for 48 h (152.51 mg/L). Furthermore, fermentation was conducted using Saccharomyces cerevisiae. The bioethanol yield after fermentation was 62.29 mg/L. Bioethanol was extracted by distillation process to obtain solid bioethanol. The solid bioethanol was produced by using stearic acid as the additive. In order to get high-quality solid bioethanol, the calorific value was optimized using the response surface methodology (RSM) model. This model provided the factor variables of bioethanol concentration (vol %), stearic acid (g), and bioethanol (mL) with a minus result error. The highest calorific value was obtained with 7 g stearic acid and 5 mL bioethanol (43.17 MJ/kg). Burning time was tested to observe the quality of the solid bioethanol. The highest calorific value resulted in the longest burning time. The solid bioethanol has a potential as solid fuel due to the significantly higher calorific value compared to the liquid bioethanol.

Introduction
Greenhouse gas emissions, due to the impact of excessively used fossil fuel [1], have created negative impacts on the environment [2,3]. The primary emission of greenhouse gas is carbon dioxide [4], ethanol 55% was employed as the organic solvent [29]. Enzymatic hydrolysis was conducted in this study to hydrolyze cellulose and hemicellulose into reducing sugars. This hydrolysis has the advantage of producing more sugar yield compared to acid hydrolysis. However, the cost of the enzymatic hydrolysis is significantly higher than acid hydrolysis [30,31]. Moreover, studies about enzymatic hydrolysis have been successfully done using various forms of treated biomass such as corn stover, rice straw [32], rice hull [33], sugarcane bagasse [34][35][36], and palm empty fruit bunch (PEFB) [37][38][39]. Different researchers have used different pretreatment processes and optimization to produce bioethanol from different feedstocks. For example, Sebayang et al. [40] used Manihot glaziovii starch as a feedstock to produce bioethanol through the optimization of enzymatic hydrolysis. Kim et al. [41] used a sequential pretreatment process with diluted acid and then alkali to treat empty palm fruit bunch fiber biomass for producing bioethanol. However, there is not much research found on the optimization of the organosolv pretreatment process to produce bioethanol from PEFB through enzymatic catalyzation. The novelty of this study is to highlight the EFBs as a less expensive second-generation feedstock for bioethanol production using the organosolv pretreatment process and enzymatic hydrolysis in Malaysia. In this study, the production and optimization of solid bioethanol were carried out using stearic acid as an additive. Liquid bioethanol was produced from palm empty fruit bunches (PEFBs) via organosolv pretreatment and enzymatic hydrolysis. The optimization of solid bioethanol production was determined by response surface methodology (RSM) to get the highest calorific value. Table 1. Pros and cons of organosolv pretreatment process compared to other processes [42].

Pretreatment Method Pros Cons
Milling -Reduces cellulose crystallinity -Reduces polymerization degree -Reduces particle size -Increases specific surface area and pore size -Significantly increases power and energy consumption

Materials
The palm empty fruit bunch (PEFB) biomass, which was supplied from palm plantations in Indonesia, was used as a feedstock for bioethanol production. Aqueous ethanol with a concentration of 55 vol % was proposed as a solvent in order to break lignin content, known as the delignification process; this aqueous ethanol was supplied from Fisher Scientific (Waltham, MA, USA). Enzyme cellulose (cellulose from Trichoderma reesei ATCC 26921) and β-glucosidase (β-glucosidase from Caldocellum saccharolyticum) for the hydrolysis process, and the yeast extract, peptone, and glucose as a medium for the fermentation process, were supplied from Sigma-Aldrich (St. Louis, MO, USA). Furthermore, the bioethanol was solidified with stearic acid (Sigma-Aldrich, St. Louis, MO, USA), which has a melting point of 67-72 • C.

Preparation of Bioethanol
The palm empty fruit bunch (PEFB) biomass was dried under the sun and crushed to a small size of approximately 1 cm width. Furthermore, it was dried in an oven at a temperature of 105 • C for 24 h in order to evaporate all the water content. The dried biomass was then ground for the further homogenous processing of the material, into the 710-500 µm size range, and then this biomass was stored in a desiccator (NS 24/29 Duran, Darmstadt, Germany). The same procedure of drying and grinding was conducted in the previous study [29].
The pretreatment method, used to synthesize sugars from the dried biomass, was organosolv pretreatment and further enzymatic hydrolysis processes. The organosolv pretreatment method was performed chemically as a delignification process by dissolving dried PEFB with aqueous ethanol at a solid-liquid ratio of 1:10 (10 g in 100 mL) in an Erlenmeyer flask. The condition was settled at 55 vol % ethanol, a temperature of 120 • C, and a reaction time of 60 min [29]. The units of enzymes were found as an adequate amount to obtain a high sugar yield [43]. The temperature and reaction time were varied at 35 • C, 70 • C, and 90 • C for 2, 6, 12, 18, and 24 h, respectively, in a water bath shaker. The hydrolyzed PEFBs were carried out to measure total sugar yield using the phenol-sulfuric acid dinitrosalicylic (DNS) colorimetric method on the obtained samples of acid pretreatment, as well as on the hydrolyzed PEFBs after and without acid pretreatment. The test was conducted by mixing 1 mL of 0.1% DNS reagent solution with 1 mL diluted solution of treated PEFB. The mixed solutions were then put into a water bath at 90 • C for 5 min and allowed to cool down to room temperature before spectrophotometer reading at 540 nm.
The optimum sugar yield of hydrolysate was selected to be fermented into bioethanol. The fermentation was carried out with media consisting of 1% (w/v) yeast extract and 2% (w/v) peptone and 3% (w/v) glucose. The media was poured into the hydrolysate and was autoclaved in 121 • C for 30 min in order to sterilize it. After the medium was cooled down to room temperature, the yeast culture was added to be inoculated in a shaker for 24 h in order to activate the yeast cultures' enzymatic activity. After the inoculation, treated PEFB was added into media containing 1% (w/v) yeast extract, 2% (w/v) peptone, and 10% (v/v) yeast cultures at a solid-to-liquid ratio of 2:5 in a 250 mL volume Erlenmeyer flask. The fermentation was performed in a shaker incubator at 35 • C and 150 rpm for 84 h. Ethanol yield was determined every 12 h. Bioethanol was distilled using a control rotary evaporator (230 VAC, IKA, Staufen, Germany) at 79 • C in order to remove the water content. The concentration of bioethanol was determined using a density meter (DMA 35, Anton Paar, Graz, Austria).

Solid Ethanol Production and Optimization of Solid Bioethanol Calorific Value
Solid ethanol production was carried out by mixing melted stearic acid with ethanol. Calorific value was determined using a calorimeter (Oxygen Bomb Calorimeter 6200, Paar, MO, USA) for optimization of the solid ethanol quality. The composition of 70%, 80%, and 90% (v/v) ethanol and stearic acid was varied in order to optimize the calorific value of the solid ethanol.
Response surface methodology (RSM) with Box-Behnken experimental design was used to optimize the calorific value of the solid bioethanol [44,45]. The design is available in Design-Expert software 9.0.4.1 (Stat-Ease Inc., Minneapolis, MN, USA). The independent variables chosen were bioethanol concentration (%) represented as A, stearic acid (g) represented as B, and bioethanol (mL) represented as C. The coded levels of independent variables resulting from the Box-Behnken experimental design are presented in Table 2. Furthermore, the combustion test was carried out to observe the burning time of the solid bioethanol ( Figure 1). The four highest calorific value samples were selected to be tested. The diameter of each sample to be burned was 3 in with a length of 4 cm.

Sugars and Bioethanol Yield of PEFB
The organosolv pretreatment was used as a delignification method in this study. Aqueous ethanol was used as the solvent solution to degrade lignin in the palm empty fruit bunch (PEFB); this method was successfully used in a previous work, which was able to break down the lignin content in the biomass by producing less residue [29], hence this will not be a major problem for further processes of bioethanol production [45,46]. However, the organosolv pretreatment has been commonly used in softwood and hardwood synthesis processes, which are able to obtain 90% of the sugars' efficiency by incorporating an enzymatic hydrolysis method [47][48][49]. In the present study, enzymatic hydrolysis was performed by using the enzyme 60 FPUg −1 of cellulose to derive the sugar yield from the source biomass with varying temperature conditions of 35, 70, and 90 °C, where the enzyme cellulose 60 FPU was the standard unit of cellulose used for the enzymatic hydrolysis process in bioethanol production [50][51][52][53].
Moreover, several enzymes have been used in hydrolysis processes and are categorized based on their activities' temperature range, that is, 20-50 °C, 50-80 °C, and above 80 °C, which are known as mesozyme, thermozyme, and hyperthermozyme, respectively [54,55]. Hence, the cellulose enzyme used in this work was thermozyme, which has an activation temperature of about 39-90 °C. For industrial purposes such as pulp and paper processes, the thermozyme type of enzyme is used due to the low possibility of contamination within a short, extended process.

Sugars and Bioethanol Yield of PEFB
The organosolv pretreatment was used as a delignification method in this study. Aqueous ethanol was used as the solvent solution to degrade lignin in the palm empty fruit bunch (PEFB); this method was successfully used in a previous work, which was able to break down the lignin content in the biomass by producing less residue [29], hence this will not be a major problem for further processes of bioethanol production [45,46]. However, the organosolv pretreatment has been commonly used in softwood and hardwood synthesis processes, which are able to obtain 90% of the sugars' efficiency by incorporating an enzymatic hydrolysis method [47][48][49]. In the present study, enzymatic hydrolysis was performed by using the enzyme 60 FPUg −1 of cellulose to derive the sugar yield from the source biomass with varying temperature conditions of 35, 70, and 90 • C, where the enzyme cellulose 60 FPU was the standard unit of cellulose used for the enzymatic hydrolysis process in bioethanol production [50][51][52][53].
Moreover, several enzymes have been used in hydrolysis processes and are categorized based on their activities' temperature range, that is, 20-50 • C, 50-80 • C, and above 80 • C, which are known as mesozyme, thermozyme, and hyperthermozyme, respectively [54,55]. Hence, the cellulose enzyme used in this work was thermozyme, which has an activation temperature of about 39-90 • C. For industrial purposes such as pulp and paper processes, the thermozyme type of enzyme is used due to the low possibility of contamination within a short, extended process. Figure 2 shows the rate of sugar yields obtained from the enzymatic hydrolysis process with the enzyme 60 FPUg −1 of cellulose in different combinations of temperature, namely, 35 • C, 70 • C, and 90 • C. The result shows that the optimum sugar yield at the first 2 h of the hydrolysis process is obtained at a temperature of 90 • C as 72.81 mg·L −1 , which is much higher than the sugar yield achieved at temperatures of 35 • C and 70 • C as 7.45 mg·L −1 and 10.92 mg·L −1 , respectively. Subsequently, these sugar yields at 35 • C and 70 • C presented a significant increment once 6 h of the fermentation process was reached, and afterwards simultaneously increased as the fermentation times were increased. On the other hand, the sugar yield at 90 • C increased from 2 h of fermentation time onward, which can be attributed to the higher temperature. The increasing sugar yield occurred because of the high temperature that affected the enzyme's activity. The optimal temperature for enzyme activity is 35-37 • C. However, the higher temperature possibly increased the kinetic energy of the enzyme. At the temperature of 90 • C, the enzyme works better for a certain time before enzyme denaturation. However, overall, and once the 48 h fermentation processes ended, the optimum yield of sugars reaching 152.51 mg·L −1 was obtained at a temperature of 90 • C. This optimum yield was due to the increasing kinetic energy, which increased the interaction between the substrate and enzyme, thereby achieving a better reaction rate for a higher sugar yield [56].
Processes 2019, 7, x FOR PEER REVIEW 7 of 16 Figure 2 shows the rate of sugar yields obtained from the enzymatic hydrolysis process with the enzyme 60 FPUg −1 of cellulose in different combinations of temperature, namely, 35 °C, 70 °C, and 90 °C. The result shows that the optimum sugar yield at the first 2 h of the hydrolysis process is obtained at a temperature of 90 °C as 72.81 mg·L −1 , which is much higher than the sugar yield achieved at temperatures of 35 °C and 70 °C as 7.45 mg·L −1 and 10.92 mg·L −1 , respectively. Subsequently, these sugar yields at 35 °C and 70 °C presented a significant increment once 6 h of the fermentation process was reached, and afterwards simultaneously increased as the fermentation times were increased. On the other hand, the sugar yield at 90 °C increased from 2 h of fermentation time onward, which can be attributed to the higher temperature. The increasing sugar yield occurred because of the high temperature that affected the enzyme's activity. The optimal temperature for enzyme activity is 35-37 °C. However, the higher temperature possibly increased the kinetic energy of the enzyme. At the temperature of 90 °C, the enzyme works better for a certain time before enzyme denaturation. However, overall, and once the 48 h fermentation processes ended, the optimum yield of sugars reaching 152.51 mg·L −1 was obtained at a temperature of 90 °C. This optimum yield was due to the increasing kinetic energy, which increased the interaction between the substrate and enzyme, thereby achieving a better reaction rate for a higher sugar yield [56]. This sugar yield was thus carried out for the further fermentation process using Saccharomyces cerevisiae in order to produce bioethanol over a period of 84 h, and was then analyzed every 12 h to determine the yield of bioethanol that was converted from the sugars. Figure 3 presents the comparison of rate yield ethanol production and sugar decomposition from an analysis carried out every 12 h. These results show that the rate of ethanol yield obtained increased when the fermentation time increased, where a significant increment appears at 24 h and shows a continuous slight increase afterwards, whereas it is vice versa for the sugar decomposition. However, sugars acted as a carbon (C) source to grow the S. cerevisiae cells within the fermentation processes [57].
Meanwhile, as the boiling point of the bioethanol (78.3 °C) was lower than the boiling point of water (100 °C), the remaining water content in the bioethanol solution was not removed completely and formed an azeotropic solution (i.e., a mixture of 95% ethanol and 5% water), which had a boiling point of 78.15 °C [58]. However, the azeotropic percentage between bioethanol and water obtained in this study was 90% ethanol and 10% water. This sugar yield was thus carried out for the further fermentation process using Saccharomyces cerevisiae in order to produce bioethanol over a period of 84 h, and was then analyzed every 12 h to determine the yield of bioethanol that was converted from the sugars. Figure 3 presents the comparison of rate yield ethanol production and sugar decomposition from an analysis carried out every 12 h. These results show that the rate of ethanol yield obtained increased when the fermentation time increased, where a significant increment appears at 24 h and shows a continuous slight increase afterwards, whereas it is vice versa for the sugar decomposition. However, sugars acted as a carbon (C) source to grow the S. cerevisiae cells within the fermentation processes [57].
Meanwhile, as the boiling point of the bioethanol (78.3 • C) was lower than the boiling point of water (100 • C), the remaining water content in the bioethanol solution was not removed completely and formed an azeotropic solution (i.e., a mixture of 95% ethanol and 5% water), which had a boiling point of 78.15 • C [58]. However, the azeotropic percentage between bioethanol and water obtained in this study was 90% ethanol and 10% water.

Quadratic Regression Model for Solid Bioethanol Optimization
Optimization was performed using the response surface methodology (RSM) model. This method is a technique based on the design of experiments (DOE). RSM is used to analyze the changes of the dependent variable and has been widely used to optimize the experiment process due to the advantage that it can predict the maximum result, which is not carried out in the experiment [44,45,59]. The variation of data was estimated by Box-Behnken experimental design ( The polynomial equation above is used to predict the calorific value, and the results of the prediction are presented in Table 2, where CV represents the calorific value, A represents bioethanol concentration, B represents stearic acid, and C represents bioethanol. Analysis of variance (ANOVA) was conducted to calculate the statistical significance of the quadratic regression model and the effects of significant individual correlation of the chosen responses. The results are presented in Table 3. The p-value corresponds to the probability of error and is expressed to determine whether each regression coefficient is significant. A p-value less than 0.0001 indicates that the model is significant at the 95% level of confidence. The model F-value of 95.5% implies that the model is significant. There is only a 0.01% chance that an F-value this large can occur due to noise. The value of "Prob>" less than 0.0500 shows the model terms are significant. For this case, A, B, C, and A 2 are significant model terms. If the value exceeds 0.1000, it indicates that the model terms are insignificant. In addition, if there are many insignificant model terms (not counting those required to support hierarchy), model reduction by reducing the parameter range may improve the model.
The coefficient of determination (R 2 ) represents the variety of dependent variables (calorific value) and its relationship with the predicted variables. A high R 2 indicates the higher viability of the data due to the accordance of model-predicted data and experimental data. The relationship of those variables is presented in Figure 4. It shows that the value of R 2 is 0.9919, which indicates that 99.19% of the variability in experimental calorific value is explained by the quadratic regression model.

Quadratic Regression Model for Solid Bioethanol Optimization
Optimization was performed using the response surface methodology (RSM) model. This method is a technique based on the design of experiments (DOE). RSM is used to analyze the changes of the dependent variable and has been widely used to optimize the experiment process due to the advantage that it can predict the maximum result, which is not carried out in the experiment [44,45,59]. The variation of data was estimated by Box-Behnken experimental design (Table 2).
The polynomial equation above is used to predict the calorific value, and the results of the prediction are presented in Table 2, where CV represents the calorific value, A represents bioethanol concentration, B represents stearic acid, and C represents bioethanol.
Analysis of variance (ANOVA) was conducted to calculate the statistical significance of the quadratic regression model and the effects of significant individual correlation of the chosen responses. The results are presented in Table 3. The p-value corresponds to the probability of error and is expressed to determine whether each regression coefficient is significant. A p-value less than 0.0001 indicates that the model is significant at the 95% level of confidence. The model F-value of 95.5% implies that the model is significant. There is only a 0.01% chance that an F-value this large can occur due to noise. The value of "Prob>" less than 0.0500 shows the model terms are significant. For this case, A, B, C, and A 2 are significant model terms. If the value exceeds 0.1000, it indicates that the model terms are insignificant. In addition, if there are many insignificant model terms (not counting those required to support hierarchy), model reduction by reducing the parameter range may improve the model.
The coefficient of determination (R 2 ) represents the variety of dependent variables (calorific value) and its relationship with the predicted variables. A high R 2 indicates the higher viability of the data due to the accordance of model-predicted data and experimental data. The relationship of those variables is presented in Figure 4. It shows that the value of R 2 is 0.9919, which indicates that 99.19% of the variability in experimental calorific value is explained by the quadratic regression model.   The signal-to-noise ratio is determined by statistical parameter adequate precision. A preferable adequate precision is higher than 4. The adequate precision in this study was 36.330, as presented in Table 3. It indicates that the signal is adequate, and the quadratic regression model is viable to be used to navigate the design space. The standard deviation of experimental and predicted data is determined as residual. If residuals follow a normal distribution, it means that the experimental errors are random. Therefore, the residuals were normalized with the estimate of standard deviations, which presented as externally studentized residuals. The studentized residuals should be The signal-to-noise ratio is determined by statistical parameter adequate precision. A preferable adequate precision is higher than 4. The adequate precision in this study was 36.330, as presented in Table 3. It indicates that the signal is adequate, and the quadratic regression model is viable to be used to navigate the design space. The standard deviation of experimental and predicted data is determined as residual. If residuals follow a normal distribution, it means that the experimental errors are random. Therefore, the residuals were normalized with the estimate of standard deviations, which presented as externally studentized residuals. The studentized residuals should be according to the normal distribution function [44]. The standard deviation for the runs is within ±3.00 interval, which shows the approximation of model prediction and experimental results.

Effects of Independent Variables on Calorific Value
Solid bioethanol is an alternative for easily transported fuel. It was produced by adding stearic acid as the additive. The addition of stearic acid into different concentrations of bioethanol (70 vol %, 80 vol %, 90 vol %) was studied in order to optimize the calorific value of the solid bioethanol. Calorific value is one of the most important properties for fuel. Bioethanol in a liquid state has significantly lower calorific value compared to the solid state. The theoretical calorific value of ethanol is 29.3 MJ/kg. The liquid bioethanol in this study had different calorific values in each concentration. Bioethanol 70 vol %, 80 vol %, and 90 vol % had calorific values of 26.6 MJ/kg, 27.5 MJ/kg, and 28.3 MJ/kg, respectively. After the addition of stearic acid, the calorific value significantly increased above 30 MJ/kg due to the ratio of stearic acid and bioethanol and the concentration of bioethanol. The highest calorific value was 43.17 MJ/kg at a solid-liquid ratio of stearic acid and bioethanol 5:7 for 90% of bioethanol concentration. The increasing calorific value was due to the addition of stearic acid, which has a higher calorific value than liquid bioethanol (40.08 MJ/kg). The calorific value is affected by the number of carbonyl atoms (C) in molecules. Stearic acid (CH 3 (CH 2 ) 16 COOH) has 18 carbon atoms, whereas bioethanol (C 2 H 5 OH) has 2 carbon atoms. Hydrocarbon chains of those molecules reacted with oxygen (O 2 ) with heat/flame.
The effects of bioethanol concentration and the ratio of stearic acid-bioethanol are presented in three-dimensional surface plots (see Figure 5a-c). Figure 5a,b illustrates that bioethanol concentration had a more significant influence on the results of calorific value compared with bioethanol volume and stearic acid. Furthermore, for a fixed bioethanol concentration, the increase of either bioethanol volume or stearic acid only increases the calorific value by a slight amount. In both cases, the highest calorific value was achieved for 90% ethanol. The higher bioethanol concentration regularly increased calorific value due to the volatility of bioethanol, which allows it conveniently to be burned. However, the solid-liquid ratio of stearic acid and bioethanol support the effect of bioethanol concentration. Figure 5c illustrates that stearic acid significantly affects the calorific value compared to bioethanol volume. For a fixed stearic acid, a variation of bioethanol volume has an insignificant effect on calorific value. The lack of stearic acid inhibits the formation of solid bioethanol, whereas the high amount inhibits the bioethanol vapor to be burned by heat. In this study, the highest calorific value was obtained at 90 vol % bioethanol concentration with 5 mL bioethanol and 7 g stearic acid.
However, the high calorific value of the solid bioethanol should be supported by shape and solidity for an efficient solid fuel. The solidity was observed before the optimization of the calorific value. The variables for solidity used are the same as the calorific value optimization variables using the Box-Behnken design. According to the study results, the solidity of the solid bioethanol was affected by the bioethanol concentration and the ratio of bioethanol and stearic acid. The best solidity form as well as the highest calorific value were obtained at a concentration of 90% bioethanol and at the solid-liquid ratio of 5:7 of stearic acid and bioethanol. The lowest solidity is shown in number 17, with a concentration of 70% bioethanol. It formed a water layer at the bottom with the ratio of stearic acid higher than bioethanol (3:5). The water layer seems to come from the higher water content bioethanol azeotrope in 70% of the bioethanol concentration.
The solid-liquid ratio of stearic acid and bioethanol also affected the solidity of the solid bioethanol. Sample numbers 1, 2, 3, 8, and 16 were produced using 80% bioethanol concentration in the different solid-liquid ratio of stearic acid and bioethanol, as presented in the experimental design in Table 2. The higher stearic acid content of sample 8 resulted in a too dense form which inhibited the vaporization of bioethanol to be burned out [60]. Sample number 3 containing 7:7 stearic acid and bioethanol produced the calorific value of 42.53 MJ/kg. It is insignificantly different from the highest calorific value (43.171 MJ/kg), which was obtained from sample number 11. The burning test was performed in order to prove the feasibility of the solid bioethanol as solid fuel. The four highest calorific value samples were selected to be investigated because of the reference that calorific value is the main factor of flame temperature. The samples to be burned had a diameter of 3 in and a length of 4 cm. Table 4 shows that the longest burning time of the solid bioethanol, namely 177 min, was obtained from sample number 11, which had the highest calorific value. This The burning test was performed in order to prove the feasibility of the solid bioethanol as solid fuel. The four highest calorific value samples were selected to be investigated because of the reference that calorific value is the main factor of flame temperature. The samples to be burned had a diameter of 3 in and a length of 4 cm. Table 4 shows that the longest burning time of the solid bioethanol, namely 177 min, was obtained from sample number 11, which had the highest calorific value. This shows that the highest calorific value profoundly influences the burning time of solid bioethanol. However, concentrations of 80% and 90% bioethanol seem to have insignificant burning time with different bioethanol-stearic acid ratios. Nonetheless, the higher concentration of bioethanol has a better effect on the flame as well as the addition of stearic acid. According to its chemical reaction, one mole of bioethanol produces less CO 2 than LPG (that mainly contains propane and butane) in combustion due to the lower carbon content per mole [61]. Those gases produce high carbon dioxide during the combustion. Burning bioethanol produced 2 moles of carbon dioxide in every reaction. Meanwhile, combustion reactions of the propane and butane content in LPG produce significantly higher carbon dioxide [62].
The combustion of solid bioethanol has the same reaction as liquid bioethanol. However, stearic acid increases the calorific value as well as the burning time of bioethanol in the solid state. According to the burning time test results in Table 4, samples 8 and 15, which have slightly different calorific value as compared to samples 4 and 11, generate a significantly lower burning time. It is due to the disproportionate amount of stearic acid content in the sample. As for samples 8 and 15, the stearic acid and bioethanol ratio is 7:3 and 5:7, respectively. A stearic acid content higher than bioethanol provided a better solid shape of the solid bioethanol. This affects the rate of bioethanol vaporization due to the tight polymerization of stearic acid, which inhibits the vaporization of bioethanol. Inversely, the lack of stearic acid leads to a shorter burning time due to the easy vaporization of bioethanol. In addition, adding a proper amount of stearic acid is an essential factor in obtaining a quality solid bioethanol. In this study, sample 11 generated the longest burning time as well as the highest calorific value. The burning of the solid bioethanol is shown in Figure 6. shows that the highest calorific value profoundly influences the burning time of solid bioethanol. However, concentrations of 80% and 90% bioethanol seem to have insignificant burning time with different bioethanol-stearic acid ratios. Nonetheless, the higher concentration of bioethanol has a better effect on the flame as well as the addition of stearic acid. According to its chemical reaction, one mole of bioethanol produces less CO2 than LPG (that mainly contains propane and butane) in combustion due to the lower carbon content per mole [61]. Those gases produce high carbon dioxide during the combustion. Burning bioethanol produced 2 moles of carbon dioxide in every reaction. Meanwhile, combustion reactions of the propane and butane content in LPG produce significantly higher carbon dioxide [62].
The combustion of solid bioethanol has the same reaction as liquid bioethanol. However, stearic acid increases the calorific value as well as the burning time of bioethanol in the solid state. According to the burning time test results in Table 4, samples 8 and 15, which have slightly different calorific value as compared to samples 4 and 11, generate a significantly lower burning time. It is due to the disproportionate amount of stearic acid content in the sample. As for samples 8 and 15, the stearic acid and bioethanol ratio is 7:3 and 5:7, respectively. A stearic acid content higher than bioethanol provided a better solid shape of the solid bioethanol. This affects the rate of bioethanol vaporization due to the tight polymerization of stearic acid, which inhibits the vaporization of bioethanol. Inversely, the lack of stearic acid leads to a shorter burning time due to the easy vaporization of bioethanol. In addition, adding a proper amount of stearic acid is an essential factor in obtaining a quality solid bioethanol. In this study, sample 11 generated the longest burning time as well as the highest calorific value. The burning of the solid bioethanol is shown in Figure 6.

Conclusions
This study was aimed at producing solid bioethanol from liquid bioethanol obtained from palm empty fruit bunches (PEFBs). The production of liquid bioethanol was carried out by organosolv pretreatment, enzymatic hydrolysis, and fermentation. Cellulose was employed to hydrolyze treated PEFBs into sugars. It resulted in 152.51 mg/L of sugar yield. Furthermore, the fermentation process resulted in 62.29 mg/L of bioethanol yield. The bioethanol was distilled by rotary evaporator 4 or 5 times at 70 • C to obtain concentrated bioethanol. The bioethanol was collected to produce solid bioethanol using stearic acid. The solid bioethanol was successfully produced and its calorific value was optimized by RSM. The optimization resulted in the highest calorific value of 43.17 MJ/kg at 5 mL bioethanol and 7 g stearic acid for a bioethanol concentration of 90 vol %. This value is significantly higher than the highest liquid bioethanol calorific value (28.3 MJ/kg). The viability of solid bioethanol was also proven by a burning test. The highest calorific value and the proper shape of the solid bioethanol affect its quality. Therefore, it can be concluded that solid bioethanol using stearic acid has the potential to be solid fuel with a high calorific value. However, further study is needed to determine and optimize the storage time of solid bioethanol.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

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Yield Attributes and Yield of Urdbean as Influenced by Foliar Application of Salicylic Acid under Rainfed Condition

Blackgram is one of the most highly prized pulse crop, cultivated in almost all parts of India. It has inevitably marked itself as the most popular pulse and can be most appropriately referred to as the “king of the pulses” due to its mouth watering taste and numerous other nutritional qualities. Whether it be the very special “Dal makhni” of Punjab or the “Vada Sambhar” of South India, the taste rules the hearts of one and all alike. Indian immigrants have popularized the taste worldwide as well.

Introduction
Blackgram is one of the most highly prized pulse crop, cultivated in almost all parts of India. It has inevitably marked itself as the most popular pulse and can be most appropriately referred to as the "king of the pulses" due to its mouth watering taste and numerous other nutritional qualities. Whether it be the very special "Dal makhni" of Punjab or the "Vada Sambhar" of South India, the taste rules the hearts of one and all alike. Indian immigrants have popularized the taste worldwide as well.
In Japan, the health conscious people eat these seeds by soaking them in water overnight and then serving them as fresh bean sprout salad which is highly nutritious. Blackgram is perfect combination of all nutrients, which The experiment was conducted in the department of Plant Physiology C S Azad University of Agriculture & Technology. Kanpur in field during kharif session 2017 adopted Randomized Block Design with 10 treatments and Azad -3 variety including control and each treatment was replicated three times with three levels of salicylic acid i.e. 50, 100 and 150 ppm at the time of 20 DAS, 40 DAS and 60 DAS which was given through foliar application. Results maximum increase in yield and yield attributes viz., number of pods/plant, dry weight/plant, seed yield/plant, 100 seed weight (g), seed yield q/ha and harvest index(%) was recorded under the treatment of salicylic acid @ 150 ppm followed by 100 and 50 ppm of salicylic acid over control. Thus, in general it may be concluded that the foliar application of salicylic acid @ 150 ppm was found optimum concentration in enhancing yield of Black gram over rest of treatment under rain fed conditions. includes proteins (25-26%), carbohydrates (60%), fat (1.5%), minerals, amino acids and vitamins. The decreasing per capita availability of pulses from 1951in 60.7 g and 2000 in 35.9 g of great concern in the India contest where most of the peoples are vegetarian. An average adult man would require bog and adult woman 50 g of protein per day.
Black gram also known as urd, mash in India. In India total black gram production was estimated 14.76 million tonnes from 23.63 million hectare area with productivity of 642 kg/ha. Grain legumes occupy a unique position in Indian agriculture as they provide vegetable protein for human diet, pulse are also important in our agriculture system because of their nitrogen fixing ability about 40% of the total nitrogen in the world in added by leguminous plant. They can also suit to dry farming condition because of their ability to trap moisture from deep layers of the soil by virtue of their deep penetrating root system.
In India, it is the fourth important pulse crop after chickpea and pigeon pea, green gram is cultivated in state of Rajasthan, Madhya Pradesh, Punjab, Haryana, U.P., Maharashtra, Karnataka, Andhra Pradesh and Tamil Nadu. In Uttar Pradesh, it is being cultivated in an area of 3.91 lakhs hectare with 1.72 lakhs tonnes production Anonymous (2014).
The main reason of low productivity of the green gram is cultivation on marginal and submarginal lands with no use of recommended dose of fertilizers. Flowering is important parameter that is directly related to yield and productivity of plants. Salicylic acid has been reported to in duce flowering in a number of plants. Different plant species including ornamental plant Sinningia speciosa flowered much earlier as compared to the untreated control, when they received an exogenous foliar spray of salicylic acid.

Materials and Methods
The experiment was conducted geographically, Kanpur is situated in the central part of U.P. and subtropical tract of North India between latitude ranging from 25 0 56' to 28 0 58' North and longitude 79 0 31' to 80 0 34' East and is located on an elevation of about 125.9 meters above mean sea level in gangetic plain. The seasonal rainfall of about 816 mm received mostly from II nd Fortnight of June or first Fortnight of July to mid-October with a few showers in winter season.
The experiment was conducted in randomized block design with 3 replications. Seed of Azad-3 variety were used in this experiment.
Pure seeds of variety were obtained from the Economic botanist (Legume section) of this University. Foliar application of salicylic acid @ 150 ppm (60 DAS) -T 10

Application of fertilizers
The experimental field was fertilized with recommended dose of NPK /ha @ 20 Kg N, 40 Kg P 2 O 5 and 20 Kg K 2 O. Basal manuring experimental field was ploughed once with a soil turning plough and two ploughings with desi plough. Finally, it was planked to give a uniform level. The sowing was done in plot with plant to plant 40 cm, row to row 10 cm and depth 5-8 cm below. All the data analysis related to yield and yield contributing characters as well as chemical analysis were statistically analyzed by the methods suggested by Fisher (1935). Plant sampling method randomly three plants were taken out from each urdbean variety at different growth stages, and the data on different traits were recorded on these plants.

Effect of salicylic acid on yield attributes of urdbean
Above observations were recorded at different stages as 35 DAS, 70 DAS, and harvesting and sprayed of the salicylic acid at 20 DAS 40 DAS and 60 DAS on the foliar application. All level of foliar application of salicylic acid better response in yield attributes viz. that number of pods, 100 seed economic yield, biological yield, harvest index and yield varied significantly due to foliar application of salicylic acid treatment and their significantly.
Maximum value of all traits obtained in T 10 under salicylic acid (150 ppm) comparison of all other treatments as well as control. Yield and yield attributes were reported significant results in salicylic acid T 10 , T 9 , T 8 in higher comparison to salicylic acid 100 ppm T 7 , T 6 , T 5, and salicylic acid 50 ppm T 4 , T 3 , T 2 and all treatments give significant results than control (T 1 ). Among the different levels of salicylic acid, application @ 150 ppm was found significantly better and positively influenced all traits under rain fed conditions. Coefficient variance revealed that yield possessed significantly positive yield attributes and yields indicates involvement of these traits in yield formation process under stress environment condition.

Number of pods/plant
A perusal of data on number of pods as presented in Table 1 and Figure 1 showed that different levels of treatments significantly increased the number of pods over control at harvest stage. Maximum number of pods was recorded in the treatment of salicylic acid 150 ppm 60 DAS closely followed by salicylic acid -150 ppm. 40 DAS and over controls. It was inferred that application of salicylic acid similar result was also reported Sujatha

Dry weight of pods
Data as depicted in Table 1 and Figure 1 showed that foliar application of salicylic acid @ 150 ppm influenced dry weight of pods at maturity stages of crop growth. Significantly higher dry weight was observed in (T

Seed yield per plant
The data presented in Table 2 and Figure 2 revealed that the seed yield per plant significantly increased. Maximum seed yield per plant was recorded by the treatment (T 10 ) and other all treatment over control. The application of salicylic acid conferred an increase in leaf area. An improvement in leaf area following SA application in plants grown under saline conditions was also reported by Sathiyamurthy et al., (2017).

Seed test weight (g)
Different foliar application of salicylic acid treatments significantly increased the seed index weight over the control. The maximum 100 seed weight was obtained treatment (T 10 ) wt. 4.41g followed by all other treatment. Similar result was also reported Sujatha (2001), Ali and Mahmoud (2013), Sathiyamurthy et al., (2017) number of pods per plant components of urdgbean crops.

Effect of salicylic acid on yields and harvest index of urdbean
The seed yield per unit area is an important scale for agronomic assessment of the crop. The suitability of the treatment is best judged by their efficiency to produce higher yield. Yield of any crop is the ultimate result of interaction of the biotic and environment factors. The superiority or inferiority of any treatment could be judged by magnitude of changes brought in the productivity.

Biological yield q/ha
A perusal of data on harvest index as presented in Table 3 and Figure 3 the data recorded on biological yield in kg/ha was converted into seed yield in quintals/ha the data revealed that all the foliar application of salicylic acid gave significant response on seed yield over control treatments (T 1 ). The maximum grain yield (21.97 q/ha) in treatment (T 10 ) was recorded with the foliar application of salicylic acid @ 150 ppm at 60 DAS. It was found significantly superior than all the other treatments the minimum biological yield (12.35 q/ha) was recorded in control treatment (T 1 ). Similar result was also reported Ali and Mahmoud (2013) and Sathiyamurthy et al., (2017) seed yield and yield components of urdgbean.

Seed yield q/ha
A perusal of data on harvest index as presented in Table 3 and Figure 3 the data recorded on seed yield in kg/ha were converted into seed yield in quintals/ha the data revealed that all the foliar application of salicylic acid gave significant response on seed yield over control treatments (T 1 ). The maximum grain yield (10.35 q/ha) in treatment (T 10 ) was recorded with the foliar application of salicylic acid @ 150 ppm at 60 DAS. It was found significantly superior than all the other treatments the minimum seed yield (8.20 q/ha) was recorded in control treatment (T 1 ). Foliar application of different treatment of salicylic acid had significant variation in the biological and seed yield of black gram. Among the treatments, foliar application of salicylic acid 150 ppm at 60 DAS recorded significantly higher both yield. Similar result was also reported Ali and Mahmoud (2013) and Sathiyamurthy et al., (2017) seed yield and yield components of urdgbean.

Harvest index
A perusal of data on harvest index as presented in Table 3 and Figure 2 showed that it varied significantly due to foliar application of salicylic acid treatment. Harvest index increased significantly in treatment (T 10 ) over each other treatment. Further, it also appears that application of salicylic acid @ 150 ppm at 60 DAS resulted in significantly higher 30.30 % over control 18.21 % over rest of treatments.
Harvest index as depicted in us that it varied significantly due to variation, treatment of foliar application of salicylic acid @ 150 ppm recorded high yield and harvest index from the result it appears application of salicylic acid @ 150 ppm had positive effect on yield and harvest index. A similar result was also established by Ali and Mahmoud (2013) and Sathiyamurthy et al., (2017).
Above observations were recorded at different stages as 35 DAS, 70 DAS, and harvesting and sprayed of the salicylic acid at 20 DAS 40 DAS and 60 DAS on the foliar application. All level of foliar application of salicylic acid better response in yield attributes viz. that number of pods, 100 seed economic yield, biological yield, harvest index and yield varied significantly due to foliar application of salicylic acid treatment and their significantly. The results obviously suggest and it may be concluded that foliar application of salicylic acid on urdbean variety Azad -3 significantly change in plant morphology, improvement in vegetative and reproductive growth to enable them for higher yield build up and the affects initiated once were carried over through the life cycle of plant. These substances judiciously work out can be employed successfully for yield maximization of urdbean.


extfieldsofstudy: Biology Medicine

text:
Effect of acorn flour on the physico-chemical and sensory properties of biscuits

The aim of the work was to explore the feasibility of using acorn flour as a novel and healthy ingredient in biscuits. The physico-chemical characteristics of acorn flour obtained from three different Quercus species were compared. Acorns of Quercus coccifera L. were the most antioxidant and were therefore used for preparing biscuits at two levels of addition, 30 and 60 g 100 g−1 on wheat flour basis. The physico-chemical and sensory characteristics of the obtained biscuits were then assessed. Acorn-added biscuits showed significantly (p < 0.05) higher content of phenolics, antioxidant activity and oxidative stability than control biscuits, prepared without acorn flour. These features improved as the level of acorn flour increased. As for appearance, the acorn-added biscuits were darker, larger, more voluminous and more friable than control biscuits. Higher levels of fermentative alcohols and esters, as well as Maillard reaction volatile compounds (particularly furans), were observed in the acorn-added biscuits.

Introduction
Throughout the Mediterranean basin, the acorns, the fruits of the oak, have been used for centuries for edible purposes as a staple food (Hoeche et al., 2014). Collected from wild trees, and milled to flour, acorns were used for the production of traditional flat breads such as pan 'ispeli (Pinna, 2013), talo and ogi (Ayerdi et al., 2016) in Italy (Sardinia region) and Spain (Basque region), respectively. The production of a coffee substitute was another use of acorn in the same area (Pignone and Laghetti, 2010). Being a fundamental high-energy nutritional resource in subsistence economies and during times of shortage, acorns were considered a food for the poor, and were abandoned when the economic conditions improved. Therefore, the culture of using acorns in human nutrition practically disappeared after the Second World War, and these fruits are now barely used for feeding pigs (Pignone and Laghetti, 2010).
Most acorn types, indeed, show a high content of tannins, which are astringent and behave as anti-nutrients (Papoti et al., 2018). In the past, local populations identified and selected the trees bearing sweeter acorns, directly used for edible purposes, whereas the excessively astringent or even bitter acorns, which resulted unpalatable, were subjected to appropriate heat treatments and leaching (Papoti et al., 2018;Pignone and Laghetti, 2010;Raki c et al., 2007) or detoxification with clay (Johns and Duquette, 1991). However, nowadays the knowledge related to the use of acorns as food remains only in the memories of the elderly and is being lost.
In the context of the increased awareness of the nutritional properties of all nuts, acorns have recently become the object of renewed interest being nutritious fruits, with good contents of calcium, iron, magnesium, potassium, phosphorus, as well as A and E vitamins (Li et al., 2015). The lipid fraction is characterized by high levels of unsaturated fatty acids (Al-Rousan et al., 2013;€ Ozcan, 2007). In addition, recent studies highlighted that acorns have a rich phytochemical potential, mostly represented by phenolic compounds (Papoti et al., 2018;Vinha et al., 2016), and display interesting biological activities (Vinha et al., 2016) such as anti-inflammatory and anti-asthma effect (Moon et al., 2013), and a role in the prevention of obesity, dementia and liver diseases (Kang et al., 2004;Lee et al., 2005). Therefore, a "new age" is going to start for the use of acorns and for their reintegration into the human diet (Vinha et al., 2016). The extraction of the lipid fraction has been proposed in consideration of the good level of antioxidants such as tocopherols, carotenoids and phenolics observed in acorn oil (Makhlouf et al., 2018). Furthermore, due to the absence of gluten, acorn flour is being proposed as a new ingredient for producing gluten-free foods, such as bread and biscuits (Korus et al., 2015;Korus et al., 2017). The use of acorn flour has been proposed also in the production of wheat-and barley-based bread ( Svec et al., 2018) and sponge cakes (Molavi et al., 2015). However, the addition of acorn flour worsened the sensory attributes by increasing density, hardness and gumminess of sponge cakes and decreasing the specific volume of bread. These findings were related to a negative effect on the viscoelastic properties of dough, mostly due to the absence of gluten and presence of fiber in the added flour. We hypothesize that biscuits, on the contrary, could tolerate the addition of acorn flour better than bread and sponge cakes, because their characteristic friability requires a weak gluten network. Moreover, biscuits are popular, daily consumed, bakery items and have long shelf-life, all features that make them suitable for the addition of functional ingredients.
In this frame, the aim of the work was to verify our hypothesis, i.e. to explore the feasibility of using acorn flour as a novel and healthy ingredient in wheat-based biscuits. After comparing the physicochemical characteristics of acorn flour obtained from three different Quercus species, the most antioxidant flour was used at two levels of addition (30 and 60 g 100 g À1 on wheat flour basis) and its effect on the physico-chemical and sensory characteristics of biscuits was assessed.

Preparation of acorn flour
Mature acorn fruits were manually harvested from three Quercus species, namely Quercus ilex L., Quercus suber L., and Quercus coccifera L. (indicated as QI, QS and QC, respectively) in a forest located in Eastern Algeria (Oum El Bouaghi region) in November 2017. The acorns were shelled and ground using an electric grinder (Moulinex, Groupe SEB, Ecully, France) to obtain wholemeal flour whose particle size, determined by a vibratory sieve shaker (Retsch AS200, Haan, Germany), is shown in Table 1. The moisture content of flours accounted for 24.3 AE 0.5, 20.9 AE 0.1 and 22.7 AE 0.7 g 100 g À1 for QI, QS and QC, respectively. The obtained flours were slightly sweet, not bitter at all and showed limited levels of astringency (being QC slightly more astringent that QS and QI), as assessed by tasting them. Therefore, the flours did not need any debittering pretreatment and were directly used for preparing biscuits as they were. All flour samples were stored at -20 C until being used.

Preparation of biscuits
Ingredients for the preparation of biscuits were wholemeal flour of acorns (Q. coccifera) prepared as above described, refined flour of soft wheat (Triticum aestivum L.) having protein ¼ 10.8 g 100 g À1 , fat ¼ 1.1 g 100 g À1 , carbohydrates ¼ 72.7 g 100 g À1 , fiber ¼ 2.2 g 100 g À1 , ash ¼ 0.5 g 100 g À1 , moisture ¼ 12.7 g 100 g À1 (Molini Amoruso, Cerignola, Italy), sucrose (Eridania, Bologna, Italy), extra-virgin olive oil (Olearia de Santis, Bitonto, Italy) and sodium bicarbonate (Solvay, Bollate, Italy). Biscuits enriched of acorn flour at the level of 30 and 60 g 100 g À1 on wheat flour basis (coded Q30 and Q60, respectively) and control biscuits, made up of soft wheat refined flour, without acorn flour, were prepared. Preliminary trials were made to establish empirically the amount of water to be used for obtaining a similar dough consistency in all three types of biscuits. The amount of water needed was very similar in control and acorn-enriched dough. In fact, wholemeal acorn flour contained more fiberswhich absorb a relevant amount of waterthan refined wheat flour, therefore potentially requiring more water, however acorn flour was much moister than wheat flour, then reducing the amount of water to be actually added. Therefore the same amount of water was used for all biscuit types. The process consisted of: i) mixing flour (wheat flour 100%, or a blend of wheat flour and 30 or 60 g 100 g À1 of acorn flour) (100 g), sugar (35 g), extra virgin olive oil (14 g) and sodium bicarbonate (0.5 g) by a spiral kneader (mod. KM 398 CB, Bomann, Kempen, Germany) for 3 min; ii) adding water (25 mL for all biscuit types), and kneading again for 10 min; iii) sheeting the dough by a rolling pin to the thickness of 6 mm, and shaping as round biscuits by means of a biscuit cutter (4.5 cm diameter); iv) transferring the shaped pieces of dough in a metal tray by mixing all three types of biscuits according to a Latinsquare design, to minimize any effect of tray location during subsequent baking; v) baking in a preheated electric oven (Smeg SI 850 RA-5 oven, Smeg S.p.A., Guastalla, Italy) for 12 min at 160 C. The baking temperature was selected according to other studies (Arshad et al., 2007;Pasqualone et al., 2015;Sharma and Zhou, 2011;Supski, 2006), then cooking time was set up by means of preliminary trials in order to obtain perfectly cooked biscuits without burns. Two independent replicate baking experiments were carried out. Biscuits were finely crushed for all the analyses with the exception of colorimetric, sensory, and textural determinations.

Basic physico-chemical determinations
Moisture content was determined at 105 C by means of an automatic moisture analyzer (Radwag Wagi Elektroniczne, Radom, Poland). Water activity (a w ) was determined by using the water activity meter Aqua Lab 4TE (Meter Group Inc., Pullman, WA, USA) according to the manufacturers' instructions. Protein content (N Â 5.7) was analyzed according to the AACC approved method 46-11.02 (AACC, 2000), whereas fat was extracted and determined by Soxhlet apparatus using diethyl ether as solvent, and total dietary fiber was determined by the enzymatic-gravimetric procedure (AOAC, 1995). Carbohydrates were calculated by difference.
Colorimetric evaluations of red index (a*), yellow index (b*) and brown index (BI, defined as 100-L*) were carried out under D65 illuminant by using a spectro-colorimeter CM-700d (Konica Minolta Sensing, Osaka, Japan) equipped with a pulsed xenon lamp and granular materials attachment CR-A50 (Konica Minolta Sensing, Osaka, Japan).
Chemical determinations were carried out in triplicate, whereas colorimetric evaluations were made with five replications.

Extraction of phenolic and flavonoid compounds
Five g of sample (acorn flour or powdered biscuits) were mixed with 50 mL of ethanol/water 30:70 (v/v). The mixture was subjected for 35 min to ultrasound treatment (CEIA, Viciomaggio, Italy) at room temperature. The obtained extracts were centrifuged at 6000 Â g for 10 min at 4 C (SL 16R Centrifuge, Thermo Fisher Scientific Inc., Waltham, MA, USA), then were filtered with nylon filters having pores of 0.45 μm (Sigma Aldrich).

Determination of phenolic compounds
The content of total phenolic compounds (TPC) was determined as in Table 1 Particle size distribution of acorn flour obtained from three different Quercus species (Quercus ilex L., Quercus suber L. and Quercus coccifera L., indicated as QI, QS and QC, respectively).

Type of flour
Particle size class (g 100 g À1 )  Pasqualone et al. (2016). Each flour extract (100 μL) was mixed with 100 μL of Folin-Ciocalteu reagent. After 4 min, the mixture was added of 800 μL of 5% Na 2 CO 3 and incubated for 20 min in a water bath at 40 C. The absorbance was then measured at 750 nm by an Agilent Cary 60 spectrophotometer (Agilent Technologies, Santa Clara, USA). The total phenolic content was expressed as mg of gallic acid equivalents per 100 g of dry matter based on a gallic acid standard curve made in the same conditions (R 2 ¼ 0.997). The analysis was carried out in triplicate.

Determination of flavonoid compounds
The content of total flavonoid compounds was determined as reported in Makhlouf et al. (2018). Each flour extract (500 μL) was mixed with 2 mL of distilled water and 150 μL of NaNO 2 solution (5% w/v). After 6 min, 150 μL of a 10% solution of AlCl 3 was added and allowed to stand further 6 min; thereafter, NaOH solution (2 mL, 1M) was added to the mixture. Then, distilled water was added to bring the final volume to 5 mL. After incubation at room temperature for 15 min, the absorbance of the reaction mixture was read at 510 nm. The results were expressed as mg of catechin equivalent per 100 g of dry matter based on a catechin standard curve made in the same conditions (R 2 ¼ 0.999). The analysis was carried out in triplicate.

Determination of antioxidant activity
The antioxidant activity, compared with the standard antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), was evaluated by the assay based on using the radical of the 2,2 0 -azinobis (3ethylbenzothiazoline-6-sulphonic acid) (ABTS), as described in Pasqualone et al. (2014a;. Briefly, 50 μL of each extract were added to 950 μL of ABTS reagent previously prepared. After 8 min, the decrease of absorbance was measured at 734 nm. A calibration curve of Trolox was used, and the results were expressed as μmol Trolox equivalents (TE) per g of dry matter.
The antioxidant activity of the extracts was analyzed also by means of the assay based on evaluating the capacity to scavenge the 1,1-diphenyl 2-picrylhydrazyl radical (DPPH). In cuvettes for spectrophotometry, 50 μL of each sample were added to 950 μL of DPPH solution (0.08 mM in ethanol). After 30 min in the dark, the decrease of absorbance was read at 517 nm. The results were expressed as μmol TE per g of dry matter.
The analyses were carried out in triplicate.

Determination of induction time
The oxidative stability of biscuitsnamely, the oxidative stability of their lipid fractionwas evaluated by measuring the induction time (IT) by an automatic testing device (Rapidoxy, Anton Paar, Blankenfelde-Mahlow, Germany). The instrument induces a forced oxidation of the sample (3 g) by subjecting it to conditions of high temperature and high oxygen pressure. According to manufacturer's instructions, IT was defined as the time needed for a 10% drop of the oxygen pressure under the following conditions: T ¼ 140 C, initial O 2 pressure ¼ 700 kPa. The analysis was carried out in triplicate.

Determination of volatile compounds
Volatile compounds of biscuits were determined by headspace solid phase micro-extraction (HS-SPME) coupled with gas-chromatography/ mass spectrometry (GC/MS). After weighting the samples (500 AE 0.05 mg) in 20 mL vials, the extraction of volatile compounds was carried out by exposing a 75 μm carboxen/polydimethylsiloxane (CAR/PDMS) SPME fiber (Supelco, Bellefonte, PA, USA) in the headspace of the sample at 40 C for 15 min. The fiber was then desorbed in the GC injector (2 min) and the volatile compounds were separated by using an Agilent 6850 gas-chromatograph equipped with an Agilent 5975 massspectrometer (Agilent Technologies Inc., Santa Clara, CA, USA) and a HP-Innowax (Agilent Technologies Inc., Santa Clara, CA, USA) polar capillary column (60 m length Â 0.25 mm i.d. Â 0.25 μm film thickness), in the conditions reported in Pasqualone et al. (2014a). Peak identification was performed by computer matching with the reference mass spectra of National Institute of Standards and Technology (NIST) and Wiley libraries. The results were expressed as percentage of the integrated area on the total area. The analysis was carried out in triplicate.

Determination of dimensional indices of biscuits
Diameter (D) and thickness (T) of biscuits before and after baking were determined by a caliper and used to calculate surface (S) and volume (V). The percentage increases in D, T, S and V due to baking were calculated as follows: Spread factor was calculated as the ratio between diameter and thickness of baked biscuits, according to the AACC method 10-50.05 (AACC, 2000). Six biscuits were analyzed for each parameter.

Texture profile analysis of biscuits
The textural properties of biscuits, in terms of fracture force (N mm À2 ), were determined by a 3-point bending test as in Giarnetti et al. (2015). A Texture Analyzer (Z1.0 TN, Zwick GmbH & Co. KG, Ulm, Germany), equipped with a 1000 N load-cell, was used. The biscuits were placed on supports with their top surface down. The distance between the support bars was 4 cm. The downward movement of the probe, set at a speed of 5 mm min À1 , was continued until the biscuit was broken. At least eight biscuits were tested per each type.

Sensory evaluation of biscuits
Quantitative Descriptive Sensory Analysis of biscuits was performed by a trained panel consisting of 8 members in the conditions described in a previous work (Pasqualone et al., 2011). The list of sensory terms included descriptors of visual-tactile characteristics (color, friability), odor (bran-like, caramel), taste (sweet, bitter) and texture attributes perceived in the mouth (dryness, astringency, graininess). The descriptors were rated on an anchored line scale that provided a 0-9 score range (0 ¼ minimum; 9 ¼ maximum intensity), as reported in Table 2. Three sensory analyses were carried out.

Statistical analysis
Statistical analysis was carried out using XLSTAT software (Addinsoft SARL, New York, NY, USA). Significant differences were determined at p 0.05 by one-way analysis of variance (ANOVA) followed by Tukey's HSD test. 3. Results and discussion

Characteristics of acorn flour
The chemical analyses carried out on the acorn flour obtained from three different Quercus species, namely Q. ilex, Q. suber and Q. coccifera, confirmed that acorns are a highly energetic food, rich in carbohydrates, fats, and with relatively low contents of proteins, although the latter two nutrients are more largely affected by environmental and genetic factors (Vinha et al., 2016) (Table 3). Significant differences were observed among species. The highest fiber content was observed in the QI acorn flour, whereas, conversely, the QC flour was the richest in protein and fat. Fat content was abundant in all the flours (7.78-8.88 g 100 g -1 d.m.), but it has to be considered that acorn fat has a nutritionally favorable fatty acid profile, as previously reported (Al-Rousan et al., 2013;€ Ozcan, 2007). The examined flours showed also relevant contents of phenolic and flavonoid compounds, which were significantly more abundant in QC. These findings determined a higher antioxidant activity in the QC flour which, therefore, was selected for enriching the biscuits in bioactive compounds. Overall, the TPC levels of the examined flours were much lower than the range 7.3-11.7 g 100 g À1 TPC, reported for extremely astringent acorns (Shimada and Saitoh, 2003). In the present study all the flours were slightly sweet, not bitter at all and showed limited levels of astringency, as assessed by tasting them (data not shown), although other authors reported that acorns of QC and QS are very bitter and unpalatable (Ayerdi et al., 2016). Environmental effects and/or genetic causes could not be excluded, being our sampling area (Eastern Algeria) far from that of the study of Ayerdi et al. (2016), i.e. Spain. In addition, differences in the ripening degree of acorns could also occur among different studies, being unripe acorns more bitter and astringent than mature acorns.
As regards their visual appearance, all the acorn flours examined were yellow-brownish. QC flour, however, was lighter in color than the other two flour types.

Characteristics of biscuits
The QC flour was used for preparing biscuits at 30 and 60 g 100 g À1 on wheat flour basis, inducing a significant increase of the phenolic content and antioxidant activity of the end products (Table 4). The Q30 biscuits were significantly richer of phenolics and flavonoids than control without acorn flour, and showed higher antioxidant activity. These positive features further increased in Q60 biscuits. Accordingly, the oxidative stability of biscuits, evaluated by measuring the time needed to oxidize biscuits under forced conditions (induction time), was significantly longer as the level of acorn flour increased.
The addition of acorn flour resulted also in a relevant increase in red and brown indices, whereas the yellow index decreased compared to control (Table 4). This browning effect was imputable to the phenolic fraction of the composite flour, which is known to easily undergo oxidation by polyphenoloxidase developing brown quinones and related compounds (Pasqualone et al., 2014b;Taranto et al., 2012). Other authors similarly reported a significant browning effect of the addition of acorn flour in gluten-free bread (Korus et al., 2015). Color was evaluated because of its great influence on consumer acceptance. Therefore, the observed color alterations, with respect to control biscuits, should be properly communicated to consumers together with the positive antioxidant features, to avoid rejection.
As in other bakery products, several dimensional indices can be measured in biscuits to evaluate the extent of thermal expansion occurred during baking, which is due to the thermal increase in volume of the carbon dioxide developed by leavening agents, dough moisture, and air entrapped in the dough during kneading. Subsequent baking to dryness renders biscuits having a greater degree of expansion more friable than those less expanded and more compact. The expansion is influenced by dough extensibility and strength, therefore its measurement highlights any variation of dough induced by newly added ingredients.
The increase of diameter induced by baking varied significantly with the addition of acorn flour (Table 4). Biscuits appeared progressively larger as the level of acorn enrichment increased, probably due to the effect of gluten dilution with a gluten-free flour. Other authors found that gluten-free biscuits increased in diameter more than conventional biscuits (Hossain et al., 2017). The addition of acorn flour, however, did not significantly influence the increase in thickness due to baking.
As a consequence of large increase in diameter, the acorn-enriched biscuits showed a greater increase of surface compared to control biscuits. The expansion in volume, although significantly higher in the acorn-enriched biscuits than in the control, was less influenced by the amount of added acorn flour (30 or 60 g 100 g À1 ), because of the reduced effect on thickness.
The spread factor is another physical indicator of biscuit quality; higher values of this index are appreciated owing to a positive influence on the acceptability of biscuits (Klunklin and Savage, 2018). The spread factor of acorn flour-added biscuits was higher than in the control.
If gluten is abundant and elastic (which is undesired in biscuits), it can inhibit biscuit expansion. High values of spread factor were observed in gluten-free biscuits, compared to conventional wheat biscuits (Sharma et al., 2016). Further, the addition of non gluten containing ingredients, such as rice flour or minor millet flour, increased the spread factor of wheat-based biscuits (Chung et al., 2014;Sharma et al., 2016). However, opposite results were observed by adding flours of bean and sesame (Hoojjat and Zabik, 1984), or lentil flour (Zucco et al., 2011). Especially if complex ingredients are added to the dough, a number of interactions occur among proteins, starches, fiber, lipids and minor compounds, thus the results obtained could not be exhaustively explained only by the dilution of gluten. In our case, being acorn flour a relatively fatty ingredient, the additional fat component contributed by this flour could have had a positive effect on spread ratio (Sudha et al., 2007).
The measure of the friability of biscuits was carried out through a three-point bending test ("snap test") until the break, simulating the breaking force exerted during chewing. The fracture force, lower for more friable biscuits, was significantly greater in control biscuits and progressively decreased as the level of acorn flour increased (Table 4). Also these findings were mainly imputable to gluten dilution, although a contribution by the lipid fraction of the acorn flour cannot be excluded. Lipids are known to interfere with gluten formation and play an essential role in determining the friability of biscuits (Caponio et al., 2006).
The sensory properties showed significant differences among the examined biscuits for the majority of descriptors (Table 4). The sensory evaluation of color surface paralleled the colorimetric measurement, with acorn-enriched biscuits darker than the control. A significantly more pronounced friability of biscuits enriched of acorn flour was also observed, compared to control, in agreement with the instrumental data Table 5 Volatile compounds of biscuits enriched of increasing levels of acorn flour (Quercus coccifera L.). Values are expressed as percentage of the integrated area on the total area (mean of three replications AESD). Control ¼ biscuits without acorn flour; Q30 and Q60 are biscuits prepared with 30 and 60 g 100 g À1 acorn flour on wheat flour basis, respectively. of breaking force. The odor sensations prevailing in biscuits were bran-like and caramel notes, both much more intense when acorn flour was added. The branlike note was associated to the typical odor of bran and wholemeal products, therefore more perceivable in biscuits from acorn-wheat composite flours, being acorns milled to wholemeal flour whereas refined wheat flour was used in control biscuits. The caramel note, particularly intense in Q60 biscuits, was imputable to Maillard reaction during baking.
The sweet taste of biscuits was perceived significantly more intense in control, whereas bitterness was absent in all biscuit types.
Dryness was not different among biscuits. The biscuits added of acorn flour showed higher graininess than control, i.e. they were more friable and crumbly due to fat contributed by acorns, but the crumbs were granular and slightly heterogeneous in size having this flour also a high fiber content. The addition of acorn flour caused the appearance of an astringent sensation, although the latter was scored very low.
The volatile compounds were significantly different among biscuits ( Table 5). The acorn-enriched biscuits showed higher levels of fermentative alcohols and their esters, particularly ethanol and ethyl acetate, probably due to some fermentative activity in the raw material. 2-Methylbutanal and 3-methylbutanal, Strecker aldehydes deriving from Maillard reaction, were more present in the volatile pattern of biscuits added of acorns. On the contrary, the amount of hexanal was lower in Q60 than in the other types of biscuits, indicating a lower level of lipid oxidation, which agreed with higher contents of phenolic compounds. 2-Butanone, with no significant differences among biscuits, was also abundant. This compound was already detected in biscuits in previous researches  and its origin was attributed to β-chetoacids, arose from thermal treatment of tryglicerides, via decarboxylation (Chung et al., 1993). The furan compounds were mostly represented by furfural, a caramel-like odorant deriving by Maillard reaction, significantly more abundant in acorn-added biscuits than in control. Furfural is typically present in biscuits, being reported in several studies (Giarnetti et al., 2015;Mohsen et al., 2009;Pasqualone et al., 2014aPasqualone et al., , 2015. Little amounts of pyrazines, produced by Maillard reaction, were also detected.

Conclusions
Our study aimed at rediscovering endangered knowledge on the use of acorns for edible purposes, while at the same time taking into account the expectations of the modern consumer, used to foods with good sensory quality. The characteristics of biscuits are known to be less affected than those of bread from gluten dilution. In particular, the most important quality feature for biscuits is friability. After baking, the QC acornenriched biscuits were darker, larger, more voluminous and more friable than control biscuits. Only a very slight astringency was perceived. Therefore, according to our starting hypothesis, the enrichment of biscuits with QC acorn flour proved to be a very effective strategy for conferring antioxidant properties to biscuits, without excessively altering the sensory features.
Nowadays, the keyword is sustainability. Bringing light on the use of acorns for edible purposes could limit the waste of a nutritious and healthy food source. Acorns are an edible wild food, largely underutilized, which could supplement cultivated plants and help facing the increasing demand of food for a rapidly growing global population.

Declarations
Author contribution statement Antonella Pasqualone: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper. Fatima Makhlouf: Conceived and designed the experiments; Performed the experiments; Wrote the paper.
Malika Barkat, Carmine Summo, Francesco Caponio: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data. Graziana Difonzo, Giacomo Squeo: Performed the experiments; Analyzed and interpreted the data.


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Coating of Seeds with Collagen Hydrolysates from Leather Waste

The subject of this paper is improvement in the growth and yield of three different types of legumes and rape in drought conditions by coating seeds with hydrolysed collagen from tanning waste. In addition, the impact of various additives in the seed shell on the growth of the plant was investigated. The encapsulation process of seeds was conducted on a disc granulator. A centrally placed seed was first coated with a layer of fungicides. The next layer was collagen hydrolysate, collagen hydrolysate with latex or a solution of yellow dextrin and polyvinyl alcohol. The outer layer was a mineral additive e.g. dolomite or kaolin. After the end of the encapsulation process on the disk granulator, all of the seeds tested were sown into soils. Seeds without coating were also sown as control seeds. Seedlings were maintained for 29 days with cultivation without irrigation. The length of the seedlings was analysed for all of the seeds sown. Higher seedling growth values were obtained for seeds coated with collagen hydrolysate in comparison with control seeds (without coating). The use of collagen hydrolysate gave slightly better results than in the case of a solution of dextrin with polyvinyl alcohol.

Introduction
The foremost grain legume species contributors to human nutrition are the soybean (although the species is often classified for statistical purposes as an oil crop), common bean, chickpea, bean and pea [1][2][3]. Among crop plants, legumes have great potential as a protein production platform because of their naturally high protein content, nutritional value, independence of N-nutrition, pollen containment, availability of processing technology, storage stability, etc. [4]. In addition, proteins from legume seeds have been widely studied as regards their functional and bioactive properties, important for novel food development and for human health [5][6][7][8]. Oilseed rape has become one of the most important oil crops within the last decades. The main reason behind this trend is its multifunctional usage [9]. Unfortunately, crop losses due to drought and soil salinity are becoming increasingly severe and commonplace. Drought, one of the environmental stresses, is the most significant factor restricting plant growth and crop productivity in the majority of agricultural fields in the world [10]. Water scarcity, declining quality of water for irrigation, and soil salinity are problems which are becoming more and more acute [11,12].
The coating technique can be used to cover the seeds of agricultural products with the objective of achieving the adherence of macro and micronutrients, inoculants (bacteria) and fungicides to their surface or modifying their format and size with inert material [13][14][15][16].
Seed coating is a mechanism involving the application of necessary materials in such a way that they affect the seed or soil at the seed-soil interface. Coating can protect the seed from many kinds of diseases and pests [17]. Seed-coating agents are widespread and used in crop protection [18,19]. In the previous paper, we found that it is possible to form a collagen-based seed coating using a disk granulator. Preliminary studies also confirmed that the addition of collagen hydrolysates during the process of pea encapsulation improves the growth of seedlings [20,21]. A collagen-based product used to coat the surface was first produced and used in the EU and Russia. Importantly, processing collagen-based materials into valuable materials for agriculture will help solve the problem of solid waste generated by the leather industry. The fibre and textile industry is a diverse sector that covers the entire production chain of transforming natural and chemical fibres into end-use products [22]. Tannery waste with the addition of relevant mineral substances may then be used for the production of leathery materials, composition leather, and composite materials based on fragmented collagen fibres [23][24][25][26].
The subject of this paper is improvement in the growth and yield of three different types of legumes (peas, soybean, broad bean) and rape in drought conditions by coating seeds with hydrolysed collagen from tanning waste. In addition, the impact of various additives in the seed shell on the growth of the plant was investigated.

Materials and methods
The research was conducted with three legumes cultivars (peas, soybean and broad bean) and rape grown in laboratory conditions at the Łukasiewicz Research Network -Institute of Leather Industry. (Poland) IHAR Group. The germination capacity of the seeds of pea, soybean, broad bean and rape was 97%, 83%, 98% and 93%, respectively. Seedlings were maintained for 29 days with cultivation without irrigation.
The bovine shavings used as raw material showed the typical characteristics of semi-finished leather: moisture 51%, total ash 8.6%, total nitrogen 16.5%, chromium oxide 4.4%, and pH of aqueous extract 4.2. Collagen hydrolysates were obtained for the tests in various process conditions (Figure 1). On the basis of the results obtained, one preparation was selected that was characterised by the best properties in terms of its use in agriculture.
The encapsulation process took place at the laboratory of the Lodz University of Technology using a disk granulator [27][28][29] at optimum process conditions. Basic elements may be distinguished in the design of the granulator used for the granulation tests described: the granulator disk, the electric motor with rotational speed regulation and the granulated deposit moistening system. A binding liquid is fed directly onto the granulated material using spraying nozzles. A precise description of the process parameters and the test station are provided in [30,31]. The individual layers of the coating were formed in a specific order. A centrally placed seed was first coated with a layer of fungicides. The process of seed coating usually involves the use of adhesives to bind materials to the surface of seeds. Such procedures are ade-quate for about 90% of small seed species [32]. Therefore, the next layer was collagen hydrolysate (clear, yellow liquid, density 1.1125 g/ml, water content 67.19%, pH 7.5) or a solution of yellow dextrin and polyvinyl alcohol. Dextrin was used as a classical encapsulation reference liquid [33]. The outer layer of the coating was a mineral additive fostering germination (dolomite and kaolin, size of grains < 250 µm).
Dolomite flour (magnesium -calcium carbonate) and kaolin were chosen as typical fine grained materials [34][35][36]. What is more, encapsulation using latex (CAS: 25085-39-6) was attempted, as latex is considered a material improving the stability of a collagen preparation [37,38]. The formulation was developed in collaboration with Pestila II sp. z o.o. S.k. After the end of the encapsulation process in the disk granulator, all of the seeds tested were sown into soils (sowing in stable climatic conditions, without access to water). Seeds without coating were also sown as control seeds. The growth was observed for 29 days. The length of the seedlings was analysed for all of the seeds sown and compared in terms of the impact of having a coating and using additives in the coating on the plants' growth.

Results and discussion
Seeds are usually coated for ease of handling, singulation, precise placement, and the incorporation of beneficial chemicals or microbials. Quality demands for  seeds suitable for coating have improved knowledge of physiological seed quality. The incorporation of nutrients into seed coatings provides a unique opportunity to supply each seedling sown with an accurately controlled quantity of nutrients that may be preferentially available to the species sown [39]. The solution proposed is to increase the yield of various legume species using by-products of the leather industry.
The parameters of collagen hydrolysates obtained from tanning waste are listed in Table 1. A CHR1AEC collagen preparation was used for coating, since it has optimum parameters in terms of the properties of the binding liquid in encapsulation processes. Table 2 contains chromatography results of the amino-acid composition of the collagen preparation. By comparing the retention times of chromatography peaks of the sample tested with those of amino-acids present in the standard, 7 amino-acids were identified, i.e. glutamic acid, arginine with NH 3 , alanine, proline, leucine, lysine and hydroxyproline.
Thus, the presence of small, free amino-acids in the collagen hydrolysate was confirmed. This is beneficial as coating containing collagen will constitute a valuable source of nutrients necessary for plant growth. Significantly, the specific amino-acids of collagen, such as proline and hydroxyproline, act mainly as a hydric balance of the plant, strengthening the cellular walls in such a way that they increase resistance to unfavourable climatic conditions [40]. Collagen hydrolysates with an average molecular weight below 13000 Dalton, with large polydispersity, contain peptides, olygopeptides and a mixture of free amino-acids (glutamic acid, arginine, alanine, proline, leucine, lysine and hydroxyproline). Free amino-acids, organic nitrogen and film forming properties will protect and nurture the seeds in the long term.
After the end of the encapsulation process, we obtained a full, closed coating. Granules were of a homogeneous spherical shape and did not form agglomerates. The coatings were characterised by high durability. Stabilisation of an average mass of 1 coated seed was observed after 3 days from the coating process (saturation with moisture from the environment). As a result of the sprout growth, the shell naturally cracks in the ground (in the case of peas and broad beans) or above the ground (in the case of soybeans). It was also observed that the coating does not dissolve in contact with wet soil. Despite cracking, the collagen coating continues to adhere to the seed for a long time, which is why the seed is kept protected and supplied with nutrients ( Figure 2).
In order to determine the impact of a collagen preparation on plant growth, a comparative analysis of the average length of seedlings in relation to another reference binding liquid used in seed encapsulation processes was performed [33]. In this series of tests, seeds were coated only with a binding liquid, i.e. a collagen preparation, as well as yellow dextrin and polyvinyl alcohol (without a mineral additive). The coated seeds were sown in stable climatic conditions. After 7 days following sowing, the length of the seedlings was measured and compared in terms of the binding liquid applied. When analysing the average seedling length for collagen preparations, slightly longer seedlings (in the case of broad beans, peas and rape) or seedlings comparable (in the case of soybeans) with the control seeds were observed (Figure 3). Coating containing a collagen preparation thus provides a plant with favourable conditions at the early stage of sprout growth, which translates into an effective growth of the plant.
Legume breeding is difficult considering the scarce and complex nature of resistance in legumes in general [41]. Their cultivation is strongly hampered in the Mediterranean and Middle East farming systems by the occurrence of broomrape, causing significant yield losses [42,43]. Considering the above, the average seedling length after 29 days following sowing into the soil without access to water in socalled laboratory drought conditions was determined. Seedling length for seeds coated with a collagen preparation and addition of dolomite, as well as seeds without coating, i.e. control seeds were analysed (Figure 4). Higher seedling growth values were obtained for seeds coated with collagen hydrolysate in comparison with control seeds. That is why collagen coating rich in micronutrients necessary for seed growth improves the condition of plants in laboratory drought    After 29 days following sowing, all of the legume species tested originating from coated seeds were characterised by greater flexibility and elasticity than those originating from noncoated seeds. Therefore it may be concluded that the use of collagen hydrolysate for seed coating improves the growth and condition of legumes in difficult cultivation conditions (drought conditions). This coincides with the results provided by Gaidau et al., who confirmed that the use of collagen hydrolysate for coating wheat seeds has a significant impact on the density and growth of a plant's biomass at extreme soil pH conditions [40].
Type I collagen is an important biopolymer and has been widely used in biomaterials due to its excellent biocompatibility and biodegradable properties [44].
Inherent chemical or physical differences in the properties of collagen vary depending on the species of collagen. In order to enhance the durability of collagen-based biomaterials, a cross-linking procedure becomes necessary to improve their biological stability [45]. In order to provide proper durability and stability of a collagen preparation made from tanning waste, an optimum composition of a seed coating formulation was determined that contained latex (20%) and collagen hydrolysate (80%). Seeds coated with collagen hydrolysate and dolomite, as well as coated using the latex and dolomite formulation tested were sown into the ground in laboratory drought conditions. The seedling length was measured at regular intervals and compared in terms of the coating additives applied. Having analysed the average seedling length for seeds coated with a collagen preparation and the formulation developed, it was found that the addition of latex to the collagen preparation improves the growth of plants in the days following sowing in the case of all of the types of seeds tested, i.e. for peas, broad beans and soybeans ( Figure 5). What is more, the average length of seedlings originating from non-coated seeds was marked on diagrams on the 29 th day. In this way it was confirmed yet again that the addition of coating containing a collagen preparation significantly improves plant growth, which coincides with observations found in references [20,21,40].

Conclusions
Collagen hydrolysate contains specific amino-acids, such as proline, hydroxyproline and glutamic acid, which are nutrients and irrigation agents for plants. Therefore, increased resistance to unfavourable climatic conditions will be provided. Also, the compatibility of protein hydrolysate with many other chemical materials (dolomite, kaolin, latex) used in seed and plant treatment due to the high    0 (soyben) Seedling length, mm miscibility in different solvents, adhesivity increased absorption at leaf level, and biodegradability opens the way for toxicity reduction and plant health stimulation. The higher and better-defined quality standards in the seed and coating industry, combined with additional quality demands for enhanced seeds, will continue to improve stand establishment potential for growers. Due to interdisciplinary approaches through research and technological innovations in biosciences, biotechnology and engineering, it is possible to design eco-friendly specialty chemicals from nature's abundant renewable resources. With increasing public awareness of the potential environmental and health hazards of both agrochemicals and fertilisers, as well as advances in biotechnology to improve the performance of agricultural products, the application of collagen to seeds is likely to increase in the future.