Volume60 2018 CANADIANBIOSYSTEMSENGINEERING 3.1

Effect of Conditioning Treatment Parameters of Cellulases Solution on

Milling Characteristics of Brown Rice

Qiang Zhang1,2*, Nian Liu1,2, Ming Tu1,2 and Shuangshuang Wang1,2 1College of Engineering, Huazhong Agricultural University, Wuhan 430070, China 2Key Laboratory of Agricultural Equipment in Mid-lower Yangtze River, Ministry of Agriculture, Wuhan 430070, China Corresponding Author: Qiang Zhang (zq604@mail.hzau.edu.cn)

ABSTRACT A new enzymatic pre-treatment method for improving milling characteristics was studied by spraying cellulases solution on brown rice. The effects of enzymatic pre-treatment parameters on milling characteristics of brown rice were analyzed by response surface methodology. The optimum milling characteristic was obtained under cellulases concentration of 117.4 mg/mL, interval time of 77.2 min and temperature of 35.5 °C. Validation experiments indicated that head rice yield and milling energy consumption were respectively 4.59% higher and 32.95% lower than the values for untreated samples, 1.83% higher and 8.10% lower than those of moisture conditioning treatment. The breaking force and surface structure of the brown rice by enzymatic pre-treatment were studied to reveal the mechanism of change for milling characteristics.

CITATION Zhang, Q., N. Liu, M. Tu and S. Wang. 2018. Effect of conditioning treatment parameters of cellulases solution on milling characteristics of brown rice.

https://doi.org/10.7451/CBE.2018.60.3.1

KEYWORDS Brown rice, cellulases, head rice yield, milling energy consumption, breaking force.

MOTS CLÉS Riz brun, cellulases, rendement en grains sans bris, consommation d’énergie pour la mouture, force de bris.

RÉSUMÉ Brown rice, cellulases, head rice yield, milling energy consumption, breaking force Une nouvelle méthode de prétraitement enzymatique pour le riz a été étudiée. Elle consiste en l’aspersion d’une solution de cellulases sur le riz brun qui vise à améliorer les caractéristiques de mouture du riz. Les effets des paramètres du prétraitement enzymatique sur les caractéristiques de mouture du riz brun ont été analysés par une méthodologie de surface de réponse. Les caractéristiques optimales de mouture ont été obtenues avec une concentration de cellulases de 117,4 mg/mL, un intervalle de temps de 77,2 minutes et une température de 35,5 °C. Les expériences de validation ont indiqué que le rendement en grains sans bris avait augmenté de 4,59 % et que la consommation d’énergie pour les opérations de mouture avait été réduite de 32,95 % comparativement aux résultats obtenus avec les échantillons non traités. Le rendement et la consommation d’énergie pour les échantillons traités ont respectivement augmenté de 1,83 % et diminué de 8,10 % comparativement à ceux ayant subi un traitement d’humidité de conditionnement. La force de bris et la structure de surface du riz brun traité avec le prétraitement enzymatique ont été étudiées pour révéler le mécanisme de changement des caractéristiques de mouture.

Submitted: 2018 March 20 Revision received: 2018 June 14 Accepted: 2018 June 28 Published online: 2018 July 20

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INTRODUCTION Rice, as one of the commonly consumed cereals, has been extensively accepted by more than half of the world’s population. Freshly-harvested paddy rice with high moisture content needs to be dried to a safe moisture content (below 14.5% (w.b.)) for storing (Nimmol and Devahastin 2010; Zheng et al. 2011). Artificial drying immediately after harvesting generates virtually invisible fissures within kernels and leads to high breakage during milling (Abud-Archila et al. 2000; Dong et al. 2010), which obviously reduce the market value of polished rice to 30%-50% level of the head rice yield (HRY). Most grinding and cutting energy to remove the cortex covering such brown rice is consumed during milling, which leads to high milling energy consumption (MEC). Therefore, considering HRY and MEC as the key indicators of milling characteristics, they are significant factors for the maximum profitability of the rice industry (Marchezan 1991; Correa et al. 2007).

HRY and MEC are crucial indexes to evaluate rice quality, which depends on many factors such as variety, harvest, drying, storage, pre-treatment before milling and milling processing (Sajawan et al. 1990; Jodari and Linscombe 1996; Wiset et al. 2001; Wongpornchai et al. 2004; Aquerreta et al. 2007). Pre-treatment and milling processing obviously influence the milling characteristics of rice. Roy et al. (2008) reported that with the increase of milling degree, MEC increased and HRY decreased in the milling processing. Yadav and Jindal (2008) found that the changes of HRY and degree of milling were related to the physicochemical properties of rice. Some researchers have studied the energy consumption during individual operations of rice processing (Roy et al. 2004; Mohapatra and Bal 2007). Other researchers have also discovered the effect of the enzyme solution parameters on the HRY and MEC of brown rice. Das et al. (2008a) observed that the HRY of rice would increase when the brown rice was milled after pre-treatment by immersion in an enzyme solution. Arora et al. (2007) noted that spraying cellulases on brown rice might make the broken percentage vary from 3.23% to 4.58% as compared to 4.72% in an untreated brown rice sample. Das et al. (2008b) found that the brown rice treated by enzyme solution immersion was nutritionally better than milled rice and had better cooking qualities.

The major constituents of the bran layer are carbohydrates (26–27%) comprising mainly of cellulose and arabinoxylans (Arora et al. 2007). Cellulose enzyme has a special catalytic hydrolysis reaction to cellulose. With the application of the cellulose enzyme, the hydrolysis of the bran layer is possible in a selective manner.

However, few studies considered the effects of the conditioning treatment parameters of cellulases solution on the milling characteristics of brown rice (Arora et al. 2007; Das et al. 2008b). In this study, brown rice has been sprayed with cellulases solution under various

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concentrations until the moisture content was suitable for milling. The objective of this study was to systematically investigate the effect of cellulases concentration (CC), interval time of cellulases treatment (ITCT) and temperature of cellulases treatment (TCT) on HRY and MEC by response surface methodology to optimize these parameters for developing milling quality. This study also attempted to explain the changing mechanism of milling characteristics of brown rice using enzymatic pre-treatment. MATERIALS and METHODS Brown rice samples Paddy, variety of Early 944 variety, was provided by the experiment station of the Huazhong Agricultural University, Wuhan, China. The collected paddy rice was stored for 3 months. After hulling by a sheller (THU-35B type, Satake, Tokyo, Japan), the brown rice samples were obtained. Then the brown rice was sieved through a 16- mesh screen to remove the upper layer of paddy rice husk, light impurity, gravel and shrunken grain. Discolored grains, mildew grains, embryo free grains and immature grains were removed by manual selection. The brown rice samples were stored in relative humidity of 55%-65% and at a temperature of 20±2 °C. The moisture content of the brown rice samples was measured based on the AOAC (1995b) official method (oven dry method, 135 °C, 3 h). The initial moisture content of the brown rice was 12.5% (w.b.). Enzyme Commercially available solid cellulases in food grade (activity: filter FPA ≥ 40000 U/g) supplied by Imperial Jade Bio-technology Co., Ltd. (Yinchuan, China) were used for the enzymatic pre-treatment of the brown rice samples. Solid cellulases were dissolved in the buffer of acetic acid (AR, Hengxing Chemical Reagent Co., Ltd., Tianjin, China) - sodium acetate (AR, Bodi Chemical Co., Ltd., Tianjin, China) solution. The buffer pH was 5.0. The prepared cellulases solution was used for enzymatic pretreatment experiment. Enzymatic pre-treatment The enzyme solution (pH 5.0) was prepared in a buffer (acetic acid - sodium acetate solution) and sprayed on brown rice with uniform distribution. Then, the samples were placed into a humidity chamber (CTHI-150(A)B type, temperature fluctuation ± 0.2 °C, Shi Dukai Equipment Co., Ltd., Shanghai, China), in order to reach the appropriate milling moisture content 15.5% (w.b.) according to the CC, ITCT and TCT experimental designs (Jia et al. 2010). Considering the moisture conditioning process, once moist, spray moisture conditioning was added with enzyme solution at 1.5% (Jia et al. 2006). Then, the samples were milled by an automatic rice mill (SY95-PC�PAE5 type, Sangyong machinery industry community, Co., Ltd., Seoul, Korea). Experimental design and statistical analysis The parameters chosen for enzymatic pre-treatment were CC (x1), ITCT (x2), and TCT (x3). The factors and the

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levels (shown in Table 1) were determined according to single factor experiment results from preliminary experiments and previous research (Jia et al. 2006; Arora et al. 2007; Das et al. 2008a; Das et al. 2008b; Jia et al. 2010). Enzymatic pre-treatment experiments of brown rice were performed by a central composite rotatable design (CCRD) employing Design Expert software program (V6.0.4) of the Stat-Ease software (Stat-Ease, Inc., Minneapolis, MN, USA) with five levels for each parameter. The significant probability was at P < 0.05. Fig.1-3 indicated the variable level combinations and results of the experiments.

The second-order polynomial response surface model (Eq. (1)) was used to fit the response variables (Yk) as the following equation:

𝑌! = 𝛽!! +! 𝛽!"𝑋!

!

!!!

+! 𝛽!""𝑋!!!

!!!

+ ! 𝛽!"#𝑋!𝑋!

!

!!!!!

(1) where, βk0, βki, βkii, βkij are the constant coefficients and xi are the coded independent variables of x1 (CC), x2 (ITCT) and x3 (TCT).

Both test data were analyzed using ANOVA to perform the significance of all test conditions for the objective variables. The results of variance analysis at p < 0.05 level and p < 0.01 were retained. The reliability of the equations was determined by variance analysis, lack-of-fit tests, and R2 analysis. The non-significant F values confirm the validity of the models for the lack of fit (P > 0.05), and the R2 is defined as the ratio of explained variation to the total variation. To obtain the optimal process parameters of the enzymatic pre-treatment, a numerical optimization module in the software was employed. The numerical optimization process is shown in Part 3.4. Head rice yield (HRY) The degree of milling (DOM) (90%) was determined by gravimetric methods with Eqn. (2). D!" = !1 − !!

!!!×100 (2)

where, mM (g) and mB (g) are the mass of milled rice and brown rice, respectively (Mohapatra and Bal 2007). After milling at setting DOM, HRY was defined as the mass of head rice to the mass of raw rice samples taken for milling, as shown in Eqn. (3).

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y! =!!!!×100 (3)

where, y1 (%) is HRY, mH (g) and mS (g) are the masses of head rice and raw rice samples, respectively. Milling energy consumption (MEC) The pre-treated brown rice samples were milled by an automatic rice mill. Then the MEC was calculated with Eqn. (4), in which the milling time was recorded by a timer (Fisher Scientific, Control Company, China) during the milling process. y! =

!∙!!!×100 (4)

where, ω (kJ/s) is the power of the automatic rice processor (400 kJ/s), t (s) is the milling time, and mS (kg) is the mass of rice sample. Breaking force (BF) Enzymatic pre-treatment process may cause partial degradation of the brown rice cortex, which may be leading to a reduction in mechanical strength. During the milling process, the grinding/cutting force for brown rice was revealed by BF in the compression test. BF is defined as the force that occurs when the macrostructure of the material is damaged, which is the first peak on the force-deformation curve during the extrusion deformation process. After enzymatic pre-treatment, BF of brown rice was determined by a Stable Micro System TA-XT2 texture analyzer (Texture Technologies Co., Surrey, UK) with a 2.5 mm cylindrical probe. The settings for pre-test, test and post-test speeds were 1.0, 0.2, 0.5 mm/s, respectively, and a 40% deformation ratio. A force vs time curve was recorded and analyzed by the software of the TA-XT2 texture analyzer, which reflected the mechanical strength of the material. Scanning electron microscopy studies Scanning electron microscopy (SEM) study was conducted after optimization of enzymatic pre-treatment, using an SEM (S-3000N, Hitachi Ltd., Japan) coupled with E-1010 ion sputtering instrument (Hitachi Ltd., Japan). In this study, brown rice samples were prepared in the optimal enzymatic pre-treatment condition. The same amount of moisture content was given to the brown rice at each time in moisture conditioning treatment to obtain the same optimum moisture content for the further milling process. Then the microstructure changes of brown rice were observed under the optimal enzymatic pre-treatment condition and the moisture conditioning treatment condition. The added moisture amounts of the optimal enzymatic pre-treatment and the moisture conditioning pretreatment were both 1.5% (Jia et al. 2006). After humidification, the conditioning pretreatment was carried out at the same temperature and the same interval time until the moisture content of brown rice reached the appropriate milling moisture content of 15.5%. The added moisture amount was defined as the increased percentage of brown rice moisture content.

Table 1. Factors and levels of experiment factors Code Value x1 (mg/mL) x2 (min) x3 (°C)

1.682 142.04 110.5 43.4 1 125.00 90.0 40.0 0 100.00 60.0 35.0 -1 75.00 30.0 30.0

-1.682 57.96 9.5 26.6 Note: x1 represents cellulases concentration; x2 represents the interval time of cellulases treatment; x3 represents the temperature of cellulases treatment.

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RESULTS and DISCUSSION The variable level combinations and results of the enzymatic pre-treatment experiments are presented in Fig.1-3. The ANOVA results showed that the equations were highly significant (P < 0.05) for all the responses. The low F values for the lack of fit and the high R2 values indicated that a large proportion of the variability was explained by the variables tested. Thus, during the conditioning treatment by cellulases solution, the behaviors of input variables on the head rice yield, milling energy consumption and breaking force of brown rice were reasonably described by Eqn. (5)- Eqn. (7), respectively.

(5)

(6)

(7)

Effect of variables on head rice yield The HRY values of the brown rice varied from 61.78% to 78.76% (Fig. 1). The regression of HRY model, R2 of 0.9248 and non-significant lack of fit of 0.1379, had high credibility. The results indicated that the effects of monomial CC, ITCT and TCT, as well as the quadratic terms of ITCT and TCT on the HRY were extremely significant at P < 0.01. The interaction of CC and TCT was significant at P < 0.05. The main factor affecting the HRY was TCT among three parameters (factor contribution ratio of 2.5361).

Fig. 1a-c presents the variation of HRY with CC, ITCT and TCT. First, the HRY tardily increased with CC nearly up to 115 mg/mL, which was attributed to the raised activity of enzyme and degree of cortex degradation. Then it nearly reached a constant (Fig. 1a). The enzymatic pre-treatment softened the bran layer and was easily removed in the rice mill. The observed result from Thakur and Gupta (2006) showed that the bran layer was a significant barrier in the water absorption process of brown rice. The degradation of the bran layer made water easily penetrate into the brown rice and decreased mechanical strength, therefore HRY increased. However, no significant effect was found on HRY when CC was increased beyond 125 mg/mL, with the reason that the increase of CC might keep the active sites of the enzyme at saturated level with substrate, leading to lower activity (Sarker et al. 1998). The rate of enzymatic hydrolysis reached the maximum and the degree of enzymatic hydrolysis kept constant. Then HRY tended towards stability.

Fig. 1. (a) Effect of cellulases concentration (CC) and interval time of cellulases treatment (ITCT) on head rice yield (HRY) (TCT at 35 °C). (b) Effect of cellulases concentration (CC) and temperature of cellulases treatment (TCT) on head rice yield (HRY) (ITCT at 60 min). (c) Effect of interval time of cellulases treatment (ITCT) and temperature of cellulases treatment (TCT) on head rice yield (HRY) (CC at 100 mg/mL).

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Under the ITCT of 60 min, the interaction of CC and TCT on the HRY of brown rice is shown in Fig. 1b. The values of HRY increased with the increase of CC, and then levelled off. TCT higher than 35 °C inhibited the increase of HRY. The high TCT accelerated moisture penetration, which resulted in the generation of many stress cracks within rice kernels and reduced the hydrolysis time. Therefore, stress cracks within kernels and the shortened ITCT caused the reduction of HRY.

As seen in Fig. 1c, under the high level of TCT, HRY increased up to the highest value and then gradually decreased with the ITCT proceeding. When ITCT was short, similarly to excessive amount of moisture added, moisture quickly penetrated into the kernel. Stress cracks caused by too large moisture gradient reduced HRY (Xiao and Ma 2007). The time of enzymatic hydrolysis was shortened. The degradation degree of the bran layer was low, and the hardness was high, so the rice milling needed larger grinding and cutting forces. Thus, HRY decreased. With the prolonging of ITCT, the rate of moisture penetration decreased, causing the reduction of moisture gradient and the prolonging of the time of enzymatic hydrolysis, respectively. The degradation degree increased, and the mechanical strength decreased, so HRY increased gradually. With the prolonging of ITCT proceeding, enzymolysis function and moisture penetration resulted in a very high degree of degradation for the brown rice layers, which reduced HRY. Effect of variables on milling energy consumption Fig. 2 indicates that the MEC values of the brown rice varied from 295 kJ/kg to 312 kJ/kg. The regression equation of MEC had a high reliability with R2 of 0.9540 and lack of fit of 0.3388. The ANOVA results indicated that MEC was extremely significant at the 1% level for CC and ITCT on linear terms as well as CC, ITCT and TCT on quadratic terms. In addition, the TCT on linear term was significant at P < 0.05. However, interaction term was not significant at the 5% level. The CC of factor contribution ratio 2.6786 was the main factor affecting the MEC among all parameters.

Fig. 2a-c describes the variations in MEC of brown rice with different combinations of the process parameters. As shown in Fig. 2b, the increase of CC caused the decrease of MEC of brown rice until it reached 115 mg/mL, and then subsequently tended to be constant. However, the MEC decreased with the TCT up to 35 °C, and then increased. The reduction of MEC was attributed to the decrease of mechanical strength of kernels with water penetrating into the brown rice and the strengthening of enzymolysis function. The increase of MEC was attributed to stress cracks caused by accelerated moisture penetration and relatively weakened enzymolysis function. The MEC slowly declined with the ITCT up to 100 min, and then slightly increased until the end. With the prolonging of ITCT proceeding, enzymolysis function and moisture penetration resulted in a very high degree of degradation for layer of brown rice, so the MEC increased.

Fig. 2. (a) Effect of cellulases concentration (CC) and interval time of cellulases treatment (ITCT) on milling energy consumption (MEC) (TCT at 35 °C). (b) Effect of cellulases concentration (CC) and temperature of cellulases treatment (TCT) on milling energy consumption (MEC) (ITCT at 60 min). (c) Effect of interval time of cellulases treatment (ITCT) and temperature of cellulases treatment (TCT) on milling energy consumption (MEC) (CC at 100 mg/mL).

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It was explained that high CC, long ITCT and feasible TCT had a significantly positive impact on the milling characteristics of brown rice. However, considering the economic cost of enzymes as the pre-treatment material for the brown rice (Passos et al. 2009), a suitable CC of less than 120 mg/mL was selected under the appropriate TCT and ITCT level. Inverse relationships between HRY and MEC were found from the above-mentioned results. Bran layer was softened to be easily removed by enzymatic pre-treatment in a mechanical polisher (Arora et al. 2007). Further, shortening milling time was helpful in improving the milling quality of brown rice. HRY, as a milling characteristic, was improved. MEC was saved through the enzymatic pre-treatment process. Effect of variables on breaking force The BF regression equation of R2 of 0.9419 and lack of fit of 0.0878 had high reliability. The results showed that the changes of the BF during the compression test were affected by CC and ITCT on linear terms, CC and TCT on the quadratic terms, as well as interaction term (CC and TCT) for BF (significant at 1%). The CC of factor contribution ratio 2.4148 was the main factor affecting the BF among all parameters.

The variations in BF of brown rice with different combinations of parameters are shown in Fig. 3a-c. The increase of CC caused the decrease of BF of brown rice, and the BF values subsequently tended to reach a constant level, as shown in Fig. 3a. The degree of the bran layer softness was higher because of the increased degradation caused by the increase of CC. The BF of brown rice decreased to a certain level and then increased with the TCT increasing as shown in Fig. 3b and Fig. 3c, which indicated the slight reduction of BF with ITCT proceeding. The interaction of CC and TCT affected the BF of brown rice under the ITCT of 60 min (Fig. 3b). It was found that BF had a negative relation with HRY, whose variation was similar to that of MEC. Different results were obtained by the experiment. It could be explained by the fact that BF of brown rice decreased with the increase of the brown rice moisture content, which improved the toughness of the brown rice. In the rice milling process, broken rice and crackle rice were decreased, so HRY increased. The compression force subjected to the milling process caused the rice grain rupture (Correa et al. 2007), and the degradation of cellulose in the cortex might have caused the decrease of BF. In this way, HRY increased and MEC reduced gradually. Owing to a positive relationship between MEC and BF of brown rice, the appropriate mechanical strength helped to increase HRY and cut down MEC in rice milling. Determination and validation of the optimal parameters Based on equations of HRY and MEC, as well as the above-mentioned optimization goals, each parameter and response was chosen to optimize the conditions of the enzymatic pre-treatment. The upper ⁄ lower bounds, different weights and importance for factors and responses

Fig. 3. (a) Effect of cellulases concentration (CC) and interval time of cellulases treatment (ITCT) on breaking force (BF) (TCT at 35 °C). (b) Effect of cellulases concentration (CC) and temperature of cellulases treatment (TCT) on breaking force (BF) (ITCT at 60 min). (c) Effect of interval time of cellulases treatment (ITCT) and temperature of cellulases treatment (TCT) on breaking force (BF) (CC at 100 mg/mL).

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of enzymatic pre-treatment were shown in Table 2. The optimal conditions were generated by running the solutions menu of the numerical optimization program in the Design Expert software. The optimum region in vicinity was as follows: CC of 117.4 mg/mL, ITCT of 77.2 min and TCT of 35.5 °C. The optimum results were HRY of 78.85% and MEC of 295.49 kJ/kg.

Validation experiments were performed in triplicate employing the same source of brown rice sample to test the optimum conditions of enzymatic pre-treatment. The results showed that the HRY of 78.38% obtained by enzymatic pre-treatment was 4.59% higher than for the un-treatment process and 1.83% higher than for the moisture conditioning treatment in the optimum condition of added moisture in the amount of 1.5%, interval time of 77.2 min, temperature of 35.5 °C and milling moisture amount of 15.5% (w.b.) (Jia et al. 2006). A MEC value of 295 kJ/kg by enzymatic pre-treatment was 32.95% lower than for the un-treated samples, and 8.10% lower than for the moisture conditioning treatment. These experimental results agreed well with the prediction values. Observation of brown rice degradation through scanning electron microscopy A comparison of the microstructure of brown rice cortex was conducted to reveal the mechanism of the enzymatic pre-treatment. The SEM studies were conducted at a magnification of 1 000x. Fig. 4a and 4b shows the changes outside the structures of the cortex by different pre-treatments, which indicated the degradation by cellulases. Fig. 4a presents a much complete and smooth surface structure of brown rice after moisture conditioning treatment under the mentioned optimum condition. The enzymatic pre-treatment of rice samples in the optimum condition of CC of 117.4 mg/mL, ITCT of 77.2 min and TCT of 35.5 °C, presented in Fig. 4b, shows a surface with numerous pits and large cracks. Das et al. (2008a) reported that in case of enzymatic treated brown rice, a clear thinning of the fibre in bran layer was observed. The difference in microstructure intuitively explained the reason why the brown rice treated by enzymatic pre-treatment got higher HRY and lower MEC than that by moisture conditioning treatment. The pits and cracks structure generated in the optimum conditions of enzymatic pre-treatment made the cortex easily removed in a short milling time. Therefore, the degree of injury to the brown rice was reduced, which contributed to high HRY.

Fig. 4. Scanning electron microscopy (SEM) micrographs of cortex of brown rice at (a) moisture conditioning treatment (once moisture adding amount at 1.5%, time at 77.2 min, temperature at 35.5 °C, milling moisture amount at 15.5% (w.b.)) and (b) enzymatic pre-treatment (cellulases concentration at 117.4 mg/mL, interval time of cellulases treatment at 77.2 min, temperature of cellulases treatment at 35.5 °C). Magnification is 2000 times. Bar is 50.0 µm.

Table 2. Optimization criteria for different factors and responses of enzymatic pre-treatment. Name Goal Lower Limit Upper Limit Lower Weight Upper Weight Importance

x1 is in range 75.00 mg/mL 125.00 mg/mL 1 1 3 x2 is in range 30.0 min 90.0 min 1 1 3 x3 is in range 30.0 °C 40.0 °C 1 1 3 y1 maximize 61.78% 100.00% 1 1 5 y2 minimize 0 kJ/kg 312 kJ/kg 1 1 5

Note: x1 represents cellulases concentration; x2 represents the interval time of cellulases treatment; x3 represents the temperature of cellulases treatment; y1 represents head rice yield; y2 represents the milling energy consumption.

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CONCLUSIONS The study of the effect of enzymatic pre-treatment for brown rice on milling characteristics was undertaken with the aim of maximizing HRY and minimizing MEC. The experimental values in terms of HRY of 78.85% and MEC of 295.49 kJ/kg inferred that enzymatic pre-treatment had an obvious advantage for improving the milling characteristics, as compared with moisture conditioning treatment and un-treated samples. The BF determined by a texture analyzer showed that the enzymatic pre-treatment method reduced the mechanical strength of brown rice, and microstructure changes observed by SEM indicated that the surface was degraded by cellulases. Furthermore, the overall process of enzymatic degradation was evidenced from the microscopy analysis establishing the relationship between the degradation amounts of cellulose and milling characteristics. Hence, it is possible for the rice industry to scale up enzymatic pre-treatment process with the corresponding equipment for maximum profitability under optimum conditions. ACKNOWLEDGMENTS The authors express their acknowledgment to the Fundamental Research Funds for the Central Universities (2662015QD043), Opening Subject for Key Laboratory of Modern Agriculture, Tarim University (TDNG20160102) and Industrial Science and Technology Project of Xinjiang Production and Construction Corps (2015AB039) for financial support and all of the persons who assisted in this writing. REFERENCES Abud-Archila, M., F. Courtois, C. Bonazzi, and J.J.

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