Simultaneous effect of vacuum and ultrasound assisted enzymatic extraction on the recovery of plant protein and bioactive compounds from sesame bran

Journal of Food Composition and Analysis 87 (2020) 103424

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Original Research Article

Simultaneous effect of vacuum and ultrasound assisted enzymatic extraction on the recovery of plant protein and bioactive compounds from sesame bran

T

Ahmet Görgüç, Pınar Özer, Fatih Mehmet Yılmaz* Aydın Adnan Menderes University, Engineering Faculty, Food Engineering Department, 09010, Efeler, Aydın, Turkey

ARTICLE INFO

ABSTRACT

Keywords: Vacuum Extraction Ultrasound Food by-product Plant protein Sesame bran

The latest studies on the extraction of bio-components from plant materials have elaborated on novel approaches to increase yield. This study evaluated the effect of vacuum operation parameters on the recovery of protein and antioxidant compounds from sesame bran. In this context, vacuum-ultrasound assisted extraction (VU) and vacuum-ultrasound assisted enzymatic extraction (VUE) methods were carried out, and the vacuum parameters were investigated using central composite design by response surface methodology (RSM). The independent parameters were found effective on protein yield, total phenolic content and antioxidant capacity values. Optimum conditions for VU were 21 min vacuum time, 72 min restoration time and 539 mmHg vacuum pressure; and for VUE were 1.82 AU/100 g enzyme concentration, 8 min vacuum time, 68 min restoration time and 238 mmHg vacuum pressure. The protein yields increased by 31.0 and 41.6 % with VU and VUE, respectively compared to standard alkaline extraction. SEM images revealed deteriorative effects of applied extraction methods. SDS-PAGE analysis was conducted to examine detailed protein fractionation and alcalase assisted extraction revealed small protein sub-units (< 18 kDa) which implies the presence of potential bioactive peptides. Additionally, LC/Q-TOF/MS analysis was performed and organic compounds in sesame bran extracts were identified.

1. Introduction Extraction is the first and foremost important step in the acquisition of target compounds from various materials. There are a number of studies regarding the extraction of macro/micromolecules, especially from plant materials by different techniques. Among them, ultrasound technology is a widely used method for the extraction procedures (Kentish and Ashokkumar, 2011). Ultrasonic waves improve the penetration rate of bulk solvent by cellular disruption mechanism (Show et al., 2007). In the extraction process, ultrasound waves pass through a liquid medium and create very small bubbles and cavitation. When bubbles explode, they create high temperature (∼5000 K) and pressure (∼1000 MPa). As a result of local sudden changes in temperature and pressure, the cellular structure deteriorates and the mass transfer rate of target compounds to the extraction solvent increases (Show et al., 2007). Enzyme assisted extraction is another common approach for the recovery of high value-added compounds. In particular, proteases provide an increase in the yield by releasing the protein bound to the polysaccharide matrix (Goula et al., 2018). Some advantages of enzymatic extraction over other techniques are processing at low temperatures for the protection of heat-sensitive compounds, and non-

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generation of hazardous wastes (Hanmoungjai et al., 2001). In the past 10 years, vacuum technology has been adapted to various food processes including drying, dehydration, frying, cooking, and cooling. Vacuum has also been used for the acceleration of the osmotic dehydration processes and for enrichment of foods by altering their pore structures (Yılmaz and Ersus Bilek, 2017). However, the sole or combined usage of vacuum has not been evaluated in the extraction procedures to date. When vacuum is applied, capillaries expand by pressure difference (Chiralt and Fito, 2003). In the restoration step at which the vacuum is switched off, the capillaries shrink to an even greater extent than before the start of the process (RadziejewskaKubzdela et al., 2014). In this stage, an intensive inflow of extraction solvent to the inside of the capillaries is observed due to the decompression and sudden increase in pressure of the capillaries (Derossi et al., 2013). This case may also facilitate the release of compounds to be extracted within the cells. The increasing demand for food proteins has led to the development of novel protein sources such as insects, fungi and algae as well as emerging wastes from food processing. Wastes of oily seeds and cereals are commonly used sources for the recovery of proteins. In most cases, these materials are reported to have 15–50 % protein after defatting or

Corresponding author. E-mail address: [email protected] (F.M. Yılmaz).

https://doi.org/10.1016/j.jfca.2020.103424 Received 1 October 2019; Received in revised form 19 December 2019; Accepted 18 January 2020 Available online 20 January 2020 0889-1575/ © 2020 Elsevier Inc. All rights reserved.

Journal of Food Composition and Analysis 87 (2020) 103424

A. Görgüç, et al.

de-hulling processes (Görgüç et al., 2019a; Pojić et al., 2018). Today, the search for alternative food based protein sources has increased, and plant proteins stand out amongst others by providing more sustainability and low cost (Yavuz and Özçelik, 2016). Plant proteins have always been important in human daily diet and it is reported that they do not contain cholesterol and trans-fatty acids, and their saturated fat ratio is low compared to animal proteins (Mangels, 2018). Besides, epidemiological studies suggest that the high animal based protein intake increases the risk of type 2 diabetes, cardiovascular diseases and colorectal cancer, whereas plant proteins provide significant protective effect against such diseases (Mattila et al., 2018). Sesame bran is an unevaluated food waste of sesame oil, tahini and roasted sesame industry, and emerges as a potential protein source with 15–20 % protein content (Görgüç et al., 2019b). Sesame contains sulphur containing amino acids unlike many of the other plant protein sources (Raza et al., 2018). The presence of flavonoids and flavonols as phenolic compounds in the sesame was also reported (Şahin et al., 2018). Annually, 6.2 million tons of sesame is processed in the world (FAOSTAT, 2018) and bran which accounts for 14–17 % of the whole sesame is separated from process as waste. From this point of view, the sesame bran could be regarded as a promising plant based protein source. Based on the given information, the objective of this study was to investigate the effect of vacuum processing parameters on the recovery of protein and bioactive compounds from sesame bran. In particular, ultrasound and ultrasound assisted enzymatic extraction methods were carried out under vacuum conditions and effectiveness of independent variables i.e. vacuum time, restoration time, vacuum pressure and enzyme concentration were investigated using response surface methodology (RSM). Additionally, SDS-PAGE, SEM and LC/Q-TOF/MS analyses were carried out at optimum extraction conditions.

pressure (100–650 mmHg) were selected as independent variables. Preliminary data were considered for the selected ranges. Operations were carried out using a vacuum - ultrasonic equipment (Ermaksan, ULT 50-S, Turkey) with an integrated temperature controller system. According to the preliminary results, VU procedures were conducted at constant ultrasound power (550 W) and temperature (54 °C). The central composite design (CCD) consisted of 18 experimental runs with four centre points, and obtained results were evaluated using response surface methodology (RSM). The sesame bran and dH2O were mixed at a ratio of 1:10 (w/v) and extractions were performed according to the experimental design at different vacuum time, restoration time and vacuum pressures. Afterwards, the pH of the mixture was adjusted to 9.5 and the mixture was placed in a rotating water bath at 45 °C and 50 rpm for 30 min. Then, the mixture was centrifuged at 4000g for 30 min. Finally, the supernatant fraction was collected and stored at -20 °C prior to analyses. 2.2.3. Ultrasound assisted enzymatic extraction under vacuum (VUE) For the combined vacuum ultrasound assisted enzymatic extraction, the effect of enzyme (alcalase) concentration together with three factors belonging to VU i.e. vacuum time, restoration time and vacuum pressure was evaluated. The experimental plan of VUE consisted of 28 runs including four centre points and the experimental ranges were as follows: Enzyme concentration (0.12–2.40 AU/100 g), vacuum time (1–30 min), restoration time after vacuum application (1–90 min), and vacuum pressure (100–650 mmHg). Operations were carried out at 550 W ultrasound power and at 43 °C, based on preliminary results. In the VUE procedures, sesame bran and dH2O were firstly mixed at ratio of 1:10 (w/v). The pH value of the mixture was then adjusted to 9.8 with 2 N NaOH by considering optimal enzyme activity. After addition of enzyme, extractions were performed according to experimental design. Then, the pH value of the mixture was adjusted to 9.5 and the mixture was placed in rotating water bath at 45 °C and 50 rpm for 30 min. Finally, the mixture was centrifuged at 4000g for 30 min, and the supernatant fraction was stored at -20 °C prior to analyses.

2. Materials and methods 2.1. Materials and chemicals Sesame (Sesamum indicum L.) bran was kindly provided from a roasted sesame and tahini manufacturing company (Tunas & Çelikler Food Ltd. Co., Turkey). During the multiple-stage processing, approximately 15 % (by weight) of whole sesame is separated as bran using full automated equipment. Sesame bran was collected from this dry-line process and transferred to the laboratory. The bran was dried at 60 °C by a tray dryer at 1.4 m/s air flow rate and < 10 % relative humidity until the moisture content of the bran reached below 5 %. The sesame bran was then sieved to 212 μm particle size to eliminate residual sesame seed particles and stored in airtight polyethylene bags at 4 °C prior to extraction procedures. Alcalase 2.4 L was kindly supplied from Novozymes (Denmark). All other chemicals used were of analytical grade and purchased from Sigma-Aldrich (United States).

2.3. Analyses 2.3.1. Protein yield Total protein content was determined according to Kjeldahl procedure (AOAC, 1998) using a semi-automatic Kjeldahl system (Velp Scientifica, UDK 132, Italy). The protein yields were calculated by Eq. (1) for alkaline extraction and VU, and by Eq. (2) for VUE.

2.2. Extraction methods

Protein yield (%) =

(E )(P , extract ) x 100 (B )(P , bran)

(1)

Protein yield (%) =

(E )(P , extract ) (A)(P , enzyme ) x 100 (B )(P , bran)

(2)

Here, E: Amount of extract (mL), P,extract: Protein content of extract (%), B: Amount of sesame bran (g), P,bran: Protein content of sesame bran (%), A: Amount of alcalase enzyme (mL) and P, enzyme: Protein content of enzyme (%).

2.2.1. Alkaline extraction Alkaline extraction was performed as the control method on the extraction of plant protein from sesame bran. Firstly, sesame bran was mixed with deionized water (1:10, w/v). Then, the pH value of the mixture was adjusted to 9.5 by 2 N NaOH solution and the mixture was placed in a rotating water bath (Daihan Scientific, Maxturdy-30, Korea) for 2 h at 45 °C and at 50 rpm stirring rate. The supernatant fraction was collected after centrifugation at 4000g (30 min), and stored at −20 °C until analyses.

2.3.2. Total phenolic content Total phenolic content (TPC) was determined according to FolinCiocalteu method as described by Yılmaz et al. (2015). Briefly, 30 μL extract, 2.37 mL dH2O and 150 μL Folin-Ciocalteu reagent were mixed and kept in the dark for 8 min. Then, 450 μL of saturated Na2CO3 was added and the mixture was stored at 40 °C for 30 min. Subsequently, the absorbance value was measured by a UV–VIS spectrophotometer (Shimadzu, V-1800, Japan) at 750 nm. The results were expressed as mg gallic acid equivalent per gram (mg GAE/g).

2.2.2. Ultrasound assisted extraction under vacuum (VU) In the vacuum ultrasound assisted extraction of plant protein and antioxidant compounds from sesame bran, vacuum time (1–30 min), restoration time after vacuum application (1–90 min), and vacuum 2

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Table 1 RSM experimental design and responses belonging to vacuum - ultrasound assisted extraction. Independent variables

Dependent variables*

Vacuum time (min)

Restoration time (min)

Vacuum pressure (mmHg)

Protein yield (%)

TPC (mg GAE/g)

ACDPPH (μmol TE/g)

ACABTS (μmol TE/g)

16 24 7 7 16 16 7 16 16 16 30 16 24 1 7 24 24 16

90 19 72 72 46 46 19 46 46 1 46 46 72 46 19 72 19 46

375 211 211 539 375 100 211 375 375 375 375 375 211 375 539 539 539 650

52.7 ± 0.4 53.7 ± 0.6 51.6 ± 0.4 57.0 ± 0.7 49.4 ± 0.6 53.7 ± 0.4 62.3 ± 0.8 49.4 ± 0.4 48.4 ± 0.9 55.9 ± 0.7 49.4 ± 0.2 48.1 ± 0.2 48.4 ± 0.7 55.3 ± 0.4 55.9 ± 0.9 55.9 ± 0.8 47.3 ± 0.4 54.8 ± 0.7

5.47 ± 0.04 5.89 ± 0.16 4.53 ± 0.01 5.98 ± 0.02 5.21 ± 0.10 5.17 ± 0.02 3.94 ± 0.03 5.23 ± 0.05 5.27 ± 0.10 4.37 ± 0.01 6.12 ± 0.07 5.24 ± 0.04 5.90 ± 0.09 4.47 ± 0.03 4.54 ± 0.04 6.24 ± 0.07 5.26 ± 0.19 6.08 ± 0.11

1.99 ± 0.08 1.64 ± 0.03 1.88 ± 0.06 1.64 ± 0.07 1.80 ± 0.08 2.05 ± 0.03 1.75 ± 0.08 1.78 ± 0.01 1.83 ± 0.10 1.73 ± 0.02 1.41 ± 0.02 1.82 ± 0.04 2.06 ± 0.14 1.47 ± 0.11 1.68 ± 0.05 1.81 ± 0.03 1.50 ± 0.03 1.84 ± 0.16

41.6 ± 0.69 40.5 ± 1.44 38.6 ± 0.70 40.3 ± 1.11 40.7 ± 0.66 38.8 ± 1.61 35.3 ± 1.00 41.4 ± 0.63 40.8 ± 0.78 37.3 ± 1.05 42.6 ± 0.56 41.2 ± 1.17 42.3 ± 0.15 37.6 ± 1.34 35.3 ± 0.84 41.5 ± 0.25 38.8 ± 1.47 37.4 ± 1.16

* The results were expressed as “Mean ± Standard deviation”.

acetonitrile: 0 min (5 % B), 8th min (15 % B), 10th min (20 % B), 13th min (25 % B), 18th min (30 % B), 20th min (45 % B), 24th min (60 % B), 27th min (80 % B), 30th min (90 % B) and 32nd min (5 % B). The column temperature was kept constant at 35 °C and 3 μL of sample was loaded into the column. Mass spectrometry was conducted using a Q-TOF/MS system (Agilent, 6550 iFunnel, United States) with electro spray ionization under the following conditions: Desiccant gas flow of 14.0 L/ min, nebuliser gas pressure of 35 psi, drying gas temperature of 290 °C, sheath gas temperature of 400 °C and sheath gas flow of 12 L/min. Mass spectra were recorded in both positive and negative ionization modes in a mass range of 50–1700 m/z.

2.3.3. Antioxidant capacity Antioxidant capacity (AC) values were determined by both DPPH and ABTS methods and the results were expressed in terms of micromole trolox equivalent per gram (μmol TE/g). For the DPPH assay, 2.9 mL of 0.1 mM DPPH solution (dissolved in EtOH) and 0.1 mL extract were mixed and stored at dark for 30 min. The absorbance value was then measured at a wavelength of 517 nm (Grajeda-Iglesias et al., 2016). Antioxidant capacity by ABTS method was determined according to Sarkis et al. (2014). Firstly, 7 mM ABTS radical solution containing 2.45 mM K2S2O8 was diluted with PBS (phosphate buffer saline, pH 7.4) until the absorbance value reaches 0.7 ± 0.02 at 734 nm. Then, 2.98 mL of this solution was mixed with 20 μL extract and the absorbance values were recorded after 0th and 6th min at 734 nm.

2.3.7. Statistical analysis Extraction procedures were duplicated and all analyses were carried out in triplicate. Obtained data were subjected to analysis of variance (ANOVA) using SPSS v15.0 software (IBM, United States) at P < 0.05, and the optimizations were performed by response surface methodology (RSM) using Design Expert v11.0 (Stat-Ease, United States). The validity of models was assessed by the determination coefficient (R2), lack of fit and F-values.

2.3.4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) Protein fractions of the extracts were determined according to Laemmli (1970). Extracts were also subjected to denaturation at 95 °C for 5 min and analysed in both denatured and native forms. The extracts were mixed with sample buffer (containing 95 % Laemmli buffer and 5 % β-mercaptoethanol) at a ratio of 1:1 (v/v). Subsequently, 25 μL of this mixture was loaded into the electrophoresis system (Bio-Rad, PowerPac Basic, United States) and the system was run at 90 V for 1 h. Then, the running gel was stained with Coomassie brilliant blue and further destained with HAc:MeOH:H2O (1:4:5, v/v/v). Finally, the gels were photographed using G:Box Chemi-XRQ software (Syngene, India).

3. Results and discussion 3.1. Effect of vacuum parameters on the extraction efficiency The effectiveness of vacuum was examined with its processing parameters which are vacuum time, restoration time and vacuum pressure. In each treatment, independent variables of vacuum parameters were tested using two different experimental designs which are vacuum - ultrasound assisted extraction (VU) and vacuum - ultrasound assisted enzymatic extraction (VUE). According to the created design of VU, 18 experiments were conducted and obtained results were presented in Table 1. The results were in the range of 47.3–62.3 % for protein yield, 3.94–6.24 mg GAE/g for TPC, 1.41–2.06 μmol TE/g for ACDPPH and 35.3–42.6 μmol TE/g for ACABTS. ANOVA results, quadratic model coefficients and statistical significance levels of the model parameters were presented in Table 2. All of the independent variables had significant effects on the responses with varying confidence intervals except for vacuum pressure on protein yield and vacuum time on ACDPPH value, but second order effect of these variables affected the mentioned responses significantly (P < 0.001). The coefficient of determination (R2) values were 0.981, 0.997,

2.3.5. Scanning electron microscopy (SEM) The cellular microstructure of sesame brans was examined by a scanning electron microscope (Zeiss Gemini Sigma, 300 V P, Germany). The samples were coated with a thin layer of gold prior to imaging and the images were obtained at 90 V with 3000X magnification level. 2.3.6. Quadrupole-time of flight liquid chromatography/mass spectrometry (Q-TOF LC/MS) Q-TOF LC/MS analysis was performed according to the method described by Sengel et al. (2018). Chromatographic separation was conducted using an HPLC system (Agilent, 1260 Infinity, United States) with a C18 column (3.0 × 50 mm, 2.7 μm; Agilent, Poroshell 120, United States). The linear gradient program was used with two mobile phase solvents where A is 0.1 % formic acid in water, and B is 3

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Table 2 Quadratic model coefficients and ANOVA results belonging to vacuum - ultrasound assisted extraction. Model parameters

Intercept Linear Vacuum time, A Restoration time, B Vacuum pressure, C Interaction AxB AxC BxC Second order A2 B2 C2 R2 Adjusted R2 p-value F-value Lack of fit

Vacuum ultrasound assisted extraction Coefficient

Protein yield

TPC

ACDPPH

ACABTS

β0

92.89***

2.88***

1.79***

25.53***

β1 β2 β3

−1.18*** −0.67*** −0.09

0.16*** 0.02*** −2 × 10-3***

0.04 −1.3 × 10-3*** −1.4 × 10-3***

0.56*** 0.13*** 0.03*

β12 β13 β23

0.01*** 2 × 10−4 7 × 10−4***

−6 × 10-4*** −2 × 10-4*** 5 × 10−5***

4 × 10−4*** −7.1 × 10-6 −8.1 × 10-6*

−0.002** −4 × 10-4** 1 × 10−4*

β11 β22 β33

0.02*** 0.003*** 7 × 10−5*** 0.9810 0.9596 < 0.001 45.82 0.3509

3 × 10−4 −2 × 10-4*** 5.14 × 10−6*** 0.9965 0.9926 < 0.001 252.69 0.0556

−0.002*** 3 × 10−5 1.8 × 10−6*** 0.9856 0.9693 < 0.001 60.73 0.2171

−0.004* −8 × 10-4*** −4 × 10-5*** 0.9866 0.9715 < 0.001 65.48 0.3277

Significance levels: *P < 0.05; **P < 0.01; ***P < 0.001. Model equation: Y = β0 + β1A + β2B + β3C + β12(AxB) + β13(AxC) + β23(BxC) + β11A2 + β22B2 + β33C2.

0.986 and 0.987 for protein yield, TPC, ACDPPH and ACABTS values, respectively. In addition, the lack of fit values which imply adequacy of the fitted models were found to be non-significant (P > 0.05). The mutual effect of vacuum pressure and restoration time accelerated the release of protein and phenolic compounds from sesame bran as illustrated in Fig. 1A for protein yield and Fig. 1B for TPC. It was noteworthy that the increase in vacuum pressure under short restoration times did not increase the responses, i.e. protein yield, TPC and AC values, but the extraction yields of protein and phenolics were highest at elevated restoration times in VU. Depending on the degree of vacuum, capillaries undergo deformation and in the restoration period, extraction solvent replace with the released gas in the capillaries (Erihemu et al., 2015) which may facilitate extraction. The mass transfer rates of immobilized compounds positioned at the remotest part of cellular structure thus would be increased with the effect of vacuum. In addition, simultaneous increase of vacuum time and restoration time to some extents yielded more antioxidant capacity. Fig. 1C represents the interactive effect of vacuum time and restoration time for ACABTS response in VU. In the ultrasound assisted extraction, ultrasonic waves cause softening of plant tissues. Due to the weakening of the cell wall integrity, especially bound phenolic compounds such as in the phenol-protein or phenol-polysaccharide complexes are being hydrolysed and solubility of phenolics in the solvent increases (Tabaraki and Nateghi, 2011). Ultrasound assisted extraction was reported to increase the recovery rate of phenolics and flavonoids compared to conventional solid-liquid extraction (Zeković et al., 2017) and results of the current study showed that vacuum parameters have also facilitating effects on the ultrasound assisted extraction. Apart from VU, combined enzymatic and ultrasound assisted extraction was also examined to see the effect of vacuum on VUE procedure. A total of 28 runs with independent variables and corresponding responses belonging to VUE were shown in Table 3. The results were in the range of 44.9–69.2 % for protein yield, 3.53–5.84 mg GAE/g for TPC, 1.85–3.01 μmol TE/g for ACDPPH and 35.6–48.1 μmol TE/g for ACABTS. Table 4 presents the quadratic model coefficients with coded variables for the VUE procedure. All independent variables had significant effects on all of the responses at different levels, either by linear or second order interactions. The p-values (< 0.001) indicated the suitability of the models to precisely predict the variations. Additionally, the quality of the models could be determined based on R2 values which were higher than 0.95 for all of the responses.

It was possible to obtain high recovery rates of protein and phenolics with increasing antioxidant capacity values at prolonged restoration time, even at low vacuum pressures (Fig. 1D, E and G) in alcalase assisted treatment. The further increase in the release of protein and antioxidant compounds could be achieved after a certain vacuum pressure. At moderate vacuum pressures, the penetration of enzyme into the cellular matrix would be limited due to the existence of relatively narrow pores. The elevated vacuum pressure would cause higher deformation and would act the pores as a result of higher pressure difference as driving force. Thus, the interaction of enzyme and target substrates would increase and led to higher recovery rates. The increase in enzyme concentration linearly increased the antioxidant capacity values. Fig. 1F depicts the interactive effect of enzyme concentration and restoration time. The higher responses could be achieved at prolonged restoration time with relatively lower enzyme concentration or at shorter restoration time but with higher enzyme concentration. Many of the published reports showed that enzymatic extraction treatments combined with novel techniques such as ultrasound and microwave increase the extraction efficiency of both protein and antioxidant compounds (Görgüç et al., 2019b). However, Moczkowska et al. (2019) found that the standard alkaline extraction was more efficient than ultrasound assisted enzymatic extraction on the recovery of protein from flaxseed gum. Only recently, Xu et al. (2015) reported that ultrasound and vacuum assisted extraction followed by a multi-stage clean-up procedure enabled the removal of lipid from freeze–dried food samples up to 40 %. The current study handled a detailed examination of vacuum processing parameters. The effect of vacuum application in extraction procedures especially in food and bio–mass processing needs to be evaluated with further studies. 3.2. Optimization and comparison of extraction techniques Optimum conditions for the extraction of protein and antioxidant compounds from sesame bran by VU and VUE methods were determined by maximizing the amount of the responses i.e. protein yield, TPC, and AC value by DPPH and ABTS assays. The optimal process conditions, program outputs and experimental validation results were demonstrated in Table 5. The results of the measured responses were in close agreement with predicated values, and the deviations were found to be insignificant (P > 0.05). The optimal extraction parameters of 4

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Fig. 1. Response surface 3D plots of independent variables on the responses (A, B and C belong to vacuum - ultrasound assisted extraction; D, E, F and G belong to vacuum - ultrasound assisted enzymatic extraction).

5

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Table 3 RSM experimental design and responses belonging to vacuum - ultrasound assisted enzymatic extraction. Independent variables

Dependent variables**

Enzyme concentration (AU*/100 g)

Vacuum time (min)

Restoration time (min)

Vacuum pressure (mmHg)

Protein yield (%)

TPC (mg GAE/g)

ACDPPH (μmol TE/g)

ACABTS (μmol TE/g)

1.27 1.27 0.69 1.27 1.27 1.82 1.27 1.82 1.27 0.69 1.82 0.69 1.82 1.27 0.69 1.27 1.82 0.69 0.69 0.69 0.69 1.82 0.12 1.82 1.27 1.27 2.40 1.82

16 16 23 16 16 23 16 8 16 8 8 8 8 30 23 1 23 23 8 8 23 23 16 8 16 16 16 23

46 1 23 46 46 68 46 23 46 23 68 68 23 46 68 46 68 23 68 23 68 23 46 68 46 90 46 23

375 375 513 375 375 238 375 513 100 513 238 513 238 375 238 375 513 238 238 238 513 513 375 513 650 375 375 238

55.4 ± 0.6 51.1 ± 0.7 44.9 ± 0.8 56.5 ± 0.9 56.5 ± 0.5 56.3 ± 0.4 55.4 ± 0.9 69.2 ± 0.5 61.9 ± 0.7 59.9 ± 1.0 67.0 ± 0.5 65.4 ± 0.4 62.7 ± 0.7 50.9 ± 0.4 55.0 ± 0.4 68.3 ± 0.7 57.4 ± 0.4 50.2 ± 0.7 64.5 ± 0.2 53.5 ± 0.7 53.5 ± 0.6 55.2 ± 0.3 50.5 ± 0.4 70.2 ± 0.4 67.5 ± 1.0 63.6 ± 0.8 63.6 ± 0.9 57.4 ± 0.9

4.76 ± 0.04 4.76 ± 0.06 4.20 ± 0.14 4.89 ± 0.13 4.71 ± 0.08 5.39 ± 0.21 4.93 ± 0.23 5.47 ± 0.04 4.96 ± 0.08 4.90 ± 0.15 5.25 ± 0.07 5.84 ± 0.11 5.25 ± 0.04 4.91 ± 0.01 4.58 ± 0.10 5.45 ± 0.11 5.31 ± 0.16 3.53 ± 0.08 5.23 ± 0.05 4.04 ± 0.04 5.15 ± 0.06 5.09 ± 0.10 3.70 ± 0.08 5.52 ± 0.16 5.58 ± 0.12 5.70 ± 0.18 4.61 ± 0.03 4.94 ± 0.11

2.56 ± 0.12 2.66 ± 0.04 2.31 ± 0.09 2.67 ± 0.03 2.59 ± 0.04 2.55 ± 0.04 2.59 ± 0.12 2.77 ± 0.07 2.61 ± 0.05 2.81 ± 0.04 2.97 ± 0.09 2.46 ± 0.06 3.01 ± 0.10 1.85 ± 0.07 1.96 ± 0.04 2.59 ± 0.13 2.26 ± 0.04 2.10 ± 0.09 2.59 ± 0.06 2.61 ± 0.07 1.95 ± 0.05 2.38 ± 0.08 2.37 ± 0.05 2.48 ± 0.07 2.42 ± 0.09 2.43 ± 0.08 2.97 ± 0.13 2.56 ± 0.01

37.4 ± 0.36 37.2 ± 1.49 35.8 ± 1.46 38.4 ± 1.40 36.6 ± 0.82 48.1 ± 1.22 38.8 ± 0.70 39.3 ± 1.76 44.8 ± 0.97 37.5 ± 1.70 44.7 ± 1.06 42.4 ± 1.25 43.1 ± 1.70 41.3 ± 0.47 41.3 ± 1.77 43.3 ± 1.55 47.2 ± 1.42 37.2 ± 1.80 42.4 ± 1.64 38.4 ± 1.10 41.3 ± 1.03 38.3 ± 1.85 35.6 ± 1.27 47.7 ± 1.69 45.4 ± 1.86 46.0 ± 0.97 42.2 ± 1.19 42.7 ± 1.85

* AU: Anson unit. ** The results were expressed as “Mean ± Standard deviation”.

VU were 21 min vacuum time, 72 min restoration time and 539 mmHg vacuum pressure; and predicted values of the quadratic model was determined as 55.5 % for protein yield, 6.16 mg GAE/g for TPC, 1.84 μmol TE/g for ACDPPH and 41.2 μmol TE/g for ACABTS values. Optimum

process conditions belonging to VUE were determined as 1.82 AU/100 g enzyme concentration, 8 min vacuum time, 68 min restoration time and 238 mmHg vacuum pressure. The predicted values for VUE were 66.1 % for protein yield, 5.30 mg GAE/g for TPC, 2.97 μmol TE/g for ACDPPH

Table 4 Quadratic model coefficients and ANOVA results belonging to vacuum - ultrasound assisted enzymatic extraction. Model parameters

Intercept Linear Enzyme concentration, A Vacuum time, B Restoration time, C Vacuum pressure, D Interaction AxB AxC AxD BxC BxD CxD Second order A2 B2 C2 D2 R2 Adjusted R2 p-value F-value Lack of fit

Vacuum ultrasound assisted enzymatic extraction Coefficient

Protein yield

TPC

ACDPPH

ACABTS

β0

56.32***

2.91***

2.07***

58.98***

β1 β2 β3 β4

18.98*** −0.36*** 0.27*** −0.06*

6.57*** −0.11*** 0.02*** 8 × 10−5***

0.72*** 0.004*** 0.003*** 0.002***

−0.24*** −0.77 −0.22*** −0.08

β12 β13 β14 β23 β24 β34

−0.11 −0.27** 0.02 −3 × 10−3 −2 × 10−3** −4 × 10−5

0.06** −0.04*** −0.004*** 2 × 10−4 −4 × 10−5 −1 × 10−5

0.02** 0.005 −0.003*** 3 × 10−5 2 × 10−5 −2 × 10−5***

0.25 0.07 −0.007 0.002 −3.2 × 10−4 2.5 × 10−4*

β11 β22 β33 β44

2.56 0.01* 4 × 10−4 1 × 10−4*** 0.9726 0.9430 < 0.001 32.92 0.0579

−2.91*** 0.002** 2 × 10−4*** 6.1 × 10−6*** 0.9791 0.9566 < 0.001 43.51 0.4486

0.29 −0.002*** −3 × 10−5 −1.1 × 10−6 0.9850 0.9688 < 0.001 60.89 0.4762

4.22 0.02*** 0.002** 1 × 10−4*** 0.9587 0.9143 < 0.001 21.57 0.4551

Significance levels: *P < 0.05; **P < 0.01; ***P < 0.001. Model equation: Y = β0 + β1A + β2B + β3C + β4D+ β12(AxB) + β13(AxC) + β14(AxD) + β23(BxC) + β24(BxD) + β34(CxD) + β11A2 + β22B2 + β33C2 + β44. 6

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Table 5 Optimum process conditions and responses of vacuum - ultrasound assisted (VU) and vacuum - ultrasound assisted enzymatic extraction (VUE). Extraction method

Enzyme concentration (AU/100 g)

Vacuum time (min)

Restoration time (min)

Vacuum pressure (mm Hg)

Protein yield (%)

TPC (mg GAE/g)

ACDPPH

Predicted

Experimental

Predicted

Experimental

Predicted

Experimental

Predicted

Experimental

VU VUE

– 1.82

21 8

72 68

539 238

55.5 66.1

58.0 ± 3.04 65.9 ± 3.04

6.16 5.30

6.10 ± 0.09 5.31 ± 0.07

1.84 2.97

1.83 ± 0.02 2.97 ± 0.02

41.2 45.7

41.3 ± 0.59 43.5 ± 2.64

and 45.7 μmol TE/g for ACABTS. Conducted extraction methods were observed to increase the protein yield, TPC and ACABTS values significantly (P < 0.05) compared to those of alkaline procedure. Alkaline extraction procedure resulted an average of 24.5 % protein yield, 3.45 mg GAE/g TPC, 2.53 μmol TE/g ACDPPH and 35.1 μmol TE/g ACABTS, respectively. VUE procedure was more efficient than VU. According to the predicted responses at optimum points, combined enzymatic treatment resulted in 19.1 % and 61.4 % more protein yield and ACDPPH value, respectively than VU. Furthermore, it was possible to obtain such increments at lower vacuum pressure, vacuum time and restoration time in VUE. Ultrasound and enzyme assisted extraction studies have mainly focused on the effects of process variables such as solid to solvent ratio, ultrasound power, enzyme concentration, extraction temperature and time (Dabbour et al., 2018; Zhu and Fu, 2012). However, to our best

(μmol TE/g)

ACABTS

(μmol TE/g)

knowledge there has been no study directly addressing the vacuum as a process variable on the extraction of bio-components. 3.3. Effects of extraction procedures on the microstructure of sesame bran Cellular structure of the sesame bran before (Fig. 2A) and after alkaline extraction (Fig. 2B), VU (Fig. 2C) and combined VUE (Fig. 2D) were investigated in order to examine the effects of extraction techniques in morphological basis. As shown in Fig. 2A, untreated sesame bran was in a flat and tight form. After alkaline extraction a mild stage of destruction on the microstructure of sesame bran was observed (Fig. 2B). In the case of VU, higher destruction in bran tissues was observed as seen in Fig. 2C, whereas after extraction with VUE most of the cells were crimped, shattered and had hollow structures (Fig. 2D). Due to the formation of cavitation bubbles during the ultrasound

Fig. 2. SEM images of sesame bran after extraction procedures (3000X); A: Untreated bran, B: After alkaline extraction, C: After vacuum - ultrasound assisted extraction, D: After vacuum - ultrasound assisted enzymatic extraction. 7

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Fig. 3. SDS-PAGE protein fragmentations of extracts obtained under optimal process conditions; M: Protein marker, 1: Denatured protein by alkaline extraction, 2: Native protein by alkaline extraction, 3: Denatured protein by vacuum - ultrasound assisted extraction, 4: Native protein by vacuum - ultrasound extraction, 5: Denatured protein by vacuum - ultrasound assisted enzymatic extraction, 6: Native protein by vacuum - ultrasound assisted enzymatic extraction.

treatment, a greater penetration of solvent into the cellular materials occurs. Moreover, capillaries encounter deformation with vacuum treatment. Then, the cell walls tend to disrupt more easily, and the release rate of intracellular compounds increases (Chittapalo and Noomhorm, 2009). Similar to our findings, the applications of combined enzyme and ultrasound treatments were shown to increase the presence of deformed structures compared to control method and raw material (Goula et al., 2018; Liao et al., 2015). Based on the cellular disruption levels of treated sesame brans, SEM images were compatible with extraction yield results.

were presented with respect to their main classes as protein, phenolic, vitamin, carbohydrate, lipid and organic acid. Results revealed that the sesame bran had 17 different amino acids: Alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Among them, methionine has a special importance due to the fact that the plant-based foods are generally poor in sulphurcontaining amino acids (Gorissen and Witard, 2018). The identified phenolic compounds in the extracts were 3,4-dihydroxybenzoic acid, psalicylic acid, caffeic acid, sinapic acid, m-coumaric acid, 3-hydroxyanthranilic acid, prim-o-glucosylcimifugin, dioonflavone and diosmetin. In addition, sesame bran contained some other bioactive compounds such as vitamin B5 and α-linolenic acid (an ω-3 fatty acid). Similar to our findings, the presence of phenolic acids and flavonoids in dry sesame seed was reported (Ghisoni et al., 2017). According to a recent study, different phenolic compounds in sesame bran e.g. gallic acid, procatechuic acid, a ferulic acid derivative, p-coumaric acid and ferulic acid was detected (Ortega-Hernández et al., 2018). Nep et al. (2016) studied the polysaccharide characterization of sesame gum obtained from sesame leaves (Sesamum radiatum L.) using high performance anion exchange chromatography, and identified arabinose, galactose, glucose, glucuronic acid, mannose, rhamnose and xylose as carbohydrate fraction. Wu et al. (2016) identified a total of 23 phenolic compounds in the sesame oil. In general, the identification of different compounds is possibly due to the differences in the parts of the plant materials and extraction procedures.

3.4. Identification of protein fractions by SDS-PAGE Fig. 3 demonstrates the fractions of sesame bran protein extracts obtained after optimum process conditions. Each extract was analysed in both native, and denatured form by denaturation at 95 °C for 5 min. No difference was observed among denatured and native samples, possibly denaturation by heat treatment caused no further hydrolysis of proteins considering the β-mercaptoethanol already breaks down proteins into the primary structure (Besic and Minteer, 2011). The most predominant bands were observed between 30 and 41 kDa as three different fractions. However, no presence of protein bands over 18 kDa was detected in the VUE extract. This could be due to the effect of alcalase, a proteolytic enzyme which hydrolyzes large proteins into the small subunits (Sun et al., 2016). Also, a 53 kDa of protein was detected in four bands belonging to VU and alkaline extraction. According to literature findings, 80–90 % of the total protein of sesame consists of water-soluble 11S globulins linked by disulphide bonds, and 2S albumin with a molecular weight of 13 kDa (Orruño and Morgan, 2007). In addition, the 7S globulins were reported to constitute the 1–2 % of total sesame proteins (Tai et al., 2001). Achouri et al. (2012) reported the presence of protein bands in the range of 10–100 kDa in aqueous sesame extract. These latter authors also stated that the extracts obtained by different concentration of ammonium sulphate solutions contained 140 kDa 7S globulin and 2S albumin bands. Additional techniques such as western blotting and mass spectrometry should be used for the detailed identification of protein subunits of sesame bran.

4. Conclusion The effect of simultaneous application of vacuum to ultrasound and ultrasound assisted enzymatic extraction procedures was successfully investigated. Vacuum parameters and enzyme concentration had significant effects on the extraction of protein and bioactive compounds from food waste sesame bran. The results revealed that the implemented extraction techniques increased the protein yield, total phenolic content and antioxidant capacity values compared to standard alkaline procedure. The highest protein yield and antioxidant capacity values were obtained by combined vacuum - ultrasound assisted enzymatic extraction with lower vacuum pressure and shorter process time. Scanning electron microscopy images revealed that the microstructure of sesame bran has highly been affected by applied extraction processes. Based on SDS-PAGE analysis, no presence of a protein unit over 18 kDa was observed in alcalase bearing extractions. According to the Q-TOF/MS spectra, 17 different amino acids including seven essential amino acids with sulphur containing methionine, and nine different phenolic compounds were detected. The results of this study could be an insight to the future researches seeking new protocols for high yield extraction involving vacuum application.

3.5. Identification of protein sub-units, phenolics and other compounds by LC/Q-TOF/MS The extracts obtained after optimal conditions of the VU and VUE methods, and alkaline extract were analysed by LC/Q-TOF/MS for the identification of compounds in sesame bran. Table 6 shows the molecular formula, polarity (ionization mode), score values, m/z value, retention time and errors (ppm) of identified compounds based on their chemical group classification. Compounds were tentatively identified in both positive and negative ionization modes. The identified compounds

8

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Table 6 Identified compounds belonging to alkaline extraction, VU and VUE methods by LC/Q-TOF/MS. Group

Sub-group

Component

Molecular formula

Polarity

Score

m/z

Retention time (min)

Error (ppm)

Method**

Protein

Amino acid

L-Methionine L-Leucine L-Tyrosine L-Phenylalanine D-Tryptophan His Leu Val Ala Ser Leu Gly Leu Thr Leu Ile Asp Asn Phe Gln Phe Phe Glu Pro Glu Met Phe 3,4-Dihydroxybenzoic acid p-Salicylic acid Caffeic acid Sinapic acid m-Coumaric acid 3-Hydroxyanthranilic acid

C5H11NO2S C6H13NO2 C9H11NO3 C9H11NO2 C11H12N2O2 C12H20N4O3 C8H16N2O3 C9H18N2O4 C8H16N2O3 C10H20N2O4 C10H18N2O5 C13H17N3O4 C14H19N3O4 C14H18N2O5 C10H16N2O5 C14H20N2O3S C7H6O4 C7H6O3 C9H804 C11H12O5 C9H8O3 C7H7NO3

+ – + + + + + + + + + + + + + + – – – – – +

97,17 44,32 96,82 99,01 99,56 97,60 98,97 93,30 93,37 96,70 96,44 95,42 97,22 82,08 94,08 90,77 98,27 99,62 99,82 98,93 96,35 87,47

150,0590 130,0888 182,0796 166,0863 205,0974 269,1618 189,1243 219,1350 189,1243 233,1500 247,1297 280,1301 294,1461 295,1292 245,1143 297,1277 153,0209 137,0256 179,0366 223,0629 163,0414 154,0500

1,450 1,609 1,746 2,113 3,356 1,439 1,563 1,993 2,128 2,230 2,298 2,795 3,032 4,022 5,291 6,183 3,822 5,505 7,380 8,114 9,853 3,062

−4,36 −10,97 −4,34 −0,20 −1,04 −3,79 −4,74 −5,07 −4,74 −1,70 −3,33 −3,38 −4,18 −1,03 −4,40 −3,19 −10,11 −8,82 −8,93 −7,72 −7,17 −1,07

VUE VUE A, VUE A, VU, VUE A, VU, VUE A, VUE A, VUE A, VUE A, VUE A, VUE A, VUE A, VUE A, VUE A, VU, VUE A, VUE A, VUE A, VUE A, VU, VUE VUE VU VU, VUE A, VU

Prim-o-glucosylcimifugin Dioonflavone Diosmetin Pantothenic Acid Folinic acid α,β-Trehalose Sucrose Raffinose Maltotriose 3-Hydroxycapric acid Stearidonic acid Linoelaidic acid 3-hydroxy-tetradecanoic acid 16-hydroxy hexadecanoic acid cis-9,10-Epoxystearic acid 2-Hydroxyhexadecanoic acid Δ2-trans-Hexadecenoic acid α-Linolenic acid Elaidic acid Petroselinic acid Oleic acid Phytosphingosine Palmitoyl Ethanolamide 19(R)-hydroxy-PGE1 DL-pipecolic acid D-(+)-3-Phenyllactic acid DL-b-Hydroxycaprylic acid Corosolic acid

C22H28O11 C36H30O10 C16H12O6 C9H17NO5 C20H23N7O7 C12H22O11 C12H22O11 C18H32O16 C18H32O16 C10H20O3 C18H28O2 C18H32O2 C14H28O3 C16H32O3 C18H34O3 C16H32O3 C16H30O2 C18H30O2 C18H34O2 C18H34O2 C18H34O2 C18H39NO3 C18H37NO2 C20H34O6 C6H11NO2 C9H10O3 C8H16O3 C30H48O4

+ + – + + + – – + – + + – – – – – – – – + + + + + – – –

83,92 49,08 97,64 83,64 89,29 97,97 92,50 93,79 99,80 94,93 88,39 83,15 96,99 97,85 92,79 99,94 40,81 97,35 63,63 84,3 91,35 94,78 81,54 100 97,17 94,09 91,09 62,12

469,1684 623,1906 299,0585 220,1181 474,1724 365,1063 341,1121 503,1660 527,1593 187,1356 277,2165 281,2478 243,1982 271,2302 297,2459 271,2302 253,2193 277,2196 281,2509 281,2513 283,2635 318,3009 300,2900 393,2255 130,0870 165,0571 159,1037 471,3508

6,168 17,643 18,392 2,610 5,569 1,198 1,224 1,213 1,221 19,126 22,602 24,432 24,762 24,920 25,485 26,185 27,427 27,484 28,196 28,196 28,261 22,884 26,973 2,343 1,379 6,058 11,435 25,892

4,46 0,97 −8,07 −0,56 1,58 −2,42 −9,13 −8,38 −2,07 −8,53 −1,07 −0,29 −6,73 −8,58 −7,84 −8,56 −7,73 −8,16 −7,97 −9,60 −1,29 −1,99 −1,11 −2,07 −5,81 −8,50 −6,25 −6,03

A, VU VU, VUE VUE VU A, VU A, VU,VUE VUE A, VU,VUE A, VU,VUE A, VU, VUE A, VU, VUE VU, VUE VU, VUE A, VU, VUE A, VU, VUE A, VU, VUE VU VUE A, VUE VU A, VU A, VU VU VUE A, VU A, VU, VUE A, VU VU, VUE

Dipeptide*

Phenolic

Phenolic acid

Intermediate of tryptophan Flavonoid Vitamin Carbohydrate

Vitamin B5 Folic acid derivative Disaccharide Trisaccharide

Lipid

Fatty acid

Organic acid

Sphingolipid Fatty acid amide Prostaglandin Organic acid

* Ala: Alanine, Asn: Asparagine, Asp: Aspartic acid, Gln: Glutamine, Glu: Glutamic acid, Gly: Glycine, His: Histidine, Ile: Isoleucine, Leu: Leucine, Met: Methionine, Phe: Phenylalanine, Pro: Proline, Ser: Serine, Thr: Threonine, Trp: Tryptophan, Tyr: Tyrosine, Val: Valine. ** A: Alkaline extraction, VU: Vacuum ultrasound assisted extraction, VUE: Vacuum ultrasound assisted enzymatic extraction.

CRediT authorship contribution statement

References

Ahmet Görgüç: Methodology, Validation, Investigation, Formal analysis, Writing - original draft. Pınar Özer: Investigation. Fatih Mehmet Yılmaz: Conceptualization, Methodology, Writing - original draft, Supervision, Project administration.

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