Enzyme-assisted extraction processing from oilseeds: Principle, processing
and application

Jun-jun Liu, Mohammed Abdalbasit A. Gasmalla, Pengfei Li, Ruijin

Yang

PII: S1466-8564(16)30078-9
DOI: doi: 10.1016/j.ifset.2016.05.002
Reference: INNFOO 1523

To appear in: Innovative Food Science and Emerging Technologies

Received date: 27 October 2015

Revised date: 28 April 2016
Accepted date: 3 May 2016

Please cite this article as: Liu, J.-, Gasmalla, M.A.A., Li, P. & Yang, R., Enzyme-assisted
extraction processing from oilseeds: Principle, processing and application, Innovative Food
Science and Emerging Technologies (2016), doi: 10.1016/j.ifset.2016.05.002

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Enzyme-assisted Extraction Processing from Oilseeds: Principle, Processing and

Application

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Jun-jun Liua, Mohammed Abdalbasit A. Gasmallaa,b, Pengfei Lia, Ruijin Yanga*

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State Key Laboratory of Food Science & Technology and School of Food Science and Techn

ology, Jiangnan University, No. 1800 Lihu Road, Wuxi 214122, China
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b
Department of Nutrition & Food technology, Faculty of Science and Technology,
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Omdurman Islamic University, P.O. Box 382, 14415, Khartoum, Sudan

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*Corresponding author: Email yrj@jiangnan.edu.cn Tel: + 86-510-85919150; Fax:

+86510-85919150.

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Abstract

Enzyme-assisted extraction processing (EAEP), feasible alternative to screw pressing

and organic solvent extraction technologies, is a promising method for the

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simultaneously extraction of oil and protein from oilseed. This method incorporates

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comminuting, extraction buffers and enzymes to allow production of a range of oils

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and proteins although different challenges appear during the process. The demand for

acceptable and high free oil yields and purities of protein are always incompatible in

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many processes. This review article covers technological aspects of EAEP, and
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discusses the application of enzymes in pretreatment, extraction and demulsification,

and explores the quality characteristics and safety of the oils and proteins obtained,
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focusing particularly on recent application of EAEP at the laboratory and industrial

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scale.
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Keywords: Enzyme-assisted extraction processing, Technical aspects, Enzyme

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application, Quality; safety, Application

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1. Introduction

Enzyme-assisted extraction processing (EAEP) is a promising method for using

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enzymes to facilitate the recovery of oils and proteins from oilseeds, in which water is

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used as the extraction solvent which has many advantages compared to conventional

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extraction. Conventionally, edible oil is produced by extraction with an organic

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solvent alone or by screw pressing prior to solvent extraction.

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Mechanical oil expression from oil-bearing materials is a solid-liquid phase separation

process which involves the application of pressure by hydraulic or screw press with or
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without heat (Ogunsina, Olatunde, & Adeleye, 2014). Although mechanical oil
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expression has advantages of wide scope of application and low equipment cost, the
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oil yield is relatively low and the protein loses economic value due to degeneration

during mechanical processing.

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Although n-hexane is the most prevalent solvent used in the chemical/food industries
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and achieves crude oil yield of over 96% (JulianaMariaLeiteNobrega de Moura &

Johnson, 2009), its highly flammable, explosive and acute inhalation exposure of

humans to high levels of hexane causes mild central nervous system effects , such as

dizziness and headache rendering it unsafe to both plant and humans (Wu, Johnson, &

Jung, 2009). For instance, it was earlier reported that when fifteen workers in an

adhesive bandage factory were exposed to n-hexane, eleven of them showed macular

changes and one had central retinopathy (Raitta, Sepplinen, & Huuskonen, 1978).

In comparison with solvent extraction, the use of an aqueous medium is much safer,

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environmental-friendly and economical. In addition, it contributes to a much safer and

flexible operation, lower energy consumption and operational costs, and lower capital

investment. The EAEP products are of superior quality and highly suitable for human

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consumption.

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Oil obtained by EAEP has better quality than that produced using other technologies.

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Because free fatty acids are neutralized by alkaline conditions during extraction

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processing, oils produced by EAEP have good oxidative stability and low

phospholipid concentration, thus reducing losses of oil during refining

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processing(Abdulkarim, Long, Lai, Muhammad, & Ghazali, 2005; Bocevska,

Karlovi, Turkulov, & Pericin, 1993; S. B. Zhang, Lu, Yang, Li, & Wang, 2011).
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Due to the denaturizing of protein or residual solvent, the protein residue is mainly

used in the manufacture of compound feed stuffs or fertilizers (Jiang, Hua, Wang, &
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Xu, 2010). Since the plant protein resource is not utilized optimally, the use of new

technologies like EAEP can improve extraction yield, nutrition benefit and
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functional characteristics of plant proteins.

Despite the advantages, the application of EAEP in industry is still limited due to long

processing time and the high disbursement for the drying process after the enzyme

treatment (Dominguez, Sineiro, Nunez, & Lema, 1995; Shah, Sharma, & Gupta,

2005b). Compared to mechanical oil expressing processing and solvent extraction,

EAEP consumes more time and has a higher cost of production since the price of

enzymes is quiet high. In addition, downstream steps, such as centrifugation for

separation, emulsification and drying for protein and residues, are quiet difficult and
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time-consuming. At last, emulsification is an unavoidable part of the extracted oil,

which faces a number of problems.

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2. Technical aspects of EAEP

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2.1 Principle of EAEP from oilseeds

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Extraction processing of oilseeds mainly consists of dispersing the ground oilseeds

into water and providing a motive force to free oil from cellular confines(L_A

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Johnson & Lusas, 1983; Rosenthal, Pyle, & Niranjan, 1996). The microstructure of
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soybean which is similar to most oilseeds is shown in Figure 1(Li, Gasmalla, Zhang,

Liu, Bing, & Yang, 2016). Protein bodies are distributed all over the cell and present
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different particle sizes depending on the component and structure of cell wall oilseeds.
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These protein bodies contain approximately 60-70% of the total protein present in
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oilseeds(Snyder & Kwon, 1987). The process of EAEP is illustrated in Figure 2.

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Centrifugation was indispensable in subsequent steps to separate the free oil from
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cream, skim and solids. However, the extraction technology still brings problems such

as forming stable emulsion during the process, high cost of enzymes and high effluent

generation.

2.2 Factors affecting of EAEP from oilseeds

2.2.1 Grinding approaches

The first and critical step during EAEP to improve oil extraction from oilseeds is

operations used to rupture cell walls and release the oil so that it can be recovered as

free oil and oil-rich cream.

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Enzyme-assisted mechanical grinding

In recent years, the application of enzymes as pretreatment during EAEP has been

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proved to increase oil yields (F. Li, et al., 2012). Use of enzymes as pretreatment

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breaks cell wall and facilitates oil extraction. In addition, employing this approach is

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conducive to avoiding the formation of an oil-in-water emulsion. According to Ziga

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et al, significantly lower residual oil in the meal was found in the case of Chilean

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hazelnuts (Ziga, Soto, Mora, Chamy, & Lema, 2003). Besides, these studies

indicated that enzyme pretreatment is applicable to various oilseeds and can be

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employed prior to both mechanical and solvent extraction methods. The enhancement

of oil yield owes to the hydrolytic action of the enzymes on the cell wall and
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membrane components which facilitate subsequent oil release.

Different enzymes degraded different ingredients of oilseeds, thus affecting oil yields.
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Enzymes such as cellulase, hemicellulase, pectinase, protease, amylase, glucanase and

polygalacturonase have been used in previous studies(J. N. de Moura, Campbell,

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Mahfuz, Jung, Glatz, & Johnson, 2008; L_A Johnson, et al., 1983; H. Li, Song, Zhou,

Wang, & Cao, 2011; A. Sharma & Gupta, 2001). For instance, polygalacturonase,

amylase and protease were previously used in extraction of coconut oil, while

pectinase and cellulase were applied in the extraction of (Aliakbarian, De Faveri,

Converti, & Perego, 2008; Najafian, Ghodsvali, Khodaparast, & Diosady, 2009)

(Chen & Diosady, 2003; Tano-Debrah & Ohta, 1997).

A better oil extraction yield can be expected when a judiciously chosen mixture of

enzymes is used owing to possible synergy. For example, when acidic cellulase and
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alkaline protease were used in combination, 80-90% oil extraction was obtained from

corn germ (Moreau, Dickey, Johnston, & Hicks, 2009). Lamsal and Murphy et al

(2006) reported extrusion facilitated protease (0.5% wt/wt) action resulting in oil

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recovery increasing from 71 to 88% (extrusion temperature at 100 C, EAEP was

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carried out at 50C, pH 8, 15 min for extruded flakes). A combination of pectinase

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with cellulase and -glucanase were employed in a ratio of 4:1:1 to result in the

highest yield (91.6% emulsified oil). This marginal enhancement in yield was

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attributed to the elimination of other barriers to the release of oil (S. B. Zhang, Wang,
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& Xu, 2007b) . In addition, a mixture of three enzymes (pectinase, cellulose and

hemicellulase) formulations was also reported to improve the oil yield (Aliakbarian, et
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al., 2008). Similar results were also reported by earlier studies (Chiacchierini, Mele,
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Restuccia, & Vinci, 2007; De Faveri, Aliakbarian, Avogadro, Perego, & Converti,
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2008). Oil yield increases with enzyme concentration but commercial enzyme
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concentration is limited to 1% with enzyme activity above 100,000 U/g.

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Mechanical grinding

Extrusion is a mechanical process exposing material to high temperature, shear force

and pressure over a short period of time. Extruding flakes provide vigorous

thermal/mechanical treatment that may be helpful in rupturing cell walls (cell

distortion) and expelling some free oil.

In general, the smaller the particle size of the materials, the better is the oil extraction.

Theoretically, the path length between enzymes and cellular components is shortened

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with disruption of the cell wall during size reduction. Particle size of the oil-bearing

materials is one important factor that affects the enzyme function and oil yield(R.

Sharma, Sharma, Rana, & Joshi, 2015). Nevertheless most of the early studies did not

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consider the particle size of the oil-bearing materials as a key factor influencing

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extraction efficiency (Passos, Yilmaz, Silva, & Coimbra, 2009; Rosenthal, Pyle,

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Niranjan, Gilmour, & Trinca, 2001). The grape seeds used by Passos et al. (2009)

were grouped into different particle size ranges (in mm): <0.50, 0.50-0.60,0.60-0.71,

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0.71-1.0, 1.0-1.4, 1.4-2.0, and >2.0, and increment in oil yield was observed at lower
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particle sizes. According to Passos et al (2009), oilseeds with weak structure collapse

when exposed to the flow of solvents, then losing their marcoporosity, which prevents
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a uniform and convenient percolation. In addition, the yields improve when the
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linseeds are crushed to form different particle sizes including fine powders (Gros,
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Lanoisell, & Vorobiev, 2003). The oil yield was similar to the result by Freitas et al
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who also applied extrusion to disrupt the soybeans cell wall without flaking (Freitas,
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Hartman, Couri, Jablonka, & de Carvalho, 1997). Sineiro and Dominguez et al. (1998)

optimized the extraction parameters of sunflower oil during enzyme assisted aqueous

extraction processing. They reported that particle size substantially influences the oil

extraction yield.

Thereforematerials with very low particle size are not recommended and there

appear to be an optimum size. But grinding of materials with high oil content into

very low particle sizes may cause the particles to adhere.

2.2.2 Operational variables

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The optimum pH for enzymatic hydrolysis varies from enzyme to enzyme

(Abdulkarim, et al., 2005). The pH does not only affect enzyme activity but also

separation of oil and protein. It is reported that motivation mechanism of oil

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extraction is contacted closely with that of protein extraction. Oil extraction rate is

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directly proportional to protein extraction rate. Oil extraction rate is lowest at

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the protein isoelectric point (pI).

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Enzymes can simultaneously solubilize and hydrolyzed the proteins and disrupt

polysaccharide constituents, which facilitates oil extraction (Latif & Anwar, 2011;
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Passos, et al., 2009; Rovaris, et al., 2012). When mixed enzymes of cellulose,

pectinase, and hemicellulose (1:1:1) were used at pH 4.5-5.0 by Long et al (Long, et

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al., 2011), the flaxseed oil yield (73.9%) was higher than the oil yield of each

individual enzyme. Rovaris et al. (2012) used a mixture of Alcalase 2.4L and
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Celluclast 1.5L with oil yield of 26.82%, which was higher than 20.63% at

uncontrolled pH in the case of soy-bean oil. Higher oil and protein extraction yields
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were also observed by Tabtabaei & Diosady (2013) when the mixture of cellulose,

pectinase and hemicellulosae (1:1:1) was used at pH of 4.5 - 5.0 during the EAEP of

yellow mustard flour compared to the oil and protein extraction yields obtained with

each individual enzyme.

Besides pH, temperature is a parameter that should not be neglected during processing.

Optimum incubation temperature varies for different oilseeds and enzymes. Thus,

besides the oil yield, the oil and protein quality characteristics must also be taken into

consideration when selecting EAEP temperature. High temperatures not only cause a
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gradual loss of enzyme activity but also darkening of oil and inactivation of proteins.

However, low temperatures lead to slow reaction rates of enzymes and extraction rate

of oil and protein. The optimum temperature range for enzymatic hydrolysis is

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between 45 and 55 (Rui, Zhang, Li, & Pan, 2009). Considering energy, the

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lowest possible temperature is employed to satisfy the activity demands (Passos, et al.,

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2009). A lower temperature of 30 was found to be favorable especially to preserve

the oil quality in the case of olive fruits (Aliakbarian, et al., 2008; De Faveri, et al.,

arc a, renes, os Mo ano,

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Sgaramella, & Surricchio, 1999). In addition, Gros et al. (2003) also used a

temperature of 34 for similar reason in linseed oil extraction. It is reported that a

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significant effect of temperature on oil yield was observed at 40, but the oil yield
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decreased with the decrease of temperature to 37 (A. Sharma, Khare, & Gupta,
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2002). Enzymatic hydrolysis begins to decrease at temperatures greater than 45,

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due to enzyme inactivation as reported by Ziga et al. (2003).

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During processing of extraction, improving liquid to solid ratio is beneficial to

separation of oil and protein while reducing amount of liquid waste. High liquid to

solid ratio is conducive to increase extraction rate and product yield. But high liquid

to solid ratio may lead to increased liquid waste and difficulty of demulsification in

follow-up processing, which leads to increasing costs of waste water treatment and

demulsification. Therefore, oil yield, amount of liquid waste and demulsification

should be taken into considered when determining liquid to solid ratio.

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Zhang et al. (2007b) reported that very low moisture content results in the formation

of thick suspensions which can prevent the enzymes from effectively penetrating into

the substrate. According to Sineiro et al. (1998), Onl certain areas in sunflower

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kernels were degraded by enzymes at low moisture content. Although, higher water

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activity demonstrated higher oil yield, excessive moisture content in oilseeds can

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decrease the concentration of enzymes and substrates (Dominguez, et al., 1995; S. B.

Zhang, et al., 2007b). Therefore, appropriate liquid to solid ratio needs to be

determined during EAEP processing.

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Incubating time also varies for different oilseeds and enzymes. Oil yield, protein

quality, recovery rate and economic costs should be taken into consideration during
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determination of the optimal incubating time. Long incubating time increases

difficulty in demulsification against oil extraction. Short incubation time reduces

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efficiency of enzymatic hydrolysis of oil materials hence leading to low oil yields.

Prolonging the incubation time can enhance the degradation of cell wall components
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(Abdulkarim, Lai, Muhammad, Long, & Ghazali, 2006; Dominguez, et al., 1995;

Jiang, et al., 2010). Passos et al. (2009) reported that an enzyme mixture of cellulose,

protease, xylanase, and pectinase used for 120 h resulted in 3.8% oil yield which was

higher than that used for 24 h. However, it is unacceptable for industry to employ very

long time durations (Passos, et al., 2009) since this would cause lower oil quality and

high energy usage (Abdulkarim, et al., 2006; Jiang, et al., 2010). Zhang et al. (2007b)

reported that the oil yield decreased after a certain incubation period, and this was

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attributed to the possibility that all the substrates reacted with the enzymes and left

negligible substrates for further enzymatic reaction to take place.

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3. Enzyme application in EAEP

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3.1 Enzymatic treatment

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The main components of the cell wall are cellulose, hemicellulose and pectin. With

the help of cell wall, compartmentalization between the cells is possible and this aids

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in protection of the cell and maintenance of the cell shape(P. D. Fullbrook, 1983;
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Lanzani, Petrini, Cozzoli, Gallavresi, Carola, & Jacini, 1975; Sosulski, Sosulski, &

Coxworth, 1988). However, the cotyledon cells contain a large amount of oils and
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proteins as energy reserves and are wrapped in the cell wall which may obstruct the
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release of oils and proteins. The enzymes involved in the hydrolysis of fruit and
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oilseed cell walls are mainly pectinases, cellulases, and hemicellulases, all of which
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act in concert. Cellulases, hemicellulases and pectinases affect oil and protein
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extraction yields as well as dissolution of non-lipid material, but the degradation of

cellulose is generally very limited (Aliakbarian, et al., 2008; Gros, et al., 2003).

Researchers attempted different combinations of cell wall degrading enzymes

(carbohydrases and proteases) for individual seeds or fruits. Moreau(Moreau,

Johnston, Powell, & Hicks, 2004) reported that corn oil yield was greater than 90% by

EAEP(Moreau, et al., 2004). However, when the most effective cellulase enzymes,

Multifect GC and Celluclast, were used in the study, oil yields of about 93% and 91%

were obtained, respectively. Dominguez (1995) applied Celluclast 1.5L and

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Multifactor to assist hexane to extract soybean oil. There were 5% and 8-10%

increases after adding enzymes which were simultaneously carried out with the oil

extraction or treated prior to the solvent extraction, respectively. A mixture of amylase

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(80 U), protease (368 U) and cellulase (380 U) was employed by Sharma (2002).The

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highest oil yield in their report was 77% under pH 7.0, temperature 65C, extraction

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time 18 h with constant shaking at 80 rpm. The oil yields with both processes were

almost independent of enzyme concentrations in the range they investigated. Besides,

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adding enzymes reduces the oil extraction time by 20-30 minutes.
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Perez (2013) evaluated the effect of pectinase on the oil solvent extraction. A fine

meal of sunflower seeds was prepared for the test and 2% (w/w) pectinase was added
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to the meal prior to solvent extraction. The effect of the enzyme on the fiber structure

was observed, allowing a higher oil release and a quick extraction process. At the
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same time, they found that pectinase treatment was highly effective in the tocopherol

extraction from sunflower seeds, obtaining a 32.3% increase on average. EAEP can be
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used in some new ingredients and biofuel production. Cellulolytic, hemicellulolytic

enzymes, as well as proteases were studied in enzyme-supported oil extraction from

Jatropha curcas seeds to improve oil yield and aqueous extraction a maximum yield

of 86% was obtained using an alkaline protease(Shah, Sharma, & Gupta, 2003). In

addition, cellulase, neutral protease and alkaline protease were investigated by Liang

and Zhang et al. (2012) and signicantl higher lipid recover of 49.82% was

achieved by snailase and trypsin combined sonication. Both studies help designing an

economically viable EAEP.

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3.2 Cream demulsification

Since extraction of oil from oil-bearing material and obtaining free oil are almost

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equally important for EAEP processes, the evaluation of these enzyme preparations

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could ignore the aspect of their contribution to stabilization of emulsions. More details

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about emulsion which formed in the EAEP were discussed (Ramon Morales Chabrand,

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Kim, Zhang, Glatz, & Jung, 2008; JMLN De Moura, Mahfuz, Campbell, Jung, Glatz,

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& Johnson, 2008; Lamsal & Johnson, 2007). Emulsions formed during aqueous or

enzyme-assisted aqueous extraction of vegetable oil were the major obstacles to

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releasing free oil. Fang and Moreau investigated demulsification of cream formed in

EAEP of rice bran, the oil recovery from emulsion reached 87.8% with Protex
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6L(Fang & Moreau, 2014). So far, the common methods such as ultrasound treatment,

heating treatment, ethanol treatment, phase inversion method, freezing-thawing and

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enzymatic treatment were widely used in cream demulsification(Fang, Fei, Sun, & Jin,

2015; FENG, et al., 2013; Tabtabaei, et al., 2013; Y. WANG, JIA, & ZHANG, 2008b;
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Wu, et al., 2009; S. B. Zhang, Wang, & Xu, 2007a). Freezing-thawing method can

lower the stability of emulsion and achieve the highest oil recovery in many oilseeds.

Fat crystals may form and pierce into the water phase during freezing-thawing process.

However, the challenges of high energy consumption and difficulty in scaling up,

were quite obvious.

It is investigated that the emulsions can be destabilized by organic solvents. Tabtabaei

and Diosady (2012) tested many organic solvents, such as isopropyl alcohol,

tetrahydrofuran, and dioxane, to destabilized the yellow mustard emulsions and over
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97 and 95% of the oil in the emulsion was successfully recovered using 4:1 THF:oil

and 9:1 dioxane:oil weight ratios, respectively. In addition, ethanol is regarded as

green solvent for food industr , ut the manufacturers and customers still worr

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about the residual solvent in the product. And this limits the application of ethanol for

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demulsification.

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The effectiveness of enz mes demulsication was found to e related to the

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characteristics of emulsions, such as contents, acidity and alkalesce, concentration of

protein and phosphatide. It could be very specific due to the difference in starting
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material as well as EAEP processes (Fang, et al., 2014; Lawrence A Johnson, 2008;

Latif, Diosady, & Anwar, 2008). With advantages such as mild reaction temperature,
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less byproducts and good quality of oil, the EAEP industrial application is promising

especially if the cost of enzyme decreases, the recycling utilization of water and
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development of new ingredients/foods with the process could be sorted out.

Protease can catalyze hydrolysis peptide bond in the interior of the polypeptide chain,
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which resulting exposition of shorter fragments of protein or peptides the emulsifying

property and aggregation of oil droplets. Wu and Johnson reported that protease

released more free oil than phospholipase at concentrations less than 2%. At 0.2%

concentration, 88% and 48% of the free oil were obtained by using protease and

phospholipase, respectively (Wu, et al., 2009). Protease was considered as a viable

option for demulsification by Zhang and Wang (2007b). In their study, Alcalase 2.4L

was employed for demulsification at 60C for 1h and free oil yield of 7376% was

obtained.
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With catalysis of phospholipase, phosphatide can be degraded and major substance

stabilize the emulsion was destroyed. It is reported that a cocktail of LysoMaxTM

(phospholipase A2) and G-ZYME 999 (lysophospholipase) resulted in considerable oil

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release from the EAEP soybean cream(Lamsal, et al., 2007). Compared with soybean,

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peanut shows significant difference in cream demulsification with phospholipase. It

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was found that phospholipase A2 has a marginal effect on releasing free oil from

peanut cream. The possible reason could be that peanut contained much lower content

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of phospholipids than soybean. Therefore, phospholipids might not be a major
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contributor to the stability of peanut emulsion. In the case of yellow mustard flour,

surprising observation that the Lipomode (Phospholipase A2) treatment at pH 7.5 and
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40C not only decreased free oil recoveries from all of the emulsions (Table 8), but
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also increased the stability of the emulsions was found(Tabtabaei, et al., 2013),which
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may be attributed to role of Lipomode (Phospholipase A2) in cleaving the ester bond
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at the sn-2 position in the glycerophosphatide, to produce lysophospholipids that are

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more effective emulsiers than the diacylglycerophosphatide(Ramn Morales

Chabrand & Glatz, 2009).

4. Quality and safety of the product

4.1 Quality of oil by EAEP compared with other methods

Generally, oils extracted by EAEP are of better quality than other oils extracted by

traditional methods when analyzed based on parameters such as free fatty acid

contents, fatty acids composition, iodine value, saponification number, unsaponifiable

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matter, peroxide value, phosphorus amount, nutrient content (amount of tocopherols,

etc.), refractive index, density, color, etc(Lawrence A Johnson, Johnson, White, &

Galloway, 2008; L_A Johnson, et al., 1983; Latif & Anwar, 2009; Libo, Yaqin, Yu, &

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Xin, 2011; Nyam, Tan, Man, Yaakob, Lai, & Long, 2009; T. Wang & Johnson, 2001a,

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2001b).

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The byproducts obtained by EAEP include proteins, polysaccharides, oils, phenolic

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compounds and their fractions can be extracted with good yields while preserving

their intrinsic qualities.

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One of the indicators used to estimate the quality of oil is the amount of free fatty
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acids contained in the oil after extraction. Previously, oils extracted by

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enzyme-assisted processing had problems of oxidation stability. As a result, the oils

showed a lower quality than those obtained by cold pressing, while the oils had better
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quality than those extracted by solvent according to Sosulski et al. (1993). But

comparing the two types of processes, there was no significant variance in acidity and
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peroxide or iodine values (Najafian, et al., 2009). According to Tano-Debrah (1997),

no quality loss was observed by EAEP used in coconut oil. Soybean oil extracted by

EAEP has less free fatty acid (Latif, et al., 2011; Towa, Kapchie, Hauck, & Murphy,

2010) and imilar results were also reached by Zhang et al. (2007b). The rapeseed oil

extracted enzymatically in this study shows more free fatty acids but has a lower

peroxide value, and the color is darker than that obtained by solvent extraction.

According to Raghavendra (2011), the quality of virgin oil extracted by enzyme

assisted processing is enhanced and better than commercial virgin oil for free fatty
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acids and peroxides due to enzymes. In this study contents of lauric acid and vitamin

E were higher in virgin oil compare to others oils (commercial virgin coconut oil and

commercial standard coconut oil). Moreover, coconut oil extracted with enzymes

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gives better scores than other oils in terms of sensory attributes like freshness and

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nutty odor.

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The use of enzymes was reported to induc lauric, eicosapentaenoic and adrenic acids

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formation in Lophira lanceolata seed oil(Nonviho, Paris, Muniglia, Sohounhlou, &

Brosse, 2015). Rakesh Sharma et al. (2015) reported that enzymatic treatment
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significantly increased the oil yield and quality. Enzymatic treatment also increases

free fatty acid contents (FFAs) (0.82-2.95%) and peroxide value (7.30-16.50 mg/kg)
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while decrease in iodine value (80.0-78.0) and total phenols(165-139 mg/kg) during

storage. The chemical composition, physicochemical properties and antioxidant

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activities demonstrated that yellow horn oil obtained by EAEP can be high-quality

edible oil for the food industry(J. Li, et al., 2013). Generally, oils extracted by EAEP
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are of better quality than that by other methods.

4.2 Quality of by-products

The process used to fractionate oilseeds to valuable oil and proteins has a direct

impact on end-product quality. This is particularly true for the functional properties of

proteins that can be affected by the operational conditions of extraction associated

with denaturation (organic solvent, pH, temperature, salts, or ionic strength).

Fan et al. (2011) compared the traditional hot water extraction of polysaccharides

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from Grifola frondosa with enzyme-assisted aqueous extractions. The results

indicated that extraction with enzymes mixtures for a period (varied from 15 min to 2

h) contributes to improve extraction yields and antioxidant efficacy of extracted

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polysaccharides when enzymes were tested for activity and used alone or in mixtures.

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The reason may be that enzyme hydrolysis helps to reduce the molecular weight of

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polysaccharides, a physical characteristic that is in favor of the antioxidant activity.

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A recent study from Bagnasco et al. (2013) presents an enzyme assisted aqueous

extraction method performed on rice to value all different fractions obtained after
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conversion of raw rice into white rice like rice hull and bran mainly.
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Hanmoungjai et al investigated the effect of enzyme concentration, incubation time

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and temperature on oil and protein extraction yields (Hanmoungjai, Pyle, & Niranjan,

2001). The maximal extraction yields of oil and protein were 79 and 68%,
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respectively.
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And the quality of oil recovered from the process in terms of free fatty acid, iodine

value, and saponification value was comparable with solvent-extracted oil and

commercial rice bran oil, though the peroxide value was higher.

Hydrothermal pretreatment was employed before germ milling and they found that

hydrothermal pretreatment has positive effects on the quality of oil. Times and pH of

hydrothermal pretreatment had an obvious effect on the free fatty acid. Meanwhile,

peroxide value was not significantly increased. Various enzymes (Gamanase 1.5 L,

Pextinex Ultra SP-L, Celluclast 15 and SP-348) were used individually or in mixtures

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and the effects of oil yield were studied.

Rosenthal et al. (2001) reported extraction yield of protein and oil significantly higher

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than normal processes from 27.8 to 66.2%, and 41.8 to 58.7% by using protease in

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heated soybean flours, respectively.

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Compared with traditional solvent extraction methods, the meal obtained by EAEP

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had lighter color. This could be attributed to the removal of sugar and phenolic

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compounds into skim, thus the reaction between phenolics and protein was reduced.
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Considering other fractions obtained from the plant from enzyme-assisted aqueous

extraction, co-products with a higher added value than those obtained by methods
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using organic solvents are produced. Thus, proteins, polysaccharides, or active

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compounds such as polyphenols can be extracted with good yields while preserving
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their intrinsic qualities.

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5. Application of EAEP at the laboratory and industrial scale

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EAEP has been used widely for reactions at the laboratory scale up to industrial

processes. Due to its advantages of being eco-friendly and effective, EAEP has been

encouraged as an emerging technology. The cosmetics market is increasingly

demanding for natural extracts produced in a greener way and is one area where

alternative technologies like EAEP can be considered.

5.1 Laboratory scale

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Because of its excellent accessibility and possibilities, EAEP is easily applicable at

the laboratory scale. Comparing with conventional techniques, EAEP not only can

generate oils but also can be produced by-products such as peptides, protein, tea

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saponin, polysaccharides, or active compounds et al. In the laboratory, enzymes are

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available which make the study easier to continue with the aid of appropriate tools.

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Previously, it was reported that under optimized conditions the total free peanut oil

and protein hydrolysates, yields were increased to 91.98% and 88.21% respectively

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(Jiang, et al., 2010). Elsewhere, the use of protease resulted in significantly higher
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yields over the control (protein yield increased from 27.8 to 66.2%, oil yield increased

from 41.8 to 58.7%)(Rosenthal, et al., 2001). Because of its innovation and

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environmental conservationEAEP as a green technology is encouraged and studied

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generally worldwide.
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With the use of enzymes, the aqueous extraction processing is getting more and more

studied for the alternative solutions of hexane and other harmful solvents. Substrates
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of EAEP are extensive such as peanut, soybean, sunflower, rapeseed, and olive seed.

Because of the large quantities of these substrates at industrial scale, traditional

extraction processing implies large volumes of organic solvents which are limited by

VOC emissions(Kapchie, Wei, Hauck, & Murphy, 2008; A. Sharma, et al., 2002).

Patents, on applications of enzymes to oilseeds for the extraction of oils and protein

are available which provides opportunity to transform from laboratory scale to

industrial scale(P. Fullbrook, 1984; Penet, et al., 2007; T Jr, 1972).

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5.2 An industrial scale

Enzymes are widely used on an industrial scale but mainly as catalysts, and the

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applications mainly concerning the synthesis of pharmaceuticals, compounds for

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chemistry of specialty, polymers, etc (Rolle, 1998; A Schmid, Dordick, Hauer, Kiener,

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Wubbolts, & Witholt, 2001; Andreas Schmid, Hollmann, Park, & Bhler, 2002).

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At industrial scale, one advantage of EAEP is environment benefit because it can

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avoid the risk of organic solvents and particularly hexane. Additionally, even the

extraction yields of EAEP are lower than those of conventional solvent extraction
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processing, the quality of the products produced by the smooth process is usually
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higher. It is the selectivity and the mild reaction conditions that preserve the integrity
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of the products. At an industrial scale it is very important for profits concerning the

high quality of the products.

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One of the limitations of EAEP is the high cost of the production. On industrial scale,
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the investments of enzymes for the large volumes of products are not always available,

which creates the biggest bottleneck of application. However, another limitation of

EAEP is purification of the products (Puri, Sharma, & Barrow, 2012). The purification

processing of the products is usually more complex than that obtained from the

conventional solvent extraction. Its higher diversities of the molecules and phases

cause the complexity. However, another limitation of EAEP is purification of the

products. The purification processing of the products is usually more complex than

that obtained from the conventional solvent extraction.

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A theoretical model can be built for the application of EAEP. Even with the high cost

of enzymes, the costs of the production can be reduced on a large scale. Moreover,

enzymatic reactions are usually more difficult to scale up for large volumes since

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enzymes behave differently depending on parameters. Some alternative methods are

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using immobilized enzymes which can be used several times with no loss of

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activity(Bornscheuer, 2003; Sheldon, 2007). In that case, the life of enzyme is

prolonged and the costs are reduced.

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6. Concluding remarks and future prospects
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Nowadays, EAEP has already made the transformation from the laboratory scale to
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industrial processes. This technique is considered as an alternative method to produce

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valuable products without loss of quality at moderate conditions. Because of the

variance of enzymes, the most appropriate enzymes for the extraction should be found
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to increase the production yield and quality. Compared with solvent extraction, EAEP

is more eco-friendly. Besides that, one of the main advantages of EAEP is the
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specificity of enzymes. The most suitable enzyme can be used for different raw

materials to derive the desired reaction. Nowadays, EAEP is widely used at the

laboratory scale, and many research studies are conducted on the topic. With

the ecological imbalance going on, and a lot of the problems in the environment, such

a technique is encouraged.

However, the high cost of enzymes and products of lower oil yield than that of solvent

extraction method has been a major weakness of EAEP. Despite the weaknesses,

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productions of oil and protein with high quality have increased owing to the perceived

environmental advantages of EAEP.

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7. Acknowledgements

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The authors gratefully acknowledge the supported by the National High Technology

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Research and Development Program of China (2013AA102103). This study was also

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supported by the Natural Science Foundation of Jiang Su Province (BK2012116).

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Figure 1 Confocal micrograph of raw peanut(Li, Gasmalla, Zhang, Liu, Bing, & Yang,
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2016)

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Figure 2 Schematic process of enzyme-aided aqueous extraction.

Typical steps in EAEP are: (1) mechanical disruption of oilseeds by grinding; (2)

extraction of oil and protein with or without enzymes; (3) separation of an oil-rich

emulsion, insoluble solids, and a liquid containing water soluble; and (4)

de-emulsification of the oil-rich cream fraction to recover free oil.

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Table 1 Enzymes used for aqueous enzymatic extraction

oilseeds enzymes conditions Oil yields Reference
olive seeds pectinase, 25 mL/kgpaste; 15.72goil/100 (Aliakbarian,
cellulase, 60 min gpaste De Faveri,
hemicellulase Converti, &

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Perego, 2008)
soybean cellulase, Particle size 1 .5m 74.64% (Rosenthal,

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hemicellulase, Liquid-to-solid ratio Pyle, Niranjan,
pectinase, 0.05; Gilmour, &

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protease enzyme concentration Trinca, 2001)

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0.1%;
time of hydrolysis 30
min

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seed kernels pectinase, Temperature 50 6.94g/100g (Rui, Zhang,
of white cellulase, Time 60 min seed kernels Li, & Pan,
pitaya acid protease 2009)
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linseed pectinases, Temperature 34 70% (Gros,
hemicellulases pH 7 Lanoisell, &
cellulases 10:1 water-seed ratio Vorobiev,
2003)
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Moringa Termamyl 120L, Seed to water ratio 1:6 - (Abdulkarim,

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oleifera seed TypeL(a-Amylase), w/v; Long, Lai,

Neutrase 0.8L pH 6.8; Muhammad,
(Neutral protease), incubating & Ghazali,
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Celluclast 1.5 L FG temperature 45; 2005)

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(Cellulase) , incubating time 24 h;

Pectinex Ultra SP-L shaking rate 120 rpm;
(Pectinase)
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Soybean Protex7L(protease) Seed to water ratio 98.8% (Stephanie

1:10 w/v; Jung &
pH 7.0; Mahfuz, 2009)
incubating
temperature 50;
incubating time 1h;
shaking rate 300 rpm;
Grape seed cellulase, Time 24 h; 13.7% (Passos,
protease, pH 4; Yilmaz, Silva,
xylanase, temperature 3040 C; & Coimbra,
pectinase particle diameters 2009)
1.01.4 mm

Sesame seed Protex 7L, Seed to water ratio 1:6 57.4% (Latif &
Alcalase 2.4L, w/v ; Anwar, 2011)
Viscozyme L, Time 120;

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Natuzyme, temperature 45 C;
Kemzyme shaking rate 120 rpm

Bayberry Cellulose, enzyme amount 31.15% (Y.-l. Zhang,

kernels protease 3.17%(w/w), Li, Yin, Jiang,

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liquid/solid ratio 4.91 Yan, & Xu,
ml/g, 2012)

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time 4 h,
temperature 51.6 C.

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pumpkin seeds cellulase, enzyme concentration 64.17% (Gai, Jiao, Mu,

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pectinase , (1.4%, w/w), et al., 2013)
proteinase temperature (44 C),
time (66min) ,

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irradiation power
(419 W)
sunflower cellulases treatment time 2 h, 35.65 (Sineiro,
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seeds water/seeds ratio Dom nguez,
(7.5-8):1, Nunez, &
enzyme/seed ratio Lema, 1998)
1.25-1.4%
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soybean Alcalase pH 8, 29.48 % (Rovaris, et

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Celluclast , incubating time 4 h, al., 2012)

Alcalase incubating
Viscozyme L temperature 60
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Isatis Cellulase Enzyme concentration 59.27% (Jiao, et al.,

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indigotica Proteinase 1.82% (w/w), 2014)

seeds pectinase temperature 43,
time 83 min
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irradiation power 375

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Forsythia Cellulase enzyme cocktail 1.5% 21.62% (Gai, Jiao,
suspense Pectinase (w/w) Wei, et al.,
seed proteinase time 90 min 2013)

Lophira Pectinases Temperature 50 38% (Nonviho,

lanceolata cellulases Time 4 h Paris,
seed shaking rate 350 rpm Muniglia,
Sohounhlou,
& Brosse,
2015)

Peanut seed Alcalase 2.4L Temperature 60, 79.32% (Jiang, Hua,

pH 9.5, Wang, & Xu,
ratio of material to 2010)

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water 1:5 (w/w),

enzyme amount 1.5%
(w/w)
hydrolysis time 5 h
Jatropha Pectinex Ultra Temperature 50 47%, (Shah,

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seeds SP-L pH 9 Sharma, &
Promozyme time 18 h Gupta, 2005)

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Soybean Protex 51FP, Temperature 50, 88% (Wu, Johnson,

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Protex 6L pH 9.0, & Jung, 2009)

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Protex 7L time1 h
Soybean Protex 6L pH 8.0, 95.47% (S Jung,
Lyso MaxTM temperature 50 Maurer, &

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water/seeds ratio10:1 Johnson,
time 1 h 2009)
palm fruits Celluclast 1.5 L Time 1 h 80% (Teixeira,
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Pectinase Temperature 50 Macedo,
Multieffect FE Shaking rate 200rpm Macedo, da
Silva, &
Rodrigues,
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2013)
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Olive seed Pectinex Ultra pH 3.5 72.13% (Najafian,

SP-L temperature 35 Ghodsvali,
Pectinase 1.6021 time 60 min Khodaparast,
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shaking rate 80 rpm & Diosady,

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2009)
Peanut seed alcalase2.4L pH 8.5 92.2% (WANG, JIA,
temperature 60 & ZHANG,
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time 8 h 2008)
enzyme level 1.5%
Irvingia Alcalase, kernel-to-water ratio 90.0%. (Womeni,
gabonensis Pectinex 0.110.19, Ndjouenkeu,
seed kernels Viscozyme concentration of Kapseu,
enzyme 1.42.0%, Mbiapo,
time of incubation Parmentier, &
1418 h. Fanni, 2008)
C. oleifera protease a ratio of 1:7 w/v 83% (W. g. Zhang,
seeds Enzyme (2% w/v) Zhang, &
temperature 50 Chen, 2012)
time 3 h

Egyptian Ben Protease from ratio of material to - (Ahamd,

Seed Bacillus sp. water 1:6 w/v Abdel-Razik,
temperature 50 Hozayen, &

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time 24 h Shokry, 2015)

Perilla seeds Cellulase, Time 2h 81.74% (Li, Zhang,
Viscozyme L, Temperature 50 Sui, Zhang,
Alcalase 2.4L, ratio of material to Feng, & Jiang,
Protex 6L, water 1:6 w/v 2014)

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Protex 7L
Sesame seed papain, pH 7.0, 87.58 % (Hou, Shang,

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trypsin, temperature 50 C , Wang, & Liu,
cellulase time 3 h, 2013)

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shaking rate 80 rpm.

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Rice bran Alcalase pH 9.0, 79% (Hanmoungjai,
temperature 40-60C , Pyle, &
time 1-3 h Niranjan,

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2001)
Rice bran ProtizymeTM Temperature 65C, 77% (Sharma,
(protease), Shaking rate 80 rpm, Khare, &
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PalkodexTM Time 18 h Gupta, 2001)
(-amylase)
soybeans Protease, pH 8.0, 88% (Lamsal,
Cellulase temperature 50 C , Murphy, &
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ratio of material to Johnson,

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water 1:10 w/v, 2006)

shaking rate 200 rpm,
time 1 h
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Rapeseed pectinase, Temperature 60, 8890% (S. B. Zhang,

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cellulase, pH 11 Wang, & Xu,

-glucanase time 1 h 2007)
enzyme concentration
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of 2.5% (v/w)
Sunflower Protex 7L Temperature 45 C 87.25% (Latif &
seed Alcalase2.4L time 2 h Anwar, 2009)
Viscozyme L shaking rate 120 rpm
Soybean Protex 6L solids-to-liquid 96% (de Moura
ratio1:6, Bell, Maurer,
pH 9.0, Yao, Wang,
temperature 65C Jung, &
shaking rate 180 rpm Johnson,
time 1.5 h 2013)
Wheat Germ Cellulase, the ratio of material to 63.8% (Sharma &
Protease, water was 2.5:10, Gupta, 2001)
Amylase temperature 50 C,
time12 h,
the amount of single
enzyme 2 % (V/W)

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Rice Bran Cellulase, the ratio of material to 39.3% (Fang &

Protease, water was 2.5:10, Moreau, 2014)
Amylase temperature 50 C,
time12 h,

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the amount of single
enzyme 2 % (V/W)

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Wild apricot Cellulase, Time 2 h , 47.33 % (Bisht, et al.,
kernels Protease temperature 50C, 2015)

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enzyme concentration

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0.30.4 %
Moringa Neutrase 0.8L pH 7.5, 74.0% (Abdulkarim,
oleifera seed (neutral protease), temperature 60C Lai,

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Termamyl 120L, enzyme concentration Muhammad,
type L (a-amylase), 2% (v/w) Long, &
Pectinex Ultra SP-L time 36 h Ghazali, 2006)
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(pectinase), shaking speed 180
Celluclast 1.5L FG rpm
(cellulase)
Canola seeds Protex 7L, the ratio of material to 26.0% (Latif,
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Multifect Pectinase water was 1:6, Diosady, &

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FE, temperature 45C, Anwar, 2008)

Multifect CX 13L, shaking speed 120rpm
Natuzyme
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Yellow Viscozyme L 1st stage pH 4.8 3 h 76.3% (Tabtabaei &

mustard Pectinex Ultra SP-L 40 Diosady,
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Celluclast 1.5 L 2nd stage pH 11 30min 2013)

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Industrial Relevance

1. Enzymes are widely used on an industrial scale but mainly as catalysts, and

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the applications mainly concerning the synthesis of pharmaceuticals,

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compounds for chemistry of specialty, polymers, etc

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2. At the industrial scale, one advantage of EAEP is environment benefit because

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it can avoid the risk of organic solvents and particularly hexane

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3. The quality of the products produced by the smooth process is usually higher.

4. EAEP technique is considered as an alternative method to produce valuable

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products without loss of quality at moderate conditions.
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5. Compared with solvent extraction, EAEP is more eco-friendly. Besides that,

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one of the main advantages of EAEP is the specificity of enzymes.

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Highlights

1> EAEP is an efficient, environmentally friendly method to produce edible oil.

2> Process of EAEP is notified constantly by users.

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3> Application of EAEP is transforming from laboratory scale to industrial scale.

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