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Effect of freeze-thaw pretreatment on yield and quality of perilla seed oil
Journal Pre-proof Effect of freeze-thaw pretreatment on yield and quality of perilla seed oil Kyo-Yeon Lee, M. Shafiur Rahman, Ah-Na Kim, Yejin Son, Suyeon Gu, Myoung-Hee Lee, Jung In Kim, Tae Joung Ha, Doyeon Kwak, Hyun-Jin Kim, William L. Kerr, SungGil Choi PII:
S0023-6438(20)30014-1
DOI:
https://doi.org/10.1016/j.lwt.2020.109026
Reference:
YFSTL 109026
To appear in:
LWT - Food Science and Technology
Received Date: 17 June 2019 Revised Date:
29 November 2019
Accepted Date: 4 January 2020
Please cite this article as: Lee, K.-Y., Rahman, M.S., Kim, A.-N., Son, Y., Gu, S., Lee, M.-H., Kim, J.I., Ha, T.J., Kwak, D., Kim, H.-J., Kerr, W.L., Choi, S.-G., Effect of freeze-thaw pretreatment on yield and quality of perilla seed oil, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/ j.lwt.2020.109026. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Author statement
Authors Kyo-Yeon Lee
Contributions Experimental design, Conducted experiments and Methodology, data curation, primary draft, and reviewer (s) responses
M. Shafiur Rahman
Data curation, Methodology, writing origin draft, and reviewer (s) responses
Ah-Na Kim
Conducted experiments and Methodology
Yejin Son, Suyeon
Conducted experiments
Gu Myoung-Hee Lee,
Ideas, Planning, Funding acquisition, Project administration
Jung In Kim, Tae Joung Ha, and Doyeon Kwak Hyun-Jin Kim
Ideas, Planning, Data curation, and Statistical analysis
William L. Kerr
Review and editing
Sung-Gil Choi
Ideas, Planning, Study design, Review and editing, and supervision whole study
Effect of freeze-thaw pretreatment on yield and quality of perilla seed oil
1 2 3
Kyo-Yeon Leea, M. Shafiur Rahmanb,c, Ah-Na Kima, Yejin Sona, Suyeon Gua, Myoung-Hee
4
Leed, Jung In Kimd, Tae Joung Had, Doyeon Kwakd, Hyun-Jin Kimc, William L. Kerre and
5
Sung-Gil Choic*
6 7
a
8
South Korea
9
b
Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828,
Department of Food Engineering and Technology, State University of Bangladesh, Dhaka 1205,
10
Bangladesh
11
c
12
Gyeongsang National University, Jinju 52828, South Kore
13
d
14
e
15
GA 30602–2610, USA
Department of Food Science and Technology (Institute of Agriculture and Life Sciences),
Department of Southern Area Crop Science, NICS, RDA, Miryang, Korea
Department of Food Science and Technology, University of Georgia, 100 Cedar Street, Athens,
16 17 18
*Corresponding Author: Sung-Gil Choi
19
Email: [email protected]
20
Tel: +82-55-772-1906; Fax: +82-55-772-1909
21 22
1
23
Abstract
24
This work investigated the effect of freeze-thaw (FT) pretreatment on the yield and quality of oil
25
cold-pressed from perilla seeds. FT pretreatment ruptured the perilla seed coat and internal
26
structure, resulting in an oil yield of 78.71%, about 2.5-times greater than the yield from
27
untreated control perilla seeds. Acid values were relatively low (0.54 mg KOH/g) and not
28
different in the FT-treated and control oil. Likewise, peroxide values were low (1.34 meq/kg)
29
and not different amongst the treatment groups. Viscosity values (96.5 mPa s) were indicative of
30
a light oil while color values (L*a*b*) indicated a light yellow-green color. The major
31
unsaturated fatty acids were identified as linolenic acid (C18:3, ω−3), linoleic acid (C18:2, ω−6),
32
and oleic acid (C18:1, ω−9). The most abundant volatiles were 1-(furan-2-yl)-4-methylpentan-1-
33
one and 3-(4-methyl-3-pentenyl)-furan in both of the oil samples. However, the normalized
34
relative intensity of the volatile compounds was reduced in the FT-treated PO. The acylglycerol
35
profile in FT-treated and control PO was not different, yet another indication that FT
36
pretreatment did not increase oil rancidity or oxidative instability. FT pretreatment on oil seeds
37
before cold-pressing is an attractive technique for obtaining high oil yield without deteriorative
38
effects on the quality characteristics of edible oil.
39 40
Keywords: Perilla oil, freeze-thaw pretreatment, seeds microstructure, extraction yield, volatile
41
compounds, glycerides profile
42
2
43
1.
Introduction
44
Perilla (Perilla frutescens) belongs to the family Labiatae and is an aromatic vegetable that
45
is widely used for cooking and medicinal purposes in Asian countries, particularly India, China,
46
Japan and Korea (Li, Zhang, Hou, Li, & Chen, 2015; Zhang et al., 2018). Perilla seeds have 39 to
47
58% oil content (Zhao, Hong, Lee, Lee, & Kim, 2012), of which 53.6 to 64% of the fatty acids
48
are of the omega-3 variety (Yoon, & Noh, 2011). In addition to the high content (76.2%) of
49
polyunsaturated fatty acids (Li et al., 2015), other compounds such as vitamin E, sterols,
50
flavonoids, and phenolic compounds have been identified in perilla oil (PO) (Lee et al., 2013). It
51
has been reported that PO may potentially lower the risk of chronic diseases, prevent abnormal
52
clotting, relax blood vessels, reduce inflammation, and has antioxidant and anticancer properties
53
(Li et al., 2014).
54
It is difficult to extract all of the oil from seeds, particularly by mechanical method, so it
55
would be beneficial to develop methods for obtaining high oil yield from perilla seeds while
56
maintaining nutritional and quality characteristics (Li et al., 2015). Typically, solvent extraction
57
or mechanical pressing is used to obtain oil from oilseeds (Wroniak, Rękas, Siger, & Janowicz,
58
2016). Extraction with organic solvents has become less popular as some consumers are
59
concerned with the health implications and effects of solvent disposal on the environment
60
(Rahman et al., 2019). Mechanically expelled oil may be pressed by cold or hot methods. In the
61
former, the cleaned seeds are pressed directly. In the latter, the seeds are mashed and heated to
62
100-120°C prior to expelling. Hot pressing leads to greater oil yield but degrades heat-labile
63
compounds both in the oil and defatted meal. As a result, cold pressing is more popular in the
64
market for high-quality oil. Despite the many advantages of cold-pressing, low oil yield has
65
hindered this technique from becoming commercially widespread (Wroniak et al., 2016). 3
66
Researchers have studied several pretreatments for improving oil yields such as hot-air
67
roasting of the seeds, microwave irradiation and ultrasound-assisted hexane extraction (Jung et
68
al., 2012; Zhao et al. 2012; Li et al., 2015). Roasting seeds prior to cold-pressing can increase oil
69
yield (Wroniak et al., 2016) but reduce quality and stability as witnessed by greater acidity and
70
peroxide values (Bakhshabadi et al., 2017). PO has distinctive volatiles with characteristic odor
71
originated from raw seeds or roasting process, and that volatiles in PO are important quality
72
attribute affecting consumer acceptance (Park, Seol, Chang, Yoon, & Lee, 2011). In addition,
73
thermal pretreatments of oil seeds lead to meal proteins denaturation and oil oxidation, as well as
74
changes in fatty acids, sterols, phenolic compounds and tocopherol (Koubaa et al., 2016).
75
Freeze-thaw (FT) pretreatment of oil seeds could be a promising technique for improving
76
the yield and quality of the oil. Freezing and thawing can cause substantial structural changes in
77
plant tissues through the formation of large ice crystals and changes in ionic strength and pH
78
caused by freeze-concentration. These results in the disruption of membranes and breakdown of
79
high molecular weight molecules (Zhao, Dong, Li, Kong, & Liu, 2015) resulting in increased
80
surface hydrophobicity. It has been shown that freeze-thaw pretreatment can enhance the release
81
of bound bioactive compounds in corn (Jiao, Li, Chang, & Xiao, 2018). In addition, FT cycling
82
is an efficient and inexpensive method that induces the maximum degree of cell membrane
83
permeabilization (Meyer & Richter, 2001). Thus, it is reasonable that the softening of tissue from
84
FT treatment could help facilitate the release of oil from lipid vacuoles within the cells.
85
The objective of this study was to investigate the effect of FT pretreatment on the yield and
86
quality characteristics of oil obtained from perilla seeds. The yield was measured after 1-5 FT
87
cycles and compared with sample receiving no pretreatment. Oil quality was assessed in a
88
variety of ways including oil viscosity, acid value, and peroxide values. In addition, 4
89
triacylglycerol and fatty acid profiles were determined along with measurements of volatile
90
compounds present in the oils.
91 92
2.
Materials and methods
93
2.1. Materials
94
Perilla seeds (Perilla frutescens var. Daewoo) were collected from Chungbuk Agricultural
95
Research and Extension Service in the Republic of Korea. The perilla seeds were cleaned using
96
tap water to remove impurities and dried at 35 °C for 48 h using a laboratory convection dryer.
97
The dried seeds (moisture content 2%) were packaged in plastic bags and stored at 4 °C.
98 99
2.2. Reagents
100
The reagents including BF3-MeOH, 2-methyl-1-pentanol, divinylbenzene-carboxen-
101
polydimethylsiloxane (DVB/Carboxen/ PDMS), anhydrous sodium sulfate, sodium thiosulfate
102
and sodium hydroxide were of analytical grade and purchased from Sigma-Aldrich (Sigma-
103
Aldrich Corp., St. Louis, MO, USA). The HPLC grade methanol, acetonitrile and
104
dichloromethane were purchased from Dae Jung Chemical & Metal Co., Ltd., Shi-heung, South
105
Korea.
106 107
2.3. Freeze-thaw cycles
108
For the freeze-thaw treatment, 500 g of perilla seeds were placed in LLDPE zipper bags (SC
109
Johnson, Seoul, Korea) and frozen at -20 °C for 48 h. Subsequently, 500 mL of distilled water
110
was added to the frozen seeds inside the bag and maintained at 4 ± 1 °C inside a refrigerator for
111
24 h. The seeds were refrozen and thawed at the same temperatures and times, and the process 5
112
repeated for 1, 3, or 5 cycles. After freezing and thawing, the FT-treated seeds were dried at 35
113
°C to a moisture content of 2%. Subsequently, the treated seeds were packaged in LLDPE bags
114
and stored at 4 °C.
115 116
2.4. Hydraulic pressing of oil
117
Prior to pressing, the untreated control and FT-treated perilla seeds were kept in desiccators
118
containing P2O5 at room temperature to equilibrate the moisture content. This helped limit
119
variation in seed moisture content that might influence the oil yield. After that, 500 g of perilla
120
seeds were transferred into the oil expeller (Oil Love Premium, NATIONAL ENG CO., LTD,
121
Goyang, South Korea). The resulting PO was centrifuged for 15 min at 9500×g to remove
122
gummy layers. The oil in the upper layer was collected into glass containers and stored at -20 °C
123
until further analysis. The oil yield was calculated gravimetrically as: Oil yield % = W /W × 100
124 125
(1)
where W1 is the weight of oil obtained and W2 is the total oil content in the perilla seeds.
126 127
2.5. Perilla seed microstructure
128
A scanning electron microscope (SEM) (JSM-6701F, Jeol, Japan) was used to investigate
129
changes in perilla seeds microstructure before and after FT treatment. Samples were prepared
130
following a method modified by Wroniak et al. (2016). The sample was placed on SEM stub
131
using double-sided carbon tape and then sputter-coated with gold to make the sample conductive.
132
Images were collected using an accelerating voltage of 15 kV and back-scatter mode and
133
recorded digitally.
134 6
135
2.6. Physicochemical properties of perilla oil
136
2.6.1. Total oil in perilla seeds
137
The total oil content in perilla seeds was determined using AOAC Method 948.22 (2000).
138
The lipid was determined gravimetrically after samples were extracted with ether for 16 h in a
139
Soxhlet extractor.
140 141
2.6.2. Viscosity
142
Viscosity of the PO was measured using a rotational viscometer (DV II +, Brookfield
143
Engineering Labs, MA, USA). PO (25 mL) was placed in a 50 mL centrifuge tube and allowed
144
to equilibrate to room temperature. Prior to analyses, the viscometer with the SC4-34 cylindrical
145
spindle was calibrated using distilled water. The oil viscosity was determined at 20 rpm,
146
corresponding to a shear rate of 20.4 s-1.
147 148
2.6.3. Color value
149
The color profile of the control and FT-treated PO was determined using a colorimeter
150
(Minolta CR–300, Japan) to evaluate L* value (light–dark), a* value (red–green) and b* value
151
(yellow–blue). Before analysis, a standard white plate (Y=93.5, X=0.3132, y=0.3198) was used
152
for calibration of the machine.
153 154
2.6.4. Acid value and peroxide value
155
The acid value (AV) and peroxide value (PV) of control and FT-treated PO were measured
156
according to the method adopted by Lee et al. (2019). In short, the AV is the mass of KOH
157
required to neutralize 1 g of the oil. PV measures primary oxidation products, namely peroxides 7
158
that liberate iodine from potassium iodide. These are determined by titrating with sodium
159
thiosulfate in the presence of a starch indicator. AV was calculated by:
160
/
=
×
×
.
(2)
161
where V1 is the volume and N1 is the normality of KOH, and m is the sample mass. PV was
162
calculated as: !
163
"#/$
=
%& '
×(× )))×*
(3)
164
where V2 and Vo are the volumes of titrated sodium thiosulfate solution for the sample and blank,
165
respectively; c is the molar concentration of sodium thiosulfate; and T is the titre of thiosulfate
166
solution.
167 168
2.7. Fatty acid composition
169
The fatty acid composition of the oil samples was measured using gas chromatography (GC,
170
Agilent 7890A, Agilent Co., Palo Alto, CA, USA) equipped with a flame ionization detector
171
(FID). Fatty acid methyl esters (FAME) were prepared according to the method of Morrison and
172
Smith (1964) by methyl esterification using BF3-MeOH complex as a catalyst. The samples (2–6
173
µL) were injected into the GC. The FAMEs were separated using a highly polar, fused-silica
174
capillary column (CP-Sil 88; 100 m × 0.25 mm i.d, 0.2-µm film thickness; Agilent Technologies,
175
Santa Clara, CA, USA) and 100:1 split injection. The carrier gas helium was used with 300 kPa
176
head pressure. The temperatures were 220 and 250°C for the injector and FID detector,
177
respectively. The oven temperature was kept at 140 °C for 35 min. After that, the oven was
178
heated at 4 °C/min to 230 °C. The different fatty acids in the PO were identified based on their
179
retention time and quantified using their relative area percentage as compared to internal
180
standard. 8
181 182
2.8. Volatile compounds in PO
183
To analysis volatile compounds, PO samples (1 g) with 2-methyl-1-pentanol as an internal
184
standard were placed into a vial with a septum cap and heated at 50 ºC for 5 min. After heating, a
185
solid-phase microextraction (SPME) fiber (50/30 µm DVB/CAR/PDMS Stableflex, Sigma-
186
Aldrich, Saint Louis, MO, USA) was inserted into the vial cap to absorb volatile compounds in
187
the head space at the same temperature for 5 min. The absorbed volatile compounds were
188
immediately analyzed using gas chromatography-mass spectrometer (GC-MS) (Shimadzu,
189
Tokyo, Japan) equipped with a DB-WAX capillary column (30 m × 0.25 mm i.d. × 0.25 µm film
190
thickness, Agilent J&W, Santa Clara, CA, USA) with split ratios of 1:10. Helium was used as the
191
carrier gas at 1 mL/min. The injector temperature was set to 250 ºC, the oven temperature
192
program was initiated at 40 ºC for 3 min, increased at a rate of 5 ºC/min to 90 ºC, increased at a
193
rate of 19 ºC/min to 230 ºC, and held for 5 min. The GC column effluent was detected using a
194
Shimadzu GCMS-TQ 8030 MS with an electron ionization source (70 eV). The ion source and
195
interface temperatures were 230 ºC and 250 ºC, respectively. Data monitoring was performed in
196
the full scan mode (m/z 45-550). The scan event time and velocity were 0.3 sec and 2000
197
amu/sec, respectively. The volatiles was identified using retention indices (RIs, calculated using
198
a series of n-alkanes) and fragmentation spectra from GC-MS databases (NIST 11 and Wiley 9
199
mass spectral libraries). The normalized relative intensities were calculated on the basis of peak-
200
area ratios.
201 202 203
2.9. Glyceride profile The composition of mon-, di- and tri-glycerides was analyzed via ultra-performance liquid 9
204
chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS) (Waters,
205
Milford, MA, USA). The oil sample (0.5 g) was treated by mixing with 20 mL of chloroform:
206
methanol (2:1) and sonicated for 10 min. The diluted sample was transferred to the sample vials
207
for UPLC-Q-TOF MS analysis, then 1 µL injected onto the column. The different glycerides
208
were separated on an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm, Waters, USA).
209
The mobile phase consisted of solvent A (0.1% formic acid water) and solvent B (acetonitrile
210
containing 0.1% formic acid), with a flow rate of 0.4 mL/min at a temperature of 40 °C for 10
211
min. The components eluted were detected by a Xevo® G2–S Q–TOF MS in positive–ion mode.
212
The temperatures were 130 and 450 °C for the source and desolvation respectively, and the
213
voltages were set to 2000, 42, and 4 V for the capillary, sampling cone, and extraction cone,
214
respectively. The cone and desolvation gas flow rate were 10 and 900 L/h respectively, and the
215
collision energy was raised from 15 to 55 eV with a scan range of 50–1500 m/z.
216 217
2.10. Statistical analysis
218
All experiments were replicated three times and the data are presented as mean ± standard
219
deviation (n=3). The data for oil yield, and physicochemical properties and relative abundances
220
of the compounds were analyzed by analysis of variance (ANOVA) using SAS 9.4 statistical
221
software package (SAS Institute Inc, Cary, NC, USA). The Duncan test was used for the analysis
222
of significant differences (p < 0.05).
223
The processed GC-MS data were analyzed by multivariate statistics using SIMCA-P+
224
version 12.0.1 (Umetrics, Umeå, Sweden). For visualization of the analyzed data, partial least
225
squares discriminant analysis (PLS-DA) was used. The three parameters of R2X, R2Y, and Q2Y
226
were used to evaluate the quality of PLS-DA models, validated by permutation tests. The relative 10
227
abundances of the compounds were statistically analyzed using one-way analysis of variance
228
(ANOVA) with Duncan's test (p <0.05) by SPSS 17.0 (SPSS Inc., Chicago, IL).
229 230
3. Results and discussion
231
3.1. Oil yield
232
The total oil content of perilla seeds used in this study was 43.70 g/100 g. Oil yields from
233
untreated control and FT-treated perilla seeds are presented in Table 1. The oil yield substantially
234
increased with each FT-cycle, ranging from 33.11% with no treatment up to 78.71% after 5- FT
235
cycles, the latter of which was 2.5-times higher than the yield from untreated perilla seeds. It has
236
been reported that FT treatment of plant tissues ruptures the cell membranes and cell walls
237
(Phothiset & Charoenrein, 2014), allowing oil to be more easily released from vacuoles and
238
travel through the loosed plant tissue. Baby and Ranganathan (2016) reported that higher oil
239
yield could be directly related to the level of cell rupture. Zhao, Baik, Choi, & Kim (2014)
240
reported on a variety of methods for improving extraction of bioactive compounds from plant-
241
based materials such as high-pressure processing, pulsed electric fields or FT cycling. They
242
discussed how large ice crystals may compress membranes. In addition, freezing rate determines
243
ice crystal size, the extent of formation of intra- and extra-cellular ice crystals, and the degree to
244
which solutes are concentrated causing desiccation of cells. Jiao et al. (2018) found that freezing
245
temperature and consequently the freezing rate influenced the extraction efficiency of lutein and
246
zeaxanthin from corn. Greatest efficiency was attained at the slowest freezing rate, which was
247
also associated with the development of the largest ice crystals.
248
A few studies have examined the use of pretreatments for improving the yield of oil from
249
perilla seeds. Jung et al. (2012) found yields of 31.74, 39.56, and 41.76 g/100 g seeds for 11
250
samples extracted by mechanical pressing after roasting, supercritical-CO2 extraction, and
251
hexane-solvent extraction, respectively. Zhao et al. (2012) reported that PO yield from unroasted
252
seeds was 38.4 g/100 g seeds, increasing to as high as 44 g/100 g if the seeds were extensively
253
roasted prior to expelling. Wroniak et al. (2016) found that heat pretreatments such as roasting
254
oil seeds by convection oven or microwave irradiation are an effective means of increasing oil
255
yield. However, thermal treatments were shown to decrease oil quality as they create trans fatty
256
acids and certain tocopherols, and lead to more phosphorus in the oil (Zhao et al., 2012). It has
257
also been shown that heating changes compounds such as fatty acids, sterols, phenolics,
258
tocopherol, and meal proteins, and decreases the oxidative stability of oil (Koubaa et al., 2016;
259
Bakhshabadi et al., 2017). Thus, non-thermal pretreatments such as FT cycling of perilla seeds
260
may be an attractive method for providing high yield while maintaining oil quality.
261 262
3.2. Seed microstructure
263
The microstructure of FT-treated and untreated perilla seeds was investigated using scanning
264
electron microscopy (Fig. 1). A uniform spheroidal particle with an intact fibrous testa was
265
observed for untreated control perilla seeds (Fig. 1A). In contrast, the perilla seeds subjected to
266
FT had damaged seed coats, showing signs of splitting at the seams and exposing the inner
267
cotyledon, hypocotyl and radicle. The degree of testa separation increased with the number of FT
268
cycles. The inner seed portions also showed greater signs of splitting and fracturing after FT,
269
particularly after 5 FT cycles (Fig. 1D). This fracturing would expose more of the inner regions
270
of the seed, making it easier for oil to be transported to the surface when the seed was subject to
271
mechanical forces. Indeed, after 5 FT-cycles the surface of the endosperm took on an oily
272
appearance. At the cellular level, it is known that freeze-thaw cycling can disrupt cell walls, cell 12
273
membranes, lead to changes in cell size and cause rupture of the cells themselves (Phothiset &
274
Charoenrein, 2014). A similar report has been demonstrated in the microstructure of FT
275
pretreated corn (Jiao et al., 2018). They also reported that FT treatments convert polysaccharides
276
and glycoproteins present in the cell wall into shorter fragments, making them easier to rupture.
277 278
3.3. Physicochemical properties of perilla oil
279
Physicochemical properties including color, viscosity, AV, and PV of the POs are presented
280
in Table 2. The oils obtained from FT-treated and untreated perilla seeds were golden yellow in
281
color with similar viscosity (96.5 mPa s). This is one indication that the oil was not degraded by
282
the FT process, likely because the seeds were not subject to high temperature at any time. Min
283
and Jeong (1993) reported that viscosity of PO decreased with increasing roasting temperatures
284
when roasting was used to process perilla seeds. Similar observation on the effects of heat and
285
PO viscosity has been reported by Zhao et al. (2012). In our processing, the perilla seeds or oil
286
were never exposed to greater than ~25°C.
287
Likewise, no significant differences were found in the color values L*, a* or b* amongst the
288
oils extracted from the control and FT pretreated perilla seeds. Park et al. (2011) reported that
289
color changes occur in the PO due to chemical reactions including Maillard browning and
290
caramelization, and these reactions are more likely when perilla seeds are subjected to thermal
291
treatment. It was also reported that PO color gradually changed from light yellow to deep brown
292
with greater seed roasting time and temperature (Zhao et al., 2012) because of increased
293
browning byproducts and degradation of phospholipids in the oil (Anjum, Anwar, Jamil, & Iqbal,
294
2006).
295
The acid value (AV) and peroxide value (PV) are other important parameters for assessing 13
296
the quality of edible oils. High AV values are an indication of the hydrolytic rancidity of oil and
297
oil products (Cao, Ruan, Chen, Hong, & Cai, 2017). Free fatty acids arise from the breakdown of
298
triglycerides, catalyzed by inherent lipase enzymes. Peroxide values (PV) show the level of
299
primary autoxidation of double bonds in edible oils (Cao et al., 2017). As shown in Table 2,
300
there were no significant effects of FT-cycles on either AVs or PVs. AVs were 0.54 mg KOH/g,
301
while PVs were 1.34-1.35 meq/kg. These are relatively low values, although specific critical
302
values depend on the commodity and industry. For example, common industry standards for
303
almonds are AV<1.5 mg KOH/g and PV<5.0 meq/kg (Almond Board of California, 2014). The
304
facts that were no differences in AVs or PVs amongst the treatment groups again suggest that the
305
relatively low-temperature conditions of the process limited oxidative changes.
306 307
3.4. Fatty acid composition
308
Some pretreatments of oil seeds may lead to changes in fatty acid composition of the oil due
309
to the sensitivity of polyunsaturated fatty acids (Koubaa et al. 2016). For that reason, fatty acid
310
composition of the FT-treated PO was measured (Table 3). The major unsaturated fatty acids
311
were identified as linolenic acid (C18:3, ω−3), linoleic acid (C18:2, ω−6), and oleic acid (C18:1,
312
ω−9), with concentrations of ∼60%, ∼13%, and ∼16%, respectively. Similar results were
313
reported by Zhao et al. (2012) and Yoon and Noh (2011), who found that PO had 53.6 to 65%
314
omega-3 fatty acids.
315
No significant differences were found between the fatty acid composition of the control and
316
FT-treated POs. Again, this can be related to the relatively low temperature processing of the PO.
317
In contrast, several researchers (Anjum et al., 2006; Uquiche, Jeréz, & Ortíz, 2008; Wroniak et
318
al., 2016) have shown that polyunsaturated fatty acids in vegetable oils are reduced, and trans 14
319
fatty acids increased, after microwave or convection oven pretreatments. Moreover, oils
320
containing greater polyunsaturated fatty acids are more susceptible to oxidation during thermal
321
processing and storage (Sayyad et al., 2017), thus low temperature treatments such as FT may
322
not influence oxidative deterioration of oil.
323 324
3.5. Volatile compounds
325
A total of 17 volatile compounds were detected as major flavor compounds of PO by GC-
326
MS analysis. Among these volatiles, the 13 compounds were identified (Table 4). The major
327
volatile compounds identified in control and FT-treated PO were 1-(furan-2-yl)-4-methylpentan-
328
1-one, 3-(4-methyl-3-pentenyl)-furan, 3-methyl-butanal, 2,4-dimethylheptane, 2-methylbutanal,
329
2,3-dimethylpentanal,
330
methylpentan-1-one and 3-(4-methyl-3-pentenyl)-furan were the most abundant in both of the oil
331
samples. The normalized intensities of the identified compounds were statistically analyzed and
332
it was found that six compounds (2-methyl-2-butane, 2,4-dimethylheptane, 3-methylbutanal,
333
hexanal, 2-methyl-2-heptanol, 1-penten-3-ol) were significantly reduced by FT pretreatment. The
334
actual reasons for these differences are unclear. It might be expected that the damaged cellular
335
components of FT-treated perilla seeds led to the release of bound compounds. As a result, some
336
of the volatiles could be released from FT-treated seeds prior to pressing, thus the concentrations
337
of that volatiles were reduced or absent in FT-treated PO. By contrast, the tight seed coat for the
338
control sample fractured during pressing leading to the release of volatiles, which could be
339
transferred to the extracted oil. It was reported that FT pretreatment facilitated the release of
340
bound bioactive components from corn (Jiao et al. 2018).
trans,trans-2,4-heptadienal.
15
Among
volatiles,
1-(furan-2-yl)-4-
341
The types of volatile compounds and their intensities in PO may vary with perilla seed
342
species, pretreatments and extraction processes and their parameters (Liu, Wan, Zhao, & Chen
343
2013). Similar volatile compounds including 2,4-heptadienal, hexanal, butanone, and furan were
344
detected by Kim, Yoon and Rhee (2000) in untreated PO. However, the volatile components of
345
oil that have been subject to high temperatures are often quite different. Park et al. (2011)
346
reported that pyrazine (2,5-dimethylpyrazine) and 2-furancarboxaldehyde were two major
347
volatiles in PO from roasted seeds. Kim, Yoon and Rhee (2000) and Lee, Lee, Sung and Shin
348
(2015) reported that pyrazines and ketones are the most dominant volatile compounds in PO
349
obtained from roasted perilla seeds, and those volatiles were not detected in that of the unroasted
350
control PO. In our analyses, we did not find evidence of pyrazine or ketone-based volatile
351
compounds.
352 353
3.6. Acylglycerol profile
354
The profiles of acylglycerol composition of fats and oils can be changed by various factors,
355
including esterification and lipase activity during processing and storage, and the changes of the
356
profiles are directly associated with the quality of fats and oils. In particular, free fatty acids
357
produced by the decomposition of the acylglycerols can be easily oxidized compared to intact
358
acylglycerols and accumulation of the oxidized fatty acids are the main factors of fat and oil
359
rancidity (Gao, Wu, & Feng, 2019; Cao et al., 2017). Changes in the acylglycerol composition
360
may be evidence of decomposition, interesterification or lipase activity.
361 362
Acylglycerol composition of the PO used in this study was determined by UPLC-Q-TOF-
363
MS analysis. The chromatographic peaks of the control and FT-treated PO are shown in Fig. 2. 16
364
A total of 16 fragments were identified for mono-, di- and tri-acylglycerols in each oil sample.
365
The mass spectrum of the PO showed that PO was mainly composed of triacylglycerols (TGs).
366
The TG profiles were detected from mass-to-charge (m/z) 868 to 931, while m/z of the
367
monoacylglycerol (MGs) were 360 and 425, the diacylglycerol (DGs) were m/z 554, 628, and
368
702. However, there was no difference between acylglycerol profiles of control PO and FT-
369
treated PO except DG profile. Although the DGs (m/z 554 and 628) were only detected from
370
control PO, DG intensities were too lower than those of the TGs to affect the quality of the PO.
371
These results supported the AV and PV were not affected by the FT pretreatment. This also may
372
be attributed to the low-temperature processing that limits any substantial chemical changes in
373
the PO (Koubaa et al., 2016). It was reported that hydrolysis process in oil converts
374
triacylglycerols to monoacylglycerols, diacylglycerols, and free fatty acids (Cao et al. 2017), and
375
the conversion is hastened by higher temperature, water activity, and longer storage, resulting in
376
increased AV (Son et al., 2019). However, the FT pretreatment on perilla seeds had no effect on
377
the degradation of TGs.
378 379
3.
Conclusions
380
Cold pressing is a popular technique for high-quality edible oil. But, it gives low oil
381
recovery from oil seeds and further treatment is needed to extract residual oil using hot pressing
382
or solvent extraction which deteriorates oil quality. The FT pretreatment used in this study gave
383
about 2.5-times higher oil yield than that of the untreated control. The greater yield by FT
384
pretreatment was realized without causing significant changes in the chemical constituents and
385
physicochemical characteristics of the oil. When comparing oils from untreated and FT-treated
386
perilla seeds, there were no differences in acid values, primary oxidation products, oil viscosity, 17
387
fatty acid profiles, and glyceride composition. In addition, FT pretreatment did not influence on
388
increasing odor characteristic volatiles in the extracted oil, which may be an important
389
characteristic for consumer acceptance. Thus, FT pretreatment of perilla seeds could be
390
considered an attractive treatment for high yield with better quality oil that could be desirable for
391
oil industries.
392 393
Conflict of interest
394
There are no conflicts of interest to declare.
395 396
Acknowledgements
397
This study was carried out with the support of "Cooperative Research Program for
398
Agriculture Science & Technology Development (Project No. PJ0125012018)" Rural
399
Development Administration, Republic of Korea.
400 401
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Yoon, S. H., & Noh, S. (2011). Positional distribution of fatty acids in perilla (Perilla frutescens L.) oil. Journal of the American Oil Chemists' Society, 88(1), 157-158.
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Zhao, T., Hong, S. I., Lee, J., Lee, J. S., & Kim, I. H. (2012). Impact of roasting on the chemical
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Zhao, S., Baik, O. D., Choi, Y. J., & Kim, S. M. (2014). Pretreatments for the efficient extraction
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of bioactive compounds from plant-based biomaterials. Critical reviews in food science and
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nutrition, 54(10), 1283-1297.
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Zhao, J., Dong, F., Li, Y., Kong, B., & Liu, Q. (2015). Effect of freeze–thaw cycles on the
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emulsion activity and structural characteristics of soy protein isolate. Process Biochemistry,
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Zhang, Y., Mei, X., Zhang, Q., Li, M., Wu, X., & Ma, C. (2018). Protective effect of a
487
rosmarinic acid–rich extract from cold‐pressed Perilla frutescens seed flour on oxidative
488
hepatotoxicity in vitro and in vivo. Journal of Food Biochemistry, 42(2), e12492.
22
489
Figure captions
490 491 492
Fig. 1. Perilla seeds microstructure as affected by freeze-thaw (FT) pretreatment. (A) Control; (B), (C), and (D), 1-, 3-, and 5-times FT-treated seeds microstructure, respectively.
493 494 495
Fig. 2. Triacylglycerol profile in control (a) and freeze-thaw-pretreated (b) perilla oil analyzed by UPLC-Q-TOF MS. A; monoacylglycerols, B; diacylglycerols, C; triacylglycerols (TG).
23
Table 1 Oil yield from perilla seeds with and without pretreatment by freeze-thaw cycles Freeze-thaw cycles
Oil yield (%)
0
33.11±0.09d
1 60.01±0.11c 3 75.11±0.09b 5 78.71±0.04a All values are mean ± SD (n=3). Values not followed by the same superscript letter within a column are significantly different (p < 0.05) by Duncan’s test.
Table 2 Physicochemical properties of control and freeze-thaw treated perilla oil Color
Freeze-
Viscosity
Acid value
Peroxide value
thaw cycles
(mPa s)
L*
a*
b*
(mg KOH/g)
(meq/kg)
0
96.55±0.00a
48.21±0.01a
-1.45±0.01a
4.57±0.04a
0.54±0.01a
1.35±0.01a
1
96.55±0.00a
48.20±0.01a
-1.43±0.02a
4.60±0.01a
0.54±0.00a
1.34±0.00a
3
96.55±0.00a
48.21±0.01a
-1.45±0.01a
4.61±0.00a
0.54±0.00a
1.34±0.00a
5
96.54±0.01a
48.22±0.00a
-1.45±0.01a
4.61±0.00a
0.54±0.00a
1.34±0.00a
All values are mean ± standard deviation (n=3); Different letters in a column indicate significant differences (p < 0.05). L* = Lightness; a* = Redness; b* =Yellowness
Table 3 Fatty acid composition of control and freeze-thaw treated perilla oils Fatty acid composition (%)
Freeze-thaw cycles
C16:0
C18:0
C18:1
C18:2
C18:3
0
5.66±0.04a
2.41±0.10a
16.00±0.00a
13.20±0.01a
60.01±0.01a
1
5.67±0.04a
2.42±0.08a
15.99±0.01a
13.18±0.03a
59.91±0.10a
3
5.69±0.01a
2.41±0.10a
16.02±0.03a
13.16±0.04a
59.94±0.07a
5
5.69±0.00a
2.43±0.07a
16.03±0.03a
13.17±0.03a
59.96±0.04a
All values are mean ± SD (n=10). Different letters superscript within the same column that means are significantly different (p < 0.05) by Duncan’s test.
Table 4 Volatile compounds in control and FT-treated perilla oil analyzed using GC-MS RT1 (min)
Volatile Compounds
1.47
2-methyl-2-butane
2.05 2.18 3.11 3.16 3.75 6.54 7.36 8.81 10.20 14.15 14.88 15.23 15.83 16.46 16.51 18.92
2,3-dimethylpentanal 2,4-dimethylheptane 2-methylbutanal 3-methyl-butanal unknown 1 hexanal 2-methyl-2-heptanol 1-penten-3-ol unknown 2 unknown 3 3-hexan-1-ol 3-(4-methyl-3-pentenyl)-furan trans,trans -2,4-heptadienal 2,5-dihydroxybenzaldehyde unknown 4 1-(furan-2-yl)-4-methylpentan-1-one
Normalized Relative intensity Control FT-treated PO
p-value2
Identified2
Similarity
8.26±0.00a
0.00±0.00b
0.013
RI, MS
97
a
a
0.834
RI, MS
86
b
0.028
RI, MS
92
a
0.139
RI, MS
86
1.64±0.16
b
0.032
RI, MS
84
1.41±0.00
b
0.003
-
-
0.00±0.00
b
0.003
RI, MS
93
0.00±0.00
b
0.005
RI, MS
82
0.00±0.00
b
0.0001
RI, MS
98
1.70±0.00
b
0.019
-
-
1.20±0.00
b
0.008
-
-
0.00±0.00
a
0.069
RI, MS
94
3.19±0.22
a
0.200
RI, MS
93
1.42±0.37
a
0.846
RI, MS
91
0.86±0.01
a
0.064
RI, MS
76
b
0.0001
-
-
2.11±0.00
2.53±0.75
a
2.22±0.22
a
3.67±1.00
a
3.40±0.00
a
1.57±0.43
a
0.92±0.12
a
1.87±0.24
a
3.60±0.00
a
1.91±0.00
a
0.29±0.31
a
4.18±0.08
a
1.44±1.33
a
1.70±0.88
a
2.06±0.00
a
20.77±0.37
a
1.94±0.18 0.00±0.00
1.29±1.14
1.26±0.00
18.24±2.27
a
0.321
RI, MS 1
95 2
All values are mean ± standard deviation (n=3); Different superscript letters in a row indicate significant differences (p < 0.05). RT retention time, p-values were analyzed by t test, 2The volatiles were identified by standards of mass spectrum (MS) and/or retention indices (RI) values, FT=freeze-thaw.
Fig. 1.
Fig. 2 (a)
(b)
Highlights •
Freeze-thaw (FT) pretreatment on perilla seeds led to ruptured cellular structure.
•
FT treatment resulted in about 2.5-times higher oil yield (78.71%) than control sample.
•
Physicochemical properties and fatty acid profile in oil was not affected by FT treatment.
•
The volatile compounds were slightly reduced in FT-treated perilla oil.
•
Glyceride profiles in FT-treated oil were not changed by FT pretreatment.
S0023-6438(20)30014-1
DOI:
https://doi.org/10.1016/j.lwt.2020.109026
Reference:
YFSTL 109026
To appear in:
LWT - Food Science and Technology
Received Date: 17 June 2019 Revised Date:
29 November 2019
Accepted Date: 4 January 2020
Please cite this article as: Lee, K.-Y., Rahman, M.S., Kim, A.-N., Son, Y., Gu, S., Lee, M.-H., Kim, J.I., Ha, T.J., Kwak, D., Kim, H.-J., Kerr, W.L., Choi, S.-G., Effect of freeze-thaw pretreatment on yield and quality of perilla seed oil, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/ j.lwt.2020.109026. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Author statement
Authors Kyo-Yeon Lee
Contributions Experimental design, Conducted experiments and Methodology, data curation, primary draft, and reviewer (s) responses
M. Shafiur Rahman
Data curation, Methodology, writing origin draft, and reviewer (s) responses
Ah-Na Kim
Conducted experiments and Methodology
Yejin Son, Suyeon
Conducted experiments
Gu Myoung-Hee Lee,
Ideas, Planning, Funding acquisition, Project administration
Jung In Kim, Tae Joung Ha, and Doyeon Kwak Hyun-Jin Kim
Ideas, Planning, Data curation, and Statistical analysis
William L. Kerr
Review and editing
Sung-Gil Choi
Ideas, Planning, Study design, Review and editing, and supervision whole study
Effect of freeze-thaw pretreatment on yield and quality of perilla seed oil
1 2 3
Kyo-Yeon Leea, M. Shafiur Rahmanb,c, Ah-Na Kima, Yejin Sona, Suyeon Gua, Myoung-Hee
4
Leed, Jung In Kimd, Tae Joung Had, Doyeon Kwakd, Hyun-Jin Kimc, William L. Kerre and
5
Sung-Gil Choic*
6 7
a
8
South Korea
9
b
Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828,
Department of Food Engineering and Technology, State University of Bangladesh, Dhaka 1205,
10
Bangladesh
11
c
12
Gyeongsang National University, Jinju 52828, South Kore
13
d
14
e
15
GA 30602–2610, USA
Department of Food Science and Technology (Institute of Agriculture and Life Sciences),
Department of Southern Area Crop Science, NICS, RDA, Miryang, Korea
Department of Food Science and Technology, University of Georgia, 100 Cedar Street, Athens,
16 17 18
*Corresponding Author: Sung-Gil Choi
19
Email: [email protected]
20
Tel: +82-55-772-1906; Fax: +82-55-772-1909
21 22
1
23
Abstract
24
This work investigated the effect of freeze-thaw (FT) pretreatment on the yield and quality of oil
25
cold-pressed from perilla seeds. FT pretreatment ruptured the perilla seed coat and internal
26
structure, resulting in an oil yield of 78.71%, about 2.5-times greater than the yield from
27
untreated control perilla seeds. Acid values were relatively low (0.54 mg KOH/g) and not
28
different in the FT-treated and control oil. Likewise, peroxide values were low (1.34 meq/kg)
29
and not different amongst the treatment groups. Viscosity values (96.5 mPa s) were indicative of
30
a light oil while color values (L*a*b*) indicated a light yellow-green color. The major
31
unsaturated fatty acids were identified as linolenic acid (C18:3, ω−3), linoleic acid (C18:2, ω−6),
32
and oleic acid (C18:1, ω−9). The most abundant volatiles were 1-(furan-2-yl)-4-methylpentan-1-
33
one and 3-(4-methyl-3-pentenyl)-furan in both of the oil samples. However, the normalized
34
relative intensity of the volatile compounds was reduced in the FT-treated PO. The acylglycerol
35
profile in FT-treated and control PO was not different, yet another indication that FT
36
pretreatment did not increase oil rancidity or oxidative instability. FT pretreatment on oil seeds
37
before cold-pressing is an attractive technique for obtaining high oil yield without deteriorative
38
effects on the quality characteristics of edible oil.
39 40
Keywords: Perilla oil, freeze-thaw pretreatment, seeds microstructure, extraction yield, volatile
41
compounds, glycerides profile
42
2
43
1.
Introduction
44
Perilla (Perilla frutescens) belongs to the family Labiatae and is an aromatic vegetable that
45
is widely used for cooking and medicinal purposes in Asian countries, particularly India, China,
46
Japan and Korea (Li, Zhang, Hou, Li, & Chen, 2015; Zhang et al., 2018). Perilla seeds have 39 to
47
58% oil content (Zhao, Hong, Lee, Lee, & Kim, 2012), of which 53.6 to 64% of the fatty acids
48
are of the omega-3 variety (Yoon, & Noh, 2011). In addition to the high content (76.2%) of
49
polyunsaturated fatty acids (Li et al., 2015), other compounds such as vitamin E, sterols,
50
flavonoids, and phenolic compounds have been identified in perilla oil (PO) (Lee et al., 2013). It
51
has been reported that PO may potentially lower the risk of chronic diseases, prevent abnormal
52
clotting, relax blood vessels, reduce inflammation, and has antioxidant and anticancer properties
53
(Li et al., 2014).
54
It is difficult to extract all of the oil from seeds, particularly by mechanical method, so it
55
would be beneficial to develop methods for obtaining high oil yield from perilla seeds while
56
maintaining nutritional and quality characteristics (Li et al., 2015). Typically, solvent extraction
57
or mechanical pressing is used to obtain oil from oilseeds (Wroniak, Rękas, Siger, & Janowicz,
58
2016). Extraction with organic solvents has become less popular as some consumers are
59
concerned with the health implications and effects of solvent disposal on the environment
60
(Rahman et al., 2019). Mechanically expelled oil may be pressed by cold or hot methods. In the
61
former, the cleaned seeds are pressed directly. In the latter, the seeds are mashed and heated to
62
100-120°C prior to expelling. Hot pressing leads to greater oil yield but degrades heat-labile
63
compounds both in the oil and defatted meal. As a result, cold pressing is more popular in the
64
market for high-quality oil. Despite the many advantages of cold-pressing, low oil yield has
65
hindered this technique from becoming commercially widespread (Wroniak et al., 2016). 3
66
Researchers have studied several pretreatments for improving oil yields such as hot-air
67
roasting of the seeds, microwave irradiation and ultrasound-assisted hexane extraction (Jung et
68
al., 2012; Zhao et al. 2012; Li et al., 2015). Roasting seeds prior to cold-pressing can increase oil
69
yield (Wroniak et al., 2016) but reduce quality and stability as witnessed by greater acidity and
70
peroxide values (Bakhshabadi et al., 2017). PO has distinctive volatiles with characteristic odor
71
originated from raw seeds or roasting process, and that volatiles in PO are important quality
72
attribute affecting consumer acceptance (Park, Seol, Chang, Yoon, & Lee, 2011). In addition,
73
thermal pretreatments of oil seeds lead to meal proteins denaturation and oil oxidation, as well as
74
changes in fatty acids, sterols, phenolic compounds and tocopherol (Koubaa et al., 2016).
75
Freeze-thaw (FT) pretreatment of oil seeds could be a promising technique for improving
76
the yield and quality of the oil. Freezing and thawing can cause substantial structural changes in
77
plant tissues through the formation of large ice crystals and changes in ionic strength and pH
78
caused by freeze-concentration. These results in the disruption of membranes and breakdown of
79
high molecular weight molecules (Zhao, Dong, Li, Kong, & Liu, 2015) resulting in increased
80
surface hydrophobicity. It has been shown that freeze-thaw pretreatment can enhance the release
81
of bound bioactive compounds in corn (Jiao, Li, Chang, & Xiao, 2018). In addition, FT cycling
82
is an efficient and inexpensive method that induces the maximum degree of cell membrane
83
permeabilization (Meyer & Richter, 2001). Thus, it is reasonable that the softening of tissue from
84
FT treatment could help facilitate the release of oil from lipid vacuoles within the cells.
85
The objective of this study was to investigate the effect of FT pretreatment on the yield and
86
quality characteristics of oil obtained from perilla seeds. The yield was measured after 1-5 FT
87
cycles and compared with sample receiving no pretreatment. Oil quality was assessed in a
88
variety of ways including oil viscosity, acid value, and peroxide values. In addition, 4
89
triacylglycerol and fatty acid profiles were determined along with measurements of volatile
90
compounds present in the oils.
91 92
2.
Materials and methods
93
2.1. Materials
94
Perilla seeds (Perilla frutescens var. Daewoo) were collected from Chungbuk Agricultural
95
Research and Extension Service in the Republic of Korea. The perilla seeds were cleaned using
96
tap water to remove impurities and dried at 35 °C for 48 h using a laboratory convection dryer.
97
The dried seeds (moisture content 2%) were packaged in plastic bags and stored at 4 °C.
98 99
2.2. Reagents
100
The reagents including BF3-MeOH, 2-methyl-1-pentanol, divinylbenzene-carboxen-
101
polydimethylsiloxane (DVB/Carboxen/ PDMS), anhydrous sodium sulfate, sodium thiosulfate
102
and sodium hydroxide were of analytical grade and purchased from Sigma-Aldrich (Sigma-
103
Aldrich Corp., St. Louis, MO, USA). The HPLC grade methanol, acetonitrile and
104
dichloromethane were purchased from Dae Jung Chemical & Metal Co., Ltd., Shi-heung, South
105
Korea.
106 107
2.3. Freeze-thaw cycles
108
For the freeze-thaw treatment, 500 g of perilla seeds were placed in LLDPE zipper bags (SC
109
Johnson, Seoul, Korea) and frozen at -20 °C for 48 h. Subsequently, 500 mL of distilled water
110
was added to the frozen seeds inside the bag and maintained at 4 ± 1 °C inside a refrigerator for
111
24 h. The seeds were refrozen and thawed at the same temperatures and times, and the process 5
112
repeated for 1, 3, or 5 cycles. After freezing and thawing, the FT-treated seeds were dried at 35
113
°C to a moisture content of 2%. Subsequently, the treated seeds were packaged in LLDPE bags
114
and stored at 4 °C.
115 116
2.4. Hydraulic pressing of oil
117
Prior to pressing, the untreated control and FT-treated perilla seeds were kept in desiccators
118
containing P2O5 at room temperature to equilibrate the moisture content. This helped limit
119
variation in seed moisture content that might influence the oil yield. After that, 500 g of perilla
120
seeds were transferred into the oil expeller (Oil Love Premium, NATIONAL ENG CO., LTD,
121
Goyang, South Korea). The resulting PO was centrifuged for 15 min at 9500×g to remove
122
gummy layers. The oil in the upper layer was collected into glass containers and stored at -20 °C
123
until further analysis. The oil yield was calculated gravimetrically as: Oil yield % = W /W × 100
124 125
(1)
where W1 is the weight of oil obtained and W2 is the total oil content in the perilla seeds.
126 127
2.5. Perilla seed microstructure
128
A scanning electron microscope (SEM) (JSM-6701F, Jeol, Japan) was used to investigate
129
changes in perilla seeds microstructure before and after FT treatment. Samples were prepared
130
following a method modified by Wroniak et al. (2016). The sample was placed on SEM stub
131
using double-sided carbon tape and then sputter-coated with gold to make the sample conductive.
132
Images were collected using an accelerating voltage of 15 kV and back-scatter mode and
133
recorded digitally.
134 6
135
2.6. Physicochemical properties of perilla oil
136
2.6.1. Total oil in perilla seeds
137
The total oil content in perilla seeds was determined using AOAC Method 948.22 (2000).
138
The lipid was determined gravimetrically after samples were extracted with ether for 16 h in a
139
Soxhlet extractor.
140 141
2.6.2. Viscosity
142
Viscosity of the PO was measured using a rotational viscometer (DV II +, Brookfield
143
Engineering Labs, MA, USA). PO (25 mL) was placed in a 50 mL centrifuge tube and allowed
144
to equilibrate to room temperature. Prior to analyses, the viscometer with the SC4-34 cylindrical
145
spindle was calibrated using distilled water. The oil viscosity was determined at 20 rpm,
146
corresponding to a shear rate of 20.4 s-1.
147 148
2.6.3. Color value
149
The color profile of the control and FT-treated PO was determined using a colorimeter
150
(Minolta CR–300, Japan) to evaluate L* value (light–dark), a* value (red–green) and b* value
151
(yellow–blue). Before analysis, a standard white plate (Y=93.5, X=0.3132, y=0.3198) was used
152
for calibration of the machine.
153 154
2.6.4. Acid value and peroxide value
155
The acid value (AV) and peroxide value (PV) of control and FT-treated PO were measured
156
according to the method adopted by Lee et al. (2019). In short, the AV is the mass of KOH
157
required to neutralize 1 g of the oil. PV measures primary oxidation products, namely peroxides 7
158
that liberate iodine from potassium iodide. These are determined by titrating with sodium
159
thiosulfate in the presence of a starch indicator. AV was calculated by:
160
/
=
×
×
.
(2)
161
where V1 is the volume and N1 is the normality of KOH, and m is the sample mass. PV was
162
calculated as: !
163
"#/$
=
%& '
×(× )))×*
(3)
164
where V2 and Vo are the volumes of titrated sodium thiosulfate solution for the sample and blank,
165
respectively; c is the molar concentration of sodium thiosulfate; and T is the titre of thiosulfate
166
solution.
167 168
2.7. Fatty acid composition
169
The fatty acid composition of the oil samples was measured using gas chromatography (GC,
170
Agilent 7890A, Agilent Co., Palo Alto, CA, USA) equipped with a flame ionization detector
171
(FID). Fatty acid methyl esters (FAME) were prepared according to the method of Morrison and
172
Smith (1964) by methyl esterification using BF3-MeOH complex as a catalyst. The samples (2–6
173
µL) were injected into the GC. The FAMEs were separated using a highly polar, fused-silica
174
capillary column (CP-Sil 88; 100 m × 0.25 mm i.d, 0.2-µm film thickness; Agilent Technologies,
175
Santa Clara, CA, USA) and 100:1 split injection. The carrier gas helium was used with 300 kPa
176
head pressure. The temperatures were 220 and 250°C for the injector and FID detector,
177
respectively. The oven temperature was kept at 140 °C for 35 min. After that, the oven was
178
heated at 4 °C/min to 230 °C. The different fatty acids in the PO were identified based on their
179
retention time and quantified using their relative area percentage as compared to internal
180
standard. 8
181 182
2.8. Volatile compounds in PO
183
To analysis volatile compounds, PO samples (1 g) with 2-methyl-1-pentanol as an internal
184
standard were placed into a vial with a septum cap and heated at 50 ºC for 5 min. After heating, a
185
solid-phase microextraction (SPME) fiber (50/30 µm DVB/CAR/PDMS Stableflex, Sigma-
186
Aldrich, Saint Louis, MO, USA) was inserted into the vial cap to absorb volatile compounds in
187
the head space at the same temperature for 5 min. The absorbed volatile compounds were
188
immediately analyzed using gas chromatography-mass spectrometer (GC-MS) (Shimadzu,
189
Tokyo, Japan) equipped with a DB-WAX capillary column (30 m × 0.25 mm i.d. × 0.25 µm film
190
thickness, Agilent J&W, Santa Clara, CA, USA) with split ratios of 1:10. Helium was used as the
191
carrier gas at 1 mL/min. The injector temperature was set to 250 ºC, the oven temperature
192
program was initiated at 40 ºC for 3 min, increased at a rate of 5 ºC/min to 90 ºC, increased at a
193
rate of 19 ºC/min to 230 ºC, and held for 5 min. The GC column effluent was detected using a
194
Shimadzu GCMS-TQ 8030 MS with an electron ionization source (70 eV). The ion source and
195
interface temperatures were 230 ºC and 250 ºC, respectively. Data monitoring was performed in
196
the full scan mode (m/z 45-550). The scan event time and velocity were 0.3 sec and 2000
197
amu/sec, respectively. The volatiles was identified using retention indices (RIs, calculated using
198
a series of n-alkanes) and fragmentation spectra from GC-MS databases (NIST 11 and Wiley 9
199
mass spectral libraries). The normalized relative intensities were calculated on the basis of peak-
200
area ratios.
201 202 203
2.9. Glyceride profile The composition of mon-, di- and tri-glycerides was analyzed via ultra-performance liquid 9
204
chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS) (Waters,
205
Milford, MA, USA). The oil sample (0.5 g) was treated by mixing with 20 mL of chloroform:
206
methanol (2:1) and sonicated for 10 min. The diluted sample was transferred to the sample vials
207
for UPLC-Q-TOF MS analysis, then 1 µL injected onto the column. The different glycerides
208
were separated on an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm, Waters, USA).
209
The mobile phase consisted of solvent A (0.1% formic acid water) and solvent B (acetonitrile
210
containing 0.1% formic acid), with a flow rate of 0.4 mL/min at a temperature of 40 °C for 10
211
min. The components eluted were detected by a Xevo® G2–S Q–TOF MS in positive–ion mode.
212
The temperatures were 130 and 450 °C for the source and desolvation respectively, and the
213
voltages were set to 2000, 42, and 4 V for the capillary, sampling cone, and extraction cone,
214
respectively. The cone and desolvation gas flow rate were 10 and 900 L/h respectively, and the
215
collision energy was raised from 15 to 55 eV with a scan range of 50–1500 m/z.
216 217
2.10. Statistical analysis
218
All experiments were replicated three times and the data are presented as mean ± standard
219
deviation (n=3). The data for oil yield, and physicochemical properties and relative abundances
220
of the compounds were analyzed by analysis of variance (ANOVA) using SAS 9.4 statistical
221
software package (SAS Institute Inc, Cary, NC, USA). The Duncan test was used for the analysis
222
of significant differences (p < 0.05).
223
The processed GC-MS data were analyzed by multivariate statistics using SIMCA-P+
224
version 12.0.1 (Umetrics, Umeå, Sweden). For visualization of the analyzed data, partial least
225
squares discriminant analysis (PLS-DA) was used. The three parameters of R2X, R2Y, and Q2Y
226
were used to evaluate the quality of PLS-DA models, validated by permutation tests. The relative 10
227
abundances of the compounds were statistically analyzed using one-way analysis of variance
228
(ANOVA) with Duncan's test (p <0.05) by SPSS 17.0 (SPSS Inc., Chicago, IL).
229 230
3. Results and discussion
231
3.1. Oil yield
232
The total oil content of perilla seeds used in this study was 43.70 g/100 g. Oil yields from
233
untreated control and FT-treated perilla seeds are presented in Table 1. The oil yield substantially
234
increased with each FT-cycle, ranging from 33.11% with no treatment up to 78.71% after 5- FT
235
cycles, the latter of which was 2.5-times higher than the yield from untreated perilla seeds. It has
236
been reported that FT treatment of plant tissues ruptures the cell membranes and cell walls
237
(Phothiset & Charoenrein, 2014), allowing oil to be more easily released from vacuoles and
238
travel through the loosed plant tissue. Baby and Ranganathan (2016) reported that higher oil
239
yield could be directly related to the level of cell rupture. Zhao, Baik, Choi, & Kim (2014)
240
reported on a variety of methods for improving extraction of bioactive compounds from plant-
241
based materials such as high-pressure processing, pulsed electric fields or FT cycling. They
242
discussed how large ice crystals may compress membranes. In addition, freezing rate determines
243
ice crystal size, the extent of formation of intra- and extra-cellular ice crystals, and the degree to
244
which solutes are concentrated causing desiccation of cells. Jiao et al. (2018) found that freezing
245
temperature and consequently the freezing rate influenced the extraction efficiency of lutein and
246
zeaxanthin from corn. Greatest efficiency was attained at the slowest freezing rate, which was
247
also associated with the development of the largest ice crystals.
248
A few studies have examined the use of pretreatments for improving the yield of oil from
249
perilla seeds. Jung et al. (2012) found yields of 31.74, 39.56, and 41.76 g/100 g seeds for 11
250
samples extracted by mechanical pressing after roasting, supercritical-CO2 extraction, and
251
hexane-solvent extraction, respectively. Zhao et al. (2012) reported that PO yield from unroasted
252
seeds was 38.4 g/100 g seeds, increasing to as high as 44 g/100 g if the seeds were extensively
253
roasted prior to expelling. Wroniak et al. (2016) found that heat pretreatments such as roasting
254
oil seeds by convection oven or microwave irradiation are an effective means of increasing oil
255
yield. However, thermal treatments were shown to decrease oil quality as they create trans fatty
256
acids and certain tocopherols, and lead to more phosphorus in the oil (Zhao et al., 2012). It has
257
also been shown that heating changes compounds such as fatty acids, sterols, phenolics,
258
tocopherol, and meal proteins, and decreases the oxidative stability of oil (Koubaa et al., 2016;
259
Bakhshabadi et al., 2017). Thus, non-thermal pretreatments such as FT cycling of perilla seeds
260
may be an attractive method for providing high yield while maintaining oil quality.
261 262
3.2. Seed microstructure
263
The microstructure of FT-treated and untreated perilla seeds was investigated using scanning
264
electron microscopy (Fig. 1). A uniform spheroidal particle with an intact fibrous testa was
265
observed for untreated control perilla seeds (Fig. 1A). In contrast, the perilla seeds subjected to
266
FT had damaged seed coats, showing signs of splitting at the seams and exposing the inner
267
cotyledon, hypocotyl and radicle. The degree of testa separation increased with the number of FT
268
cycles. The inner seed portions also showed greater signs of splitting and fracturing after FT,
269
particularly after 5 FT cycles (Fig. 1D). This fracturing would expose more of the inner regions
270
of the seed, making it easier for oil to be transported to the surface when the seed was subject to
271
mechanical forces. Indeed, after 5 FT-cycles the surface of the endosperm took on an oily
272
appearance. At the cellular level, it is known that freeze-thaw cycling can disrupt cell walls, cell 12
273
membranes, lead to changes in cell size and cause rupture of the cells themselves (Phothiset &
274
Charoenrein, 2014). A similar report has been demonstrated in the microstructure of FT
275
pretreated corn (Jiao et al., 2018). They also reported that FT treatments convert polysaccharides
276
and glycoproteins present in the cell wall into shorter fragments, making them easier to rupture.
277 278
3.3. Physicochemical properties of perilla oil
279
Physicochemical properties including color, viscosity, AV, and PV of the POs are presented
280
in Table 2. The oils obtained from FT-treated and untreated perilla seeds were golden yellow in
281
color with similar viscosity (96.5 mPa s). This is one indication that the oil was not degraded by
282
the FT process, likely because the seeds were not subject to high temperature at any time. Min
283
and Jeong (1993) reported that viscosity of PO decreased with increasing roasting temperatures
284
when roasting was used to process perilla seeds. Similar observation on the effects of heat and
285
PO viscosity has been reported by Zhao et al. (2012). In our processing, the perilla seeds or oil
286
were never exposed to greater than ~25°C.
287
Likewise, no significant differences were found in the color values L*, a* or b* amongst the
288
oils extracted from the control and FT pretreated perilla seeds. Park et al. (2011) reported that
289
color changes occur in the PO due to chemical reactions including Maillard browning and
290
caramelization, and these reactions are more likely when perilla seeds are subjected to thermal
291
treatment. It was also reported that PO color gradually changed from light yellow to deep brown
292
with greater seed roasting time and temperature (Zhao et al., 2012) because of increased
293
browning byproducts and degradation of phospholipids in the oil (Anjum, Anwar, Jamil, & Iqbal,
294
2006).
295
The acid value (AV) and peroxide value (PV) are other important parameters for assessing 13
296
the quality of edible oils. High AV values are an indication of the hydrolytic rancidity of oil and
297
oil products (Cao, Ruan, Chen, Hong, & Cai, 2017). Free fatty acids arise from the breakdown of
298
triglycerides, catalyzed by inherent lipase enzymes. Peroxide values (PV) show the level of
299
primary autoxidation of double bonds in edible oils (Cao et al., 2017). As shown in Table 2,
300
there were no significant effects of FT-cycles on either AVs or PVs. AVs were 0.54 mg KOH/g,
301
while PVs were 1.34-1.35 meq/kg. These are relatively low values, although specific critical
302
values depend on the commodity and industry. For example, common industry standards for
303
almonds are AV<1.5 mg KOH/g and PV<5.0 meq/kg (Almond Board of California, 2014). The
304
facts that were no differences in AVs or PVs amongst the treatment groups again suggest that the
305
relatively low-temperature conditions of the process limited oxidative changes.
306 307
3.4. Fatty acid composition
308
Some pretreatments of oil seeds may lead to changes in fatty acid composition of the oil due
309
to the sensitivity of polyunsaturated fatty acids (Koubaa et al. 2016). For that reason, fatty acid
310
composition of the FT-treated PO was measured (Table 3). The major unsaturated fatty acids
311
were identified as linolenic acid (C18:3, ω−3), linoleic acid (C18:2, ω−6), and oleic acid (C18:1,
312
ω−9), with concentrations of ∼60%, ∼13%, and ∼16%, respectively. Similar results were
313
reported by Zhao et al. (2012) and Yoon and Noh (2011), who found that PO had 53.6 to 65%
314
omega-3 fatty acids.
315
No significant differences were found between the fatty acid composition of the control and
316
FT-treated POs. Again, this can be related to the relatively low temperature processing of the PO.
317
In contrast, several researchers (Anjum et al., 2006; Uquiche, Jeréz, & Ortíz, 2008; Wroniak et
318
al., 2016) have shown that polyunsaturated fatty acids in vegetable oils are reduced, and trans 14
319
fatty acids increased, after microwave or convection oven pretreatments. Moreover, oils
320
containing greater polyunsaturated fatty acids are more susceptible to oxidation during thermal
321
processing and storage (Sayyad et al., 2017), thus low temperature treatments such as FT may
322
not influence oxidative deterioration of oil.
323 324
3.5. Volatile compounds
325
A total of 17 volatile compounds were detected as major flavor compounds of PO by GC-
326
MS analysis. Among these volatiles, the 13 compounds were identified (Table 4). The major
327
volatile compounds identified in control and FT-treated PO were 1-(furan-2-yl)-4-methylpentan-
328
1-one, 3-(4-methyl-3-pentenyl)-furan, 3-methyl-butanal, 2,4-dimethylheptane, 2-methylbutanal,
329
2,3-dimethylpentanal,
330
methylpentan-1-one and 3-(4-methyl-3-pentenyl)-furan were the most abundant in both of the oil
331
samples. The normalized intensities of the identified compounds were statistically analyzed and
332
it was found that six compounds (2-methyl-2-butane, 2,4-dimethylheptane, 3-methylbutanal,
333
hexanal, 2-methyl-2-heptanol, 1-penten-3-ol) were significantly reduced by FT pretreatment. The
334
actual reasons for these differences are unclear. It might be expected that the damaged cellular
335
components of FT-treated perilla seeds led to the release of bound compounds. As a result, some
336
of the volatiles could be released from FT-treated seeds prior to pressing, thus the concentrations
337
of that volatiles were reduced or absent in FT-treated PO. By contrast, the tight seed coat for the
338
control sample fractured during pressing leading to the release of volatiles, which could be
339
transferred to the extracted oil. It was reported that FT pretreatment facilitated the release of
340
bound bioactive components from corn (Jiao et al. 2018).
trans,trans-2,4-heptadienal.
15
Among
volatiles,
1-(furan-2-yl)-4-
341
The types of volatile compounds and their intensities in PO may vary with perilla seed
342
species, pretreatments and extraction processes and their parameters (Liu, Wan, Zhao, & Chen
343
2013). Similar volatile compounds including 2,4-heptadienal, hexanal, butanone, and furan were
344
detected by Kim, Yoon and Rhee (2000) in untreated PO. However, the volatile components of
345
oil that have been subject to high temperatures are often quite different. Park et al. (2011)
346
reported that pyrazine (2,5-dimethylpyrazine) and 2-furancarboxaldehyde were two major
347
volatiles in PO from roasted seeds. Kim, Yoon and Rhee (2000) and Lee, Lee, Sung and Shin
348
(2015) reported that pyrazines and ketones are the most dominant volatile compounds in PO
349
obtained from roasted perilla seeds, and those volatiles were not detected in that of the unroasted
350
control PO. In our analyses, we did not find evidence of pyrazine or ketone-based volatile
351
compounds.
352 353
3.6. Acylglycerol profile
354
The profiles of acylglycerol composition of fats and oils can be changed by various factors,
355
including esterification and lipase activity during processing and storage, and the changes of the
356
profiles are directly associated with the quality of fats and oils. In particular, free fatty acids
357
produced by the decomposition of the acylglycerols can be easily oxidized compared to intact
358
acylglycerols and accumulation of the oxidized fatty acids are the main factors of fat and oil
359
rancidity (Gao, Wu, & Feng, 2019; Cao et al., 2017). Changes in the acylglycerol composition
360
may be evidence of decomposition, interesterification or lipase activity.
361 362
Acylglycerol composition of the PO used in this study was determined by UPLC-Q-TOF-
363
MS analysis. The chromatographic peaks of the control and FT-treated PO are shown in Fig. 2. 16
364
A total of 16 fragments were identified for mono-, di- and tri-acylglycerols in each oil sample.
365
The mass spectrum of the PO showed that PO was mainly composed of triacylglycerols (TGs).
366
The TG profiles were detected from mass-to-charge (m/z) 868 to 931, while m/z of the
367
monoacylglycerol (MGs) were 360 and 425, the diacylglycerol (DGs) were m/z 554, 628, and
368
702. However, there was no difference between acylglycerol profiles of control PO and FT-
369
treated PO except DG profile. Although the DGs (m/z 554 and 628) were only detected from
370
control PO, DG intensities were too lower than those of the TGs to affect the quality of the PO.
371
These results supported the AV and PV were not affected by the FT pretreatment. This also may
372
be attributed to the low-temperature processing that limits any substantial chemical changes in
373
the PO (Koubaa et al., 2016). It was reported that hydrolysis process in oil converts
374
triacylglycerols to monoacylglycerols, diacylglycerols, and free fatty acids (Cao et al. 2017), and
375
the conversion is hastened by higher temperature, water activity, and longer storage, resulting in
376
increased AV (Son et al., 2019). However, the FT pretreatment on perilla seeds had no effect on
377
the degradation of TGs.
378 379
3.
Conclusions
380
Cold pressing is a popular technique for high-quality edible oil. But, it gives low oil
381
recovery from oil seeds and further treatment is needed to extract residual oil using hot pressing
382
or solvent extraction which deteriorates oil quality. The FT pretreatment used in this study gave
383
about 2.5-times higher oil yield than that of the untreated control. The greater yield by FT
384
pretreatment was realized without causing significant changes in the chemical constituents and
385
physicochemical characteristics of the oil. When comparing oils from untreated and FT-treated
386
perilla seeds, there were no differences in acid values, primary oxidation products, oil viscosity, 17
387
fatty acid profiles, and glyceride composition. In addition, FT pretreatment did not influence on
388
increasing odor characteristic volatiles in the extracted oil, which may be an important
389
characteristic for consumer acceptance. Thus, FT pretreatment of perilla seeds could be
390
considered an attractive treatment for high yield with better quality oil that could be desirable for
391
oil industries.
392 393
Conflict of interest
394
There are no conflicts of interest to declare.
395 396
Acknowledgements
397
This study was carried out with the support of "Cooperative Research Program for
398
Agriculture Science & Technology Development (Project No. PJ0125012018)" Rural
399
Development Administration, Republic of Korea.
400 401
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402
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Figure captions
490 491 492
Fig. 1. Perilla seeds microstructure as affected by freeze-thaw (FT) pretreatment. (A) Control; (B), (C), and (D), 1-, 3-, and 5-times FT-treated seeds microstructure, respectively.
493 494 495
Fig. 2. Triacylglycerol profile in control (a) and freeze-thaw-pretreated (b) perilla oil analyzed by UPLC-Q-TOF MS. A; monoacylglycerols, B; diacylglycerols, C; triacylglycerols (TG).
23
Table 1 Oil yield from perilla seeds with and without pretreatment by freeze-thaw cycles Freeze-thaw cycles
Oil yield (%)
0
33.11±0.09d
1 60.01±0.11c 3 75.11±0.09b 5 78.71±0.04a All values are mean ± SD (n=3). Values not followed by the same superscript letter within a column are significantly different (p < 0.05) by Duncan’s test.
Table 2 Physicochemical properties of control and freeze-thaw treated perilla oil Color
Freeze-
Viscosity
Acid value
Peroxide value
thaw cycles
(mPa s)
L*
a*
b*
(mg KOH/g)
(meq/kg)
0
96.55±0.00a
48.21±0.01a
-1.45±0.01a
4.57±0.04a
0.54±0.01a
1.35±0.01a
1
96.55±0.00a
48.20±0.01a
-1.43±0.02a
4.60±0.01a
0.54±0.00a
1.34±0.00a
3
96.55±0.00a
48.21±0.01a
-1.45±0.01a
4.61±0.00a
0.54±0.00a
1.34±0.00a
5
96.54±0.01a
48.22±0.00a
-1.45±0.01a
4.61±0.00a
0.54±0.00a
1.34±0.00a
All values are mean ± standard deviation (n=3); Different letters in a column indicate significant differences (p < 0.05). L* = Lightness; a* = Redness; b* =Yellowness
Table 3 Fatty acid composition of control and freeze-thaw treated perilla oils Fatty acid composition (%)
Freeze-thaw cycles
C16:0
C18:0
C18:1
C18:2
C18:3
0
5.66±0.04a
2.41±0.10a
16.00±0.00a
13.20±0.01a
60.01±0.01a
1
5.67±0.04a
2.42±0.08a
15.99±0.01a
13.18±0.03a
59.91±0.10a
3
5.69±0.01a
2.41±0.10a
16.02±0.03a
13.16±0.04a
59.94±0.07a
5
5.69±0.00a
2.43±0.07a
16.03±0.03a
13.17±0.03a
59.96±0.04a
All values are mean ± SD (n=10). Different letters superscript within the same column that means are significantly different (p < 0.05) by Duncan’s test.
Table 4 Volatile compounds in control and FT-treated perilla oil analyzed using GC-MS RT1 (min)
Volatile Compounds
1.47
2-methyl-2-butane
2.05 2.18 3.11 3.16 3.75 6.54 7.36 8.81 10.20 14.15 14.88 15.23 15.83 16.46 16.51 18.92
2,3-dimethylpentanal 2,4-dimethylheptane 2-methylbutanal 3-methyl-butanal unknown 1 hexanal 2-methyl-2-heptanol 1-penten-3-ol unknown 2 unknown 3 3-hexan-1-ol 3-(4-methyl-3-pentenyl)-furan trans,trans -2,4-heptadienal 2,5-dihydroxybenzaldehyde unknown 4 1-(furan-2-yl)-4-methylpentan-1-one
Normalized Relative intensity Control FT-treated PO
p-value2
Identified2
Similarity
8.26±0.00a
0.00±0.00b
0.013
RI, MS
97
a
a
0.834
RI, MS
86
b
0.028
RI, MS
92
a
0.139
RI, MS
86
1.64±0.16
b
0.032
RI, MS
84
1.41±0.00
b
0.003
-
-
0.00±0.00
b
0.003
RI, MS
93
0.00±0.00
b
0.005
RI, MS
82
0.00±0.00
b
0.0001
RI, MS
98
1.70±0.00
b
0.019
-
-
1.20±0.00
b
0.008
-
-
0.00±0.00
a
0.069
RI, MS
94
3.19±0.22
a
0.200
RI, MS
93
1.42±0.37
a
0.846
RI, MS
91
0.86±0.01
a
0.064
RI, MS
76
b
0.0001
-
-
2.11±0.00
2.53±0.75
a
2.22±0.22
a
3.67±1.00
a
3.40±0.00
a
1.57±0.43
a
0.92±0.12
a
1.87±0.24
a
3.60±0.00
a
1.91±0.00
a
0.29±0.31
a
4.18±0.08
a
1.44±1.33
a
1.70±0.88
a
2.06±0.00
a
20.77±0.37
a
1.94±0.18 0.00±0.00
1.29±1.14
1.26±0.00
18.24±2.27
a
0.321
RI, MS 1
95 2
All values are mean ± standard deviation (n=3); Different superscript letters in a row indicate significant differences (p < 0.05). RT retention time, p-values were analyzed by t test, 2The volatiles were identified by standards of mass spectrum (MS) and/or retention indices (RI) values, FT=freeze-thaw.
Fig. 1.
Fig. 2 (a)
(b)
Highlights •
Freeze-thaw (FT) pretreatment on perilla seeds led to ruptured cellular structure.
•
FT treatment resulted in about 2.5-times higher oil yield (78.71%) than control sample.
•
Physicochemical properties and fatty acid profile in oil was not affected by FT treatment.
•
The volatile compounds were slightly reduced in FT-treated perilla oil.
•
Glyceride profiles in FT-treated oil were not changed by FT pretreatment.