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\begin{document}

\title{Impact of steaming and roasting heat-treatment on physico-chemical,
functional and digestibility of walnut kernel}



\author[1]{Xiaoyan Zhao}%
\author[2]{Xiangrui Ren}%
\author[2]{Hongkai Liu}%
\author[1]{Meng Wang}%
\author[2]{Xiaowei Zhang}%
\author[3]{Haifang Hu}%
\affil[1]{University of Jinan}%
\affil[2]{Affiliation not available}%
\affil[3]{Xinjiang Academy of Forestry Sciences}%


\vspace{-1em}



  
  \date{May 21, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
In order to develop the application of walnut kernel, the effect of
steaming and roasting treatment on the physicochemical and functional
properties of walnut kernel at 95 \selectlanguage{ngerman}ºC for different time (15, 20 and 30
min) was investigated, and compared to those of untreated sample.
Scanning electron microscopy suggested that heating treatment had a
notable effect on the microstructure of walnut kernel, especially the
steam heating. Both treatments significantly increased the enthalpy,
vitro protein digestibility, viscosity, G\selectlanguage{english}' and G\selectlanguage{english}" (P \textless{} 0.05),
the order from high to low was steaming \textgreater{} roasting
\textgreater{} untreated. All samples contained the amounts of essential
amino acids, the amino acid score (AAS) of samples by steaming was the
highest compared to that of the untreated and roasting, and the only
limiting amino acid of walnut kernel before or after heat treatment was
lysine. In addition, the protein of walnut kernel after heating
treatment with the extension of time contained more \selectlanguage{greek}α-\selectlanguage{english}helix and random
coil structures compared to the untreated sample, while \selectlanguage{greek}β-\selectlanguage{english}sheet and
\selectlanguage{greek}β-\selectlanguage{english}turns structures decreased. Moreover, the thermal treatment could
cause the changes of the water/oil holding capacity, foaming and
emulsifying properties of walnut kernel flour. When there were
differences between the results of steaming and roasting samples, it was
concluded that the water played an important role in steaming. These
results suggested that the thermal treatment as an effective approach
could improve the physico-chemical, structural and functional properties
of walnut kernel and be potentially applied in the food processing.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Walnut (\emph{Juglans regia L.} ) belongs to a tree nut due to
consisting of a hard nutshell protecting the kernel. It is
widely\textbf{c} oncerned and popular for its high nutritional value.
Walnut is distinguished by high oil content (43.3-78.48\%) of
unsaturated fatty acids (about 70\% total fatty acids) {[}1{]}. Besides,
walnut also an excellent source of protein, carbohydrate and
polyphenolic compounds {[}2,3{]}. Especially, the defatted walnut meal
is discarded as a by-product of oil extraction. It consists of the
abundant protein (about 55 d.w\%) and carbohydrate (about 28 d.w\%),
which can act as the high nutritional components in food products
{[}4{]}. Food and Drug Administration and European Food Safety Authority
declares that the walnut kernel can decrease the risk of heart disease,
cardiovascular disease, cancer, hypertension and all-cause {[}5{]}. In
short, the walnut kernel has the characteristics of improving human
health {[}5{]}. The references reported that thermal treatment had
significant influence on polyphenols content, vitamin E content and fat
acid profile {[}6{]}, and also improved the utilization and availability
of protein and reduced the anti-nutritional factors {[}7{]}. In
addition, the studies proved that the quality of walnut meal flour was
not damaged under mild thermal treatment procedures {[}6{]}, and applied
as a functional ingredient in food industry {[}3{]}.

However, the utilization of walnut in the food system is often limited
partially because of the digestibility and allergy of walnut protein
{[}8{]}. The thermal processing can conquer these shortcomings, which
was proved that it could be used to process beans and cereals, such as
soybean, common bean {[}9,10{]}. In general, the thermal processing
method is a common processing, which includes wet-heat and dry-heat
treatment. The wet-heat method transfers heat to food through steam or
water, such as boiling, steaming and cooking in simmering liquid; while
the dry-heat transfers to food by hot air, such roasting and air frying
{[}9{]}.

Roasting method is a kind of typical food hot processing, which can
improve the digestibility, eating qualities and bioavailability of food
products by modifying the physicochemical properties and structure of
food components {[}11{]}. The reason is due to biochemical changes in
process of roasting, such as hydrolysis, oxidation, reduction, and
resulting reactions from pyrolysis {[}11{]}. These changes also will be
helpful to enhance the color, flavor, taste active compounds, and
antioxidants and decrease the microbiological pollution, natural poison
and enzyme inhibitors in food {[}11{]}. The steaming method is
considered as one of the most common mechanical heating methods with the
features of low cost and convenience in food industry, which can affect
the texture, color, digestibility, structure and antioxidant compounds
in food material {[}12-14{]}. Moreover, it can also avoid the leaching
of nutritional compositions {[}12{]}.

Meanwhile, there is absent from the systematic report on the effects of
roasting and steaming methods on the physicochemical and functional
properties of walnut meal flour {[}6{]}. Such information is needed to
strengthen the study for this species from walnut meal as a major source
of protein and other nutritional requirements for healthy growth.
Therefore, the purpose of this study was to compare the effects of
roasting and steaming methods under different treatment time at the same
heating temperature on the vitro digestibility, physico-chemical and
functional properties of walnut meal flour.

\textbf{Procedures}

\textbf{Material}

Dried walnut (Xinxin No. 2) was supported by Xinjiang Academy of
Forestry Sciences (Urumqi, China) and harvested in September of 2022.
The shelled walnut kernel formed from a proximate composition of
20.08\selectlanguage{ngerman}±1.38\% protein, 5.11±0.18\% moisture content, 63.22±5.12\% oil and
1.71±0.06\% ash {[}15{]}. The norleucine was bought from Sigma Chemical
Co. (St Louis, MO, USA). The pepsin (3000 U mg\textsuperscript{-1}) and
pancreatin from porcine pancreas (4000 U mg\textsuperscript{-1}) were
purchased from the Novozymes (Bagsværd, Denmark). All other reagents
were analytical grade, and bought from Sinopharm Chemical Reagent Co.,
Ltd (Beijing, China).

\textbf{Sample Preparation}

The obtained shelled walnut kernel was thermally processed according to
a previously described method {[}9{]}. The moist-heat processing method
was as following: five hundred grams of shelled walnut kernel was
steamed in boiled water for 15, 20 and 30 min (S15, S20 and S30) at a
temperature of 95 ± 1.5 ºC, which were cooled to room temperature,
frozen for 24 h at -18 ºC, and lyophilized for 48 h at -55 ºC. The dry
heated processing method was as following: five hundred grams of shelled
walnut kernel was roasted in an air fryer (HD9252, Philips, Qingdao,
Shandong, China) at 95 ± 1.5 ºC for 15, 20 and 30 min (R15, R20 and
R30). The raw was as the untreated sample. Total samples were 7 (1
untreated + three moisture heated + three dry heated). The defatted
walnut kernel flour (100 mesh screen) by ground processing contained
about protein 44.85\%±1.21\%, oil 15.65\%±1.08\%, moisture content
5.32±0.53\% and ash 3.09±0.51\%.

\textbf{Scanning Electron Microscopy (SEM) Analysis}

The surface morphological characteristics of samples with different
thermal treatment were observed using a SU8100 scanning electron
microscope (Hitachi High- Technologies Corporation, Tokyo, Japan). The
sample powder was stuck on a specimen holder using a double-sided
conductive adhesive and then coated with a 10 nm gold film by ion
sputtering. An accelerating voltage of 15 kV was used during
observation.\textsuperscript{16} The sample images were obtained at a
magnification of 3000 times.

\textbf{Amino Acid Composition}

The determination of total amino acids of the walnut kernel flours (WKF)
was performed by high performance liquid chromatography (HPLC, Agilent
1260, Agilent Technologies Co., Ltd., Palo Alto, CA, USA, USA) according
to the described previously method of Trigui et al {[}17{]}. The HPLC
system was equipped with two columns (Styragel HMW7 and HMW6; Styragel,
Waters, Milford, MA, USA). Two hundred milligrams of samples were
hydrolyzed by 15 mL 6 M HCl for 24 h at 120 ºC. Then, the hydrolyzed
samples were mixed with 40 mL of 0.2 M citrate buffer (pH 2.2).
Norleucine was added as the standard solution with the amount of 1mL.
The mixture solution was diluted to 100 mL by citrate buffer. After the
samples were filtered by a 0.2 \selectlanguage{greek}μ\selectlanguage{english}m nylon filter, which was detected by
HPLC. The amino acid contents were expressed as g
100g\textsuperscript{-1} protein. Amino Acid Score (AAS) of amino acid
composition in samples was expressed as following the equation {[}18{]}.

\(\text{AAS\ }\left(\%\right)=\frac{mg\ of\ amino\ acid\ in\ 1\ g\ of\ test\ protein}{\text{mg\ of\ amino\ acid\ in\ requirement\ pattern}}\times 100\)(1)

\textbf{Fourier Transform Infrared (FTIR) Spectroscopy}

FTIR spectra of samples were scanned at room temperature by a Bruker
Vertex 70 FTIR spectrometer (Bruker Daltonics, Bremen, Germany) as
reported previously {[}9{]}. Different testing samples were acquired in
KBr tablets. The spectra were collected with a scanning range of
4000-500 cm\textsuperscript{-1} at a resolution 4
cm\textsuperscript{-1}and 32 scans. The secondary structure of protein
in untreated, moist-heated, and dry-heated walnut kernel was estimated
by a second derivative in the amide I region (1700--1600
cm\textsuperscript{-1}). Deconvolution of the second derivative of FTIR
was obtained by Savitsky--Golay algorithm from Origin Pro 2021 software
(Hearne Scientific Software).

\textbf{Thermal Properties}

The thermal properties of walnut kernel flour were measured by a
differential scanning calorimeter (DSC, TA Q20, New Castle, USA). Around
3.0 mg of sample was blended with 12 \selectlanguage{greek}μ\selectlanguage{english}L distilled water and locked in an
aluminum pan. The pans were kept at room temperature overnight before
determining. The samples were heated from 20 to 120 \selectlanguage{ngerman}ºC. Heating rate was
10 ºC min\textsuperscript{-1}. The reference was the empty
pan.\textsuperscript{19} The onset temperature(T\textsubscript{0}), peak
temperature(T\textsubscript{p}), conclusion temperature
(T\textsubscript{c}) and enthalpy change (\selectlanguage{english}^H) were used to reflect the
thermal properties of samples {[}19{]}.

\textbf{Rheological Properties}

Rheological properties of samples were investigated according to a
previous study {[}20{]}. Samples were prepared in 10 mg
mL\textsuperscript{-1} deionized water at 95 oC for 20 min, and kept
mixing for 60 min to ensure complete solubilization, then equilibrated
30 min at ambient temperature. After processing, the sample was laid in
an aluminum parallel plate and analyzed by a Discovery HR-2 rheometer
(TA Instruments Ltd., New Castle, DE). The experimental conditions were
as following: 40 mm diameter, 1 mm gap and 25 oC temperature. The strain
sweeping from 0.1 to 10\% was performed at 1 Hz frequency. The frequency
sweeps with a fixed strain of 1\% were measured by the range from 100 to
0.01 rad s\textsuperscript{-1} at room temperature within the linear
viscoelastic range. The storage modulus (G\selectlanguage{english}') and loss modulus (G\selectlanguage{english}'\selectlanguage{english}') were
automatically recorded. Data were collected according to the logarithmic
mode and performed in triplicate.

\textbf{Functional Properties}

Changes of water holding capacity (WHC) and oil holding capacity (OHC)
were determined according to the methods described by Choe et al. with
minor modifications {[}9{]}. The sample (1.0 g) was placed in
pre-weighed 25 mL centrifuge tubes and dispersed in 10 mL of distilled
water/soybean oil under magnetic stirring with occasional vortex
agitations for 10 min at room temperature. Afterwards, the samples were
centrifuged at 4000 x g for 15 min at ambient temperature. Then, the
centrifuge tube containing sediment was weighed. The WHC or OHC was
appraised as g water or oil per g dry sample, respectively.

The emulsifying capacity (EC) and emulsifying stability (ES) were
evaluated according to the described method of Di et al. with small
modifications {[}21{]}. Briefly, 5 mL of soybean oil and 5 mL of sample
solution (80 mg mL\textsuperscript{-1}) was mixed. Then the mixture was
homogenized by a high-speed shearing blender (PT-10-35GT, BUCHI
Labortechnik AG, Switzerland) at 10, 000 rpm for 2 min at room
temperature and centrifuged at 3000 xg for 5 min at room temperature.
The emulsion formed was admitted to keep for some minutes until the
emulsion layer was stable. EC was calculated as follows Di et
al:\textsuperscript{21}

\(\text{EC\ }\left(\%\right)=\frac{H_{2}}{H_{1}}\times 100\) (2)

Where H\textsubscript{1} was the total height of liquid, cm;
H\textsubscript{2} was the height of the emulsified layer, cm.

The above emulsion was centrifuged again after heating at 80 degC for 30
min and then cooled at room temperature. ES was expressed by the
following equation Di et al {[}21{]}:

\(\text{ES\ }\left(\%\right)=\frac{H_{4}}{H_{3}}\times 100\) (3)

Where H\textsubscript{3} was the total height of the liquid after
heating; cm; H\textsubscript{4} was the height of emulsion layer after
heating, cm.

The foaming capacity (FC) and foaming stability (FS) were evaluated
according to the described method of Di et al. with small modifications
{[}21{]}. Sample solutions were prepared at 10 mg
mL\textsuperscript{-1}and pH of the phosphate buffer was adjusted to
7.0. The solutions were whipped in graduated plastic tubes using a
homogenizer (PT-10-35GT, BUCHI Labortechnik AG, Switzerland) at 10, 000
rpm for 3 min at room temperature. The volume was recorded before and
after whipping. FC and FS were reported by the formula (4).

\(\text{FC\ }\left(\%\right)=\frac{V_{1}-V_{0}}{V_{0}}\times 100\)(4)

Where V\textsubscript{0} represented the volume prior to homogenization,
mL; V\textsubscript{1} represented the volume after homogenization, mL.

Following, the determination of FS was performed by a similar procedure
and the residual foam volume was measured after sample keeping for 30
min at ambient temperature. The formula (5) was applied to express the
FS value.

\(\text{FS\ }\left(\%\right)=\frac{V_{3}-V_{0}}{V_{0}}\times 100\)(5)

Where V\textsubscript{3} represented the residual volume after standing,
mL.

\textbf{In Vitro Digestibility (IVPD)}

The vitro digestibility of samples by thermal treatment conditions was
determined using the method of Hao et al {[}22{]} and Di et al.
{[}21{]}. The vitro gastric and intestinal digestion were simulated by
the pepsin and trypsin, respectively. The digestive base fluid
(simulated intestinal fluid (SIF) and simulated gastric fluid (SGF)) was
prepared according to Hao et al. before the experiment {[}22{]}. The
experimental steps were as follows: the sample solution (1g
100mL\textsuperscript{-1}) and an equal amount of SGF solution and
pepsin (2000 U mL\textsuperscript{-1}) were mixed, then the pH of
mixture solution was adjusted to 2.0 with 1 M HCl. Thereafter, the
mixture was incubated in Shaking Water Bath with 150 rpm at 37 oC. The
digestive solution was collected at different time including 0, 1, 5,
10, 30, 60 and 120 min. The gastric digestion was terminated by boiling
water bath immediately for 10 min. The digestion solution was
centrifugated at 4700 x g for 15 min at 8 degC. Then, the bicinchoninic
acid (BCA) method was used to determine the protein content in
supernatant {[}21{]}. The vitro digestibility rate of walnut meal flour
was expressed according to the following equation.

\(\text{IVPD}\left(\%\right)=\frac{PC_{0}-PC_{t}}{PC_{0}}\times 100\)(6)

Where PC\textsubscript{0} was the initial protein content;
PC\textsubscript{t} was the protein content after simulated in vitro
digestion.

SIF solution and sample solution digested by pepsin was mixed in a ratio
of 1:1 (v/v) and the trypsin solution with the activity of 1000 U
mL\textsuperscript{-1} was added at the same time. The pH of mixture
solution was adjusted to 7.0 with 1 M NaOH. Then, the mixture was
incubated and oscillated in a water bath at 37 oC for 0, 1, 5, 10, 30,
60 and 120 min. Finally, the intestinal digestion was ended in ice bath
for 30 min. After centrifugation, the protein content of supernatant was
measured as above described. All data was done in triplicate.

\textbf{Statistical Analysis}

The analysis and experiments of samples were expressed in triplicate.
The data were shown as mean +- standard deviation and analyzed by the
statistical software SPSS 19.0 (IBM, New York, USA). The differences
between means were performed by a one-way analysis of variance (ANOVA).
The significant differences of means were confirmed by the Tukey's test
with a significance value \emph{P} \textless{} 0.05.

\textbf{Results and Discussion}

\textbf{Morphological Characterization of Heat-treated Walnut Kernel}

The samples had shown a significant variation in microstructure and they
varied in the sizes and shapes (Fig. 1). SEM micrographs indicated that
the thermal processing could modify the microstructure of samples, which
varied in the sizes and shapes compared to the untreated sample (Fig.
1). The morphology of untreated walnut kernel flour presented a complex
spherical to oblong/elliptical granular structures together with the
smaller particles and irregularly shaped cellular fragments. The
granular structures might be starch granules, while the protein bodies
were displayed by the shape of smaller spheres particles {[}24{]}.
Generally, the protein, starch, cellulose molecules etc. were partly or
completely embedded together, for example the protein body substance was
surrounded by the smaller particles with spherical or irregular in shape
{[}24{]}. Here, the roasting and steaming at 100 oC for 15, 20 and 30
min had marked influence on the surface of granules compared to the
untreated sample. The clumping phenomenon in samples of thermal
treatment began to appear, and the change extent varied with heating
time (Fig. 1). The part granule integrity in WKF was kept after thermal
treatment for 15 min, whereas it was observed to significantly change at
20 and 30 min (\emph{P} \textless{} 0.05). Both flours treated by
steaming and roasting showed aggregated microstructure with irregular
surfaces. However, the aggregate of sample processed by steaming was
obviously larger than that of roasting (\emph{P} \textless{} 0.05). The
results indicated that the steaming had maximally influence on walnut
kernel compared to roasting treatment, which was likely attributed that
the gelatinization was easy to occur due to the presence of water in
steaming {[}25{]}.

\textbf{Amino Acid Composition Analysis}

Amino acid (AA) composition is an important chemical property of
protein. The reason is that it can determine the nutritional value of
protein {[}26{]}. AA composition of the untreated and heat-processed
samples of walnut kernel is exhibited in Table 1. The data of
experiments revealed that the protein in different WKF samples included
16 amino acids, including 7 essential amino acids (EAA) and 9
nonessential amino acids (NEAA) (Table 1). No significant difference was
found in the total content of AA between untreated, roasting and
steaming samples for 15 and 20 min (\emph{P}\textgreater{} 0.05). The AA
content of the sample was significantly higher than that of other
samples when it was steamed for 30 min (\emph{P} \textless{} 00.05).
From the Table 1, it was observed that the amount of acidic AA in
samples was higher than alkaline AA, which suggested that the WKF had
the acidic characteristic {[}27{]}. These findings were in agreement
with those previously reported for walnut meal flour {[}27{]}. Glutamic
(Glu), aspartic (Asp), and Arginine (Arg) in all WKF were relatively
abundant (Table 1), Asp contents in untreated, S15-S30 and R15-30 were
7.13, 5.77-7.24 and 6.04-5.10 g 100g\textsuperscript{-1}, respectively;
Glu contents were 19.15, 15.83-15.50 and18.60-17.66 g 100g
\textsuperscript{-1}, respectively; Arg contents were13.26, 13.48-15.18
and 13.90-13.31 g 100g\textsuperscript{-1}, respectively.

In addition, EAA content could reflect the nutritional value of protein
{[}28{]}. All samples contained EAA contained (except Tryptophan). The
total EAA contents in untreated, S15, S20, S30, R15, R20 and R30 were
26.72, 26.93, 27.89, 30.19, 27.07, 26.11 and 25.74 g
100g\textsuperscript{-1}, respectively, which occupied 30.42, 33.33,
32.12, 33.23, 31.08, 31.12 and 31.2 \%of the total AA content,
respectively. Therefore, the walnut kernel obtained by steam heating
could be regarded as high quality of natural proteins due to being
composed of the most of the EAA (Table 1). After different heat
treatments, the content of EAA in protein of walnut kernel changed to a
certain extent compared to the untreated sample. The steam treated
samples were significantly higher than the untreated and roasted samples
(\emph{P} \textless{} 0.05). After steaming, the total amount of total
EAA in protein increased significantly (\emph{P} \textless{} 0.05) with
the increase of heating time. Roasting treatment had no significant
effect on the total EAA content of walnut protein (\emph{P}
\textgreater{} 0.05). The results could be explained by their different
processing methods. The thermal stability of protein was comprehended by
the composition of AA.\textsuperscript{17} The higher the content of
hydrophobic AA in the protein, the more stable the protein was, which
suggested that the more stable protein contained the higher total
hydrophobic AA content. The hydrophobic AA content of the steaming
treated walnut kernel gradually increased compared with the untreated,
while the roasting treated walnut kernel gradually decreased, which was
due to a thermodynamically less stable for the protein of walnut kernel.
However, there was the small difference for total hydrophilic AA among
samples.

It is generally believed that the closer the Amino Acid Score (AAS) is
to 100, the closer the AA pattern in samples is to the recommended
value,\textsuperscript{18} which suggests that the nutritional value of
sample is higher. AAS of untreated and heated walnut kernels is observed
in Table 1. For walnut kernel samples before and after heat treatment,
the AAS of rest AA except lysine was closest to 100 or more. It was
illustrated that these amino acids could basically meet the recommended
requirements {[}18{]}. Lysine was the first limiting amino acid in
walnut kernel samples according to the AA score (Table 1) {[}18{]}. It
was worth noting that the lysine AAS by steaming treatment increased
from 39.78 to 47.56\%, while roasting treatment decreased from 39.78 to
37.11\%. This showed that the steaming treatment could improve the
nutritional value of protein in kernel. These results proved that the
AAS of walnut kernels by steaming processing significantly increased
compared to the untreated and roasting processing (\emph{P} \textless{}
0.05). Although the AAS of histidine, valine, leucine, isoleucine,
phenylalanine and threonine had decreased after roasting treatment, it
was still close to the recommended value of WHO/FAO/UNU Expert
Consultation {[}18{]}, and AAS of methionine by roasting treatment for
15 min increased from 71.76 to 77.65\%. These data indicated that the
two kinds of heat treatment could improve the nutritional value of
protein in walnut kernel. The amino acid content of each component of
protein in walnut kernel changed after different heat treatment, but
most EAA could still meet the recommended requirements. Therefore, the
walnut protein by different thermal processing could be used as a
high-quality protein ingredient in food {[}29{]}.

In addition, the different heat treatments also changed the amount of AA
composition of the protein in walnut kernel. The results showed that the
content of other AA contents except for glutamic gradually increased
with the extension of steaming treatment time. It indicated that the
steaming processing led to a significant increase in major AA contents,
which might be contributed to the release of bound amino acids during
steaming with water {[}30{]}. The increase in the amino acid levels
after the steaming of walnut kernel could inspire utilization in food
formulations. It was worth noting that the content of AA components
changed little when the roasting time was 15 min; when the roasting time
was extended to 20 and 30 min, other AA contents except for arginine
reduced. This phenomenon could interpret the higher concentration of
hydrophobic AA in steamed walnut kernel. Compared with the untreated and
steaming processing samples, the major AA content and total AA of
samples in roasting processing from 15 to 30 min gradually reduced. This
might be due to little water in roasting processing, which would lead to
the partial degradation or loss of protein or the result of Maillard
reaction between amino acids and sugars {[}31{]}.

\textbf{FTIR Analysis of Walnut Kernel During Thermal Treatment}

The untreated and heated walnut kernel samples were analyzed by FTIR
spectroscopy to study the change in amide I frequency ((1600--1700
cm\textsuperscript{-1}), which chiefly implies the C = O stretch
vibration of the amide group {[}28{]}. The corresponding secondary
structure of protein includes \selectlanguage{greek}α-\selectlanguage{english}helix (1648 and 1664
cm\textsuperscript{-1}), \selectlanguage{greek}β-\selectlanguage{english}sheet (1615--1637 cm\textsuperscript{-1} and
1682--1700 cm\textsuperscript{-1}), \selectlanguage{greek}β-\selectlanguage{english}turn (1664--1681
cm\textsuperscript{-1}) and random coil (1637 to 1648
cm\textsuperscript{-1}) {[}32{]}. Table 2 shows the percentage content
of the secondary structure of protein in walnut kernel treated with
different time of steaming and roasting. With the processing time
continued to increase, the change in the \selectlanguage{greek}β-\selectlanguage{english}sheet and \selectlanguage{greek}β-\selectlanguage{english}turns structures
gradually slowed down, while the \selectlanguage{greek}α-\selectlanguage{english}helix and random coil gradually rose
up. The percentage of \selectlanguage{greek}β-\selectlanguage{english}sheet structure (47.16\%) was the highest in
untreated sample, and the random coil occupied the lowest percentage
(12.86\%). It was interesting that the protein secondary structure in
walnut kernel significantly changed after the steaming and roasting
treatment. As shown Table 2, the relative contents of \selectlanguage{greek}β-\selectlanguage{english}sheet (39.91\%
and 35.67\%) and \selectlanguage{greek}β-\selectlanguage{english}turns (19.98\% and 19.19\%) of the samples by
steaming and roasting treatment for 30 min decreased, respectively,
which decreased by 18.17\% and 36.79\%,28.61\% and 41.79\% compared with
the native, respectively. Furthermore, the relative contents of \selectlanguage{greek}α-\selectlanguage{english}helix
(24.44\% and 25.92\%) and random coil (14.47\% and 16.85\%) of samples
by steaming and roasting treatment for 30 min increased, respectively,
which increased by 50.91\% and 54.44\%, 11.13\% and 23.68\% compared
with the untreated, respectively. The results exhibited that the \selectlanguage{greek}β-\selectlanguage{english}sheet
and \selectlanguage{greek}β-\selectlanguage{english}turns structure of protein in heat-treated walnut kernel were
transformed to \selectlanguage{greek}α-\selectlanguage{english}helix and random coil structures. It was supported by
the investigation of Vanga et al. {[}33{]}, which found that the \selectlanguage{greek}α-\selectlanguage{english}helix
and random coil structures of protein in peanut with an increase in the
heat treatment time increased. The reason for this phenomenon was that
the original rigid structure of the protein was destroyed to a certain
extent. Therefore, the flexibility of protein increased and the
conformation transformed from order to disorder {[}28,34{]}. On the
other hand, the hydrogen bonds of protein might be destroyed by heat,
which led to a greater degree of random distribution of molecular free
energy {[}28,35{]}. As a consequence, it was concluded that a set of
reactions in walnut kernels after steaming and roasting treatment
occurred a set of reactions, such as protein denaturation and
aggregation, which would cause the interconversion between \selectlanguage{greek}α-\selectlanguage{english}helix,
\selectlanguage{greek}β-\selectlanguage{english}sheet, \selectlanguage{greek}β-\selectlanguage{english}turn and random coil structures {[}28{]}.

At the same time, it was also found that the different secondary
structures of protein was presented in steaming and roasting walnut
kernels due to two distinct thermal processing (moisture and dry heating
method) (Table 2). It was obvious that the content of \selectlanguage{greek}β-\selectlanguage{english}sheets was
higher in walnut kernel by roasting treatment for 30 min compared to the
steaming treatment (36.67\%), which accounted for 39.91\% of the whole
secondary structure (Table 2). On the contrary, a low content of the
a-\selectlanguage{english}helix and random coil was determined for walnut kernel by roasting
treatment for 30 min, which was 24.44\% and 14.47\%, respectively, while
that of walnut kernel by steaming treatment was higher (25.92\% and
16.85\%, respectively). It could be inferred that the steaming and
roasting heating would cause the structural changes of protein in walnut
kernel, which was attributed to the denaturation, cross-linking and
aggregation of protein through heating treatment.\textsuperscript{36}

\textbf{Differential Scanning Calorimetry (DSC) Analysis}

The thermal properties of the protein in both heating treated (steaming
and roasting) and untreated walnut kernel samples are carried out by
DSC. The denaturation peak temperature (Tp), onset temperature (To),
endset temperature (Te) and denaturation enthalpy (\selectlanguage{greek}Δ\selectlanguage{english}H) of protein in
walnut kernel before and after heating treatment are summarized in Table
3. Tp, To, Te and \selectlanguage{greek}Δ\selectlanguage{english}H play the important roles in the thermal properties
of proteins, which is attributed to being closely connected with the
protein conformation. Tp is the temperature at which protein denatures,
To represents the initial temperature of the protein denaturation, Te is
the temperature of at which protein denaturation ends, while \selectlanguage{greek}Δ\selectlanguage{english}H
represents the energy required during protein denaturation {[}21,37{]}.
As shown Table 3, there was a significant difference for the Te and Tp
of protein in walnut kernel before and after heating treatment
(\emph{P}\textless{} 0.05). The results showed that the heating
processing increased the Te and Tp values of protein, the highest Tp
values were observed after 15 min of roasting and 30 min of steaming,
which was 102.83 \selectlanguage{ngerman}°C and 102.22 °C, respectively, the highest Te values
were found after 15 min of roasting and 20 min of steaming, which was
116.04 °C and 114.50 °C, respectively. While T\textsubscript{onset} in
all samples slightly changed. With increasing steaming and roasting
time, there is no significant change in Tp (\emph{P} \textgreater{}
0.05)\textsubscript{,} and To and Te of other samples except roasting
for 15 min also had not the significant difference
(\emph{P}\textgreater{} 0.05). It was also found that the denaturation
enthalpy (\selectlanguage{greek}Δ\selectlanguage{english}H) of protein in walnut kernel increased after steaming and
roasting treatment (Table 3). The increase of random coil and a-\selectlanguage{english}helix
proportions in protein can reflect the changes of \selectlanguage{greek}Δ\selectlanguage{english}H {[}38{]}. As can be
shown in Table 3, there are an increase in the \selectlanguage{greek}Δ\selectlanguage{english}H values of roasted and
steamed walnut kernel compared to that of untreated sample, which
suggested that the heating treatment could increase the unfolding
molecular structure of protein and change the stable structure of
protein {[}39{]}. The value of \selectlanguage{greek}Δ\selectlanguage{english}H was the highest in roasting and
steaming for 20 min, which was 7.07 and 8.69 J/g, respectively. The
result indicated that heating (roasting and steaming) treatment at 95 \selectlanguage{ngerman}°C
improved the denaturation temperature and \selectlanguage{greek}Δ\selectlanguage{english}H values of protein in walnut
kernel. However, it was obvious that \selectlanguage{greek}Δ\selectlanguage{english}H was lower in dry-heated
(roasting) samples compared to the moist--heat (steaming) samples as a
result of both different thermal processing methods.

\textbf{Rheological Properties}

The rheological properties of walnut kernel flours by roasting and
steaming treatment with different time were accessed as shown in Fig. 2.
The storage and loss modulus were G' and G'', respectively, which could
reflect the elasticity and viscosity of samples {[}40{]}. Fig. 2 A and B
exhibit the G' and G'' changes of untreated and heating processing
samples. It could be observed that the G' of all samples was greater
than the G'', which reflected the dominant viscoelastic solid-like
behavior in all samples {[}41{]}. As shown in Fig.2 (A and B), the G'
and G'' values of other samples except for the untreated and R15
increased slightly with increasing angular frequency, suggesting if the
samples formed a gel, it could be stable and strong {[}42{]}. However,
the G' and G'' of untreated and R15 samples gradually with increasing
frequency increased, which suggested that the gel network structure of
two samples was more fragile {[}43{]}. Compared with untreated walnut
kernel, heating treatment resulted in the increase of G' and G'' values,
which was explained that the protein molecular interaction was improved
due to the denaturation and exposure of hydrophobic groups during heat
processing {[}44{]}. It was worthy of remark that G' and G" values of
steaming treatment were higher than that of roasting processing, which
was attributed that the steam contained water during heating. de Vries
et al. reported that the addition of a small amount of water in protein
could promoted the hydrated protein aggregates during heating {[}45{]},
which would cause the increase of G' and G'' values. These results
indicated that the rheological properties of walnut kernel were
significantly altered by different heat treatments {[}43{]}.

Fig. 2 C describes the flow behavior of the walnut kernel flours with
different heating methods and time. As can be seen in Fig. 2 C, with the
increase of shear rate, all sample viscosity gradually reduced, which
showed the shear thinning behavior (pseudoplastic) {[}21{]}. It was
explained that the partial destruction of the protein network structure
in walnut kernel was caused by the increase of shear rate, which would
decrease the fluidity of the protein and the viscosity {[}21{]}.
Compared to the untreated walnut kernel, the initial viscosity of
R15-R30 and S15-S30 was increased, the order of viscosity values from
high to low was steaming \textgreater{} roasting \textgreater{}
untreated sample. In addition, with the extension of steaming and
roasting time, the viscosity of the samples increased, this might be
related with the changes in the protein structure after heating. It was
supposed that the heating treatment for 15-30 min could cause the
crosslinking of internal molecules {[}43{]}, namely, the formation of
three-dimensional network structure occurred due to the changes of
protein conformation in samples. on the other hand, the heat treatment
could increase the number of active molecules per unit volume, which
enhanced the effective collisions and interactions between molecules
{[}43{]}. It was concluded that the appropriate heat treatment could
lead to the unfolding of protein molecules and enhance the interaction
of molecules {[}43{]}. These findings further proved that the heat
processing could be helpful to result in the more interaction behavior
and improve the elastic properties of samples {[}43{]}.

\textbf{In Vitro Protein Digestibility (IVPD)}

The vitro protein digestion rate of untreated, steaming and roasting
walnut kernel was evaluated. The results of in vitro protein
digestibility of walnut kernel before and after heating are shown in
Fig. 3. For all samples, the rate of digestion in the intestine were
higher than those in the stomach because of the higher number of
catalytic sites on trypsin, which was supported by described finding of
Fang et al.\textsuperscript{40} Compared with untreated walnut kernel,
the IVPD of protein increased after heating, which indicated that the
heat-processing could enhance IVPD to some extent, which was agreement
with the results of Chinma et al. {[}46{]}. The reason might be that
heating treatment could improve the hydrolysis activity of intrinsic
protease and the solubility of protein in walnut kernel, and remove the
protease inhibitors, tannins, and phytic acid {[}46,47{]}. This also
indicated that the heat-processing could potentially improve the
digestibility of protein in food systems {[}48{]}. The digestion of
protein is also related to its secondary structure {[}49{]}. The
reference reported that the \selectlanguage{greek}α-\selectlanguage{english}helix content of proteins with heating
treatment increased and the \selectlanguage{greek}β-\selectlanguage{english}sheet content decreased, which would lead
to an increase of the food digestibility {[}50{]}. The results in this
study were agreement with the finding of Carbonaro et al. {[}50{]}. R30
and S30 presented the highest percentage of protein digestibility
(gastric digestion: 15.42\% and 17.83\%, respectively; intestinal
digestion: 22.37\% and 27.31\%, respectively), the lowest IVPD was
identified in untreated sample (gastric digestion: 4.18\%; intestinal
digestion: 7.58\%). It was attributed to the unfolding of protein
structure during heating treatment, that was that the more hydrophobic
groups were exposed, which would result in proteinase to produce the
additional binding sites. Then, the protein skeleton was decomposed and
the IVPD increased at last {[}21{]}. It was interesting that the
digestibility of samples by steaming treatment was significantly higher
than that of roasting treatment (\emph{P} \textless{} 0.05). One of the
main contributing factors to this phenomenon might be the water in
steaming {[}30{]}. Protein application value can be reflected by IVPD,
which also influences the quality of protein absorbed by the body. In
general, the high-quality proteins have high IVPD {[}21{]}. As more
amino acids from the protein backbone are released by proteolysis, which
can be better digested and absorbed by the body {[}21{]}. Consequently,
these results proved that the proper heat treatment could influence the
structural \emph{c} haracteristic (tertiary and secondary structure) of
protein, which would be helpful to enhancing the occupating coefficient
of protein in walnut kernel and expanding the applications in food
products.

\textbf{Functional Properties}

\textbf{Water and Oil Holding Capacity}

Because the water holding capacity (WHC) and oil holding capacity (OHC)
affect the application of samples in food, it is necessary to study the
functionality {[}51{]}. In this study, the effect of different heat
treatments on WHC and OHC of walnut meal flours were assessed. WHC of
untreated and treated WKF is depicted in Table 4. Compare to the
untreated and roasting samples, WHC values of steaming samples increased
significantly (\emph{P} \textless{} 0.05). The results were supported by
finding of Choe et al. {[}9{]}, they found that moist--heat treatment
could significantly increase WHC of whole seeds. The first reason
possibly was the structural changes of components in walnut kernel
during heating, which would lead to absorbing more water and improving
the hydration property {[}9{]}. Another reason was that the phenomenon
of starch swelling or gelatinization or damaging in walnut kernel
occurred during steaming treatment, which would cause the increase of
WHC due to more exposed hydroxyl regions with interacting with water
{[}9{]}. Finally, it also probably was due to the higher content of
hydrophilic amino acids in walnut kernel during steam processing
{[}9{]}. On the contrary, the roasting walnut kernel showed the higher
OHC than that of untreated or steaming treatment (Table 4), which was
attributed to the protein denaturation as well as the exposure of more
hydrophobic regions {[}52{]}.

However, WHC of R30 was significantly lower than untreated, while there
was non-significantly (\emph{P} \textgreater{} 0.05) between untreated
and R15. Additionally, with the extending of heating time, WHC gradually
reduced. Untreated sample had WHC of 2.84 g g\textsuperscript{-1} while
as steaming and roasting treated flours had WHC ranging from 4.43 to
3.45 g g\textsuperscript{-1} and 3.32 to 1.85 g g\textsuperscript{-1},
respectively. It was interesting that the walnut kernel by roasting
treatment showed the highest OHC, which was 5.67-5.73 g
g\textsuperscript{-1}, the untreated sample exhibited the lowest OHC of
1.21 g g\textsuperscript{-1}. However, no significant differences in OHC
were observed between roasting samples for 15, 20 and 30 min. In
steaming samples for 15, 20 and 30 min, the decrease in OHC might be
related with the low concentration of hydrophobic amino acids (Table 1)
{[}46{]}, which had a high attraction for oil. Therefore, OHC of S15
significantly was higher than that of S20 and S30. Because WHC and OHC
altered with heat processing, which should be thought over when the food
formulas were developed.

\textbf{Foaming Capacity and Stability}

The foaming capacity (FC) and stability (FS) of the samples are reported
in Table 4. The results clearly showed that the FC and FS of the samples
by roasting and steaming treatment were higher than that of the
untreated sample (Table 4). Untreated sample had a FC (62.13\%) and FS
(43.30\%) significantly lower than heating samples (\emph{P} \textless{}
0.05). The steaming and roasting samples both had good foaming
properties with FC (65.44-68.60\% and 69.17-71.11\%, respectively) and
FS (80.07- 67.48\% and 80.11-48.18\% respectively). With the changes of
heating methods, the FC and FS of the samples changed. The results
showed that the FC and FS of samples by steaming treatment were slightly
different than the roasting. When the roasting time was 30 min, the FC
of sample reached the maximum value, while FS with the extending of time
gradually reduced. However, FC during the steaming was not significantly
different between S15, S20 and S30, but the FS gradually decreased with
time from 15 to 30 min. The functional properties of protein can be
altered due to the changes of the secondary and tertiary structure
{[}22{]}. The conformation changes of protein also would cause the
changes of foaming properties {[}53{]}. some researchers have reported
that the partial unfolding of proteins can improve the FC and FS of
protein {[}22{]}. In the present study, the infrared data had shown that
the number of unfolded structures in protein increased. Therefore, the
application of walnut kernel flour depended on the processing
conditions, ingredient formulation during heating treatment.

\textbf{Emulsifying Capacity and Stability}

The above study showed that different heating methods and time affected
the conformation of protein in samples. It was trusted that the
structure changes could influence the efficacy of evolving oil droplets
due to the different of morphology and surface groups, thus, the
functional properties of protein might be affected {[}54{]}. The
emulsion capacity (EC) reflected the adsorption capacity of protein at
the water-oil interface, and the emulsion stability (ES) indicated the
retention ability of protein at the oil-water interface after emulsion
{[}54{]}. Table 4 shows the emulsion capacity (EC) and emulsion
stability (ES) of untreated and treated walnut kernel. Untreated sample
had the EC of 44.32\%. EC of roasting and steaming treated walnut kernel
samples ranged from 55.42 to 56.82\% and from 52.37 to 56.38\%,
respectively. EC of walnut kernel heated for 15, 20 and 30 min increased
significantly (\emph{P} \textless{} 0.05). However, EC of untreated
walnut kernel exhibited a significant (\emph{P} \textless{} 0.05)
difference compared to the heated samples. It was attributed that the
heating treatment could cause the changes of native structure or surface
properties of proteins in walnut kernel, which promoted more dissolution
and the adsorption at the interfacial area {[}22{]}. Furthermore, no
differences were found between the R15, R20, R30, S15 and S30. In
contrast, there was a significant but minor decrease in EA (52.37\%) for
S30 compared to other samples. In general, the emulsifying properties of
samples are due to depending on the strength of the formed interfacial
films of protein at the oil-water interface {[}55{]}. The increase of
protein adsorption and decrease of interfacial energy would hinder the
separation of two phases, which resulted in the high EC {[}56{]}.

Table 4 also shows the variation of the ES at different treatment
methods and time. The ES of samples with heating treatment was
significantly (\emph{P} \textless{} 0.05) higher compared to untreated
sample. It was further proved that the samples by heating treatment had
better ES due to the better surface characteristics that could be
beneficial to hinder the aggregation of droplet {[}57{]}. Untreated
sample represented ES of 74.21\% (Table 4). The ES of R15-R30 and
S15-S30 increased by 92.46 - 96.39\% compared to untreated samples,
which indicated a greater capability of the protein polymer in
heat-treated samples to adsorb on the surface of the oil droplet. It was
concluded that the heating treatment had the ability to enhance the ES
of protein in walnut kernel. It was farther confirmed that the unfolding
of proteins would be helpful to improving the surface assimilation and
maintenance of processing proteins at the oil-water interface, which
would lead to the better diffusion and ES {[}57{]}. On the whole, the
protein from heated walnut kernel might more reliably accumulate liquids
on the surface, which lead to a relatively stable emulsion. Therefore,
the samples obtained by heat treatment of walnut kernel would improve
the stability in applications of food products {[}57{]}.

\textbf{Conclusion}

The thermal treatment significantly influenced the physicochemical,
structural, functional and digestibility properties of protein in walnut
kernel flour in a temperature-dependent action. SEM micrographs reveled
the walnut kernel flour by roasting and steaming treatment more
sensitive than the untreated, while the steam treatment had more
influence on walnut kernel flour than the roasting treatment. The
secondary structures of protein in walnut kernel flours were affected by
heating as measured by FTIR, the protein structure changes of samples
for steamed processing happened to a greater extent than that of roasted
ones. It was found that the heat treatment could result in unfolding of
protein, which had significantly affected the functionality of samples.
The results showed that the roasting and steaming treatment affected the
amino acid composition and amino acid score (AAS), such as the roast
processing decreased the AAS for essential amino acids compared to the
untreated sample, while the steam processing increased the AAS. But the
lysine was the only limiting amino acid for all samples.

It was noted that the digestibility, water holding capacity and oil
holding capacity, foaming and emulsion capacities and stabilities of
walnut kernel flour during heat processing increased compared to
untreated, however, with the extension of heating time, these functional
properties would produce the different changes. At the same time, the
walnut kernel flours prepared by heating treatment were characterized by
high enthalpy (^H), storage modulus (G'), loss modulus (G'') and
viscosity compared to that of untreated sample, the order of these
values from high to low was the steaming \textgreater{} roasting
\textgreater{} untreated. Therefore, it could conclude that when the
heating technique would be used to alter the functional properties and
quality of walnut kernel flour and considered to have good utilization
promises in food industry. Further, it was proved that the moisture
pretreatment due to containing water in the modifications of functional
properties and quality of protein based on food materials was more
effective than dry-heating.

\textbf{Author Contribution Xiaoyan Zhao} : Project administration,
Resources, Supervision, Funding acquisition, Writing -- review \&
editing. \textbf{Haifang Hu} : Project administration, Resources,
Supervision, Funding acquisition. \textbf{Xiangrui Ren} : Investigation,
Writing -- original draft. \textbf{Xiaowei Zhang} : Data curation,
Methodology, Writing - original draft. \textbf{Hongkai Liu} :
Conceptualization, Software. \textbf{Meng Wang} : Validation, Formal
analysis.

\textbf{Acknowledgments} Financial support of this work by National
Natural Science Foundation of China (Grant No. 21406133) and Xinjiang
Key Scientific and Technological Project (Grant No. 2021A02002-2).

\textbf{Data availability} Xiaoyan Zhao can be contacted for the data

\textbf{Conflict of Interest} The authors declare that they have no
known competing financial interests or personal relationships that could
have appeared to influence the work reported in this paper.

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Figure captions:

Fig. 1. Scanning electron microscopy (SEM) images of the flours from
walnut kernel with roasting and steaming treatment. A: untreated walnut
kernel flour; B-D: walnut kernel treated by steaming for 15, 20 and 30
min, respectively; E-G: walnut kernel treated by roasting for 15, 20 and
30 min, respectively.

Fig. 2. The rheology properties of the walnut kernel flours with
roasting and steaming treatment. A: Plots of storage modulus G\selectlanguage{english}'; B:
Plots of loss modulus G\selectlanguage{english}'\selectlanguage{english}'; C: viscosity.

Fig. 3. The determined digestibility (\%) curves of walnut kernel flours
with roasting and steaming treatment during (A) gastric digestion and
(B) intestinal digestion.

Fig. 1.\selectlanguage{english}
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Fig. 3\selectlanguage{english}
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Table 1

The amino acid composition (g 100g\textsuperscript{-1} protein) and
amino Acid Score for adults (\%) of the untreated and heat-processed
walnut kernel with high content of protein.

Sample\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllllllll@{}}
\toprule
& & Untreated & Untreated & S15 & S15 & S20 & S20 & S30 & S30 & R15 &
R15 & R20 & R20 & R30 & R30\tabularnewline
& AA & AAS\textsuperscript{*} & AA & AA & AAS & AA & AAS & AA & AAS & AA
& AAS & AA & AAS & AA & AAS\tabularnewline
Essential amino acids & Essential amino acids & Essential amino acids &
Essential amino acids & Essential amino acids & Essential amino acids &
Essential amino acids & Essential amino acids & Essential amino acids &
Essential amino acids & Essential amino acids & Essential amino acids &
Essential amino acids & Essential amino acids & Essential amino acids &
Essential amino acids\tabularnewline
Histidine & 2.74\selectlanguage{ngerman}±0.03\textsuperscript{b} &
182.67\selectlanguage{ngerman}±0.53\textsuperscript{C} & 2.87\selectlanguage{ngerman}±0.07\textsuperscript{c} &
2.87\selectlanguage{ngerman}±0.07\textsuperscript{c} & 191.33\selectlanguage{ngerman}±0.62\textsuperscript{D} &
2.88\selectlanguage{ngerman}±0.03\textsuperscript{c} & 192.00\selectlanguage{ngerman}±0.18\textsuperscript{D} &
3.18\selectlanguage{ngerman}±0.05\textsuperscript{d} & 212.00\selectlanguage{ngerman}±0.32\textsuperscript{E} &
2.74\selectlanguage{ngerman}±0.14\textsuperscript{b} & 182.67\selectlanguage{ngerman}±0.61\textsuperscript{C} &
2.62\selectlanguage{ngerman}±0.05\textsuperscript{a} & 174.67\selectlanguage{ngerman}±0.43\textsuperscript{A} &
2.66\selectlanguage{ngerman}±0.04\textsuperscript{a} &
177.33\selectlanguage{ngerman}±0.28\textsuperscript{B}\tabularnewline
Lysine & 1.79\selectlanguage{ngerman}±0.01\textsuperscript{bc} & 39.78\selectlanguage{ngerman}±0.60 \textsuperscript{BC}
& 1.79\selectlanguage{ngerman}±0.02\textsuperscript{bc} & 1.79\selectlanguage{ngerman}±0.02\textsuperscript{bc} &
39.78\selectlanguage{ngerman}±0.56 \textsuperscript{BC} & 1.80\selectlanguage{ngerman}±0.02\textsuperscript{bc} &
40.00\selectlanguage{ngerman}±0.52 \textsuperscript{BC} & 2.14\selectlanguage{ngerman}±0.05\textsuperscript{d} &
47.56\selectlanguage{ngerman}±0.51\textsuperscript{D} & 1.79\selectlanguage{ngerman}±0.04\textsuperscript{bc} &
39.78\selectlanguage{ngerman}±0.62\textsuperscript{BC} & 1.74\selectlanguage{ngerman}±0.03\textsuperscript{b} &
38.64\selectlanguage{ngerman}±0.62\textsuperscript{B} & 1.67\selectlanguage{ngerman}±0.01\textsuperscript{a} &
37.11\selectlanguage{ngerman}±0.32\textsuperscript{A}\tabularnewline
Valine & 3.93\selectlanguage{ngerman}±0.04\textsuperscript{c} & 100.77\selectlanguage{ngerman}±0.35\textsuperscript{B} &
3.97\selectlanguage{ngerman}±0.02\textsuperscript{c} & 3.97\selectlanguage{ngerman}±0.02\textsuperscript{c} &
101.79\selectlanguage{ngerman}±0.31\textsuperscript{C} & 4.05\selectlanguage{ngerman}±0.01\textsuperscript{d} &
103.85\selectlanguage{ngerman}±0.48\textsuperscript{D} & 4.32\selectlanguage{ngerman}±0.06\textsuperscript{e} &
110.77\selectlanguage{ngerman}±0.43\textsuperscript{E} & 3.97\selectlanguage{ngerman}±0.03\textsuperscript{c} &
101.79\selectlanguage{ngerman}±0.29\textsuperscript{C} & 3.87\selectlanguage{ngerman}±0.02\textsuperscript{b} &
99.23\selectlanguage{ngerman}±0.26\textsuperscript{B} & 3.82\selectlanguage{ngerman}±0.02\textsuperscript{a} &
97.95\selectlanguage{ngerman}±0.33\textsuperscript{A}\tabularnewline
Leucine & 6.95\selectlanguage{ngerman}±0.04\textsuperscript{b} & 117.80\selectlanguage{ngerman}±0.58\textsuperscript{A}
& 7.01\selectlanguage{ngerman}±0.06\textsuperscript{b} & 7.01\selectlanguage{ngerman}±0.06\textsuperscript{b} &
118.81\selectlanguage{ngerman}±0.37\textsuperscript{A} & 7.33\selectlanguage{ngerman}±0.08\textsuperscript{c} &
124.24\selectlanguage{ngerman}±0.63\textsuperscript{B} & 7.86\selectlanguage{ngerman}±0.04\textsuperscript{d} &
133.22\selectlanguage{ngerman}±0.41\textsuperscript{C} & 7.04\selectlanguage{ngerman}±0.03\textsuperscript{d} &
119.32\selectlanguage{ngerman}±0.52\textsuperscript{A} & 6.80\selectlanguage{ngerman}±0.03\textsuperscript{ab} &
115.25\selectlanguage{ngerman}±0.53\textsuperscript{A} & 6.74\selectlanguage{ngerman}±0.04\textsuperscript{a} &
114.24\selectlanguage{ngerman}±0.38\textsuperscript{A}\tabularnewline
Isoleucine & 3.00\selectlanguage{ngerman}±0.02\textsuperscript{c} &
100.00\selectlanguage{ngerman}±0.51\textsuperscript{C} & 3.03\selectlanguage{ngerman}±0.04\textsuperscript{c} &
3.03\selectlanguage{ngerman}±0.04\textsuperscript{c} & 101.00\selectlanguage{ngerman}±0.21\textsuperscript{C} &
3.13\selectlanguage{ngerman}±0.02\textsuperscript{d} & 104.33\selectlanguage{ngerman}±0.26\textsuperscript{D} &
3.41\selectlanguage{ngerman}±0.01\textsuperscript{e} & 113.67\selectlanguage{ngerman}±0.26\textsuperscript{E} &
3.05\selectlanguage{ngerman}±0.03 & 101.67\selectlanguage{ngerman}±0.26\textsuperscript{C} &
2.91\selectlanguage{ngerman}±0.02\textsuperscript{b} & 97.00\selectlanguage{ngerman}±0.19\textsuperscript{B} &
2.87\selectlanguage{ngerman}±0.01\textsuperscript{a} &
95.67\selectlanguage{ngerman}±0.16\textsuperscript{A}\tabularnewline
Phenylalanine & 4.19\selectlanguage{ngerman}±0.03\textsuperscript{c} &
220.53\selectlanguage{ngerman}±0.66\textsuperscript{C} & 4.11\selectlanguage{ngerman}±0.01\textsuperscript{b} &
4.11\selectlanguage{ngerman}±0.01\textsuperscript{b} & 216.32\selectlanguage{ngerman}±0.52\textsuperscript{B} &
4.40\selectlanguage{ngerman}±0.04\textsuperscript{e} & 231.58\selectlanguage{ngerman}±0.56\textsuperscript{E} &
4.75\selectlanguage{ngerman}±0.02\textsuperscript{f} & 250.00\selectlanguage{ngerman}±0.78\textsuperscript{F} &
4.28\selectlanguage{ngerman}±0.02\textsuperscript{d} & 225.26\selectlanguage{ngerman}±0.81\textsuperscript{D} &
4.12\selectlanguage{ngerman}±0.03\textsuperscript{b} & 216.84\selectlanguage{ngerman}±0.63\textsuperscript{B} &
4.04\selectlanguage{ngerman}±0.02\textsuperscript{a} &
212.63\selectlanguage{ngerman}±0.57\textsuperscript{A}\tabularnewline
Threonine & 2.90\selectlanguage{ngerman}±0.02\textsuperscript{c} &
126.09\selectlanguage{ngerman}±0.47\textsuperscript{B} & 2.94\selectlanguage{ngerman}±0.06\textsuperscript{cd} &
2.94\selectlanguage{ngerman}±0.06\textsuperscript{cd} & 127.82\selectlanguage{ngerman}±0.39\textsuperscript{B} &
3.08\selectlanguage{ngerman}±0.02\textsuperscript{d} & 133.91\selectlanguage{ngerman}±0.44\textsuperscript{C} &
3.30\selectlanguage{ngerman}±0.05\textsuperscript{e} & 143.48\selectlanguage{ngerman}±0.53\textsuperscript{D} &
2.88\selectlanguage{ngerman}±0.03\textsuperscript{c} & 125.22\selectlanguage{ngerman}±0.48\textsuperscript{B} &
2.80\selectlanguage{ngerman}±0.01\textsuperscript{b} & 121.74\selectlanguage{ngerman}±0.46\textsuperscript{A} &
2.76\selectlanguage{ngerman}±0.02\textsuperscript{a} &
120.00\selectlanguage{ngerman}±0.52\textsuperscript{A}\tabularnewline
Methionine & 1.22\selectlanguage{ngerman}±0.06\textsuperscript{b} &
71.76\selectlanguage{ngerman}±0.23\textsuperscript{B} & 1.21\selectlanguage{ngerman}±0.08\textsuperscript{b} &
1.21\selectlanguage{ngerman}±0.08\textsuperscript{b} & 71.18\selectlanguage{ngerman}±0.19\textsuperscript{B} &
1.22\selectlanguage{ngerman}±0.02\textsuperscript{b} & 71.76\selectlanguage{ngerman}±0.27\textsuperscript{B} &
1.23\selectlanguage{ngerman}±0.05\textsuperscript{b} & 72.35\selectlanguage{ngerman}±0.16\textsuperscript{C} &
1.32\selectlanguage{ngerman}±0.06\textsuperscript{c} & 77.65\selectlanguage{ngerman}±0.31\textsuperscript{D} &
1.25\selectlanguage{ngerman}±0.03\textsuperscript{b} & 73.53\selectlanguage{ngerman}±0.22\textsuperscript{C} &
1.18\selectlanguage{ngerman}±0.03\textsuperscript{a} &
69.41\selectlanguage{ngerman}±0.17\textsuperscript{A}\tabularnewline
Non- essential amino acids & Non- essential amino acids & Non- essential
amino acids & Non- essential amino acids & Non- essential amino acids &
Non- essential amino acids & Non- essential amino acids & Non- essential
amino acids & Non- essential amino acids & Non- essential amino acids &
Non- essential amino acids & Non- essential amino acids & Non- essential
amino acids & Non- essential amino acids & Non- essential amino acids &
Non- essential amino acids\tabularnewline
& & AA & AA & AA & AA & AA & AA & AA & AA & AA & AA & AA & AA & AA &
AA\tabularnewline
Aspartic & Aspartic & 6.13\selectlanguage{ngerman}±0.16\textsuperscript{e} &
6.13\selectlanguage{ngerman}±0.16\textsuperscript{e} & 5.77\selectlanguage{ngerman}±0.14\textsuperscript{c} &
5.77\selectlanguage{ngerman}±0.14\textsuperscript{c} & 6.16\selectlanguage{ngerman}±0.16\textsuperscript{e} &
6.16\selectlanguage{ngerman}±0.16\textsuperscript{e} & 7.24\selectlanguage{ngerman}±0.13\textsuperscript{f} &
7.24\selectlanguage{ngerman}±0.13\textsuperscript{f} & 6.04\selectlanguage{ngerman}±0.14\textsuperscript{d} &
6.04\selectlanguage{ngerman}±0.14\textsuperscript{d} & 5.48\selectlanguage{ngerman}±0.14\textsuperscript{b} &
5.48\selectlanguage{ngerman}±0.14\textsuperscript{b} & 5.10\selectlanguage{ngerman}±0.13\textsuperscript{a} &
5.10\selectlanguage{ngerman}±0.13\textsuperscript{a}\tabularnewline
Glutamic & Glutamic & 18.15\selectlanguage{ngerman}±0.34\textsuperscript{c} &
18.15\selectlanguage{ngerman}±0.34\textsuperscript{c} & 15.83\selectlanguage{ngerman}±0.17\textsuperscript{a} &
15.83\selectlanguage{ngerman}±0.17\textsuperscript{a} & 15.76\selectlanguage{ngerman}±0.13\textsuperscript{a} &
15.76\selectlanguage{ngerman}±0.13\textsuperscript{a} & 15.50\selectlanguage{ngerman}±0.12\textsuperscript{a} &
15.50\selectlanguage{ngerman}±0.12\textsuperscript{a} & 17.60\selectlanguage{ngerman}±0.25\textsuperscript{c} &
17.60\selectlanguage{ngerman}±0.25\textsuperscript{c} & 17.59\selectlanguage{ngerman}±0.37\textsuperscript{b} &
17.59\selectlanguage{ngerman}±0.37\textsuperscript{b} & 17.66\selectlanguage{ngerman}±0.35\textsuperscript{b} &
17.66\selectlanguage{ngerman}±0.35\textsuperscript{b}\tabularnewline
Arginine & Arginine & 13.26\selectlanguage{ngerman}±0.22\textsuperscript{a} &
13.26\selectlanguage{ngerman}±0.22\textsuperscript{a} & 13.48\selectlanguage{ngerman}±0.31\textsuperscript{a} &
13.48\selectlanguage{ngerman}±0.31\textsuperscript{a} & 14.10\selectlanguage{ngerman}±0.25\textsuperscript{c} &
14.10\selectlanguage{ngerman}±0.25\textsuperscript{c} & 15.18\selectlanguage{ngerman}±0.15\textsuperscript{d} &
15.18\selectlanguage{ngerman}±0.15\textsuperscript{d} & 13.90\selectlanguage{ngerman}±0.33\textsuperscript{ab} &
13.90\selectlanguage{ngerman}±0.33\textsuperscript{ab} & 13.85\selectlanguage{ngerman}±0.26\textsuperscript{ab} &
13.85\selectlanguage{ngerman}±0.26\textsuperscript{ab} & 13.31\selectlanguage{ngerman}±0.33\textsuperscript{a} &
13.31\selectlanguage{ngerman}±0.33\textsuperscript{a}\tabularnewline
Serine & Serine & 5.55\selectlanguage{ngerman}±0.03\textsuperscript{b} &
5.55\selectlanguage{ngerman}±0.03\textsuperscript{b} & 5.62\selectlanguage{ngerman}±0.02\textsuperscript{bc} &
5.62\selectlanguage{ngerman}±0.02\textsuperscript{bc} & 5.92\selectlanguage{ngerman}±0.02\textsuperscript{d} &
5.92\selectlanguage{ngerman}±0.02\textsuperscript{d} & 5.30\selectlanguage{ngerman}±0.04\textsuperscript{a} &
5.30\selectlanguage{ngerman}±0.04\textsuperscript{a} & 5.59\selectlanguage{ngerman}±0.04\textsuperscript{b} &
5.59\selectlanguage{ngerman}±0.04\textsuperscript{b} & 5.39\selectlanguage{ngerman}±0.06\textsuperscript{a} &
5.39\selectlanguage{ngerman}±0.06\textsuperscript{a} & 5.44\selectlanguage{ngerman}±0.03\textsuperscript{a} &
5.44\selectlanguage{ngerman}±0.03\textsuperscript{a}\tabularnewline
Tyrosine & Tyrosine & 2.23\selectlanguage{ngerman}±0.06\textsuperscript{c} &
2.23\selectlanguage{ngerman}±0.06\textsuperscript{c} & 2.39\selectlanguage{ngerman}±0.05\textsuperscript{d} &
2.39\selectlanguage{ngerman}±0.05\textsuperscript{d} & 2.48\selectlanguage{ngerman}±0.04\textsuperscript{e} &
2.48\selectlanguage{ngerman}±0.04\textsuperscript{e} & 2.81\selectlanguage{ngerman}±0.02\textsuperscript{f} &
2.81\selectlanguage{ngerman}±0.02\textsuperscript{f} & 1.95\selectlanguage{ngerman}±0.04\textsuperscript{b} &
1.95\selectlanguage{ngerman}±0.04\textsuperscript{b} & 1.88\selectlanguage{ngerman}±0.03\textsuperscript{a} &
1.88\selectlanguage{ngerman}±0.03\textsuperscript{a} & 1.86\selectlanguage{ngerman}±0.04\textsuperscript{a} &
1.86\selectlanguage{ngerman}±0.04\textsuperscript{a}\tabularnewline
Cysteine & Cysteine & 0.35\selectlanguage{ngerman}±0.02\textsuperscript{c} &
0.35\selectlanguage{ngerman}±0.02\textsuperscript{c} & 0.27\selectlanguage{ngerman}±0.03\textsuperscript{b} &
0.27\selectlanguage{ngerman}±0.03\textsuperscript{b} & 0.47\selectlanguage{ngerman}±0.04\textsuperscript{d} &
0.47\selectlanguage{ngerman}±0.04\textsuperscript{d} & 0.54\selectlanguage{ngerman}±0.02\textsuperscript{e} &
0.54\selectlanguage{ngerman}±0.02\textsuperscript{e} & 0.42\selectlanguage{ngerman}±0.03\textsuperscript{d} &
0.42\selectlanguage{ngerman}±0.03\textsuperscript{d} & 0.31\selectlanguage{ngerman}±0.01\textsuperscript{b} &
0.31\selectlanguage{ngerman}±0.01\textsuperscript{b} & 0.22\selectlanguage{ngerman}±0.03\textsuperscript{a} &
0.22\selectlanguage{ngerman}±0.03\textsuperscript{a}\tabularnewline
Alanine & Alanine & 4.14\selectlanguage{ngerman}±0.02\textsuperscript{b} &
4.14\selectlanguage{ngerman}±0.02\textsuperscript{b} & 4.14\selectlanguage{ngerman}±0.03\textsuperscript{b} &
4.14\selectlanguage{ngerman}±0.03\textsuperscript{b} & 4.30\selectlanguage{ngerman}±0.04\textsuperscript{c} &
4.30\selectlanguage{ngerman}±0.04\textsuperscript{c} & 4.67\selectlanguage{ngerman}±0.02\textsuperscript{d} &
4.67\selectlanguage{ngerman}±0.02\textsuperscript{d} & 4.17\selectlanguage{ngerman}±0.05\textsuperscript{b} &
4.17\selectlanguage{ngerman}±0.05\textsuperscript{b} & 4.04\selectlanguage{ngerman}±0.02\textsuperscript{a} &
4.04\selectlanguage{ngerman}±0.02\textsuperscript{a} & 4.04\selectlanguage{ngerman}±0.03\textsuperscript{a} &
4.04\selectlanguage{ngerman}±0.03\textsuperscript{a}\tabularnewline
Proline & Proline & 4.04\selectlanguage{ngerman}±0.02\textsuperscript{ab} &
4.04\selectlanguage{ngerman}±0.02\textsuperscript{ab} & 4.03\selectlanguage{ngerman}±0.02\textsuperscript{ab} &
4.03\selectlanguage{ngerman}±0.02\textsuperscript{ab} & 4.18\selectlanguage{ngerman}±0.04\textsuperscript{c} &
4.18\selectlanguage{ngerman}±0.04\textsuperscript{c} & 4.49\selectlanguage{ngerman}±0.03\textsuperscript{d} &
4.49\selectlanguage{ngerman}±0.03\textsuperscript{d} & 4.05\selectlanguage{ngerman}±0.03\textsuperscript{ab} &
4.05\selectlanguage{ngerman}±0.03\textsuperscript{ab} & 3.93\selectlanguage{ngerman}±0.03\textsuperscript{a} &
3.93\selectlanguage{ngerman}±0.03\textsuperscript{a} & 3.99\selectlanguage{ngerman}±0.06\textsuperscript{a} &
3.99\selectlanguage{ngerman}±0.06\textsuperscript{a}\tabularnewline
Glycine & Glycine & 5.28\selectlanguage{ngerman}±0.13\textsuperscript{b} &
5.28\selectlanguage{ngerman}±0.13\textsuperscript{b} & 5.32\selectlanguage{ngerman}±0.12\textsuperscript{b} &
5.32\selectlanguage{ngerman}±0.12\textsuperscript{b} & 5.56\selectlanguage{ngerman}±0.13\textsuperscript{c} &
5.56\selectlanguage{ngerman}±0.13\textsuperscript{c} & 5.93\selectlanguage{ngerman}±0.09\textsuperscript{d} &
5.93\selectlanguage{ngerman}±0.09\textsuperscript{d} & 5.31\selectlanguage{ngerman}±0.12\textsuperscript{b} &
5.31\selectlanguage{ngerman}±0.12\textsuperscript{b} & 5.16\selectlanguage{ngerman}±0.07\textsuperscript{a} &
5.16\selectlanguage{ngerman}±0.07\textsuperscript{a} & 5.14\selectlanguage{ngerman}±0.12\textsuperscript{a} &
5.14\selectlanguage{ngerman}±0.12\textsuperscript{a}\tabularnewline
Total EAA & Total EAA & 26.72\selectlanguage{ngerman}±0.12\textsuperscript{a} &
26.72\selectlanguage{ngerman}±0.12\textsuperscript{a} & 26.93\selectlanguage{ngerman}±0.03\textsuperscript{a} &
26.93\selectlanguage{ngerman}±0.03\textsuperscript{a} & 27.89\selectlanguage{ngerman}±0.09\textsuperscript{b} &
27.89\selectlanguage{ngerman}±0.09\textsuperscript{b} & 30.19\selectlanguage{ngerman}±0.04\textsuperscript{c} &
30.19\selectlanguage{ngerman}±0.04\textsuperscript{c} & 27.07\selectlanguage{ngerman}±0.02\textsuperscript{ab} &
27.07\selectlanguage{ngerman}±0.02\textsuperscript{ab} & 26.11\selectlanguage{ngerman}±0.12\textsuperscript{a} &
26.11\selectlanguage{ngerman}±0.12\textsuperscript{a} & 25.74\selectlanguage{ngerman}±0.11\textsuperscript{a} &
25.74\selectlanguage{ngerman}±0.11\textsuperscript{a}\tabularnewline
TAA & TAA & 85.85\selectlanguage{ngerman}±2.32\textsuperscript{a} &
85.85\selectlanguage{ngerman}±2.32\textsuperscript{a} & 83.78\selectlanguage{ngerman}±1.23\textsuperscript{a} &
83.78\selectlanguage{ngerman}±1.23\textsuperscript{a} & 86.82\selectlanguage{ngerman}±1.25\textsuperscript{a} &
86.82\selectlanguage{ngerman}±1.25\textsuperscript{a} & 91.85\selectlanguage{ngerman}±1.21\textsuperscript{b} &
91.85\selectlanguage{ngerman}±1.21\textsuperscript{b} & 87.10\selectlanguage{ngerman}±1.33\textsuperscript{a} &
87.10\selectlanguage{ngerman}±1.33\textsuperscript{a} & 83.74\selectlanguage{ngerman}±1.26\textsuperscript{a} &
83.74\selectlanguage{ngerman}±1.26\textsuperscript{a} & 82.5\selectlanguage{ngerman}±1.25\textsuperscript{a} &
82.5\selectlanguage{ngerman}±1.25\textsuperscript{a}\tabularnewline
EAA/TAA\selectlanguage{english}(\%\selectlanguage{english}) & EAA/TAA(\%) & 30.42\selectlanguage{ngerman}±0.01\textsuperscript{a} &
30.42\selectlanguage{ngerman}±0.01\textsuperscript{a} & 33.33\selectlanguage{ngerman}±0.07\textsuperscript{c} &
33.33\selectlanguage{ngerman}±0.07\textsuperscript{c} & 32.12\selectlanguage{ngerman}±0.06\textsuperscript{c} &
32.12\selectlanguage{ngerman}±0.06\textsuperscript{c} & 33.23\selectlanguage{ngerman}±0.05\textsuperscript{c} &
33.23\selectlanguage{ngerman}±0.05\textsuperscript{c} & 31.08\selectlanguage{ngerman}±0.08\textsuperscript{b} &
31.08\selectlanguage{ngerman}±0.08\textsuperscript{b} & 31.12\selectlanguage{ngerman}±0.08\textsuperscript{b} &
31.12\selectlanguage{ngerman}±0.08\textsuperscript{b} & 31.2\selectlanguage{ngerman}±0.05\textsuperscript{b} &
31.2\selectlanguage{ngerman}±0.05\textsuperscript{b}\tabularnewline
Hydrophobic AA & Hydrophobic AA & 32.75\selectlanguage{ngerman}±0.17\textsuperscript{b} &
32.75\selectlanguage{ngerman}±0.17\textsuperscript{b} & 32.82\selectlanguage{ngerman}±0.12\textsuperscript{b} &
32.82\selectlanguage{ngerman}±0.12\textsuperscript{b} & 34.17\selectlanguage{ngerman}±0.08\textsuperscript{c} &
34.17\selectlanguage{ngerman}±0.08\textsuperscript{c} & 36.29\selectlanguage{ngerman}±0.11\textsuperscript{d} &
36.29\selectlanguage{ngerman}±0.11\textsuperscript{d} & 33.19\selectlanguage{ngerman}±0.09\textsuperscript{b} &
33.19\selectlanguage{ngerman}±0.09\textsuperscript{b} & 32.08\selectlanguage{ngerman}±0.13\textsuperscript{b} &
32.08\selectlanguage{ngerman}±0.13\textsuperscript{b} & 31.82\selectlanguage{ngerman}±0.12\textsuperscript{a} &
31.82\selectlanguage{ngerman}±0.12\textsuperscript{a}\tabularnewline
Hydrophilic AA & Hydrophilic AA & 11.03\selectlanguage{ngerman}±0.11\textsuperscript{b} &
11.03\selectlanguage{ngerman}±0.11\textsuperscript{b} & 11.22\selectlanguage{ngerman}±0.13\textsuperscript{b} &
11.22\selectlanguage{ngerman}±0.13\textsuperscript{b} & 11.95\selectlanguage{ngerman}±0.18\textsuperscript{c} &
11.95\selectlanguage{ngerman}±0.18\textsuperscript{c} & 11.95\selectlanguage{ngerman}±0.15\textsuperscript{c} &
11.95\selectlanguage{ngerman}±0.15\textsuperscript{c} & 10.84\selectlanguage{ngerman}±0.03\textsuperscript{a} &
10.84\selectlanguage{ngerman}±0.03\textsuperscript{a} & 10.38\selectlanguage{ngerman}±0.03\textsuperscript{a} &
10.38\selectlanguage{ngerman}±0.03\textsuperscript{a} & 10.28\selectlanguage{ngerman}±0.04\textsuperscript{a} &
10.28\selectlanguage{ngerman}±0.04\textsuperscript{a}\tabularnewline
Alkaline AA & Alkaline AA & 17.79\selectlanguage{ngerman}±0.12\textsuperscript{a} &
17.79\selectlanguage{ngerman}±0.12\textsuperscript{a} & 18.14\selectlanguage{ngerman}±0.09\textsuperscript{a} &
18.14\selectlanguage{ngerman}±0.09\textsuperscript{a} & 18.78\selectlanguage{ngerman}±0.12\textsuperscript{a} &
18.78\selectlanguage{ngerman}±0.12\textsuperscript{a} & 20.50\selectlanguage{ngerman}±0.07\textsuperscript{b} &
20.50\selectlanguage{ngerman}±0.07\textsuperscript{b} & 18.43\selectlanguage{ngerman}±0.08\textsuperscript{a} &
18.43\selectlanguage{ngerman}±0.08\textsuperscript{a} & 18.21\selectlanguage{ngerman}±0.08\textsuperscript{a} &
18.21\selectlanguage{ngerman}±0.08\textsuperscript{a} & 17.64\selectlanguage{ngerman}±0.11\textsuperscript{a} &
17.64\selectlanguage{ngerman}±0.11\textsuperscript{a}\tabularnewline
Acidic AA & Acidic AA & 24.28\selectlanguage{ngerman}±0.06\textsuperscript{a} &
24.28\selectlanguage{ngerman}±0.06\textsuperscript{a} & 21.60\selectlanguage{ngerman}±0.05\textsuperscript{b} &
21.60\selectlanguage{ngerman}±0.05\textsuperscript{b} & 21.92\selectlanguage{ngerman}±0.08\textsuperscript{b} &
21.92\selectlanguage{ngerman}±0.08\textsuperscript{b} & 22.74\selectlanguage{ngerman}±0.03\textsuperscript{c} &
22.74\selectlanguage{ngerman}±0.03\textsuperscript{c} & 23.64\selectlanguage{ngerman}±0.11\textsuperscript{d} &
23.64\selectlanguage{ngerman}±0.11\textsuperscript{d} & 23.07\selectlanguage{ngerman}±0.07\textsuperscript{cd} &
23.07\selectlanguage{ngerman}±0.07\textsuperscript{cd} & 22.76\selectlanguage{ngerman}±0.06 & 22.76\selectlanguage{ngerman}±0.06\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

The values were presented as the mean ± standard deviation in
triplicate. AA: amino acid; AAS: amino acid score (mg of amino acid in 1
g of test protein/mg of amino acid in requirement pattern) × 100; EAA:
essential amino acid; TAA: total amino acid; R: roasting; S: steaming.

\textsuperscript{*}WHO/FAO/UNU (2007) Expert Consultation Report for
adults.

\textsuperscript{a-g} Means within a row with different letters were
significant difference of the AA composition between samples for each
amino acid (\emph{P} \selectlanguage{english}<0.05).

\textsuperscript{A--F} Means within a row with different letters were a
significant difference of the AAS between samples for each essential
amino acid (\emph{P} \selectlanguage{english}<0.05)

Table2

Effect of steaming and roasting treatment on secondary structure of
protein in walnut kernel.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Sample & Secondary structure content (\%) & Secondary structure content
(\%) & Secondary structure content (\%) & Secondary structure content
(\%)\tabularnewline
\midrule
\endhead
& a-\selectlanguage{english}helix & \selectlanguage{greek}β-\selectlanguage{english}sheets & \selectlanguage{greek}β-\selectlanguage{english}turns & Random coil\tabularnewline
Untreated & 11.81\selectlanguage{ngerman}±0.28\textsuperscript{a} &
47.16\selectlanguage{ngerman}±0.72\textsuperscript{c} & 27.21\selectlanguage{ngerman}±0.41\textsuperscript{d} &
12.86\selectlanguage{ngerman}±0.33\textsuperscript{a}\tabularnewline
R15 & 14.48\selectlanguage{ngerman}±0.32\textsuperscript{b} & 47.57\selectlanguage{ngerman}±0.29\textsuperscript{c} &
15.40\selectlanguage{ngerman}±0.31\textsuperscript{a} &
12.52\selectlanguage{ngerman}±0.26\textsuperscript{a}\tabularnewline
R20 & 19.75\selectlanguage{ngerman}±0.34\textsuperscript{d} & 48.56\selectlanguage{ngerman}±0.48\textsuperscript{c} &
20.42\selectlanguage{ngerman}±0.35\textsuperscript{b} &
11.26\selectlanguage{ngerman}±0.23\textsuperscript{a}\tabularnewline
R30 & 24.44\selectlanguage{ngerman}±0.41\textsuperscript{e} & 39.91\selectlanguage{ngerman}±0.32\textsuperscript{b} &
19.98\selectlanguage{ngerman}±0.25\textsuperscript{b} &
14.47\selectlanguage{ngerman}±0.32\textsuperscript{b}\tabularnewline
S15 & 15.38\selectlanguage{ngerman}±0.43\textsuperscript{b} & 47.21\selectlanguage{ngerman}±0.37\textsuperscript{c} &
22.48\selectlanguage{ngerman}±0.24\textsuperscript{c} &
12.72\selectlanguage{ngerman}±0.36\textsuperscript{a}\tabularnewline
S20 & 18.87\selectlanguage{ngerman}±0.36\textsuperscript{c} & 46.21\selectlanguage{ngerman}±0.63\textsuperscript{c} &
20.69\selectlanguage{ngerman}±0.27\textsuperscript{b} &
13.15\selectlanguage{ngerman}±0.23\textsuperscript{a}\tabularnewline
S30 & 25.92\selectlanguage{ngerman}±0.32\textsuperscript{f} & 36.67\selectlanguage{ngerman}±0.33\textsuperscript{a} &
19.19\selectlanguage{ngerman}±0.19\textsuperscript{b} &
16.85\selectlanguage{ngerman}±0.22\textsuperscript{c}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

The values were presented as the mean ± standard deviation in
triplicate; R: roasting; S: steaming.

The different superscript letter of values in same column suggested
significantly different (\emph{P} \textless{} 0.05) according to Tukey's
test.

Table 3

Thermal behaviour of untreated, steaming and roasting walnut kernel
flour\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Sample & ^H (J g\textsuperscript{-1}) & T\textsubscript{onset} (\selectlanguage{ngerman}°C) &
T\textsubscript{peak} (\selectlanguage{ngerman}°C) & T\textsubscript{end} (\selectlanguage{ngerman}°C) & ^T
(\selectlanguage{ngerman}°C)\tabularnewline
\midrule
\endhead
Untreated & 4.53\selectlanguage{ngerman}±0.02\textsuperscript{a} & 92.76\selectlanguage{ngerman}±1.03\textsuperscript{a}
& 98.83\selectlanguage{ngerman}±0.27\textsuperscript{a} & 109.08\selectlanguage{ngerman}±0.2\textsuperscript{a} &
16.32\selectlanguage{ngerman}±0.09\textsuperscript{a}\tabularnewline
R15 & 6.69\selectlanguage{ngerman}±0.12\textsuperscript{b} & 94.34\selectlanguage{ngerman}±0.41\textsuperscript{b} &
102.83\selectlanguage{ngerman}±0.2\textsuperscript{b} & 116.04\selectlanguage{ngerman}±0.26\textsuperscript{c} &
21.70\selectlanguage{ngerman}±0.11\textsuperscript{c}\tabularnewline
R20 & 7.07\selectlanguage{ngerman}±0.11\textsuperscript{c} & 91.76\selectlanguage{ngerman}±0.06\textsuperscript{a} &
100.37\selectlanguage{ngerman}±0.86\textsuperscript{b} & 114.35\selectlanguage{ngerman}±0.11\textsuperscript{b} &
22.59\selectlanguage{ngerman}±0.13\textsuperscript{e}\tabularnewline
R30 & 6.24\selectlanguage{ngerman}±0.12\textsuperscript{b} & 92.24\selectlanguage{ngerman}±1.02\textsuperscript{a} &
101.76\selectlanguage{ngerman}±0.31\textsuperscript{b} & 115.12\selectlanguage{ngerman}±0.25\textsuperscript{b} &
22.88\selectlanguage{ngerman}±0.11\textsuperscript{e}\tabularnewline
S15 & 7.73\selectlanguage{ngerman}±0.12\textsuperscript{d} & 91.52\selectlanguage{ngerman}±0.43\textsuperscript{a} &
100.70\selectlanguage{ngerman}±0.56\textsuperscript{b} & 113.51\selectlanguage{ngerman}±0.02\textsuperscript{b} &
22.01\selectlanguage{ngerman}±0.08\textsuperscript{d}\tabularnewline
S20 & 8.69\selectlanguage{ngerman}±0.13\textsuperscript{e} & 93.45\selectlanguage{ngerman}±1.12\textsuperscript{a} &
101.97\selectlanguage{ngerman}±0.63\textsuperscript{b} & 114.50\selectlanguage{ngerman}±0.13\textsuperscript{b} &
21.05\selectlanguage{ngerman}±0.16\textsuperscript{c}\tabularnewline
S30 & 7.11\selectlanguage{ngerman}±0.11\textsuperscript{c} & 93.39\selectlanguage{ngerman}±0.92\textsuperscript{a} &
102.22\selectlanguage{ngerman}±0.15\textsuperscript{b} & 114.18\selectlanguage{ngerman}±0.08\textsuperscript{b} &
20.79\selectlanguage{ngerman}±0.05\textsuperscript{b}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

The values were presented as the mean ± standard deviation in
triplicate. R: roasting; S: steaming.

The different superscript letter of values in same column suggested
significantly different (\emph{P} \textless{} 0.05) according to Tukey's
test.

T\textsubscript{onset}: Onset temperature; T\textsubscript{peak}: peak
temperature; T\textsubscript{end}: conclusion temperature; \selectlanguage{greek}Δ\selectlanguage{english}T:
T\textsubscript{end} - T\textsubscript{onset}; \selectlanguage{greek}Δ\selectlanguage{english}H: enthalpy; R:
roasting; S: steaming.

Table 4

Functional properties of untreated and heated walnut kernel flours (n =
3).\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
Sample & WHC (g g\textsuperscript{-1}) & OHC (g g\textsuperscript{-1}) &
EC (\%) & ES (\%) & FC (\%) & FS (\%)\tabularnewline
\midrule
\endhead
Untreated & 2.84\selectlanguage{ngerman}±0.03\textsuperscript{b} & 1.21\selectlanguage{ngerman}±0.01\textsuperscript{a}
& 44.32\selectlanguage{ngerman}±1.26\textsuperscript{a} & 74.21\selectlanguage{ngerman}±1.15\textsuperscript{a} &
62.13\selectlanguage{ngerman}±1.13\textsuperscript{a} &
43.30\selectlanguage{ngerman}±1.23\textsuperscript{a}\tabularnewline
R15 & 3.32\selectlanguage{ngerman}±0.16\textsuperscript{c} & 5.67\selectlanguage{ngerman}±0.12\textsuperscript{c} &
55.42\selectlanguage{ngerman}±2.21\textsuperscript{c} & 96.24\selectlanguage{ngerman}±2.41\textsuperscript{b} &
65.44\selectlanguage{ngerman}±1.15\textsuperscript{b} &
80.07\selectlanguage{ngerman}±2.06\textsuperscript{e}\tabularnewline
R20 & 2.81\selectlanguage{ngerman}±0.01\textsuperscript{b} & 5.73\selectlanguage{ngerman}±0.09\textsuperscript{c} &
56.82\selectlanguage{ngerman}±1.77\textsuperscript{c} & 96.39\selectlanguage{ngerman}±2.08\textsuperscript{b} &
64.36\selectlanguage{ngerman}±1.11\textsuperscript{b} &
80.13\selectlanguage{ngerman}±1.97\textsuperscript{e}\tabularnewline
R30 & 1.85\selectlanguage{ngerman}±0.04\textsuperscript{a} & 5.68\selectlanguage{ngerman}±0.03\textsuperscript{c} &
56.51\selectlanguage{ngerman}±1.42\textsuperscript{c} & 95.64\selectlanguage{ngerman}±3.68\textsuperscript{b} &
68.60\selectlanguage{ngerman}±0.53\textsuperscript{c} &
77.48\selectlanguage{ngerman}±1.82\textsuperscript{c}\tabularnewline
S15 & 4.43\selectlanguage{ngerman}±0.08\textsuperscript{e} & 5.34\selectlanguage{ngerman}±0.11\textsuperscript{c} &
56.34\selectlanguage{ngerman}±1.31\textsuperscript{c} & 95.62\selectlanguage{ngerman}±2.75\textsuperscript{b} &
69.17\selectlanguage{ngerman}±0.36\textsuperscript{d} &
80.11\selectlanguage{ngerman}±1.52\textsuperscript{e}\tabularnewline
S20 & 3.86\selectlanguage{ngerman}±0.03\textsuperscript{d} & 4.55\selectlanguage{ngerman}±0.13\textsuperscript{b} &
56.38\selectlanguage{ngerman}±1.27\textsuperscript{c} & 92.46\selectlanguage{ngerman}±3.13\textsuperscript{b} &
71.11\selectlanguage{ngerman}±1.12\textsuperscript{d} &
78.39\selectlanguage{ngerman}±1.37\textsuperscript{d}\tabularnewline
S30 & 3.45\selectlanguage{ngerman}±0.01\textsuperscript{c} & 4.62\selectlanguage{ngerman}±0.11\textsuperscript{b} &
52.37\selectlanguage{ngerman}±1.08\textsuperscript{b} & 96.35\selectlanguage{ngerman}±2.95\textsuperscript{b} &
69.32\selectlanguage{ngerman}±0.58\textsuperscript{d} &
75.18\selectlanguage{ngerman}±2.07\textsuperscript{b}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Values expressed were mean ± standard deviation in triplicate. WHC and
OHC: water holding capacity and oil holding capacity, respectively; FC
and FS: foaming capacity and foaming stability, respectively; EC and ES:
emulsifying capacity and emulsifying stability, respectively; R:
roasting; S: steaming.

Means in the column with different superscript were significantly
different at \emph{P} \textless{} 0.05 according to Tukey's test.

\selectlanguage{english}
\FloatBarrier
\end{document}

