- Email: [email protected]
Extraction of low methoxylated pectin from pea hulls via RSM
Journal Pre-proof Extraction of low methoxylated pectin from pea hulls via RSM Friederike Gutöhrlein, Stephan Drusch, Sebastian Schalow PII:
S0268-005X(19)32208-8
DOI:
https://doi.org/10.1016/j.foodhyd.2019.105609
Reference:
FOOHYD 105609
To appear in:
Food Hydrocolloids
Received Date: 23 September 2019 Revised Date:
26 November 2019
Accepted Date: 17 December 2019
Please cite this article as: Gutöhrlein, F., Drusch, S., Schalow, S., Extraction of low methoxylated pectin from pea hulls via RSM, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/j.foodhyd.2019.105609. 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. © 2019 Published by Elsevier Ltd.
Reactions during acidic extraction of pectic polysaccharides from pea hulls Solubilisation
Degradation
Temperature / Time /pH Citric acid x
x x
x x
x x
x
Nitric acid Temperature + Time Galacturonic acid
Rhamnose
x Xylose
RGI side Chains (Ara,Gal)
1
Extraction of low methoxylated pectin from pea hulls via RSM
2
Friederike Gutöhrlein, Stephan Drusch*, Sebastian Schalow
3 4
Technische Universität Berlin, Food Technology and Food Material Science
5
Königin-Luise-Strasse 22, D-14195 Berlin, Germany
6
*Corresponding author
7
E-mail-address: [email protected]
8 9 10
Abstract
11
Nowadays, low methoxylated pectin (LMP) is generated in a multi-step process from high
12
methoxylated pectin using fruit by-products as a raw material, although various other plant
13
sources naturally contain LMP. In this study, we prove that LMP may be directly extracted
14
from pea hulls. Extraction was conducted according to a central composite design (CCD) and
15
evaluated via response surface methodology (RSM). The influence of different parameters
16
(pH, temperature, time) on yield and composition of the extracted pectic polysaccharides
17
(PPS) was investigated using nitric acid and citric acid as extraction media. Citric acid
18
yielded higher amounts of PPS (3.5 - 9.8%) compared to nitric acid (1.4 - 8.0%). However,
19
there is a conflict of aims between a high yield and the purity of the extracted PPS.
20
Composition analysis suggests that under ‘mild’ extraction conditions (pH 2, 70 °C) PPS
21
consist of homogalacturonan, xylogalacturonan and rhamnogalacturonan with arabinose and
22
galactose side chains (RG-I). With increasing temperature (90 °), yield is maximised due to
23
an increased solubilisation of cell wall polysaccharides. Under ‘harsh” conditions (pH 1,
24
90 °C) the purity of PPS increases in terms of a relatively higher content of uronic acids, but
25
yield decreases. This is attributed to a cleavage of non-GalA components and an ongoing
26
depolymerisation of the pectic galacturonan. PPS extracted under these conditions is
27
characterised by a low degree of acetylation (4%) and a relatively high protein content (7%).
28 29
Keywords
30
Pea hulls, extraction, citric acid, nitric acid, pectin
31
1
32
1. Introduction
33
Low methoxylated pectin (LMP) is defined by a degree of methoxylation (DM) lower than
34
50 %, which enables LMP to form gels by cross-linking via divalent cations (e. g. calcium),
35
even in the absence of sugar (Endreß & Christensen, 2009; Willats, Knox, & Mikkelsen,
36
2006). This makes it a widely used stabiliser in various food matrices with a reduced sugar
37
content such as low-calorie jams or jellies. LMP gels are pumpable, remeltable and
38
reformable and may be further used as fillings for bakery products in industrial fabrication
39
(BeMiller, 2019). Moreover, LMP can be applied as a texturiser in yoghurt, fruit preparations
40
or desserts, respectively (Everett, & McLeod, 2005; Van Buggenhout, Sila, Duvetter, Van
41
Loey, & Hendrickx, 2009).
42
Industrially, pectin is extracted from citrus peels and apple pomace or in minor quantities
43
from sugar beet pulp (Thibault & Ralet, 2003). Extraction is executed in an acidic milieu using
44
water as a solvent, which is commonly acidified with nitric acid to provide a pH value
45
between 1 and 3 in the final slurry. Extraction temperature and time may vary in broad
46
ranges (50 – 100 °C; 3 – 12 h) (Rolin, 2002). The slurry is filtrated, the liquid phase (thin
47
juice) is concentrated by evaporation and subsequently precipitated with ethanol. The
48
resulting precipitate then is dried and milled (Shan, 2016). The industrial process provides a
49
high methoxylated pectin (HMP; DM ≥ 50 %) which typically shows a galacturonic acid
50
(GalA) content of 65 % or higher and thus meets legislation requirements of the FAO and EU
51
for food application (Endreß & Christensen, 2009). A more ecological approach during pectin
52
extraction may consider a replacement of inorganic acids by organic acids such as citric acid.
53
Various studies revealed, that even higher yields of pectin were obtained if citric acid was
54
applied instead of nitric acid using apple pomace, (Canteri-Schemin, Fertonani,
55
Waszczynskyj, & Wosiacki, 2005), cacao pod husks (Vriesmann, Teófilo, & de Oliveira
56
Petkowicz, 2011 and 2012) or passion fruit peel (Kliemann et al., 2009), respectively, as a
57
raw material. However, these studies mainly focused on yield optimisation and little is known
58
about the composition of such citric acid extracted pectins.
59
To generate LMP from the respective source another step has to be introduced into the
60
process of pectin extraction, which is the demethoxylation of the inherent HMP. This step
61
may be directly realised during pectin extraction or after precipitation of HMP using acid or
62
alkali in an alcoholic medium. The resulting solution is further neutralised, washed several
63
times with alcohol and then dried and milled to generate a powder of LMP (Thibault & Ralet,
64
2003). Such methods using chemical demethoxylation usually result in a random distribution
65
of the methoxyl groups within the homogalacturonan chain and may additionally promote
66
depolymerisation of GalA backbone to a certain extent. Enzymatic demethoxylation has been
67
reported as an alternative route to alter the pectin fine structure in terms of its degree of
68
methoxylation (Rolin, Chrestensen, Hansen, Staunstrup, J., & Sørensen, 2010). 2
69
Depending on the type of pectin-degrading enzyme used for demethoxylation (plant or
70
microbial), the resulting LMP may differ with regard to its distribution pattern of methoxyl
71
groups (blockwise or random) (Fraeye et al., 2009; Limberg et al., 2000a,b; Shan, 2016).
72 73
Unlike the above mentioned raw materials, pectic polysaccharides (PPS) with a low DM have
74
been proven as a natural constituent in vegetables as shown for cell wall materials prepared
75
from carrots (Pickardt, Dongowski, & Kunzek, 2004). Jafari, Khodaiyan, Kiani, & Hosseini
76
(2017) directly extracted LMP from carrot pomace without a demethoxylation step, resulting
77
in pectin samples with a DM that ranged from 22.1 % to 51.8 % depending on the extraction
78
conditions. Furthermore, other raw materials such as passion fruit rind, pistachio peels,
79
pomegranate peels, water melon peels or cacao pod husks might be used for the extraction
80
of PPS with a low DM in principal (Abid et al., 2017; Chaharbaghi, Khodaiyan, & Hosseini,
81
2017; Raji, Khodaiyan, Rezaei, Kiani, & Hosseini, 2017; Vriesmann et al., 2012; Yapo, 2009).
82
However, none of them found its way into the commercial process of pectin extraction so far.
83 84
Pea hulls may be regarded as a promising raw material for the recovery of LMP. Pea hulls
85
are a by-product during commercial pea processing, that are removed by mechanical steps
86
prior to the further valorization of the cotyledons in terms of protein and starch extraction.
87
Therefore, unlike other by-products from fruit or vegetable processing, pea hulls may be
88
recovered and collected in the dry-state and then handled centrally by a pea processing plant
89
during the campaign. Although pea hulls account for approximately 7 to 12 % of the total pea
90
mass (Ali-Khan, 1993; Igbasan, Guenter, & Slominski, 1997;) their commercial valorisation is
91
still under-utilised and only minor quantities are used for fibre enrichment in foods (Dalgetty &
92
Baik, 2006). Around 75 % of the pea hull mass consist of cellulose and hemicellulose
93
(Reichert, 1981). Furthermore, pea hulls contain 9 - 17 % of uronic acids, mainly present as
94
galacturonic acid (~97 %) with a low DM and a degree of acetylation (DAc) of approximately
95
10 % (Gutöhrlein, Drusch, & Schalow, 2018; Reichert, 1981; Weightman, Renard, & Thibault,
96
1994). In the past, several studies showed that an acidic extraction of PPS from pea hulls is
97
possible in general. In any case, the step of acidic extraction, however, was integrated into a
98
sequence of other steps using different extraction media such as chelators (CDTA) or strong
99
alkali (NaOH/KOH) and resulting in PPS fractions with different yield and with variable
100
composition (Le Goff, Renard, Bonnin, & Thibault, 2001; Renard, Weightman, & Thibault,
101
1997, Weightman et al., 1994). Using pea hulls as a raw material for commercial pectin
102
production firstly requires a systematic approach using common extraction media under
103
conditions typically applied in industrial processing, in order to identify significant factors and
104
interactions that affect pectin yield and purity. Comparable to other raw materials, it must be
105
assumed that pectin extraction from pea hulls under acidic conditions is governed by two
3
106
reactions overlapping each other: the solubilisation of the insoluble protopectin from the cell
107
wall and the degradation of the solubilised pectin in terms of the cleavage of non-GalA side
108
chains and the depolymerisation of the GalA backbone.
109
Therefore, the aim of this study was to evaluate pectin extraction from pea hulls with regard
110
to yield and composition of the extracted pectin or PPS depending on several factors:
111
extraction medium (nitric and citric acid), pH, temperature and extraction time. A central
112
composite design (CCD) was established and evaluated using response surface
113
methodology (RSM) which may be generally regarded as a useful tool for process
114
optimisation in terms of pectin yield and purity (Jafari et al., 2017; Vriesmann et al., 2011 and
115
2012). Composition was evaluated with regard to the content of uronich acids (UA), neutral
116
sugars and protein as well the DM and the degree of acetylation (DA).
117 118
2. Materials and methods
119
2.1. Material
120
Pea hulls were provided by Emsland Stärke GmbH (Germany) in form of commercially
121
available pea hull fibre PH1000 (Lot 52140). All chemicals were analytical grade and
122
supplied by Carl Roth GmbH & Co. KG (Germany), VWR International GmbH (Germany) and
123
PSS Polymer Standards Service GmbH (Germany). Enzyme preparations were provided by
124
Novozymes Switzerland AG (Switzerland) and Erbsloeh Geisenheim AG (Germany).
125 126
2.2. Experimental design for pectin extraction
127
A powerful tool for process optimisation, in general, is the response surface methodology
128
(RSM). Instead of monitoring the effects of one factor at time, a multivariate statistic
129
technique is used. Experimental data are fitted to a polynomial equation. In this way, the
130
influence of several numeric factors can be identified at the same time. A central composite
131
design (CCD) is often used to design experiments that are evaluated by RSM. A CCD
132
consists of (1) a full factorial design, which can be described as a cube and factors are varied
133
on two levels, (2) a central point that is termed with "0" and (3) star points that vary in a
134
defined distance α from the central point. In this experimental design, only the central point is
135
repeated several times. Therefore, all factors are studied at five levels (-α, -1, 0, +1, +α) with
136
a reduced number of experiments compared to a one factor at time design. The respective
137
levels of the numeric factors "extraction time", "temperature" and "pH", used in the present
138
study, are shown in Table 1. These factor levels were chosen according to the parameters
139
typically used in industrial pectin extraction. Central points (0) were performed in triplicate. All
140
experiments were performed in a randomised order to avoid systematic errors. The design
141
was used for both types of extraction media: nitric acid (-1) and citric acid (+1), which were 4
142
categorical factors. The main effects and factor interactions were evaluated by ANOVA for
143
the different responses: yield, content of uronic acid (UA), degree of methoxylation (DM),
144
degree of acetylation (DAc), content of total and individual neutral sugars and content of
145
protein. Furthermore, polynomial regression equation was calculated as described by
146
Bezerra, Santelli, Oliveira, Silveira Villar, & Escaleira (2008). Model equations with minimum
147
Predicted Residual Sum of Squares (PRESS) were chosen after non-significant factors had
148
been excluded which resulted in a quadratic model that was used for further statistical
149
evaluation using Design Expert 8.0 (Stat Ease Inc., USA). Moreover, the respective model
150
was used to create contour plots with gnuplot (Version5 patchlevel 5), in which the calculated
151
responses were printed as isolines, supported by a colour gradient to facilitate the survey of
152
experimental results (Kleppmann, 2013). Once a significant lack of fit occurred, experiments
153
were anyway evaluated and discussed, with the restriction that the respective result should
154
not be used for any further prediction. As described before, CCD varies numeric factors on
155
three levels. If a categoric factor is added, e. g. the type of acid, the CCD needs to be
156
executed for both nitric and citric acid. To identify the effect of this categoric factor, all data
157
have to be calculated within the same ANOVA.
158 159
2.3. Extraction procedure
160
For each extraction, 50 g of pea hulls were processed as shown in Fig. 1. Pre-tests revealed
161
that the raw pea hulls consisted of a fraction < 50 µm that was characterized by a high
162
amount of residual starch (~30 %) and protein (~18 %). Hence, this fraction was separated
163
by dry-sieving prior to extraction. Extraction procedures using nitric acid were conducted as
164
follows: pea hulls were suspended in 1000 g of distilled water at the respective pH (adjusted
165
by the addition of HNO3 (65% w/w)) and temperature according to the CCD (Table 1). The
166
suspension was stirred and temperature was kept constant for the specific extraction time.
167
Losses due to evaporation were compensated by the addition of distilled water. The pH-
168
value was monitored, however, no changes were detected neither due to evaporation nor
169
due to the addition of water. After extraction, the suspension was filtered through a filter cloth
170
(P4033, Winkler GmbH, Germany). The retentate was washed with 400 g of distilled water
171
and filtered again. Both filtrates were combined and cooled in an ice water bath (< 20 °C).
172
The PPS was precipitated from the filtrate by adding the two-fold amount of ethanol (95%
173
v/v) and allowing the suspension to stand for another 30 minutes at room temperature. The
174
precipitate was separated by filtration and then washed again (three times in total with
175
intermediate filtration) with ethanol (95% v/v). After the last filtration step and mechanically
176
squeezing off the ethanol, PPS samples were dried at 50 °C for 3 hours in a drying-oven and
177
allowed to cool down in a desiccator overnight. Dry PPS samples exhibited a dry substance
178
of at least 92 °% and were then milled smaller than 250 µm with a centrifugal mill (ZM1, 5
179
Retsch, Haan, Germany). Samples were stored in sealed glass jars in a refrigerator until
180
further analysis. Extractions using citric acid were conducted as described above with the
181
following modifications. The pH was adjusted by firstly adding citric acid monohydrate to the
182
pre-heated dist. water. After addition of pea hulls, pH and temperature were adjusted again
183
and extraction was conducted for the respective period of time (Table 1).
184 185
2.4. Determination of PPS yield
186
Yield (%) was determined by dividing the mass of dried PPS by the initial mass of pea hulls
187
used for the specific extraction.
188 189
2.5. Determination of PPS composition
190
The content of uronic acids (UA) and the degree of methoxylation (DM) were analysed
191
spectrophotometrically via the m-hydroxybiphenyl method (Blumenkrantz & Asboe-Hansen,
192
1973) after sample pre-treatment as described by Gutöhrlein et al. (2018) and the
193
chromotropic acid method (Bäuerle, Otterbach, Gierschner, & Baumann, 1977), respectively.
194
The degree of acetylation (DAc) was analysed with a Megazyme Acetic Acid Assay Kit (ACS
195
Manual Format, Megazyme u.c., Ireland) in a multistage degradation of acetic acid to NADH,
196
which was determined spectrophotometrically at 340 nm. Release of acetic acid from PPS
197
was conducted by saponification (Levigne, Thomas, Ralet, Quemener, & Thibault, 2002).
198
DAc (%) was calculated as the molar ratio of acetic acid and UA. Neutral sugar analysis was
199
carried out by HPAEC-PAD (Shimadzu LC20AD SP; columns: CarboPac™ Bio LC™ 4 x
200
50 mm Guard, CarboPac™ PA10 4 x 250 mm, CarboPac™ PA1 4 x 250 mm; detector:
201
Dionex PAD-2) after liberation of neutral sugar monosaccharides from PPS by a combined
202
enzymatic and chemical digestion. To this end, PPS was firstly treated by a mixture of cell
203
wall degrading enzymes (1% Vegazyme M (Erbsloeh Geisenheim AG, Germany) and 1%
204
Ultrazyme AFP L (Novozymes Switzerland AG, Switzerland)) in distilled water for 48 h at
205
35 °C. Subsequently, a treatment with TFA (0.2 M) at 80 °C for 96 h was applied. The
206
digestion was completed by a second enzymatic step at pH 5 and 35 °C for 24 h using the
207
same enzyme mixture. After centrifugation, sample solution was injected to HPAEC-PAD.
208
NaOH (0.025 M) was used as eluent with a flow rate of 0.7ml/min. The content of individual
209
sugars (fucose, rhamnose, arabinose, galactose, glucose and xylose) was calculated by an
210
external calibration with defined concentration of the respective sugar standard. Molecular
211
weight distribution was performed using gel permeation chromatography (GPC) (Degasser,
212
Degasys DG 1310; pump, Shimadzu LC-10ADVP; autosampler, Merck AS-4000; guard
213
column, Agilent PL Aquagel OH, 3 mm; 1st column, Agilent PL Aquagel OH Mixed-H, 8 mm;
214
2nd column, Agilent PL Aquagel Mixed, 8 mm; software, Shimadzu LabSolutions v5.71 SP1))
215
as described by Wegener, Kaufmann, & Kroh (2017) with the following modifications: 6
216
samples were solubilised in distilled water overnight and purified by centrifugation and
217
filtration (0.45µm syringe filter). Signal detection was realised via a refractive index detector.
218
All measurements were conducted at least in duplicate.
219 220
3. Results and discussion
221
3.1. Influence of extraction parameters on yield of PPS
222
PPS yield varied between 1.4 and 8.0 % for an extraction with nitric acid and between 3.5
223
and 9.8 % if citric acid was used (Fig. 2). ANOVA tables showing the results of statistical
224
evaluation are presented within the supplementary material (appendix). PPS yield was
225
significantly affected by pH, temperature and extraction time (see appendix, Table A1). In
226
general, a higher temperature and a higher pH increased PPS yield. Therefore, highest
227
yields were achieved at a temperature of 90 °C and pH 2 (Fig. 2). Furthermore, a significant
228
interaction between pH and temperature was detected (p < 0.0001) (Table A1), which
229
indicates that the effect of temperature also depends on the pH at which extraction was
230
executed. More specific, at pH 1 temperature less affects PPS yield, whereas at pH 2 PPS
231
yield increases more strongly with increasing temperature. Moreover, a longer extraction
232
time (6 h) led to a higher yield under ‘mild’ conditions (pH 2, 70 °C), but resulted in a lower
233
yield at ‘harsh’ conditions (pH 1, 90 °C).
234 235
Generally, a low pH as well as a high temperature support the solubilisation of pectin from
236
insoluble cell-wall-located protopectin (Andersen et al., 2017; Methacanon, Krongsin, &
237
Gamonpilas, 2014; Renard, Voragen, Thibault, & Pilnik, 1990). Independently from the raw
238
material, this main reaction may be overlapped by another reaction during pectin extraction,
239
which is the acidic hydrolysis of pectin already extracted from the cell wall. Hydrolysis is
240
related to the depolymerisation of pectin via backbone degradation or a cleavage of neutral
241
sugars from the side chains of PPS (Einhorn-Stoll, Kastner, Urbisch, Kroh, & Drusch, 2019).
242
Interestingly, both reactions seem to affect extraction of PPS from pea hulls with a strong
243
dependence from extraction time and temperature in a very narrow pH range (pH = 1 – 2).
244
Hence, under ‘mild’ conditions solubilisation of protopectin is favored at longer extraction
245
time, whereas depolymerisation during extraction under ‘harsh’ conditions would be the
246
predominant reaction. Practically, a lower yield after extraction under ‘harsh’ conditions is a
247
result of a higher amount of shorter PPS fragments, which will not precipitate during ethanol
248
washing.
249 250
If citric acid is used, PPS yield is higher than by using nitric acid (Fig. 2). The effect of higher
251
yields using citric acid have been previously discussed by Canteri-Schemin et al. (2005) and
7
252
Kliemann et al. (2009) using the example of apple pomace or passion fruit peel extraction.
253
The latter explained a higher yield by a weaker hydrolysis of extracted pectins. In contrast,
254
Kermani et al. (2014) proposed that the extraction of LMP from mango peels is attributed to
255
the chelating activity of citric acid. Citric acid may chelate cations and thus promote the
256
extraction of calcium bound pectin. However, two carboxylic acid groups in their dissociated
257
form would be necessary for complexation of divalent cations. Ravn and Meyer (2014) stated
258
a pKa of 3.09 for citric acid. Therefore, citric acid molecules should be mostly undissociated
259
at the strong acidic conditions (pH 0.8 - 2.2) used in the present study which makes a
260
complexation of calcium rather unlikely. The higher yield of pea hull PPS using citric acid
261
may be also attributed to a weaker hydrolysis of extracted pectin under the conditions used
262
in here. Nevertheless, the yield alone cannot reveal any information on the purity or the
263
molecular fine structure of the extracted PPS, e. g. with regard to its UA content or the
264
proportion of single neutral sugars. For a better understanding of any potentially occurring
265
reaction we analysed several molecular parameters which will be shown and discussed in
266
section 3.2.
267 268
3.2. Influence of extraction parameters on composition of PPS
269
The content of UA in extracted PPS was significantly affected by the type of acid (p < 0.0001)
270
(Table A2). PPS consisted of 45 to 77% UA when extracted with nitric acid and 36 to 67% if
271
citric acid was applied (Fig. 3).
272
For both types of acid, an increase in temperature also led to an increase of UA in the
273
resulting PPS (Fig. 3), which has already been described for an extraction of cacao pod
274
husks with nitric acid (Vriesmann et al., 2011) and an extraction of banana peels with citric
275
acid (Oliveira et al., 2016) at conditions similar to those used in the present study. This effect
276
may be generally attributed to the removal of neutral sugars, which will be discussed in the
277
following section. Furthermore, UA was significantly influenced by pH (p = 0.0226) and a
278
significant interaction between pH and extraction time (p = 0.0157) was identified (Table A2).
279
Accordingly, on average, a prolongation of extraction time from three to six hours increased
280
the UA content in PPS at pH 2, whereas UA decreased with increasing extraction time at
281
pH 1. Moreover, a significant interaction between pH and type of acid (p = 0.0444) was found
282
This is attributed to the fact, that pH hardly affected UA in case of citric acid (averaged over
283
extraction time and temperature) but resulted in a considerable increase in UA if pH was
284
lowered using nitric acid. Thus, highest UA contents (> 70 %) were found in PPS samples
285
that were extracted under harsh conditions (e. g. pH 1, 90 °C) using nitric acid. This result
286
discloses a conflict of aims with regard to the yield of PPS extracted from pea hulls and its
287
purity in terms of the content of UA. Hence, using nitric acid, highest UA contents were
288
achieved under conditions at which yields were only moderate. It must be assumed that 8
289
under harsh conditions non-UA components are largely removed, but concurrently the
290
depolymersisation of the pectic backbone is taking place leading to a lower total yield.
291
Gel permeation chromatography (GPC) was additionally used to monitor decomposition of
292
pea hull PPS under variable extraction conditions. Fig. 4 shows a representative GPC profile
293
of extracted PPS using nitric acid as extraction medium.
294 295
Molecular weight decreases with increasing extraction temperature as well as with prolonged
296
extraction time, which is illustrated by a shift of the GPC profiles towards longer elution time.
297
No clear differences between both types of acid were noticed (results for citric acid not
298
shown); therefore the same hydrolytic effect on the pectic backbone has to be assumed.
299
However, a citric acid treatment at prolonged extraction time under ‘mild’ conditions (pH 2,
300
70 °C), contrary to nitric acid, resulted in highest yields as well as in highest purity (UA)
301
(Fig. 2, Fig. 3). Most likely, the depolymerisation of the GalA backbone is less dominant as
302
citric acid exhibits lower acid strength at same pH conditions compared to nitric acid.
303
However, these conditions may be adequate to remove non-UA components such as neutral
304
sugars to a great extent even though maximum UA contents only range at about 62 % (Fig
305
3d). Thus, longer extraction times using citric acid may be favorable to extract PPS from pea
306
hulls, contrary to e. g. other raw materials such as pomegranate peels at which extraction
307
time virtually had no impact on GalA content as shown by Pereira et al. (2016).
308 309
Fig. 5 depicts an overview of the content of single neutral sugars (rhamnose, arabinose,
310
galactose, xylose) in pea hull PPS extracted by nitric acid for three hours. A prolonged
311
extraction time of six hours compared to three hours yielded similar amounts of rhamnose,
312
arabinose and galactose, respectively, whereas xylose levels in PPS were increased (Fig.
313
A1). Moreover, the content of rhamnose, arabinose, and galactose was reduced with
314
increasing temperature and decreasing pH (Fig. 5a–c). Consequently, the total amount of
315
neutral sugars was markedly reduced which was accompanied by a relatively increasing
316
amount of UA under the respective conditions (Table 2). Typically, these neutral sugars are
317
known to be located in pectic rhamnogalacturonan I (RG-I) sequences as well as in
318
arabinogalactan side chains (Voragen, Coenen, Verhoef, & Schols, 2009; Yapo, 2011).
319
Results of the present study suggest a hydrolysis of these RG-I sequences and a cleavage
320
of pectic side chains in extracted PPS (Axelos & Branger, 1993; Van Buren, 1979), and
321
finally reveal a pectin purification with increasingly drastic extraction conditions as already
322
stated above. Hence, pea hull PPS that may designated as ‘pectin’ according to legal
323
regulations, can be only provided under ‘harsh’ extraction conditions (pH 1, 90 °C) (Table 2).
324
In contrast to rhamnose, arabinose and galactose, the content of xylose increased with
325
increasing temperature and decreasing pH (Fig. 5d). Based on the literature (Yapo, 2011) 9
326
one may suggest that extracted PPS thus was rich in xylogalacturonan, but experimental
327
proof e.g. by NMR is required. Generally, xylose in legume cell walls is known to be a
328
constituent of hemicellulosic xylan or xyloglucan (Shiga & Lajolo, 2006). Furthermore,
329
xylogalacturonan has been previously isolated from pea hulls under acidic conditions,
330
indicating short side chains of xylose linked to GalA backbone in pea hull pectin (Le Goff et
331
al. 2001). The results of the present study confirm that xylogalacturonan in PPS may not be
332
degraded by conditions of acidic extraction, neither by nitric nor by citric acid. Extraction
333
procedures using citric acid were characterised by a relatively higher amount of rhamnose,
334
arabinose and galactose in pea hull PPS in comparison with nitric acid, and thus, exhibited a
335
relatively lower xylose content (see appendix, Fig. A1). It must be assumed that less
336
hydrolysis of arabinogalactan in this case is a consequence of the lower acid strength of citric
337
acid compared to nitric acid as already discussed above.
338 339
As expected, all extracted PPS samples were characterised by a low or medium degree of
340
methoxylation (DM). DM values of PPS extracted from pea hulls using nitric or citric acid at
341
pH 1 and 2 and at a temperature of 70 and 90 °C are shown in Table 2. Contour plots for DM
342
are summarised within the supplementary material (Fig. A2). Generally, the DM values of
343
PPS make clear that acid extraction using nitric or citric acid offer the opportunity to isolate
344
LMP from pea hulls. DM was higher when using citric acid (35.9 to 66.4%) compared to nitric
345
acid (17.0 to 46.3 %) (Table 2). Former investigations of Weightman et al. (1994) revealed
346
that a two-stage extraction using a chelating agent (CDTA) and hydrochloric acid resulted in
347
pea hull pectin fractions with a DM ranging from 24 to 51%. In the present study, the DM was
348
significantly affected by the extraction temperature (Table A3). Higher temperature and
349
longer extraction time decreased the DM, independently from the used acid (Fig. A2). This
350
result is in agreement with studies on extraction of PPS from complex cell wall matrix such
351
as passion fruit peel in a one factor at time experiment (Kulkarni & Vijayanand, 2010) or from
352
banana peels in studies using RSM (Happi Emaga, Ronkart, Roebrt, Wathelet, & Paquot,
353
2008; Oliveira et al., 2016;). Furthermore, demethoxylation of isolated pectin with increasing
354
temperature and time has been described in various studies (Constenla & Lozano, 2003;
355
Diaz, Anthon, & Barret, 2007), which is related to the hydrolytic cleavage of methoxyl groups
356
from the respective GalA building block under acidic conditions. Comparable to the DM, the
357
degree of acetylation (DAc) was markedly lowered with increasingly drastic conditions.
358
Hence, DAc decreased from 10.6 to 3.9 % changing extraction conditions from ‘mild’ (pH 2,
359
70 °C,) to ‘harsh’ (pH 1, 90 °C,) in case of nitric acid, respectively from 11.7 to 1.9 % in case
360
of citric acid (Table 2). A prolonged extraction time of six hours further decreased the DAc of
361
pea hull PPS for both types of acid compared to a treatment for three hours (Fig. A3).
362
Weightman et al. (1994) measured a DAc of 12% for pea hull PPS extracted with
10
363
hydrochloric acid at pH 1.5 and a temperature of 85 °C for 3*0.5 hours. With this much
364
shorter extraction time, their extraction procedure may be regarded comparable to an
365
extraction under ‘mild’ conditions as applied in our study. Finally, results of the present study
366
prove an exceptionally high content of protein up to 11.8 % in pea hull PPS (Table 2), which
367
is in the range or even higher than the protein content of e. g. sugar beet pectin as shown in
368
several studies (Funami et al., 2011; Li et al., 2015; Nakauma et al., 2008; Yapo, Robert,.
369
Etienne, Wathelet, & Paquot, 2007). Although not investigated it detail, it is assumed that this
370
fraction in pea hull PPS belongs to the group of cell wall (glyco-)proteins (Waldron, Parker, &
371
Smith, 2003).
372
Apart from their role as an important structural component within the cell wall in situ, this
373
protein fraction may additionally act as a functional constituent as discussed for sugar beet
374
pectin showing emulsifying properties in a recent study (Ngouémazong, Christiaens,
375
Shpigelman, Van Loey, & Hendrickx, 2015).
376 377
4. Conclusion
378
Acid extraction of pectin generally includes the solubilisation of pectin from cell-wall bound
379
protopectin and a subsequent degradation of the solubilised pectin. The results of the
380
present study suggest that both processes occur simultaneously during pectin extraction
381
from pea hulls in dependence of the parameters pH, temperature, time, and type of acid,
382
respectively. In this regard, a conflict of aims arises between a high yield and the purity of the
383
extracted pectic polysaccharides (PPS). ‘Harsh’ conditions during extraction in terms of a low
384
pH (pH 1) and a high temperature (90 °C) result in a high purity (high UA content) but yield
385
decreases as a consequence of an ongoing depolymerisation of PPS. This effect is even
386
more pronounced at prolonged extraction time. A higher purity is attributed to the cleavage of
387
RG-I sequences and a removal of pectic side chains such as arabinogalactan as shown by a
388
lower amount of rhamnose, arabinose and galactose in the resulting PPS. Neutral sugar
389
analysis moreover revealed that pea hull PPS are rich in xylogalacturonan, which obviously
390
resists degradation under acidic conditions. Furthermore, this study confirms a higher yield in
391
PPS if citric acid is applied as extraction medium, which is attributed to weaker acid strength
392
of citric acid compared to nitric acid and a reduced degradation of non-GalA components,
393
particularly neutral sugars. In conclusion, pH seems to be the driving force with regard to
394
pectin solubilisation and depolymerisation. Hence, less acidic conditions (pH 2) in
395
combination with an elevated extraction temperature and a prolonged extraction time are
396
recommended to extract pea hull PPS in adequate amounts and purity (>65 %) which may
397
be in line with the legal guidelines for low-methoxyated pectin (LMP). Gelling properties of
398
the LMP might be affected by process-induced structural alterations and minor constituents.
399
Largely independent from the extraction conditions, pea hull PPS is characterised by a high 11
400
amount of cell wall protein. Thererfore, we assume that protein-rich pea hull PPS exhibit
401
surface active properties which might be useful for the stabilisation of disperse food systems
402
such as emulsions or foams. This will be part of our ongoing research on pea hull PPS
403
functionality.
404 405
Acknowledgements
406
This IGF Project (18678 N) of the FEI is supported via AiF within the programme for
407
promoting the Industrial Collective Research (IGF) of the German Ministry of Economics and
408
Energy (BMWi), based on a resolution of the German Parliament. The authors thank Astrid
409
Kiegel, Christina Härter, Alexandra Urbisch and Pramita Devi for sample preparation and/or
410
analytical support.
12
411
References
412
Ali-Khan, S. T. (1993). Seed hull content in field pea. Canadian Journal of Plant Science, 73,
413
611-613.
414
Andersen, N., Cognet, T., Santacoloma, P., Larsen, J., Armagan, I., Larsen, F., Gernaey, K.,
415
Abildskov, J., & Huusom, J. (2017). Dynamic modelling of pectin extraction describing
416
yield and functional characteristics. Journal of Food Engineering, 192, 61-71.
417
Axelos, M., & Branger, M. (1993). The effect of the degree of esterification on the thermal
418
stability and chain conformation of pectins. Food Hydrocolloids, 7, 91-102.
419
Bäuerle, G., Otterbach, G., Gierschner, K., & Baumann, G. (1977). Bestimmung des
420
Polyuronidgehaltes
421
Deutsche Lebensmittel-Rundschau, 73, 281-286.
422 423
und
des
Veresterungsgrades
von
Handelspektinpräparaten.
BeMiller, J. N. (2019). Pectins. In J. N. BeMiller (Ed.), Carbohydrate Chemistry for Food Scientists (pp. 303-312). AACC International Press.
424
Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Silveira Villar, L., & Escaleira, L. A. (2008).
425
Response surface methodology (RSM) as a tool for optimization in analytical chemistry.
426
Talanta, 76, 965-977.
427 428
Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54, 484-489.
429
Canteri-Schemin, M. H., Fertonani, H. C. R., Waszczynskyj, N., & Wosiacki, G. (2005).
430
Extraction of pectin from apple pomace. Brazilian Archives of Biology and Technology,
431
48, 259-266.
432 433 434 435 436 437
Chaharbaghi, E., Khodaiyan, F., & Hosseini, S. S. (2017). Optimization of pectin extraction from pistachio green hull as a new source. Carbohydrate Polymers, 173, 107-113. Constenla, D., & Lozano, J. (2003). Kinetic model of pectin demethylation. Latin American Applied Research, 33, 91-95. Dalgetty, D. D. & Baik, B.-K. (2006). Fortification of Bread with Hulls and Cotyledon Fibres Isolated from Peas, Lentils, and Chickpeas. Cereal Chemistry, 83, 269-274.
438
Diaz, J. V., Anthon, G. E., & Barrett, D. M. (2007). Nonenzymatic degradation of citrus pectin
439
and pectate during prolonged heating: effects of pH, temperature, and degree of methyl
440
esterification. Journal of Agricultural and Food Chemistry, 55, 5131-5136.
441
Einhorn-Stoll, U., Kastner, H., Urbisch, A., Kroh, L. W., & Drusch, S. (2019). Thermal
442
degradation of citrus pectin in low-moisture environment – Influence of acidic and
443
alkaline pre-treatment. Food Hydrocolloids, 86, 104-115.
444 445
Endreß, H.-U., & Christensen, S. (2009) Pectins. In G. O. Phillips, & P. A. Williams (Eds.), Handbook of Hydrocolloids (pp. 274-297). Oxford: Woodhead Publishing Ltd.
446
Everett, D. W., & McLeod, R. E. (2005). Interactions of polysaccharide stabilisers with casein
447
aggregates in stirred skim-milk yoghurt. International Dairy Journal, 15, 1175–1183. 13
448
Fraeye, I., Doungla, E., Duvetter, T., Moldenaers, P., Loey, A. V., & Hendrickx, M. (2009).
449
Influence of intrinsic and extrinsic factors on rheology of pectin-calcium gels. Food
450
Hydrocolloids, 23, 2069-2077.
451
Funami, T., Nakauma, M., Ishihara, S., Tanaka, R., Inoue, T., & Phillips, G. O. (2011).
452
Structural modifications of sugar beet pectin and the relationship of structure to
453
functionality. Food Hydrocolloids, 25, 221-229.
454 455
Gutöhrlein, F., Drusch, S., & Schalow, S. (2018). Towards By-Product utilization of Pea Hulls: Isolation and Quantification of Galacturonic Acid. Foods, 7, 203.
456
Happi Emaga, T., Ronkart, S. N., Robert, C., Wathelet, B., & Paquot, M. (2008).
457
Characterisation of pectins extracted from banana peels (Musa AAA) under different
458
conditions using an experimental design. Food Chemistry, 108, 463-471.
459
Igbasan, F. A., Guenter, W., & Slominski, B. A. (1997). Field peas: Chemical composition
460
and energy and amino acid availabilities for poultry. Canadian Journal of Animal
461
Science, 77, 293-300.
462
Jafari, F., Khodaiyan, F., Kiani, H., & Hosseini, S. S. (2017). Pectin from carrot pomace:
463
Optimization of extraction and physicochemical properties. Carbohydrate Polymers,
464
157, 1315-1322.
465
Kermani, Z. J., Shpigelman, A., Kyomugasho, C., Buggenhout, S. V., Ramezani, M., Loey, A.
466
M. V., & Hendrickx, M. E. (2014). The impact of extraction with a chelating agent under
467
acidic conditions on the cell wall polymers of mango peel. Food Chemistry, 161, 199-
468
207.
469
Kleppmann, W. (2013). Versuchsplanung. (8. Aufl.). München: Carl Hanser Verlag.
470
Kliemann, E., Nunes de Simas, K., Amante, E. R., Schwinden Prudêncio, E., Teófilo, R. F.,
471
Ferreira, M. M. C., & Amboni, R. D. M. C. (2009). Optimisation of pectin acid extraction
472
from passion fruit peel (Passiflora edulis flavicarpa) using response surface
473
methodology. International Journal of Food Science & Technology, 44, 476-483.
474
Kulkarni, S., & Vijayanand, P. (2010). Effect of extraction conditions on the quality
475
characteristics of pectin from passion fruit peel (Passiflora edulis f. flavicarpa L.). LWT -
476
Food Science and Technology, 43, 1026-1031.
477
Le Goff, A., Renard, C. M. G. C., Bonnin, E., & Thibault, J.-F. (2001). Extraction, purification
478
and chemical characterization of xylogalacturonans from pea hulls. Carbohydrate
479
Polymers, 45, 325-334.
480
Levigne, S., Thomas, M., Ralet, M.-C., Quemener, B., & Thibault, J.-F. (2002). Determination
481
of the degrees of methylation and acetylation of pectins using a C18 column and
482
internal standards. Food Hydrocolloids, 16, 547-550.
483
Li, D., Du, G., Jing, W., Li, J., Yan, J., & Liu, Z. (2015). Combined effects of independent
484
variables on yield and protein content of pectin extracted from sugar beet pulp by citric
14
485
acid. Carbohydrate Polymers, 129, 108-114.
486
Limberg, G., Körner, R., Buchholt, H. C., Christensen, T. M. I. E., Roepstorff, P., &
487
Mikkelsen, J. D. (2000a). Analysis of different de-esterifcation mechanisms for pectin by
488
enzymatic fingerprinting using endopectin lyase and endopolygalacturonase II from A.
489
niger. Carbohydrate Research, 327, 293-307.
490
Limberg, G., Körner, R., Buchholt, H. C., Christensen, T. M. I. E., Roepstorff, P., &
491
Mikkelsen, J. D. (2000b). Quantification of the amount of galacturonic acid residues in
492
blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and
493
endo-polygalacturonase II from Aspergillus niger. Carbohydrate Research, 327, 321-
494
332.
495
Methacanon, P., Krongsin, J., & Gamonpilas, C. (2014). Pomelo (Citrus maxima) pectin:
496
Effects of extraction parameters and its properties. Food Hydrocolloids, 35, 383-391.
497
Nakauma, M., Funami, T., Noda, S., Ishihara, S., Al-Assaf, S., Nishinari, K., & Phillips, G. O.
498
(2008). Comparison of sugar beet pectin, soybean soluble polysaccharide, and gum
499
arabic as food emulsifiers. 1. Effect of concentration, pH, and salts on the emulsifying
500
properties. Food Hydrocolloids, 22, 1254-1267.
501
Ngouémazong, E. D., Christiaens, S., Shpigelman, A., Van Loey, A., & Hendrickx, M. (2015).
502
The emulsifying and emulsion-stabilizing properties of pectin: A review. Comprehensive
503
Reviews in Food Science and Food Safety, 14, 705-718.
504
Pereira, P. H. F., Oliveira, T. I. S., Rosa, M. F., Cavalcante, F. L., Moates, G. K., Wellner, N.,
505
Waldron, K. W., & Azeredo, H. M. (2016). Pectin extraction from pomegranate peels
506
with citric acid. International Journal of Biological Macromolecules, 88, 373-379.
507
Pickardt, C., Dongowski, G., & Kunzek, H. (2004). The influence of mechanical and
508
enzymatic disintegration of carrots on the structure and properties of cell wall materials.
509
European Food Research & Technology, 219, 229-239.
510
Raji, Z., Khodaiyan, F., Rezaei, K., Kiani, H., & Hosseini, S. S. (2017). Extraction
511
optimization and physicochemical properties of pectin from melon peel. International
512
Journal of Biological Macromolecules, 98, 709-716.
513 514 515 516
Ravn, H. C., & Meyer, A. S. (2014). Chelating agents improve enzymatic solubilization of pectinaceous co-processing streams. Process Biochemistry, 49, 250-257. Reichert, R. (1981). Quantitative isolation and estimation of cell wall material from dehulled pea (Pisum sativum) flours and concentrates. Cereal Chemistry, 58, 266-270.
517
Renard, C. M. G. C., Voragen, A. G. J., Thibault, J. F., & Pilnik, W. (1990). Studies on apple
518
protopectin: I. Extraction of Insoluble Pectin by Chemical Means. Carbohydrate
519
Polymers, 12, 9-25.
520 521
Renard, C. M. G. C., Weightman, R. M., & Thibault, J.-F. (1997). The xylose-rich pectins from pea hulls. International Journal of Biological Macromolecules, 21, 155-162.
15
522 523
Rolin, C. (2002). Commercial pectin preparations. In G. B. Seymour, & J. B. Knox (Eds.), Pectins and their manipulation (pp 222-241). Oxford: CRC Press Blackwell Publishing.
524
Rolin, C., Chrestensen, I. B., Hansen, K. M., Staunstrup, J., & Sørensen S. (2010). Tailoring
525
pectin with specific shape, composition and esterifaction pattern. In P. A. Williams, & G.
526
O. Phillips (Eds.), Gums and Stabilisers for the Food Industry 15 (pp 13-25). Cambridge:
527
RSC Publishing.
528
Oliveira, T. I. S., Rosa, M. F., Cavalcante, F. L., Pereira, P. H. F., Moates, G. K., Wellner, N.,
529
Mazzetto, S. E., Waldron, K. W., & Azeredo, H. M. C. (2016). Optimization of pectin
530
extraction from banana peels with citric acid by using response surface methodology.
531
Food Chemistry, 198, 113-118.
532
Shan, Y. (2016). Extraction processes of functional components from citrus peel. In Y. Shan
533
(Ed.), Comprehensive Utilization of Citrus By-Products (pp 31-58). London: Academic
534
Press.
535 536
Shiga, T. M., & Lajolo, F. M. (2006). Cell wall polysaccharides of common beans (Phaseolus vulgaris L.) composition and structure. Carbohydrate Polymers, 63, 1-12.
537
Thibault, J.-F., & Ralet, M.-C. (2003). Physico-chemical properties of pectins in the cell walls
538
and after extraction. In F. Voragen, H. Schols, & R. Visser (Eds.), Advances in Pectin
539
and Pectinase Research (pp 91-105). Dordrecht: Springer Netherlands.
540
Van Buggenhout, S, Sila, D. N., Duvetter, T., Van Loey, A., & Hendrickx, M. (2009). Pectins
541
in Processed Fruits and Vegetables. Comprehensive Reviews in Food Science and
542
Food Safety, 8, 105-117.
543 544 545 546
Van Buren, J. P. (1979). The chemistry of texture in fruits and vegetables. Journal of Texture Studies, 10, 1-23. Voragen, A. G. J., Coenen, G.-J., Verhoef, R. P., & Schols, H. A. (2009). Pectin, a versatile polysaccharide present in plant cell walls. Structural Chemistry, 20, 263-275.
547
Vriesmann, L. C., Teófilo, R. F., & de Oliveira Petkowicz, C. L. (2011). Optimization of nitric
548
acid-mediated extraction of pectin from cacao pod husks (Theobroma cacao L.) using
549
response surface methodology. Carbohydrate Polymers, 84, 1230-1236.
550
Vriesmann, L. C., Teófilo, R. F., & de Oliveira Petkowicz, C. L. (2012). Extraction and
551
characterization of pectin from cacao pod husks (Theobroma cacao L.) with citric acid.
552
LWT - Food Science & Technology, 49, 108-116.
553 554
Waldron, K., Parker, M., & Smith, A. (2003). Plant cell walls and food quality. Comprehensive Reviews in Food Science and Food Safety, 2, 128-146.
555
Wegener, S., Kaufmann, M., & Kroh, L. W. (2017). Influence of L-pyroglutamic acid on the
556
color formation process of non-enzymatic browning reactions. Food Chemistry, 232,
557
450-454.
558
Weightman, R. M., Renard, C. M. G. C., & Thibault J.-F. (1994). Structure and properties of
16
559
the polysaccharides from pea hulls. Part 1: Chemical extraction and fractionation of the
560
polysaccharides. Carbohydrate Polymers, 24, 139-148.
561 562
Willats, W. G. T., Knox, J. P., & Mikkelsen, J. D. (2006). Pectin: new insights into an old polymer are starting to gel. Trends in Food Science & Technology, 17, 97-104.
563
Yapo, B. M., Robert, C., Etienne, I., Wathelet, B., & Paquot, M. (2007). Effect of extraction
564
conditions on the yield, purity and surface properties of sugar beet pulp pectin extracts.
565
Food Chemistry, 100, 1356-1364.
566
Yapo, B. M. (2009). Biochemical characteristics and gelling capacity of pectin from yellow
567
passion fruit rind as affected by acid extractant nature. Journal of Agricultural and Food
568
Chemistry, 57, 1572-1578.
569 570
Yapo, B. M. (2011). Pectic substances: From simple pectic polysaccharides to complex pectins - a new hypothetical model. Carbohydrate Polymers, 86, 373-385.
571 572
17
573
Figures and Tables - overview
574
Fig. 1. Schematic overview of PPS extraction from pea hulls.
575
Fig. 2. Contour plots for pea hull PPS yield (scale ranging from 1 to 10 %) in dependence of
576
pH and temperature using nitric acid (upper row) or citric acid (lower row) for two different
577
extraction times (left column (a, c): three hours; right column (b, d): six hours).
578
Fig. 3. Contour plots for UA content in pea hull PPS (scale ranging from 40 to 80 %) in
579
dependence of pH and temperature using nitric acid (upper row) or citric acid (lower row) for
580
two different extraction times (left column (a, c): three hours; right column (b, d): six hours).
581
Fig. 4. GPC profile of PPS extracted with nitric acid in dependence of extraction conditions
582
exemplarily shown for nitric acid (absorption normalised to maximal absorption of the
583
respective sample).
584
Fig. 5. Contour plots for the content of selected neutral sugars in pea hull PPS extracted with
585
nitric acid for 3 hours in dependence of pH and temperature.
586 587
Table 1 CCD for extraction process of PPS from pea hulls using nitric acid or citric acid.
588
Table 2 Composition of pea hull PPS shown for a 3 h extraction with nitric or citric acid at
589
different temperature and pH levels.
590
18
591 592
Table 1
593
CCD for extraction process of PPS from pea hulls using nitric acid or citric acid. Factor
-1
-α (-1.353)
Temperature Time
0
+1
+α (+1.353)
[°C]
66
70
80
90
94
[h]
2.5
3.0
4.5
6.0
6.5
0.8
1.0
1.5
2.0
2.2
pH
594 595 596
Table 2
597
Composition of pea hull PPS exemplarily shown for a 3 h extraction with nitric or citric acid at
598
different temperature and pH levels. Type of acid Nitric
Citric
599
Temperature [°C]
pH
Yield [%]
UA [%]
DM [%]
DAc [%]
Neutral sugars total [%]
Protein [%]
70
1
4.8
56.0
44.2
3.0
26.1
9.1
70
2
3.7
47.5
47.5
10.6
34.0
7.6
90
1
2.8
69.8
38.1
3.9
15.4
7.3
90
2
8.0
63.7
37.8
n.d.
25.3
7.2
70
1
4.2
49.1
45.5
8.0
32.7
9.1
70
2
3.3
45.5
48.9
11.7
35.7
8.9
90
1
5.6
67.4
41.1
1.9
19.6
7.6
90
2
9.8
61.0
36.4
5.4
27.5
8.8
n.d. – not detected
600 601
19
Pea hulls
Sieving
Fraction < 50µm
Fraction > 50µm
Extraction
Filtration
Precipitation
Drying
PPS
Fibre
(a) 3h Nitric Acid
(c) 3h Citric Acid
(b) 6h Nitric acid
(d) 6h Citric Acid
Fig. 2. Contour plots for pea hull PPS yield (scale ranging from 1 to 10 %) in dependence of pH and temperature using nitric acid (upper row) or citric acid (lower row) for two different extraction times (left column (a, c): three hours; right column (b, d): six hours).
(a) 3h Nitric Acid
(c) 3h Citric Acid
(b) 6h Nitric acid
(d) 6h Citric Acid
Fig. 3. Contour plots for UA content in pea hull PPS (scale ranging from 40 to 80 %) in dependence of pH and temperature using nitric acid (upper row) or citric acid (lower row) for two different extraction times (left column (a, c): three hours; right column (b, d): six hours).
1,0 0,9
70°C, 3 h
0,8 90°C, 3 h
Detector [a.u.]
0,7 0,6
90°C, 6 h
0,5 0,4 0,3 0,2 0,1 0,0 0
5
10
15
Elution time (min)
20
25
(a) Rhamnose
(c) Galactose
(b) Arabinose
(d) Xylose
Fig. 5. Contour plots for the content of selected neutral sugars in pea hull PPS extracted with nitric acid for 3 hours in dependence of pH and temperature.
Highlights • Extraction with nitric or citric acid generates low methoxylated pectin. • Pea hull pectin is low acetylated and rich in xylose and protein. • Increasing temperature and decreasing pH promote pectin purity but reduce yield. • Citric acid extraction increases yield due to a higher amount of neutral sugars. • Prolonged extraction with nitric acid at increased pH and temperature is recommended.
Extraction of low methoxylated pectin from pea hulls via RSM Friederike Gutöhrlein, Stephan Drusch*, Sebastian Schalow
Friederike Gutöhrlein: Conceptualisation, Investigation, formal analysis, scientific discussion Stephan Drusch: Writing – review and editing, supervision, scientific discussion Sebastian Schalow: Writing-orignal draft, project administration, scientific discussion
Extraction of low methoxylated pectin from pea hulls via RSM Friederike Gutöhrlein, Stephan Drusch*, Sebastian Schalow
Compliance with ethical standards: The authors declare no conflict of interest.
S0268-005X(19)32208-8
DOI:
https://doi.org/10.1016/j.foodhyd.2019.105609
Reference:
FOOHYD 105609
To appear in:
Food Hydrocolloids
Received Date: 23 September 2019 Revised Date:
26 November 2019
Accepted Date: 17 December 2019
Please cite this article as: Gutöhrlein, F., Drusch, S., Schalow, S., Extraction of low methoxylated pectin from pea hulls via RSM, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/j.foodhyd.2019.105609. 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. © 2019 Published by Elsevier Ltd.
Reactions during acidic extraction of pectic polysaccharides from pea hulls Solubilisation
Degradation
Temperature / Time /pH Citric acid x
x x
x x
x x
x
Nitric acid Temperature + Time Galacturonic acid
Rhamnose
x Xylose
RGI side Chains (Ara,Gal)
1
Extraction of low methoxylated pectin from pea hulls via RSM
2
Friederike Gutöhrlein, Stephan Drusch*, Sebastian Schalow
3 4
Technische Universität Berlin, Food Technology and Food Material Science
5
Königin-Luise-Strasse 22, D-14195 Berlin, Germany
6
*Corresponding author
7
E-mail-address: [email protected]
8 9 10
Abstract
11
Nowadays, low methoxylated pectin (LMP) is generated in a multi-step process from high
12
methoxylated pectin using fruit by-products as a raw material, although various other plant
13
sources naturally contain LMP. In this study, we prove that LMP may be directly extracted
14
from pea hulls. Extraction was conducted according to a central composite design (CCD) and
15
evaluated via response surface methodology (RSM). The influence of different parameters
16
(pH, temperature, time) on yield and composition of the extracted pectic polysaccharides
17
(PPS) was investigated using nitric acid and citric acid as extraction media. Citric acid
18
yielded higher amounts of PPS (3.5 - 9.8%) compared to nitric acid (1.4 - 8.0%). However,
19
there is a conflict of aims between a high yield and the purity of the extracted PPS.
20
Composition analysis suggests that under ‘mild’ extraction conditions (pH 2, 70 °C) PPS
21
consist of homogalacturonan, xylogalacturonan and rhamnogalacturonan with arabinose and
22
galactose side chains (RG-I). With increasing temperature (90 °), yield is maximised due to
23
an increased solubilisation of cell wall polysaccharides. Under ‘harsh” conditions (pH 1,
24
90 °C) the purity of PPS increases in terms of a relatively higher content of uronic acids, but
25
yield decreases. This is attributed to a cleavage of non-GalA components and an ongoing
26
depolymerisation of the pectic galacturonan. PPS extracted under these conditions is
27
characterised by a low degree of acetylation (4%) and a relatively high protein content (7%).
28 29
Keywords
30
Pea hulls, extraction, citric acid, nitric acid, pectin
31
1
32
1. Introduction
33
Low methoxylated pectin (LMP) is defined by a degree of methoxylation (DM) lower than
34
50 %, which enables LMP to form gels by cross-linking via divalent cations (e. g. calcium),
35
even in the absence of sugar (Endreß & Christensen, 2009; Willats, Knox, & Mikkelsen,
36
2006). This makes it a widely used stabiliser in various food matrices with a reduced sugar
37
content such as low-calorie jams or jellies. LMP gels are pumpable, remeltable and
38
reformable and may be further used as fillings for bakery products in industrial fabrication
39
(BeMiller, 2019). Moreover, LMP can be applied as a texturiser in yoghurt, fruit preparations
40
or desserts, respectively (Everett, & McLeod, 2005; Van Buggenhout, Sila, Duvetter, Van
41
Loey, & Hendrickx, 2009).
42
Industrially, pectin is extracted from citrus peels and apple pomace or in minor quantities
43
from sugar beet pulp (Thibault & Ralet, 2003). Extraction is executed in an acidic milieu using
44
water as a solvent, which is commonly acidified with nitric acid to provide a pH value
45
between 1 and 3 in the final slurry. Extraction temperature and time may vary in broad
46
ranges (50 – 100 °C; 3 – 12 h) (Rolin, 2002). The slurry is filtrated, the liquid phase (thin
47
juice) is concentrated by evaporation and subsequently precipitated with ethanol. The
48
resulting precipitate then is dried and milled (Shan, 2016). The industrial process provides a
49
high methoxylated pectin (HMP; DM ≥ 50 %) which typically shows a galacturonic acid
50
(GalA) content of 65 % or higher and thus meets legislation requirements of the FAO and EU
51
for food application (Endreß & Christensen, 2009). A more ecological approach during pectin
52
extraction may consider a replacement of inorganic acids by organic acids such as citric acid.
53
Various studies revealed, that even higher yields of pectin were obtained if citric acid was
54
applied instead of nitric acid using apple pomace, (Canteri-Schemin, Fertonani,
55
Waszczynskyj, & Wosiacki, 2005), cacao pod husks (Vriesmann, Teófilo, & de Oliveira
56
Petkowicz, 2011 and 2012) or passion fruit peel (Kliemann et al., 2009), respectively, as a
57
raw material. However, these studies mainly focused on yield optimisation and little is known
58
about the composition of such citric acid extracted pectins.
59
To generate LMP from the respective source another step has to be introduced into the
60
process of pectin extraction, which is the demethoxylation of the inherent HMP. This step
61
may be directly realised during pectin extraction or after precipitation of HMP using acid or
62
alkali in an alcoholic medium. The resulting solution is further neutralised, washed several
63
times with alcohol and then dried and milled to generate a powder of LMP (Thibault & Ralet,
64
2003). Such methods using chemical demethoxylation usually result in a random distribution
65
of the methoxyl groups within the homogalacturonan chain and may additionally promote
66
depolymerisation of GalA backbone to a certain extent. Enzymatic demethoxylation has been
67
reported as an alternative route to alter the pectin fine structure in terms of its degree of
68
methoxylation (Rolin, Chrestensen, Hansen, Staunstrup, J., & Sørensen, 2010). 2
69
Depending on the type of pectin-degrading enzyme used for demethoxylation (plant or
70
microbial), the resulting LMP may differ with regard to its distribution pattern of methoxyl
71
groups (blockwise or random) (Fraeye et al., 2009; Limberg et al., 2000a,b; Shan, 2016).
72 73
Unlike the above mentioned raw materials, pectic polysaccharides (PPS) with a low DM have
74
been proven as a natural constituent in vegetables as shown for cell wall materials prepared
75
from carrots (Pickardt, Dongowski, & Kunzek, 2004). Jafari, Khodaiyan, Kiani, & Hosseini
76
(2017) directly extracted LMP from carrot pomace without a demethoxylation step, resulting
77
in pectin samples with a DM that ranged from 22.1 % to 51.8 % depending on the extraction
78
conditions. Furthermore, other raw materials such as passion fruit rind, pistachio peels,
79
pomegranate peels, water melon peels or cacao pod husks might be used for the extraction
80
of PPS with a low DM in principal (Abid et al., 2017; Chaharbaghi, Khodaiyan, & Hosseini,
81
2017; Raji, Khodaiyan, Rezaei, Kiani, & Hosseini, 2017; Vriesmann et al., 2012; Yapo, 2009).
82
However, none of them found its way into the commercial process of pectin extraction so far.
83 84
Pea hulls may be regarded as a promising raw material for the recovery of LMP. Pea hulls
85
are a by-product during commercial pea processing, that are removed by mechanical steps
86
prior to the further valorization of the cotyledons in terms of protein and starch extraction.
87
Therefore, unlike other by-products from fruit or vegetable processing, pea hulls may be
88
recovered and collected in the dry-state and then handled centrally by a pea processing plant
89
during the campaign. Although pea hulls account for approximately 7 to 12 % of the total pea
90
mass (Ali-Khan, 1993; Igbasan, Guenter, & Slominski, 1997;) their commercial valorisation is
91
still under-utilised and only minor quantities are used for fibre enrichment in foods (Dalgetty &
92
Baik, 2006). Around 75 % of the pea hull mass consist of cellulose and hemicellulose
93
(Reichert, 1981). Furthermore, pea hulls contain 9 - 17 % of uronic acids, mainly present as
94
galacturonic acid (~97 %) with a low DM and a degree of acetylation (DAc) of approximately
95
10 % (Gutöhrlein, Drusch, & Schalow, 2018; Reichert, 1981; Weightman, Renard, & Thibault,
96
1994). In the past, several studies showed that an acidic extraction of PPS from pea hulls is
97
possible in general. In any case, the step of acidic extraction, however, was integrated into a
98
sequence of other steps using different extraction media such as chelators (CDTA) or strong
99
alkali (NaOH/KOH) and resulting in PPS fractions with different yield and with variable
100
composition (Le Goff, Renard, Bonnin, & Thibault, 2001; Renard, Weightman, & Thibault,
101
1997, Weightman et al., 1994). Using pea hulls as a raw material for commercial pectin
102
production firstly requires a systematic approach using common extraction media under
103
conditions typically applied in industrial processing, in order to identify significant factors and
104
interactions that affect pectin yield and purity. Comparable to other raw materials, it must be
105
assumed that pectin extraction from pea hulls under acidic conditions is governed by two
3
106
reactions overlapping each other: the solubilisation of the insoluble protopectin from the cell
107
wall and the degradation of the solubilised pectin in terms of the cleavage of non-GalA side
108
chains and the depolymerisation of the GalA backbone.
109
Therefore, the aim of this study was to evaluate pectin extraction from pea hulls with regard
110
to yield and composition of the extracted pectin or PPS depending on several factors:
111
extraction medium (nitric and citric acid), pH, temperature and extraction time. A central
112
composite design (CCD) was established and evaluated using response surface
113
methodology (RSM) which may be generally regarded as a useful tool for process
114
optimisation in terms of pectin yield and purity (Jafari et al., 2017; Vriesmann et al., 2011 and
115
2012). Composition was evaluated with regard to the content of uronich acids (UA), neutral
116
sugars and protein as well the DM and the degree of acetylation (DA).
117 118
2. Materials and methods
119
2.1. Material
120
Pea hulls were provided by Emsland Stärke GmbH (Germany) in form of commercially
121
available pea hull fibre PH1000 (Lot 52140). All chemicals were analytical grade and
122
supplied by Carl Roth GmbH & Co. KG (Germany), VWR International GmbH (Germany) and
123
PSS Polymer Standards Service GmbH (Germany). Enzyme preparations were provided by
124
Novozymes Switzerland AG (Switzerland) and Erbsloeh Geisenheim AG (Germany).
125 126
2.2. Experimental design for pectin extraction
127
A powerful tool for process optimisation, in general, is the response surface methodology
128
(RSM). Instead of monitoring the effects of one factor at time, a multivariate statistic
129
technique is used. Experimental data are fitted to a polynomial equation. In this way, the
130
influence of several numeric factors can be identified at the same time. A central composite
131
design (CCD) is often used to design experiments that are evaluated by RSM. A CCD
132
consists of (1) a full factorial design, which can be described as a cube and factors are varied
133
on two levels, (2) a central point that is termed with "0" and (3) star points that vary in a
134
defined distance α from the central point. In this experimental design, only the central point is
135
repeated several times. Therefore, all factors are studied at five levels (-α, -1, 0, +1, +α) with
136
a reduced number of experiments compared to a one factor at time design. The respective
137
levels of the numeric factors "extraction time", "temperature" and "pH", used in the present
138
study, are shown in Table 1. These factor levels were chosen according to the parameters
139
typically used in industrial pectin extraction. Central points (0) were performed in triplicate. All
140
experiments were performed in a randomised order to avoid systematic errors. The design
141
was used for both types of extraction media: nitric acid (-1) and citric acid (+1), which were 4
142
categorical factors. The main effects and factor interactions were evaluated by ANOVA for
143
the different responses: yield, content of uronic acid (UA), degree of methoxylation (DM),
144
degree of acetylation (DAc), content of total and individual neutral sugars and content of
145
protein. Furthermore, polynomial regression equation was calculated as described by
146
Bezerra, Santelli, Oliveira, Silveira Villar, & Escaleira (2008). Model equations with minimum
147
Predicted Residual Sum of Squares (PRESS) were chosen after non-significant factors had
148
been excluded which resulted in a quadratic model that was used for further statistical
149
evaluation using Design Expert 8.0 (Stat Ease Inc., USA). Moreover, the respective model
150
was used to create contour plots with gnuplot (Version5 patchlevel 5), in which the calculated
151
responses were printed as isolines, supported by a colour gradient to facilitate the survey of
152
experimental results (Kleppmann, 2013). Once a significant lack of fit occurred, experiments
153
were anyway evaluated and discussed, with the restriction that the respective result should
154
not be used for any further prediction. As described before, CCD varies numeric factors on
155
three levels. If a categoric factor is added, e. g. the type of acid, the CCD needs to be
156
executed for both nitric and citric acid. To identify the effect of this categoric factor, all data
157
have to be calculated within the same ANOVA.
158 159
2.3. Extraction procedure
160
For each extraction, 50 g of pea hulls were processed as shown in Fig. 1. Pre-tests revealed
161
that the raw pea hulls consisted of a fraction < 50 µm that was characterized by a high
162
amount of residual starch (~30 %) and protein (~18 %). Hence, this fraction was separated
163
by dry-sieving prior to extraction. Extraction procedures using nitric acid were conducted as
164
follows: pea hulls were suspended in 1000 g of distilled water at the respective pH (adjusted
165
by the addition of HNO3 (65% w/w)) and temperature according to the CCD (Table 1). The
166
suspension was stirred and temperature was kept constant for the specific extraction time.
167
Losses due to evaporation were compensated by the addition of distilled water. The pH-
168
value was monitored, however, no changes were detected neither due to evaporation nor
169
due to the addition of water. After extraction, the suspension was filtered through a filter cloth
170
(P4033, Winkler GmbH, Germany). The retentate was washed with 400 g of distilled water
171
and filtered again. Both filtrates were combined and cooled in an ice water bath (< 20 °C).
172
The PPS was precipitated from the filtrate by adding the two-fold amount of ethanol (95%
173
v/v) and allowing the suspension to stand for another 30 minutes at room temperature. The
174
precipitate was separated by filtration and then washed again (three times in total with
175
intermediate filtration) with ethanol (95% v/v). After the last filtration step and mechanically
176
squeezing off the ethanol, PPS samples were dried at 50 °C for 3 hours in a drying-oven and
177
allowed to cool down in a desiccator overnight. Dry PPS samples exhibited a dry substance
178
of at least 92 °% and were then milled smaller than 250 µm with a centrifugal mill (ZM1, 5
179
Retsch, Haan, Germany). Samples were stored in sealed glass jars in a refrigerator until
180
further analysis. Extractions using citric acid were conducted as described above with the
181
following modifications. The pH was adjusted by firstly adding citric acid monohydrate to the
182
pre-heated dist. water. After addition of pea hulls, pH and temperature were adjusted again
183
and extraction was conducted for the respective period of time (Table 1).
184 185
2.4. Determination of PPS yield
186
Yield (%) was determined by dividing the mass of dried PPS by the initial mass of pea hulls
187
used for the specific extraction.
188 189
2.5. Determination of PPS composition
190
The content of uronic acids (UA) and the degree of methoxylation (DM) were analysed
191
spectrophotometrically via the m-hydroxybiphenyl method (Blumenkrantz & Asboe-Hansen,
192
1973) after sample pre-treatment as described by Gutöhrlein et al. (2018) and the
193
chromotropic acid method (Bäuerle, Otterbach, Gierschner, & Baumann, 1977), respectively.
194
The degree of acetylation (DAc) was analysed with a Megazyme Acetic Acid Assay Kit (ACS
195
Manual Format, Megazyme u.c., Ireland) in a multistage degradation of acetic acid to NADH,
196
which was determined spectrophotometrically at 340 nm. Release of acetic acid from PPS
197
was conducted by saponification (Levigne, Thomas, Ralet, Quemener, & Thibault, 2002).
198
DAc (%) was calculated as the molar ratio of acetic acid and UA. Neutral sugar analysis was
199
carried out by HPAEC-PAD (Shimadzu LC20AD SP; columns: CarboPac™ Bio LC™ 4 x
200
50 mm Guard, CarboPac™ PA10 4 x 250 mm, CarboPac™ PA1 4 x 250 mm; detector:
201
Dionex PAD-2) after liberation of neutral sugar monosaccharides from PPS by a combined
202
enzymatic and chemical digestion. To this end, PPS was firstly treated by a mixture of cell
203
wall degrading enzymes (1% Vegazyme M (Erbsloeh Geisenheim AG, Germany) and 1%
204
Ultrazyme AFP L (Novozymes Switzerland AG, Switzerland)) in distilled water for 48 h at
205
35 °C. Subsequently, a treatment with TFA (0.2 M) at 80 °C for 96 h was applied. The
206
digestion was completed by a second enzymatic step at pH 5 and 35 °C for 24 h using the
207
same enzyme mixture. After centrifugation, sample solution was injected to HPAEC-PAD.
208
NaOH (0.025 M) was used as eluent with a flow rate of 0.7ml/min. The content of individual
209
sugars (fucose, rhamnose, arabinose, galactose, glucose and xylose) was calculated by an
210
external calibration with defined concentration of the respective sugar standard. Molecular
211
weight distribution was performed using gel permeation chromatography (GPC) (Degasser,
212
Degasys DG 1310; pump, Shimadzu LC-10ADVP; autosampler, Merck AS-4000; guard
213
column, Agilent PL Aquagel OH, 3 mm; 1st column, Agilent PL Aquagel OH Mixed-H, 8 mm;
214
2nd column, Agilent PL Aquagel Mixed, 8 mm; software, Shimadzu LabSolutions v5.71 SP1))
215
as described by Wegener, Kaufmann, & Kroh (2017) with the following modifications: 6
216
samples were solubilised in distilled water overnight and purified by centrifugation and
217
filtration (0.45µm syringe filter). Signal detection was realised via a refractive index detector.
218
All measurements were conducted at least in duplicate.
219 220
3. Results and discussion
221
3.1. Influence of extraction parameters on yield of PPS
222
PPS yield varied between 1.4 and 8.0 % for an extraction with nitric acid and between 3.5
223
and 9.8 % if citric acid was used (Fig. 2). ANOVA tables showing the results of statistical
224
evaluation are presented within the supplementary material (appendix). PPS yield was
225
significantly affected by pH, temperature and extraction time (see appendix, Table A1). In
226
general, a higher temperature and a higher pH increased PPS yield. Therefore, highest
227
yields were achieved at a temperature of 90 °C and pH 2 (Fig. 2). Furthermore, a significant
228
interaction between pH and temperature was detected (p < 0.0001) (Table A1), which
229
indicates that the effect of temperature also depends on the pH at which extraction was
230
executed. More specific, at pH 1 temperature less affects PPS yield, whereas at pH 2 PPS
231
yield increases more strongly with increasing temperature. Moreover, a longer extraction
232
time (6 h) led to a higher yield under ‘mild’ conditions (pH 2, 70 °C), but resulted in a lower
233
yield at ‘harsh’ conditions (pH 1, 90 °C).
234 235
Generally, a low pH as well as a high temperature support the solubilisation of pectin from
236
insoluble cell-wall-located protopectin (Andersen et al., 2017; Methacanon, Krongsin, &
237
Gamonpilas, 2014; Renard, Voragen, Thibault, & Pilnik, 1990). Independently from the raw
238
material, this main reaction may be overlapped by another reaction during pectin extraction,
239
which is the acidic hydrolysis of pectin already extracted from the cell wall. Hydrolysis is
240
related to the depolymerisation of pectin via backbone degradation or a cleavage of neutral
241
sugars from the side chains of PPS (Einhorn-Stoll, Kastner, Urbisch, Kroh, & Drusch, 2019).
242
Interestingly, both reactions seem to affect extraction of PPS from pea hulls with a strong
243
dependence from extraction time and temperature in a very narrow pH range (pH = 1 – 2).
244
Hence, under ‘mild’ conditions solubilisation of protopectin is favored at longer extraction
245
time, whereas depolymerisation during extraction under ‘harsh’ conditions would be the
246
predominant reaction. Practically, a lower yield after extraction under ‘harsh’ conditions is a
247
result of a higher amount of shorter PPS fragments, which will not precipitate during ethanol
248
washing.
249 250
If citric acid is used, PPS yield is higher than by using nitric acid (Fig. 2). The effect of higher
251
yields using citric acid have been previously discussed by Canteri-Schemin et al. (2005) and
7
252
Kliemann et al. (2009) using the example of apple pomace or passion fruit peel extraction.
253
The latter explained a higher yield by a weaker hydrolysis of extracted pectins. In contrast,
254
Kermani et al. (2014) proposed that the extraction of LMP from mango peels is attributed to
255
the chelating activity of citric acid. Citric acid may chelate cations and thus promote the
256
extraction of calcium bound pectin. However, two carboxylic acid groups in their dissociated
257
form would be necessary for complexation of divalent cations. Ravn and Meyer (2014) stated
258
a pKa of 3.09 for citric acid. Therefore, citric acid molecules should be mostly undissociated
259
at the strong acidic conditions (pH 0.8 - 2.2) used in the present study which makes a
260
complexation of calcium rather unlikely. The higher yield of pea hull PPS using citric acid
261
may be also attributed to a weaker hydrolysis of extracted pectin under the conditions used
262
in here. Nevertheless, the yield alone cannot reveal any information on the purity or the
263
molecular fine structure of the extracted PPS, e. g. with regard to its UA content or the
264
proportion of single neutral sugars. For a better understanding of any potentially occurring
265
reaction we analysed several molecular parameters which will be shown and discussed in
266
section 3.2.
267 268
3.2. Influence of extraction parameters on composition of PPS
269
The content of UA in extracted PPS was significantly affected by the type of acid (p < 0.0001)
270
(Table A2). PPS consisted of 45 to 77% UA when extracted with nitric acid and 36 to 67% if
271
citric acid was applied (Fig. 3).
272
For both types of acid, an increase in temperature also led to an increase of UA in the
273
resulting PPS (Fig. 3), which has already been described for an extraction of cacao pod
274
husks with nitric acid (Vriesmann et al., 2011) and an extraction of banana peels with citric
275
acid (Oliveira et al., 2016) at conditions similar to those used in the present study. This effect
276
may be generally attributed to the removal of neutral sugars, which will be discussed in the
277
following section. Furthermore, UA was significantly influenced by pH (p = 0.0226) and a
278
significant interaction between pH and extraction time (p = 0.0157) was identified (Table A2).
279
Accordingly, on average, a prolongation of extraction time from three to six hours increased
280
the UA content in PPS at pH 2, whereas UA decreased with increasing extraction time at
281
pH 1. Moreover, a significant interaction between pH and type of acid (p = 0.0444) was found
282
This is attributed to the fact, that pH hardly affected UA in case of citric acid (averaged over
283
extraction time and temperature) but resulted in a considerable increase in UA if pH was
284
lowered using nitric acid. Thus, highest UA contents (> 70 %) were found in PPS samples
285
that were extracted under harsh conditions (e. g. pH 1, 90 °C) using nitric acid. This result
286
discloses a conflict of aims with regard to the yield of PPS extracted from pea hulls and its
287
purity in terms of the content of UA. Hence, using nitric acid, highest UA contents were
288
achieved under conditions at which yields were only moderate. It must be assumed that 8
289
under harsh conditions non-UA components are largely removed, but concurrently the
290
depolymersisation of the pectic backbone is taking place leading to a lower total yield.
291
Gel permeation chromatography (GPC) was additionally used to monitor decomposition of
292
pea hull PPS under variable extraction conditions. Fig. 4 shows a representative GPC profile
293
of extracted PPS using nitric acid as extraction medium.
294 295
Molecular weight decreases with increasing extraction temperature as well as with prolonged
296
extraction time, which is illustrated by a shift of the GPC profiles towards longer elution time.
297
No clear differences between both types of acid were noticed (results for citric acid not
298
shown); therefore the same hydrolytic effect on the pectic backbone has to be assumed.
299
However, a citric acid treatment at prolonged extraction time under ‘mild’ conditions (pH 2,
300
70 °C), contrary to nitric acid, resulted in highest yields as well as in highest purity (UA)
301
(Fig. 2, Fig. 3). Most likely, the depolymerisation of the GalA backbone is less dominant as
302
citric acid exhibits lower acid strength at same pH conditions compared to nitric acid.
303
However, these conditions may be adequate to remove non-UA components such as neutral
304
sugars to a great extent even though maximum UA contents only range at about 62 % (Fig
305
3d). Thus, longer extraction times using citric acid may be favorable to extract PPS from pea
306
hulls, contrary to e. g. other raw materials such as pomegranate peels at which extraction
307
time virtually had no impact on GalA content as shown by Pereira et al. (2016).
308 309
Fig. 5 depicts an overview of the content of single neutral sugars (rhamnose, arabinose,
310
galactose, xylose) in pea hull PPS extracted by nitric acid for three hours. A prolonged
311
extraction time of six hours compared to three hours yielded similar amounts of rhamnose,
312
arabinose and galactose, respectively, whereas xylose levels in PPS were increased (Fig.
313
A1). Moreover, the content of rhamnose, arabinose, and galactose was reduced with
314
increasing temperature and decreasing pH (Fig. 5a–c). Consequently, the total amount of
315
neutral sugars was markedly reduced which was accompanied by a relatively increasing
316
amount of UA under the respective conditions (Table 2). Typically, these neutral sugars are
317
known to be located in pectic rhamnogalacturonan I (RG-I) sequences as well as in
318
arabinogalactan side chains (Voragen, Coenen, Verhoef, & Schols, 2009; Yapo, 2011).
319
Results of the present study suggest a hydrolysis of these RG-I sequences and a cleavage
320
of pectic side chains in extracted PPS (Axelos & Branger, 1993; Van Buren, 1979), and
321
finally reveal a pectin purification with increasingly drastic extraction conditions as already
322
stated above. Hence, pea hull PPS that may designated as ‘pectin’ according to legal
323
regulations, can be only provided under ‘harsh’ extraction conditions (pH 1, 90 °C) (Table 2).
324
In contrast to rhamnose, arabinose and galactose, the content of xylose increased with
325
increasing temperature and decreasing pH (Fig. 5d). Based on the literature (Yapo, 2011) 9
326
one may suggest that extracted PPS thus was rich in xylogalacturonan, but experimental
327
proof e.g. by NMR is required. Generally, xylose in legume cell walls is known to be a
328
constituent of hemicellulosic xylan or xyloglucan (Shiga & Lajolo, 2006). Furthermore,
329
xylogalacturonan has been previously isolated from pea hulls under acidic conditions,
330
indicating short side chains of xylose linked to GalA backbone in pea hull pectin (Le Goff et
331
al. 2001). The results of the present study confirm that xylogalacturonan in PPS may not be
332
degraded by conditions of acidic extraction, neither by nitric nor by citric acid. Extraction
333
procedures using citric acid were characterised by a relatively higher amount of rhamnose,
334
arabinose and galactose in pea hull PPS in comparison with nitric acid, and thus, exhibited a
335
relatively lower xylose content (see appendix, Fig. A1). It must be assumed that less
336
hydrolysis of arabinogalactan in this case is a consequence of the lower acid strength of citric
337
acid compared to nitric acid as already discussed above.
338 339
As expected, all extracted PPS samples were characterised by a low or medium degree of
340
methoxylation (DM). DM values of PPS extracted from pea hulls using nitric or citric acid at
341
pH 1 and 2 and at a temperature of 70 and 90 °C are shown in Table 2. Contour plots for DM
342
are summarised within the supplementary material (Fig. A2). Generally, the DM values of
343
PPS make clear that acid extraction using nitric or citric acid offer the opportunity to isolate
344
LMP from pea hulls. DM was higher when using citric acid (35.9 to 66.4%) compared to nitric
345
acid (17.0 to 46.3 %) (Table 2). Former investigations of Weightman et al. (1994) revealed
346
that a two-stage extraction using a chelating agent (CDTA) and hydrochloric acid resulted in
347
pea hull pectin fractions with a DM ranging from 24 to 51%. In the present study, the DM was
348
significantly affected by the extraction temperature (Table A3). Higher temperature and
349
longer extraction time decreased the DM, independently from the used acid (Fig. A2). This
350
result is in agreement with studies on extraction of PPS from complex cell wall matrix such
351
as passion fruit peel in a one factor at time experiment (Kulkarni & Vijayanand, 2010) or from
352
banana peels in studies using RSM (Happi Emaga, Ronkart, Roebrt, Wathelet, & Paquot,
353
2008; Oliveira et al., 2016;). Furthermore, demethoxylation of isolated pectin with increasing
354
temperature and time has been described in various studies (Constenla & Lozano, 2003;
355
Diaz, Anthon, & Barret, 2007), which is related to the hydrolytic cleavage of methoxyl groups
356
from the respective GalA building block under acidic conditions. Comparable to the DM, the
357
degree of acetylation (DAc) was markedly lowered with increasingly drastic conditions.
358
Hence, DAc decreased from 10.6 to 3.9 % changing extraction conditions from ‘mild’ (pH 2,
359
70 °C,) to ‘harsh’ (pH 1, 90 °C,) in case of nitric acid, respectively from 11.7 to 1.9 % in case
360
of citric acid (Table 2). A prolonged extraction time of six hours further decreased the DAc of
361
pea hull PPS for both types of acid compared to a treatment for three hours (Fig. A3).
362
Weightman et al. (1994) measured a DAc of 12% for pea hull PPS extracted with
10
363
hydrochloric acid at pH 1.5 and a temperature of 85 °C for 3*0.5 hours. With this much
364
shorter extraction time, their extraction procedure may be regarded comparable to an
365
extraction under ‘mild’ conditions as applied in our study. Finally, results of the present study
366
prove an exceptionally high content of protein up to 11.8 % in pea hull PPS (Table 2), which
367
is in the range or even higher than the protein content of e. g. sugar beet pectin as shown in
368
several studies (Funami et al., 2011; Li et al., 2015; Nakauma et al., 2008; Yapo, Robert,.
369
Etienne, Wathelet, & Paquot, 2007). Although not investigated it detail, it is assumed that this
370
fraction in pea hull PPS belongs to the group of cell wall (glyco-)proteins (Waldron, Parker, &
371
Smith, 2003).
372
Apart from their role as an important structural component within the cell wall in situ, this
373
protein fraction may additionally act as a functional constituent as discussed for sugar beet
374
pectin showing emulsifying properties in a recent study (Ngouémazong, Christiaens,
375
Shpigelman, Van Loey, & Hendrickx, 2015).
376 377
4. Conclusion
378
Acid extraction of pectin generally includes the solubilisation of pectin from cell-wall bound
379
protopectin and a subsequent degradation of the solubilised pectin. The results of the
380
present study suggest that both processes occur simultaneously during pectin extraction
381
from pea hulls in dependence of the parameters pH, temperature, time, and type of acid,
382
respectively. In this regard, a conflict of aims arises between a high yield and the purity of the
383
extracted pectic polysaccharides (PPS). ‘Harsh’ conditions during extraction in terms of a low
384
pH (pH 1) and a high temperature (90 °C) result in a high purity (high UA content) but yield
385
decreases as a consequence of an ongoing depolymerisation of PPS. This effect is even
386
more pronounced at prolonged extraction time. A higher purity is attributed to the cleavage of
387
RG-I sequences and a removal of pectic side chains such as arabinogalactan as shown by a
388
lower amount of rhamnose, arabinose and galactose in the resulting PPS. Neutral sugar
389
analysis moreover revealed that pea hull PPS are rich in xylogalacturonan, which obviously
390
resists degradation under acidic conditions. Furthermore, this study confirms a higher yield in
391
PPS if citric acid is applied as extraction medium, which is attributed to weaker acid strength
392
of citric acid compared to nitric acid and a reduced degradation of non-GalA components,
393
particularly neutral sugars. In conclusion, pH seems to be the driving force with regard to
394
pectin solubilisation and depolymerisation. Hence, less acidic conditions (pH 2) in
395
combination with an elevated extraction temperature and a prolonged extraction time are
396
recommended to extract pea hull PPS in adequate amounts and purity (>65 %) which may
397
be in line with the legal guidelines for low-methoxyated pectin (LMP). Gelling properties of
398
the LMP might be affected by process-induced structural alterations and minor constituents.
399
Largely independent from the extraction conditions, pea hull PPS is characterised by a high 11
400
amount of cell wall protein. Thererfore, we assume that protein-rich pea hull PPS exhibit
401
surface active properties which might be useful for the stabilisation of disperse food systems
402
such as emulsions or foams. This will be part of our ongoing research on pea hull PPS
403
functionality.
404 405
Acknowledgements
406
This IGF Project (18678 N) of the FEI is supported via AiF within the programme for
407
promoting the Industrial Collective Research (IGF) of the German Ministry of Economics and
408
Energy (BMWi), based on a resolution of the German Parliament. The authors thank Astrid
409
Kiegel, Christina Härter, Alexandra Urbisch and Pramita Devi for sample preparation and/or
410
analytical support.
12
411
References
412
Ali-Khan, S. T. (1993). Seed hull content in field pea. Canadian Journal of Plant Science, 73,
413
611-613.
414
Andersen, N., Cognet, T., Santacoloma, P., Larsen, J., Armagan, I., Larsen, F., Gernaey, K.,
415
Abildskov, J., & Huusom, J. (2017). Dynamic modelling of pectin extraction describing
416
yield and functional characteristics. Journal of Food Engineering, 192, 61-71.
417
Axelos, M., & Branger, M. (1993). The effect of the degree of esterification on the thermal
418
stability and chain conformation of pectins. Food Hydrocolloids, 7, 91-102.
419
Bäuerle, G., Otterbach, G., Gierschner, K., & Baumann, G. (1977). Bestimmung des
420
Polyuronidgehaltes
421
Deutsche Lebensmittel-Rundschau, 73, 281-286.
422 423
und
des
Veresterungsgrades
von
Handelspektinpräparaten.
BeMiller, J. N. (2019). Pectins. In J. N. BeMiller (Ed.), Carbohydrate Chemistry for Food Scientists (pp. 303-312). AACC International Press.
424
Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Silveira Villar, L., & Escaleira, L. A. (2008).
425
Response surface methodology (RSM) as a tool for optimization in analytical chemistry.
426
Talanta, 76, 965-977.
427 428
Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54, 484-489.
429
Canteri-Schemin, M. H., Fertonani, H. C. R., Waszczynskyj, N., & Wosiacki, G. (2005).
430
Extraction of pectin from apple pomace. Brazilian Archives of Biology and Technology,
431
48, 259-266.
432 433 434 435 436 437
Chaharbaghi, E., Khodaiyan, F., & Hosseini, S. S. (2017). Optimization of pectin extraction from pistachio green hull as a new source. Carbohydrate Polymers, 173, 107-113. Constenla, D., & Lozano, J. (2003). Kinetic model of pectin demethylation. Latin American Applied Research, 33, 91-95. Dalgetty, D. D. & Baik, B.-K. (2006). Fortification of Bread with Hulls and Cotyledon Fibres Isolated from Peas, Lentils, and Chickpeas. Cereal Chemistry, 83, 269-274.
438
Diaz, J. V., Anthon, G. E., & Barrett, D. M. (2007). Nonenzymatic degradation of citrus pectin
439
and pectate during prolonged heating: effects of pH, temperature, and degree of methyl
440
esterification. Journal of Agricultural and Food Chemistry, 55, 5131-5136.
441
Einhorn-Stoll, U., Kastner, H., Urbisch, A., Kroh, L. W., & Drusch, S. (2019). Thermal
442
degradation of citrus pectin in low-moisture environment – Influence of acidic and
443
alkaline pre-treatment. Food Hydrocolloids, 86, 104-115.
444 445
Endreß, H.-U., & Christensen, S. (2009) Pectins. In G. O. Phillips, & P. A. Williams (Eds.), Handbook of Hydrocolloids (pp. 274-297). Oxford: Woodhead Publishing Ltd.
446
Everett, D. W., & McLeod, R. E. (2005). Interactions of polysaccharide stabilisers with casein
447
aggregates in stirred skim-milk yoghurt. International Dairy Journal, 15, 1175–1183. 13
448
Fraeye, I., Doungla, E., Duvetter, T., Moldenaers, P., Loey, A. V., & Hendrickx, M. (2009).
449
Influence of intrinsic and extrinsic factors on rheology of pectin-calcium gels. Food
450
Hydrocolloids, 23, 2069-2077.
451
Funami, T., Nakauma, M., Ishihara, S., Tanaka, R., Inoue, T., & Phillips, G. O. (2011).
452
Structural modifications of sugar beet pectin and the relationship of structure to
453
functionality. Food Hydrocolloids, 25, 221-229.
454 455
Gutöhrlein, F., Drusch, S., & Schalow, S. (2018). Towards By-Product utilization of Pea Hulls: Isolation and Quantification of Galacturonic Acid. Foods, 7, 203.
456
Happi Emaga, T., Ronkart, S. N., Robert, C., Wathelet, B., & Paquot, M. (2008).
457
Characterisation of pectins extracted from banana peels (Musa AAA) under different
458
conditions using an experimental design. Food Chemistry, 108, 463-471.
459
Igbasan, F. A., Guenter, W., & Slominski, B. A. (1997). Field peas: Chemical composition
460
and energy and amino acid availabilities for poultry. Canadian Journal of Animal
461
Science, 77, 293-300.
462
Jafari, F., Khodaiyan, F., Kiani, H., & Hosseini, S. S. (2017). Pectin from carrot pomace:
463
Optimization of extraction and physicochemical properties. Carbohydrate Polymers,
464
157, 1315-1322.
465
Kermani, Z. J., Shpigelman, A., Kyomugasho, C., Buggenhout, S. V., Ramezani, M., Loey, A.
466
M. V., & Hendrickx, M. E. (2014). The impact of extraction with a chelating agent under
467
acidic conditions on the cell wall polymers of mango peel. Food Chemistry, 161, 199-
468
207.
469
Kleppmann, W. (2013). Versuchsplanung. (8. Aufl.). München: Carl Hanser Verlag.
470
Kliemann, E., Nunes de Simas, K., Amante, E. R., Schwinden Prudêncio, E., Teófilo, R. F.,
471
Ferreira, M. M. C., & Amboni, R. D. M. C. (2009). Optimisation of pectin acid extraction
472
from passion fruit peel (Passiflora edulis flavicarpa) using response surface
473
methodology. International Journal of Food Science & Technology, 44, 476-483.
474
Kulkarni, S., & Vijayanand, P. (2010). Effect of extraction conditions on the quality
475
characteristics of pectin from passion fruit peel (Passiflora edulis f. flavicarpa L.). LWT -
476
Food Science and Technology, 43, 1026-1031.
477
Le Goff, A., Renard, C. M. G. C., Bonnin, E., & Thibault, J.-F. (2001). Extraction, purification
478
and chemical characterization of xylogalacturonans from pea hulls. Carbohydrate
479
Polymers, 45, 325-334.
480
Levigne, S., Thomas, M., Ralet, M.-C., Quemener, B., & Thibault, J.-F. (2002). Determination
481
of the degrees of methylation and acetylation of pectins using a C18 column and
482
internal standards. Food Hydrocolloids, 16, 547-550.
483
Li, D., Du, G., Jing, W., Li, J., Yan, J., & Liu, Z. (2015). Combined effects of independent
484
variables on yield and protein content of pectin extracted from sugar beet pulp by citric
14
485
acid. Carbohydrate Polymers, 129, 108-114.
486
Limberg, G., Körner, R., Buchholt, H. C., Christensen, T. M. I. E., Roepstorff, P., &
487
Mikkelsen, J. D. (2000a). Analysis of different de-esterifcation mechanisms for pectin by
488
enzymatic fingerprinting using endopectin lyase and endopolygalacturonase II from A.
489
niger. Carbohydrate Research, 327, 293-307.
490
Limberg, G., Körner, R., Buchholt, H. C., Christensen, T. M. I. E., Roepstorff, P., &
491
Mikkelsen, J. D. (2000b). Quantification of the amount of galacturonic acid residues in
492
blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and
493
endo-polygalacturonase II from Aspergillus niger. Carbohydrate Research, 327, 321-
494
332.
495
Methacanon, P., Krongsin, J., & Gamonpilas, C. (2014). Pomelo (Citrus maxima) pectin:
496
Effects of extraction parameters and its properties. Food Hydrocolloids, 35, 383-391.
497
Nakauma, M., Funami, T., Noda, S., Ishihara, S., Al-Assaf, S., Nishinari, K., & Phillips, G. O.
498
(2008). Comparison of sugar beet pectin, soybean soluble polysaccharide, and gum
499
arabic as food emulsifiers. 1. Effect of concentration, pH, and salts on the emulsifying
500
properties. Food Hydrocolloids, 22, 1254-1267.
501
Ngouémazong, E. D., Christiaens, S., Shpigelman, A., Van Loey, A., & Hendrickx, M. (2015).
502
The emulsifying and emulsion-stabilizing properties of pectin: A review. Comprehensive
503
Reviews in Food Science and Food Safety, 14, 705-718.
504
Pereira, P. H. F., Oliveira, T. I. S., Rosa, M. F., Cavalcante, F. L., Moates, G. K., Wellner, N.,
505
Waldron, K. W., & Azeredo, H. M. (2016). Pectin extraction from pomegranate peels
506
with citric acid. International Journal of Biological Macromolecules, 88, 373-379.
507
Pickardt, C., Dongowski, G., & Kunzek, H. (2004). The influence of mechanical and
508
enzymatic disintegration of carrots on the structure and properties of cell wall materials.
509
European Food Research & Technology, 219, 229-239.
510
Raji, Z., Khodaiyan, F., Rezaei, K., Kiani, H., & Hosseini, S. S. (2017). Extraction
511
optimization and physicochemical properties of pectin from melon peel. International
512
Journal of Biological Macromolecules, 98, 709-716.
513 514 515 516
Ravn, H. C., & Meyer, A. S. (2014). Chelating agents improve enzymatic solubilization of pectinaceous co-processing streams. Process Biochemistry, 49, 250-257. Reichert, R. (1981). Quantitative isolation and estimation of cell wall material from dehulled pea (Pisum sativum) flours and concentrates. Cereal Chemistry, 58, 266-270.
517
Renard, C. M. G. C., Voragen, A. G. J., Thibault, J. F., & Pilnik, W. (1990). Studies on apple
518
protopectin: I. Extraction of Insoluble Pectin by Chemical Means. Carbohydrate
519
Polymers, 12, 9-25.
520 521
Renard, C. M. G. C., Weightman, R. M., & Thibault, J.-F. (1997). The xylose-rich pectins from pea hulls. International Journal of Biological Macromolecules, 21, 155-162.
15
522 523
Rolin, C. (2002). Commercial pectin preparations. In G. B. Seymour, & J. B. Knox (Eds.), Pectins and their manipulation (pp 222-241). Oxford: CRC Press Blackwell Publishing.
524
Rolin, C., Chrestensen, I. B., Hansen, K. M., Staunstrup, J., & Sørensen S. (2010). Tailoring
525
pectin with specific shape, composition and esterifaction pattern. In P. A. Williams, & G.
526
O. Phillips (Eds.), Gums and Stabilisers for the Food Industry 15 (pp 13-25). Cambridge:
527
RSC Publishing.
528
Oliveira, T. I. S., Rosa, M. F., Cavalcante, F. L., Pereira, P. H. F., Moates, G. K., Wellner, N.,
529
Mazzetto, S. E., Waldron, K. W., & Azeredo, H. M. C. (2016). Optimization of pectin
530
extraction from banana peels with citric acid by using response surface methodology.
531
Food Chemistry, 198, 113-118.
532
Shan, Y. (2016). Extraction processes of functional components from citrus peel. In Y. Shan
533
(Ed.), Comprehensive Utilization of Citrus By-Products (pp 31-58). London: Academic
534
Press.
535 536
Shiga, T. M., & Lajolo, F. M. (2006). Cell wall polysaccharides of common beans (Phaseolus vulgaris L.) composition and structure. Carbohydrate Polymers, 63, 1-12.
537
Thibault, J.-F., & Ralet, M.-C. (2003). Physico-chemical properties of pectins in the cell walls
538
and after extraction. In F. Voragen, H. Schols, & R. Visser (Eds.), Advances in Pectin
539
and Pectinase Research (pp 91-105). Dordrecht: Springer Netherlands.
540
Van Buggenhout, S, Sila, D. N., Duvetter, T., Van Loey, A., & Hendrickx, M. (2009). Pectins
541
in Processed Fruits and Vegetables. Comprehensive Reviews in Food Science and
542
Food Safety, 8, 105-117.
543 544 545 546
Van Buren, J. P. (1979). The chemistry of texture in fruits and vegetables. Journal of Texture Studies, 10, 1-23. Voragen, A. G. J., Coenen, G.-J., Verhoef, R. P., & Schols, H. A. (2009). Pectin, a versatile polysaccharide present in plant cell walls. Structural Chemistry, 20, 263-275.
547
Vriesmann, L. C., Teófilo, R. F., & de Oliveira Petkowicz, C. L. (2011). Optimization of nitric
548
acid-mediated extraction of pectin from cacao pod husks (Theobroma cacao L.) using
549
response surface methodology. Carbohydrate Polymers, 84, 1230-1236.
550
Vriesmann, L. C., Teófilo, R. F., & de Oliveira Petkowicz, C. L. (2012). Extraction and
551
characterization of pectin from cacao pod husks (Theobroma cacao L.) with citric acid.
552
LWT - Food Science & Technology, 49, 108-116.
553 554
Waldron, K., Parker, M., & Smith, A. (2003). Plant cell walls and food quality. Comprehensive Reviews in Food Science and Food Safety, 2, 128-146.
555
Wegener, S., Kaufmann, M., & Kroh, L. W. (2017). Influence of L-pyroglutamic acid on the
556
color formation process of non-enzymatic browning reactions. Food Chemistry, 232,
557
450-454.
558
Weightman, R. M., Renard, C. M. G. C., & Thibault J.-F. (1994). Structure and properties of
16
559
the polysaccharides from pea hulls. Part 1: Chemical extraction and fractionation of the
560
polysaccharides. Carbohydrate Polymers, 24, 139-148.
561 562
Willats, W. G. T., Knox, J. P., & Mikkelsen, J. D. (2006). Pectin: new insights into an old polymer are starting to gel. Trends in Food Science & Technology, 17, 97-104.
563
Yapo, B. M., Robert, C., Etienne, I., Wathelet, B., & Paquot, M. (2007). Effect of extraction
564
conditions on the yield, purity and surface properties of sugar beet pulp pectin extracts.
565
Food Chemistry, 100, 1356-1364.
566
Yapo, B. M. (2009). Biochemical characteristics and gelling capacity of pectin from yellow
567
passion fruit rind as affected by acid extractant nature. Journal of Agricultural and Food
568
Chemistry, 57, 1572-1578.
569 570
Yapo, B. M. (2011). Pectic substances: From simple pectic polysaccharides to complex pectins - a new hypothetical model. Carbohydrate Polymers, 86, 373-385.
571 572
17
573
Figures and Tables - overview
574
Fig. 1. Schematic overview of PPS extraction from pea hulls.
575
Fig. 2. Contour plots for pea hull PPS yield (scale ranging from 1 to 10 %) in dependence of
576
pH and temperature using nitric acid (upper row) or citric acid (lower row) for two different
577
extraction times (left column (a, c): three hours; right column (b, d): six hours).
578
Fig. 3. Contour plots for UA content in pea hull PPS (scale ranging from 40 to 80 %) in
579
dependence of pH and temperature using nitric acid (upper row) or citric acid (lower row) for
580
two different extraction times (left column (a, c): three hours; right column (b, d): six hours).
581
Fig. 4. GPC profile of PPS extracted with nitric acid in dependence of extraction conditions
582
exemplarily shown for nitric acid (absorption normalised to maximal absorption of the
583
respective sample).
584
Fig. 5. Contour plots for the content of selected neutral sugars in pea hull PPS extracted with
585
nitric acid for 3 hours in dependence of pH and temperature.
586 587
Table 1 CCD for extraction process of PPS from pea hulls using nitric acid or citric acid.
588
Table 2 Composition of pea hull PPS shown for a 3 h extraction with nitric or citric acid at
589
different temperature and pH levels.
590
18
591 592
Table 1
593
CCD for extraction process of PPS from pea hulls using nitric acid or citric acid. Factor
-1
-α (-1.353)
Temperature Time
0
+1
+α (+1.353)
[°C]
66
70
80
90
94
[h]
2.5
3.0
4.5
6.0
6.5
0.8
1.0
1.5
2.0
2.2
pH
594 595 596
Table 2
597
Composition of pea hull PPS exemplarily shown for a 3 h extraction with nitric or citric acid at
598
different temperature and pH levels. Type of acid Nitric
Citric
599
Temperature [°C]
pH
Yield [%]
UA [%]
DM [%]
DAc [%]
Neutral sugars total [%]
Protein [%]
70
1
4.8
56.0
44.2
3.0
26.1
9.1
70
2
3.7
47.5
47.5
10.6
34.0
7.6
90
1
2.8
69.8
38.1
3.9
15.4
7.3
90
2
8.0
63.7
37.8
n.d.
25.3
7.2
70
1
4.2
49.1
45.5
8.0
32.7
9.1
70
2
3.3
45.5
48.9
11.7
35.7
8.9
90
1
5.6
67.4
41.1
1.9
19.6
7.6
90
2
9.8
61.0
36.4
5.4
27.5
8.8
n.d. – not detected
600 601
19
Pea hulls
Sieving
Fraction < 50µm
Fraction > 50µm
Extraction
Filtration
Precipitation
Drying
PPS
Fibre
(a) 3h Nitric Acid
(c) 3h Citric Acid
(b) 6h Nitric acid
(d) 6h Citric Acid
Fig. 2. Contour plots for pea hull PPS yield (scale ranging from 1 to 10 %) in dependence of pH and temperature using nitric acid (upper row) or citric acid (lower row) for two different extraction times (left column (a, c): three hours; right column (b, d): six hours).
(a) 3h Nitric Acid
(c) 3h Citric Acid
(b) 6h Nitric acid
(d) 6h Citric Acid
Fig. 3. Contour plots for UA content in pea hull PPS (scale ranging from 40 to 80 %) in dependence of pH and temperature using nitric acid (upper row) or citric acid (lower row) for two different extraction times (left column (a, c): three hours; right column (b, d): six hours).
1,0 0,9
70°C, 3 h
0,8 90°C, 3 h
Detector [a.u.]
0,7 0,6
90°C, 6 h
0,5 0,4 0,3 0,2 0,1 0,0 0
5
10
15
Elution time (min)
20
25
(a) Rhamnose
(c) Galactose
(b) Arabinose
(d) Xylose
Fig. 5. Contour plots for the content of selected neutral sugars in pea hull PPS extracted with nitric acid for 3 hours in dependence of pH and temperature.
Highlights • Extraction with nitric or citric acid generates low methoxylated pectin. • Pea hull pectin is low acetylated and rich in xylose and protein. • Increasing temperature and decreasing pH promote pectin purity but reduce yield. • Citric acid extraction increases yield due to a higher amount of neutral sugars. • Prolonged extraction with nitric acid at increased pH and temperature is recommended.
Extraction of low methoxylated pectin from pea hulls via RSM Friederike Gutöhrlein, Stephan Drusch*, Sebastian Schalow
Friederike Gutöhrlein: Conceptualisation, Investigation, formal analysis, scientific discussion Stephan Drusch: Writing – review and editing, supervision, scientific discussion Sebastian Schalow: Writing-orignal draft, project administration, scientific discussion
Extraction of low methoxylated pectin from pea hulls via RSM Friederike Gutöhrlein, Stephan Drusch*, Sebastian Schalow
Compliance with ethical standards: The authors declare no conflict of interest.