Journal Pre-proof

Lupin protein: Isolation and techno-functional properties, a review

Billy Lo, Stefan Kasapis, Asgar Farahnaky

PII: S0268-005X(20)31735-5
DOI: https://doi.org/10.1016/j.foodhyd.2020.106318
Reference: FOOHYD 106318

To appear in: Food Hydrocolloids

Received Date: 28 June 2020

Revised Date: 3 September 2020
Accepted Date: 7 September 2020

Please cite this article as: Lo, B., Kasapis, S., Farahnaky, A., Lupin protein: Isolation and
techno-functional properties, a review, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/
j.foodhyd.2020.106318.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier Ltd.

 Authors statement:

Billy Lu contributed to conceptualization, methodology, literature review, paper collection

and classification, compiling tables, writing original draft, review and editing. Asgar
Farahnaky contributed through conceptualization, methodology, resources, supervision,
writing, review and editing. Stefan Kasapis contributed through conceptualisation, review
and editing

of
ro
-p
re
lP
na
ur
Jo
Jo
ur
na
lP
re
-p
ro
of
 1 Lupin Protein: Isolation and Techno-Functional Properties, A Review

2 Billy Lo, Stefan Kasapis, Asgar Farahnaky*

3 Biosciences and Food Technology, RMIT University, Bundoora West Campus, Plenty Road,
4 Melbourne, Vic 3083, Australia

5 *Corresponding Author: email address: asgar.farahnaky@rmit.edu.au

6 Abstract

7 Plant proteins are rapidly becoming more of a prime interest to food manufactures as
8 consumers are shifting away from meat-based diets. Therefore, food manufactures need to

of
9 incorporate functional plant proteins as ingredients into their products. Lupin has a high
protein (~40%) and fibre (~40%) content, is low in starch and gluten free hence, it can have

ro
10
11 an important role. Despite its nutritional and proven health benefits, lupin is underutilised and
12
-p
still does not play a major role in the human diet. Concentrating its protein and producing
re
13 lupin protein isolate with suitable properties would make it attractive as a high protein and
14 functional ingredient for food manufacturing.
lP

15 Over the recent years the industry has shown interest in large scale production of lupin
na

16 protein isolate and improving its techno-functional properties. This paper reviews the
17 literature on different lupin protein isolation techniques and conditions. The impact of protein
ur

18 isolation technique on chemical composition, yield and physiochemical properties including

Jo

19 solubility, water holding capacity, molecular weight and particle size distribution, of the
20 resultant protein isolates, are compared and contrasted. Moreover, major functional properties
21 such as viscosity, gelling, emulsification, and thermal properties of the isolated proteins are
22 reviewed.

23 Varied extraction yields and largely different properties and functionalities are achieved for
24 different lupin protein extraction techniques. In the light of the unique composition and
25 nutritional properties of lupin seed and its protein component, future research for improving
26 the protein isolation process for lupin and improving its physical functionalities using
27 innovative technologies are discussed.

28 Keywords: Lupin protein, Chemical characteristics, Protein functionality, Protein isolation

29

30

1
31

32 1. Introduction

33 Lupin is a type of legume which is native to the Mediterranean region and Latin
34 America. There are many varieties of lupin, each having distinct chemical compositions
35 (Villarino et al., 2016). This is due to their genetic differences and the environment in which
36 they are grown. These attributes in turn affect their respective functional properties (Trugo,
37 Baer & Baer, 2003). For instance, the lupinus albus variety of lupin proliferate in
38 Mediterranean climates, where the presence of slightly acidic soil is beneficial for its growth
39 (Trugo, Baer & Baer, 2003). The most common variety of lupins are the abovementioned

of
40 lupinus albus, more commonly recognised as large white lupin, and the lupinus angustifolius
41 otherwise known as the narrow leaf (Villarino et al., 2016).

ro
42 Australia is one of the biggest growers of lupin, being responsible for 80-85%, or 1.6
43
-p
million tonnes of total world production (Kohajdová, Karovičová & Schmidt, 2011; Villarino
re
44 et al., 2016). The cost to farm lupin is low, compared to other legumes, particularly in
Australia, where the production cost of lupin is half that of soy. Currently, however, lupin is
lP

45
46 exclusively used as animal feed rather than for human consumption (Duranti et al. 2008;
na

47 Khan et al. 2015).

48 1.1. Composition
ur

49 Lupin has a low starch content (gluten free) but is high in fibre (30%-41%), of which
Jo

50 mainly is insoluble, and protein (Kohajdová, Karovičová & Schmidt, 2011; Trugo, Baer &
51 Baer, 2003; Villarino et al., 2016). With a high fibre and low starch content, it has been
52 suggested that lupin consumption can provide many health benefits such as lowering blood
53 cholesterol and improving bowel health (Arnoldi et al., 2015, Mazumder et al., 2020).

54 The protein content of lupin, as reported in existing literature, varies between 30%-
55 42%, with the fluctuation attributable to the variety of the lupin in question (Fudiyansyah et
56 al., 1995; Kohajdová, Karovičová & Schmidt, 2011). Depending on the species of lupin, the
57 composition may vary, especially the protein content (Ruiz-López et al., 2019). The protein
58 of lupin is stored in globulins, or commonly known as conglutins, which hold the majority of
59 the lupin protein (87%) (Mane et al., 2018). Compared to other food ingredients such as
60 wheat (12%) and soy (35%), lupin has a higher protein content (Duranti et al., 2008; Villarino
61 et al., 2016). This indicates that lupin could be used in many food products (e.g. bakery

2
62 products like bread making) to improve their protein level and possible nutraceutical
63 properties. Moreover, lupin has similar properties to soybeans such as size, appearance and
64 protein composition (Fudiyansyah et al., 1995).

65 The proteins of a food ingredient attract significant attention due to their impact on
66 the functionality and nutritional quality of the final food product. As stated by Zayas (1997),
67 protein functionality refers to its physicochemical properties, which influence food
68 manufacturing decisions due to its impact on the structure and sensory properties of the final
69 products. Some of these functional properties include the protein solubility, swelling,
70 foaming and gelling propensity. The high protein content of lupin renders the legume a
71 promising candidate as a functional ingredient in human food production; for it to be usable

of
72 in this context, the protein needs to be extracted, the extraneous content separated, and have

ro
73 high functional properties.

74 1.2 Significance of this paper -p

re
75 Despite the confirmed nutritional and health promoting benefits of lupin, its
applications in food manufacturing is still rather limited. This paper will examine the
lP

76
77 applicability of this important legume in food manufacturing as a unique source of protein
na

78 through exploring its functional properties. It reviews the techniques examined for effective
79 protein isolation and their impact on the chemical and functional qualities of the lupin
ur

80 protein, including its composition, molecular, emulsification, thermal and rheological

81 properties. Opportunities for modifying lupin protein functionalities and using lupin protein
Jo

82 in food applications as well as future studies are also discussed.

83 2. Chemical composition and protein solubility of lupin proteins isolated by

84 different methods

85 There are numerous studies which have prepared lupin protein isolates/concentrates from
86 different lupin seeds including some of the main lupin varieties (i.e. lupinus albus and lupinus
87 angustifolius) and have reported protein extraction yield and protein solubility of different
88 isolation techniques (Table 1).

89 2.1. Lupin protein: extraction and yield

90 Lupin flour has a relatively high protein level, but it contains large quantities of non-
91 protein compounds such as fibre and fat. As a result, to be considered as a high value
92 ingredient its protein needs to be concentrated through separation of major non-protein

3
 93 compounds. Table 1 presents a summary of different lupin extraction methods and their
94 isolation conditions and compares protein extraction yields. The latter studies revealed that
95 the yield of protein isolates was affected by factors including the variety of lupin and the
96 method and conditions of isolation processes as detailed below and Table 1.

97 Looking into the isolation techniques in Table 1, it indicates that the most common
98 reported method of lupin protein isolation is the isoelectric precipitation as outlined Figure 1.
99 This procedure requires lupin seeds or their flours to be suspended in an aqueous solution, the
100 pH of the solution is then adjusted, usually to a point well above the isoelectric point of the
101 protein (i.e. alkaline pH) for maximum solubilization of lupin proteins, centrifuged, pellet
102 (mainly containing non protein biopolymers) is discarded before the pH of supernatant being

of
103 adjusted back to the isoelectric point of 4.5. Isoelectric point is where the proteins are

ro
104 neutrally charged (i.e. zero charge) causing the lupin proteins to aggregate and precipitate so
105
106
-p
that they can easily be separated through further centrifugation or filtration (Novák &
Havlíček, 2016). The precipitate obtained at the isoelectric point is mainly protein but may
re
107 include impurities of non-protein components that can be further purified with rewashing as
lP

108 well as moving pH away from 4.5 followed by centrifugation and removing the remaining
109 non-protein components. Examples of isolated lupin protein paste and powder before and
na

110 after freeze drying are given in Figure 2, A and B. The resulting lupin protein isolate is a
111 shelf stable yellowish powder.
ur

112 Due to its prevalence within the food industry, simplicity and high efficacy for protein
Jo

113 extraction, many studies have investigated the conventional isoelectric precipitation method
114 as a means of protein extraction from lupin. For instance, Aguilera et al. (1983),
115 demonstrated that acid precipitation produced a protein isolate yield ranging from 69-82%
116 when applied to lupin seeds. Chew et al. (2003) further examined the effect of acid
117 precipitation on lupin protein extracts, as well as the recovery of the protein. It was revealed
118 that acid precipitation produced a 59% protein recovery rate, which is a similar result with
119 those returned from the study conducted by Aguilera, Gerngross & Lusas (1983). These
120 studies indicate that isoelectric precipitation is a viable method for lupin protein extraction.

121 In contrast, however, some later studies have produced less favourable results in regard to the
122 protein yield from acid precipitation. For instance, the study by D'Agostina et al. (2006)
123 focused on protein isolation from lupin in the context of large pilot scale production, and
124 adjusting the spray drying conditions. This study indicated that the recovery of proteins by

4
125 acid precipitation had a low yield of 2-10%. These results were similarly unfavourable to
126 those returned from the study conducted by Alu’datt et al. (2017), where acid precipitation
127 produced a protein extract yield of 19.12%.

128 In addition to acid precipitation, research groups have explored the use of salt
129 solutions as a means of protein isolation (Lqari et al. 2002; Muranyi et al. 2016). For
130 instance, Lqari et al. (2002) compared the use of alkaline extraction with or without salt
131 extraction using Na2SO3 followed by acid precipitation at pH 4.3 to examine their relative
132 efficacy in protein extraction. The results indicated that alkaline extraction had a higher
133 protein recovery rate (79%) compared to salt extraction (72%) and the use of Na2SO3 was not
134 able to improve extraction yield. In contrast, however, Muranyi et al. (2016) conducted a

of
135 similar study that returned a yield of protein recovery that was much lower for both protein

ro
136 isolation methods; the protein yield from alkaline extraction was 26-31%, while that of salt
137
138
-p
extraction (using NaCl) was 19-30%, and combined (acid and salt) extraction produced 23-
25% protein recovery yield. In comparison, the study by Lqari et al. (2002) had a ~50%
re
139 increase in yield for an acid precipitation method assisted with alkaline pre-treatments, and a
lP

140 ~40% increase in the salt extraction relative to the results of Muranyi et al. (2016). The
141 disparity in the yield from the salt extraction method could be attributed to the different salt
na

142 solutions used in the respective studies. In another study, Sussmann et al. (2013) investigated
143 the use of dialysis with high salt solutions at different concentrations as another method of
ur

144 protein extraction. The recovery of protein in this study was 35-54%, which is a higher
Jo

145 recovery rate than those of the studies by Muranyi et al. (2016) and Lqari et al. (2002).

146 Bader et al. (2011b) also examined the recovery of protein extracted from lupin after
147 using a range of solvents for lupin fat extraction. The study showed that the recovery of
148 protein extracted was 36-44%. The lowest yield of 36% was obtained by using ethanol to
149 extract the fat before isolating the protein but the highest recovery of 44% yield was obtained
150 through the use of diethyl-ether ethanol to extract fat before protein extraction.

151 Ultrafiltration and diafiltration are food processing technologies that have been
152 commonly used to separate proteins in milk (Cheang & Zydney, 2004; Gavazzi-April et al.
153 2018; Rajagopalan & Cheryan, 1991). The current literature includes studies that examined
154 the application of these technologies in preparing protein isolate from lupin. Chew et al.
155 (2003) studied acid precipitation followed by diafiltration/ultrafiltration for lupin protein
156 extraction. In this study, the acid precipitation alone produced a 59% protein recovery rate,

5
157 and further processing via diafiltration/ultrafiltration increased this result to a 92% protein
158 recovery rate and by adding an additional processing step, the recovery had an increase of
159 33% of yield. The efficacy of diafiltration/ultrafiltration was further demonstrated in
160 D'Agostina et al. (2006) where the recovery of proteins by acid precipitation alone produced
161 a yield of 2-10%, which increased to 20-50% upon the application of ultrafiltration, thereby
162 producing a ~40% increase in yield. This study is similar to the study conducted by Chew et
163 al. (2003) where diafiltration/ultrafiltration increased the yield of proteins recovered. Further
164 to acid precipitation and ultrafiltration, Fontanari et al. (2012) combined these isolation
165 methods with the addition of salt solutions (Na2SO3 and NaCl). The yield of protein
166 recovered was 31-53%. In this study, ultrafiltration was used before the proteins were

of
167 precipitated at pH 4.5, whereas it was used after the proteins has been precipitated in the

ro
168 studies by Chew et al. (2003) and D'Agostina et al. (2006).

169
170
-p
Micellization is another protein isolation method that has been investigated such as
the study conducted by Rodríguez-Ambriz, et al. (2005) in which lupin flour 10% (w/v) was
re
171 suspended in NaCl at pH 7 and centrifuged at 5000 g for 10 minutes, the supernatant was
lP

172 then washed and freeze-dried. The results produced a protein recovery rate of 30% which was
173 lower than the acid precipitation (60%).
na

174 Overall, acid precipitation has been the major method investigated by different groups
175 and attempts have been made to couple it with other techniques including heating regimes,
ur

176 salt extraction, diafiltration and ultrafiltration in order to improve the protein level,
Jo

177 production yield and product techno-functionality with some degree of success. Given the
178 technical difficulties involved with all wet extraction techniques for upscaling, the dry
179 electrostatic separation reported by Wang et al. (2016) presents a good potential for large
180 scale processing but would require further investigation to examine its efficacy to improve
181 yield and protein level.

182 2.2. Protein Solubility

183 Proteins solubility is affected by various protein extraction factors and testing
184 conditions, including the extraction method and conditions, the pH of the protein system (Lo,
185 2017), solvent type, salt and temperature (Zayas, 1997). The existing literature suggests that
186 protein solubility is higher at alkaline and low acidic pH environments. This is because there
187 are less protein-protein interactive forces which cause the proteins to associate with each
188 other (Lo, 2017).

6
189 Lupin protein extracts are usually dried at the final processing step and the most
190 common drying method at the laboratory scale is either freeze-drying or spray-drying (Table
191 1). The dried lupin protein extracts must then be rehydrated or reconstituted for it to be used
192 as a functional ingredient, and therefore, the dried extracts must have a high solubility as a
193 driver for many physical functionalities. Protein solubility of lupin protein extracts has been
194 investigated in existing literature due to this fundamental step in food processing that
195 involves lupin as a functional ingredient. From these studies (Table 1), the Kjeldahl method
196 was the most commonly used to analyse protein solubility (Aguilera, Gerngross & Lusas,
197 1983; Bartkiene, et al., 2018; Chew, Casey & Johnson, 2003; D'Agostina et al., 2006)
198 followed by Dumas combustion method (Bader et al., 2011; Berghout et al., 2014).

of
199 Aguilera, Gerngross & Lusas (1983) used isoelectric precipitation to extract the lupin

ro
200 protein, which had a solubility of approximately 84-91%. These results are similar to those
201 -p
produced by Bader et al. (2011), where the protein solubility was 92.2-98.4%.

Prior studies have investigated the solubility of lupin protein extracts at a range of pH.
re
202
203 The study by Berghout, Boom & Van Der Goot (2014) produced the highest protein
lP

204 solubility results of 90%, where the protein extract was first obtained at 4°C by isoelectric
205 precipitation. In contrast, the solubility was the lowest (76%) where the protein was extracted
na

206 in an environment with the same pH but at 90°C. This result could be attributed to the heat
207 denaturation of the proteins. The isolates at pH 4.5 had the highest solubility at 4°C (68%)
ur

208 while at 20°C, the solubility was decreased to 42%. These tests indicate that pH is not the
Jo

209 only factor that has an effect on protein solubility; the thermal conditions also have an
210 influence on solubility. Berghout et al. (2015a) also studied solubility at a range of pH after
211 extracting the proteins by acid precipitation followed by freeze drying or membrane
212 concentration using a membrane with a 5 kDa pore size. The results demonstrated that
213 solubility was the lowest at pH 4.5 at both methods (freeze drying: 0%; membrane
214 concentration: 5%). Given that the isoelectric point of lupin protein is close to 4.5, its
215 solubility will be the lowest at this point. If the pH is adjusted to acidic or alkaline
216 environment away from the isoelectric point, the protein solubility is increased as shown in
217 Table 1. Bartkiene et al. (2018) also studied the solubility at a range of pH 2-10, which is a
218 similar study to Berghout, et al. (2015b). In this study the protein extracted by isoelectric
219 precipitation had the highest solubility at pH 10 (70-90%) while the lowest at pH 4 (10-25%).
220 Schlegel, Sontheimer, Eisner, & Schweiggert‐Weisz. (2019) also studied the protein
221 solubility after extracting the protein from lupin by acid precipitation. The solubility profile

7
222 determined at a pH range of 4-9 showed that pH 4-5 had the lowest solubility (9-10%), while
223 by increasing the pH towards pH 9 the solubility increased. This solubility profile in this
224 study is similar to the studies by Bartkiene et al. (2018) and Berghout, et al. (2015b). Sousa et
225 al. (1996) also studied the solubility of the extracted protein at a range of pH and reported
226 that the lowest solubility was at pH 4 (<10%). The solubility increased again (80%) by
227 decreasing the pH to 2 and increasing the pH to 5-8 (10-75%). Lqari et al. (2002) also studied
228 the protein solubility of the isolated protein at the isoelectric point pH 4-4.8. The solubility
229 was analysed at the isoelectric point pH 4-4.8 and was found to be around 20-25% which
230 seems high given that it is close to the isoelectric point of lupin.

231 Pozani et al. (2002) studied the solubility of the protein extracted from lupin by acid

of
232 precipitation. The authors in this study investigated the solubility with the addition of

ro
233 dithiothreitol (DTT) and temperature. The findings from this study showed that the solubility
234
235
-p
of the control lupin protein was 80%. By heating the protein to 80°C for 15 minutes, it had a
20% reduction in the solubility. The addition of DTT had no effect on the solubility of the
re
236 protein. However, a combination of DTT addition and heating to 80°C decreased the
lP

237 solubility by 10%.

238 There are studies that have used other methods to extract protein; this has also
na

239 influenced the solubility properties. Bader et al. (2011a) investigated the protein solubility
240 after extracting the protein by acid precipitation, followed by cross flow membrane filtration.
ur

241 The study showed that the solubility obtained from acid precipitation was 67% and cross flow
Jo

242 filtration (150 MPa) was able to increase it to 73% at 35 °C. At 60°C the protein extracted by
243 acid precipitation had a solubility of 67% and cross flow filtration (150 MPa) 78%. It
244 suggests that an additional processing step was able to increase the solubility of lupin protein.
245 Chew et al. (2003) also studied the impact on the solubility of extracted lupin proteins by acid
246 precipitation and ultrafiltration with a membrane pore size of 10 kDa. The proteins extracted
247 by acid precipitation indicated that the solubility at pH 2 was 55% and pH 4-8 was 20-75%.
248 The study showed that ultrafiltration increased the solubility at pH 2 to 80% and pH 4-8 to
249 50-80%. The trend from these findings is similar to Bader et al. (2011a) and D'Agostina et al.
250 (2006), suggesting that ultrafiltration assisted in improving the solubility of the extracted
251 lupin proteins. Hojilla‐Evangelista et al. (2004) also conducted a similar study, where acid
252 precipitation and ultrafiltration (with a membrane pore size of 5 kDa) were used to extract the
253 proteins. In this study ultrafiltration was not used as an additional step. It still showed that
254 ultrafiltration was able to increase the solubility by 10% compared to acid precipitation.

8
255 Fontanari et al. (2012) also used ultrafiltration (with a membrane pore size of 5 kDa) to assist
256 in extracting protein in lupin. In this study, instead of using acid precipitation salt solutions
257 (Na2SO3 or NaCl) were used as the first step of isolation before using ultrafiltration at pH of
258 10 and 11. The findings showed that extraction using Na2SO3 had a higher solubility
259 compared to NaCl. Wäsche et al. (2001) also studied the extraction of proteins by acid
260 precipitation as assisted by cross flow membrane filtration (pore size of 15 kDa). The study
261 showed that the solubility was 84-100% which is relatively high. A range of pore size has
262 been used in different membrane filtration studies and this could be behind the different yield
263 outcomes.

264 Another processing method of extracting proteins from lupin was high pressure. This

of
265 study conducted by Chapleau et al. (2003) found that high pressure processing at 400-600

ro
266 MPa showed a 20% reduction in solubility. However, below 400 MPa high pressure
267 treatment showed no effect on solubility. -p
A few authors have investigated the solubility of the protein extracted by
re
268
269 micellization El-Adawy et al. (2001). This study showed that solubility was found to be the
lP

270 lowest at pH 4 (15%). This trend is similar to the results found by other authors mentioned
271 above where solubility is the lowest at the isoelectric point but higher outside the isoelectric
na

272 point (70-85%). Rodríguez-Ambriz et al. (2005) also studied the protein solubility of the
273 extracted protein by acid precipitation and micellization. The results show that acid
ur

274 precipitation at pH 2 had a high solubility (85%), while at pH 4-10 the solubility range was
Jo

275 15-95%. These results are similar to micellization where pH 2 had a solubility of 75% while
276 at pH 4-10, the solubility range was at 10%-95%.

277 Yoshie-Stark et al. (2004) used acid precipitation to extract the protein from lupin.
278 However, before the extracting process, the lupin flakes were deoiled using hexane or carbon
279 dioxide. The results showed that the hexane deoiled protein isolate had a protein solubility of
280 64-71% while carbon dioxide deoiled isolate had a solubility of ~60-68%. It suggests that the
281 use of carbon dioxide or hexane in protein extraction was similar.

282 Overall, the solubility values reported for lupin proteins in Table 1 have a wide range
283 which predominantly are driven by the pH of the solubility test. By setting pH well away
284 from the isoelectric point of lupin protein substantial solubility changes (more than 50%)
285 have been recorded. From the extraction point of view, lupin solubility is impacted by the
286 protein extraction method, using combination extraction methods such as acid precipitation-

9
287 ultrafiltration, presence of salts and degree of heat denaturation. Based on these, adequate
288 strategies and practical approaches can be developed to minimise protein damage during
289 extraction and final lupin protein with improved solubility is obtained. In addition, drying and
290 its impact on protein solubility must be considered. Freeze drying followed by spray drying
291 are likely to result in lupin protein isolates with higher solubility as compared to other harsher
292 drying techniques such as high temperature oven drying.

293 3. Molecular weight, particle size and zeta potential of lupin proteins isolated by
294 different methods

295 There are numerous studies which investigate the molecular properties of lupin

of
296 protein concentrates or isolates that have been extracted using the various extraction
297 techniques described above. These properties include molecular weight, particle size and zeta

ro
298 potential (Table 2).

299 3.1. Molecular weight

-p
re
300 One of the most common methods used to determine the molecular weight of a
lP

301 polypeptides is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

302 which has been explored for lupin protein by many authors as per Table 2. Alu’datt et al.
na

303 (2017) discovered that lupin proteins contain three globulin proteins, including 12 protein
304 subunits of b-conglutin (7S), 4 protein subunits of a-conglutin (11S) and a minor c-conglutin
ur

305 (2S); these globulin proteins were found to have molecular weights of 170, 315 and 17 kDa,
Jo

306 respectively. It was also discovered that the molecular composition of the lupin varied
307 depending on the species of lupin from which the protein was extracted.

308 Bader, Bez, & Eisner (2011) focussed on lupinus angustifolius and the lupinus albus
309 varieties of lupin and discovered that they contain 22 and 19 bands, respectively. This study
310 also demonstrated that different species of lupin have different molecular weights. The study
311 also indicated that lupinus angustifolius had protein fractions with molecular weights of 71 to
312 88 kDa. These results correspond to the findings by Berghout, Boom, & Van Der Goot,
313 (2014) where the molecular weight of the lupin was measured to be 20–90 kDa, which is a
314 common range for lupin proteins. Fontanari et al. (2012) also produced corroborating
315 molecular weight results of lupin that fall within this range. Furthermore, this study further
316 discovered that eight of the subunits had a molecular weight between 14 and 71 kDa.
317 Berghout, Boom, & Van Der Goot, (2014) found that at pH 7, 2 hours heating at 90°C
318 treatment had less noticeable protein bands in the higher molecular weight region. This result

10
319 could be attributed to the denaturation and aggregation of proteins at high temperatures which
320 consequently affected the protein solubility.

321 Burgos-Díaz et al. (2019a) indicated that lupin proteins have a molecular weight of
322 25–100 kDa which is similar to what Bader, Bez, & Eisner, (2011) and Berghout, Boom, &
323 Van Der Goot, (2014) has mentioned. It was also discovered that there were some bands
324 which had faded at approximately 98 kDa along some low molecular weight bands. The
325 study suggested that this outcome may be due to the inability of the proteins to precipitate at
326 pH 4.5; in such relatively acidic conditions, the γ-conglutin, does not precipitate. Hojilla‐
327 Evangelista, Sessa, & Mohamed, (2004) revealed that the acid precipitation method of
328 protein extraction resulted in streaking in gel patterns which suggests protein denaturation. It

of
329 was found that the bands were concentrated between 31-66.2 kDa. These results, however,

ro
330 did not correlate with the lower molecular weights results of 21.4-31 kDa found by Muranyi
331 et al. (2016). -p
re
332 Schlegel, Sontheimer, Eisner, & Schweiggert‐Weisz. (2019) found that hydrolysis
333 caused the polypeptides to break into smaller fragments with molecular sizes below 23 kDa.
lP

334 The results also suggest that by combining serine and cysteine endopeptidase, the α-conglutin
335 and β-conglutin subunit proteins of lupin can be hydrolysed.
na

336 It was found that acid precipitation showed a decrease in α-conglutin and γ-conglutin
ur

337 subunits compared to salt extraction. Rodríguez-Ambriz et al. (2005) also found that the
338 method of protein extraction could influence the molecular weight of lupin proteins. In this
Jo

339 study, lupin proteins were extracted using acid precipitation and micellization, and the
340 resultant protein samples had slight differences in their molecular weights. This outcome is
341 suggestive of protein denaturation from the extraction process at pH 4.5 that may, in turn,
342 cause changes in protein composition and structure. Another possible reason of this
343 difference may be the heating of the proteins prior to loading on gels to measure the
344 molecular weight using SDS-PAGE.

345 Chapleau, & De Lamballerie-Anton, (2003) examined the impact of high pressure
346 upon molecular weight of the lupin proteins and discovered that the 7S subunit was more
347 versatile under high pressure than the 11S. This observation is due to the number of -SH
348 residues present in the 11S subunits compared to 7S.

349 Overall, molecular weight studies of lupin proteins isolated by different techniques reveal
350 moderate variations among different isolation techniques. Temperature, pH and the presence

11
351 of different salts can affect the molecular weight distribution of the resulting isolates. Fine
352 tuning these extraction parameters towards minimising protein conformational changes can
353 help retaining natural lupin protein structure and functionalities.

354 3.2. Particle size of lupin protein dispersions

355 When lupin protein is mixed with water or incorporated in formulated liquid foods,
356 protein is not completely solubilised and hence produces a dispersion. Particle size of lupin
357 protein dispersions is another important property of lupin protein due to its influence on the
358 functional properties, such as its emulsifiability and chemical stability, and therefore has been
359 the subject of numerous studies. Berghout et al. (2015b) investigated the impact of the protein

of
360 extraction method upon particle size, focussing on acid precipitation and ultrafiltration.
361 Extraction via acid precipitation alone resulted in a particle size of 39.6 µm, which is larger

ro
362 than the particle size of 11.3 µm when the lupin protein isolates from acid precipitation were
363 -p
further processed via ultrafiltration. The study suggested that the difference in particle size
could be due to the pore size of the membrane used for the filtration process. The pore size of
re
364
365 the membrane determines the size of the molecules that can pass through the membrane
lP

366 which affects the particle size (Fellows, 2017).

na

367 Bader, Bez, & Eisner, (2011) focussed on determining the protein particle size using
368 high pressure homogenization, which involves the solution being homogenised at 0, 50, 100,
ur

369 150 MPa at 35°C. The findings from this study revealed that, at 50-100 MPa, there was a
370 particle size distribution reduction 0.1-10 µm; however, at 150 MPa, the formation of
Jo

371 aggregates was observed which resulted in an increase in the particle size distribution to 10-
372 100 µm. High pressure may provide opportunities for opening the lupin protein molecular
373 structure and as a result increasing the chance of favoured collisions between protein
374 molecules for aggregation. In another study, Chapleau, & De Lamballerie-Anton (2003)
375 examined the use of high pressure in processing lupin proteins and its impact on protein
376 particle size. The study produced findings that the control had a particle size of 1.17 µm, and
377 at 600 MPa, the particle size was reduced to 0.74 µm. This study indicates the presence of
378 aggregates being formed at 200 MPa thereby resulting in an increase in particle size, a result
379 that is similar to the findings of Bader, Bez, & Eisner (2011).

380 Electrostatic separation is another processing method that has been applied to the
381 lupin protein in existing literature. The purpose of this process in this context is to reduce the
382 protein particle size of the lupin powder when processed at 2500, 4000, 6000 or 8000 rpm. It

12
383 was shown that the particle size distribution at 8000 rpm was much narrower compared to the
384 2500 rpm (Wang et al., 2016). Berghout, Boom, & Van Der Goot, (2015) examined the
385 impact of heat treatment at 75oC-90oC on particle size of extracted lupin protein. The results
386 indicate no difference in particle size across this temperature range, which lead the authors to
387 suggest that the unchanging particle size is indicative of lupin protein having high thermal
388 stability. In another study that also explored the impact of heat exposure on lupin protein,
389 Burgos-Díaz et al. (2019) demonstrated an increase in the particle size distribution at 90°C
390 compared to 25°C. This latter study attributed this result of increased particle size to protein
391 denaturation, which caused the protein to aggregate.

392 3.3. Zeta Potential

of
393 Protein molecules can be neutral, negatively or positively charged depending on pH

ro
394 among other factors including the presence of ionizing salts. Zeta potential is a measure of
395 -p
the net charge (surface charge) of a food system, which is usually analysed in a soluble form.
Limited studies have investigated the zeta potential of lupin protein extract due to extraction
re
396
397 conditions, presence of salts or pH change. Berghout, Boom, & Van Der Goot, (2015b)
lP

398 examined the difference in zeta potential between wet (saturated) and freeze fried lupin
399 proteins. This study revealed two main findings, namely, at pH 7, the protein suspension had
na

400 a zeta potential of _35 mV and that both concentrated and freeze-dried proteins are
401 electrostatically stable.
ur

402 Burgos-Díaz et al. (2019b) also sought to discover the correlation between pH and the
Jo

403 zeta potential of lupin protein by exposing lupin protein to a range of pH. The findings were
404 obtained at pH 2 (+35), 4.6 (_10), and 10 (_30) mV, and show that at pH 4.6, which is close to
405 the isoelectric point, the charge is close to zero, it becomes positive below the isoelectric
406 point, and negative above the isoelectric point.

407

408 4. Foaming, emulsification and water absorption capacity properties of lupin

409 proteins isolated by different methods

410 Protein functionality is an important consideration when used in food production, and
411 therefore, has been the subject of numerous studies. Existing literature has focussed on
412 functional properties including foaming, emulsification and water absorption capacity of
413 extracted protein isolate/concentrate from lupin (Table 3).

13
414 4.1. Foaming properties of lupin protein

415 Foaming is an important functional property that relates to the ability of an ingredient
416 to create air bubbles, which influences the textual properties of the final food product (Zayas,
417 1997). Such foaming ability in this context is usually measured according to its foaming
418 capacity and stability. Due to its significance in food production, there are studies which have
419 investigated the foaming properties of lupin proteins. For instance, Alu’datt et al. (2017)
420 examined the foaming stability of various legumes, including lupin. The study demonstrated
421 that the foaming stability of lupin was similar to chickpea protein (50%); however, broad
422 beans were found to have a higher foaming stability than lupin. Rodríguez-Ambriz et al.
423 (2005) focussed on the foaming capacity of lupin protein extracted by micellization (LMI)

of
424 and acid precipitation (LPI) and investigated how foaming capacity is affected by pH. The

ro
425 study revealed that LPI produced lupin protein extracts that had the highest foaming capacity
426
427
-p
at pH 2 (500%) compared to LMI (350%). In contrast, LMI had the highest foam capacity at
pH 10 (450%) compared to LPI (~350%). At pH 4-8, foaming capacity was similar for both
re
428 LPI and LMI. These results suggest that high alkaline or low acidic environments have
lP

429 greater impacts on the foaming properties of lupin proteins.

430 In addition, El-Adawy et al. (2001) examined the foaming capacity of lupin protein.
na

431 In this study, micellization (LMI) and acid precipitation (LPI) techniques were also used to
432 extract proteins, but from two different species of lupin: lupinus termis (bitter) and lupinus
ur

433 albus (sweet). This study revealed that the foam capacity of bitter lupin MI (120%) and sweet
Jo

434 lupin MI (146%) that were first processed via micellization, was higher than the bitter lupin
435 PI (104%) and sweet lupin PI (106%) samples produced from acid precipitation. This
436 indicates that the technique of protein extraction has a significant impact on the functional
437 properties of the resultant lupin protein isolates.

438 The results of El-Adawy et al. (2001) were corroborated by D'Agostina et al. (2006),
439 where the foaming properties of protein extracted via acid precipitation (LPI-E) and
440 diafiltration/ultrafiltration (LPI-F) were studied. It was revealed that the foaming capacity
441 (%) of LPI-F (1800-2080%) sample was higher than that of the LPI-E (1100-1470%)
442 iteration of the study. The greater foaming properties of LPI-F was demonstrated in relation
443 to foaming stability (%) where for LPI-F (90-96%) was higher than LPI-E (70-90%).

444 Further studies focussed on the correlation between protein extraction method and
445 foaming properties of the extracted protein. Hojilla‐Evangelista et al. (2004) produced one

14
446 such study that investigated the influence of acid precipitation and ultrafiltration. This study,
447 however, produced different results compared to D'Agostina et al. (2006). Hojilla‐Evangelista
448 et al. (2004) revealed that the foaming stability of protein extracted via acid precipitation
449 (16.8%) was greater than that of ultrafiltration (2.6%). This difference could be attributable to
450 the method of protein isolation which impacts on the functional properties. Moreover,
451 ultrafiltration technique separates proteins based on their size, but acid participation acts
452 based on charge, therefore the products of these two techniques are likely to have different
453 protein subunit compositions as clearly indicated in the molecular weight studies reviewed
454 (Section 3, Table 2).

455 In addition to the protein extraction method, studies have also examined the impact of

of
456 subsequent treatment of the protein isolates upon its foaming properties. Schlegel et al.

ro
457 (2019) for instance, investigated the difference in relation to the foaming properties between
458
459
-p
hydrolysed lupin protein and an untreated control. This study revealed that the hydrolysed
proteins had a higher foaming activity compared to untreated lupin protein.
re
460 In addition to the protein extraction method, other factors that are salient in a food
lP

461 production context have been examined for their impact upon the foaming properties of
462 proteins. For instance, Raymundo, Empis & Sousa (1998) studied the effects of thermal
na

463 characteristics on foaming stability of lupin proteins. This study demonstrated that the highest
foaming stability of 16% was observed when the protein system was exposed to thermal
ur

464
465 conditions of 60°C. When the temperature was increased to 90°C, foaming stability
Jo

466 decreased by 7.5%. This negative correlation between temperature and foaming stability
467 could be attributed to the denaturation of protein when exposed to high temperatures that in
468 turn compromises the foaming properties. This study also examined the use of a
469 hydrocolloid, xanthan gum, to improve foaming stability. This segment of the study
470 demonstrated that incorporating 0.06% of xanthan gum to the lupin protein suspension
471 produced the highest foam stability (25%).

472 Overall, the reported values for lupin protein isolate in Table 3 indicate low to moderate
473 foaming capacity and stability for this plant-based protein.

474 4.2. Emulsification Properties

475 Emulsification properties refer to the propensity of the proteins to create stable
476 emulsions by emulsifying protein solutions in oil (Zayas, 1997). The significant role that the
477 emulsification properties have on a food system has warranted studies to examine this

15
478 element in particular. There have been several studies which have investigated the
479 emulsification properties of lupin protein; a summary of these studies is produced in Table 3.
480 For instance, Bader, Bez & Eisner (2011) investigated the emulsification properties of
481 proteins extracted from two main varieties of lupin, lupinus angustifolius and lupinus albus,
482 as well as the impact of homogenisation upon their emulsification properties. The study
483 revealed that lupinus angustifolius (780mL oil.g-1) had a higher emulsification properties than
484 lupinus albus (580 oil.g-1). Upon processing via homogenisation, no effect could be observed
485 on emulsification capacity at 50-100 MPa. At a higher pressure level of 150 MPa, however,
486 lupinua angustifolius proteins had increased emulsification propensity (780 oil.g-1).

487 In another study, Chapleau & De Lamballerie-Anton (2003) also investigated the

of
488 correlation between high pressure treatment and emulsification stability; in particular, this

ro
489 study examined the creaming index (fat separation) and flocculation properties. In relation to
490
491
-p
the creaming index, the results revealed that high pressure homogenisation had the effect of
decreasing the creaming index of the lupin emulsion. The control had a high creaming index
re
492 (97.36%), however by using high pressure homogenisation, the creaming decreased, which is
lP

493 indicative of increased emulsion stability. Upon the application of 200 MPa of pressure
494 treatment, the creaming index decreased to 95.69%, which was then further reduced to
na

495 90.36% upon increasing the pressure to 600 MPa which is a 7% decrease in creaming. In
496 relation to the flocculation properties that were investigated, the study indicated that high
ur

497 pressure homogenisation reduced flocculation. This was demonstrated where the control had
Jo

498 a flocculation of 14.15 (µm), which decreased to 6.31 (µm) upon high pressure treatment of
499 the system at 600 MPa.

500 In addition, Burgos-Díaz et al. (2019b) investigated the creaming properties of lupin
501 emulsified at different oil concentrations and revealed that the creaming properties are
502 affected by various factors including thermal treatment, oil concentration and lupin protein
503 particle aggregation. The study also demonstrated that no creaming occurs at 5% LPI for all
504 oil concentrations and revealed that there was an increase of creaming for all emulsions over
505 the first 2-4 days of storage at all concentrations.

506 In addition to their investigation of protein treatment upon its foaming properties
507 referred to above, Schlegel et al. (2019) also examined the relationship between hydrolysis
508 treatment of protein and its emulsion capacity. It was revealed that hydrolysed lupin protein
509 had a higher emulsification capacity (620 mL/g) compared to untreated lupin protein isolate.

16
510 Further studies have examined the impact of the protein extraction method used and
511 the emulsification properties of lupin protein. For instance, Chew, Casey & Johnson (2003)
512 investigated this relationship, focussing on the protein extraction method of
513 diafiltration/ultrafiltration. This study indicated that proteins extracted via ultrafiltration had a
514 lower emulsion capacity compared to acid precipitation across pH 4-8 conditions. At pH 2,
515 lupin proteins produced via acid precipitation had a lower emulsion capacity (450 oil/g of
516 protein) compared with those extracted from ultrafiltration (470 oil/g of protein). These
517 results were corroborated by D'Agostina et al. (2006) who demonstrated that the LPI-F (380-
518 450%) had a higher emulsification capacity than LPI-E (370-570%); however, for lupin
519 proteins in particular, LPI-F had a higher emulsion stability (75-90%) than LPI-E (61-63%).

of
520 Hojilla‐Evangelista, Sessa & Mohamed (2004) also investigated the relationship

ro
521 between emulsification properties of lupin protein and protein extract method. In particular,
522
523
-p
the study examined the methods of acid precipitation and ultrafiltration finding that the
emulsion stability was similar for both acid precipitation and ultrafiltrated lupin protein
re
524 samples, i.e. 23.4 and 25.5 minutes, respectively.
lP

525 In addition, acid precipitation and micellization were the focus of El-Adawy et al.
526 (2001) when investigating protein extraction methods and emulsion capacity of lupin protein.
na

527 In this study, micellization (bitter lupin 196.2 oil ml/ g protein, sweet lupin 210.2 oil ml/ g
protein) of both lupins had a higher emulsification capacity than acid precipitated lupin (bitter
ur

528
529 lupin 164 oil ml/g, sweet lupin 169.4 oil ml g).
Jo

530 Moderately high emulsion capacity values have been reported for lupin protein isolates from
531 different isolation techniques and under different conditions. This may provide an
532 opportunity for expanding the use of this plant-based protein for new food and non-food
533 applications.

534 4.3. Water Absorption Capacity

535 Water Holding Capacity (WHC) or Water Absorption Capacity (WAC) is a measure
536 of the amount of a powdered substance that can be dissolved in water until the saturation
537 point is reached (per gram) (Aryee, Agyei & Udenigwe, 2018). Several studies have
538 investigated the WHC/WAC of lupin protein. For instance, Alu’datt et al. (2017) investigated
539 and compared the respective WHC of the proteins isolated from lupin, chickpeas and broad
540 beans. The study revealed that broad beans have the highest WHC (44%) followed by
541 chickpea protein (42%), with lupin protein having the lowest WHC of 33%. This finding

17
542 indicates that the variety of legume, and their unique chemical compositions, affect how its
543 proteins interact with water.

544 Berghout, Boom & Van Der Goot (2014) also examined the WHC of the isolated
545 lupin protein but in relation to how this functional property is affected by the thermal
546 conditions in which the proteins were extracted. This study demonstrated that the WHC was
547 the highest for the proteins isolated in conditions of pH 7 at 90°C (1.2-4.3 mL water/g
548 protein). The WHC of lupin proteins isolated at 4, 20 and 50°C with pH 7, however, could
549 not be determined. The authors speculated that this was because at pH 7, the proteins did not
550 sediment during centrifugation and was consequently dispersed, resulting in the inability to
551 measure WHC. It has also been suggested that this outcome could be related to the

of
552 conformational structure of the protein which prevents the measurement of WHC.

ro
553 Furthermore, D'Agostina et al. (2006) studied whether the protein extraction
554 -p
technique used has an impact on the WHC of the resultant lupin protein isolates, focussing on
the techniques of acid precipitation (LPI-E) and ultrafiltration (LPI-F). This study revealed
re
555
556 that the ultrafiltrated proteins had a higher WHC of 1-1.8 mL/g of protein than those obtained
lP

557 via acid precipitation (0-0.8 mL/g of protein). This indicates that ultrafiltration served to
558 optimise the WHC of lupin proteins more effectively compared to acid precipitation.
na

559 El-Adawy et al. (2001) investigated the WAC (g/100g isolate) of the isolated protein
ur

560 from the bitter and sweet varieties of lupin, where the proteins were extracted by acid
561 precipitation and micellization. This study produced results where the sweet lupin proteins
Jo

562 extracted by acid precipitation (225.7g/100g of isolate) and micellization (229.4g/100g of

563 isolate) had a higher WAC than the protein isolates of bitter lupin extracted by acid
564 precipitation (209.6g/100g of isolate) and micellization (212.5g/100g of isolate). This
565 demonstrates that different species of lupin produce proteins with different water holding
566 properties, which may be due to the variation in chemical composition and molecular
567 conformation.

568 Rodríguez-Ambriz et al. (2005) investigated and compared the effect of acid
569 precipitation and micellization on the WAC (mL water/g protein) of the protein isolates from
570 the lupin campestris variety of lupin and soybean. This study revealed that the WAC of
571 soybean extracted by acid precipitation (2.2 mL) and micellization (3.5 mL) was higher than
572 that of lupin counterparts when extracted by acid precipitation (1.7mL) and micellization
573 (1.3mL). This validates the finding by El-Adawy et al. (2001) that different compositions

18
574 associated with the type of legume impacts the WHC/WAC of the protein isolates extracted
575 therefrom.

576 Water absorption capacity of lupin protein isolates varies, to some extent, depending on the
577 isolation technique and the testing media conditions such as pH and the presence of salts.
578 From Table 3, it can be concluded that compared to many other functional food ingredients,
579 lupin protein isolate has a low water absorption capacity, therefore this deficiency may be
580 considered a hurdle against its extensive application in food systems and requires further
581 investigation.

582 5. Viscosity and gelling properties of lupin proteins isolated by different methods

of
583 Several studies have investigated the rheological properties of viscosity and gelation

ro
584 of isolated lupin protein as outlined in Table 5.

585 5.1. Viscosity -p

re
586 Viscosity is a measure of resistance to flow of a liquid system (Cronin & Fitzpatrick,
587 2011). According to Chapleau & De Lamballerie-Anton (2003) liquids that show a decrease
lP

588 in viscosity with increased shear rate are non-Newtonian fluids with shear thinning
589 behaviour. Berghout, Boom & Van Der Goot (2015) revealed that the isolated lupin protein
na

590 had shear thinning viscosity, with the protein being deformed at high shearing. This confirms
ur

591 that the aqueous dispersions of isolated lupin protein are non-Newtonian.

Berghout et al. (2015b) investigated the impact of the lupin protein processing method
Jo

592
593 upon the viscosity of the resultant isolate. Method 1 involved the freeze drying of the isolate
594 after extraction and demonstrated that the viscosity was higher than the viscosity of the
595 isolates produced in Method 2 isolation, where the lupin protein was extracted via
596 ultrafiltration after acid precipitation and processed by applying 10% heat treatment. This
597 study also indicated that the isolates produced from Method 1 had viscosity that showed a
598 higher shear thinning trend compared to Method 2. This finding indicates that the method of
599 protein isolation is likely to influence the viscosity and rheological behaviour of the protein.

600 Chapleau & De Lamballerie-Anton (2003) also examined the properties of lupin
601 isolates, focussing primarily on the viscoelastic behaviour – (the viscosity and elasticity) – of
602 the protein system. This study revealed that the viscoelasticity behaviour of the lupin protein
603 after high pressure treatment remained unchanged, and therefore, indicated that high pressure
604 treatment has little to no effect on the viscoelastic properties of lupin proteins. The study also

19
605 found that high pressure treatment of the protein system at 400 MPa resulted in higher
606 viscosity (10 Pa.s) compared to other pressure treatment levels (i.e. control, 200 and 600
607 MPa).

608 In another study, Chew, Casey & Johnson (2003) investigated the effect of thermal
609 exposure on the viscosity of the lupin proteins isolated by acid precipitation and
610 ultrafiltration. In this study, at pH 8 the lupin protein isolated via ultrafiltration and acid
611 precipitation had similar viscosities (shear thinning) across the temperature range of 10-70°C.
612 At pH 4, ultrafiltrated lupin protein had a higher viscosity compared to acid precipitated lupin
613 protein. It was further revealed that acid precipitation had an increase in viscosity (3.5 Pa.s) at
614 20°C and then decreased as the temperature increased. Ultrafiltrated protein also followed

of
615 this trend of a negative correlation between viscosity and temperature increase, which is

ro
616 similar to pH 8. At pH 6.7, the ultrafiltrated system had a higher viscosity (12 mPa.s)
617
618
-p
compared to the acid precipitated protein (6 mPa.s) at 10°C. The ultrafiltrated isolate had a
decrease in viscosity (shear thinning) as temperature increased; however, acid precipitation at
re
619 50-70°C showed an increase in viscosity. This increase in the viscosity at 50-70°C could be
lP

620 due to the high sensitivity of protein denaturation at this temperature range.

621 In a further study, Raymundo, Empis & Sousa (1998) investigated the impact on
na

622 viscosity from the use of a hydrocolloid (xanthan gum) in an isolated lupin solution. The
623 results demonstrated that an increased concentration of xanthan gum resulted in increased
ur

624 viscosity of the overall protein system, where the addition of 0.16% xanthan gum to a control
Jo

625 of 1 cP increased the viscosity to 20 cP. This finding is consistent with the fact that xanthan
626 gum is a known thickener, and therefore, the increase in viscosity following its incorporation
627 into the protein system aligns with previous reports.

628 Overall, lupin protein does not produce high viscosity (with or without heating) when mixed
629 with water or incorporated into formulated liquid food systems due to its limited water
630 interactions, low solubility and close conformational structure. However, it performs
631 differently in various products depending on parameters such as pH and concentration.

632 5.2. Gelation Properties

633 Gelation is an important functional property in food application as many food

634 products are produced in a gelatinous form, and therefore, require gelling ingredients to
635 achieve the desired texture and consistency. Such gelling ingredients are usually a variety of

20
636 protein or polysaccharides (Zayas, 1997). As a result, there have been studies that have
637 investigated the gelling properties of isolated lupin protein.

638 One such study was conducted by Bader, Bez & Eisner (2011) that investigated the
639 impact of high-pressure homogenisation on the gelling properties of lupin protein isolates. In
640 this study, no gel formation was observed for the untreated and treated isolated lupin proteins
641 of the lupinus angustifolius variety at 15% (w/w); therefore, gel strength could not be
642 determined. When the test was repeated for the lupinus albus variety of lupin, however, the
643 protein isolates formed a gel at 15% (w/w). This indicates that different lupin protein isolates
644 from different varieties could have different functional properties, and that gelling properties
645 could be enhanced with high-pressure homogenisation treatment. In particular, the study

of
646 revealed that homogenisation at 0-150 MPa at 35°C could increase the gel strength of lupin

ro
647 proteins to 0.15-0.55 N.cm-2. Further, this trend can also be seen with homogenisation at 0-
648
649
-p
150 MPa at higher thermal conditions of 60°C where gel strength of the lupin increased to
0.13-0.6 N.cm-2. This demonstrates that homogenisation can improve the gel strength of the
re
650 lupin protein at different temperatures.
lP

651 Berghout, Boom & Van Der Goot (2015) investigated the gelation properties of lupin
652 protein and soy protein. This study found that the storage modulus (G’) of lupin protein at
na

653 high protein concentration (30% w/v) was similar to that of soy protein with lower
654 concentration (24% w/v). This study suggested that the lupin protein produces weaker and
ur

655 less elastic gels compared to soy protein. In another study, Chapleau & De Lamballerie-
Jo

656 Anton (2003) examined the gelling properties of the emulsions created from high pressure
657 treatments. The study demonstrated that high pressure treatment enhanced the gelling
658 properties of the lupin emulsions (G’ > G").

659 Further, Kiosseoglou et al. (1999) studied the gelling properties of lupin protein. The
660 study observed the formation of pseudogels at 80°C, where the cohesion of the gel increased
661 when the system was cooled down to 10°C. This result was explained by the disulphide
662 bonding that was formed when the lupin solution was heated to 90°C, thereby producing an
663 observable gelatinous structure. It was further shown that these gels were insoluble in sodium
664 dodecyl sulphate and urea solutions.

665 Prior studies have also investigated the impact of the protein extraction method upon
666 the gelation properties of the resultant isolate. Rodríguez-Ambriz et al. (2005) conducted one
667 such study, focussing on the difference in the gelation properties of the isolates extracted via

21
668 acid precipitation, and another protein sample extracted via micellization. The study revealed
669 that the micellised lupin protein produced gels with a minimum protein concentration of 8%
670 (w/v) whereas the same gelling propensity was observed for acid precipitated lupin protein at
671 6% concentration. However, no gel could be formed at pH 6-8 and this suggests that there
672 were no crosslinks in the structure at this pH range.

673 In summary, lupin protein isolates produce weak gels with insoluble swollen micro
674 particles that cannot withstand their weight against gravity, hence being unable to compete
675 with other structure forming proteins such as soy protein. Therefore, to find widespread
676 application for lupin protein its gelation properties require improvement through
677 modification.

of
678

ro
679 6. Thermal properties of lupin proteins isolated by different methods

680
-p
The denaturation of proteins has a significant impact on their functionality and is of
re
681 great importance in food processing. Therefore, it is important to understand the properties
lP

682 and changes of lupin when exposed to heat. The most common method of measuring the
683 thermal properties of proteins is differential scanning calorimetry (DSC), and was used by
na

684 Berghout, Boom & Van Der Goot (2015) to analyse the impact of heat exposure on lupin
685 protein. This study revealed that protein isolation did not cause complete protein
ur

686 denaturation. The finding is important because it indicates that lupin protein retains largely its
Jo

687 native structure even after the acid precipitation process.

688 These results were corroborated by Fontanari et al. (2012), who demonstrated that the
689 protein had high thermal stability (195-208°C). It was observed that the lupin protein
690 experienced only one broad endothermic peak between 63 and 74°C, indicating that protein
691 denaturation commences in thermal conditions of this range, although the range of
692 temperature denaturation would be moisture dependent. The study also revealed that addition
693 of NaCl produced the highest value of ∆H (313-335 J.g-1). This is indicative of high levels of
694 protein denaturation.

695 Numerous studies have also investigated the relationship between protein extraction
696 method selection and thermal properties of the protein system. For instance, Hojilla‐
697 Evangelista, Sessa & Mohamed (2004) revealed that ultrafiltrated lupin protein had a higher
698 thermal stability (58.8%) than the acid precipitated lupin protein (48.3%). The inferior

22
699 thermal stability of proteins extracted via acid precipitation could be attributed to the
700 denaturation of the protein when the protein is isolated through exposure to high acid and low
701 pH environment. This observation was validated by Berghout, Boom & Van Der Goot
702 (2015).

703 The pH conditions in which protein extraction occurs also impact upon the thermal
704 properties of proteins, and therefore, was the subject of study for Kiosseoglou, Doxastakis,
705 Alevisopoulos, & Kasapis. (1999) in the context of lupin protein. This study indicated that
706 protein extraction conducted at pH 4.5 impacted upon the structural integrity of the protein
707 isolates and in turn, affected its molecular weight. Moreover, it was revealed that thermal
708 treatment at 90°C caused the formation of disulphide bonds in the molecular structure which

of
709 plays a vital role in creating the three-dimensional structure of the protein.

ro
710 Sirtori et al. (2010) found that there were two endothermic peaks during denaturation
711 -p
of the control lupin protein. However, after heating the proteins for 15 minutes at 100°C, and
5 minutes or longer at 200 and 300°C only one peak was observed. Harsher thermal
re
712
713 treatments of 15-30 minutes at 200°C showed that there was an impact on the native protein
lP

714 structure as no peaks could be found for heating at 200 °C for 30 minutes.
na

715 It appears that lupin protein isolates obtained from different isolation techniques (reported in
716 Tables 1 and 5) have maintained their native form as confirmed by their endothermic DSC
ur

717 peaks. This also reveals that pH modifications (acidic or alkaline) during protein isolation do
718 not cause substantial changes of lupin protein conformation.
Jo

719 7. Summary and Future work

720 Lupin is a type of legume that has various nutritional qualities that are highly valued in
721 food production. The most notable of these characteristics include its high protein and fibre
722 content, as well as its low levels of starch. These favourable qualities render lupin a
723 promising alternative to existing food ingredient. In reality, however, very few food
724 production processes have incorporated lupin due to its relatively inadequate functional
725 properties compared to more established ingredients. Many of these deficiencies relate to
726 limited availability of high quality lupin based food ingredients and the poor techno-
727 functional properties of lupin proteins, such as its low solubility and inferior viscosity, gelling
728 and emulsification properties.

23
729 The potential of lupin has prompted research into its chemical composition and the cause
730 of its inadequacies. This paper sets out to compile and present the key findings of the
731 literature to date in this field and to identify the deficiencies to inform the direction of future
732 research endeavours. In general terms, numerous papers have investigated the impact of the
733 various existing methods of protein extraction to determine whether the application of such
734 technologies influence the functional elements of lupin, including its chemical composition,
735 emulsification, rheology and thermal properties. This review dealt with this area and can
736 confirm that the selection of the extraction method has a significant impact on the functional
737 qualities of lupin proteins.

738 The protein isolation methods reviewed in this work include time consuming and energy

of
739 intensive multiple step protocols that consume large quantities of chemicals with

ro
740 environmental concerns. Therefore, the industry is looking into innovative clean and cost-
741
742
-p
effective protein isolation techniques for producing high protein isolates. While the natural
polypeptide structure of the lupin protein offers potentials for acting as viscosifying,
re
743 emulsifying, gelling agent, evaluation of the functional properties of lupin clearly indicated
lP

744 its poor functional properties. At present, therefore, this is a major hurdle against using lupin
745 protein products in manufactured foods and future research is required on lupin protein
na

746 modification preferably using physical and clean technologies.

747 In the light of this information, it is suggested that future research in this field should
ur

748 focus on how these protein isolation techniques could be altered or applied differently to
Jo

749 enhance the efficiency of protein isolation process and the overall functional properties of
750 lupin protein isolate. The purpose of doing so would be to augment the nutraceutical elements
751 of lupin for the benefit of consumers, while providing greater operational flexibility for food
752 manufactures in their ingredient procurement decisions.

753 Overall, this review of the literature clearly indicates the poor physical functionality of lupin
754 protein isolates as compared to other proteins such as soy protein that can be related to their
755 limited hydration properties, low levels of protein-water and protein-food biopolymer
756 interactions.

757 Novel biopolymer modification technologies such as power ultrasound and dry and moist
758 extrusion and their combinations are to be employed to unveil their potential for radical
759 change in lupin protein conformation and properties. Fractionation of lupin protein into its

24
760 subunits and study of their techno-functionality and possible health benefits are other areas
761 worth exploring.

762 Lupin protein also has great potential to act as a sustainable and environmentally friendly
763 plant-based polypeptide substrate for producing low molecular weight protein hydrolysates
764 and nutraceuticals. Green and clean biophysical technologies are to be investigated to
765 generate and test these products. Furthermore, the nutraceutical properties of the hydrolysates
766 are to be explored and examined.

767 This research did not receive any specific grant from funding agencies in the public,
768 commercial, or not-for-profit sectors.

of
769 References

ro
770 Aguilera, J., Gerngross, M., & Lusas, E. (1983). Aqueous processing of lupin seed.
771 -p
International Journal of Food Science & Technology, 18(3), 327-333.
re
772 Alamanou, S., & Doxastakis, G. (1995). Thermoreversible size selective swelling
773 polymers as a means of purification and concentration of lupin seed proteins (Lupinus albus
lP

774 ssp. Graecus). Food Hydrocolloids, 9(2), 103-109.

na

775 Alu’datt, M., Rababah, H., Alhamad, T., Ereifej, M., Gammoh, N., Kubow, K., &
776 Tawalbeh, S. (2017). Preparation of mayonnaise from extracted plant protein isolates of
ur

777 chickpea, broad bean and lupin flour: Chemical, physiochemical, nutritional and therapeutic
Jo

778 properties. Journal of Food Science and Technology, 54(6), 1395-1405.

779 Arnoldi, A., Boschin, G., Zanoni, C., & Lammi, C. (2015). The health benefits of sweet
780 lupin seed flours and isolated proteins. Journal of Functional Foods, 18, 550-563.

781 Aryee, A., Agyei, D., & Udenigwe, C. (2018). Impact of processing on the chemistry
782 and functionality of food proteins. In Proteins in Food Processing (Second ed., pp. 27-45).
783 Elsevier.

784 Bader, S., Bez, J., & Eisner, P. (2011). Can protein functionalities be enhanced by high-
785 pressure homogenization? – A study on functional properties of lupin proteins. Procedia
786 Food Science, 1(C), 1359-1366.

787 Bader, S., Czerny, M., Eisner, P., & Buettner, A. (2009). Characterisation of odour‐
788 active compounds in lupin flour. Journal of the Science of Food and Agriculture, 89(14),
789 2421-2427.

25
790 Bader, S., Oviedo, J., Pickardt, C., & Eisner, P. (2011). Influence of different organic
791 solvents on the functional and sensory properties of lupin (Lupinus angustifolius L.) proteins.
792 LWT - Food Science and Technology, 44(6), 1396-1404.

793 Bartkiene, E., Sakiene, V., Bartkevics, V., Rusko, J., Lele, V., Juodeikiene, G., Claudia,
794 W., & Braun, P. (2018). Lupinus angustifolius L. lactofermentation and protein isolation:
795 Effects on phenolic compounds and genistein, antioxidant properties, trypsin inhibitor
796 activity, and protein digestibility. European Food Research and Technology, 244(9), 1521-
797 1531.

798 Berghout, J., Boom, R., & Van Der Goot, A. (2014). The potential of aqueous

of
799 fractionation of lupin seeds for high-protein foods. Food Chemistry, 159, 64-70.

ro
800 Berghout, J., Boom, R., & Van Der Goot, A. (2015). Understanding the differences in
801 gelling properties between lupin protein isolate and soy protein isolate. Food Hydrocolloids,
802 43(C), 465-472.
-p
re
803 Berghout, J., Marmolejo-Garcia, C., Berton-Carabin, C., Nikiforidis, C., Boom, R., &
lP

804 Van Der Goot, A. (2015a). Aqueous fractionation yields chemically stable lupin protein
805 isolates. Food Research International, 72(C), 82-90.
na

806 Berghout, J., Venema, P., Boom, R., & Van Der Goot, A. (2015b). Comparing
ur

807 functional properties of concentrated protein isolates with freeze-dried protein isolates from
808 lupin seeds. Food Hydrocolloids, 51, 346-354.
Jo

809 Burgos-Díaz, C., Opazo-Navarrete, M., Wandersleben, T., Soto-Añual, M., Barahona,
810 T., & Bustamante, M. (2019a). Chemical and nutritional evaluation of protein-rich ingredients
811 Obtained through a technological process from yellow lupin seeds (Lupinus luteus). Plant
812 Foods for Human Nutrition, 74(4), 508-517.

813 Burgos-Díaz, C., Wandersleben, T., Olivos, M., Lichtin, N., Bustamante, M., &
814 Solans, C. (2019b). Food-grade Pickering stabilizers obtained from a protein-rich lupin
815 cultivar (AluProt-CGNA®): Chemical characterization and emulsifying properties. Food
816 Hydrocolloids, 87, 847-857.

817 Chapleau, N., & De Lamballerie-Anton, M. (2003). Improvement of emulsifying

818 properties of lupin proteins by high pressure induced aggregation. Food Hydrocolloids, 17(3),
819 273-280.

26
820 Chew, P., Casey, A., & Johnson, S. (2003). Protein quality and physico-functionality
821 of Australian sweet lupin (Lupinus angustifolius cv. Gungurru) protein concentrates prepared
822 by isoelectric precipitation or ultrafiltration. Food Chemistry, 83(4), 575-583.

823 Cronin, K., & Fitzpatrick, J. (2011). Plant and Equipment | Agitators in Milk
824 Processing Plants. In Encyclopedia of Dairy Sciences (Second ed., pp. 160-165). Elsevier.

825 D'Agostina, A., Antonioni, C., Resta, D., Arnoldi, A., Bez, J., Knauf, U., & Wäsche,
826 A. (2006). Optimization of a pilot-scale process for producing lupin protein isolates with
827 valuable technological properties and minimum thermal damage. Journal of Agricultural and
828 Food Chemistry, 54(1), 92-98.

of
829 Duranti, M., Consonni, A., Magni, C., Sessa, F., & Scarafoni, A. (2008). The major

ro
830 proteins of lupin seed: Characterisation and molecular properties for use as functional and
831 nutraceutical ingredients. Trends in Food Science and Technology, 19(12), 624-633.

832
-p
El-Adawy, T., Rahma, E., El-Bedawey, A., & Gafar, A. (2001). Nutritional potential
re
833 and functional properties of sweet and bitter lupin seed protein isolates. Food Chemistry,
lP

834 74(4), 455-462.

835 Fellows P.J. (2017). Extraction and Separation of Food Components. In Food
na

836 Processing Technology - Principles and Practice (4th ed., pp. 1-2). Elsevier.
ur

837 Figura, L. & Teixeira, A. A. (2007). Food Physics Physical Properties - Measurement
Jo

838 and Applications (1st ed. 2007. ed.). Berlin, Heidelberg: Springer Berlin Heidelberg: Imprint:
839 Springer.

840 Fontanari, G., Martins, G., Kobelnik, J., Pastre, M., Arêas, M., Batistuti, I., &
841 Fertonani, A. (2012). Lupinus albus Thermal studies on protein isolates of white lupin seeds
842 (Lupinus albus). Journal of Thermal Analysis and Calorimetry, 108(1), 141-148.

843 Fudiyansyah, N., Petterson, D., Bell, R., & Fairbrother, A. (1995). A nutritional,
844 chemical and sensory evaluation of lupin (L. angustifolius) tempe. International Journal of
845 Food Science & Technology, 30(3), 297-305.

846 Gavazzi-April, C., Benoit, S., Doyen, A., Britten, M., & Pouliot, Y. (2018).
847 Preparation of milk protein concentrates by ultrafiltration and continuous diafiltration: Effect
848 of process design on overall efficiency. Journal of Dairy Science, 101(11), 9670-9679.

27
849 Hojilla‐Evangelista, M., Sessa, D., & Mohamed, A. (2004). Functional properties of
850 soybean and lupin protein concentrates produced by ultrafiltration‐diafiltration. Journal of the
851 American Oil Chemists' Society, 81(12), 1153-1157.

852 Kiosseoglou, A., Doxastakis, G., Alevisopoulos, S., & Kasapis, S. (1999). Physical
853 characterization of thermally induced networks of lupin protein isolates prepared by
854 isoelectric precipitation and dialysis. International Journal of Food Science & Technology,
855 34(3), 253-263.

856 Khan, M., Karnpanit, W., Nasar‐Abbas, S., Huma, Z., & Jayasena, V. (2015).
857 Phytochemical composition and bioactivities of lupin: A review. International Journal of

of
858 Food Science and Technology, 50(9), 2004-2012.

ro
859 Kohajdová, Z., Karovičová, J., & Schmidt, S. (2011). Lupin composition and possible
860 use in bakery – a review. Czech Journal of Food Sciences, 29(3), 203-211.

861
-p
Lo, B. (2017). Effect of Low Frequency Ultrasound on the Solubility and Physical
re
862 Characteristics of Reconstituted Milk Protein Powders.
lP

863 Lqari, H., Vioque, J., Pedroche, J., & Millán, F. (2002). Lupinus angustifolius protein
864 isolates: Chemical composition, functional properties and protein characterization. Food
na

865 Chemistry, 76(3), 349-356.

ur

866 Mane, S., Johnson, S., Duranti, M., Pareek, V., & Utikar, R. (2018). Lupin seed γ-
Jo

867 conglutin: Extraction and purification methods - A review. Trends in Food Science and
868 Technology, 73, 1-11.

869 Muranyi, I., Otto, C., Pickardt, C., Koehler, P., & Schweiggert-Weisz, U. (2013).
870 Microscopic characterisation and composition of proteins from lupin seed (Lupinus
871 angustifolius L.) as affected by the isolation procedure. Food Research International, 54(2),
872 1419-1429.

873 Muranyi, I., Volke, D., Hoffmann, R., Eisner, P., Herfellner, T., Brunnbauer, M.,
874 Koehler, P., & Schweiggert-Weisz, U. (2016). Protein distribution in lupin protein isolates
875 from Lupinus angustifolius L. prepared by various isolation techniques. Food Chemistry,
876 207, 6-15.

877 Pozani, S., Doxastakis, G., & Kiosseoglou, V. (2002). Functionality of lupin seed
878 protein isolate in relation to its interfacial behaviour. Food Hydrocolloids, 16(3), 241-247.

28
879 Rajagopalan, N., & Cheryan, M. (1991). Total protein isolate from milk by
880 ultrafiltration: factors affecting product composition. Journal of Dairy Science, 74(8), 2435-
881 2439.

882 Raymundo, A., Empis, J., & Sousa, I. (1998). White lupin protein isolate as a foaming
883 agent. Zeitschrift Für Lebensmitteluntersuchung Und -Forschung A, 207(2), 91-96.

884 Rodríguez-Ambriz, S., Martínez-Ayala, L., Millán, A., & Dávila-Ortíz, F. (2005).
885 Composition and functional properties of lupinus campestris protein isolates. Plant Foods for
886 Human Nutrition, 60(3), 99-107.

887 Ruiz-López, M., Barrientos-Ramírez, L., García-López, P., Valdés-Miramontes, E.,

of
888 Zamora-Natera, J., Rodríguez-Macias, R., Salcedo-Pérez, E., Bañuelos-Pineda, J., & Vargas-

ro
889 Radillo, J. (2019). Nutritional and bioactive compounds in Mexican lupin beans species: a
890 mini-review. Nutrients, 11(8), 1785.

891
-p
Schlegel, K., Sontheimer, K., Eisner, P., & Schweiggert-Weisz, U. (2019). Effect of
re
892 enzyme-assisted hydrolysis on protein pattern, technofunctional, and sensory properties of
lP

893 lupin protein isolates using enzyme combinations. Food Science and Nutrition, 00, 1-11.

894 Sirtori, E., Resta, D., Brambilla, F., Zacherl, C., & Arnoldi, A. (2010). The effects of
na

895 various processing conditions on a protein isolate from Lupinus angustifolius. Food
ur

896 Chemistry, 120(2), 496-504.

Jo

897 Sousa, I., Morgan P., Mitchell, J., Harding, S., & Hill, S. (1996). Hydrodynamic
898 characterization of lupin proteins:  solubility, intrinsic viscosity, and molar mass. Journal of
899 Agricultural and Food Chemistry, 44(10), 3018-3021.

900 Sussmann, D., Pickardt, C., Schweiggert, U., & Eisner, P. (2013). Influence of
901 different processing parameters on the isolation of lupin (Lupinus Angustifolius L.) protein
902 isolates: a preliminary study. Journal of Food Process Engineering, 36(1), 18-28.

903 Villarino, C., Jayasena, V., Coorey, R., Chakrabarti-Bell, S., & Johnson, S. (2016).
904 Nutritional, health, and technological functionality of lupin flour addition to bread and other
905 baked products: benefits and challenges. Critical Reviews in Food Science and Nutrition,
906 56(5), 835-857.

29
907 Wang, J., Zhao, J., De Wit, M., Boom, R., & Schutyser, M. (2016). Lupine protein
908 enrichment by milling and electrostatic separation. Innovative Food Science and Emerging
909 Technologies, 33, 596-602.

910 Wäsche, A., Müller, K., & Knauf, U. (2001). New processing of lupin protein isolates
911 and functional properties. Food / Nahrung, 45(6), 393-395.

912 Yoshie-Stark, Y., Bez, J., Wada, Y., & Wäsche, A. (2004). Functional properties,
913 lipoxygenase activity, and health aspects of Lupinus albus protein isolates. Journal of
914 Agricultural and Food Chemistry, 52(25), 7681-7689.

915 Zayas, J. (1997). Functionality of Proteins in Food (1st ed. 1997. ed.). Berlin,

of
916 Heidelberg: Springer Berlin Heidelberg: Imprint: Springer.

ro
-p
re
lP
na
ur
Jo

30
 Table 1. Chemical composition and protein solubility of lupin proteins isolated by different methods.

Moisture
Protein Method of protein solubility Protein solubility Reference
Method of extraction content Yield (%)
content (%) analysis (%) Title
(%)
N estimated by AOCS method
-Lupin Seeds (lupinus albus var Tifwhite) dehulled, milled, water
Kjeldahl method used to measure
extraction (1/1.5 w/w) performed at 65°C for 40 min. pH adjusted A: 69% Aguilera,
the protein solubility using a 6.25
to either pH 8 or 9, centrifuged and supernatant separated. B: 75% Gerngross
5.1-7.3 72-79 factor. Soluble N calculated as the 84-91
-Suspension pH adjusted to 4.4 or 4.5, 32°C then centrifuged. D: 81% & Lusas
ratio between the N in the
-Pellet was neutralised to pH 6.8 before spray drying at inlet E: 82% (1983)
supernatant after extraction and
temperature of 193°C and outlet of 90°C

f
the total N.

o
Alamanou &
Protein was isolated from lupin (lupinus albus) using isoelectric

ro
40-92% Doxastakis
precipitation, dialysis and ultrafiltration
(1995)

-p
Isoelectric precipitation: lupin flour was suspended in water, then
Alu’datt et

re
centrifuged and filtered in filter paper. pH adjusted to 4.5-4.6 using 19.12% 58.75%
al. (2017)
acetic acid (20% v/v), then centrifuged before freeze drying

lP
-At 35°C, solubility
Lupin protein isolate dispersed in
increased from 67%
Protein was extract from lupin (lupinus albus and lupinus 50 mL 0.1 mol.L-1 NaCl at pH 7.0 Bader, Bez

na
to 73% at 150 MPa
angustifolius). Acid precipitation was used followed by cross flow for 60 min, after centrifugation at & Eisner
-At 60°C, solubility
membrane filtration 20000 g for 15min, the N of the (2011)
increased from 67%
ur 36-44%
soluble proteins was determined.
to 78% at 150 MPa
Jo
-First stage- 200g lupin flakes (lupinus angustifolius) (defatted and
Lowest Protein solubility was measured
full fat flakes) suspended in acidified water (pH 4.5) for 45 min.
protein based on N with Dumas
Centrifuged at 3300g for 5 min and supernatant discarded. This was
recoveries 92-98% combustion method with 6.25 Bader et al.
repeated again for the pellet. 92.2-98.4
found for factor. 1g sample is mixed in (2011)
-Second stage- follows the first stage but the pellet was resuspended
defatted 50mL water, pH adjusted to 7,
in water at pH 7.2 at 30°C for 35 min. pH adjusted to 4.5 and
sample centrifuged at 20000 g for 15 min.
centrifuged at 3300g, 5 min. The pellet neutralised to 6.8.
pH 10 had the highest
Lupin flakes (lupinus angustifolius S. 1700, 1701, 1800, and 1072)
Protein solubility was determined solubility (70-90%)-
suspended in water or NaCl solutions (1:8 w/v) at pH 8 and 80.43 to Bartkiene, et
using the Kjeldahl method with a pH 8 (50-75%), pH 6
centrifuged at 3300g, 10 min. pH of pellet adjusted to 4.5 and freeze 89.08% al. (2018)
5.7 factor. (10-40%), pH 4 (10-
dried after centrifugation
25%), pH 2 (50-70%)
 pH 7 isolate at 4°C
Isolate was suspended in water
-Full fat lupin (lupinus angustifolius) dispersed in water (1:15) at had the highest
80-85% (0.01g/mL), shaken for 1 h and Berghout,
pH 9, kept at 4, 20, 50 or 90°C for 2 h. Centrifuged 30 min, 20°C, solubility (90%) but
protein content centrifuged (3000g, 25°C for 15 Boom & Van
11000g. pH adjusted 4.5 and kept at 4, 20, 50 or 90 °C, 1h, 36.7-43.5% 90°C had the lowest
for all min). Supernatant was dried and N Der Goot
centrifuged for 30 min, 11000g. Pellet washed twice with water and (76%). pH 4.5 isolate
extractions determined by Dumas combustion (2014)
freeze dried, for the pH 7 isolate, adjusted to pH 7 before drying. had the highest at 4°C
method.
68%, 20°C was 42%

-Lupin flour (lupinus angustifolius) suspended in water, pH adjusted

4°C 88%,
to 9.0 and heated to 80°C (40 min) then kept at 30°C, centrifuged at
20°C 87-90%
11000g at 4°C. pH reduced to 4.5 while stirring at 4°C or 20 °C for Berghout et
5.9-6.7 4°C & heat

f
1 h, centrifuged at 11000g for 30 min. Pellet washed twice at 40°C al. (2015a)

o
treated 82-
or 20°C with water and maintained at pH 4.5 or neutralized to pH 7,

ro
84%
before freeze drying.

-p
Acid Precipitation:

re
Acid Precipitation: pH of lupin flour (lupinus angustifolius) in tap pH 3-4 (45-70%)
N Solubility Index. Freeze dried
water adjusted to 9 and stirred at 4°C, 1 h, before centrifuging. The pH 4.5-6 (0-80%)
isolate dispersed in 1% water,

lP
supernatant is adjusted to pH 4.5 while stirring, for 1 h at 4°C, pH 7-8 (80-85%)
centrifuged (3000 g, 25°C for 15 Berghout, et
centrifuged. Pellet washed and neutralised to pH 7, freeze dried. 5% 86-89% Ultrafiltration:
min), freeze dried. The protein al. (2015b)
Ultrafiltration: Same as above, after pH neutralisation to 7 the pH 3-4 (50-85%)

na
content was measured using
pellet was concentrated through a membrane (5kDa pores, 4atm, pH 4.5-6 (5-98%)
Dumas combustion method
stirring speed at 500rpm) at 4°C. pH 7-8 (80-95%)

ur -Initial solubility
Chapleau &
Jo
Protein content was determined 100%. 200MPa
-Protein was isolated from lupin (lupin llbus) using Tris–HCl buffer De
using bicinchoninic acid (BCA) treatment had no
at adjusted at pH 7.0 and it was treated with high pressure (200-600 Lamballerie-
procedure (Sigma procedure no. effect on solubility.
MPa) at 15 g/L. Anton
TPRO-562). - 400-600 MPa had a
(2003)
20% loss in solubility
Lupin seeds (Australian Sweet lupinus angustifolius) soaked in
water (1:3 w/v) for 3 h with additional water (1:10 w/v) before
UF: pH 2: 80%
homogenising for 1 min at high speed. pH adjusted to 8-9, kept for
Acid pH 4-8: 50-80%
30 min and centrifuged at 2060g, 30 min at 4°C. Pellet resuspended
recovery: Acid: pH 2: 55%
in water (1:5 w/v) and centrifuged, supernatant used to prepare The protein solubility was Chew, Casey
59% pH 4-8: 20-75%
lupin protein concentrate. 93-95% determined using Kjeldahl & Johnson
Ultrafiltratio UF isolate had a
Acid Precipitation (AP): Supernatant was acidified to 4.5 using method. (2003)
n: 92% higher protein
HCl, centrifuged at 2060g for 30 min at 4°C.
recovery solubility than acid
Ultra/Diafiltrated (UFDF) Protein isolate: Protein extract heated to
precipitation
40°C, passed through the filter (membrane size 10000 Da,
membrane area 0.1m2) 5 times, water added to the retentate each
 time. The protein concentrates prepared by AP and UFDF adjusted
to pH 7 before spray drying.

Acid Precipitation: Lupin seeds (lupinus albus Typ Top, lupinus

Protein solubility was determined
albus Ares) soaked in water in pH 4.5. Seeds grounded, soaked in T1: 10-50%
by determining the N content of Solubility ranged D'Agostina
water at pH 7. Protein precipitated at pH 4.5 and then spray dried. 4-8% T3: 3-55% 70-90%
soluble proteins by using the from 64-75% et al. (2006).
Ultrafiltration: Protein was precipitated and underwent T4:5-57%
Kjeldahl method.
ultrafiltration before spray drying.
Kjeldahl method, N to protein Protein solubility
-Two methods of protein isolation for two types of lupin (lupinus
96-97% conversion of 6.25 used. Isolate ranged from 70-85%. El-Adawy et
termis and lupinus albus): isoelectric precipitation (P1) and by Bitter 2-4%
protein content mixed with 20mL, centrifuged at pH 4 had the lowest al. (2001).
micellization (MI) methods
4000 rpm for 20 min. solubility (15%)

o f
Isolate mixed with water with and
-Lupin flakes (lupinus albus) put in water (1:20/w/v) with Na2SO3 or Isolated without NaCl

ro
without NaCl, pH 1-12.
NaCl. pH adjusted (7, 11) and centrifuged, 10000g 30 min. the highest solubility
Dispersion (1:20w/w) stirred, Fontanari et
-Supernatant ultra-filtrated with pore size of 5kDa at pH of 10 and 31-53% 80-92% at pH 2-12 (65-70%)

-p
centrifuged (10,000 g, 60 min). N al. (2012).
11, then adjusted to pH 4.5 and 5, pellet resuspended in water and pH -However lowest at
of supernatant determined,
was adjusted to 7 before freeze drying pH 4-5 (5-10%)

re
Kjeldahl method, 6.25 factor.
UFDF Protein isolate: Lupin meal (lupinus albus) homogenized in

lP
water (1 g/34 mL ratio) at 5000 rpm, 15 min, pH 6.6, 25°C.
Centrifuged (15344g for 25 min, 18oC), filtered using filter paper.
Acid Solubility ~63% at
Precipitates washed with water, homogenized, centrifuged, and

na
precipitation: The protein solubility measured pH 7 but much lower Hojilla‐
filtered as indicated above. Supernatant treated by ultrafiltration
96.3% using Biuret method with the pH at pH 4, highest found Evangelista,
(5kDa membrane with a flux rate of 2.7 L/h), the concentrated
Diafiltration ranged at 3-10. The soluble at pH 10. Sessa &
retentate freeze dried.
Acid Precipitation (AP): Lupin meal homogenized in water (1 ur and
Ultrafiltration:
protein calculated with a N factor
of 6.25 in the supernatant.
Ultrafiltration isolate
had a higher solubility
Mohamed
(2004)
Jo
g/20mL ratio) at 5000 rpm, 15 min and centrifuged (15344g for 20
73.2% than AP by 10%
min at 2-5°C). pH of supernatant adjusted to 4.5 and centrifuged
again. Pellet neutralised before centrifuging (15344g, 20 min).
Supernatant desalted by dialyzing and extract freeze dried.
-Protein isolation of lupin (lupinus albus) follows acid precipitation
(pH 7 then to pH 4.5) and then freeze dried.
A1: 5.8% A1: 92.5%
-For dialysis protein extraction, solution placed in membranes with
A2: 7.3% A2: 92% Kiosseoglou
1.3 X 104 Da then submerged in water, kept refrigerated for 48 h
B1: 6.4% B1: 83.2% et al. (1999)
and the material in the membranes was then freeze-dried.
B2: 6.1% B2: 87%
(A1) and defatted (A2), and dialysed full fat (B1) and defatted (B2)
lupin protein isolates.
Isolate A: lupin powder (lupinus angustifolius) suspended in water 250mg of lupin homogenised in
Lupin
(10%) at pH 12, 1 h, then centrifuged at 8000g. pH adjusted t 4.3 20mL of water at pH 7. N Solubility around 20-
Flour: 7.4% A: 79% Lqari et al.
and centrifuged at 8000g, washed again and adjusted back to pH 4.3 determined for solubility using a 25%
A: 3.4& B: 72% (2002)
before freeze drying. Kjeldahl. The pH range of for the 4-4.8 pH range
B: 9.4%
Isolate B: same as above but powder suspended in 0.25%Na2SO3 at solubility test is 4-4.8
pH 10.5. Precipitate obtained at the IEP was successively washed
with water and adjusted to pH 4.3 before drying.

Lupin protein isolated using acid precipitation and salt induced Muranyi et
39-45% 90-95%
extraction followed by dilutive precipitation (Lupin Cultivar). al. (2013)

-Alkaline Extraction (EA): lupin flakes (lupinus angustifolius)

suspended in water (1:8 w/v). pH adjusted to 8 with NaOH (0.5M).
-Salt Extraction (ES) prepared by suspending lupin flakes in NaCl Alkaline

f
(0.5M) (1:8 w/v). The combination of ES and EA the lupin flakes extraction:

o
prepared by suspending the lupin flakes in NaCl (0.5M) solution 26-31%. Salt
Muranyi et

ro
(1:8 w/v) and pH adjusted to 8. Stirred for 1 h, sieved, centrifuged extraction:
al. (2016)
at 3300g, 10 min and supernatant filtered. 19-30%.

-p
Isoelectric precipitation by acidifying solution to pH 4.5. The EAS Combined:
and EA lupin protein was first adjusted to pH 5.5 before adjusting to 23-25%

re
pH 4.5. After the final centrifugation the pH of the pellet measured
to be pH 5.5. The protein isolate was then freeze dried.

lP
-Solubility of control
Isolate (1% w/v) suspended in LPI was 80%
phosphate buffer (0.15M) at pH 7 -After heating to Pozani,

na
-Seeds (lupinus albus) milled and reconstituted in water (1/10) with heat treatment and/ or the 80°C, 15 min, it Doxastakis
-pH adjusted to 9.5, kept for 3 h before centrifuged at 3000g. pH of addition of DTT. Centrifuged at decreased to 60% &
supernatant adjusted to 4.5 before freeze drying the pellet.
ur 3000g for 30 min, supernatant
collected, and solubility
-Addition of DTT had
no effect
Kiosseoglou
(2002)
Jo
determined using the Kjeldahl. -DTT and heat gave
70% solubility
Raymundo,
Commercial white lupin protein isolate (L9000), obtained from
90% Empis &
Mittex, Weingarten (Germany).
Sousa (1998)
Isoelectric precipitation: 10% (w/v) of lupin suspension (Wild lupin
campestris seeds) with pH 9 centrifuged at 10,000g, 30 min. pH of Isoelectric
supernatant adjusted to 4.6 before storing at 4°C overnight. Then Precipitation -ISO-pH 2 (85%)
Protein solubility measured using Rodríguez-
centrifuged at 10,000g, 10 min at 4°C. Pellet resuspended in water (60%) pH 4-10 (15-95%)
93.2% the Kjeldahl method. pH range Ambriz, et
before freeze drying. MPI- pH2 (75%)
was 2-10. N × 6.25 was used. al. (2005)
Micellization: Lupin 10% (w/v) suspended in NaCl at pH 7 and Micellization pH 4-10 (10%-95%)
centrifuged at 5000g, 10 min. Supernatant washed with water, left (30%)
30 min, centrifugation at 10.000gfor 10 min and then freeze-dried
Seeds (lupinus angustifolius) were dehulled and milled. Flakes Lupin protein isolate powder pH 4-10%
suspended in 0.5 M HCl (1:8). Acid pre-extracted flakes rehydrated homogenized, pH adjusted. It pH 5-9%
in water (1:8 w/w) and pH adjusted to 8 using while stirring for 1 h. centrifuged at 20,000g for 30 min. pH 6- 45%
Schlegel et
The suspension centrifuged at 5600g at 4°C for 1 h and the pH of 92% N of soluble fraction determined pH 7- 70%
al. (2019)
the supernatant adjusted to 4.5. The suspension centrifuged at 5,600 by Kjeldahl. Solubility expressed pH 8-75%
g, 130 min, then neutralised by NaOH and pasteurized (70°C for 10 as % of total N of original sample. pH 9-75%
min) before spray drying. Protein measured at pH range 4-9
-Solubility the lowest
Lupin seeds (lupinus luteus) milled and rehydrated in water (1/10). Solubility measured with Kjeldahl
at pH 4 (<10%)
pH adjusted to 9 while stirring, 2 h before centrifuging at 5000g. pH at pH range 2-8. Protein solution Sousa et al.
-Highest at pH 2
of supernatant adjusted to 4.5 and centrifuged. Pellet washed with diluted then centrifuged at 42000g (1996)
(80%)
water before freeze drying. for 40 min before measurement.
-pH 5-8 (10-75%)

o f
- Lupin flakes (lupinus angustifolius) suspended in water at pH 6
(1/8 w/v ratio) at 30°C, 60 min, the mixture separated in a sieve. 35-54%

ro
Sussmann, et
Suspension centrifuged at 3300g, 10 min. Supernatant underwent protein
al. (2013)
dialysis/dilution of the high-salt protein extract (HSPE). Solution extracted

-p
centrifuged at 3300g, 10 min before washing the pellet.
Milling and electrostatic separation used for protein enrichment.

re
Lupin flour (lupinus angustifolius) added to the screw feeder and it Protein content
Wang et al.
was processed. The electric field strength was kept at 200 kV/m 62-69% ranged from

lP
(2016)
with a voltage of 20 kV to the positive electrode. The powder 40-70%
collected in four nodes GE, GC, PC and PE.

na
Isolate E:
Solubility determined at pH 2-10 Wäsche,
5%
Acid precipitation of lupin (lupinus albus and lupinus angustifolius) by stirring isolate in water and Isolate E: 98-100% Müller &
Isolate F:
followed by cross flow membrane filtration (15 kDa).
ur 5%
centrifugation. N measured with
Kjeldahl and the factor 6.25.
Isolate F: 84-90% Knauf
(2001)
Jo
Hexane Isolate Solubility at pH 3-8 determined by Hexane Isolate
First stage extraction: The deoiled lupin flakes crushed in cold
E: 100% stirring 1g powder in 50mL water, E: 64%
water in pH 4.5. The protein from lupin suspension was extracted
F: 93.53% 2 h at 30°C. Suspension F: 71% Yoshie-Stark
using cross-flow membrane filtration unit (15 kDa) (Isolate F).
CO2 Isolate centrifuged at 10,000g, 20 min. CO2 Isolate et al. (2004).
Second stage: Protein w extracted at neutral pH by acid
E: 95.83% Protein solubility measured by E: 59.8%
precipitation (protein isolate E).
F: 94.43% Kjeldahl method, 6.25 N factor. F: 68.3%
 Table 2. Molecular weight, particle size and zeta potential of lupin proteins isolated by different methods.

Method of extraction Method of analysis Molecular Weight Particle Size Zeta Potential Reference

3 globulin protein fractions

including 12 protein
20µl protein in the buffer solution loaded on SDS-
Isoelectric precipitation subunits of β-conglutin (7S),
PAGE gel and operated at 80 V for 30 min then at Alu’datt et al.
method was reported in 4 protein subunits of α-
160 V for 45 min. Gel was fixed in 50% ethanol and (2017)
table 1. conglutin (11S) and a minor
12% acetic acid solution followed by silver staining.
c-conglutin (2S) of 170, 315

f
and 17 kDa

o
-Lupinus angustifolius -Particle size measured after high

ro
A standard protein marker with molecular weight contains 22 bands, and pressure homogenization at 0, 50,
Isoelectric precipitation fractions in the 10kDa-250kDa range used for SDS- lupinus albus19 bands 100, 150 MPa at 35°C. At 50-

-p
Bader, Bez &
method was reported in PAGE analysis. The gels were stained with -lupinus angustifolius had 100MPa there was a particle size
Eisner (2011)
table 1. Coomassie Blue and the molecular weight of each protein fractions of 71-88 reduction compared to the control

re
band was compared to the standard Mw marker. kDa. (0.1-10µm). At 150MPa, there was
aggregates formation of 10-100µm.

lP
-Majority of the proteins
-1 ° were in 20–90 kDa range,
A protein solution (0.01 g.mL ) heat treated to 95 C

na
most common for lupin. Berghout,
Isoelectric precipitation for 4 min. 15µl was added to the SDS-PAGE gel and
-The 90°C and pH 7 Boom & Van
method was reported in operated at 200 V for 45 min. The gel washed and
isolation had less protein Der Goot
table 1. stained with Coomassie blue.
ur bands in the higher Mw
region indicating solubility
(2014)
Jo
reduction due to heating
Dispersing full fat lupin
in water (1:15) at pH 9,
stirred at 4, 20, 50 or
90°C, 2 h, centrifuged for
30 min, 20°C, 11000g, There was no change in particle size Berghout,
pH adjusted to 4.5 and of lupin protein with temperature. Boom & Van
stirred at 4, 20, 50 or 90 This indicates that the particles have Der Goot
°C, 1h, centrifuged again. a high thermal stability (2015)
Pellet washed twice,
freeze dried. For the pH 7
isolate, pH adjusted to 7
before drying.
 Minor differences in
zeta potential between
for wet and freeze
Acid Precipitation method had a
dried LPI found. At
larger particle size (39.6µm)
pH 7 LPI had a zeta
Isoelectric precipitation compared to ultrafiltration (11.3
potential of -35 mV Berghout et al.
method was reported in µm) Freeze drying resulted in
indicating an (2015b)
Table 1. formation of large heat stable lupin
electrostatically
protein isolate particles as compared
stabilized dispersion
to membrane filtration
for both concentrated
and freeze-dried
proteins

o f
Lupin protein
concentrate (LPC): lupin

ro
flour (lupinus luteus)
Most within the 25–100 kDa
suspended in water (1:10

-p
range, which is similar for
w/v). pH adjusted to 8,
lupin proteins
then centrifuged and the

re
The LPI had a bleached
pH of the supernatant
band of proteins at 98 kDa
adjusted to 7. The proteins were diluted with buffer (20 mM 2-

lP
and some other low
Lupin protein isolate mercaptoethanol), 10 μg sample was loaded into the
molecular weight bands. Burgos-Díaz et
(LPI): the method follows SDS-PAGE gel, then it stained with Coomassie blue.
Suggests that the proteins al. (2019a)

na
the same as LPC. After Molecular weights of the bands were estimated by
did not precipitate at pH 4.5
the first centrifugation, comparing them with standard Mw markers.
In LPI, conglutin γ could not
the pH adjusted to 4.5.
Precipitated protein was
then separated using
ur be recovered because it does
not precipitate at pH 4.5.
Jo
However, it is present in
centrifugation. The pellet
LPC.
resuspended in water
(1:5), pH adjusted to 7.
-LPI & LPC spray dried.
AluProt-CGNA® cultivar
-Zeta potential
acid precipitated. pH of
showed the charge
the suspension (1:10)
There was an increase in particle was positive at pH 2
adjusted to 8, then
size distribution at 90°C compared (+35), 4.6 (-10) to pH
centrifuged at 3200g for Burgos-Díaz et
to 25°C (horizontal shift to the 10 as negative (-30)
15 min. Supernatant al (2019b)
right). This is due to protein -Increase of NaCl
adjusted to pH 4.5. The
denaturation. showed a decrease in
pellet resuspended in
zeta potential. 0-
water & pH adjusted to
200mM (-33, -8)
7.4 and then spray dried.
 With pressure increase an
increase of proteins
Molecular weight determined using SDS-PAGE
composition at the O/W Control: 1.17 µm Chapleau &
-Isoelectric precipitation analysis. Protein solution (2 mg/ml) in the denaturing
interface observed. 7S 200 MPa: 1.14 µm De
method was reported in buffer heated for 5 min in boiling water before
globulin was more pressure 400 MPa: 0.75 µm Lamballerie-
Table 1. loaded into the gel (0.3 µl) along with the protein
sensitive compared to the 600 MPa: 0.74µm Anton (2003)
standards. After running silver stained.
11S due to number of –SH
residues in the 11S subunits
Mw determined by SDS-PAGE. Protein solutions in
Isoelectric precipitation Eight protein bands were in
the denaturing buffer were heated for 5 min in Fontanari et al.
method was reported in the range of 14 and 71 kDa.
boiling water before. Gel ran at 30 mA, 5 h. Protein (2012)
Table 1.
bands stained with Coomassie brilliant blue.

o f
Lupin isolate showed
Hojilla‐
SDS-PAGE: Dialysed and freeze-dried protein streaking in gel pattern

ro
Isoelectric precipitation Evangelista,
extracts weighed out (1-2.5mg). Then heated for 5 suggesting denaturation
method was reported in Sessa &
min in boiling water before loading into the gel along Bands heavily concentrated

-p
Table 1. Mohamed
with the protein standards. at 31-66.2kDa but less
(2004).
concentrated at 21.4-31 kDa

re
Mw determined using 2D SDS-PAGE. 30mg protein

lP
isolate dissolved in chaotropic solution, centrifuged
at 12,000 for 1 min. 5 µl of samples were transferred Alkaline extraction
Isoelectric precipitation to 120 µl multi-chaotropic solutions. Treated for 2 h indicated the subunits of α
Muranyi et al.

na
method was reported in at 50 V at 20°C which then increased to 250 V for 15 conglutin and γ conglutin
(2016)
Table 1. min, and then 4000 V until 20,000 Vh. The strips was lower compared to salt-
placed in DTT solution, then put on the gels and run induced extraction
ur
at 200 V. The gel washed and stained with colloidal
Coomassie.
Jo
Protein samples were reconstituted in 0.75 M Tris Only slight differences
Isoelectric precipitation HCl (pH 8.8), 20% glycerol/0.05% (w/v). between the lupin proteins Rodríguez-
method was reported in Suspension centrifuged at 1000 g, 5 min. Supernatant obtained from the isoelectric Ambriz et al.
Table 1. decanted, used to load into the SDS-PAGE native precipitation and (2005)
gel. Gel stained with Coomassie Brilliant Blue. micellization.
Seeds (lupinus -Serine and cysteine
angustifolius) dehulled, endopeptidase are able to
milled. Flakes suspended hydrolyze the α-conglutin
in 0.5 M HCl (1:8). The 10 μg of sample was loaded into the gel SDS-PAGE and β-conglutin fractions of
extracted flakes dispersed along with the standard markers (molecular weight of lupin protein (13 kDa) Schlegel et al.
in water (1:8 w/w) and 10–250 kDa). -All treatments were able to (2019)
the pH adjusted to 8, kept hydrolyse the polypeptides
for 1 h. Suspension into smaller units (23 kDa)
centrifuged at 5600g, which breaks the
4°C, 1 h. pH of polypeptides
supernatant adjusted to
4.5. Proteins separated by
centrifuge at 5,600 g, 130
min, then neutralised by
NaOH, pasteurized
(70°C) and spray died.
Classifier wheel speed were 2500,
Isoelectric precipitation
4000, 6000 or 8000 rpm. 8000 rpm Wang et al.
method was reported in
had the smaller particle size (2016)
Table 1.
distribution compared to 2500 rpm

f
Table 3. Foaming, emulsification and water absorption capacity properties of lupin proteins isolated by different methods.

o
ro
Emulsification Water Absorption
Method of extraction Method of Analysis Foaming Properties Reference Title
Properties Capacity

-p
-Water holding capacity expressed as water
preserved in the residue after centrifugation. Lupin had a 50% foam stability,

re
Isoelectric precipitation Lupin Protein: 33%
-Foaming: 2 g of lupin protein was added with 40 ml similar to chickpea protein Alu’datt et al.
method was reported in Chickpea Protein: 42%
water at 30° C while mixing. isolate, but less than broad bean (2017).

lP
table 1. Broad Bean Protein: 44%
-Foam stability measured as the ratio of foam volume protein isolate
obtained at 0 min to foam volume at 60 min.

na
-L. Angustifolius (780mL
oil.g-1) had a higher
emulsification properties
Isoelectric precipitation ur
1 g of lupin protein sample was reconstituted in 99 g
water and then homogenized while adding 125 mL of
than L. Albus (580 oil.g-
1
) Homogenization had Bader, Bez &
Jo
method was reported in corn oil to the protein solution. The emulsification
no effect on Eisner, (2011)
table 1. capacity was calculated by mL of oil per g protein
emulsification at 50-
isolate.
100MPa.
-At 150MPa, had an
increased emulsification
1.2-4.3 mL water/g protein
found, highest was at pH 7,
90°C isolate. WHC for 4°C,
20°C & 50°C at pH 7 could
Isoelectric precipitation The method AACC 56-30 was used which is based Berghout, Boom
not be determined.
method was reported in on how much water that lupin protein (1g) will be & Van Der
It is suggested that at pH 7
table 1. absorb by centrifugation (2000g, 20°C for 10 min). Goot, (2014)
does not sediment by
centrifugation and stays
dispersed that may be related
to the conformational
 structure of the protein.

Creaming stability:
varied with heat
treatment, oil
concentration and lupin
0-5% LPI used to prepare emulsions containing 5- protein aggregate
Isoelectric precipitation
20% oil. 5 ml of each emulsion added into glass particles concentrations. Burgos-Díaz et
method was reported in
tubes, which helps prevent moisture loss. The No creaming for 5% LPI al. (2019)
table 2.
samples monitored for 14 days. for all oil concentration.

fo
An increased creaming

ro
for all emulsions over
first 2-4 days across all

-p
concentrations.
High pressure caused

re
decrease of flocculation
and creaming.

lP
Flocculation index was determined by comparing the Creaming Index (%):
particle size (d4.3) of the emulsion in Tris HCl buffer Control:97.36
Isoelectric precipitation at pH 7 (0.1 M) with or without SDS separation of 200 MPa: 95.69 Chapleau & De

na
method was reported in the oil droplet, which causes flocculation in the 400 MPa: 91.06 Lamballerie-
table 1. emulsion. Samples stored at room temperature for 24 600 MPa: 90.36 Anton, (2003)
h before centrifuging at 2000g, 30 min. Volume
above the supernatant was the volume of cream.ur Flocculation Index (µm)
Control: 14.15
Jo
200 MPa: 11.51
400 MPa: 5.78
600 MPa: 6.31
Acid precipitation
protein:
pH 2 (450), pH 4(220)
pH 6 (190)
Samples were suspended in water (2% w/v) and pH
pH 8 (220) mL oil/g of
Isoelectric precipitation was adjusted using HCl or NaOH. The suspension
protein Chew, Casey &
method was reported in was stirred and homogenised for 1 minute. The
UF lupin protein: Johnson, (2003)
table 1. volume of the foam layer was recorded after 30
pH 2 (470)
seconds of homogenisation.
pH4 (250)
pH 6 (220)
pH 8 (200);
mL oil/g of protein
 Foaming:
Emulsifying capacity
Foams created by using an agitating mixer. Protein
Foam capacity (%) LPI-E (mL oil/g of protein)
solutions (5%) obtained by comparing the foam Water-binding capacity
(1100-1470) LPI-Type E (380-450)
Isoelectric precipitation volume after 8 min of whipping with the volume of (mL/g of protein)
LPI-F (1800-2080) LPI-Type F (370-570) D'Agostina et al.
method was reported in the starting protein solution. -LPI-E (0-0.8)
Foam stability (%) Emulsion stability (%) (2006)
Table 1. Emulsification: LPI-F (1-1.8)
LPI-E (70-90) LPI-Type E (61-63)
Protein solution (1% w/w) stirred at 20°C while
LPI-F (90-96) LPI-Type F (75-90)
adding oil until it reached the inversion point to
calculate the emulsifying capacity
Foaming:
Foam capacity measured by mixing 1% protein
solution using a blender. The change in volume

o f
recorded after 15, 30, 45, 60, 90, 120 min and
reported as foam stability. Water absorption

ro
Emulsification capacity
Emulsification: capacity
Bitter Lupin (PI)- 104% (oil ml/ g protein):
Isoelectric precipitation 2 g of lupin protein suspended in 23 mL of either (g/100g isolate):

-p
Bitter Lupin (MI)- 120% Bitter Lupin (PI)- 164 El-Adawy et al.
method was reported in water or 0.5 M NaCl. The suspension blended for 30 Bitter Lupin (PI)- 209.6
Sweet Lupin (PI)- 106% Bitter Lupin (MI)- 196.2 (2001)
table 1. seconds. Peanut oil added to the suspension from a Bitter Lupin (MI)- 212.5

re
Sweet Lupin (MI)- 146% Sweet Lupin (PI)- 169.4
burette until the emulsion breakpoint was achieved. Sweet Lupin (PI)- 225.7
Sweet Lupin (MI)- 210.2
Breakpoint defined where the emulsion coalescence Sweet Lupin (MI)- 229.4

lP
separated and substantial loss in consistency.
WAC:

na
Water and fat absorption capacities estimated
and expressed as g of water or oil bound/ g isolate.
Foaming properties:
ur
5mL protein solution (1%) adjusted to pH 7 and air
added to in a graduated column at 100 mL/min at 20
AP (16.8%) had a higher
The emulsion stability
was similar for both AP
Jo
foaming stability compared to Hojilla‐
psi. When the bubbles begin to appear, the timing and UFDF (23.4/25.5)
Isoelectric precipitation UFDF (2.6%) Evangelista,
started and the foam volume measured after 1 min as
method was reported in Sessa &
foam capacity. Foam stability obtained from the % of Emulsification capacity
Table 1. Foaming Activity Mohamed
the original foam remaining after 15 min. Acid precipitation: 45.4
Acid Precipitation: 104mL (2004).
Emulsification: (m2/g)
UFDF: 98mL
Protein samples (2mL) homogenised in corn oil UFDF: 71.5 (m2/g)
(6mL) for 1 minute (20,000rpm)
60°C had the highest foaming
stability (16%). With
Foams were produced by air-dispersion by using 15 temperature increase the
Isoelectric precipitation Raymundo,
ml of protein solutions (3%w/v) in 2.5 cm diameter foaming stability decreased
method was reported in Empis & Sousa
graduated cylindrical vessels operated at a single (7.5% at 90°C). Among the
Table 1. (1998)
speed. additives, xanthan gum added to
LPI had the highest foam
stability (25%)
 -Foaming depends on pH, LPI
WAC (water/g protein)
Foaming properties: 50mL of lupin protein solution had the highest capacity (500%)
-Lupin, acid precipitation
homogenized for 1 min at 25 °C. The foam volume at pH 2 compared to LMI
(1.7mL)
recorded after 30 s. (350%).
Isoelectric precipitation -Micellization, lupin protein Rodríguez-
WAC: 0.5 g of lupin protein homogenized into 5 ml -LMI had the highest foam
method was reported in isolate (1.3mL) Ambriz et al.
water or corn oil for 1 min and 30 min at 25 ◦C. The capacity at pH 10 (450%)
Table 1. -Soybean, acid precipitation (2005)
suspension centrifuged at 1600g for 25 min. The compared to LPI (~350%).
(2.2mL)
volume of free liquid measured, residual volume -Foam capacity at pH 4-8 was
-Soybean, micellization
expressed as mL water or oil absorbed /g protein similar for LMI and LIP
protein (3.5)
Foaming properties: Lupin protein solution (5%) at
pH 7 whipped for 8 min. The increase in volume

f
-All protein hydrolysates LPI was the highest in

o
used for foam activity (g/L). % of the remaining
Isoelectric precipitation showed a significant increase in emulsification capacity
foam volume after 1 h defined as foaming stability. Schlegel et al.

ro
method was reported in foaming activity compared to than most of the
Emulsifying capacity: Lupin protein solution (2019).
Table 1. untreated LPI hydrolysates (620 ml/g)
(1%w/w) at pH 7.0 titrated until a phase inversion.

-p
Volume of oil required to obtain phase inversion
used to calculate the emulsifying capacity

re
lP
Table 4. Viscosity, swelling and gelling properties of lupin proteins isolated by different methods.

na
Method of
Method for Analysis Viscosity Gelling Properties Reference Title
extraction
Gelling: Lupin protein (15% w/w) -Gelling was not determined for the untreated and treated lupin
Isoelectric
suspended in water with continuous
stirring for 30 min while adjusting the pH
ur protein isolates of L. angustifolius L. at 15% (w/w)
-Albus protein formed gels at 15% (w/w).
Jo
precipitation Bader, Bez &
to 7.0. Gels placed in a glass tube, heated -Homogenization (0-150MPa) at 35°C showed an increase
method was -2 Eisner (2011)
to 95°C for 1 h, then cooled down and in gel strength (0.15-0.55 N cm )
reported in Table 1.
stored at 1°C. The gel strength measured -Homogenization (0-150MPa) at 60°C showed an increase
using a texture analyzer in gel strength (0.13-0.6 N cm-2)
Viscosity/Gelling: Viscosity and gelling
-At high protein concentration (30% w/v) the G’ of lupin
Isoelectric was measured using a cone-plate
Lupin protein isolate showed a shear protein gels was similar to soy protein isolate (24% w/v) Berghout, Boom
precipitation geometry (CP-20-2). The samples were
thinning matrix which suggests that high gel, however lupin protein gel was weak. & Van Der Goot
method was measured with the flow rate range from 1
shear rates broke down the network. -It suggests that lupin protein formed weaker, less elastic (2015)
reported in Table 2. to 100 s-1 at 25° C.
gels than soy protein isolate
Viscosity measured using cone-plate By applying heat to a 10% method 1 LPI
Isoelectric
geometry at 25°C at a shear rate range of dispersion the viscosity was higher (2
precipitation Berghout et al.
0.01-100 s-1. The hydrated lupin protein factor) than the method 2 LPI dispersion
method was (2015b)
dispersion heated to 121° C and was -Freeze dried LPI dispersion showed
reported in Table 1.
subjected to a frequency sweep with a more shear thinning compared to the wet
 constant strain (0.1% within linear LPI dispersion
regime) from 0.1 to 10 rad/s at 25°C.

To disrupt of the emulsion flocculation

Isoelectric Viscoelasticity: All pressure treatments
shear stress of 10 Pa was applied for 2 -Rheological behavior of Chapleau & De
precipitation had the same viscoelastic behavior.
min. The emulsion left 15 min so the emulsions from pressure treated protein was Lamballerie-
method was -Viscosity: 400MPa had a higher
droplets can interact. Finally, the characteristic of a strong gel Anton (2003)
reported in Table 1. viscosity compared to other pressures
viscosity of emulsion measured.
-At pH 8 UF and acid precipitated lupin
had a similar viscosity at a range of
temperatures (10-70°C). The results also

o f
show that as the temperature increases,

ro
the viscosity becomes shear thinning

-p
- At pH 4 UF had a higher viscosity (6.5
Pa.s) compared to acid precipitated lupin

re
(2.5 mPa.s) at 10oC. UF lupin showed
shear thinning trend as the temperature
Apparent viscosity of the lupin protein

lP
Isoelectric increases. Acid precipitation protein had
was measured using a Brookfield DV-1+
precipitation an increase viscosity at 20oC (3.5 mPa.s) Chew, Casey &
viscometer at a shear rate 61.2 s-1. Lupin
method was then deceases (shear thinning) as the Johnson. (2003)

na
protein suspensions measured at 10, 20,
reported in Table 1. temperature increases.
30, 50 and 70 °C at pH 4, 6, 7 or 8.

ur
- At pH 6.7 UF had a higher viscosity
(123 mPa.s) compared to acid precipitated
Jo
lupin protein (6 mPa.s) at 10°C. UF
protein showed at decrease in viscosity
(shear thinning) as temperature increases.
Acid precipitation showed that the
viscosity increased (4-7 mPa.s) as the
temperature increase (50-70oC).

1 mL lupin protein suspension (20% w/w)

A pseudogel produced at 80 °C where the cohesion of the
Isoelectric sealed in tubes and heated at 80°C, 1 h,
gel increased when it cooled to 10 °C. Heating to 90 °C Kiosseoglou et al.
precipitation then cooled down to 10°C, 1 h. Three
indicated extensive formation of disulphide bonds as it (1999)
reported in Table 1. tubes were heated at 90°C for another 10
produced gels insoluble in SDS and urea solutions.
min.
Isoelectric
All viscosity measurements performed -By having an increase of xanthan gum Raymundo, Empis
precipitation
using a Brookfield viscometer. concentration, the viscosity increased. & Sousa. (1998)
reported in Table 1.
Isoelectric Protein suspensions (6, 8, and 10%)
precipitation prepared and heated for 1 h in boiling
reported in Table 1. water. The tubes then cooled to 4°C
Table 5. Thermal properties of lupin proteins isolated by different methods.
Method of extraction
Isoelectric precipitation method was
reported in Table 2.
Isoelectric precipitation method was
reported in Table 1.
Isoelectric precipitation method was
reported in Table 1.
Isoelectric precipitation method was
reported in Table 1.
Lupin seeds (lupinus angustifolius)
dehulled, flaked then defatted and
dissolving in water (1:10 w/v). pH
adjusted to 8 and centrifuged. pH of
solution adjusted 4.6 and again
centrifuged. Pellet resuspended in water
(pH 7) and spray dried.
For gelation in LMI the minimum protein concentration Rodríguez-
was 8% w/v, while for lupin protein was 6%. No gelling Ambriz, et al.
occurred at pH 6 and 8 (2005)
Method of Analysis Thermal Characteristics Reference Title
Berghout, Boom
10 mg lupin protein sample weighed into the DSC pans. The DSC analysis showed that lupin protein was not fully
& Van Der Goot
f
samples were scanned at 20 to 130°C at a rate of 10°C/min denatured after protein isolation.
(2015)
o
ro
Thermal characteristics analysed using a DSC calorimeter. The isolate measured in powder form, initial temperature
-DSC: 4.0 mg protein weighed and treated under a nitrogen flow suggests a high thermal stability (195-208°C)
-p
(50 mL min-1) and run at a heating rate of 10°C min-1, 20-140°C. -DSC showed the presence of only one broad endothermic Fontanari et al.
Denaturation temperatures and enthalpy determined. peak (63°C and 74°C), indicating denaturation temperature (2012)
re
-TG-DTG: 4.0 mg of protein analysed by TG–DTG in an a- -The presence of NaCl showed the highest value of ∆H
alumina crucible, heating rates of 10°C/min-1. (313-335 J g-1), which suggests high denaturation
lP
A 2% protein dispersion adjusted to pH 7, then centrifuged at
Hojilla‐
10000g for 15 min. 10mL of the supernatant was heated to 90-
Evangelista,
100°C for 20 min and then cooled down. Then centrifuged at UFDF showed a higher thermal stability (58.8%) compared
na
Sessa &
10000g for 15 min. The supernatant is filtered in filter paper. Heat to AP (48.3%) lupin protein isolate
Mohamed
coagulability expressed as the percentage loss in solubility after
(2004).
heating.
ur
Thermal properties were measured using DSC scanned from 20 to Isoelectric precipitation at pH 4.5 caused structural changes
Jo
98°C at a rate of 0.2 °C min-1. Lupin protein sample weighed which includes a wide Mw distribution, heat treatment at 90 Kiosseoglou et
approximately 0.9 g of 12.5% w/w lupin protein/water °C shows formation of disulphide bonds which is important al. (1999)
suspensions in the DSC pans. in the formation of a three-dimensional structure.
Two endothermic peaks observed for protein denaturation in
DSC analysis: protein isolates were dissolved in distilled water at the control, but heat treatment for 15 min at 100°C or 5 min
Sirtori et al.
a concentration of 20% (w/v). or longer at 200 and 300°C eradicated only peak 1. Severer
(2010)
Heating was from 40-120°C at a heating rate of 2 K/min. thermal treatments (15–30 min. 200°C) caused a decrease in
native protein structure as no peaks observed.
 Day 1
Suspend 0.5kg of lupin flour in 3.5kg of water (12.5% w/w)

Stir the solution for 2 hours at ambient temperature

Adjust pH to 9 with NaOH

Store solution at 4°C overnight for hydration.

of
ro
Day 2
On the next day, stir the solution at ambient temperature to reintegrate the
suspension with the solution.
-p
re
Centrifuge the suspension at 3500g for 15 min
lP
na

Decant the supernatant.

ur

Adjust the pH of the supernatant to 4.0 while stirring for 30 minutes.

Jo

Centrifuge the supernatant at 3500g for 15 min

Discard the supernatant to obtain the pellet.

Freeze dry the pellet for 72 h at -40°C with a vacuum pressure of -12Pa.

Vacuum pack the lupin protein and stored at ambiant temperature for
analysis.

Figure 1. Schematic of lupin protein isolation by alkaline extraction followed by acid

precipitation performed at the RMIT Food Science Laboratory by the authors.
 A

of
B

ro
-p
re
lP
na

Figure 2. Isolated lupin protein paste before freeze drying (A) and isolated lupin protein after
freeze drying (B)
ur
Jo
Highlights

• Current methods of protein isolation from lupin was reviewed

• Isolation type and conditions affect isolate composition and protein level significantly
• Different isolation techniques produce isolates with different functional properties
• Weak techno-functionalities of lupin protein require further investigation and
innovation

of
ro
-p
re
lP
na
ur
Jo
Dear Editor In Chief,

Herewith all the authors confirm that there is no conflict of interest regarding the manuscript
submitted to Food Hydrocolloids for publication.

Lupin Protein: Isolation and Techno-Functional Properties, A Review

Billy Lo, Stefan Kasapis, Asgar Farahnaky*

Yours,

Asgar Farahnaky

On behalf of all authors

of
ro
-p
re
lP
na
ur
Jo