1 Sacha Inchi oil press-cake: Physicochemical characteristics, food-related applications and

2 biological activity

3 Erwin G. Torres Sáncheza,b, Blanca Hernández-Ledesmac, Luis-Felipe Gutiérreza,

a
4 Instituto de Ciencia y Tecnología de Alimentos, Universidad Nacional de Colombia, Carrera 30

5 No. 45-03 Edificio 500A, Bogotá D.C., 111321, Colombia.

b
6 Facultad de Ciencias Agrarias, Universidad Nacional de Colombia, Carrera 30 No. 45-03,

7 Bogotá D.C., 111321, Colombia.

c
8 Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM, CEI-

9 UAM+CSIC), Nicolás Cabrera 9, 28049, Madrid, Spain


Corresponding author. Phone: +57 1 3165000, Ext. 19227. Fax: Ext. 19206. E-mail:
lfgutierreza@unal.edu.co, https://orcid.org/0000-0001-9514-3888.

1
10 Abstract

11 Sacha Inchi (SI) (Plukenetia spp.) is an Amazonian-originary nut, used for centuries as food

12 for the native people of the Amazon rainforest that is attracting attention worldwide as a

13 functional food. Because of its high nutritional value, SI is considered as a “super food”. In

14 addition to its high content of polyunsaturated oil, SI is a good source of high-quality proteins of

15 high biological value, essential aminoacids, dietary fiber, minerals, tocopherols, phytosterols, and

16 phenolic compounds. This review summarizes the physicochemical characteristics, food-related

17 applications, and biological activity of the Sacha Inchi oil press-cake. A perspective of its

18 potential uses is provided, as a contribution to the growing increasing interest for in adding value

19 to this agro-industrial by-product. Additionally, this overview gives insights into the importance

20 of this alternative plant-based protein, which has promissory applications in the food,

21 pharmaceutical and cosmetic industries.

22 Keywords: Proteins, Sacha Inchi, functional foods, aminoacids, bioactive peptides.

2
23 1. Introduction

24 The world population growth represents a big challenge for the governments, because it needs

25 to “walk hand in hand” with various socio-economic and environmental aspects, to ensure the

26 welfare of future generations. One of the main concerns is related to guaranteeing relates to

27 guarantee the global food security to the 9 billion people expected by 2050 [1]. Consequently, the

28 search for high-nutritional foods of easy supply and reasonable cost, that meet environmental and

29 social aspects, has been a subject of an ever-growing interest for both food scientists and food

30 industries. In this context, the valorization of agro-industrial by-products has gained importance

31 in the last decades since it may allow the recovery of important nutritional compounds, while

32 contributing to environment protection and economic profit. Moreover, these by-products may be

33 a cheap and eco-friendly source of valuable compounds with promising applications in the food,

34 pharmaceutical and cosmetic industries [2].

35 Sacha Inchi (SI) (Plukenetia ssp.) is a plant of the Euphorbiaceae family with a great

36 expansion in Central and South America, and in some South East Asiatic and South Africa

37 countries [3]. Its fruits are shell-covered kernels used for centuries as food for the native people of

38 the Amazon rainforest. SI kernels are rich in oil (35-60%), proteins (25-30%), essential amino
[4]
39 acids, minerals, and vitamin E . They are mainly used for extracting their oil, rich in essential

40 fatty acids (α-linolenic acid ∼50%, and linoleic acid ∼35%) [5]. The SI oil industry has grown

41 significantly in most of the SI producer countries, because of the high demand of polyunsaturated

42 oils from the functional foods, cosmetic nutraceutical markets.

43 The oil extraction processes from oilseeds generate important amounts of by-products that

44 negatively affect the environment when they are , which if not well disposed or valorized,

45 negatively affecting the environment. In general, these by-products are important sources of

3
46 valuable compounds, such as fatty acids, amino acids, polyphenols, polysaccharides, ditary fiber,

47 and organic acids. Consequently, the interest on their valorization has grown significantly in the
[6]
48 last years . The main by-products of the SI oil processing are the shell and the press-cake

49 (SIOPC), which represent up to 70% of the raw seeds, and might reach up to 6000 ton/year in
[7]
50 Colombia . In the case of the shell, which represents 30-35% of the whole SI seeds, few data

51 about its valorization have been published [8]. Phenolic compounds with antioxidant activity were

52 recently obtained from the SI shell using a microwave-assisted extraction process with

53 hydroalcoholic mixtures of ethanol as solvent [7].

54 The SIOPC contains important amounts of bioactive compounds, such as proteins,

55 carbohydrates, minerals, and lipids, whose concentration varies, in addition to the intrinsic

56 composition of the kernels, with the oil extraction’ process efficiency. The SIOPC has been

57 generally used in animal feeding [9], as functional ingredient of various food products [10], and as

58 nonconventional source of proteins, protein isolates and hydrolysates [11].

59 With the aim of contributing to the knowledge of the agro-industrial by-products valorization,

60 and taking into account that finding feasible ways to add value to the SIOPC has great

61 importance for the SI agro-industry, this review summarizes the main physicochemical

62 characteristics of the SIOPC, its biological activities and current existing food-related

63 applications, and biological activity. A special emphasis on its proteins is given, because of their

64 importance as potential source protein concentrates and hydrolysates. This review will provide a

65 scientific basis for the development of sustainable SIOPC valorization, under a circular economy

66 approach.

67 2. Chemical composition of SIOPC

68 The chemical composition of SIOPC varies according to the own factors of the seeds-

69 associated factors (subspecies, quality, growing, geographical and climate conditions, harvesting

4
70 time, and storage environments, among others), as well as to other factors associated with the

71 extraction method and its efficiency [5b]. Cold and screw expeller pressing are the main methods

72 used for extracting oil from SI kernels at both industrial and artisanal levels. Screw pressing

73 generally gives higher oil extraction yields, but cold pressed oils are of higher quality because the
[12]
74 thermolabile compounds, such as tocopherols, are conserved in greater concentrations .

75 However, regardless the extraction method applied, the SIOPC is mainly composed by proteins,

76 lipids and carbohydrates, including dietary fiber, as observed in Table 1.

[13]
77 SIOPC contains proteins in the range of 32 to 62%. Ruiz, et al. reported higher protein

78 concentration in press cakes from Plukenetia volubilis than in those obtained from Plukenetia

79 huayllabambana (59 vs. 46%, respectively). In spite of these differences, the concentration of

80 proteins of the SIOPC is similar to that reported for soybean meal (42-50%) [14], which is the

81 most common protein source in feed formulations for different animal species, and has wide

82 applications in the food industry, including dietary supplements and functional foods.

83 Lipids are the most abundant component of SI kernels. After their extraction, 5-25% lipids

84 amounts between 5-25% may remain in the SIOPC, depending on the efficiency of the

85 extraction method applied. α-linolenic (∼50%) and linoleic (∼35%) acids are the main fatty acids

86 of the lipid fraction of the SIOPC, while minor amounts of oleic (∼9%), palmitic (∼5%) and

87 stearic (∼2%) acids have been reported [12-13]. This unique fatty profile makes SIOPC suitable for

88 enriching foods with essential fatty acids. In comparison to soybean mealsCompared to soybean

89 meals, the lipidic fraction of SIOPC is greater and richer in polyunsaturated fatty acids.

90 Total carbohydrates in SIOPC range from 4.97 to 26.80% [15]. Although these components

91 have been barely investigated, the total dietary fiber has been estimated in 25% [16], and values

92 for crude (3.0-17.3%), neutral (14.2-18.4%) and acid (5.9-15.0%) detergent fibers, including

5
 93 hemicellulose (3.0-4.2%) and lignin (1.0-1.0%), have been reported [15a, 15b, 15d, 17]. These

94 concentrations of fiber fractions are within the ranges recently reported for soybean meals [14].

95 SIOPC contains important amounts and variety of minerals (4.6 to 8.7%), especially

96 potassium, phosphorus, calcium, and magnesium. The ashes of SIOPC evaluated by Mondragón
[15]d
97 showed approximately 3180, 5600, and 42 ppm of calcium, phosphorus, and iron,
[12]
98 respectivey, while those investigated by Rawdkuen, et al. were rich in potassium (13935

99 ppm), phosphorus (13125 ppm), magnesium (8922 ppm), and calcium (7616 ppm). The

100 concentrations of calcium and phosphorus are similar to those reported for soybean meal, while

101 iron is present in a lesser amount in SIOPC [14].

102 Antinutritional factors such as tannis, saponins, alkaloids, flavonoids, triterpenes,

103 anthocyanidins, coumarins, quinones, α-amino groups, and mucilages, have been found in SIOPC

104 at low concentrations [12-13, 15a]. The astringent flavor of SIOPC could be due to the presence of

105 tannins, which are secondary metabolites that may bind and form complexes with various

106 molecules including proteins. These interactions may result in complexation, stabilization,

107 unfolding or precipitation of proteins, which has been associated with the reduction of their

108 digestibility, since the protease inhibitory effects on proteases reduce their digestion, and thus,
[18]
109 the utilization of valuable amino acids . Trypsin inhibitors are the most important group of

110 antinutritional factors present in raw beans, seeds and kernels. According to the available data,

111 the trypsin inhibitory activity of SIOPC is low in comparison to that shown by soybean meals [14,

112 16].

113 Natural antioxidants are of great interest because of their beneficial effects on human health,

114 thus being an alternative to the harmful synthetic antioxidants. The antioxidant activity of SIOPC

115 has been hardly investigated. Rawdkuen, et al. [12] reported values of 32% and 730 mol FeSO4/L

6
116 for the antioxidant capacity of SIOPC evaluated by the 2,2-diphenyl-1-picrilhidrazil (DPPH) and

117 ferric reducing antioxidant power (FRAP) assays, respectively. This antioxidant activity could be

118 related to the phenolic compunds, which content is ranged between 51 and 312 mg gallic acid

119 equivalents (GAE)/100 g [12, 16]. Further research should be required to determine if SIOPC or its

120 fractions may be a good source of antioxidant compounds.

121 The amino acid composition of SIOPC is presented in Table 2. SIOPC is rich in essential

122 amino acids (EEA), such as lysine, histidine, and leucine, and contains important amounts of

123 isoleucine, valine, tryptophan, and phenylalanine, and low concentrations of threonine and

124 methionine. This essential amino acid composition meets the recommendations of
[19]
125 FAO/WHO/ONU , except for lysine and leucine. SIOPC may be considered a good source of

126 well-balanced protein, comparable with soy protein in content of total EAA [4a, 20]. As far as the

127 non-essential amino acids (NEAA) are concerned, SIOPC contains high amounts of tyrosine,

128 glutamic acid, aspartic acid, and cysteine. The digestibility in vitro of SIOPC varied between 88.5

129 and 93.6%, being higher after its cooking at 70°C for 2 hours [21].

130 3. Technofunctional properties of SIOPC

131 Few studies have investigated the technofunctional properties of SIOPC and its fractions.
[15]a
132 According to Alcívar, et al. , the SIOPC flour presents low solubility and water adsorption

133 capacity (7.96% and 2.16 g/g, respectively). The water absorption index (3.2-4.4 g/g), water

134 solubility index (12.4-26.2%), and solubility (10.7-38.0 mg/g) of the SIOPC flour were

135 investigated by Jagersberger [16]. The albumin fraction extracted from SIOPC with saline solution

136 displayed notable protein solubility (63%), water-holding capacity (1.6 g/g), oil retention

137 capacity (1.7 g/g), and foaming (350%) and emulsifying (13.0 mL/g) abilityabilities. Heating at

138 temperatures lower than 100°C improves solubility, oil-holding capacity, foaming and
[16]
139 emulsifying capacities . The protein solubility solubility increased up to 63% as the pH

7
140 increased from 3.0 to 7.0, and then, significantly droppped (up to 18%) at with the pH increased

[22]
141 to 10.0. Addition of salt up to 2% significantly reduced the solubility of this protein fraction .
[15]c
142 Mercado, et al. reported maximal (99.4%) and minimal (18.7%) solubilities at pH values of

143 11.0 and 6.0, respectively, for proteins isolated from SIOPC. The oil absorption capacity (1.4

144 g/g), foaming capacity (55% at 1% concentration and pH 8.0), foam stability (33.7% at 1% of

145 concentration, pH 8.0 at 120 min), and emulsifying capacity (59.1%), were in general higher than

146 those obtained for soyprotein isolates. On the contrary, the water holding (1.8 g/g) and gelling

147 capacities (15%) were lower in comparison to soy protein isolates. In the study of Pezo Cuñaña

148 [23], protein isolates from SIOPC obtained by extraction with alkaline water (pH 12.0) presented

149 84.4% solubility, foam stability of 30% at pH 8.0, and emulsifying, water retention, oil

150 absorption, gelling, and foaming (pH 8.0) capacities of 53.3%, 4.7 g/g, 267.1%, 13%, and 49%,

151 respectively.

152 4. Toxicity

153 The consumption of plants parts (seeds, leaves, roots, and bark) may cause mild to severe

154 toxicity to humans by either direct or indirect ways. Alkaloids, lectins (carbohydrate-binding
[24]
155 proteins), and saponins are the main phytotoxins . Even if many plant lectins are toxic,

156 albumins isolated from SI seems to be no toxic to humans as roasted SI seeds has been consumed

157 long time ago by native people of the Amazon rainforest [25]. The concentration of phytotoxins in

158 fresh and roasted SI kernels and leaves has been investigated, as well as their cytotoxicity on

159 human peripheral blood mononuclear, embryonic kidney, and hepatic stellate cells, and mouse
[24]
160 fibroblasts, and their in vivo mutagenic properties . The presence of saponins (~27 mg/kg),

161 alkaloids (~485 mg/kg), and lectins (~0.22 ng/kg) in fresh SI kernels has been reported, but

162 roasting enables their reduction. SI kernels and leaves, especially after roasting, are considered to

8
163 be safe to for a variety of normal cell lines, and do not induce DNA mutations in Drosophila

164 expressing high biotransformation system [24]. However, heat processing must be applied before

165 their consumption to reduce phytotoxins and their potential health risks [24].

166 The acute, subchronic toxicity, and genotoxic potential of SIOPC was evaluated by oral

167 administration to rats and mice (50, 250, and 500 mg for 90 days), and its effect on the

168 appearance of toxic symptoms, hematological and biochemical parameters, organ weight and

169 histopathological examination, as well as the genotoxic potential evaluated by mouse

170 micronucleus assay. Data show neither mortality or morbidity in animals, changes in weight gain,

171 food consumption, or adverse events; nor significant changes in the evaluated hematological and

172 biochemical parameters, organ weight, histopathological findings, or micronuclei [26].

173 5. Applications of SIOPC as functional ingredient in foods

174 Developing foods with high protein content, dietary fiber and essential fatty acids is a trend

175 responding to current needings of population and specific groups of consumers such as vegans,

176 vegetarians, and people with dietary fibre intake deficiency, and weight management. Because of

177 its high nutritional value, SIOPC is suitable to enrich foods with proteins, fiber, and essential

178 fatty acids ω-3 and ω-6. In fact, various foods including SIOPC flour are elaborated at artisanal

179 level, and many others have been developed experimentally. de los Rios Carvajal [27] developed a

180 functional mango and pineapple beverage added supplemented with SIOPC flour. The obtained

181 beverage containing up to 25% protein was stable during 60 days of refrigerated storage. Granola

182 bars, extrudates, biscuits, noodles, and beverages with amounts of SIOPC and/or SIOPC flour

183 between 30 to 40% were developed by Jagersberger [16]. In the case of the extruded products, the

184 expansion index decreased and the hardness increased significantly as the concentration of

185 SIOPC flour increased. The spreading factor and breaking force of the biscuits significantly

186 increased with the addition of SIOPC. The cooking weight of the noodles decreased

9
187 as the SIOPC flour content increasedwith increasing the SIOPC flour content. Noodles with

188 10% SIOPC reached a cooking weight of about 266%, while those containing 30% SIOPC

189 reached a value of ca. 240%. According to this author, all these food products including SIOPC

190 as functional ingredient, displayed high sensory acceptance, except for the beverages, which need

191 more research to improve their taste and sensory quality.

192 One of the trends in baked goods consist on increasing the protein content and recucing

193 reducing the levels of saturated fats. SIOPC has been used as partial substitute of wheat flour

194 during the elaboration of bread leaf. The incorporation of SIOPC in the formulations significantly

195 increased the concentrations of minerals, fiber, polyunsaturated lipids, and protein, while

196 decreased the content of carbohydrates. The best rheological properties of farinography,

197 amylography and extensography were obtained with the addition of SIOPC at 6.3%, reaching

198 concentrations of protein, fiber, and α-linolenic acid of 10.3, 3.8 and 3.3%, respectively.

199 Moreover, the sensory attributes of color, appearance, aroma, flavor, and texture of breads

200 elaborated with this formulation were similar to those of the control samples [10]. Addition of

201 SIOPC at higher levels (up to 25%) negatively affects the technological properties of the flour

202 blends, batters, and breads, such as water absorption, extensibility, and texture. In the elaboration

203 of sweet bread, the partial substitution (up to 10%) of wheat flour by SIOPC resulted in good
[28]
204 sensory acceptability . SIOPC was successfully used for replacing 50% of wheat flour in the

205 formulation of cookies. The obtained products had two-fold protein content than the cookies
[29]
206 made only with wheat flour (18.3 vs. 8.9%) . The same flour from SIOPC was employed to

207 elaborate a porridge with less of 50% corn starch, 2.3% calories, and 16.3% carbohydrates, 1.4%

208 and 90.7% more fat and protein, respectively, and similar sensory characteristics in comparison

209 to the control sample [30].

10
210 Protein concentrates from SIOPC have been used as functional ingredients in various types of

211 foods. When used in pork sausages and fish fingers, tThe addition of these protein concentrates

212 improves the textural properties of the sausages, and increased the shelf life of the fish fingers [3a].

213 Moreover, SIOPC protiens has been used to prepare high quality plant-based non-soy protein

214 blends, with pea, rice, and potato proteins in effective amounts to promote satiety, maintain a

215 healthy body weight, and lean body mass. The Protein Digestibility Corrected Amino Acid Score

216 (PDCAAS) of these blends was higher than 0.95 [31].

217 6. Less conventional food-related application of SIOPC

218 SIOPC was found to be an excellent fermentation substrate for the growth of Weissella

219 cibaria, a lactic acid bacterium surviving in low pH environments, and displaying antimicrobial

220 activity against various pathogenic microorganisms such as Xanthomonas albilineans,

221 Staphylococcus aureus, and Streptococcus agalactiae, and inhibitory effect against Gram

222 negative microorganisms such as Escherichia coli and Klebsiella pneumonia [17].

223 Proteins extracted from the SIOPC under alkaline conditions (pH 12.0) were used to produce

224 biodegradable edible films as an alternative to reduce the use of residual non-biodegradable

225 solids and plastics. A combination of SIOPC proteins (8.2-9.3%) and sorbitol (1.4-1.8%) led to

226 obtain films with low solubility and water vapor permeability [32].

227 7. SIOPC as alternative source of proteins and bioactive peptides

228 Proteins are essential macronutrients because their EEA content[33]. The high content of

229 proteins and their richness in EEA, gives SIOPC a wide potential as alternative source of high

230 quality-proteins and bioactive peptides. However, Up to date, few works have investigated the

231 SIOPC proteins and their possible derived peptides. The first study related to the isolation,

232 purification, and characterization of water soluble proteins from SI (Plukenetia volubilis L.) was

233 published in 2002 by [34]. These authors found that the water-soluble proteins of SIOPC are basic

11
234 3S proteins (pI 9.4), composed by two glycosylated polypeptides (4.8% sugars) with estimated

235 molecular weights (MW) of 32.8 and 34.8 kDa. These findings were confirmed later by Moser, et
[35]
236 al. , who extracted proteins from SIOPC with distilled water at pH 7.0, obtaining dimers

237 constituted by two sub-units of about 30 kDa linked by disulfide bonds. Albumins may represent

238 25% (w/w) of the SIOPC, and its purity can be higher than 90% [34]. This fraction comprises two

239 major polypeptides, one of them with MW in the range of 25-35 kDa, and the other with MW

240 between 10-15 kDa. The secondary structures of these albumins are mainly composed of β-sheet

241 (37.2%), α-helix (24.6%), and β-turns (38.2%) [22]. The albumin fraction of SIOPC contains high

242 amounts of tryptophan residues (44 mg/g protein), and EEA (47.61%) in adequate quantities, thus

243 being a complete protein with respect to the amino acid requirements of adult people. It also

244 contains high levels of cysteine, tyrosine, threonine, and tryptophan, and low amounts of

245 phenylalanine. Moreover, it is easily digested by tosyl-L-lysyl-chloromethane hydrochloride

246 (TLCK)-chymotrypsin, N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-trypsin, and

247 pepsin upon heat denaturation [34].

248 Fractionation by the Osborne procedure resulted in the solubilization of over 90% of the

249 SIOPC proteins, composed by albumins (43.7%), globulins (27.3%), prolamins (3.0%), and
[25]
250 glutelins (31.9 %) . Althugh polypeptides in the MW range of 6-70 kDa have been found,

251 these soluble proteins are principally composed of two molecular species with MW of 32-35 kDa

252 and 60-62 kDa. The 60-62 kDa polypeptide consisted in a disulfide bond linked dimer of 32-35

253 kDa and a 60-62 kDa monomeric polypeptide, while the 32-35 kDa polypeptide was glycosylated
[12, 25]
254 . Albumins, globulins, and glutelins from SIOPC are mainly composed by polypeptides in

255 the 30-40 kDa range. However, globulins contain polypeptides slightly more basic than those

256 found in glutelins. Prolamins from SIOPC are essentially constituted by four polypeptides in the

12
257 range of 6-12 kDa [25]. Fractionation of proteins from defatted SIOPC of the Plukenetia volubilis

258 L. and Plukenetia huayllabambana species yielded respectively, globulins (39.8 vs. 21.0%),
[36]
259 albumins (37.1 vs. 34.1%), prolamins (13.9 vs. 30.1%) and glutelins (9.2 vs. 14.9%) . In both

260 species, albumins were polypeptides with MW in the range of 30-33 kDa. The globulin fractions

261 presented five polypeptides with MW of 22, 23, 31, 33, and 36 kDa, while prolamins were

262 composed by four polypetides with MW of 22-23, 23-24, 33, and 36 kDA. The glutelins were

263 constituted by five polypeptides with MW of 22-23, 23-24, 31-32, 33, and 36 kDa. Other studies

264 indicate that the major components of proteins from SIOPC are fractions with MW of 14, 30, 60,
[37] [12] [11b]
265 and 97 kDa ; 11, 17, 35, and 63 kDa ; and 15, 18, 31 and 57 kDa . According to

266 Quinteros, et al. [37], the soluble SIOPC proteins are characterized by polypeptides in the range of

267 16-70 kDa. The fractions of 45 and 73 kDa correspond to 11S globulins and 7S globulins,

268 respectively, while the fraction of 16 kDa may be assigned to 2S albumins. Moreover, SI protein

269 isolates contain a high content of globulins and proteins with isoforms.

270 In a recent study, the characterization of SI storage proteins (Plukenetia volubilis L.) from ten
[38]
271 different Peruvian localities, and the comparison of their proteomic profile was carried out .

272 The proteins were mainly composed of albumins and globulins (72.6%), glutelins (26.0%), and

273 prolamins (1.5%), which displayed MW in the range of 8-75 kDa. However, four major and

274 relatively abundant bands were detected at 20.1, 30.8, 32.2 and 36.4 kDa. Albumins and

275 globulins presented bands with MW between 22 and 36 kDa, and several bands in the region of

276 8-19 kDa, while the glutelin fraction displayed many bands between 22-36 kDa, and one band at

277 70 kDa. In agreement with previous studies, prolamins were characterized as four protein bands

278 with MW ranged from 22 to 36 kDa. Differences in the abundance of proteins between SI

279 populations were detected, and three proteins (Oleosin 2, Oleosin 3 and elongation factor 1-

280 alpha) were identified by means of nano-liquid chromatography/tandem mass spectrometry

13
281 (nLC-MS/MS). Moreover, peptides with a sequence identical to 56 proteins from the

282 Euphorbiaceae family were detected.

283 Although 0.1 M NaOH is effective for extracting proteins from SIOPC, the alkaline conditions

284 might affect some of the EEA, thereby decreasing the nutritive value of the solubilized proteins.

285 Thus, 2 M NaCl is recommended to solubilize the SIOPC proteins in the pH range of 9.0-12.0 at

286 room temperature [25]. The extracted proteins displayed the lowest solubility at pH 4.0, while the

287 highest solubility was observed at pH 11.0. This pH-dependence of protein solubility is related to

288 the dominance of acidic amino acids in the SIOPC proteins [25]. A protein isolate (92.15% protein
[15]c
289 content) from SIOPC was obtained by Mercado, et al. by alkaline extraction and isoelectric

290 precipitation under the following conditions: Solid to solvent ratio 1:30 (w/v), pH 11.0, 50°C, and

[37]
291 60 min. Quinteros, et al. reported similar extraction yields (21%) and protein content (75%)

292 in the extraction of proteins from SIOPC with water and saline solution (1 M). Alkaline

293 extraction (pH 12.0, solid to solvent ratio of 1:10, w/v) during 30 min produced a concentrate
[23] [11]a
294 with 84% protein content . Chirinos, et al. optimized the protein extraction parameters

295 from SIOPC using alkaline and enzyme-assisted extractions with Alcalase 2.4L. The enzyme-

296 assisted method resulted in a higher protein recovery than that reached with the alkaline protein

297 extraction (44.7 vs. 29.7%, respectively). The optimal alkaline extractions were found at 54.2°C,

298 solvent to solid ratio of 42:1 (v/w) ratio, NaCl concentration of 1.65 M, pH 9.5, and 30 min,

299 while for the enzyme-assisted method the optimized conditions were 50°C, solid to solvent ratio

300 of 1:50 (w/v), enzyme concentration of 5.6%, pH 9.0, and 40.4 min.

301 7.1. Preparation of hydrolysates hydrolyzates from SIOPC proteins

302 In recent years, bioactive peptides have received increased interest for pharmaceutical, food,

303 and cosmetic applications. Bioactive peptides are sequences of amino acids, inactive within the

14
304 source protein but once released, they may exert multiple biological activities. Their high

305 potency, specificity, and safety make them new and promising therapies that may fill the gap

306 between small molecules and protein drugs. Peptides possess favorable tissue penetration and the

307 capability to participate in specific and high-affinity interactions with endogenous receptors. In

308 addition, among bioactive peptides, those released from food proteins have acquired importance

309 as active components in functional foods and nutraceuticals because they are known to possess

310 regulatory functions that can lead to health benefits (Martínez-Villaluenga and Hernández-

311 Ledesma, 2020).

312 Hydrolysates Hydrolyzates from SIOPC proteins have been obtained using Alcalase 2.4L, a Comentado [DyP1]: Sería preciso revisar y poner en ingles
Americano o británico, pero no los dos

313 mixture of proteases from papaya, yielding protein content between 50 and 66%. These

314 hydrolysates stimulate the metabolism of skin cells and rejuvenate the aged skin [35]. In a recent

315 study, SIOPC was used to produce protein hydrolysates by using crude papain (pH 7.0 at 40°C)

316 and Calotropis proteases (pH 8.0 at 60°C), until reaching hydrolysis degrees of 2.7 and 11.2%,
[11b]
317 respectively . The papain hydrolysate contained higher amounts of EEA (47.2%) than that

318 obtained using Calotropis proteases (40.8%). Lysine (11.0%), leucine (7.6%), phenylalanine

319 (7.3%), and glutamic acid (6.9%) were the predominant amino acids in the papain hydrolysates,

320 while lysine (10.9%), glutamic acid (7.2%), phenylalanine (6.1%), and tyrosine (5.7%) were the

321 main amino acids of the hydrolysates obtained using Calotropis proteases. Moreover, the latter

322 hydrolysates contained lower amount of hydrophobic amino acids. Peptides with MW between

323 <8 and 57 kDa were obtained. The total soluble peptides in the papain hydrolysate and Calotropis

324 hydrolysates were 963.87 and 1022.94 μmoL/g, respectively.

325 7.2. Biological activity of proteins and peptides from SIOPC

326 Food proteins have demonstrated to exert regulatory functions and health-promoting activities
[39]
327 . Plant-based proteins have gained popularity, because of the continuous growing of the vegan

15
328 and vegetarian markets, and the interest in developing more eco-efficient food systems [40]. The

329 intake of plant-based proteins and their derived peptides has been correlated to the reduction of
[41]
330 both risk and mortality of many chronic diseases, such as cancer . Moreover, they have been

331 demonstrated to exert beneficial effects on the bone, cardiovascular, endocrine, gastrointestinal,

332 immune, and neurological systems [39, 42].

333 Few studies have evaluated the biological activity of proteins and peptides from SIOPC. The

334 antioxidant activity (determined by means of DPPH and FRAP assays) of protein hydrolysates

335 obtained with papain and Calotropis proteases, increased as a function of the hydrolysis degree.

336 The hydrolysate obtained with Calotropis proteases displayed higher antioxidant activity than

337 that the obtained with papain (DPPH: 27.46 vs. 23.15 μmoL Trolox equivalents/g; FRAP: 75.54

338 vs. 26.44 μmoL FeSO4/g). Con formato: Subíndice

339 The albumin fraction of SIOPC exhibited immunomodulatory activity by stimulating the

340 proliferation of splenic lymphocytes (at concentrations of 5-320 μg/mL), increasing the tumor

341 necrosis factor (TNF)-α secretion, enhancing the cellular lysosomal enzyme and pinocytic

342 activities, and by moderately promoting the production of nitric oxide (NO) and H2O2 of

343 RAW264.7 cells. Treatments with albumins from SIOPC at concentration of 320 μg/mL,

344 significantly reduced the RAW264.7 cells viability. Moreover, it has been stablished that this

345 albumin fraction increases lysosomal phosphatase activity, and has excellent anti-inflammatory

346 effects, and could can decrease intracellular reactive oxygen species (ROS) levels, and prevent

347 the lipopolysaccharide (LPS)-induced activation of RAW264.7 cells, thus reducing cellular stress

348 and cellular dysfunction [22].

349 The digestibility and anti-inflamatory activity in vitro of the proteins isolated from SIOPC
[43]
350 were investigated by Quinteros, et al. . The SI protein isolate was resistant to the hydrolysis

351 with pepsin at low enzyme concentrations. The 7S, 11S, and 2S globulins extracted from SIOPC

16
352 with water at a pH of 8.0 and precipitated at a pH value of 4.0, were resistant to gastric and

353 duodenal digestion. However, 11S globulins were resistant to proteolytic digestion with pepsin

354 and mixtures of pepsin and pancreatin. These isolate proteins showed significant inhibition of egg

355 albumin denaturation in a dose-dependent form, in the concentration range of 100-1000 μg/mL.

356 Moreover, the SI protein isolate at pH 4.0 (1000 μg/mL) presented an in vitro anti-inflammatory Con formato: Fuente: Cursiva

357 activity in vitro of (78.13%), evaluated with the protein denaturation method, which was

358 comparable to the the positive control (sodium diclofenac).

359 8. Concluding remarks and future perspectives

360 Proteins recovered from agroindustrial by-products can be reused as ingredient for fortified

361 foodsand dietary supplements, as techno-functional constituent due to their gelling, emulsifying,

362 foaming, and water and oil binding properties, as biopolymer material, and as source of bioactive

363 peptides [1]. SIOPC is a valuable source of important compounds with promising applications as

364 functional ingredients in the food, pharmaceutical and cosmetic industries. Moreover, under a

365 circular economy approach, new valorization alternativas for this by-product obtained during the

366 SI oil processing are required. Because of its high content of high-and quality protein content,

367 SIOPC might be an important source of bioactive peptides. The positive attributes of bioactive

368 peptides from plant-based proteins have driven research in evaluating technological process for

369 obtaining these compounds, determining their biological activity and stablishing the mechanisms

370 for their use as natural alternatives in the prevention/management of chronic diseases. However,

371 to datadate, the available evidence on the health benefits attributed to peptides released from

372 SIOPC proteins is still limited. This makes, making need for further studies, including cell

373 culture, animal assays, and human trials needed to confirm that allow confirming their properties,

374 emphasizing on their mechanisms of action, bioavailability, and interactions with other food

375 constituents. The development of new SI-based functional foods and its integration in modern

17
376 diets should be also of great value to enhance public nutrition and health using traditional

377 agricultural resources more efficiently. Comentado [DyP2]: Faltarían los agradecimientos que se me
pasó decirte de ponerlos la última vez.

378

18
379 Declaration of Competing Interest

380 The authors declare that they have no known competing financial interests or personal

381 relationships that could have appeared to influence the work reported in this paper.

382 References
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550 Pharmaceutical and Clinical Research 2016, 9 (3), 20-23. Comentado [DyP3]: He revisado la bibliografía en cuanto a los
nombres en cursiva pero el tema es que las primeras letras de las
palabras a veces están en mayúsculas y en otras en minusculas, y
habría que ponerlo todo igual, pero como está hecho con el
Endnote, si quieres, lo reviso, como me digas.

26