Isolation and characterization of nettle (Urtica dioica L.) seed proteins: Conversion of underutilized by-products of the edible oil industry into food emulsifiers

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Food Chemistry 456 (2024) 139878

Available online 3 June 2024

Isolation and characterization of nettle (Urtica dioica L.) seed proteins:

Conversion of underutilized by-products of the edible oil industry into

food emulsiers

Zeynep Aksoylu ¨

Ozbek

, Kentaro Kawata

, Hualu Zhou

, Cheryl Chung

, Jay Hoon Park

David Julian McClements

Department of Food Engineering, Manisa Celal Bayar University, Yunusemre, Manisa 45140, Turkiye

Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA

Department of Food Science and Technology, College of Agricultural and Environmental Sciences, University of Georgia, Grifn, GA 30223, USA

Department of Plastics Engineering, University of Massachusetts, Lowell, MA 01854, USA

Department of Food Science & Bioengineering, Zhejiang Gongshang University, 18 Xuezheng Street, Hangzhou, Zhejiang 310018, China

ARTICLE INFO

Keywords:

Alternative protein

Circular economy

Emulsion stability

Sustainability

ABSTRACT

This study aimed to upcycle a byproduct of the edible oil industry, cold-pressed nettle seed meal (CPNSM), into a

plant-based emulsier, thereby increasing the sustainability of the food system. The protein content of the nettle

seed protein (NSP) powder was 48.3% with glutamic acid (16.6%), asparagine (10.7%), and arginine (9.7%)

being the major amino acids. NSPs had a denaturation temperature of 66.6 ◦C and an isoelectric point of pH 4.3.

They could be used as emulsiers to form highly viscous coarse corn oil-in-water emulsions (10% oil, 4% NSP).

Nevertheless, 10-fold diluted emulsions exhibited rapid creaming under different pH (2–9), salt (0–500 mM

NaCl) and temperature (>40 ◦C) conditions, but they were relatively stable to aggregation. Our ndings suggest

that NSPs could be used as emulsiers in highly viscous or gelled foods, like dressings, sauces, egg, cheese, or

meat analogs.

1. Introduction

There is increasing interest in adopting a more plant-focused diet for

health, environmental, sustainability, and ethical reasons (World Health

Organization, 2024). Plant-based diets may reduce the risk of malnu-

trition by providing high levels of proteins, bers, minerals, vitamins,

and phytochemicals, while reducing the intake of saturated fats and

cholesterol (Ahnen, Jonnalagadda, & Slavin, 2019; Suri & Ray, 2023). In

contrast, consumption of animal-sourced foods have been linked to a

higher risk of all-cause mortality, type 2 diabetes, cardiovascular dis-

eases, overweight, and obesity (Medawar et al., 2020; Neuenschwander

et al., 2023). In terms of environmental impact, 38% of global green-

house gas emissions (GHGs) have been linked to the agriculture and food

system in 2021, with non-dairy (<40%) and dairy cattle (<18%) being

the dominant contributors (FAOSTAT, 2023). Increases in GHGs pro-

mote global warming and climate change, thereby resulting in decreased

agricultural output (Yoro & Daramola, 2020). The current global food

system lacks the ability to adequately respond to sudden impacts or to

fulll long-term demands for future generations (Fanzo et al., 2021). All

these concerns are causing policymakers and researchers to promote the

development of a more healthy, sustainable, environmentally friendly,

and affordable food system. In this context, there has been interest in

creating plant-based analogs of animal-sourced foods because their

production leads to less GHGs, pollution, and biodiversity loss (Scar-

borough et al., 2023). However, the retail market for plant-based foods

in the United States ($8.1 billion in 2023) is still much lower than that of

animal-based foods ($244.4 billion in 2023) (Good Food Institute, 2023;

USDA, 2024). A variety of next-generation plant-based food products

has been developed to meet consumer demand, including dairy, egg,

seafood, and meat analogs. The majority of consumers in developed

countries are omnivores, rather than vegetarians or vegans. Conse-

quently, the food industry is designing plant-based foods that match the

look, feel, taste, and performance of the animal-sourced products they

are designed to replace (McClements & Grossmann, 2021).

Meat analogs are currently one of the most widely consumed plant-

based foods. A wide range of plant proteins are available to assemble

* Corresponding author at: Department of Food Engineering, Manisa Celal Bayar University, Yunusemre, Manisa 45140, Turkiye.

E-mail address: zeynep.aksoylu@cbu.edu.tr (Z. Aksoylu ¨

Ozbek).

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier.com/locate/foodchem

https://doi.org/10.1016/j.foodchem.2024.139878

Received 6 March 2024; Received in revised form 10 May 2024; Accepted 27 May 2024

Food Chemistry 456 (2024) 139878

meat analogs, including those isolated from soy, pea, wheat, rice, pea-

nut, lentil, and faba bean (Sun, Fu, Chang, Li, & Fang, 2022; Wittek,

Ellwanger, Karbstein, & Emin, 2021; Yuliarti, Kiat Kovis, & Yi, 2021;

Zahari, ¨

Ostbring, Purhagen, & Rayner, 2022). These proteins can pro-

vide desirable structures and textures because of their gelling, emulsi-

fying, uid-holding, and binding properties. There is great interest in

identifying alternative sources of plants proteins that can be used for this

purpose, especially those isolated from waste streams of agricultural or

food processing operations (Aimutis, 2022). Indeed, the conversion of

waste products into value added ingredients improves the sustainability

and protability of the food industry, as well as reducing negative im-

pacts on the environment. Various waste products can be used as a

natural source of food ingredients, as well as for the production of bio-

fuels, ethanol, essential oils, biodegradable polymers, and plant fertil-

izers (Tahir, Khan, Ashraf, Rdn, & Mubarik, 2023; Waheed et al., 2020).

Thus, waste product utilization is an important element in creating a

more circular economy.

Oilseed meals are by-products of the oil processing industry that are

rich sources of protein (15–50%) (S´

a, da Silva, Pacheco, Moreno, &

Carcio, 2021). Large amounts of press cakes and residues of oil

extraction are available for utilization as global oilseed production is

forecast to reach around 661 million tons in the 2023/2024 season

(USDA, 2023). Indeed, the global production of oil crop meals and cakes

was forecast to be around 164 million tons in the 2022/2023 season

(FAO, 2023). Cold pressed oils are produced by mechanical pressing of

oilseeds without heat treatment or solvent extraction. As a result, the

native structure of the proteins may not be altered, which may improve

their functional attributes. By-products of the edible oil industry are

commonly processed into animal feed due to their relatively high pro-

tein contents and nutritional value (ˇ

Colovi´

c, Rakita, Banjac, Đuragi´

c, &

Cabarkapa, 2019). However, these by-products may also be promising

plant protein sources for utilization within the food industry.

Nettle (Urtica dioica) is an annual wild plant that is cultivated in

warm regions of Europe, North America, Asia, and North Africa (Grauso,

de Falco, Lanzotti, & Motti, 2020). Its fresh leaves can be consumed in

salads or cooked dishes, while its dried leaves can be used in teas and

spices (Engelhardt, P¨

ohnl, & Neugart, 2022). The stems, roots, and

leaves of nettles can also be used in textile, pharmaceutical, and

cosmetic products (Viotti et al., 2022). Furthermore, nettle seeds, one of

the raw materials used in the cold-pressed oil industry, are rich in oil

(>30%) and protein (>20%) (Jafari, Samani, & Jafari, 2020). During

cold-pressing, 100 kg of nettle seeds typically yields around 22.5 and

77.5 kg of cold-pressed oil and meal, respectively. Recently, researchers

have focused on producing nettle seed gum, which can be used as a

functional or nutritional ingredient in foods (Kutlu, Bozkurt, & Tornuk,

2020; Zamani & Razavi, 2021). However, there has been little research

on the isolation and characterization of nettle seed proteins. Therefore,

the aim of this research was to obtain nettle seed protein (NSP) powder

from cold-pressed nettle seed meal (CPNSM) using an isoelectric-

precipitation technique and then to characterize its composition, phys-

icochemical properties, and functionality. In particular, we evaluated

the ability of nettle seed proteins to form and stabilize oil-in-water

emulsions (O/W), as there is a growing need for plant-based emulsi-

ers in the food industry.

The results of this research may lead to a new source of plant-derived

protein ingredients that can be used as functional and nutritional in-

gredients by the food industry. Moreover, they may improve the sus-

tainability and economic viability of the food production system by

converting waste products into value added ingredients.

2. Materials and methods

2.1. Raw materials, chemicals, and reagents

CPNSM was kindly donated by Zade Vital Pharma Chemicals & Food

Inc. (Konya, Turkiye). Corn oil was purchased from a local supermarket

(Mazola, ACH Food Companies Inc., Chicago, IL, USA). Analytical grade

sodium hydroxide, n-hexane, sodium azide (NaN

), Nile red (for mi-

croscopy), and uorescein isothiocyanate isomer I (≥90%, HPLC) were

purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Hy-

drochloric acid (37%), glacial acetic acid, potassium phosphate mono-

basic anhydrous (KH

), potassium phosphate dibasic anhydrous

HPO

), sodium dihydrogen phosphate monohydrate (NaH

O), disodium hydrogen phosphate anhydrous (Na

HPO

), sodium

chloride (NaCl), and dimethyl sulfoxide (DMSO) anhydrous were pur-

chased from Fisher Scientic Inc. (Pittsburgh, PA, USA). The 4 ×

Laemmli Sample Buffer, β-mercaptoethanol (≥98%, 14.2 M), precast

protein gel (4–20%, Mini-PROTEAN TGX, 12-well, 20

L), Coomassie

Brilliant Blue R-250 protein stain powder, and Quick Start Bovine Serum

Albumin (BSA) standard set (0.125–2 mg/mL) were purchased from Bio-

Rad Laboratories Inc. (Hercules, CA, USA). PageRuler broad range

(5–250 kDa) unstained protein ladder and HPLC-grade ultrapure water

were purchased from Thermo Fisher Scientic Inc. (Rockford, IL, USA),

and Alfa Aesar (Ward Hill, MA, USA), respectively.

2.2. Extraction of NSP

Firstly, CPNSM was ground into powder using a coffee grinder

(COSORI Pulse 2-in-1, Arovast Cooperation, Anaheim, CA, USA). Then,

the proteins were isolated using an isoelectric-precipitation technique.

Initially, the proteins were extracted under mildly basic conditions to

prevent conformational changes in the protein structure, and then they

were precipitated at their isoelectric point (IEP), as described previously

(Liu, Chen, Wang, & Wang, 2013; Tang, Ying, & Shi, 2021). Briey, the

oilcake powder was mixed with double distilled water (1:20, w/v), and

the pH of the suspension was adjusted to pH 9. The suspension was then

stirred using a magnetic stirrer (RO 10P, IKA Works GmbH & Co. KG,

Staufen, Germany) at 400 rpm for 1 h at room temperature, followed by

centrifugation (Sorvall Lynx 4000, Thermo Scientic Inc., Waltham,

MA, USA) at 9000g for 45 min at 4 ◦C. The supernatant was transferred

into a beaker and the mixture was adjusted to pH 4 while continuously

stirring at 200 rpm for 30 min. Next, the suspension was stored at 4 ◦C

for 2 h to allow sufcient time for precipitation. The mixture was then

centrifuged at 10000g for 40 min at 4 ◦C. The precipitates were then

transferred into a beaker and the nal pH was adjusted to 7. The pH

values used for solubilization and precipitation of the NSPs were

determined according to the solubility and surface charge versus pH

diagrams (see Figs. 2a-b). The precipitates were then frozen overnight

followed by freeze-drying (VirTis Genesis 25 L Pilot Lyophilizer, SP

Scientic, Gardiner, NY, USA). The dried pellets were then ground into a

powder. In the remainder of this study, we refer to the ingredient ob-

tained from this process as “NSP”. However, it should be noted that it

actually contains other components, such as carbohydrates, minerals,

and lipids that can affect its functionality.

2.3. Characterization of NSPs

2.3.1. Proximate composition of cold-pressed CPNSM and NSP powder

The moisture (Method 934.01), crude fat (Method 920.39), and ash

(Method 942.05) contents of CPNSM and NSP powders were determined

gravimetrically according to ofcial methods of the Association of

Ofcial Analytical Chemists (AOAC, 2019). The protein contents of the

samples were determined using the Dumas combustion method (Ele-

mentar Rapid N-Exceed, Hanu, Germany) using a nitrogen-to-protein

conversion factor of 5.60 (Mariotti, Tom´

e, & Mirand, 2008). The total

carbohydrate contents (dry weight basis) of the samples were calculated

by subtracting crude fat, protein, and ash contents from a total of 100.

2.3.2. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-

PAGE) analysis

The molecular weight distribution of NSP polypeptides were deter-

mined using SDS-PAGE according to a method described previously

Z. Aksoylu ¨

Ozbek et al.

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Isolation and characterization of nettle (Urtica dioica L.) seed proteins: Conversion of underutilized by-products of the edible oil industry into food emulsifiers

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