Ultrasound-assisted three phase partitioning for simultaneous extraction of oil, protein and polysaccharide from pumpkin seeds
Introduction
Pumpkin (Cucurbita moschata) is a common herb in the Cucurbitaceae family and is widely grown in Asia, Europe and South America. With the increasing amount of pumpkin cultivation, the production of its by-product, pumpkin seeds, is also gradually increasing, and the effective utilization of pumpkin seeds is receiving more attention.
Pumpkin seeds are rich in oils, proteins, phytosterols, vitamins and polysaccharides, which have positive effects in lowering cholesterol, preventing prostate diseases, anticancer and anti-inflammatory properties and improving immunity (Rabrenovic, Dimic, Novakovic, ; Ferreira, Barin, Binello, Veselov, & Cravotto, 2019) and have gained attention and importance in the field of medicine and in the food, chemical and feed industries. Pumpkin seeds contain a large amount of oil that is rich in unsaturated fatty acids and bioactive substances, such as α–tocopherol, γ–tocopherol, phytosterols, β–carotene, squalene and other substances (Can-Cauich, Sauri-Duch, Moo-Huchin, Betancur-Ancona, & Cuevas-Glory, 2019). Pumpkin seed oil has excellent health benefits and is now recognized as a high-quality medicinal and edible vegetable oil (Stevenson et al., 2007). Pumpkin seed meal (a by-product of pumpkin seed oil extraction) is rich in protein and is a natural protein material (Rezig et al., 2013). Studies have shown that the proteins in pumpkin seeds have physiological functions, such as inhibiting bacterial growth, suppressing inflammation, and lowering blood sugar (Fahim, Fattah, Agha, & GAD, 1995). Polysaccharides extracted from pumpkin pulp have the ability to increase serum insulin levels, lower blood glucose levels and improve glucose tolerance and can be used as a novel anti-insulin agents (Wu, Zhu, Diao, & Wang, 2014). Polysaccharides in pumpkin seeds presented certain scavenging activity towards DPPH·, HO·, and O2−·(Wang et al., 2016).
The TPP technique is an environmentally friendly, rapid and efficient extraction method with a wide range of industrial applications and is commonly used in the food industry for the separation of bioactive ingredients. In recent years, the TPP method has been widely utilized to extract and purify enzymes, lipids, proteins, polysaccharides and other substances from natural crops (Yan, Wang, Qiu, Ma, et al., 2018). The principle of TPP is to mix the crude extracts or suspensions with solid salt and organic solvent to form three different phases simultaneously. The upper phase is the t-butanol layer, which mainly contains lipids, pigments and other nonpolar compounds. The middle precipitate phase is the protein layer, and the lower aqueous phase is the water layer, which contains some polar compounds, such as polysaccharides (Li, Jiang, Li, Zhang, & Tan, 2013). In the TPP system, the commonly used inorganic salt is ammonium sulfate and the organic solvent is t-butanol. (Tan et al., 2015; Ketnawa, Benjakul, Martínez-Alvarez, & Rawdkuen, 2014). Ammonium sulfate has been widely applied in TPP systems for its ability to significantly promote the association and crowding effects of water molecules. In addition, t-butanol had a higher boiling point and a lower flammability and was more securely than hexane, methanol or ethanol, which are used in traditional extraction methods (Pike & Dennison, 1989). In addition, both (NH4)2SO4 and t-butanol in the system could be recovered, and it was suitable for the separation and purification of temperature-sensitive bioactive molecules because of its simple operation, high efficiency, continuous operation and mild conditions (Li et al., 2013; Saxena, Iyer, & Ananthanarayan, 2007).
In recent years, the application of ultrasonic technology to the extraction of biological macromolecules has become an emerging research direction. The acoustic effect generated by the ultrasonic treatment produces cavitation bubbles, and the bursting of the bubbles will form a powerful shock wave and a strong mechanical shear force on the surrounding liquid. The cavitation phenomenon can cause local energy density changes and strong mechanical shear, thus accelerating the mass transfer between the solvent and solute (Gogate & Kabadi, 2009). The acoustic effect generated by ultrasound can significantly shorten the extraction time and increase the extraction rate, thus improving the production efficiency. Researchers have used ultrasonic-assisted three-phase extraction techniques to extract oils, bioenzymes, polysaccharides, mangiferin, curcuminoids, and other substances.
Although there has been much research work on the application of UTPP technology, there have been few studies on the simultaneous extraction of oils, proteins and polysaccharides using UTPP. Consequently, the objective of the present research was to investigate the effect of the simultaneous extraction of PSO, PSP and PSPS by the UTPP technique. The effects of (NH4)2SO4 addition, t-butanol to slurry ratio, pH, ultrasonic power, irradiation time and duty cycle on the extraction yields of PSO, PSP and PSPS were investigated. The fatty acid profiles and basic physicochemical parameters of PSO extracted by UTPP and TPP were also compared and analysed.
Section snippets
Materials
Pumpkin seeds were purchased in Harbin, Heilongjiang Province, China. Clean pumpkin seeds were placed in a vacuum dryer (DZF-6250, Shanghai Youyi Instrument Co., Ltd, Shanghai, China) and dried for 24 h to reach a moisture content of 2 g/100 g at 60 °C and 25 kPa. After grinding, the pumpkin seeds were sieved through a 60-mesh sieve, sealed in an airtight bag and stored at 4 °C until extraction.
N-hexane was chromatographically pure and other reagents (including t-butanol, ammonium sulfate,
Effects of (NH4)2SO4 addition
In TPP systems, (NH4)2SO4 is the most commonly used salting reagent because it is inexpensive, mild to proteins, and has high solubility (Chew, Ling, & Show, 2018). As shown in Fig. 1A, as the addition of (NH4)2SO4 increased from 10 g/100 mL to 50 g/100 mL, EYPSO, EYPSP and EYPSPS all showed trends of first increasing and then decreasing. This was because the salting-out effect became stronger as the salt concentration in the system increased. The optimal concentration of (NH4)2SO4 would
Conclusions
In this study, the effect of simultaneous extraction and separation of oil, protein and polysaccharides from pumpkin seeds was investigated using UTPP. The parameters of (NH4)2SO4 addition, t-butanol to slurry ratio, pH, sonication power, irradiation time and duty cycle were optimized in single-factor tests and response surface methodology to achieve efficient extractions of PSO, PSP and PSPS. The highest EYPSO, EYPSP and EYPSPS were obtained at an (NH4)2SO4 addition of 30 g/100 mL, a t-butanol
Authorship contribution statement
Hong Wang: Methodology, Investigation, Writing – original draft. Kuiren Chen: Investigation, Validation, Data curation. Jie Cheng: Investigation, Data curation. Lianzhou Jiang: Conceptualization, Supervision, Dianyu Yu: Conceptualization, Methodology, Writing – review & editing. Yingjie Dai: Supervision, Writing – review & editing. Liqi Wang: Formal analysis. Funding acquisition. All authors have read and approved the content of the manuscript.
Declaration of competing interest
There are no conflicts of interest to declare.
Acknowledgments
This work was supported by a grant from the 13th Five-year National Key Research and Development Plan: Research, development and demonstration of high-value stable processing technology and intelligent equipment for rice bran (No: 2018YFD0401101); and a grant from the National Natural Science Foundation of China (No: 32072259).
References (39)
- et al.
Application of ultrasound treatment for improving the physicochemical, functional and rheological properties of myofibrillar proteins
International Journal of Biological Macromolecules
(2018) - et al.
Ultrasound assisted three phase partitioning of a fibrinolytic enzyme
Ultrasonics Sonochemistry
(2014) - et al.
Investigation on solubility, interfacial and emulsifying properties of pumpkin (Cucurbita pepo) seed protein isolate
LWT–Food Science and Technology
(2015) - et al.
Effect of extraction method and specie on the content of bioactive compounds and antioxidant activity of pumpkin oil from Yucatan, Mexico
Food Chemistry
(2019) - et al.
Enhanced microalgal protein extraction and purification using sustainable microwave-assisted multiphase partitioning technique
Chemical Engineering Journal
(2019) - et al.
Three phase partitioning: Concentration and purification of proteins
Protein Expression and Purification
(1997) - et al.
Effect of pumpkin-seed oil on the level of free radical scavengers induced during adjuvant-arthritis in rats
Pharmacological Research
(1995) - et al.
Highly efficient pumpkin-seed extraction with the simultaneous recovery of lipophilic and hydrophilic compounds
Food and Bioproducts Processing
(2019) Aqueous methods for extraction/recovery of macromolecules from microorganisms of atypical environments: A focus on three phase partitioning
Methods in Microbiology
(2018)- et al.
A review of applications of cavitation in biochemical engineering/biotechnology
Biochemical Engineering Journal
(2009)
Kolkhoung (Pistacia khinjuk) kernel oil quality is affected by different parameters in pulsed ultrasound-assisted solvent extraction
Industrial Crops and Products
Effect of oil extraction assisted by ultrasound on the physicochemical properties and fatty acid profile of pumpkin seed oil (Cucurbita pepo)
Ultrasonics Sonochemistry
Microwave-assisted aqueous enzymatic extraction of oil from pumpkin seeds and evaluation of its physicochemical properties, fatty acid compositions and antioxidant activities
Food Chemistry
Three-phase partitioning and proteins hydrolysis patterns of alkaline proteases derived from fish viscera
Separation and Purification Technology
Simultaneously concentrating and pretreating of microalgae Chlorella spp. by three-phase partitioning
Bioresource Technology
Optimization of ultrasonic-microwave assisted extraction of oligosaccharides from lotus (Nelumbo nucifera Gaertn.) seeds
Industrial Crops and Products
Pumpkin (Cucurbita maxima) seed oil extraction using supercritical carbon dioxide and physicochemical properties of the oil
Journal of Food Engineering
Characteristics and chemical composition of date palm (Phoenix canariensis) seeds and seed oil
Industrial Crops and Products
Three phase partitioning for extraction of oil: A review
Trends in Food Science & Technology
Cited by (16)
Deep eutectic solvents as extraction media for food-derived biomacromolecules
2024, TrAC - Trends in Analytical ChemistryPumpkin seed oil: a comprehensive review of extraction methods, nutritional constituents, and health benefits
2024, Journal of the Science of Food and AgricultureUltrasound as a Techno-Functional Modifier in Food Frying and Bioactive Compounds Extraction
2024, Food Reviews International