Enhanced extraction of oleoresin from ginger (Zingiber officinale) rhizome powder using enzyme-assisted three phase partitioning
Introduction
Ginger (Zingiber officinale Roscoe) is a monocotyledonous plant belonging to the family Zingiberacea and is one of the world’s best known spices, cultivated in several countries and has been used since antiquity for its health benefits. It is generally consumed as fresh paste, dried powder, slices preserved in syrup, candy (crystallized ginger) for flavouring tea. In many countries, especially in India and China, fresh ginger is used as flavouring agent in beverages and many other food preparations (Shukla & Singh, 2007). However the major use of ground dried ginger is for domestic culinary purposes as flavouring agent, bakery products and desserts (Young, Chiang, Huang, Pan, & Chen, 2002). Ginger consists of two main fractions, the non-volatile resins and the volatile essential oil, both of which reside in the same cell but are extracted in different manner with different solvents (Azian, Kamal, & Azlina, 2004). The main non-volatile pungent constituents of oleoresin are polyphenolic compounds namely, [6]-, [8]-, and [10]-gingerols, as well as [6]-, [8]-, and [10]-shogaols (Schwertner, Rios, & Pascoe, 2006), which are responsible for its various pharmacological effects (Dugasani et al., 2010). Among other potential mechanisms, ginger has antioxidant and anti-inflammatory, analgesic, gastro-protective, antihepatotoxic, antifungal, antiemetic, antischistosomal effects (Said, Arya, Pradhan, Singh, & Rai, 2015), DNA damage and cell migration (Jayakumar & Kanthimathi, 2012).
Fresh ginger contains about 80% moisture which makes it vulnerable to microbial spoilage. Hence it is dehydrated to moisture content 10% or below and ground to a powder, and from which value added products like essential oil and oleoresins are prepared industrially. These products retain the characteristic ginger flavor and pungency. In fresh ginger, the gingerols are identified as the major active components. Shogaols are gingerol analogues with a 4, 5 double bond, resulting from elimination of the 5-hydroxy group in alkyl side chain. The shogaol series of compounds are more pungent than the gingerols (Cheng, Liu, Peng, Qi, & Li, 2011) and are virtually absent in fresh ginger, and is derived from the corresponding gingerols during thermal processing or long-term storage (Zhang, Iwaoka, Huang, Nakamoto, & Wong, 1994). Generally, the dehydration reaction of gingerol to shogaol takes place either because of the acidic environment or as a result of the increase in temperature. Gingerol is reportedly stable in the pH range 1–7 at 37 °C; however, it starts degrading at 60 °C and above in aqueous solutions (Bhattarai, Tran, & Duke, 2001).
Numerous techniques for extraction of ginger oleoresin are reported in the scientific literature. These include the conventional Soxhlet extraction and cold percolation which employ various organic solvents and high temperatures. The major disadvantages of these techniques are longer extraction time (>10 h), enormous requirement of organic solvents and exposure of the extract to a severe risk of degradation or modification of some of the constituents. In the last few decades, there has been an increasing demand for new extraction techniques with concise extraction times with minimum usage of organic solvents to avoid potential environmental pollution and to reduce the cost burden. Recently, extraction methods such as microwave-assisted extraction (Rahath Kubra, Kumar, & Rao, 2013), concurrent application of ultrasound during supercritical extraction (Balachandran et al., 2006, Supardan et al., 2011) and supercritical fluid extraction (Said et al., 2015) have been reported for ginger constituents. However these techniques require equipment with high investment and maintenance costs. In the light of these aspects, there is a continued effort globally to investigate inexpensive, rapid and greener techniques of extraction.
Three phase partitioning (TPP) is simple, scalable, rapid and relatively recent bioseparation technique involving mixing of t-butanol with aqueous antichaotropic salt, ammonium sulphate forming two phases, the aqueous and organic layers at room temperature. It has been used for extraction of wide variety of biomolecules mainly proteins. Generally t-butanol is completely miscible with water but on the addition of ammonium sulphate at adequate concentration, it separates into lower aqueous phase and an upper t-butanol phase. This upon mixing with either microbial, plant or animal cell homogenates forms three layers, separating the proteinaceous layer at the interphase of the organic and the aqueous layer (Chougle, Singhal, & Baik, 2014). It is believed that ammonium sulphate at high concentration imparts a high dielectric property to water due to which the t-butanol layer becomes hydrophobic in nature and it gets excluded from water enabling extraction of lipophilic constituents while some polar molecules get concentrated in the lower aqueous phase (Harde & Singhal, 2012). Although TPP has been extensively evaluated for simultaneous separation and purification of proteins and enzymes from crude suspensions, it has been widely employed for extraction of polar and non-polar constituents as well. Sharma, Khare, and Gupta (2002) employed TPP for extraction of oil from soybean. Later, TPP has been employed for extraction of oil from almond, apricot, and rice bran (Sharma & Gupta, 2004), edible oils (Gaur, Sharma, Khare, & Gupta, 2007), oleoresin from turmeric (Kurmudle, Bankar, Bajaj, Bule, & Singhal, 2011), forskolin (Harde & Singhal, 2012), cocoa butter (Vidhate & Singhal, 2013), astaxanthin (Chougle et al., 2014), trichosanthin from Trichosanthes kirilowii roots (Mondal, 2014) and lipids from Chlorella (Mulchandani, Kar, & Singhal, 2015). Although TPP has been employed successfully for extraction of wide range of molecules, scale up studies are scarce and more studies are also warranted as a future direction towards its economic feasibility at larger scale operations.
In this context, the present study aimed at evaluating the possible application of TPP as an efficient technique for the extraction of oleoresin from dried ginger rhizome powder. Further, the effect of various pretreatments such as enzyme and ultrasound were explored to further improve the TPP process. These processes were termed as enzyme-assisted three phase portioning (EATPP) and ultrasound-assisted three phase partitioning (UATPP).
Section snippets
Plant material and reagents
Fresh ginger rhizome roots were procured from local market, Mumbai, India. Rhizomes were cut into small pieces and dried in lab dryer at 40 ± 5 °C overnight, ground in lab mill and passed through 40 mesh sieve to get particle size of 0.420 mm and stored in an air tight container at 4 °C until completion of analysis (moisture content of rhizome powder was 10%). The portion retained on the sieve was ∼10% w/w and discarded. Gingerol standards, [6]-gingerol, [8]-gingerol, [10]-gingerol and [6]-shogaol
Conventional extraction
Conventional Soxhlet extraction of ginger using acetone, methanol, ethanol, and hexane has shown methanol to give maximum yield of oleoresin from ginger (Said et al., 2015). Although methanol is not used at an industrial scale due to the toxicity associated with the solvent residues, it was used in this work as a control to check for any increase beyond the same by employing TPP for extraction of ginger oleoresin. Alternatively, extraction can be achieved by percolation of aforementioned
Conclusion
A maximum oleoresin yield of 69 and 64 (g kg−1) was obtained with enzyme and ultrasound pretreatment, respectively from ginger rhizome powder with TPP system consisting of 10 (% w/v) ammonium sulphate concentration, 0.5:1 ratio of t-butanol to slurry and at 5% (w/v) solid loading in the slurry. Ultrasound and enzyme pretreatment had a significant impact on the yield of gingerol too. UATPP can impose constraints in scale up. EATPP can therefore be a promising approach for extraction of gingerols
Conflict of interest
There is no conflict of interest with any individual or organization.
Acknowledgements
Dr. S. Varakumar and K. V. Umesh acknowledge the University Grants Commission, New Delhi, Government of India, for providing financial support under the D. S. Kothari Postdoctoral Fellowship [Award No. F.4-2/2006 (BSR)/13-1082/2013(BSR)] and Senior Research Fellowship, respectively.
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