Isolation, purification and characterization of protein from Litchi chinensis honey and

generation of peptides

1

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Received: Dec 19, 2019Accepted: Feb 25, 2020Published Online: Mar 02, 2020Journal: Journal of Addiction and RecoveryPublisher: MedDocs Publishers LLCOnline edition: http://meddocsonline.org/Copyright: © Banerjee R (2020). This Article is distributed under the terms of Creative Commons Attribution 4.0 International License

*Corresponding Author(s): Rintu Banerjee P.K. Sinha Centre for Bioenergy and Renewables, Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, IndiaEmail: rb@iitkgp.ac.in & rb@agfe.iitkgp.ernet.in

Cite this article: Bose D, Padmavati M, Banerjee R. Isolation purification and characterization of protein from Litchi chinensis honey and generation of peptides. J Addict Recovery. 2020; 3(1): 1016.

Journal of Addiction and Recovery

Open Access | Research Article

Debalina Bose1; Manchikanti Padmavati2; Rintu Banerjee1,3* 1P.K. Sinha Centre for Bioenergy and Renewables, Advanced Technology Development Centre, Indian Institute of Technology Kharag-pur, India2Rajiv Gandhi School of Intellectual Property Law, Indian Institute of Technology Kharagpur, India3Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, India

Abstract

Objective: Food addiction is an eating disorder affecting the behavioral and neurological condition associated with BMI (Body mass index), BED (binge eating disorder) and obesity in human being. High-calorie foods, especially sug-ar, have an addictive potential. The conventional treatment processes involving cognitive behavioral therapy, mental health treatments and intake of drugs have acute side ef-fects. The objective of this study was to characterize a high calorie natural food honey, which has been reported to have addictive behavior, and further generate peptides from the protein using enzyme.

Methods: Protein from honey was concentrated by ultrafiltration, purified by ion exchange chromatography, characterized by SDS-PAGE, isoelectric focusing, sequencing and identified by MALDI-TOF/MS analysis.

Results: Ultrafiltration was found to significantly concentrate the protein and chromatographic techniques resulted in purification of protein to homogeneity. The protein having molecular weight of 55 kDa was found to have a pI of 5.5 and hydrophilic N terminal sequence. The protein was identified as Major royal jelly protein 1, most abundant protein present in honey. Peptides were generated with high antioxidant property.

Conclusion: Protein is a major biomolecule in honey ex-hibiting biological activities. The characterization of protein in this study helps to get idea of the molecular characteris-tics so that further studies on the activity can be evaluated. Moreover peptides have got high antioxidant property.

ISSN: 2637-4528

Keywords: Litchi chinensis; Protein; Purification; Characteriza-tion; Identification

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Introduction

Food addiction, a condition recognized as overeating or eat-ing disorder is related to mental health issue in which a per-son becomes addicted to food [1]. Terms like “chocoholic” and “craving” are used to describe man’s desire and fondness for food [2]. Certain foods have got addictive potential causing loss of control over food intake that may result in eating-related dis-orders (binge eating disorder, bulimia nervosa, weight gain and obesity) with alteration of behavior and neurological changes [3-5]. It has been explained that the brain response for food addiction is similar or as strong as addiction for drugs [6,7]. Though food items like coffee, bacon, milk, eggs, pizza, choco-late, cheesecake, maize have addictive potential [8,9], the crav-ing for sugar is much stronger in comparison to cocaine [10]. High calorie food has been reported to be highly addictive [5]. Honey is one such saturated solution of sugar having higher calorific value than sugar. The consumption of honey in ancient age and the addiction of sugar in modern age have evolutionary connection [10].

Honey has already been reported to have addictive proper-ties [10]. Although honey is a saturated sugar solution, protein is present in honey in minute quantity along with other bioactive components. Thus, it was felt essential to know if the protein/peptide present in honey has any role in addiction. therefore, the present article emphasizes on isolation and identification of protein present in honey along with its biochemical characte-rization. It has been successful in identifying a purified protein and is currently a fore runner of the future to address such is-sues.

CART (Cocaine- and amphetamine-regulated transcript) peptides are novel putative brain/ gut neurotransmitter and co-transmittor that probably have a role in drug abuse, the con-trol of feeding behavoir sensory processing, stress and develo-pment. They are abundant, processed and apparently released. On the other hand exogeneously applied peptide cause inhibi-tion of feeding and have neurotropic properties. Besides CART, their are certain other peptides that may act as new putative neurotransmitter and appear to have an important role in diffe-rent physiological processes including feeding, sensory proces-sing, development, stress etc [11].

In humans, increasing evidence suggests that individuals eating high calorie food have symptoms of addictive behavior [4,12]. However, understanding the addictive behavior of food necessitates its characterization [13]. Thus, it was felt essen-tial to characterize ingredients present in honey that may be responsible for addictive nature of food. In the present article Litchi chinensis honey, abundantly available in India was col-lected and protein from the honey was isolated, purified and characterized.

The structural configuration, shelf life, biological and chemi-cal stability of proteins, its efficiency and recovery are directly influenced by the purification techniques used [14]. Prior pu-rification is necessary to characterize protein [15,16]. Detailed study on purified protein is necessary to understand the char-acteristics of honey and therefore its role in addiction. Thus, based on this premise, isolation, purification and characteriza-tion of protein from Litchi chinensis honey have been consid-ered in this article.

Materials and methods

Sample

Litchi honey (Litchi chinensis) was collected from colonies of Apis mellifera in Baruipur apiculture industrial co-operative so-ciety Ltd., Dakshin Gobindapur, Kolkata, West Bengal, India. The colonies were placed in litchi plantation following standard api-cultural methods and honey was collected by beekeepers dur-ing the 2017-2018 harvest seasons (February to March 2017) using a stainless-steel honey extractor (Hi-Tech Natural Prod-ucts Limited, India). The extracted honey was filtered through a sieve to remove unwanted debris and stored in sterilized sealed glass jars at 4°C.

Isolation of protein from honey

Honey sample (200 g) was dissolved in 0.01M Tris-HCl buffer (pH 7.4) to a volume of 250 mL. Ultrafiltration process using a 10 kDa polyethersulfone membrane (Sartorious, India) was used to concentrate the solution. The obtained retentate was recirculat-ed several times till the volume was reduced to approximately one-tenth of the initial (25 mL). The protein concentration was checked after each cycle. A fraction of the concentrated reten-tate (12 mL) was subjected to ultracentrifugation at 15,000 rpm for 15 min. The process was repeated 3-4 times until the dark pellet formed on the wall of the centrifuge tube was completely removed with the collection of supernatant.

Purification of protein by BioLogic LP ion exchange chroma-tography

The supernatant (1.5 mL) from the ultrafiltration step was subjected to purification using a Q Sepharose (anion exchange) column (16/20 mm), attached to an FPLC system (Pharmacia). The cartridge was equilibrated with 0.01M Tris-HCl of pH 7.4 (buffer A), into which the concentrated protein was injected. Elution of bound proteins carried out using buffer B (0-50%, 0.5M NaCl in buffer A) at 1.5 mL/min flow rate. Absorbances of all fractions were detected at 280 nm by an online UV detector.

Estimation of protein concentration by Bradford assay

Total protein content at each step of purification was checked by the Bradford method [17]. Bovine serum albumin (BSA) was used as standard. Buffer A was used as a blank.

Characterization of purified protein

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophore-sis (SDS-PAGE)

The extent of homogeneity at each step of purification was detected by SDS-PAGE performed on 12% resolving gel and 4% stacking gel following the protocol of Laemmli [18]. Molecular weight (Mw) of the purified protein was determined by compar-ing the relative mobility of standard protein marker of 10-250 kilodaltons (kDa) (Precision Plus Protein Standard, Bio-Rad).

Determination of molecular weight by MALDI-TOF mass spectrometry

The molecular weight of the unknown protein was confirmed by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometric (MALDI-TOF/MS) analysis. Sinapic acid was used as a matrix for the analysis.

Protein sequencing

Automated Edman degradation was carried out on a protein sequencer (Model PPSQ-31A; Shimadzu Scientific, Kyoto, Japan) to determine the N-terminal amino acid sequence of the pro-tein. The purified protein was loaded onto a polyvinylidene dif-

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luoride (PVDF) membrane (Millipore) by electrophoresis which was further stained with Coomassie Brilliant Blue R-250 dye (Thermo Fisher Scientific‎), destained and washed thoroughly. Stained spots were cut off and sequence analysis was done. Ho-mology search of the obtained sequence was carried out using BLAST (Basic Local Alignment Search Tool).

Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF/MS)

MALDI-TOF/MS analysis and peptide masses were deter-mined on a mass spectrometer (UltrafleXtremeTM, Bruker, Ger-many) using α-cyano-4-hydroxycinnamic acid as matrix. The desired purified protein fraction obtained from ion-exchange chromatography was subjected to hydrolysis using a proteolytic enzyme. The reaction was carried out using sequencing grade trypsin (Promega, Madison, WI) at enzyme: purified protein ra-tio 1:5 (w/w). Samples after overnight incubation (37°C) were boiled for 5 min and centrifuged at 8000 rpm in a microcentri-fuge. The supernatant was then collected for mass spectromet-ric analysis. MASCOT search program was performed for data-base search for peptide mass fingerprinting (PMF).

Isoelectric Focusing (IEF)

Rotofor system (Bio-Rad, USA), with a mini focussing cham-ber (18 mL) equipped with 20 fractionation compartments was employed to check the isoelectric point (pI) of the puri-fied protein. 0.1 M sodium hydroxide and 0.1 M phosphoric acid were used as electrolytes in cathode and anode assembly respectively. A pH gradient was created using ampholyte (Bio-Lyte 3/10, BioRad, USA) of range 3.0-10.0. Sample solution (18 mL distilled water, 1 mL ampholyte and 0.5 mL purified protein) was prepared and loaded into the rotofor chamber. Focusing was performed at a constant power of 10W for 4 h. After the complete run, 20 fractions were collected and evaluated for pH and protein concentration. The pI value was further confirmed by MALDI-TOF mass spectrometric analysis.

Enzymatic hydrolysis

The purified protein was digested using sequencing grade trypsin (Promega, USA) to produce protein hydrolysate or pep-tides [19]. Trypsin (0.03%, w/w) was added to purified protein fraction for hydrolysis at 37°C and pH 7.4 for 24 h. The prote-olytic mixture was then boiled for 5 min and subjected to 15 min centrifugation at 8000 rpm. The supernatant was then as-sayed for antioxidant activities.

Bioactive properties

DPPH (1, 1-diphenyl-2-picrylhydrazyl) assay

Crude honey, purified protein, and protein hydrolysate/pep-tides were used for further analysis of the antioxidant property. Scavenging of free radicals by the samples was determined [20]. Samples (0.5 mL) was mixed with a solution of DPPH (4 mL, 0.5mM) in methanol and incubated for 30 min in dark. Metha-nol was used as a blank to measure the absorbance at 515 nm and results were calculated in triplicates as percent inhibition of DPPH radical using the formula:

DPPH activity (%) = [(Dcontrol - Dsample)/Dcontrol] × 100

Where, Dcontrol is the absorbance of solution without sample and, Dsample is the absorbance of sample solution.

FRAP (Ferric reducing antioxidant power) assay

Crude honey, purified protein, and protein hydrolysate/pep-tides were assayed for reducing power [21]. Sample (0.5 mL) was added to 1.5 mL FRAP reagent [acetate buffer (300mM/L, pH 3.6): TPTZ solution (10mM in 40mM/L HCl): ferric chloride (20mM FeCl3.6H2O) at 10:1:1 ratio] and incubated at 37°C for 30 min. Distilled water was used as a blank for measuring ab-sorbance at 593 nm. Calibrations were performed using ferrous sulphate solutions (0-100μM). Results were expressed as μmol of Fe (II) in triplicate values.

ABTS [2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)] antioxidant assay

Crude honey, purified protein, and protein hydrolysate/pep-tides were assayed for antioxidant activity in reaction with ABTS radical [22]. ABTS cation was produced by reacting ABTS (50 mL) in phosphate-buffered saline (2 mM, pH 7.4) with 0.2 mL of po-tassium persulphate (70 mm) in dark followed by incubation for 12-16 h. ABTS•+ solution was diluted with buffer to obtain an ab-sorbance of 0.700 ± 0.05 at 734 nm. Sample (0.5 mL) was then added to 3.5 ml ABTS•+ solution, homogenized and incubated for 10 min. Absorbance was then measured at 734 nm against an artificial honey sample as blank. The percentage decrease in the absorbance was calculated using the formula:

ABTS•+ inhibition (%) = [(Ablank - Atest)/Ablank] × 100

Where, Ablank is the absorbance of blank sample (t=0 min) and Atest is the absorbance of test sample at the end of the reaction (t=10 min).

Results & discussion

Concentration of protein through ultrafiltration and ultra-centrifugation

To isolate the desired protein from honey solution, ultrafil-tration (10 kDa cutoff membrane) process was adopted. Con-centration and fractionation of protein were carried out by passing the entire solution for several cycles. Molecular weight compounds more than 10 kDa present in honey solution was collected in the retentate whereas the low molecular weight compounds were collected in the filtrate. After several runs, ultrafiltration followed by ultracentrifugation resulted in honey protein concentration that was next employed to purification. The protein concentration at each step of purification is shown in Table 1.

Purification of protein

The concentrated protein obtained through ultrafiltration and ultracentrifugation was subjected to purification through anion exchange chromatography where a graph showing two peaks were observed, a minor hump followed by a single major peak as shown in Figure 2(A). Fractions 32 to 41 represents peak 1 while peak 2 is represented by fractions 42 to 53. Q Sepharose (Quaternary sepharose) being a strong anion exchanger, elu-tion of protein was done through a continuous gradient of salt (NaCl). The protein concentration of these fractions were mea-sured by Bradford method and the following fractions yielded the highest protein content (Table I).

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Table 1: Protein concentration at each step of purification and ion exchange fractions with corresponding protein concentrations

Purification steps Total volume (mL) Total protein (mg) Protein concentration (mg/mL)

Crude honey 250 135 0.54

Ultrafiltration (10 kDa Retentate) 25 128 5.12

Ultrafiltration (10 kDa Permeate) 225 0 0

Ion exchange chromatography fraction 1.5 0.56 0.37

Anion-exchange chromatography fraction Protein concentration (mg/mL)

39 0.04

44 0.11

45 0.28

46 0.42

47 0.56

SDS-PAGE of the above fractions showed clear bands in frac-tions 46, 47 and 48 proving the fact that the desired honey pro-tein has been effectively purified. Figure 2(B) represents a thick band of retentate (lane 1) which got reduced to thin bands in fraction 44 (lane 3), fraction 45 (lane 6), fraction 46 (lane 5), and fraction 47 (lane 7) with subsequent purification steps. Hence the protein present in the highest concentration in the honey sample was purified having a protein content of 0.56 mg/mL in fraction 47. The major peak was isolated and subjected to further characterization.

Figure 1: Chromatogram of Major Royal Jelly Protein 1 and SDS-PAGE analysis: (A) Protein purification profile by Q Sep-harose anion exchange chromatography (B) SDS-PAGE analy-sis of ion exchange fractions: Retentate (Lane 1), Fraction 38 (Lane 2), Fraction 44 (Lane 3), Fraction 39 (Lane 4), Fraction 46 (Lane 5), Fraction 45 (Lane 6), and Fraction 47 (Lane 7).

MALDI-TOF mass spectrometry technique as shown in Figure 3(B) which was almost similar to that inferred by gel electro-phoretic technique.

Figure 2: Molecular weight of purified protein: (A) SDS-PAGE analysis of crude honey (Lane 1), ultrafiltered honey (Lane 2), puri-fied protein (Lane 3), and molecular marker (Lane 4) (B) Molecular weight determination by MALDI-TOF/MS analysis.

Molecular weight of the protein

The fraction of protein showing highest concentration in ion-exchange chromatography was collected and subjected to SDS-PAGE analysis and the molecular weight was found to be 55 kDa as shown in Figure 3(A). The obtained result had similarity with the honey samples reported previously [23,24]. The molecular weight of the unknown protein was found to be 53-54 kDa by

N-terminal sequence of the protein

The protein sequence obtained contains a putative leader sequence and a long segment that contains several pairs of ba-sic amino acids which are potential cleavage sites. The obtained sequence, N-I-L-R-G-E-S-L-N-K-S-L-P-I-L was nearly identical to N(S)-I-L-R-G-E-S-L-D-K deduced from MRJP1 of Apis cerena [25] and was also identical to the previously reported N-terminal se-quence of MRJP1 deduced from honeybee Apis mellifera (Am-MRJP1) [26,27]. Obtained sequence on homology search exhib-ited 86% identity with MJRP1 of Apis cerana and Apis florea.

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The analysis showed that the number of nonpolar amino acids were higher than the polar amino acids indicating hydrophobic-ity of the protein which has an important role in determining the tertiary structure of the proteins shaping the molecule and its active sites.

Identification of protein

The purified protein of 55 kDa had significant similarity with the reported literature as Major Royal Jelly Protein 1 (MRJP1) or royalactin of Apis mellifera. This protein was further analyzed through MALDI-TOF mass spectrometric analysis. MASCOT search program showed a significant-top score of 77 as depict-ed in Figure 4(A) with protein sequence coverage of 25% and 14 peptide matches showed in Figure 4(B).

Figure 3: Mascot search results showing MRJP1 as identified protein: (A) Mascot Search program showing identified protein with significant score (B) Protein sequence coverage and peptide matches.

Major Royal Jelly Proteins (MRJPs) or yellow protein family are proteins available in honey after removing pollen protein [28]. These proteins in honey are a family of nine members MRJP 1-9 (Tamura et al., 2009). MRJP 1-5, also known as apal-bumins are the most prominent and has a molecular weight ranging from 49-87 kDa [26]. The most abundant among them

is MRJP1, comprising of an oligomer of 280-420 kDa or a mono-mer of 55 kDa [26]. Thus, the identified purified protein was a monomeric form (MRJP1) also known as apalbumin-1 or roya-lactin as depicted in Figure 4(B) where the sequence of the ma-jor peptides of the protein has been shown.

Isoelectric point (pI) of protein

Protein concentration was observed in the 7th fraction whereas the remaining fractions revealed no protein content. Thus, the pH 5.5 of the 7th fraction was the respective pI of the protein. The result was identical to the estimated pI value of MRJP1 of Apis cerena (AcMRJP1) [25]. The pI value obtained by isoelectric focusing was different from the pI (5.1) estimated by MALDI-TOF/MS analysis. The difference in the pI value may be because of the lack of true separation barriers between the rotofor chambers, salt and/or buffers transferred with the sam-ple, or minor leakage within the core of anodic and cathodic solutions affecting the gradient linearity [29]. Post-translational modifications during protein separation and characterization may also have altered the pI of protein [29].

Bioactivities

The crude honey, purified protein and protein hydrolysate/peptides obtained after tryptic digestion was subjected to bio-chemical analysis for the evaluation of their biological activities. The results obtained have been shown in Table 2.

The present study shows protein hydrolysate/peptides to have higher DPPH activity compared to that reported by Guo et al. (2005). The result revealed peptides to have a % inhibition value of 68.21 ± 4.01, which is higher than in the case of puri-fied protein (Table 2). The higher % inhibition value of peptides might be because of the increased solvent accessibility of amino acids due to disruption of the tertiary structure of protein lead-ing to free radical scavenging and metal chelation.

Reducing power is an important parameter to estimate the reductants present in a biological sample. The ability of a bio-logical sample to reduce the ferric ion to ferrous ion acting as a reducing agent is determined by FRAP assay (Alzahrani et al., 2012). The reducing capacity of peptides was noted to be high-er than the purified protein (Table 2). The difference in reducing capacity may be due to the specific composition of amino acid and the smaller size of peptides than the high molecular weight of protein. The results reveal that peptides act as good electron donors and are strong reducing agents.

The ABTS assay is one of the most frequently used analytical strategies for antioxidant activity. The present study reported ABTS scavenging activity of peptides to be higher than protein (Table 2), which may be because of the amino acid side chain, chain length, and hydrophobicity. The amino acid composition of protein hydrolysate is also an important factor contributing to its antioxidant activities (Ulagesan et al., 2018).

However, the bioactive properties of crude honey were found to be much higher than the purified protein and the peptides. The higher antioxidant property of crude honey may be contributions of other bioactive molecules in honey such as phenolics, flavonoids, ascorbic acid, enzymes such as catalase and peroxidase (Habib et al., 2014).

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Table 2: Bioactive properties of crude honey, purified protein and peptides

Sample

Bioactivity assays Crude honey Purified protein Protein hydrolysate

DPPH (%) 72.90 ± 1.23 59.36 ± 0.38 68.21 ± 4.01

FRAP (Fe [II] µM) 1000.87 ± 0.28 386.38 ± 1.23 402.91 ± 0.83

ABTS (%) 28.64 ± 2.05 11.82 ± 3.22 19.01 ± 1.74

Data represented as mean ± standard deviation based on three measurements (n=3).

Conclusion

The protein extracted from Litchi chinensis honey (monoflo-ral) was a monomer of the major protein (MRJP) present in hon-ey. The isolated major protein, 55 kDa was identified as MRJP1, SDS-PAGE examination of which confirmed it to be a monomer. The purified protein had pI of 5.5 and the N-terminal sequence suggested the protein to be hydrophilic in nature. Moreover, the protein, upon digestion with trypsin yielded hydrolysates or peptides with significant antioxidant activity. The small size, hydrophobicity, specific amino acid composition, molecular weight and chain length are factors responsible for the antioxi-dant activity of peptides, which if orally available can be used for preventing and treating chronic diseases resulting due to oxidative stress. The isolated protein confirms its non addictive nature. Thus, honey can be recommended as one of the food ingredients for regular consumption.

Acknowledgement

The authors gratefully acknowledge the Council of Scientif-ic & Industrial Research [Grant no. 38(1355)/13/EMR-II dated 14.02.2013], Govt. of India, New Delhi, India; for the financial support provided to Ms. Debalina Bose for the grant of her fel-lowship.

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