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

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 sugar, have an addictive potential. The conventional treatment processes involving cognitive behavioral therapy, mental health treatments and intake of drugs have acute side effects. 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 exhibiting biological activities. The characterization of protein in this study helps to get idea of the molecular characteristics so that further studies on the activity can be evaluated. Moreover peptides have got high antioxidant property. ISSN: 2637-4528


Introduction
Food addiction, a condition recognized as overeating or eating disorder is related to mental health issue in which a person 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 disorders (binge eating disorder, bulimia nervosa, weight gain and obesity) with alteration of behavior and neurological changes [3][4][5]. It has been explained that the brain response for food addiction is similar or as strong as addiction for drugs [6,7]. Though foods items like coffee, bacon, milk, eggs, pizza, chocolate, cheesecake, maize have addictive potential [8,9], the craving 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 addiction properties [10]. Although honey is a saturated sugar solution but protein is present in a minute quantity along with other bioactive components. Thus, it was felt essential to know if the protein/ peptide present in honey has any role on addiction. Thus, the present article emphasizes on isolation and identification of protein present in honey along with its biochemical characterization. It has been successful in identifying some purified protein and is currently a fore runner of the future to address such issues.
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 control of feeding behavoir sensory processing, stress and development. They are abundant, processed and apparently released. On the other hand exogeneously applied peptide cause inhibition of feeding and have neurotropic properties. Besides CART their are certain other peptides that may have a new putative neurotransmitters that appears to have an important role in different physiological processes including feeding, sensory processing, 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 essential 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 collected and protein from the honey was isolated, purified and characterized.
The structural configuration, shelf life, biological and chemical stability of proteins, its efficiency and recovery are directly influenced by the purification techniques used [14]. Prior purification is necessary to characterize protein [15,16]. Detailed study on purified protein is necessary to understand the characteristic of honey and therefore its role in addiction. Thus, based on this premise, isolation, purification and characterization of protein from Litchi chinensis honey have been considered in this article.

Sample
Litchi honey (Litchi chinensis) was collected from colonies of Apis mellifera in Baruipur apiculture industrial co-operative society Ltd., Dakshin Gobindapur, Kolkata, West Bengal, India. The colonies were placed in litchi plantation following standard apicultural methods and honey was collected by beekeepers during the 2017-2018 harvest seasons (February to March 2017) using a stainless-steel honey extractor (Hi-Tech Natural Products 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 recirculated several times unless 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 retentate (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 supernatants.

Purification of protein by BioLogic LP ion exchange chromatography
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 was 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.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (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 (M w ) of the purified protein was determined by comparing 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 protein. The purified protein was loaded onto a polyvinylidene dif-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. Homology 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 determined on a mass spectrometer (UltrafleXtreme TM , Bruker, Germany) 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 ratio 1:5 (w/w). Samples after overnight incubation (37°C) were boiled for 5 min and centrifuged at 8000 rpm in a microcentrifuge. The supernatant was then collected for mass spectrometric analysis. MASCOT search program was performed for database search for peptide mass fingerprinting (PMF).

Isoelectric Focusing (IEF)
Rotofor system (Bio-Rad, USA), with a mini focussing chamber (18 ml) equipped with 20 fractionation compartments was employed to check the isoelectric point (pI) of the purified 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 hr. 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 peptides [19]. Trypsin (0.03%, w/w) was added to purified protein fraction for hydrolysis at 37°C and pH 7.4 for 24 hr. The proteolytic mixture was then boiled for 5 min and subjected to 15 min centrifugation at 8000 rpm. The supernatant was then assayed for antioxidant activities.

DPPH (1, 1-diphenyl-2-picrylhydrazyl) assay
Crude honey, purified protein, and protein hydrolysate/peptides were used for further analysis of the antioxidant property. Scavenging of free radicals by the samples was determined with modifications [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. Methanol 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: Where, D control is the absorbance of solution without sample and, D sample is the absorbance of sample solution.

ABTS [2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)] antioxidant assay
Crude honey, purified protein, and protein hydrolysate/peptides were assayed for antioxidant activity in reaction with ABTS cation 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 potassium persulphate (70 mm) in dark followed by incubation for 12-16 hr. ABTS •+ solution was diluted with buffer to obtain an absorbance 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 (%) = [(A blank - A test )/A blank ] × 100
Where, A blank is the absorbance of blank sample (t=0 min) and A test is the absorbance of test sample at the end of the reaction (t=10 min).

Concentration of protein through ultrafiltration and ultracentrifugation
To isolate the desired protein from the honey solution, ultrafiltration (10 kDa cutoff membrane) process was adopted. Concentration 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, elution of protein was done through a continuous gradient of salt (NaCl). The protein concentration of these fractions was measured by Bradford method and the following fractions yielded the highest protein content (Table I). SDS-PAGE of the above fractions showed clear bands in fractions 46, 47 and 48 proving the fact that the desired honey protein has been effectively purified. Figure 2  kDa by MALDI-TOF mass spectrometry technique as shown in Figure 3(B) which was almost similar to that inferred by gel electrophoresis technique.

Molecular weight of the protein
The fraction of protein showing the highest concentration in ion-exchange chromatography was collected and subjected to SDS-PAGE analysis and the molecular weight was found to be 55 kDa 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

N-terminal sequence of the protein
The protein sequence obtained contains a putative leader sequence and a long segment that contains several pairs of basic amino acids which are potential cleavage sites. The honey peptide is the incompletely sequenced fragment where N and C terminal has some overlapping. The neuropeptide that is secreted and processed has also been identified as the leader sequence. 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 Journal of Addiction and Recovery N-terminal sequence of MRJP1 deduced from honeybee Apis mellifera (AmMRJP1) [26,27]. Obtained sequence on homology search exhibited 86% identity with MJRP1 of Apis cerana and Apis florea. The analysis showed that the number of nonpolar amino acids were higher than the polar amino acids indicating hydrophobicity 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 depicted in Figure 4(A) with protein sequence coverage of 25% and 14 peptide matches showed in Figure 4(B). 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 apalbumins 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 monomer of 55 kDa [26].Thus, the identified purified protein was a monomeric form (MRJP1) also known as apalbumin-1 or royalactin as depicted in figure 4(B) where the sequence of the major peptides of the protein has been shown.

Isoelectric point (pI) of protein
Protein concentration was observed in the 7 th fraction whereas the remaining fractions revealed no protein content. Thus, the pH 5.5 of the 7 th 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 sample, 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 biochemical 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 purified protein ( Table 2). The higher % inhibition value in peptides might be because of the increased solvent accessibility of amino acids due to disruption of the tertiary structure of protein leading 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 biological 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 higher 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 a 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). Data represented as mean ± standard deviation based on three measurements (n=3).

Conclusion
The protein extracted from Litchi chinensis honey (monofloral) was a monomer of the major protein (MRJP) present in honey. 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 activities. The small size, hydrophobicity, specific amino acid composition, molecular weight and chain length are factors responsible for the antioxidant 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.