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For an interesting point, peanut seeds contain storage proteins initially as reservoirs for seed germination while some of them are allergens having antifungal activity The first group is the proteins with trypsin inhibitor activity They are Ara h 2 which stands in both the globulin and the 2S albumin families regarding close amino acid sequence homology (≥35-39%), and the relatively homologous Ara h 7 [2,3,4,5,6] The globulin Ara h 3 is another one in this group [7] The second is nonspecific lipid transfer proteins Ara h 9 [4] The Ara h 2, Ara h 7 and Ara h 9 are in the same prolamin superfamily based on the eight cysteine conserved pattern [5,8], though very low sequence homology It has been known in the literatures that seed albumins are extractable in water or diluted salt while globulins are salt soluble extracted by high salt concentration i.e at least 1 M NaCl Prolamins are soluble in aqueous alcohol The globulin family has been so far identified for peanut drought tolerant genotypes [9], which are known as the approach to reduce aflatoxin contamination Focusing at the Ara h members, the albumin and prolamin families have potential to associate with antifungal action It is of our initial intention to extract peanut seed proteins with alternated order of solvent hopefully to

obtain fractions of albumins and prolamins having distinguished profiles Both fractions were tested against A

flavus comparing to the globulin fraction The data will be applied for screening peanut germplasms with an

advantage genotype of antifungal seed storage proteins for the breeding program

2 Materials and methods

2.1 Biological materials

Peanut seeds were purchased from a local market The aflatoxin-producing A flavus isolate was originally

from the laboratory at Suranaree University of Technology, Nakhonratchasima province, Thailand

2.2 Sample preparation

Peanut kernels were crushed in liquid N2 and defatted with hexane (15 ml/g) at -20°C overnight The

defatted meals were pooled by centrifugation (10000g, 10 min, 4°C), air-dried and stored at -20oC until used

2.3 Protein fractionation

Briefly for the whole, extractions were done by 1-h stirring at room temperature using 5-ml solvent per 1 g

defatted meal The supernatant of each extract collected by centrifugation (10000g, 30 min, 4oC) was kept as a protein fraction relating to its solubility i.e water-soluble, salt-soluble and alcohol-soluble Two methods were carried out In the first method, traditional extraction was conducted orderly by water, 2 M NaCl and 70% ethanol At each step after collecting the supernatant, the pellet was rinsed twice with a small volume of the subsequent solvent The fraction was made clear of NaCl or ethanol by washing with 50 mM Tris-HCl buffer (pH 6.8) using centrifugal filtration through a 10-kDa cutoff filter membrane In the second method, the alternated extraction was sequentially by 2 M NaCl in the buffer (pH 6.8), 70% ethanol and water The pellet from either step was rinsed twice with acetone and air-dried before used in the next step The salt-soluble fraction was desalted by centrifugal filtration as above The alcohol-soluble fraction was added with 4 volumes of acetone, then the protein precipitate was air-dried and suspended in 50 mM Tris-HCl (pH 6.8)

2.4 SDS-PAGE for seed protein profiling

All procedures and electrophoretic reagents were as described by Laemmli [10] Profiling of proteins (10 g) was made in a 13% gel with a Mini-Protean III unit (Bio-Rad) Protein bands were stained by Coomassie

brilliant blue R-250 Low MW markers (GE Healthcare) were introduced for size estimation

2.5 Inhibitory assay on A flavus

The assay was run only with protein fractions from the alternate method Freshly A flavus sporulated

culture on potato dextrose agar (PDA) medium was flooded with sterile water The culture surface was disturbed gently with a sterile loop to release spores In triplicates, 80-μl spore suspensions (8 x 105spores/ml) were taken to the 96-well plate Control wells were further added with 20-μl sterile water, while including in the tested wells with a protein extract (20 μl; 0.2 mg protein/well) Following 10 h at 30°C, O.D 550 nm was recorded by a Microplate Reader Microscopic observation for spore germination was made after 5% glutaraldehyde fixation and the percentage of germinating spores was obtained by counting totally 300 spores

Antifungal test on the mycelial growth was studied by growing A flavus on a 90-mm PDA plate until the

mycelial colony spread over 3 cm Germ-free paper discs (0.9 cm in diameter) were placed at the distance of 0.5 cm to the edge of the colony After filling the disc with aliquots of water as control and the protein solution, the plate was incubated for 24 h and inspected for growth inhibition

3 Results and discussion

From the traditional water-to-salt-to-alcohol extraction in Fig 1a, the salt-soluble fraction had polypeptide compositions from <16 kDa to >97 kDa This promising globulin fraction showed almost the same profiles as those of peanut seed total protein or mixture of globulins/albumins reported elsewhere [11,14], yet the typical major globulins were three at 20-27 kDa and four at 35-43 kDa for arachins, and at 65 kDa for con-arachins These are two globulins predominantly at least 73% [11,12] in peanut seed proteins Other bands were also apparent here which could be referred as globulins [13] at 15.5-19.5 kDa together with the minor band at around 95 kDa The bands between 27-35 kDa and below 16 kDa were unknown for peanut seed globulins Band patterns of the water-soluble (albumins) and alcohol-soluble (prolamins) fractions were similar having several bands commonly with the globulin fraction The albumin profiles were different from those previously reported [14] due to the details of extraction Regarding restrictedly to the band patterns, the traditionally sequential extraction did not give characteristic profiles of albumin and prolamin fractions SDS-gel profiles

of peanut seed prolamins have not been evident in literatures What can be searched is the molecular mass of 9-10 kDa for a prolamin Ara h 9 [4,5] The gel system in the work can not resolve such very small molecules The reproducible results from the alternate method salt-to-alcohol-to-water extraction were shown in Fig 1b The salt-soluble protein profiles resembled those in the former method The following alcohol extraction led to characteristic appearance of small polypeptide profiles disappearing large amount of the major bands of the globulin fraction Results from final water extraction could be compared with that of the salt extraction, but four new intense specific bands were recovered remarkably Therefore, this alternate method was proposed to successfully discriminate fractions of the most abundant peanut seed globulins, the minor albumins and the trace prolamins It should be reminded that the three protein families are known by their preferable solubility in solution of different ionic strength and polarity Proteins possess intermediate property can be extracted by more than one solvent There was a report for the seed of Conophor nut [15] that salt extract dissolved almost an equal amount of true albumins (41.86%) and true globulins (58.14%)

Effect on A flavus spore germination showed after 10-h incubation, simply from turbidity at O.D 550 nm

(Table 1) The fungus grew the least in the control well not containing any protein fraction The alcohol-soluble proteins (prolamins) exhibited a result very little different from that of the control, interpreting its inhibitory effect When the spores were treated with the expecting globulin or albumin fraction, fungal growth was highly detectable This evidence was consistent with the spore counting (Table 1 and Fig 2) that

percentage of spore germination in the control was lowest (74.44%) Germ tube emergence in treatment with the prolamin fraction slightly increased Meanwhile, the other two protein fractions enhanced germination to 100% and promoted mycelial extension, perhaps as an enriched nitrogen source Globulin fraction wetting with paper discs supported the mycelial spread over the discs, in an amount dependent manner (0.1-0.3 mg), which was seen by the green spore-fruiting body, comparing to the negative control disc The albumin fraction (0.3 mg) and prolamin fraction (0.2 mg) were active against the fungal invasion The inhibition was not so strong that it did not give growth clear zone

It cannot be negligible that the globulin, albumin and prolamin family can be cross-contaminated while being extracted with the specific solubilizing solution for each The experiments started peanut seed protein extraction with 2 M NaCl in buffer hopefully to separate out true globulins and intermediate globulins/albumins Then the solvent was switched to rather hydrophobicity of 70% ethanol for prolamin extraction, then back to the polarity of water for true albumins The last water extract might retain some intermediate globulins However, the present data recommend that profiles of the albumin and especially

prolamin fractions obtained by the alternate method contain A flavus resistance associated proteins having potential in identification of the A flavus resistant peanuts The globulin family is proposed weak markers due

to its excess protein compositions with strong supplementing effect on the fungal growth Besides, it contains

a large amount of potent allergens [16]

Fig 1 SDS-PAGE profiles of peanut seed proteins fractionated by (a) traditional method and (b) alternate method Lane M: low MW markers, 1: salt-soluble fraction, 2: water-soluble fraction, and 3: alcohol-soluble fraction

e f g

Fig 2 Inhibitory assay on Aspergillus flavus Spore germination assay; (a) control (b-d) treatment with prolamin fraction, albumin

fraction, and globulin fraction, respectively Mycelial growth inhibition test by; (e) prolamin fraction, (f) albumin fraction, and (g) globulin fraction Sterile water was used as the negative control The protein amount in milligrams was numerically labeled

Table 1 The effect of the protein fraction on Aspergillus flavus spore germination (n = 3)

Treatment Expecting protein family O.D 550 nm % Spore germination

Control (water)

Salt-soluble fraction

Water-soluble fraction

Alcohol-soluble fraction

- Globulin Albumin Prolamin

0.089 ± 0.005 0.555 ± 0.008 0.625 ± 0.003 0.096 ± 0.006

74.44 ± 5.01 100.00 ± 0.00 100.00 ± 0.00 81.67 ± 1.45

Acknowledgements

This work was supported by the Higher Education Research Promotion and National Research University

Project of Thailand, Office of the Higher Education Commission, through the Food and Functional Food

Research Cluster of Khon Kaen University

References

[1] Sanders TH, Cole RJ, Blankenship PD, Hill RA Relation of environmental stress duration to Aspergillus flavus invasion and

aflatoxin production in preharvest peanuts Peanut Sci 1985;12:90-3

[2] Stanley JS, King N, Burks AW, Huang SK, Sampson H, Cockrell G, Helm RM, West CM, Bannon GA Identification and

mutational analysis of the immunodominant IgE binding epitopes of the major peanut allergen Ara h 2 Arch Biochem Biophys 1997;342,

244–53

[3] Barre A, Borges J-P, Culerrier R, Rougé P Homology modelling of the major peanut allergen Ara h 2 and surface mapping of

IgE-binding epitopes Immunol Lett 2005;100:153-8

[4] Pele M Peanut allergens Romanian Biotechnol Lett 2010;15:5204-12

[5] Hauser M, Egger M, Wallner M, Wopfner N, Schmidt G, Ferreira F Molecular properties of plant food allergens: A current

classification into protein families The Open Immunol J 2008;1:1-12

[6] Schmidt H, Krause S, Gelhaus C, Peterson A, Janssen O, Becker W-M Detection and structural characterization of natural Ara h

7, the third peanut allergen of the 2S albumin family J Proteome Res 2010;9:3701-9

[7] Koppelman SJ, Knol EF, Vlooswijk RA, Wensing M, Knulst AC, Hefle SL, Gruppen H, Piersma S Peanut allergen Ara h 3:

isolation from peanuts and biochemical characterization Allergy 2003;58:1144-51

[8] Lehmann K, Schweimer K, Reese G, Randow S, Vieths S, R¨osch P Structure and stability of 2S albumin-type peanut allergens:

implications for the severity of peanut allergic reactions Biochem J 2006;395:463-72

[9] Katam R, Vasanthaiah HKN, Basha SM Proteomic Approach to Screen Peanut Genotypes with Enhanced Nutritional Qualities In: Frontiers in the Convergence of Bioscience and Information Technologies Korea, 2007

[10] Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 1970;227:239-51

[11] Liang XQ, Luo M, Holbrook CC, Guo BZ Storage protein profiles in Spanish and runner market type peanuts and potential

markers BMC Plant Biol 2006;6:6-24

[12] Marcone MF, Kakuda Y, Yada RY Salt-soluble seed globulins of various dicotyledonous and monocotyledonous plants-I

Isolation/purification and characterization Food Chem 1998;62:27-47

[13] Basha SMM, Pancholy SK Polypeptide composition of arachin and non-arachin proteins from early bunch peanut (Arachis

hypogaea L.) seed Peanut Sci 1981;8:82-8

[14] El-Beltagi HS Characterization of isoenzymes, enzyme activities and protein profiles of roasted peanut (Arachis hypogaea L.)

EJEAFChe 2010;9:1099-109

[15] Gbadamosi SO, Abiose SH, Aluko RE Solubilization, amino acid composition and electrophoretic characterization of Conophor

nut (Tetracarpidium conophorum) proteins Internat Food Res J 2012;19:651-6

[16] Nicolaou N, Custovic A Molecular diagnosis of peanut and legume allergy: Allergenic proteins Curr Opin Allergy Clin Immnol

2011;11:222-8