Peanut Cells Research Articles

Effects of Roasting Temperatures on Peanut Oil and Protein Yield Extracted via Aqueous Enzymatic Extraction and Stability of the Oil Body Emulsion.

Oil body emulsions (OBEs) affect the final oil yield as an intermediate in the concurrent peanut oil and protein extraction process using an aqueous enzyme extraction (AEE) method. Roasting temperature promotes peanut cell structure breakdown, affecting OBE composition and stability and improving peanut oil and protein extraction rates. Therefore, this study aimed to investigate the effects of pretreatment at different roasting temperatures on peanut oil and protein yield extracted through AEE. The results showed that peanut oil and protein extraction rates peaked at 90 °C, 92.21%, and 77.02%, respectively. The roasting temperature did not change OBE composition but affected its stability. The OBE average particle size increased significantly with increasing temperature, while at 90 °C, the zeta potential peaked, and the interfacial protein concentration hit its lowest, indicating OBE stability was the lowest. Optical microscopy and confocal laser scanning microscopy confirmed the average particle size findings. The oil quality obtained after roasting treatment at 90 °C did not differ significantly from that at 50 °C. The protein composition remained unaffected by the roasting temperature. Conclusively, the 90 °C roasting treatment effectively improved the yield of peanut oil extracted using AEE, providing a theoretical basis for choosing a suitable pretreatment roasting temperature.

Improving enzyme accessibility in the aqueous enzymatic extraction process by microwave-induced porous cell walls to increase oil body and protein yields

Aqueous enzymatic extraction (AEE) is a green and sustainable extraction method. However, the intact cell wall hinders the accessibility of enzymes, severely limiting the efficiency and application of the AEE method. In this study, microwave-assisted aqueous enzymatic extraction (MAAEE) was used to degrade the peanut cell wall and improve the yield of oil and protein. Transmission electron microscopy, scanning electron microscopy, BET-specific surface area, Fourier transform infrared spectroscopy, X-ray diffraction, and chemical composition analysis were used to characterize the structure and chemical changes of the peanut cell wall. The results showed that microwave treatment could break the structure of the peanut cell wall, increase the porosity of raw materials, accelerate the efficiency of enzymatic hydrolysis, and promote the extraction of oil and protein. Compared with raw peanut powder (MC-0), microwaved peanut powder (MC-4) had a higher oil body yield (>44%) and protein release rate (>76%). At the microscopic level, cell wall rupture, oil accumulation, and cell contents scattered outside the cell were observed. The mean pore size, pore volume, and specific surface area increased by 54.1%, 149.4%, and 63.5%, respectively, while the crystallinity decreased by 4.4%. The content of hemicellulose and CDTA-soluble pectin (CSP) decreased by 18.6% and 32.9%, respectively. The degradation rates of cellulase and pectinase on cell wall components were significantly increased by 8.5%–43.2% (p < 0.05). In summary, microwave-induced porous cell walls could significantly improve enzyme accessibility, accelerate enzymatic hydrolysis efficiency, and promote oil and protein extraction.

The quantity of OA and activity of cellulase and polygalacturonase are involved in variation of virulence in Sclerotium rolfsii

In order to understand the pathogenic mechanisms of Sclerotium rolfsii on peanut and to analyze the variation of virulence in S. rolfsii strains, the highly virulent strain (ZY2) and weakly virulent strain (GP3-1) were investigated under both in vivo and in vitro conditions. The results indicated that S. rolfsii directly infected peanut by producing infection cushions. ZY2 formed infection cushions earlier than GP3-1, and ZY2 produced a greater number of infection cushions compare to GP3-1. Both strains could utilize cellulose, xylose, or polygalacturonic acid in the Czapek medium. The activities of cellulase (CL) and polygalacturonase (PG) in the inoculated peanut stems increased significantly at 9 ​h after inoculation. The activities of CL and PG produced by ZY2 in the inoculated stems were significantly higher than that produced by GP3-1. Both strains could produce oxalic acid (OA), and the content of OA produced by ZY2 in the inoculated stems was higher than that produced by GP3-1. In summary, it suggested that S. rolfsii destroyed peanut cells through physical and biochemical factors by secreting a large amount of OA, CL and PG during the formation of infection cushions. The difference in OA content, activity of CL and PG produced by highly and weakly virulent strains played important roles in variation of virulence.

The microbiota and metabolites during the fermentation of intact plant cells depend on the content of starch, proteins and lipids in the cells

Intact cells, as the smallest unit of whole foods, were isolated from three legume crops and fermented with human faecal inoculum to elucidate the effect of food macro-nutrients compositional difference (starch, proteins and lipids) on in vitro colonic fermentation profiles. After 48 h of fermentation, the highest production of short-chain fatty acids (SCFAs) were observed for the pea cells, abundance in starch (64.9 %, db). In contrast, branch chain fatty acids (BCFAs) were the major metabolites for protein-enriched soybean cells (protein content 56.9 %, db). The peanut cells rich in lipids (49.2 %, db) has the lowest fermentation rate among the three varieties. Correspondingly, pea cells favoured the growth of Bifidobacterium, whereas soybean and peanut cells promoted an abundance of Bacteroides and Shigella, respectively. Furthermore, except the intact pea cells promoting the abundance of butyrate producer Roseburia, a similar fermentation pattern was found between intact and broken cells suggesting that macro-nutrient types, rather than structure, dominate the production of metabolites in colonic fermentation. The findings elucidate how the food compositional difference can modulate the gut microbiome and thus provide the knowledge to design whole food legumes-based functional foods.

The Mechanism of Extraction of Peanut Protein and Oil Bodies by Enzymatic Hydrolysis of the Cell Wall.

Degradation of the peanut cell wall is a critical step in the aqueous enzymatic extraction process to extract proteins and oil bodies. Viscozyme® L, a compound cell wall degrading enzyme, has been applied as an alternative to protease in the process of aqueous enzymatic extraction, but the mechanism of cell wall enzymolysis remains unclear. The present study aims to investigate the changes in cellulose, hemicellulose, and pectin content of the peanut cell wall hydrolyzed by Viscozyme® L. The degree to which the main components of the peanut cell wall, such as trans-1, 2-cyclohexanediamine-N,N,N',N'-acetic acid-soluble pectin (CDTA-soluble pectin), Na2CO3-soluble pectin, cellulose, and hemicellulose, are degraded is closely related to the extraction of oil bodies and peanut protein at different solid-liquid ratio of powered peanut seed in distilled water, enzyme concentration, enzyme hydrolysis temperature, and enzyme hydrolysis time. The key sites of Viscozyme® L activity on cell wall polysaccharides were explored by comparing the changes in chemical bonds under different extraction conditions using Fourier-transform infrared spectroscopy (FT-IR) absorption bands and principal component analysis (PCA). Viscozyme® L acted on the C-O stretching, C-C stretching, and CH2 symmetrical bending of cellulose, the C-O stretching and O-C-O asymmetrical bending of hemicellulose, and the C-O stretching and C-C stretching of pectin.

Size and Charge Stability of Oil Bodies from Peanut

In order to offer scientific bases for the application of oil bodies from peanut in food, this research was undertaken to study the size and charge stability of oil bodies from five peanut varieties. It showed that the mean diameter of oil bodies fromyuhua9719andyuhua9830is obviously larger thanyuhua23,yuhua27, andyuhua9502in the peanut cell. Moreover, the analysis of diameter distribution of oil bodies also showed that the median diameter of oil bodies increased dramatically in the order ofyuhua9719&gt;yuhua9830&gt;yuhua23&gt;yuhua27&gt;yuhua9502after aqueous extraction. The charge stability of oil bodies from peanut was observed with zeta (ζ) potential measurements, which indicated that charge properties and the absolute value of oil bodies from five peanut varieties were significantly affected by pH and salt concentration, but the degree of influence is different. Of the five peanut varieties,yuhua27andyuhua9830possessed excellent charge stability (ζ-potential&gt;35 mV) in neutral microenvironment without salt concentration.

Insights into the novel members of the FAD2 gene family involved in high-oleate fluxes in peanut.

The FAD2 gene family is functionally responsible for the conversion of oleic acid to linoleic acid in oilseed plants. Multiple members of the FAD gene are known to occur in several oilseed species. In this study, six novel full-length cDNA sequences (named as AhFAD2-1, -2, -3, -4, -5, and -6) were identified in peanut (Arachis hypogaea L.), an analysis of which revealed open reading frames of 379, 383, 394, or 442 amino acids. Sequence comparisons showed that AhFAD2-1 and AhFAD2-2 shared 76% identity, while AhFAD2-2, -3, and -4 displayed highly significant homology. There was only 27% identity overlap between the microsomal ω-6 fatty acid desaturase and the chloroplast ω-6 fatty acid desaturase encoded by AhFAD2-1, -2, -3, -4, and AhFAD2-5, -6, respectively. The phylogeny tree of FAD2 transcripts showed five major groups, and AhFAD2-1 was clearly separated from other groups. Analysis of AhFAD2-1 and AhFAD2-2 transcript distribution in different peanut tissues showed that the AhFAD2-1 gene showed upward of a 70-fold increase in expression of fatty acid than the AhFAD2-2 gene in peanut developing seeds, while the AhFAD2-2 gene expressed most abundantly in peanut flowers. Because the AhFAD2-1 gene played a major role in the conversion of oleic to linoleic acid during seed development, the identification of this novel member in this study would facilitate the further genetic manipulation of peanut oil quality. The implications of overall results also suggest that there may be more candidate genes controlling levels of oleate acid in developing seeds. Results also may be due to the presence of complex gene networks controlling the fluxes between the endoplasmic reticulum and the chloroplast within the peanut cells.

Investigation of cationic peanut peroxidase glycans by electrospray ionization mass spectrometry

Cationic peanut peroxidase (CP) was isolated from peanut ( Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by 1H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry.

Identification of two further cationic peroxidase isoenzymes secreted by peanut cells in suspension culture

Two cationic peroxidase isoenzymes have been identified, using ion-exchange and hydrophobic column chromatography, in the spent media of peanut cells maintained in suspension culture. One isoenzyme, designated CPz-I, has a molecular mass of 34 000 Da determined by SDS-PAGE and 33 610 ± 12 Da by MALDI-TOF mass spectrometry and a pI > 9.5 determined by isoelectric focusing. The N-terminal, and some internal amino acid sequences from trypsin-generated peptides have been obtained, but data-base sequence comparisons have identified no other peroxidases. The second isoenzyme, designated CPz-II, is a glycoprotein as determined by recognition by a monoclonal antibody (mAb 2.23) which is specific for the xylose/fucose containing complex plant N-glycan structure, and has a molecular mass of 33 500 Da determined by SDS-PAGE and 33 164 ± 17 Da determined by MALDI-TOF mass spectrometry, and a basic pI of 9.0 as determined by isoelectric focusing. N-terminal, and some internal amino acid sequences from trypsin-generated peptides, have been obtained and data-base searching revealed highest, but not complete, identity with the translated sequence of prxPNC-2, a cDNA isolated previously from the same peanut cell suspension culture system. This partial identity is confirmed by recognition of CPz-II by an antibody raised against the protein product of prxPNC-2 obtained by the expression of the cDNA in Escherichia coli.

Characterization of two class II chitinase genes from peanut and expression studies in transgenic tobacco plants.

Two different genes encoding class II chitinases from peanut (Arachis hypogaea L. cv. NC4), A.h.Chi2;1 and A.h.Chi2;2, have been cloned. In peanut cell suspension cultures, mRNA levels of A.h.Chi2;2 increased after ethylene or salicylate treatment and in the presence of conidia from Botrytis cinerea. The second gene, A.h.Chi2;1, was only expressed after treatment with the fungal spores. Transgenic tobacco plants containing the complete peanut A.h.Chi2;1 gene exhibited essentially the same expression pattern in leaves as observed in peanut cell cultures. Expression characteristics of transgenic tobacco carrying a promoter-GUS fusion of A.h.Chi2;1 are described.

Cellular composition of the angiofibromas in tuberous sclerosis.

The angiofibroma of tuberous sclerosis is associated with a proliferation of dermal spindle cells that have been considered to be fibroblasts on the basis of a variety of techniques, although some cells have a glial appearance. This study of six angiofibromas demonstrated an increase in S100-positive, peanut agglutinin-negative spindle cells in the dermis in addition to increased numbers of epidermal melanocytes. The second major spindle cell population in the dermis proved to be dermal dendrocytes based on their expression of factor XIIIa. The number of these cells decreases in older patients, in whom the lesions were generally less cellular and more fibrotic. The angiofibroma of tuberous sclerosis is a hamartoma involving increased numbers of dermal dendrocytes, neurosustentacular cells, blood vessels, and melanocytes, in addition to collagen.

Isolation of peroxidase from bean and millet compared with that from peanut

Purification of peanut (Arachis hypogaea L.) peroxidase in milligram amounts is feasible through its isolation from spent medium of cultured cells. Using the same and modified approaches for another cultured dicot and a monocot yielded only low amounts of peroxidase for bean (Phaseolus vulgaris) and millet (Setaria italica Beav.). We showed that the procedures used for millet rendered a purer fraction than the commercial crude fraction available for horseradish. The data suggest that cultured peanut cells in suspension culture are an ideal system for isolation of peroxidase in terms of the ease of purification and yield. Key words: peanut, bean, millet, peroxidase, purification, tissue.

The influence of sucrose and an elevated CO2 concentration on photosynthesis of photoautotrophic peanut (Arachis hypogaea L.) cell cultures

Using photoautotrophic cells ofArachis hypogaea (L.) growing at ambient CO2, it was shown that exogenous sucrose supplied to the liquid medium reduced14CO2 fixation (supplied as NaH14CO3). This was mostly due to a reduced labelling in P-esters, and to a lesser extent, in the serine/glycine moiety. However, radioactivity in the neutral sugar fraction was increased upon supplement of exogenous sucrose. The reduced labelling of P-esters and serine/glycine agrees with a lower concentration and specific activity of Rubisco in the sucrose supplied treatments as compared to the control. Following a transfer into a sugar free nutrient medium the concentration and activity of Rubisco is increased. The concentration of PEPCase was not influenced by sucrose application, although its specific activity was increased.

Effect of calcium, its inhibitors, and heavy metals on the growth cycle of peanut cell aggregates

During the normal growth cycle of peanut (Arachis hypogaea L.) cells, cultured in suspension medium, cell aggregates of 0.5 mm during late growth phase. Calmodulin rose to its original level during 0.5 but not of the <0.5 populations. At high (1 mM) concentrations these ions caused arrested growth. At low (10 μM) levels and in the presence of 3 mM calcium they had a synergistic effect.

Association of Calcium and Calmodulin to Peroxidase Secretion and Activation

Summary The effect of the addition of EGTA, chlorpromazine, or trifluoperazine to the medium of cultured peanut cells for 14 d caused alterations in the patterns of released proteins. The observations on the peroxidases, identified amongst these proteins, suggested that the cationic peroxidase (C.PRX) is probably calcium controlled and that the anionic peroxidase (A.PRX) is under control of calmodulin. These suggestions are further supported by in vitro experiments with EGTA lnormal enzyme activity of C.PRX at (A407nm = 0.395 x 106; Umol = 5.36 x 1011) to (A404-407nm = 0.467 x 106; Umol = 1.07 x 1012). Subsequent reconstitution of the enzyme with calcium restores activity to the basal level. Cadmium is capable at physiological ranges (µM) to substitute effectively for the calcium in C.PRX since it restores the enzyme activity as well as calcium does. Since the anionic isozyme is not so affected, it suggests a major difference between peroxidase isozymes.

Peanut peroxidase and the anionic component

Secreted peroxidase (EC 1.11.1.7) from cultured peanut (Arachis hypogaea L.) cells has two main isozymes, one cationic and one anionic. The cationic isozyme has been studied intensively owing to its greater availability. However, the several-fold lower yield of anionic peroxidase has limited study of this isozyme. In the current investigation, the yield of this isozyme from the spent medium is quantified. In addition, feedback by cationic peroxidase on its own synthesis is investigated. Key words: peanut, peroxidase, anionic, cationic, yield, feedback.

Characterization of two forms of cationic peroxidase from cultured peanut cells.

Two forms of cationic peroxidase from peanut cells were differentiated by concanavalin A affinity chromatography. They differed in molecular mass as well as concanavalin A binding, leading to the initial suggestion that they represented two isozymes of peroxidase. However, similar values for the specific activity, Soret absorption, calcium content, and peptide molecular mass were observed for each of the forms. Therefore, the binding and nonbinding fractions most likely represent two molecular forms of cationic peanut peroxidase, rather than two distinct cationic isozymes. The difference between these two forms is discussed in terms of glycosylation. Through the amino acid sequence analysis of the formic acid treated peptide, the cationic isozyme has been shown to be identical in amino acid sequence to the cDNA clone PNC1.

Preliminary crystallographic study of peanut peroxidase.

The cationic isozyme of peroxidase isolated from suspension cultures of peanut cells is a heme-containing and calcium-dependent glycoprotein having four covalently attached oligosaccharide chains. Attempts were made to crystallize the glycoprotein for X-ray diffraction analysis, and these have met with some success. Crystals have now been grown that are suitable for a full three-dimensional structural analysis. The crystals are thin plates and we have shown them to be of the orthorhombic space group P2(1)2(1)2(1) with a = 48.1, b = 97.2, c = 146.2 A. The crystals diffract to beyond 2.8 A resolution, appear to be stable to lengthy X-ray exposure, and contain two molecules of 40,000 daltons each in the asymmetric unit.

Immunological Assays on the Relation of Culture Medium Peroxidases to Those in Fractions Isolated from Peanut Cells

Summary Through the means of Ouchterlony gel diffusion, Western blot and competitive ELISA it was determined that A. PRX or C. PRX from culture medium and their corresponding isozymes from the cytoplasm and cell wall have a higher than 80% similarity. Quantitative assays indicated that the cell-wall fractions were largely represented by the major cationic peroxidase isolated from spent medium of peanut cells. Conversely the cytoplasmic- or soluble-cell fraction was mainly represented by the anionic isozyme isolated from that medium. Consequently the enriched medium peroxidases may be used for studies of events in the cytoplasm and cell wall.

Oxidation of tyrosine by peroxidase isozymes derived from peanut suspension culture medium and by isolated cell walls

A comparative study on tyrosine oxidation was made with a pure cationic and anionic peroxidase from peanut cell culture medium. The results showed that both isozymes possessed almost identical capacity to oxidize tyrosine to dityrosine, isodityrosine and polytyrosine with the main difference being the pH optimum (pH 4 for the anionic and pH 7 for the cationic isozyme). Variation of reaction time after 1.5 h incubation had little effect on the quantity and quality of the oxidation products. On the other hand, increase of enzyme units correspondingly increased tyrosine-oxidation. The removal of heme and carbohydrate moieties from the holoenzyme arrested the reaction thereby suggesting the role played by these moieties in stabilizing the active site of peroxidase isoenzymes. Isolated cell wall extracts catalyzed the tyrosine-oxidation equally well as the purified peroxidase. Even though polyclonal antibodies against anionic peroxidase inhibited the in vitro tyrosine reaction they did not affect the tyrosine oxidation by the cell walls, while the cationic antibodies did.