Effects of the Maillard Reaction on the Immunoreactivity of Amandin in Food Matrices.

Amandin is the major storage protein and allergen in almond seeds. Foods, containing almonds, subjected to thermal processing typically experience Maillard browning reaction. The resulting destruction of amino groups, protein glycation, and/or denaturation may alter amandin immunoreactivity. Amandin immunoreactivity of variously processed almond containing foods was therefore the focus of the current investigation. Commercial and laboratory prepared foods, including those likely to have been subjected to Maillard browning, were objectively assessed by determining Hunter L* , a* , b* values. The L* values for the tested samples were in the range of 31.75 to 85.28 consistent with Maillard browning. Three murine monoclonal antibodies, 4C10, 4F10, and 2A3, were used to determine the immunoreactivity of the targeted samples using immunoassays (ELISA, Western blot, dot blot). The tested foods did not exhibit cross-reactivity indicating that the immunoassays were amandin specific. For sandwich ELISAs, ratio (R) of sample immunoreactivity to reference immunoreactivity was calculated. The ranges of R values were 0.67 to 15.19 (4C10), 1.00 to 11.83 (4F10), and 0.77 to 23.30 (2A3). The results of dot blot and Western blot were consistent with those of ELISAs. Results of these investigations demonstrate that amandin is a stable marker protein for almond detection regardless of the degree of amandin denaturation and/or destruction as a consequence of Maillard reaction encountered under the tested processing conditions. PRACTICAL APPLICATION: Foods containing almond are often subjected to processing prior to consumption. Amandin, the major allergen in almond, may experience Maillard reaction. Understanding the change in amandin immunoreactivity as a result of Maillard reaction is important for amandin detection and production of hypoallergenic food products.

A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (<15% CV) and reproducible (intra- and inter assay variability <15% CV). The assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

Solubilization and electrophoretic characterization of select edible nut seed proteins.

The solubility of almond, Brazil nut, cashew nut, hazelnut, macadamia, pecan, pine nut, pistachio, walnut, and peanut proteins in several aqueous solvents was qualitatively and quantitatively assessed. In addition, the effects of extraction time and ionic strength on protein solubility were also investigated. Electrophoresis and protein determination (Lowry, Bradford, and micro-Kjeldahl) methods were used for qualitative and quantitative assessment of proteins, respectively. Depending on the seed, buffer type and ionic strength significantly affected protein solubility. The results suggest that buffered sodium borate (BSB; 0.1 M H(3)BO(3), 0.025 M Na(2)B(4)O(7), 0.075 M NaCl, pH 8.45) optimally solubilizes nut seed proteins. Qualitative differences in seed protein electrophoretic profiles were revealed. For a specific seed type, these differences were dependent on the solvent(s) used to solubilize the seed proteins. SDS-PAGE results suggest the polypeptide molecular mass range for the tree nut seed proteins to be 3-100 kDa. The results of native IEF suggested that the proteins were mainly acidic, with a pI range from >4.5 to <7.0. Western immunoblotting experiments indicated that rabbit polyclonal antibodies recognized substantially the same polypeptides as those recognized by the corresponding pooled patient sera IgE.

Starch-entrapped microspheres extend in vitro fecal fermentation, increase butyrate production, and influence microbiota pattern.

Previous research has revealed that waxy corn starch which has been entrapped in a matrix of electrostatically cross-linked alginate, shows a slow digestion rate such that much of the starch may reach the colon; thus making this a new type of resistant starch. The purpose of this research was to test the fermentative properties of starch-entrapped microspheres using a batch fecal fermentation method. Fermentation of starch-entrapped microspheres showed significantly lower rates of gas production compared to waxy corn starch, and showed significant increases in total SCFAs during the latter stages of fermentation (24-48 h), whereas waxy corn starch did not. Cooking the starch-entrapped microspheres increased the amount of SCFAs and the molar fraction of butyrate produced during fermentation. Bacterial fingerprinting revealed that uncooked starch-entrapped microspheres have a unique effect on the microbiota that is different from waxy corn starch alone, but cooking causes a shift toward a pattern more closely resembling that of the starch. Starch-entrapped microspheres may deliver slowly fermentable carbohydrate to the colon, with the ability to influence the microbiota. Further human studies are required to determine whether these characteristics occur in vivo.

Starch-entrapped biopolymer microspheres as a novel approach to vary blood glucose profiles.

BACKGROUND: With emerging knowledge of the impact of the metabolic quality of glycemic carbohydrates on human health, there is a need for novel carbohydrate ingredients that can be custom-made to deliver controlled amounts of glucose to the body and to test hypotheses on the postprandial metabolic consequences of carbohydrates. OBJECTIVE: The goal of the present study was to demonstrate the applicability and action of starch-entrapped biopolymer microspheres as customized, novel, slowly digestible carbohydrates to obtain desired glycemic responses. METHODS: Starch-entrapped microspheres were developed; and starch digestion and glucose release, subsequent to their cooking (100 degrees C, 20 min) in water, were initially monitored by measuring the rapidly digestible, slowly digestible, and resistant starch fractions using the in vitro Englyst assay. Glycemic and insulinemic responses after consumption of glucose and two different slowly digestible starch microsphere diets were compared using a crossover study in 10 healthy individuals. The mechanism of starch digestion in the microspheres was elucidated from scanning electron microscopic images of the in vitro digested microspheres. RESULTS: Factors such as biopolymer type and concentration, microsphere size, and starch type were manipulated to obtain starch materials with defined amounts of slowly digestible starch based on in vitro studies. Scanning electron microscopy showed that cooked starch entrapped in the dense biopolymer matrix is digested layer by layer from the outside to the inside of the microsphere. Glycemic and insulinemic responses to microsphere test diets were moderate as compared to a glucose diet, but more important, they showed extended glucose release. CONCLUSIONS: Starch-entrapped microspheres provide a useful tool to study the postprandial metabolic consequences of slowly digestible carbohydrates.

Effects of processing on immunoreactivity of cashew nut (Anacardium occidentale L.) seed flour proteins.

Cashew nut seeds were subjected to processing including autoclaving (121 degrees C for 5, 10, 20, and 30 min), blanching (100 degrees C for 1, 4, 7, and 10 min), microwave heating (1 and 2 min each at 500 and 1000 W), dry roasting (140 degrees C for 20 and 30 min; 170 degrees C for 15 and 20 min; and 200 degrees C for 10 and 15 min), gamma-irradiation (1, 5, 10, and 25 kGy), and pH (1, 3, 5, 7, 9, 11, and 13). Proteins from unprocessed and processed cashew nut seeds were probed for stability using anti-Ana o 2 rabbit polyclonal antibodies and mouse monoclonal antibodies directed against Ana o 1, Ana o 2, and Ana o 3 as detection agents. Results indicate that Ana o 1, Ana o 2, and Ana o 3 are stable regardless of the processing method to which the nut seeds are subjected.

Biochemical characterization of soluble proteins in pecan [Carya illinoinensis (Wangenh.) K. Koch].

Pecans (cv. Desirable) contained approximately 10% protein on a dry weight basis. The minimum nitrogen solubility (5.9-7.5%) at 0.25-0.75 M trichloroacetic acid represented the nonprotein nitrogen. Among the solvents assessed for protein solubilization, 0.1 M NaOH was the most effective, while borate saline buffer (pH 8.45) was judged to be optimal for protein solubilization. The protein solubility was minimal in the pH range of 3-7 and significantly increased on either side of this pH range. Increasing the NaCl concentration from 0 to 4 M significantly improved ( approximately 8-fold increase) protein solubilization. Following Osborne protein fractionation, the alkali-soluble glutelin fraction (60.1%) accounted for a major portion of pecan proteins followed by globulin (31.5%), prolamin (3.4%), and albumin (1.5%), respectively. The majority of pecan polypeptides were in the molecular mass range of 12-66 kDa and in the pI range of 4.0-8.3. The pecan globulin fraction was characterized by the presence of several glycoprotein polypeptides. Lysine was the first limiting essential amino acid in the defatted flour, globulin, prolamin, and alkaline glutelin fractions. Leucine and tryptophan were the first limiting essential amino acids in albumin and acid glutelin fractions, respectively. Rabbit polyclonal antibodies detected a range of pecan polypeptides in the 12-60 kDa range, of which the globulin fraction contained the most reactive polypeptides.

Enzyme-linked immunosorbent assay (ELISA) for detection of sulfur-rich protein (SRP) in soybeans (Glycine max L.) and certain other edible plant seeds.

As a result of methionine deficiency, legume proteins are considered to be incomplete, and therefore there is a need to explore ways to improve legume protein amino acid balance. Using rabbit anti-soybean sulfur-rich protein (SRP) polyclonal antibodies (pAb), sensitive immunoassays (nanogram sensitivity) were developed. The immunoassays detected SRP in all soybean seeds and soybean-based commercial samples examined. In addition, the presence of pAb cross-reactive proteins was detected in certain dry beans and oilseeds. The cross-reactive proteins were isolated using purified IgG-based immunoaffinity column chromatography. Biochemical analyses including N-terminal amino acid sequencing and amino acid composition indicated that the cross-reactive proteins were comparable to soybean SRP. The cross-reactive proteins contained methionine (1.6-2.4 residues/100 residues) and cysteine (2.4-3.6 residues/100 residues), which satisfies the FAO/WHO recommended pattern for sulfur amino acids in both adults and children (2-5 years old). The results suggest the presence of constitutive SRPs in several dry beans and oilseeds.

Biochemical composition and immunological comparison of select pecan [Carya illinoinensis (Wangenh.) K. Koch] cultivars.

On an edible portion basis, pecan moisture, protein, lipid, total soluble sugars, and ash contents ranged from 2.1% to 6.4%, 6.0% to 11.3%, 65.9% to 78.0%, 3.3% to 5.3%, and 1.2% to 1.8%, respectively. With the exception of a high tannin (2.7%) Texas seedling, pecan tannin content was in a narrow range (0.6-1.85%). Unsaturated fatty acids (>90%) dominated pecan lipid composition with oleic (52.52-74.09%) and linoleic (17.69-37.52%) acids as the predominant unsaturated fatty acids. Location significantly influenced pecan biochemical composition. Pecan lipid content was negatively correlated with protein (r = -0.663) and total sugar (r = -0.625). Among the samples tested using SDS-PAGE a common pattern, with minor differences, in subunit polypeptide profiles was revealed. Rabbit polyclonal antibody-based immunoblotting experiments (Western blot) also illustrated the similarity in polypeptide profiles with respect to immunoreactivity. All tested cultivars registered similar immunoreactivity when their protein extracts (each at 1 mg/mL) were assessed using inhibition ELISAs (mean +/- standard deviation = 0.89 +/- 0.20; n = 27) with the USDA "Desirable" cultivar as the reference standard (immunoreactivity designated as 1.0).

A circular dichroism and fluorescence spectrometric assessment of effects of selected chemical denaturants on soybean (Glycine max L.) storage proteins glycinin (11S) and beta-conglycinin (7S).

Soybean glycinin (11S) and beta-conglycinin (7S) were subjected to select chemical treatments at various concentrations and resulting changes in protein structures were investigated by circular dichroism (CD) and fluorescence spectrometry. Fluorescence quenching results indicated that urea >/=3 M caused significant unfolding of 11S, but not that of 7S. GuHCl was more effective than urea in denaturation of 11S. A two-step transition in 11S structure was observed with a possible existence of a folding intermediate at 2.5 M GuHCl. Sodium dodecyl sulfate (SDS) measurably altered secondary and tertiary structures of 11S and 7S below SDS critical micellar concentration (CMC), possibly due to formation of mixed peptide-SDS micelles. SDS treatment increased alpha-helical and unordered structures of both proteins at the expense of beta-sheet structure. NaCl and CaCl 2 caused a significant decrease in fluorescence intensity without shifting emission lambda max. Exposure of 7S and 11S to NaSCN respectively at >/=0.3 and >/=0.6 M NaSCN caused a significant increase in fluorescence intensity measured at the corresponding lambda max of the protein. beta-Mercaptoethanol (beta-ME), N-ethylmaleimide (NEM), and phytic acid caused variable red shifts, 2.5-4 nm, in the emission lambda max.

Starch with a slow digestion property produced by altering its chain length, branch density, and crystalline structure.

The hypothesis of increasing the branch density of starch to reduce its digestion rate through partial shortening of amylopectin exterior chains and the length of amylose was investigated. Starch products prepared using beta-amylase, beta-amylase and transglucosidase, maltogenic alpha-amylase, and maltogenic alpha-amylase and transglucosidase showed significant reduction of rapidly digested starch by 14.5%, 29.0%, 19.8%, and 31.0% with a concomitant increase of slowly digested starch by 9.0%, 19.7%, 5.7%, and 11.0%, respectively. The resistant starch content increased from 5.1% to 13.5% in treated starches. The total contents of the prebiotics isomaltose, isomaltotriose, and panose (Isomaltooligosaccharides) were 2.3% and 5.5%, respectively, for beta-amylase/transglucosidase- and maltogenic alpha-amylase/transglucosidase-treated starches. The molecular weight distribution of enzyme-treated starches and their debranched chain length distributions, analyzed using high-performance size-exclusion chromatography with multiangle laser light scattering and refractive index detection (HPSEC-MALLS-RI) and HPSEC-RI, showed distinctly different patterns among starches with different enzyme treatments. A larger proportion of low molecular weight fractions appeared in starches treated additionally with transglucosidase. All enzyme-treated starches showed a mixture of B- and V-type X-ray diffraction patterns, and 1H NMR spectra showed a significant increase of alpha-1,6 linkages. Both the increase of the starch branch density and the crystalline structure in the treated starches likely contribute to their slow digestion property.

Structural basis for the slow digestion property of native cereal starches.

Native cereal starches are ideal slowly digestible starches (SDS), and the structural basis for their slow digestion property was investigated. The shape, size, surface pores and channels, and degree of crystallinity of starch granules were not related to the proportion of SDS, while semicrystalline structure was critical to the slow digestion property as evidenced by loss of SDS after cooking. The high proportion of SDS in cereal starches, as compared to potato starch, was related to their A-type crystalline structure with a lower degree of perfection as indicated by a higher amount of shortest A chains with a degree of polymerization (DP) of 5-10. The A-type amorphous lamellae, an important component of crystalline regions of native cereal starches, also affect the amount of SDS as shown by a reduction of SDS in lintnerized maize starches. These observations demonstrate that the supramolecular A-type crystalline structure, including the distribution and perfection of crystalline regions (both crystalline and amorphous lamellae), determines the slow digestion property of native cereal starches.

Chemical composition of selected edible nut seeds.

Commercially important edible nut seeds were analyzed for chemical composition and moisture sorption. Moisture (1.47-9.51%), protein (7.50-21.56%), lipid (42.88-66.71%), ash (1.16-3.28%), total soluble sugars (0.55-3.96%), tannins (0.01-0.88%), and phytate (0.15-0.35%) contents varied considerably. Regardless of the seed type, lipids were mainly composed of mono- and polyunsaturated fatty acids (>75% of the total lipids). Fatty acid composition analysis indicated that oleic acid (C18:1) was the main constituent of monounsaturated lipids in all seed samples. With the exception of macadamia, linoleic acid (C18:2) was the major polyunsaturated fatty acid. In the case of walnuts, in addition to linoleic acid (59.79%) linolenic acid (C18:3) also significantly contributed toward the total polyunsaturated lipids. Amino acid composition analyses indicated lysine (Brazil nut, cashew nut, hazelnut, pine nut, and walnut), sulfur amino acids methionine and cysteine (almond), tryptophan (macadamia, pecan), and threonine (peanut) to be the first limiting amino acid as compared to human (2-5 year old) amino acid requirements. The amino acid composition of the seeds was characterized by the dominance of hydrophobic (range = 37.16-44.54%) and acidic (27.95-33.17%) amino acids followed by basic (16.16-21.17%) and hydrophilic (8.48-11.74%) amino acids. Trypsin inhibitory activity, hemagglutinating activity, and proteolytic activity were not detected in the nut seed samples analyzed. Sorption isotherms (Aw range = 0.08-0.97) indicated a narrow range for monolayer water content (11-29 mg/g of dry matter). No visible mold growth was evident on any of the samples stored at Aw < 0.53 and 25 degrees C for 6 months.

Antigenic stability of pecan [Carya illinoinensis (Wangenh.) K. Koch] proteins: effects of thermal treatments and in vitro digestion.

Rabbit polyclonal antibody-based inhibition ELISA as well as immunoblotting analyses of proteins extracted from variously processed pecans (cv. Desirable) indicate that pecan proteins are antigenically stable. Pecan antigens were more sensitive to moist heat than dry heat processing treatments. SDS-PAGE and immunoblotting analysis of the native and heat-denatured proteins that were previously subjected to in vitro simulated gastric fluid digestions indicate that stable antigenic peptides were produced. Both enzyme-to-substrate ratio and digestion time were influential in determining the stability of pecan polypeptides. The stable antigenic polypeptides may serve as useful markers in developing assays suitable for the detection of trace amounts of pecans in foods.

Almond (Prunus dulcis L.) protein quality.

Three marketing varieties of almonds; Carmel, Mission, and Nonpareil; were analyzed for proximate composition and protein nutritive quality. Moisture, lipids, protein, ash, sugars, and tannins ranges were 3.05-4.33%, 43.37-47.50%, 20.68-23.30%, 3.74-4.56%, 5.35-7.45%, and 0.12-0.18%, respectively. No detectable hemagglutinating and trypsin inhibitory activities were present in Carmel, Mission, and Nonpareil almonds. Amino acid analyses indicated the sulfur amino acids (methionine + cysteine), lysine, and threonine to be the first, second, and third limiting amino acids in almonds when compared to the recommended amino acid pattern for children 2-5-year old. However, compared to the recommended amino acid pattern for adults, sulfur amino acids were the only limiting amino acids in almonds tested. True Protein Digestibility (% TPD) values for Carmel, Mission, and Nonpareil were 88.55 +/- 1.26, 92.25 +/- 1.05, and 82.62 +/- 1.47, respectively. Protein Digestibility Corrected Amino Acid Scoring (PDCAAS) values suggested almond proteins to be of poor nutritional quality.

Phaseolin in vitro pepsin digestibility: role of acids and phenolic compounds.

Great Northern bean (Phaseolus vulgaris L.) phaseolin proteolysis at 37 degrees C, varying HCl concentrations (10 mM to 1 M), phaseolin:pepsin ratios ranging from 5:1 to 100:1 (w/w), and incubation times up to 24 h was investigated. The results suggest that phaseolin is not resistant to in vitro pepsin hydrolysis. At a phaseolin-to-pepsin ratio of 100:1 (w/w), native phaseolin was completely digested in 24 h when incubated in 50 mM HCl, while heat-denatured phaseolin (30 min at 100 degrees C, boiling water bath) was digested in 1 h under similar conditions. When incubated at 37 degrees C for 24 h, acid alone, even at as low a concentration as 10 mM, caused a partial breakdown of native phaseolin. The degree of phaseolin hydrolysis by HCl was dependent on the acid concentration used. The rate of native phaseolin hydrolysis increased with increasing HCl concentration rather than pepsin concentration. Common food acids were able to partially hydrolyze phaseolin. Among the food acids tested, oxalic acid was the most effective in hydrolyzing phaseolin. Spectroscopic studies revealed a significant change in secondary and tertiary structures when native phaseolin was incubated in dilute HCl. None of the tested phenolic compounds adversely affected phaseolin hydrolysis by pepsin.

Isolation, purification, and biochemical characterization of a novel water soluble protein from Inca peanut (Plukenetia volubilis L.).

A water soluble storage albumin from Inca peanut (IPA) accounted for approximately 25% (w/w) of defatted seed flour weight, representing 31% of the total seed protein. IPA is a 3S storage protein composed of two glycosylated polypeptides, with estimated molecular weights (MW) of 32800 and 34800 Da, respectively. IPA has an estimated sugar content of 4.8% +/- 0.92% (n = 6). IPA is a basic protein (pI of approximately 9.4) and contains all of the essential amino acids in adequate amounts when compared to the FAO/WHO recommended pattern for a human adult. The tryptophan content of IPA is unusually high (44 mg/g of protein), whereas the phenylalanine content is low (9 mg/g of protein). IPA is a highly digestible protein in vitro.

Biochemical characterization of amandin, the major storage protein in almond (Prunus dulcis L.).

The almond major storage protein, amandin, was prepared by column chromatography (amandin-1), cryoprecipitation (amandin-2), and isoelectric precipitation (amandin-3) methods. Amandin is a legumin type protein characterized by a sedimentation value of 14S. Amandin is composed of two major types of polypeptides with estimated molecular weights of 42-46 and 20-22 kDa linked via disulfide bonds. Several additional minor polypeptides were also present in amandin. Amandin is a storage protein with an estimated molecular weight of 427,300 +/- 47,600 Da (n = 7) and a Stokes radius of 65.88 +/- 3.21 A (n = 7). Amandin is not a glycoprotein. Amandin-1, amandin-2, and amandin-3 are antigenically related and have similar biochemical properties. Amandin-3 is more negatively charged than either amandin-1 or amandin-2. Methionine is the first essential limiting amino acid in amandin followed by lysine and threonine.