Mahesh Venkatachalam

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.

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.

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°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.

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.

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.

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.

Methods to slow starch digestion rate in functional cereal products.

Publisher Summary Starch, being the principal component in cereals and tubers, is the major carbohydrate related to postprandial glycemia and Glycaemic Index (GI) of foods. Carbohydrates modified for lower GI would be desirable as ingredients in functional cereal and other products for certain groups, that is, diabetics, and possibly for other improvements in general health. The current understanding of GI, particularly in relation to slowly digestible cereal starches—in terms of their structure and manufacturing approaches, is the focus of this chapter. Strategies for producing slowly digesting/low-GI functional cereal-based foods could include creating proper food forms and incorporating particular ingredients into foods, such as starches with the slow digesting property that extend glucose release, some non-digesting carbohydrates, Dietary Fiber (DF), Resistant Starch (RS), including viscosity-increasing polysaccharides that delay gastric emptying or decrease digestive enzyme access, organic acids and their salts to slow down gastric emptying, or anti-nutritional agents that inhibit digestion of starch, and other low-glycemic carbohydrates. Most importantly, human trials must show what levels of such carbohydrate-based ingredients or foods are needed to elicit a positive physiological or metabolic response. Food form will continue to be a major issue in the development of slow digesting cereal foods. Highly organized, dense food forms impede starch digestion and, thereby lower the glycemic response of starchy foods. Besides pasta, whole kernel foods, wherein the cellular layers surrounding the starch granules are intact, also present an example of an organized food form with low GI and probable slow digestion property.