Effect of roasting history and buffer composition on peanut protein extraction efficiency

Peanut is a major allergenic food. Undeclared peanut (allergens) from mis-formulation or contamination during food processing pose a potential risk for sensitized individuals and must be avoided. Reliable detection and quantification methods for food allergens are necessary in order to ensure compliance with food labelling and to improve consumer protection. The extraction of proteins from allergenic foods and complex food products is an important step in any allergen detection method. In this study, the protein extraction efficiency of various buffers prepared in-house and some extraction buffers included in some commercial allergen enzyme-linked immunosorbent assay (ELISA) test kits for peanut determination in food products were tested. In addition, the effect of roasting history on the extractability of peanut protein was investigated by the biuret and the bicinchoninic acid (BCA) assays. Elevated roasting temperatures in food processing were found to have a major impact on protein extraction efficiency by reducing protein yields of oil and dry roasted peanuts by 50-75% and 75-80%, respectively, compared with the raw material. Extraction buffers operating in the higher pH range (pH 8-11) showed best yields.     

Investigation on sequential extraction of peanut allergens for subsequent analysis by ELISA and 2D gel electrophoresis

A two-step sequential extraction method of peanut proteins was proposed with the aim to investigate the protein composition and allergen content of peanut samples. The extraction procedure reported is fully compatible with subsequent analysis by enzyme-linked immunosorbent assays (ELISA) as well as 2D gel electrophoresis (2D PAGE). This sequential extraction method was used to study three different peanut varieties and three different types of food processing. Peanuts were analysed for total protein content and the extraction efficiency of raw and processed peanuts was determined. The total protein content of the three peanut varieties was found to be comparable, but their extraction efficiency varies. The peanut extracts were characterised by employing three different ELISA test kits specific to either the allergens Ara h 1 or Ara h 2, or to soluble peanut proteins. The content of both Ara h 1 and Ara h 2 differed in the raw peanut extracts of the three varieties. However, thermal processing resulted in much larger changes in detectability. Blanching significantly increases the detectability of Ara h 2, whereas Ara h 1 detection remains almost unchanged. After roasting a clear decrease of detectability was observed for both Ara h 1 and Ara h 2, although the effect is more severe for Ara h 1. 2D PAGE was employed to compare the protein profiles and abundances of peanut extracts. Statistically relevant differences were observed for the two different protein fractions obtained by using the described method, showing the relevance of this two-step sequential extraction method.     

加工方式和蛋白提取方法对花生蛋白致敏性的影响

加工方式影响花生蛋白的致敏性。本实验系统的研究了中国传统花生制品(水煮和油炸花生)和美国常食用的烘烤花生致敏性的差异,并比较了不同蛋白提取缓冲溶液对花生致敏蛋白提取效率的影响。结果表明:不同花生制品在不同的缓冲溶液中蛋白提取效率不同,水煮和油炸并没有较烘烤方式降低致敏蛋白的数量,而降低花生蛋白的致敏性。因此加工方式并不是导致中国花生过敏发病率低的主要原因。     

Allergenicity of roasted peanuts treated with a non-human digestive protease

Peanut allergy is a severe and lifelong type of food allergy triggered by allergenic proteins and peptides in peanuts. This study investigated the effects of ultrasound-assisted alcalase treatment on the concentrations of major allergenic proteins (Ara h 1 and Ara h 2) in roasted peanut kernels and the allergenicity of treated peanut extracts. Peanut kernels were sonicated for 1 h in buffer solution, incubated with different amount of alcalase for various time, then vacuum dried. The variations of Ara h 1 and Ara h 2 contents in soluble and insoluble portions of peanuts treatments were evaluated by sandwich ELISA and SDS-PAGE, respectively. The in vitro IgE-binding capacity of treated peanut extracts was determined by a competitive inhibition ELISA using pooled plasma of 10 peanut allergic patients. Samples with lower in vitro IgE-binding were used for human skin prick tests (SPTs) in peanut allergic individuals. Results indicate that alcalase digestion of sonicated peanuts significantly increased protein solubility while decreasing Ara h 1 and Ara h 2 concentrations in both soluble and insoluble portions of peanuts relative to untreated peanuts. The maximum reductions of Ara h 1 and Ara h 2 levels were obtained following 3 hour digestion with alcalase at concentrations of 4.54 and 6.05 U/100 g. Samples obtained under these conditions showed the lowest in vitro IgE-binding and caused the least allergic response in human SPTs. The current study suggests that the allergenic potential of peanuts could be reduced by postharvest processing such as ultrasound-assisted enzymatic treatment of peanuts kernels.     

Influence of different extraction conditions on the detection of glycinin and β-conglycinin in model processed foods by ELISA

ABSTRACT

The presence of undeclared soy proteins in food can cause severe reactions in soy allergic individuals. The extraction of target proteins from processed foods is a crucial step in allergen detection by immunoassays, as only successfully extracted target proteins can be detected by the specific antibodies. The effectiveness was studied of different conditions (type of buffer, temperature and time of incubation) on the extraction of total protein, and concentration of glycinin and β-conglycinin from different food matrices. The yields were determined using a soy protein isolate and three processed foods (sausage, bread and pâté) incurred with soy proteins. The yields were affected by the processing of analysed products and the composition and pH of the extraction buffers. Neutral and alkaline buffers (pH from 7.4 to 10.6) exhibited good protein extraction capacity and detectability of the specific target proteins. Denaturing additives and highly alkaline buffer (pH 12) extracted more crude protein but they were incompatible with the ELISA assay. Overall, the best results were obtained using phosphate (pH 7.4) and Tris/HCl (pH 8.5) buffers in the presence of 0.5 M NaCl. Crude protein yield of food extracts did not correlate with that of glycinin and β-conglycinin, whereas a good relationship was found between the yields of the two proteins.     

质谱法分析烘焙对花生过敏原Ara h 1潜在致敏性的影响

为研究烘焙对花生过敏原Ara h 1潜在致敏性的影响,采用高离液序列盐溶液从鲜花生和烘焙花生中提取总蛋白,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析烘焙前后蛋白条带变化情况,并对其中的花生主要过敏蛋白Ara h 1条带进行质谱和Swiss-Model模型分析.十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示,烘焙花生蛋白出现...     

Proportion of aflatoxin B1 contaminated kernels and its concentration in imported peanut samples

Moldy and split peanut kernels were separated from peanuts exported from Brazil, Sudan, India and Taiwan by visual inspection. The remaining peanuts from Brazil, Sudan and India were roasted lightly and the skins were removed. Stained peanuts were separated from the others. Aflatoxin was detected in moldy and stained peanuts. There was a positive correlation between % of aflatoxin-contaminated peanut kernels and aflatoxin B1 concentration in whole samples. Aflatoxin concentration of moldy peanuts was higher than that of stained peanut kernels.     

Digestibility of peanut and hazelnut allergens investigated by a simple in vitro procedure

 Stability under digestion is thought to be an important prerequisite determining the allergenicity of food proteins. To test this hypothesis, two important allergenic plant-derived foods were selected for this investigation. Protein extracts from roasted peanuts and unprocessed (native) hazelnuts were digested by a static, two-step in vitro procedure with commercial enzyme tablets containing peptic and pancreatic enzymes, respectively. The food extracts were subjected to gastric digestion for 2 h followed by a 45-min treatment under duodenal conditions. Undigested control samples and the two digests were investigated by analytical sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), by SDS-PAGE immunoblotting, by an enzyme allergosorbent test (EAST) with human IgE, and by a rat basophil leukaemia (RBL) cell mediator release assay that depends on specific IgE raised in mice. Peanut proteins appeared to be more stable under digestion than hazelnut proteins, as shown by electrophoresis. These results were also underlined by immunblot data. The gastric digest from peanut contained various protein fragments that were detected by antibodies from a peanut-specific rabbit antiserum and by IgE from patients allergic to peanuts. These immunoblot reactivities decreased strongly after subsequent pancreatic digestion. In the gastric digest from hazelnuts, a rabbit antiserum with a broad reactivity against native hazelnut proteins exclusively recognized small protein fragments of <15 kDa. This serum showed no binding to blots of the pancreatic digest. Sera from hazelnut-allergic patients presented IgE reactivities to an 18-kDa major allergen with homology to major tree-pollen allergens, to a minor allergen of 12 kDa, and to multiple bands  1 30 kDa in native hazelnut extract. No binding was observed with these sera on blot strips of the two digests prepared from hazelnut extract. Under the native conditions of EAST, both digests from peanuts strongly reacted with human IgE. Their IgE binding capacity persisted at a level of about 50% when compared to undigested peanut. In the case of hazelnuts the IgE reactivity of the untreated samples was reduced to <10% by both gastric and combined gastric/duodenal digestion for a serum pool prepared from four patients and sera from three additional participants. By contrast, a constantly high immunoreactivity of the hazelnut digests was detected with serum from one patient. The results of the EAST were confirmed by dose-related mediator release experiments performed with RBL cells passively sensitized with allergen-specific murine IgE. As a whole, our results indicated that the EAST and the RBL cell assay are superior to immunoblotting for the immunologic testing of digests. In accordance with clinical observations, the allergenicity of peanut proteins was very persistent during digestion, whereas the native birch-pollen-related hazelnut allergens appeared to be relatively labile under identical conditions. Received: 6 July 1998     

Modeling of Roasted Peanut Flavor for Some Virginia-Type Peanuts from Amino Acid and Sugar Contents

Prediction of sensory scores of roasted peanuts through analysis of the chemical components of raw peanuts is a goal of current research. Thirty samples of Virginia-type peanuts selected for wide variation in flavor precursors (amino acids and sugars) were roasted and evaluated by the Critical Laboratory Evaluation Roast (CLER) technique and by a flavor profile panel. Stepwise MAXR procedure was used to develop mathematical models for predicting both CLER scores and roasted peanut flavor ratings for the flavor profile. The best 10 variable model for predicting CLER scores had an R² of 0.969. A good fit (R²= 0.762) was obtained for predicting CLER score from only raw peanut data. The best 10 variable model for predicting roasted peanut flavor gave an R² of 0.928.     

IMPROVEMENT OF OXIDATIVE STABILITY OF ROASTED PEANUTS BY EDIBLE COATINGS AND ULTRASONICATION

In this study, an innovative method was developed to improve the shelf life of roasted peanuts. Sonication was combined with edible coating for enhancing the oxidative stability of roasted peanuts. Georgia green runner peanuts were roasted, subjected to sonication and then coated with whey protein isolate (WPI), ZEIN and carboxymethyl cellulose (CMC). Relative to the control, the oxidative stability of roasted-coated samples was improved by 80, 38 and 5% for CMC, WPI and ZEIN coating, respectively, while roasted-sonicated-coated samples were improved by 91, 52 and 27% for CMC, WPI and ZEIN coating, respectively. Sonication prior to coating resulted in 11, 14 and 22% improvement beyond the CMC, WPI and ZEIN coatings, respectively. Texture analysis showed there were no significant differences ( P <  0.05) in peanut texture between the treated and the control. Color results showed the HunterLab color parameters L, a, and b for most of the treatments did not have significant differences ( P <  0.05) compared with the control.

PRACTICAL APPLICATIONS

Edible coatings used in this study (carboxymethyl cellulose, whey protein isolate and ZEIN) were capable of acting as oxygen barriers to reduce peanut lipid rancidity. This research demonstrated the potential of power ultrasound to remove lipids from the peanut surfaces and improve coating adhesion. The texture and the color of coated peanuts did not change over the storage period. This study indicated that edible coatings in combination with sonication provided an alternative way for improving the oxidative stability and eventually the shelf life and quality of roasted peanuts.     

The occurrence of aflatoxins in peanuts imported into Czechoslovakia for human consumption

Results of 492 analyses for aflatoxin in raw shelled peanuts imported into Czechoslovakia during 1982-1984 are presented. Most samples (55.3%) had aflatoxin content less than the detection limit of the radioimmunochemical screening method (0.8 micrograms/kg). Further analyses showed that 239 out of 410 samples of roasted peanuts contained aflatoxin below the detection limit. Only 1.9% of all peanut samples were found to have contamination level more than 5 micrograms/kg aflatoxin. The highest levels of aflatoxin observed were in a raw peanut sample containing 202.1 micrograms/kg and in a roasted peanut sample containing 32.6 micrograms/kg.     

Enzymatic treatment of peanut kernels to reduce allergen levels

This study investigated the use of enzymatic treatment to reduce peanut allergens in peanut kernels as affected by processing conditions. Two major peanut allergens, Ara h 1 and Ara h 2, were used as indicators of process effectiveness. Enzymatic treatment effectively reduced Ara h 1 and Ara h 2 in roasted peanut kernels by up to 100% under optimal conditions. For instance, treatment of roasted peanut kernels with α-chymotrypsin and trypsin for 1–3 h significantly increased the solubility of peanut protein while reducing Ara h 1 and Ara h 2 in peanut kernel extracts by 100% and 98%, respectively, based on ELISA readings. Ara h 1 and Ara h 2 levels in peanut protein extracts were inversely correlated with protein solubility in roasted peanut. Blanching of kernels enhanced the effectiveness of enzyme treatment in roasted peanuts but not in raw peanuts. The optimal concentration of enzyme was determined by response surface to be in the range of 0.1–0.2%. No consistent results were obtained for raw peanut kernels since Ara h 1 and Ara h 2 increased in peanut protein extracts under some treatment conditions and decreased in others.     

Effect of Peanut Butter Manufacture on Vitamin E

ABSTRACT: The effect of peanut butter manufacture on vitamin E originating from raw peanuts ( Arachis hypogaea L., runner-type) was determined. Tocopherols were quantified by normal-phase high-performance liquid chromatography. No significant differences were observed in tocopherol (T) values between 1998 and 1999 crop raw peanuts or between raw peanuts and peanut butter except for γ-T ( P > 0.05). Oil and stabilizer added to the roasted peanuts during peanut butter processing provided 4% of α-T in the finished peanut butter. Retention of total tocopherols during peanut butter manufacture was 95%. Mean α-T values (mg/100 g) of commercial peanut products ranged from 12.3 (peanut oil) to 4.1 (dry roasted peanuts).     

Comparison of Flavor Quality of Peanut Based Pastes and Peanut Butter by Sensory Methods

A bland flavored and light colored paste was developed to increase utilization of peanuts. This study compared flavor quality of two pastes prepared from whole and chopped peanuts, water extracted to remove flavor precursors, two commercial samples of peanut butter and a paste from raw peanuts (control). Cluster analysis was used to compare sensory scores of all samples. The premium brand peanut butter had the strongest roasted flavor while the pastes had significantly more of the beany flavor than the peanut butters but less than that in the raw control. Chopping of kernels results in the loss of roasted and sweet flavors and the introduction of cardboard, musty and metallic flavors.     

花生及其制品中黄曲霉毒素B1胶体金快速检测方法的建立

通过考察市售试剂盒的绝对灵敏度和最大稀释比例，选取3种效果最好的快速检测胶体金试剂盒产品，以油炸花生、烘烤花生及花生酱为代表性基质，优化样品的前处理条件和试剂盒的反应条件，根据《食品快速检测方法评价技术规范》中性能指标进行统一的技术评价，考察各品牌快检试剂盒的检测限、特异性、灵敏度、假阳性率、假阴性率、相对准确度以及参比方法一致性分析等指标，为制定花生及其制品中黄曲霉毒素B₁胶体金快速检测评价标准提供科学依据。     

Roasted Peanuts and Peanut Butter Quality are Affected by Supercritical Fluid Extraction

Roasted peanuts were extracted using supercritical CO₂ (413.6 bar, 50–60° for 0, 2 or 4 hr). Peanuts were evaluated for shatter, tristimulus color, shear-compression force, moisture, lipid content, and sensory attributes. Extracted nuts were prepared into peanut butter and evaluated for color, relative torque resistance ratio, and sensory attributes. Lipid content decreased from 51.6 to 40.6% during 4 hr extraction. SFE increased shatter, shear-compression force, and Hunter L-value, but decreased hue angle, roasted aroma intensity, moistness of mass, fracturability, and roasted flavor intensity of peanuts. SFE increased the relative torque resistance ratio, and adhesiveness of peanut butter but had little effect upon flavor. SFE may be useful to reduce peanut contaminants and lipids to produce value-added peanut products.     

氯化钙对花生中黄曲霉毒素B1提取及其快速检测结果的影响

以氯化钙为蛋白沉淀剂、黄曲霉毒素B1为靶标,探究了70％甲醇水（V/V）提取液中不同含量氯化钙对花生中黄曲霉毒素提取及其免疫检测结果的影响;采用样品加标回收的方法,研究比较了五种不同含量氯化钙对胶体金免疫层析试纸条和ELISA检测结果的影响。研究结果表明,样品经含1％CaCl2（m／V）的甲醇水处理后,用胶体金免疫层析试纸条检测时,试纸条T线颜色梯度变化,不同浓度加标回收率等参数均得到明显改善,前处理效果显著;选取20份花生实际样品,经过这种前处理之后,进行胶体金免疫层析试纸条和ELISA检测,同时将其检测结果与免疫亲和层析高效液相色谱法（IAC-HPLC）进行比对,胶体金免疫层析试纸条与IAC-HPLC检测结果符合率为90％,间接竞争ELISA与IAC-HPLC检测结果相关系数达0．996,表明本研究所改进的样品前处理方法能提高免疫检测准确度,具有很好的应用性。     

A two-site monoclonal antibody immunochromatography assay for rapid detection of peanut allergen Ara h1 in Chinese imported and exported foods

Food allergen labeling has not yet been implemented in China. Therefore, a gold immunochromatography assay (GICA) was developed using two monoclonal antibodies (mAb) against the peanut allergen Ara h1. The GICA was specific for standard peanut samples with a sensitivity of 10 ng/ml. Peanut protein traces extracted from 124 food products imported and exported by China Customs were easily and rapidly detected by GICA. 68 food samples originally labeled as containing peanuts were positive for Ara h1 and 54 food samples labeled as not containing peanuts were negative for Ara h1, indicating that the labels from the manufacturers were accurate. However, 2 food samples labeled as not containing peanuts tested positive for Ara h1. The present GICA provides a fast, simple, semi-quantitative method for the determination of peanut allergens in foods. This detection system can be used to ensure the safety of food imported and exported by China Customs.