Sensitive and specific detection of almond (Prunus dulcis) in commercial food products by real-time PCR

Almond has been widely used in all sorts of food products, mostly due to its pleasant flavor and health benefits. However almonds can become an important health problem since they are responsible for triggering adverse immune responses in allergic individuals, and since they are present in many processed foods they are considered as a potential hidden allergen. Consequently, it's important for food processors and regulatory agencies to be able to ensure accurate labeling of foods to protect the safety of the public and to avoid expensive recalls. We propose a simple and highly sensitive approach to detect almond in a wide range of processed foods. The method consists of a real-time PCR assay targeting the gene encoding for the ITS1 in almond, using a nuclease (TaqMan) probe labeled with FAM and BBQ. Sensitivity of real time PCR was determined by analysis of raw and heat treated almond-wheat flour mixtures with a range of detection of 0.1–100,000 mg/kg. The assay was successfully trialed on a total of 214 commercial foodstuffs allowing the detection of trace amounts of almond down to the level of 0.1 mg/kg, and is therefore proposed as a ready-to-use analytical tool to trace almond allergens in foods.     

Effect of heat,rutin and disulfide bond reduction on in vitro pepsin digestibility of Chinese tartary buckwheat protein fractions

Protein fractions (albumin, globulin, prolamin and glutelin) were extracted from defatted tartary buckwheat flour. The in vitro pepsin digestibilities of the four protein fractions were different, and albumin was more susceptible to pepsin hydrolysis. The native structure of the four protein fractions may be destroyed by heat treatment, and the digestibilities were all improved significantly (P < 0.05). Adding rutin to the digestion mixture of the four fractions did not cause a decrease in pepsin digestibility, although it did cause a significant increase in certain instances (P < 0.05). Treatment with β-mercaptoethanol (2-ME) only caused a higher initial proteolysis rate and did not increase the final digestibility distinctly except for prolamin. After pepsin digestion, the remaining proteins of unhydrolyzed albumin, globulin, prolamin and glutelin (untreated) shared some similarities. They also exhibited a minor band at 20,000 Da and a broad band at 10,000–14,000 Da.     

Effect of food matrix on amandin, almond (Prunus dulcis L.) major protein, immunorecognition and recovery

Amandin, the primary storage protein in almonds, contains key polypeptides recognized by almond-allergic patients. A variety of food matrices representing diverse categories of foods were analyzed to assess the effect of food matrix on amandin recognition and recovery using rabbit polyclonal antibody based immunoassays. Food matrices from dairy, nuts, and vegetables typically resulted in over-estimation of amandin. Some foods representing legumes and cereals resulted in over-estimation while others in under-estimation of amandin. The amandin recovery range was 116-198 μg/100μg (dairy) 110-292 μg/100μg (tree nuts), 43-304 μg/100μg (legumes), 106-183 μg/100μg (most cereals- with the exception of barley, whole-wheat flour, wild rice and raisin bran whole mix). Amandin recovery from spices was typically low (2-85 μg/100μg) with a few exceptions where higher recoveries were observed (121-334 μg/100μg). Salt (black and white), tea, confectionery (sugar, cocoa, dark chocolate), and fruits (1-83 μg/100μg) generally resulted in lower recoveries. Tested food matrices did not adversely affect amandin immunorecognition in Western blots. The pH and the extraction buffer type affected amandin recovery. The results suggest that food matrix effects as well as extraction conditions need to be carefully evaluated when developing immunoassays for amandin detection and quantification.     

Heat denaturation of Brazil nut allergen Ber e 1 in relation to food processing

Ber e 1, a major allergen from Brazil nuts, is very stable to in vitro peptic digestion. As heat-induced denaturation may affect protein digestibility, the denaturation behaviour of Ber e 1 was investigated. The denaturation temperature of Ber e 1 varies from approximately 80–110 °C, depending on the pH. Upon heating above its denaturation temperature at pH 7.0, the protein partly forms insoluble aggregates and partly dissociates into its polypeptides, whereas heating at pH 5.0 does neither induce aggregation, nor dissociation of the protein. The denaturation temperature of approximately 110 °C at pH values corresponding to the general pH values of foods (pH 5–7) is very high and is expected to be even higher in Brazil nuts themselves. As a result, it is unlikely that heat processing causes the denaturation of all Ber e 1 present in food products. Consequently, the allergen is assumed to be consumed (mainly) in its native form, having a high stability towards pepsin digestion.     

Stability and bioaccessibility of different forms of carotenoids and vitamin A during in vitro digestion

Vitamin A deficiency is a public health issue in developing countries and promoting dietary carotenoids as precursors is a promising strategy. However, carotenoids present in numerous fruits and vegetables are unstable and poorly bioaccessible. This study evaluated these two parameters during in vitro digestion of carotenoids and retinoids from carrot juice, raw and cooked spinach, micronutrient-fortified flour and standards without food matrix. Standards were unstable whereas vitamin A from fortified flour and native food carotenoids were generally better protected by the food matrix (30–100% remaining versus 7–30% for standards). Hydrothermal cooking did not influence spinach carotenoid digestive stability but decreased their contents, phenomenon compensated by a significantly better micellarisation from 15-fold for β-carotene to 72-fold for lutein. Finally, carrot juice provided the greatest amount of bioaccessible provitamin A with 1850 μg/100 g dry matter (DM) versus 790 and 80 μg/100 g DM in cooked and raw spinach, respectively.     

Characterization,amino acid composition and in vitro digestibility of hemp (Cannabis sativa L.) proteins

The protein constituents and thermal properties of hemp (Cannabis sativa L.) protein isolate (HPI) as well as 11S- and 7S-rich HPIs (HPI-11S and HPI-7S) were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and different scanning calorimetry (DSC), and their amino acid composition and in vitro digestibility were also evaluated, as compared to soy protein isolate (SPI). SDS-PAGE analysis showed that the edestin (consisting of acidic and basic subunits, AS and BS) was the main protein component for HPI and HPI-11S, while HPI-7S was composed of the BS of edestin and a subunit of about 4.8 kDa. DSC analysis characterized thermal transition of the edestin component and the possible present form of different subunits. Except lysine and sulfur-containing amino acids, the essential amino acids of various HPIs met the suggested requirements of FAO/WHO for 2–5 year old infants. The proportion of essential amino acids to the total amino acids (E/T) for HPI (as well as HPI-11S) was significantly higher than that of SPI. In an in vitro digestion model, various protein constituents of various HPIs were much easily digested by pepsin plus trypsin, to release oligo-peptides with molecular weight less than 10.0 kDa (under reduced condition). Only after pepsin digestion, in vitro digestibility of HPIs was comparable to that of SPI, however after pepsin plus trypsin digestion, the digestibility (88–91%) was significantly higher than that (71%) of SPI (P < 0.05). These results suggest that the protein isolates from hempseed are much more nutritional in amino acid nutrition and easily digestible than SPI, and can be utilized as a good source of protein nutrition for human consumption.     

Resistance of purified seed storage proteins from sesame (Sesamum indicum L.) to proteolytic digestive enzymes

Sesame has been increasingly associated with food allergy. The main seed storage proteins of sesame (the 2S albumin and the 7S and 11S globulins) were purified and subjected to proteolysis with pepsin, trypsin and chymotrypsin. The degree of proteolysis obtained was monitored by SDS–PAGE, followed by densitometry of the main bands. The 2S albumin was found to be stable to proteolysis, being extremely resistant to pepsin, and relatively resistant to trypsin and chymotrypsin. The 7S and 11S proteins were relatively labile to pepsin. Acidic polypeptides from the 11S protein were more susceptible to proteolysis than the basic polypeptides. Both 7S and 11S proteins generated what appeared to be stable polypeptides after proteolysis with trypsin and chymotrypsin. The results are discussed in relation to similar studies on related seed storage proteins, available structural information, and the potential allergenicity of the sesame proteins.     

Effects of food formulation and thermal processing on flavones in celery and chamomile

Flavones isolated from celery varied in their stability and susceptibility to deglycosylation during thermal processing at pH 3, 5, or 7. Apigenin 7-O-apiosylglucoside was converted to apigenin 7-O-glucoside when heated at pH 3 and 100 °C. Apigenin 7-O-glucoside showed little conversion or degradation at any pH after 5 h at 100 °C. Apigenin, luteolin, and chrysoeriol were most stable at pH 3 but progressively degraded at pH 5 or 7. Chamomile and celery were used to test the effects of glycosidase-rich foods and thermal processing on the stability of flavone glycosides. Apigenin 7-O-glucoside in chamomile extract was readily converted to apigenin aglycone after combination with almond, flax seed, or chickpea flour. Apigenin 7-O-apiosylglucoside in celery leaves was resistant to conversion by β-glucosidase-rich ingredients, but was converted to apigenin 7-O-glucoside at pH 2.7 when processed at 100 °C for 90 min and could then be further deglycosylated when mixed with almond or flax seed. Thus, combinations of acid hydrolysis and glycosidase enzymes in almond and flax seed were most effective for developing a flavone-rich, high aglycone food ingredient from celery.     

苦荞蛋白质的低消化性研究Ⅰ——热处理、还原二硫键及添加芦丁对其体外消化率的影响

采用Osborne分级分离的方法从苦荞粉中制备得到清蛋白、球蛋白、醇溶蛋白和谷蛋白。体外消化率测定结果表明,四种蛋白组分的消化率均低于对照-小麦胚分离蛋白和大豆分离蛋白,并且四种组分的体外消化率也存在不同程度的差别:清蛋白最高,谷蛋白最低。热处理可以明显提高苦荞粉四种蛋白组分的体外消化率。添加芦丁不但没有降低四种蛋白组分的体外消化率,反而均有一定程度的提高。二硫键的破坏,除醇溶蛋白得以提高之外,对于其它三种组分只是提高了初始水解速度,最终体外消化率没有明显提高。体外消化实验后,四种蛋白组分所剩的残渣蛋白SDS-PAGE分析结果表明:这些残渣蛋白的谱带存在相似之处:在20kDa处有一条很窄的谱带,在14～10kDa处的谱带较宽。     

A food matrix reduces digestion and absorption of food allergens in vivo

Scope: Food allergy is caused by primary (class 1) food allergens, e.g. Bos d 5 (cow's milk) and Cor a 8 (hazelnut) or secondary (class 2) food allergens, e.g. Mal d 1 (apple). The latter cannot sensitize susceptible individuals but can cause allergy due to immunological cross‐reactivity with homologous respiratory allergens. Here, we studied the effects of food matrix on gastrointestinal proteolysis, epithelial transport and in vivo absorption of class 1 and class 2 food allergens. Methods and results: Mal d 1 lost its IgE‐reactivity immediately after simulated gastric digestion whereas Bos d 5 and Cor a 8 did not. Only Cor a 8 maintained IgE‐binding capacity after simulated intestinal proteolysis. The presence of hazelnut and peanut extracts, which served as protein‐rich model food matrices, delayed gastrointestinal degradation and reduced epithelial transport rates of all allergens through CaCo‐2 monolayers. Finally, IgE‐reactive allergens were assessed at different time points in sera from rats fed with all three allergens with or without hazelnut extract. The levels of all allergens peaked 2 h after animals were fed without matrix and increased over 8 h after feeding. Conclusions: A protein‐rich food matrix delays gastrointestinal digestion and epithelial transport of food allergens and thereby may affect their sensitizing capacity and clinical symptoms.     

饼干模型对小麦及花生过敏原消化稳定性和免疫活性的影响

食品加工或食物基质可以不同程度地影响过敏原消化稳定性和免疫原性。然而,对食品加工和食物基质对 食物模型中过敏原的影响却知之甚少。本实验通过体外模拟胃肠消化的方式,包括模拟口腔咀嚼、胃部消化和十二 指肠消化,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹的方法,分析焙烤模型饼干中小麦过敏原和花生 过敏原的消化特性和免疫原性。结果显示：小麦和花生蛋白均可被胃蛋白酶迅速水解,醇溶蛋白、谷蛋白等致敏原 被降解成低分子质量多肽;可溶性蛋白中花生过敏原Ara h 1和Ara h 3基本消失,Ara h 2/6耐受胃肠消化;酶联免疫 吸附测定结果显示,消化后饼干中过敏原的致敏性降低。综合以上结果表明,饼干模型的消化性质基本不受焙烤加 工和其他基质的影响,免疫原性因致敏原被消化而降低。     

SOLUBILIZATION,ELECTROPHORETIC CHARACTERIZATION AND IN VITRO DIGESTIBILITY OF ALMOND (PRUNUS AMYGDALUS) PROTEINS1,2

ABSTRACT The major U.S. marketing varieties of almonds contained moisture, protein, fat, and ash in the range 4.35–5.86%, 16.42–22.17%, 53.59–56.05%, and 2.69–2.93%, respectively. Two fatty acids, oleic (range 52.44–67.07%) and linoleic (range 22.05–38.67%) accounted for up to 90% of the total fat. The majority of almond proteins (≥ 95%) are water soluble with a minimum solubility at pH ≤ 4.0. Sodium chloride (1.0 M) decreased the almond protein solubility in aqueous medium. Electrophoretic analyses indicated that one water soluble protein dominates the almond protein composition. This oligomeric major protein is made up of two kinds of polypeptides (molecular weight range 20,000–22,000 and 38,000–41,000) linked via disulfide bonds. Among the proteases tested, pepsin was the most efficient in hydrolyzing the almond proteins.     

Effect of germination and probiotic fermentation on nutrient composition of barley based food mixtures

Food mixtures formulated from non-germinated and germinated barley flour, whey powder and tomato pulp (2:1:1w/w) were autoclaved, cooled and fermented with 5% Lactobacillus acidophilus curd (10⁶ cells/ml) at 37 °C for 12 h. The cell count was found significantly higher (8.88 cfu/g) in the fermented food mixture formulated from germinated flour as compared to the non-germinated barley based food mixture. A significant drop in pH with corresponding increase in titratable acidity was found in the germinated barley flour based food mixture. Processing treatments like germination, autoclaving and probiotic fermentation did not bring about any significant change in ash and fat contents, but significant decrease was noticed in crude protein, crude fibre, starch, total and insoluble dietary fibre contents. The combined processing caused significant improvement in reducing sugar, thiamine, niacin, lysine and soluble dietary fibre contents of barley based food mixtures. In conclusion, a combination of germination and fermentation is a potential process for enhancing the nutritional quality of food mixtures based on coarse cereals.     

Food Allergens: Is There a Correlation between Stability to Digestion and Allergenicity?

Food allergy is a major health problem in the Western countries, affecting 3–8% of the population. It has not yet been established what makes a dietary protein a food allergen. Several characteristics have been proposed to be shared by food allergens. One of these is resistance to digestion. This paper reviews data from digestibility studies on purified food allergens and evaluates the predictive value of digestibility tests on the allergenic potential. We point out that food allergens do not necessarily resist digestion. We discuss how the choice of in vitro digestibility assay condition and the method used for detection of residual intact protein as well as fragments hereof may greatly influence the outcome as well as the interpretation of results. The finding that digests from food allergens may retain allergenicity, stresses the importance of using immunological assays for evaluating the allergenic potential of food allergen digestion products. Studies assessing the allergenicity of digestion products, by either IgE-binding, elicitation or sensitizing capacity, shows that digestion may abolish, decrease, have no effect, or even increase the allergenicity of food allergens. Therefore, the predictive value of the pepsin resistance test for assessing the allergenic potential of novel proteins can be questioned.     

Impact of dietary fibers on the properties and proteolytic digestibility of lactoferrin nano-particles

The application and development of biopolymer-based particulate delivery systems is receiving considerable attention from scientists and manufacturers seeking to engineer healthier and safer foods. Present work focused on the fabrication and proteolysis of bovine lactoferrin nano-particles onto which pectins, alginate and carrageenan were electrostatically deposited. DLS, CD and AFM were used to ascertain the formation of spherical structures with varying zeta potentials and modified protein folding. SDS-PAGE analyses of samples collected during simulated human gastro-duodenal digestion revealed that in the case of high and low methoxy pectin particle zeta potential values (−35.4 mV and −50.3 mV respectively) were found to be inversely linked to lactoferrin digestion rate. Moreover, the electrostatic binding of food grade iota-carrageenan onto lactoferrin nano-particles was found to yield highly charged particles (zeta = −69.2 mV) with low surface roughness and to enable a proportion of the lactoferrin to resist 1 h of simulated gastric digestion. Overall, these findings suggest that electrostatic biopolymer interactions may modulate protein digestion and possibly facilitate controlled protein delivery to the upper gastrointestinal tract or have implications to protein nutritional value.     

Effect of high-pressure treatment on in-vitro digestibility of β-lactoglobulin

The effect of high-pressure (HP)-treatment on β-lactoglobulin (β-Lg) was investigated using in-vitro pepsin digestion under simulated gastric conditions. HP-treatment of β-Lg at 400 MPa for 10 min only slightly increased its subsequent hydrolysis by pepsin. However, higher pressure treatments (600 and 800 MPa) resulted in rapid digestion of β-Lg. After these higher pressure treatments, β-Lg disappeared in less than 1 min of pepsin incubation as determined by SDS-PAGE analysis. Mass spectrometry analysis of the digestion products at corresponding incubation times revealed rapid and progressive degradation of β-Lg. Most (> 90%) of the peptide products following pepsin digestion of HP-treated β-Lg were less than 1500 Da in size. Peptide products from pepsin digestion were identified and mapped to β-strand regions (Leu₃₂–Leu₅₄ and Phe₈₂–Leu₁₀₄) and to the N- and C-terminals regions (Leu₁–Leu₁₀ and Ser₁₅₀–Leu₁₅₆) of β-Lg. While these regions corresponded to known IgE epitopes of β-Lg, the predominant peptides resulting from 60 s of incubation were short (7–10 residues) in length. These results demonstrate that HP-treatment increased the digestibility of β-Lg and represents a promising processing technology for reducing the allergenicity of known allergens in a wide variety of food materials.Industrial relevanceHigh-pressure treatment is widely used to enhance the functional attributes of food proteins. The potential for enhanced nutritional value of β-Lg was also demonstrated here by its increased digestibility. High-pressure treatment followed by incubation with proteases may represent a method for the commercial production of bioactive peptides such as inhibitors of angiotensin converting enzyme. More importantly, high-pressure-induced unfolding of milk proteins may reduce their allergenicity. Unfolded proteins are less likely to become agents of immunological sensitization because they are more readily hydrolyzed. Thus high-pressure treatment applied to food ingredients such as whey protein isolate may contribute to the development of hypoallergenic foods.     

A review of the impact of processing on nutrient bioaccessibility and digestion of almonds

Almond kernels contain phytochemicals and nutrients that potentially have positive health benefits in relation to heart disease, diabetes and obesity. One important mechanism associated with these benefits is an imposed limit on bioaccessibility (release) of nutrients, such as lipids, from almond tissue during mastication and digestion. Recent studies have demonstrated the importance of food structure during the digestion of plant foods. In particular, in the almond kernel, depending on its structure and degree of processing, the amount of lipid released from the almond tissue matrix and the fatty acids produced from lipolysis has been found to vary substantially. This review aims at discussing the commercial methods of almond processing and the different almond forms produced for human consumption, mainly with respect to their impact on nutrient composition, digestion and metabolism.     

河套小麦胚芽源多肽的抗氧化活性研究

利用中性蛋白酶、胰蛋白酶、胃蛋白酶等酶解河套小麦胚芽蛋白获得多肽,测定其体外抗氧化能力,利用聚丙烯酰胺凝胶电泳（SDS-PAGE）确定其多肽分布。结果表明,胚芽蛋白及多肽浓度与抗氧化能力呈正相关。不同浓度的中性蛋白酶酶解获得的多肽的抗氧化能力显著高于胃蛋白酶和胰蛋白酶酶解获得的多肽（P<0.05）,其还原能力、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐（ABTS+）和1,1-二苯基-2-苦基苯肼（DPPH）清除率最高达（1.17±0.004）1.0 mg/mL、（84.82%±0.87%）1.5 mg/mL和（55.01%±0.01%）1.0 mg/mL,且其ABTS+和DPPH自由基清除率均显著高于胚芽蛋白（P<0.05）。另外,不同蛋白酶水解能力不同,其中胃蛋白酶的水解能力最大,中性蛋白酶水解能力最小;胚芽蛋白多肽的抗氧化能力与其分子量大小相关,但不一定蛋白多肽的分子量越小,其抗氧化效果越好。上述结果为进一步研究河套小麦胚芽抗氧化肽提供了一定的参考依据。     

The effect of alginates on in vitro gastric digestion of particulated whey protein

The aim of the study was to investigate how microparticulated and nanoparticulated whey proteins mixed with alginate respond to simulated in vitro gastric digestion conditions at pH 3.0. Initially, particle size distributions and zeta potential were measured in all mixtures at pH 3.0. Particle size distributions as well as SDS‐PAGE were used to investigate the rate of protein degradation by pepsin during simulated in vitro gastric digestion. The complexation of nanoparticulated and microparticulated whey protein with alginates causes formation of insoluble and soluble complexes, which can resist pepsin degradation to a different degree. These results highlight the potential of developing new food products, which can enhance satiety.     

Polyphenol and nutrient release from skin of almonds during simulated human digestion

The bioaccessibility of nutrients and phytochemicals from almond skin has not been previously evaluated. We quantified the release of lipid, protein and polyphenols during simulated human digestion from natural (NS) and blanched (BS) skins, the latter being a by-product of the almond industry. Higher percentages of polyphenols were released from NS compared to BS during in vitro digestion. Most of the limited release of lipid and protein occurred during gastric digestion, with no significant differences between NS and BS. The total dietary fibre content was 45% for NS and 46% for BS, glucose and galacturonic acid being the major sugars present. No changes in dietary fibre composition and distribution of autofluorescent phenolics were observed in the cell walls of almond skin after simulated digestion. In the GI tract, the cell walls may therefore function as a useful source of fermentable fibre with beneficial implications for gut health.