Novel method for producing hypoallergenic wheat flour by enzymatic fragmentation of the constituent allergens and its application to food processing

A novel method is proposed to produce hypoallergenic wheat flour suitable for patients allergic to wheat. Wheat flour was mixed with a cellulase solution, and the mixture was incubated at 50 degrees C for 1 h to hydrolyze the carbohydrate allergens. The hydrolysate was further incubated with actinase at 40 degrees C for 1 h while gently stirring to decompose the proteinaceous allergens. The product was evaluated for its allergenicity by an enzyme-linked immunosorbent assay, the results of which suggested negative allergenicity in most cases. The product changed to a batter state that was difficult to process by the usual methods. Gelatinization of the starch in the product and the addition of a surfactant were beneficial for food processing.     

Rates of Shrinkage and Growth of Air Bubbles Entrained in Wheat Flour Dough

The rates of shrinkage at constant temperature, and growth under a temperature rise below 100°C, of bubbles entrained in wheat flour dough were analyzed and compared with those of a bubble in water. The rate of shrinkage of bubbles in flour dough was controlled by the diffusion of dissolved air from the surface of bubbles to the bulk of flour dough. The apparent diffusion coefficient of the dissolved air in wheat flour dough with the water fraction of 0.49 calculated from the shrinkage of bubbles, was (3.2 ± 1.5) × 10^1?1 m²/sec (19°C), and (6.4 ± 2.0) × 10^?11 m²/sec (42°C). However, the growth behavior of bubbles in flour dough under a temperature rise was very different from that predicted from the diffusion theory. The critical radius of bubbles to grow was larger than that estimated from the diffusion theory. The mechanism of growth of bubbles in wheat flour dough, which was different from that of a bubble in water, is a subject that needs to be clarified.     

Hydrophobicity of stored (15, 35 °C), or dry-heated (120 °C) rice flour and deteriorated breadmaking properties baked with these treated rice flour/fresh gluten flour

Rice flour was stored at 15 °C/9 months, at 35 °C/14 days, or dry-heated at 120 °C/20 min. The breadmaking properties baked with this rice flour/fresh gluten flour deteriorated. In addition, the rice flour was mixed with oil in water vigorously, and oil-binding ability was measured. Every rice flour subjected to storage or dry-heated at 120 °C showed higher hydrophobicity, owing to changes in proteins. Then, proteins in the stored rice flour were excluded with NaOH solution, and bread baked with the deproteinized rice flour showed the same breadmaking properties as unstored rice flour/fresh gluten flour. The viscoelasticity of wheat glutenin fraction decreased after the addition of dry-heated rice flour in a mixograph profile. DDD staining increased Lab in color meter, which suggested an increase in SH groups in rice protein. The increase in SH groups caused a reduction in wheat gluten protein resulting in a deterioration of rice bread quality.     

Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins

Background

Parasite-specific IgE is thought to correlate with protection against Schistosoma mansoni infection or re-infection. Only a few molecular targets of the IgE response in S. mansoni infection have been characterised. A better insight into the basic mechanisms of anti-parasite immunity could be gained from a genome-wide characterisation of such S. mansoni allergens. This would have repercussions on our understanding of allergy and the development of safe and efficacious vaccinations against helminthic parasites.

Methodology/Principal Findings

A complete medium- to high-throughput amenable workflow, including important quality controls, is described, which enables the rapid translation of S. mansoni proteins using wheat germ lysate and subsequent assessment of potential allergenicity with a humanised Rat Basophilic Leukemia (RBL) reporter cell line. Cell-free translation is completed within 90 minutes, generating sufficient amounts of parasitic protein for rapid screening of allergenicity without any need for purification. Antigenic integrity is demonstrated using Western Blotting. After overnight incubation with infected individuals'' serum, the RS-ATL8 reporter cell line is challenged with the complete wheat germ translation mixture and Luciferase activity measured, reporting cellular activation by the suspected allergen. The suitability of this system for characterization of novel S. mansoni allergens is demonstrated using well characterised plant and parasitic allergens such as Par j 2, SmTAL-1 and the IgE binding factor IPSE/alpha-1, expressed in wheat germ lysates and/or E. coli. SmTAL-1, but not SmTAL2 (used as a negative control), was able to activate the basophil reporter cell line.

Conclusion/Significance

This method offers an accessible way for assessment of potential allergenicity of anti-helminthic vaccine candidates and is suitable for medium- to high-throughput studies using infected individual sera. It is also suitable for the study of the basis of allergenicity of helminthic proteins.     

Purification and functional characterization of endo-β-mannanase MAN5 and its application in oligosaccharide production from konjac flour

MAN5, the main extracellular saccharide hydrolase from Bacillus sp. MSJ-5, is an endo-β-mannanase with a demand of at least five sugar moieties for effective cleavage. It has a pH optimum of 5.5 and a temperature optimum of 50°C and is stable at pH 5–9 or below 65°C. MAN5 has a very high ability to hydrolyze konjac flour, 10 U/mg of which could completely liquefy konjac flour gum in 10 min at 50°C. HPLC analysis showed that most glucomannan in the konjac flour was hydrolyzed into a large amount of oligosaccharides with DP of 2–6 and a very small amount of monosaccharide. With the culture supernatant as enzyme source, the optimum condition to prepare oligosaccharides from konjac flour was obtained as 10 mg/ml konjac flour incubated with 10 U/mg enzyme at 50°C for 24 h. With this condition, more than 90% polysaccharides in the konjac flour solution were hydrolyzed into oligosaccharides and a little monosaccharide (2.98% of the oligosaccharides). Konjac flour is an underutilized agricultural material with low commercial value in China. With MAN5, konjac flour can be utilized to generate high value-added oligosaccharides. The high effectiveness and cheapness of this technique indicates its potential in industry. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Min Zhang and Xiu-Lan Chen contributed equally to this work.     

羊源丁酸梭菌HDRyYB1发酵工艺的优化

【目的】目前,国内外鲜有关于羊源丁酸梭菌的报道。本课题选用羊源丁酸梭菌HDRy YB1为研究对象,对其发酵工艺进行优化,为该菌株作为饲料添加剂应用于畜牧业生产奠定基础。【方法】采用Plackett-Burman(PB)试验设计法和响应面法分析并优化显著影响HDRy YB1菌株发酵液中芽胞数的培养基成分。【结果】发酵培养基中的面粉浓度、鱼粉浓度和米粉浓度显著影响发酵液中的芽胞数,优化后的发酵培养基组分(质量体积比)为:面粉3.72%、鱼粉0.90%、米粉3.96%、酵母粉0.60%、Na Cl 0.19%、Mg SO4·7H2O 0.19%、KH2PO4 0.01%、Na HCO3 0.01%、Ca CO3 0.48%;培养参数为:37°C,初始p H为7.2-7.4,瓶装量100/250,接种量3%。在此条件下,HDRy YB1菌株发酵完全(18 h)的芽胞数为1.478×108 CFU/m L,是优化前的2.7倍。【结论】HDRy YB1菌株发酵培养基得到了优化,优化后的培养基可用于后期的扩大发酵试验,验证其在实践生产中的应用价值。     

Distribution of Zinc in Euglena gracilis Wild and Mutant Cells Cultured in Zinc Enriched Medium as Proved by X-Ray Microanalysis

As a part of the study of bubble expansion in wheat flour dough under temperature rise, the critical radius for expansion, and the time course of expansion of an isolated bubble were investigated. As the required physical properties for the calculation of the critical bubble radius for expansion and the time course of bubble expansion, the authors measured the surface tension of the liquid phase of wheat flour batter as a function of concentration and temperature, and the apparent diffusion coefficients of air in wheat flour dough as a function of temperature. The critical radius for expansion and the time course of expansion of the isolated bubble under temperature rise were compared with the theoretical values calculated from the diffusion theory. At constant temperature, the time course of bubble shrinkage in wheat flour dough was described well by diffusion theory with the surface tension and the apparent diffusion coefficient, indicating that the bubble shrinkage would be rate-limited by the diffusion in the liquid phase of wheat flour dough of air out of the bubble. Under temperature rise from 3°C to 43°C, every bubble larger than the critical radius expanded. This result is physically admissible. On the other hand, the calculated time course of the bubble radius under temperature rise was not in agreement with the experimental data.     

Xylanases from <Emphasis Type="Italic">Aspergillus niger,Aspergillus niveus</Emphasis> and <Emphasis Type="Italic">Aspergillus ochraceus</Emphasis> produced under solid-state fermentation and their application in cellulose pulp bleaching

This study describes the production of xylanases from Aspergillus niveus, A. niger, and A. ochraceus under solid-state fermentation using agro-industrial residues as substrates. Enzyme production was improved using a mixture of wheat bran and yeast extract or peptone. When a mixture of corncob and wheat bran was used, xylanase production from A. niger and A. ochraceus increased by 18%. All cultures were incubated at 30 °C at 70–80% relative humidity for 96 h. For biobleaching assays, 10 or 35 U of xylanase/g dry cellulose pulp were incubated at pH 5.5 for 1 or 2 h, at 55 °C. The delignification efficiency was 20%, the brightness (percentage of ISO) increased two to three points and the viscosity was maintained confirming the absence of cellulolytic activity. These results indicated that the use of xylanases could help to reduce the amount of chlorine compounds used in cellulose pulp treatment.     

Dilatometric Measurement of the Partial Molar Volume of Water Sorbed to Durum Wheat Flour

Moisture sorption isotherms were measured at 25 °C for untreated, dry-heated and pre-gelatinized durum wheat flour samples. The isotherms could be expressed by the Guggenheim-Anderson-de Boer equation. The amount of water sorbed to the untreated flour was highest for low water activity, with water sorbed to the pre-gelatinized and dry-heated flour samples following. The dry-heated and pregelatinized flour samples exhibited the same dependence of the moisture content on the partial molar volume of water at 25 °C as the untreated flour. The partial molar volume of water was ca. 9 cm³/mol at a moisture content of 0.03 kg-H₂O/kg-d.m. The volume increased with increasing moisture content, and reached a constant value of ca. 17.5 cm³/mol at a moisture content of 0.2 kg-H₂O/kg-d.m. or higher.     

Towards the development of innovative multi-mycotoxin reference materials as promising metrological tool for emerging and regulated mycotoxin analyses

The interest in LC-MS/MS multi-mycotoxin methods unveiled an urgent need for multi-mycotoxin reference material. A multi-fusariotoxin, including deoxynivalenol (DON); zearalenone (ZEN); T-2 toxin (T-2); HT-2 toxin (HT-2); enniatin A, A1, B, and B1 (ENNs); and beauvericin (BEA), contaminated wheat flour was obtained by inoculation Fusarium spp. strains. The candidate material has successfully passed the homogeneity test and submitted to an international interlaboratory study achieved by 19 laboratories from 11 countries using their routine analytical method. The dispersion of the results for ZEN and BEA did not allow the derivation of reliable consensus values, while the assignment was only possible for DON, HT-2, T-2, and ENN A. No link was found between the methods used by the participants and the results. Significant changes in dry matter contents (≥±1.4 % of the initial dry matter) and significant changes in ergosterol contents (≥±10 %) did not occur. Using the mycotoxin contents in wheat flour stored at ?80 °C as reference values, statistically significant decreases were observed only for T-2 contents at +24 °C, in contrast to the storage at ?20 and +4 °C. For the other involved toxins, the candidate material was found to be stable at ?20, +4, or +24 °C. Based on the T-2 decreases, a shelf life of 6 years was derived from isochronous study when the material is kept at ?20 °C. At room temperature (e.g., +24 °C) or higher, this time validity drastically decreases down to 6 months. The development of this metrological tool is an important step towards food and feed quality control using multi-mycotoxin analyses. In vivo animal experiments using multi-mycotoxin-contaminated feeds dealing with the carryover or mitigation could further benefit from the methodology of this work.     

A Novel Transformation to S-Methyl Phosphorodichloridothiolate and the Fungitoxicity of Diaryl S-Methyl Phosphorothiolates

Prior to an analysis of the shrinkage and growth of air bubbles entrained in wheat flour dough, the shrinkage and growth under a temperature rise of a small bubble in water was analysed for comparison. The rates of shrinkage and growth of the bubble were respectively controlled by the diffusion of under- and over-saturated dissolved air from and into the bubble. The diffusion coefficient of the dissolved air in water calculated from the shrinkage of the bubble was 2.10 × 10^_9m²/sec (17°C), which agrees with the literature value. On the other hand, at below 100°C, the effects on the bubble growth of the expansion of air due to the temperature rise and the increase in the saturation vapor pressure of water were negligibly small. The accompanying air entrained in flour particles suspended in water was much more stable than a free bubble in water. However, the growth under a temperature-rise of a bubble evolved from wheat flour particles was the same as the growth of a bubble in water, if many bubbles did not coexist.     

Changes in porcine,ovine, bovine and equine blood progesterone concentrations between collection and centrifugation

The aim of this study was to determine if different methods of handling porcine, ovine, bovine and equine blood between collection and centrifugation influence measurable progesterone levels. A 2 × 2 × 5 factorial experiment was conducted for each species with heparin (with or without), temperature of incubation (4 and 22°C) and time of incubation (0, 6, 12, 24 and 48 h) as the main effects. Following centrifugation, plasma and serum samples were stored at ?20°C until progesterone concentrations were determined by radioimmunoassay. Method of handling porcine and equine blood between collection and centrifugation did not affect the levels of progesterone. However, heparinized blood held at 4°C resulted in the most consistent levels of progesterone over time. Progesterone levels were fairly consistent across time in the ovine blood by all methods of handling except heparinized blood incubated at 22°C. By 24 h after collection, plasma progesterone concentrations decreased by 50% for the ovine blood incubated at 22°C with heparin. Decreases were detected by all the methods of handling the bovine blood between collection and centrifugation. The rate of decline, however, was considerably faster for blood held at 22°C than blood held at 4°C. At 12–48 h after collection, the concentrations of progesterone averaged only 5% of the time 0 sample for blood incubated at 22°C. In contrast, at least 30% of the progesterone values in the time 0 sample were detected between 12 and 48 h of incubation for the blood held at 4°C.     

Seed treatments with Vitavax for the control of loose smut of wheat and barley

Seed dressings with Vitavax—75 % w.p.—eliminated Ustilago nuda in spring barley seeds and greatly reduced infection due to U. tritici in winter wheat. Emergence and yield of these crops were not adversely affected. Seed soak treatments including 0·2 % aqueous Vitavax for 6 h at 30 °C (wheat and barley), 0·2% thiram for 24 h at 30 °C (barley) and 0·2% Vitavax for 1 h at 30 °C (barley) also rid the seeds of infection. In other tests with barley 2 h soaks in 0·2 % aqueous suspensions of Vitavax at 30 °C gave equivalent control to 12 h soaks in 0·2% thiram at 30 °C.     

Induced Heat Sensitivity of Wheat Roots and Protecting Effect of Ethanol and Kinetin

Wheat seedlings were subjected to heat shock for 2 min at 45°C. The seedlings were then incubated at 25°C or higher temperatures (usually 35°C). At 25°C the root tips survived the heat shock, but not at temperatures above 34°C, unless they had been pretreated with ethanol or kinetin, After 1 h in ethanol and after more than 15 h in kinetin the root meristem survived a high incubation temperature after the heat shock. Immediately after heat treatment the glyceride content in treated root tips was higher than in untreated roots. The same was observed after heat treatment of root tips pretreated in ethanol and kinetin. The content of ether extractable lipids was not changed by the heat shock.     

Addendum to Issue 1 - ENZITEC 2012 Cheese whey and passion fruit rind flour as substrates for protease production by Bacillus sp. SMIA-2 strain isolated from Brazilian soil

Abstract

Application of wastes from the food processing industry as carbon sources in enzyme production processes reduces the cost of production, and also helps in solving problems of their disposal. In this work, we demonstrated that sweet cheese whey, in combination with passion fruit rind flour, can be successfully used for the production of protease by Bacillus sp. SMIA-2, opening perspectives for the use of these agricultural byproducts as novel and cost-effective culture media for the production of protease. The maximum production of the enzyme was observed in a sweet cheese whey-based culture medium preparation (0.5%, w/v) containing 0.25% (w/v) passion fruit rind flour and supplemented with different metal salts at an initial pH of 7.5–8.0, incubated at 50°C for 48 h. Studies on enzymatic characterization revealed that crude protease showed maximum activity at pH 9.0 and 70°C. These characteristics presented by the protease produced by Bacillus sp. SMIA-2 could be very useful when thinking about biotechnological applications.     

Production of cellulases by Thermomucor indicae-seudaticae: characterization of a thermophilic β-glucosidase

Abstract

The current study evaluated the production and characterization of β-glucosidase by the thermophilic fungus Thermomucor indicae-seudaticae in solid-state fermentation of wheat bran. Isolated fungi have significant amounts of β-glucosidase, an enzyme that may be applied to different industrial processes, such as the production of fuels, food, and other chemical compounds. Maximal enzyme activity occurred in pH 3.5–4.5 and at 70?°C. The enzyme exhibited high thermostability, for 1?h, up to 60?°C, and good tolerance to glucose (10?mM) and ethanol (10%). The optimization of fermentative parameters on the production of β-glucosidase was carried out by evaluating the best supplementary nutrient source, pH of nutrient solution, initial substrate moisture and fermentation temperature. The optimization of the above fermentation parameters increased enzyme activity by 120.0%. The highest enzymatic activity (164.0?U/g) occurred with wheat bran containing 70% initial moisture, supplemented with 1.0% (NH₄)₂SO₄ solution at pH 5.5–6.0 and fungus incubated at 40?°C. A more detailed study of β-glucosidase suggested that Sulfur is an important component of the main amino acid present in this enzyme. The enhancer of the enzyme activity occurred when the fungus was grown on wheat bran supplemented with a sulfur-containing solution. In fact, increasing the concentration of sulfur in the solution increased its activity.     

Temperature linked degenerative tissue rots in Capsicum annuum (L.) varieties diseased with Phytophthora capsici

Five-week-old seedlings of Capsicum annuum variety SAMPEP 4, Californian Wonder and Ex Dandamasa drenched with 15,000 infectious units per ml of Phytophthora capsici were incubated at 5°C, 20°C, 30°C and 35°C in alternating light–dark cool cycle Gallenkamp incubators and monitored for root rot development. Each host–pathogen system was replicated five times. Successful disease development was contingent on been incubated at ambient temperature for not less than 3.5 ± 0.5 h. Depending on variety, degenerate tissue rots were aggravated ≤2–3 days after a preconditioning temperature treatment for 24 h possibly linked to cell wall constitution, composition and permeability. Lesion development on stem heightened (27.8%) when incubated at temperatures above 20°C. Ten days after treatment, plant mortality and disease severity were not affected significantly by post-inoculation temperature.     

Assessment of allergenicity of diploid and hexaploid wheat genotypes: identification of allergens in the albumin/globulin fraction

Wheat is an important part of the daily diet of millions of people. However, this staple food is also responsible for food allergies. Ancient cultivars of wheat are gaining interest today but nothing is known about their allergenicity. Many wheat proteins have been reported as causative food allergens, including some prolamin-type gluten proteins, and salt soluble proteins of the albumin/globulin (A/G) type. The objective of this work is to obtain information about the allergenicity of the salt soluble A/G fraction of an ancient diploid cultivar compared with a standard hexaploid bread wheat cultivar using 20 sera from patients with wheat allergy. Differences in the IgE reactivity of sera towards the two genotypes were quantified by ELISA. Qualitative differences in IgE-binding proteins were searched after 1D or 2D electrophoresis. For most of the sera, the concentration in A/G specific IgE was higher for the hexaploid T. aestivum (cv Récital) than for the diploid T. monococcum (cv Engrain). The analysis of 2D spots revealed by immunoblotting leads to the identification by mass spectrometry of 39 IgE-binding proteins, some of them unknown until now as wheat allergens. Numerous allergens were identified, differences observed between Engrain and Récital will be discussed.     

PHYSIOLOGICAL COMPARISON OF THE ISOMORPHIC LIFE HISTORY PHASES OF THE HIGH INTERTIDAL ALGA ENDOCLADIA MURICATA (RHODOPHYTA)1

The isomorphic phases of Endocladia muricata (Post. & Rupr.) J. Ag. Were compared for photosynthetic and respiratory difference in response to a variety of environmental manipulations. Photosynthetic light response during submergence at 15° C and the pattern of respiratory recovery following prolonged emergence (3 h) at either 15° or 30° C were similar between gametophytes and tetrasporophytes. The phases showed the same ability to photosynthesize and respire during emergence at each temperature tested (15°, 25°, and 35° C, fully hydrated thalli) and at various desiccation state (measured at 25° C only). Submerged rates of photosynthesis following prolonged emergence at 15° and 30° C were, however slightly greater (17%) for tetrasporophytes as compared to gametophytes. Regardless of the life history phase, plants incubated at 15° C during emergence recovered more completely than plants incubated at 30° C. Photosynthetic recovery after 1 h in plants incubated at 15° C often “spiked” and yielded rates as great as 185% of pretreatment rates. Increased photosynthetic rates during recovery were absent for the 30° C incubations. The initial photosynthetic recovery of plants collected from the upper limits of distribution was greater than that of plants collected from the lower limits. Recovered rates of respiration were highly variable over time. Respiration often exceeded pretreatment values more then threefold, and the elevated rates were sustained for 12 h. Photosynthesis and respiration in air were comparable to rates in seawater and varied slightly with increasing temperature. Photosynthetic and respiratory rates also decreased with increasing tissue water loss. Thus, only slight differences in physiological performance were observed between phases and individuals collected from different vertical positions. Metabolic differences were transient and apparent only under experimental conditions that modeled extreme environmental conditions.     

Growth and patulin formation byPenicillium urticae Bainier in pure and mixed cultures

Penicillium urticae Bainier synthesized patulin in potato-dextrose medium at temperatures ranging from 5 to 30°C. Maximum patulin yield was 2700 μg/ml of culture fluid in 14 days at 25°C. Two distinctive intervals affected patulin formation: 15 to 20°C and 30 to 35°C, the former favorable and the latter detrimental. An incubation period of 11 to 14 days made a nonsterile mixture of weathered wheat straw and soil a favorable medium for patulin formation. Autoclaved weathered wheat straw, inoculated withP. urticae alone, or in combination withTrichoderma sp., was a medium comparable to nonsterile, incubated weathered wheat straw in soil. Both carbon source and accessory growth factors were important for patulin formation. Of seven media tested, potato-dextrose was superior to potatodextrose supplemented with 70 ppm Zn-ions and 16 ppm Fe-ions, potatosucrose, Raulin-Thom, autoclaved weathered wheat straw in pure culture, weathered wheat straw in nonsterile soil, and autoclaved weathered wheat straw in mixed culture, in that order. Patulin production ranged from 337.5 to 0.2 mg/g of C in the medium. Contribution from the Northern Plains Branch, Soil and Water Conservation Research Division, Agricultural Research Service, U.S. Department of Agriculture, in cooperation with the Nebraska Agricultural Experiment Station, Lincoln. Published as Paper No.2621, Journal Series, Nebraska Agricultural Experiment Station.