Formation of nanotubes and gels at a broad pH range upon partial hydrolysis of bovine α-lactalbumin

The influence of pH (from 7.5 to 4.0) on the hydrolysis and self-assembly process of nanotubes formed from partially hydrolysed bovine α-lactalbumin was studied. The results showed that the pH had no influence on the products resulting from hydrolysis, but a decrease in pH dramatically reduced the hydrolysis rate. In contrast, the self-assembly process was favoured by decreased pH due to the reduced electrostatic repulsion between the fragments making up the nanotubes. Initially, upon adding a protease, a rapid change in conformation was indicated by circular dichroism spectroscopy. Subsequently no further change was observed. Due to slower hydrolysis and faster self-assembly at pH 6.5 and 4.0, the main building fragments were integrated quickly into nanotubes, and hence avoided further hydrolysis. Self-assembly of protease-induced α-lactalbumin nanotubes can thus be extended over a broad range of pH values. These findings might be useful for future applications in both food and pharmaceutical industries.     

Preparation of broad bean (Vicia faba L. minor) products Part,I. Broad bean protein isolates from seed flour and their acetylation in the neutral pH range

It is possible to prepare broad bean protein isolates with a high yield directly from seed flour using a wet process. This process involves an aqueous extraction at pH 7.5 followed by separation of the insoluble residue and isoelectric precipitation of the protein from the aqueous extract. Following the same principle is it possible to prepare acetylated broad bean protein isolates with any adjustable acetylation degree. For that acetic anhydride is to be added and the adjusted pH is to be maintained during the extraction. In both cases the yield of the protein is mainly determined by the used solid-liquid ratio.     

Physiology of dairy-associated Bacillus spp. over a wide pH range

Bacillus species isolated from alkaline wash solutions used for cleaning in place in South African dairy factories have been suggested to contaminate product contact surfaces of dairy processing equipment and result in post-pasteurization spoilage of milk and milk products. Growth and attachment of such Bacillus isolates under alkaline and acidic conditions have not been previously described. Therefore, the in vitro growth temperature and pH ranges, attachment abilities and hydrophobicity, and enzyme production capabilities of four Bacillus isolates (tentatively identified as B. subtilis115, B. pumilus122, B. licheniformis137 and B. cereus144) previously isolated from the alkaline wash solutions in a South African dairy were examined. Growth pH ranges were determined in buffered Standard One-like Nutrient Broth and in unbuffered 1% Milk Medium at pH values ranging from 3 to 12. Growth and attachment to stainless steel surfaces and production of protease and lipase enzymes were determined in 1% Milk Medium at pH 4, 7 and 10. Colony hydrophobicity of each isolate by the Direction of Spreading Method (DOS) was also determined at pH 4, 7 and 10. In addition, Arrhenius plots were used to examine the growth temperature ranges of the isolates. All isolates grew at pH values ranging from 4.5 to 9.5 in buffered Standard One-like Nutrient Broth, and from pH 4 to 10 in 1% Milk Medium. All isolates also attached to stainless steel at pH 4, 7 and 10 in 1% Milk Medium. Generally the attachment of B. subtilis115, B. pumilus122 and B. lichenformis137 to stainless steel surfaces was enhanced at pH 4 and 10, compared to pH 7. By contrast, the best attachment of B. cereus144 cells to stainless steel surfaces was at pH 7. Planktonic and attached cells of all isolates produced proteolytic enzymes at pH 7 and 10, but not at pH 4. Similarly, planktonic and attached cells of B. subtilis115, B. pumilus122 and B. licheniformis137 produced lipolytic enzymes at pH 7 and 10, and weak lipolysis was observed at pH 4. The Bacillus cereus144 isolate showed no lipolytic activity at pH 10. All isolates exhibited low hydrophobic properties at all pH values even though attachment to stainless steel at the same pH values occurred. None of the isolates grew below 11 degrees C or above 56 degrees C, and optimum growth temperatures were in the high mesophilic range (36-44 degrees C).     

Biochemical characterisation of MX-4, a plant cysteine protease of broad specificity and high stability

A new proteinase, mexicain-IV (MX-4), has been purified to homogeneity from the latex fruits of Jacaratia mexicana (formely Pileus mexicanus), a plant member of the Caricaceae family. MX-4 shows a Mr of 23.7 kDa, pI of 9.3 and maximum proteolytic activity on casein and BAPNA at pH 8.0–8.5 and pH 7.0–7.5, respectively. The amino acid sequence of MX-4 and its reversible inhibition by HgCl₂ show that the proteinase belongs to the family of cysteine proteinases. This enzyme exhibits rather broad substrate specificity, although there seems to be a slight preference for cleavage of peptides having certain hydrophobic residues in the P2 position. Biochemical and circular dichroism studies revealed that this enzyme belongs to the α + β class of proteins, in agreement with the results obtained by X-ray crystallographic structure determination, and showed that MX-4 has a higher pH and thermal stability than other members of the Caricaceae family, including papain. These properties make this novel protease a suitable novel analytical tool for the proteomic analysis of peptide fragments of great potential interest in the food industry and other industries.     

不同蛋白酶酶解对蚕豆蛋白生物活性的影响

以青海蚕豆为原料,通过干燥、粉碎、等电点沉淀、冻干等工艺提取蚕豆蛋白,选用酸性蛋白酶、菠萝蛋白酶、胃蛋白酶对其进行酶解,并以抗氧化活性及α-葡萄糖苷酶活性抑制率为评价指标,考察不同蛋白酶酶解对蚕豆蛋白生物活性的影响。结果表明:经蛋白酶酶解后,蚕豆蛋白多肽得率极显著升高;胃蛋白酶处理后的蚕豆蛋白多肽得率达39. 14%;经菠萝蛋白酶和酸性蛋白酶酶解后可以明显增加蚕豆蛋白中氨基酸的种类和含量。菠萝蛋白酶处理后的蚕豆蛋白酶解产物对ABTS自由基的清除率可达90. 53%(质量浓度96μg/mL),对α-葡萄糖苷酶的活性抑制率可达88. 10%(质量浓度48μg/mL);经酸性蛋白酶处理后的蚕豆蛋白酶解产物对DPPH自由基的清除率可达95. 71%(质量浓度96μg/mL)。经蛋白酶酶解后蚕豆蛋白的生物价值得以大幅度提升,具有很好的开发前景。     

曲霉型豆豉酿造中米曲霉产蛋白酶作用条件研究

研究曲霉型豆豉酿造用米曲霉沪酿3.042产蛋白酶在其酿造过程中的最适作用条件。利用大豆豆汁培养基对米曲霉沪酿3.042菌株进行培养,并用(NH4)2SO4溶液对所产蛋白酶进行沉淀,脱盐处理后,研究该酶的最适作用pH及pH稳定性、最适作用温度及热稳定性以及Na Cl浓度对酶活力的影响。结果显示:该蛋白酶最适作用pH为6.0,酶活最高为1941U/m L,在pH为6.0~8.0时稳定性较高;最适作用温度为50℃,酶活为2658U/m L,在40℃以下其酶活力较稳定;Na Cl对淀粉酶酶促反应有明显的抑制作用,随着Na Cl浓度的升高,其抑制效果越明显。     

Evaluation of indicator-based pH measurements for freshwater over a wide range of buffer intensities

Two different sulfonephthalein indicators, cresol red (CR) with a pKa of approximately 8.3 and bromothymol blue (BTB) with pKa of approximately 7.4, were tested for an analysis of freshwater over a broad range of pH and total alkalinity values. Measurements from an autonomous sensor system using a 1 cm optical path length were compared to those using a 10 cm path length on a benchtop spectrophotometer. The indicator pH perturbation was quantified with a thermodynamic model and nonlinear least-squares analysis. The laboratory study found that the perturbation-corrected pH differed between the 1 cm (large indicator perturbation) and 10 cm (small indicator perturbation) optical path length measurements from -0.017 to +0.15 with a median of +0.0041 pH units for CR and from -0.015 to +0.026 with a median of -0.0008 pH units for BTB. Precision was +0.0005-0.013 and +0.0001-0.0027 pH units for the 1 and 10-cm-path-length measurements, respectively. The autonomous sensor was deployed for 14 days in a local creek. Simultaneous glass pH electrode measurements had a large negative and drifting offset (-0.15 to -0.40 pH units) compared to the indicator-based measurements. This study is the first in situ comparison between potentiometric and spectrophotometric pH methods in a freshwater system.     

Effects of oxidase and protease treatments on the breadmaking functionality of a range of gluten-free flours

In this study, the application of glucose oxidase and protease commercial preparations was investigated in order to evaluate their impact on the breadmaking performance of four different gluten-free flours (buckwheat, corn, sorghum and teff). Bread formulas were developed without addition of hydrocolloids in order to avoid synergistic effects. Glucose oxidase improved corn (CR) and sorghum (SG) bread quality by increasing specific volume (P < 0.05) and reducing collapsing at the top. The improvements could be related to protein polymerization which resulted in enhanced continuity of the protein phase and elastic-like behavior of CR and SG batters. No significant effects were detected on buckwheat (BW) and teff breads. On the other hand, protease treatment had detrimental effects on the textural quality of BW and SG breads. The effects were related to protein degradation resulting in increased liquid-like behaviour of BW and SG batters. Overall, the results of this study suggest that protein polymerisation can improve the breadmaking performance of gluten-free flours by enhancing elastic-like behaviour of batters. However, the protein source is a key element determining the impact of the enzymes. In the absence of hydrocolloids, protein structures are important to ensure the textural quality of these types of breads.     

Some viscosity characteristics of faba bean protein isolates within a pH range relevant for foods

The rheological behaviour of concentrated aqueous dispersions of unmodified (FBPI I) and acetylated (FBPI I) faba bean (Vicia faba L. minor) protein isolates are investigated within a pH range of 4.0 up to 7.5. Besides the fact that equal concentrated dispersions of FBPI I show a much lower apparent viscosity η than those prepared with FBPI II, η and the flow behaviour index n depend also on the kind of preparation. Especially the direction of pH shifting and the mode of pH adjustment lead to marked differences of the rheological properties. In the case of FBPI II dispersions a maximum of η and n is shown at pH 5.5 to 6.5, seldom at pH 7.0, probably connected with a maximum of the hydrodynamic volume. Holding the aqueous dispersions for 24h causes mostly an increase of η and n.     

Evaluation of viral extraction methods on a broad range of Ready-To-Eat foods with conventional and real-time RT-PCR for Norovirus GII detection

Noroviruses (NoV) are a common cause of foodborne outbreaks. In spite of that, no standard viral detection method is available for food products. Therefore, three viral elution-concentration methods and one direct RNA isolation method were evaluated on a broad range of Ready-To-Eat (RTE) food products (mixed lettuce, fruit salad, raspberries and two RTE dishes) artificially seeded with a diluted stool sample contaminated with NoV genogroup II. These seeding experiments revealed two categories of RTE products, fruits and vegetables grouped together and RTE dishes (penne and tagliatelle salads) which are rich in proteins and fat formed another category. The RNA extracts were amplified and detected with two conventional RT-PCR systems (Booster and Semi-nested GII) and one real-time RT-PCR (Real-time GII) assay. A fast direct RNA isolation method detected 10(2) RT-PCRU on 10 g penne and tagliatelle salads with the conventional RT-PCR assays. However real-time RT-PCR was less sensitive for penne salad. A viral elution-concentration method, including a buffer solution for the elution step and one polyethylene glycol (PEG) precipitation step, was able to detect 10(2) RT-PCRU on 50 g frozen raspberries with conventional and real-time RT-PCR assays. Moreover the latter extraction method used no environmental hazardous chemical reagents and was easy to perform.     

Effect of stress-induced high post-mortem pH on protease activity and tenderness of beef

Forty-four Swiss Brown young bulls were stressed by regrouping unfamiliar animals before slaughter. M. longissimus thoracis (6-9th ribs) of carcasses were analysed for post-mortem pH, protease activities (m- and α-calpain, calpastatin and cathepsin B + L), Warner-Bratzler shear force and sensory tenderness and juiciness. Muscles were classified into three groups, according to ultimate pH values: > 6.3, 6.3-5.8 and < 5.8. The most significant difference related to high pH was a higher activity of m-calpain at 7th day post mortem. It was also found that meat showing the highest pH was significantly more tender and juicy. Sensory tenderness was highly correlated with activity of m-calpain at 7th day post mortem (r = 0.776) and with ultimate pH (r = 0.708). It is concluded that high ultimate pH induced by stress significantly increases m-calpain activity, and this results in a greatly enhanced tenderisation of beef meat.     

Preparation of broad bean (Vicia faba L. minor) products. Part 2. Broad bean protein isolates from seed flour in the pH range below the isoelectric point

Broad bean protein isolates in aqueous solutions form stable gels after heating at pH values below the isoelectric point (pH 2-3) if they are extracted at the same pH. But the acid extraction is a time-consuming and expensive process. This paper outlines a new and simple procedure for the preparation of broad bean protein isolates in the acid pH range. It consists of a short extraction of seed flour at pH 7.5 (30 min) followed by an after-treatment at pH 2-3 (60 min) and gives the highest yield of acid treated broad bean protein isolates after a sudden acidification.     

Characterizing and quantilying controls on arsenic solubility over a pH range of 1-11 in a uranium mill-scale experiment

A mill-scale hydrometallurgical experiment (2700 m3 of effluent treated/day) was conducted for three months at the Rabbit Lake uranium mine site located in northern Saskatchewan, Canada, to determine the controls on the solubility of dissolved arsenic over a pH range of 1-11 and to develop a thermodynamic database for the dominant mineralogical controls on arsenic in the mill and the resulting mill tailings. The arsenic concentrations in the mill ranged from 526 mg/L at pH 1.0 (initial) to 1.34 mg/L at pH 10.8 (final discharge). Geochemical modeling of the chemistry data shows that arsenic solubility is controlled by the formation of scorodite (FeAsO4-2H2O) from pH 2.4 to pH 3.1, with 99.8% of dissolved arsenic precipitated as scorodite. Model results show that scorodite is unstable (releasing arsenic back in to solution) above pH 3.1 and arsenic adsorption to the surface of 2-line ferrihydrite is the dominant controlling factor in the solubility of arsenic from pH 3.2 to pH 11.0, with 99.8% of dissolved arsenic removed from solution via this mechanism. Finally, model results show -0.2% of the total dissolved arsenic adsorbs to the surface of amorphous aluminum hydroxide from pH 5.0 to pH 8.0. Minor alterations to the thermodynamic properties of arsenite and arsenate adsorption to 2-line ferrihydrite allowed the fit between measured mill-scale and modeled concentrations for the pH range of 3.2-11.0 to be optimized.     

Catalysis and stability of an alkaline protease from a haloalkaliphilic bacterium under non-aqueous conditions as a function of pH, salt and temperature

A haloalkaliphilic bacterium, isolated from Coastal Gujarat (India) was identified as Oceanobacillus sp. (GQ162111) based on 16S rRNA gene sequence. The organism grew and secreted extra cellular protease in presence of various organic solvents. At 30% (v/v) concentration of hexane, heptane, isooctane, dodecane and decane, significant growth and protease production was evident. The alkaline protease was purified in a single step on phenyl sepharose 6 FF with 28% yield. The molecular mass as judged by SDS-PAGE was 30?kDa. The temperature optimum of protease was 50°C and the enzyme retained 70% activity in 10% (v/v) isooctane. Effect of salt and pH was investigated in combination to assess the effect of isooctane. In organic solvents, the enzyme was considerably active at pH 8-11, with optimum activity at pH 10. Salt at 2?M was optimum for activity and enzyme maintained significant stability up to 18?h even at 3?M salt concentration. Patters of growth, protease production, catalysis and stability of the enzyme are presented. The study resumes significance as limited information is available on the interaction of haloalkaliphilic bacteria and their enzymes with organic solvents.     

Gelation of a broad bean (Vicia faba L. minor) protein fraction (prepared at pH 2) in dependence on some influencing factors

A protein fraction is prepared from broad bean protein isolate at pH 2 which forms gels in the acid pH range. The gels are fluid at high temperature and become strength when cooling. The gel strength increases with increasing pH, with increasing heat treatment, and with increasing protein concentration. Addition of calcium and dextrose sirup increases the gel strength whereas sucrose decreases it. The transparency of the gels decreases with increasing pH.     

Potassium concentration and pH inter‐relationships in grape juice and wine of Chardonnay and Shiraz from a range of rootstocks in different environments

Background and Aims: pH adjustment during winemaking is a significant cost to the Australian wine industry. This study addresses potassium (K⁺) concentration and pH inter‐relationships in grape juice and wine of Chardonnay and Shiraz. Methods and Results: Chardonnay and Shiraz on own roots, and on Ramsey, 1103 Paulsen, 140 Ruggeri, K51‐40, Schwarzmann, 101‐14, Rupestris St. George and 1202 Couderc were compared at Koorlong and Merbein (Victoria), and Padthaway, Nuriootpa and Rowland Flat (South Australia). Petiole K⁺ concentrations at flowering were a poor indicator of grape juice and wine K⁺ concentrations. The concentration of H⁺ ions in grape juice and wine decreased as K⁺ concentrations increased resulting in increased pH. The relationship between H⁺ and K⁺ concentrations was linear for Chardonnay but exponential for Shiraz, where K⁺ concentrations were higher. Wine K⁺ and grape juice K⁺ concentrations exhibited a positive linear relationship, with slope for Chardonnay about half that for Shiraz, indicating a net loss of K⁺ between grape juice and wine of 58% for Chardonnay and 13% for Shiraz. Conclusions: The study has linked higher wine pH to both higher juice soluble solids and K⁺, and to poorer wine colour hue. Loss of K⁺ during fermentation and cold stabilisation appeared higher for Chardonnay than for Shiraz. Significance of the Study: Rootstocks that lead to lower K⁺ concentrations and pH in grape juice and wine are identified. Differences in the K⁺ concentration dynamics between grape juice and wine of Chardonnay and Shiraz are described and quantified.     

Isolation and properties of protease a from wheat grain

To support the sulphydryl nature of the proteolytic enzymes of wheat grain, an isolation and purification procedure was devised by which the presence of at least two different proteases in the material studied was demonstrated. Protease A was shown to contain both reactive and masked sulphydryl groups. Low molecular weight reducing compounds appeared to activate the enzyme, whereas specific sulphydryl blocking and oxidising agents had a marked inhibitory effect. Thus, the sulphydryl-dependent nature of the mechanism of activation and inhibition of wheat protease A was confirmed. When incubated with the wheat proteases, gluten appeared to be resistant to their proteolytic action. The wheat grain protease was active not only against protein substrates but also against some low molecular weight peptides. The enzyme preparation showed maximum activity at pH 5–6 for the substrates studied. All the experiments performed in the course of this study support the sulphydryl nature of wheat grain protease A.