Purification and Characterization of Extremely Thermostable Exo-oligo-1,6-glucosidase from a Caldoactive Bacillus sp. KP 1228

A p-nitrophenyl-α-D -glycopyranoside-hydrolyzing oligo-1,6-glucosidase (dextrin 6-α-glucanohydrolase, EC 3.2.1.10) of a caldoactive Bacillus sp. KP 1228 capable of growing at 51–82°C was purified to homogeneity. The molecular weight was estimated as 140,000. The enzyme consisted of two identical subunits each comprising a threonine residue at the NH₂-terminus. The enzyme was most active at 85°C and pH 5.1, and stable for 10 min up to 85°C at pH 6.8. The enzyme had no antigenic determinant common to oligo-1,6-glucosidases from Bacillus cereus ATCC 7064 (mesophile), Bacillus coagulans ATCC 7050 (facultative thermophile) and Bacillus thermoglucosidasius KP 1006 (DSM 2542) (obligate thermophile). A strong correlation between the increase in proline content and the rise in thermostability of these 4 proteins was observed.     

Improved extraction of disulphide‐rich bioactive proteins from soya hulls: characterisation of a novel aspartic proteinase

Soya hull, an underutilised coproduct of soya processing, was investigated as a source of disulphide‐rich bioactive proteins. A Viscozyme L‐assisted extraction method was developed to improve the yield of extracted proteins. The extracted proteins were identified by MALDI TOF–TOF MS, and the most abundant disulphide‐rich protein among identified proteins was purified and the enzymatic properties were evaluated. The results indicated that the Viscozyme L‐assisted extraction method extracted significantly (P < 0.05) more proteins (39.01%) than did the aqueous method (4.52%). The yield of the purified aspartic proteinase nepenthesin‐1‐like [Glycine max] (GmAPN1K) (the most abundant disulphide‐rich protein) is 570 mg Kg^?1. The specific activity of GmAPN1K was 62.1 U mg^?1 at pH 3.0 and 37 °C. The enzyme was optimally active at pH 3.0 and 55 °C. It was stable within pH range 3.0–10.0 and up to 55 °C, respectively, and was specifically inhibited by pepstatin A.     

Purification and Properties of a Thermostable Inulinase (β-D-Fructan Fructohydrolase) from Bacillus stearothermophilus KP1289

A thermophilic soil isolate, Bacillus stearothermophilus KP1289, that grew from 41 °C to 69 °C, produced extracellular inulinases in the presence of inulin. One (inulinase II) of these enzymes was purified to homogeneity. The molecular weight (M_r) and the isoelectric point of the enzyme were estimated as 54,000 and 5.0, respectively. The enzyme was active between 30 and 75 °C and at pH 4.5—8.6 with an optimum at 60 °C and pH 6.1. At 69 °C and pH 7.0 the half-life of the enzyme was 10 min. The enzyme released fructose exo-wise from the non-reducing end of inulin (M_r = 4,5000). The Michaelis constant, catalytic center activity, and specificity constant for inulin at 60 °C and pH 5.0 were 80 mM (360 mg/mL), 460 s^—1, and 5.8 s^—1 mM^—1, respectively. The ratio of specificity constants for inulin, sucrose, and raffinose was 1:0.50:0.16. The enzyme was classified as a thermophilic thermostable β-D -fructan fructohydrolase (EC 3.2.1.80).     

Modelling the survival of Enterobacter cloacae in a model olive cover brine solution

The main goal of this research was the evaluation of the survival of Enterobacter cloacae in a model olive brine. Two different assays were run; the first experiment assessed the viability of the target in brines containing NaCl (6–12%) and p‐coumaric acid (0.0–0.05%), adjusted to different pHs (4–10) and stored at 10–30 °C for 9 days. The death rate and cell levels at selected times were modelled with a polynomial equation to highlight the individual and interactive effects of NaCl/p‐coumaric acid/pH/temperature. Then, a second experiment was run for 3 months (temperature, 10 °C; pH, 4.5–5.5; NaCl, 6–8%). The survival of E. cloacae was affected mainly by pH, then by salt and temperature; however, the significance of the variables changed within the time, as salt and temperature acted in a significant way only after 1 day.     

Physicochemical properties and storage stability of spray‐dried soya bean sprout extract

Amylase is a very important enzyme due to its wide food applications. To preserve amylase activity in soya bean sprout extract (SSE), SSEs were spray‐dried with 10% maltodextrin and 0–3% alginic acids, and their physicochemical properties and storage stability were compared with freeze‐dried one. SSE exhibited maximum amylase activity at pH 7.0 and 60 °C, with the most active substrate specificity towards soluble starch. Spray‐dried SSEs exhibited higher water solubility index (WSI) and in vitro relative amylase activity but lower water vapour sorption (WVS) and smaller particle size than freeze‐dried SSE. For spray‐dried SSEs, particle size, WSI and in vitro relative amylase activity increased while WVS decreased with increasing % alginic acid. This study demonstrated that spray drying of SSE, especially with 10% maltodextrin and 2% alginic acid, was effective in keeping amylase active and stable during 7‐week storage at room temperature (25 °C).     

Physicochemical characteristics and gelation properties of collagen superfine powder from swine skin: the effects of preheating treatment

Following different preheating treatments (60 °C for 20 min; 80 °C for 20 min; or 120 °C for 10 min, referred as T₁, T₂ and T₃), edible collagen superfine powder (CSP) from swine skin was prepared by superfine grinding method. The CSPs showed a preheating‐dependent decrease in the D₅₀, with different degrees of hydrolysis (9.51–31.05%). A significant effect of preheating on rheological properties of the CSP aqueous dispersions at pH 4–9 was observed, wherein T₂ had the biggest viscosity and water holding capacity. All the 5% CSP dispersions were transformed into stable cold‐set gels after heating at 50–90 °C for 20 min, with insignificant differences in strength. These attributes were consistent with microstructures of the CSP gels detected by scanning electron microscope.     

Purification and characterisation of an acid protease from the Aspergillus hennebergii HX08 and its potential in traditional fermentation

A protease AP3 from Aspergillus hennebergii HX08 was purified by ammonium‐sulphate precipitation, followed by anion‐exchange chromatography and gel filtration. The molecular weight of acid protease AP3 was 33 kDa (SDS‐PAGE and MALDI‐TOF‐MS). The protease AP3 was identified as an acid protease with MALDI‐TOF/TOF tandem MS. Its optimal temperature and pH were 60°C and 4.0, respectively. Its K _max and V _max were 57.92 mg/mL and 32.57 U/mL, respectively. The enzyme was active over a broad pH and temperature range (pH 3.0–5.0 and 30–60°C), and exhibited high activity and stability in 2–12% (v /v) ethanol solvent. Subsequent studies suggest that the enzyme presents a relatively high substrate affinity to wheat protein (98% of total activity). Its application to solid‐state fermentation of wheat flour with Saccharomyces cerevisiae could increase the hydrolysis degree of wheat protein (28.26%) and amino acid nitrogen concentration of fermented grains (34.21%). Additionally, enhanced S. cerevisiae biomass (37.09%) and alcohol concentration (38.29%) were also observed during the process. Volatile compounds analysis of fermented grains by headspace solid‐phase micro‐extraction and GC‐MS revealed more flavour compounds. These results suggest its potential in food and alcohol industries. Copyright © 2017 The Institute of Brewing & Distilling     

Equivalent processing for pasteurization of a pineapple juice–coconut milk blend by selected nonthermal technologies

Identifying equivalent processing conditions is critical for the relevant comparison of food quality attributes. This study investigates equivalent processes for at least 5-log reduction of Escherichia coli and Listeria innocua in pineapple juice–coconut milk (PC) blends by high-pressure processing (HPP), pulsed electric fields (PEF), and ultrasound (US) either alone or combined with other preservation factors (pH, nisin, and/or heat). The two blends (pH 4 and 5) and coconut milk (pH 7) as a reference were subjected to HPP at 300–600 MPa, 20°C for 0.5–30 min; PEF at an electric field strength of 10–21 kV/cm, 40°C for 24 µs; and US at 120 µm amplitude, 25 or 45°C for 6 or 10 min. At least a 5-log reduction of E. coli was achieved at pH 4 by HPP at 400 MPa, 20°C for 1 min; PEF at 21 kV/cm, 235 Hz, 40°C for 24 µs; and US at 120 µm, 45°C for 6 min. As L. innocua showed greater resistance, a synergistic lethal effect was provided at pH 4 by HPP with 75 ppm nisin at 600 MPa, 20°C for 5 min; PEF with 50 ppm nisin at 18 kV/cm, 588 Hz, 40°C for 24 µs; and US at 45°C, 120 µm for 10 min. The total soluble solids (11.2–12.4°Bx), acidity (0.47%–0.51% citric acid), pH (3.91–4.16), and viscosity (3.55 × 10⁻³–4.0 × 10⁻³ Pa s) were not significantly affected under the identified equivalent conditions. HPP was superior to PEF and US, achieving higher ascorbic acid retention and lower color difference in PC blend compared to the untreated sample.     

Application of chitosan–alginate microspheres for the sustained release of bacteriophage in simulated gastrointestinal conditions

This study was designed to evaluate the acid stability, release property and antimicrobial efficacy of Escherichia coli O157:H7 bacteriophages encapsulated in chitosan–alginate microspheres under the simulated gastrointestinal conditions. The bacteriophages belonging to Myoviridae family were stable at the pH above 4 in trypticase soy broth. The chitosan–alginate microspheres exhibited protective effect on the viability of bacteriophages in the simulated gastric conditions at pH 2.0 and pH 2.5, showing 4.8 and 5.6 log PFU mL^‐1, respectively, after 1 h of incubation at 37 °C. The release per cent of bacteriophages from microspheres gradually increased up to 65% in the simulated intestinal condition (pH 7.5) at 37 °C for 6 h. The lytic efficacy of chitosan‐ and alginate‐encapsulated bacteriophages against E. coli O157:H7 was significantly maintained in the simulated intestinal conditions to 10 h of incubation (1.3 log reduction). The results suggest that the chitosan–alginate microspheres can be used as a reliable delivery system for bacteriophages.     

Influence of CMC‐ and guar gum‐based silver nanoparticle coatings combined with low temperature on major aroma volatile components and the sensory quality of kinnow (Citrus reticulata)

Aroma profile and organoleptic quality of CMC‐ and guar gum‐based silver nanoparticle‐coated kinnow (Citrus reticulata cv. Blanco) was evaluated for 120 days at 4 °C and 10 °C, 85–95% relative humidity. Loss in three major aroma‐active volatile compounds (limonene, linalool and γ‐terpinene) was determined after every 15 days by GC‐MS. Sensory quality of coated and uncoated fruit stored at 10 °C was declined during storage. Twenty five volatile aroma compounds were identified in fresh kinnow juice. Guar gum‐Ag coatings and 4 °C storage has significantly reduced losses of limonene, linalool and γ‐terpinene contents from 91 to 23%, 99 to 10% and 97 to 29% respectively as compared to uncoated fruit stored at 10 °C. Study suggests that CMC‐ and guar gum‐based silver nanoparticle‐coated kinnow stored at 4 °C has preserved the fruit aroma and sensory quality for 120 days.     

Optimization and Partial Purification of Bacteriocins from Enterococcus spp. Indigenous to Pakistan

This study was conducted to optimize bacteriocin producing enterococcal strains isolated from indigenous fermented dairy products of Pakistan. Isolates IJ-06, IJ-21, and IJ-31 were identified as Enterococcus faecium and IJ-11 was identified as Enterococcus faecalis. All of these enterococcal isolates were catalase, gelatinase negative, and non-hemolytic on sterile sheep blood. Bacteriocin production trials confirmed E. faecium IJ-06, IJ- 21, and IJ- 31 as the potential producer of bacteriocin showing antimicrobial activity in cell-free supernatant (CFS). E. faecalis IJ-11 displayed activity after partial purification. Optimization of culture conditions for the production of bacteriocin by the selected strains showed that maximum production was achieved at 35–37°C with 1% inoculum after 16 h of incubation and at pH 7–8. The molecular mass of the partially purified enterocins falls in the range of 4.5–4.7 kDa. These enterocins were close to class IIa of bacteriocins and were highly active against Listeria monocytogenes, Bacillus subtilis, Bacillus cereus and other closely related strains.     

Multi‐Cereal Beverage Fermented by Lactobacillus Helveticus and Saccharomyces Cerevisiae

A novel multi‐cereal‐based fermented beverage with suitable aroma, flavor, and pH fermented by lactic acid bacteria and Saccharomyces cerevisiae was developed. Twenty‐seven lactobacilli strains were screened for acid production (pH and titratable acidity) in a mixture of malt, rice, and maize substrates. It was found that Lactobacillus helveticus KLDS1.9204 had the greatest acid production among 27 lactobacilli tested. The fermentation performance of L. helveticus KLDS1.9204 was also assayed and the fermentation parameters were optimized using Plackett–Burman design and steepest ascent method. L. helveticus KLDS1.9204 showed good proteolytic capability, however, the strain could not utilize starch. The optimum substrate consisted of 50% malt (25 g/100 mL), 25% rice (20 g/100 mL), and 25% maize (30 g/100 mL). The inoculum was 5% with a ratio of S. cerevisiae to L. helveticus KLDS1.9204 of 2.5:1. The optimum temperature was 37 °C and the time was 22 h. Lastly, the quality of the multi‐cereal‐based fermented beverage was evaluated. This beverage was light yellow, transparent, and it tasted well with a pleasant acid and a unique flavor of cereals. The beverage was rich in free amino acids and organic acids. The pH and titratable acidity of the beverage were 3.5 and 29.86 °T, respectively. The soluble solids content of the beverage was 6.5 °Brix, and the alcohol content was 0.67%.     

Functional properties and antioxidative activity of protein hydrolysates from toothed ponyfish muscle treated with viscera extract from hybrid catfish

Functional properties and antioxidative activity of a protein hydrolysate prepared from toothed ponyfish (Gazza minuta) muscle, using viscera extract from hybrid catfish (Clarias macrocephalus × Clarias gariepinus), with a degree of hydrolysis (DH) of 70%, were investigated. The protein hydrolysate had a good solubility. It was soluble over a wide pH range (3–9), in which more than 77% solubility was obtained. The emulsifying activity index of the protein hydrolysate decreased with increasing concentration (P < 0.05). Conversely, the foaming abilities increased as the hydrolysate concentrations increased (P < 0.05). Protein hydrolysate exhibited the increases in 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), 2,2‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) (ABTS) radical scavenging activities, ferric reducing power (FRAP) and metal chelating activity as hydrolysate concentration increased (P < 0.05). ABTS radical scavenging activity of protein hydrolysate was stable when heated at 100 °C for 180 min and subjected to a wide pH range (1–11). Therefore, protein hydrolysate from the muscle of toothed ponyfish produced by viscera extract from hybrid catfish can be used as a promising source of functional peptides with antioxidant properties.     

Physicochemical and functional properties of flours from three Black gram (Phaseolus mungo L.) cultivars

Flours processed from three black gram cultivars (Mash1‐1, PU‐19 and T‐9) were studied for proximate composition, physicochemical and functional properties. Protein, fat, ash and crude fibre content varied significantly (P ≤ 0.05) from 24.5 – 28.4%, 1.1–1.4%, 2.7–3.3% and 2.7–3.4%, respectively, among the cultivars. Significant differences were also observed in other physicochemical properties. Pasting properties like peak viscosity, breakdown viscosity and setback viscosity were in the range of 2078.7–2473.0 cP, 644.0–863.7 cP and 588.3–804.0 cP, respectively. Textural profile analysis of flour gels displayed significant differences in hardness (13.0–18.9 g), gumminess (2.8–5.9 g), chewiness (2.0–5.7 g) and adhesiveness (21.0–40.9 g). Differential scanning calorimetry revealed starch gelatinisation from 63.5 to 75.6 °C and amylose lipid complex melting from 105.0 to 136.1 °C. Protein solubility profile in the pH range of 2–9 varied from 6.3 to 97.3%. Emulsifying activity index and Emulsifying stability index at pH 3, 5 and 7 varied correspondingly from 6.0 to 14.5 m² g^?1 and 18.8 to 64.9 min. Foaming capacity of 2% flour suspensions at pH 2, 4, 6, 8 and 10 varied significantly from 67.3 to 130% among the cultivars. Foam stability varied at different pH and was observed between 0 and 70.8% among the cultivars.     

High‐pressure processing of Manuka honey: brown pigment formation,improvement of antibacterial activity and hydroxymethylfurfural content

This work was undertaken to assess the effects of high‐pressure processing on brown pigment formation, antibacterial activity and hydroxymethylfurfural (HMF) content in Manuka honey. The honey was subjected to different pressures (200–600 MPa) at ambient temperatures (25–33 °C) and with moderate temperatures (53–74 °C) for holding times (10–30 min). The brown pigment formation in high‐pressure‐treated sample showed a significant increase (0.16–0.17, P < 0.05), whereas maximum percentage inhibition of Staphylococcus epidermidis (84.34 ± 7.62%) was achieved when honey was subjected to 600 MPa as compared to the control (64.15 ± 5.86%). The percentage inhibition in high‐pressure‐treated samples correlated linearly (r = 0.941) with brown pigment. No significant increase in HMF as affected by high pressure was found (P > 0.05). Thus, high‐pressure processing has no adverse effect on the honey quality because it can control HMF concentration during process. Increasing brown pigment formation is associated with the enhancement of antibacterial activity.     

Purification and Characterization of an Oligo-1,6-glucosidase from the Caldoactive Thermophile Bacillus caldotenax

An oligo-1,6-glucosidase (dextrin 6-α-D -glucanhydrolase, EC 3.2.1.10) of the caldoactive thermophile Bacillus caldotenax KP 1213 (YT-G, DSM406) was purified to homogeneity. Its relative molecular weight, Stokes radius, sedimentation coefficient at 20°C in water, molar absorption coefficient at 280nm and pH 6.8, and isoelectric point were estimated to be 64,000, 3.3nm, 4.8S, 122,000M ⁻¹cm⁻¹, and 4.9, respectively. The enzyme N-terminal sequence of twenty residues was determined to be Met¹-Glu-Trp-Ala-Trp-Lys-Glu-Ala-Val-Val-Tyr-Gln-Ile-Tyr-Pro-Arg-Met-Phe-Tyr²⁰. The enzyme shared its antigenic groups in part with the homologue from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile). The B. caldotenax enzyme was most active at 70°C and at pH 6.0–6.8. The best substrate for the enzyme was isomaltotriose of naturally-occurring oligo- and polysaccharides tested. The enzyme half-life at pH 6.8 was 10min at 75°C. The enzyme (Pro, 5.93mol%) fairly matched with a positive relationship between the thermostability of its six bacillary counterparts and their proline mol% contents. This relationship has been found to hold for sixteen bacterial enzymes from other four different groups (α-glucosidases, pullulanases and 3-isopropylmalate dehydrogenases).     

Interaction of β‐casein with (−)‐epigallocatechin‐3‐gallate assayed by fluorescence quenching: effect of thermal processing temperature

The interactions between the flavan‐3‐ol (?)‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐casein in phosphate‐buffered saline (PBS) of pH 6.5 subjected to thermal processing at various temperatures (25–100 °C) were investigated using fluorescence quenching. The results indicated that different temperatures had different effects on the structural changes and EGCG‐binding ability of β‐casein. At temperatures below 60 °C, the β‐casein–EGCG interaction changed little (P > 0.05) with increasing temperature. At temperatures above 80 °C, native assemblies of β‐casein in solution dissociated into individual β‐casein molecules and unfolded, as demonstrated by a red shift of the maximum fluorescence emission wavelength (λ_max) of up to 8.8 nm. The highest quenching constant (K_q) and the number of binding sites (n) were 0.92 (±0.01) × 10¹³ m ^?1 s^?1 and 0.73 (±0.02) (100 °C), respectively. These results provide insight into the potential of interactions between β‐casein–EGCG that may modulate bioactivity or bioavailability to be altered during thermal process.     

Zinc‐loaded whey protein nanoparticles prepared by enzymatic cross‐linking and desolvation

Zinc‐loaded whey protein nanoparticles were prepared by enzymatic cross‐linking whey protein followed by ethanol desolvation. Whey protein isolate (WPI) was denatured by heating (80 °C for 15 min) at pH 7.0 and then cross‐linked by transglutaminase at 50 °C for 4 h while stirring. Transglutaminase was inactivated by heating at 90 °C for 10 min, and then, ZnSO₄·7H₂O (1–10 mm ) was added. Zinc‐loaded whey protein nanoparticles were formed by adding ethanol at one to five times the volume of the protein solution at pH 9.0. The desolvated solutions were diluted by adding distilled water at ratio of 1:100 (w/v) immediately after desolvation. Dynamic light scattering (DLS) data showed that the particle size of zinc‐loaded whey protein nanoparticles increased with the amount of zinc and the volume of ethanol. Scanning electron microscopy micrographs revealed an almost spherical morphology for zinc‐loaded whey protein nanoparticles. The zinc loading efficiency was determined ranging from 76.7% to 99.2%. In vitro test data showed that the zinc release rate was low in simulated gastric fluid but high in simulated intestinal fluid. The results indicated that enzymatic cross‐linked whey protein nanoparticles may be used as a good vehicle to deliver zinc as a supplement.     

Chemical and Physical Properties of Kiwifruit (Actinidia deliciosa) Starch

Chemical and physical properties of kiwifruit (Actinidia deliciosa var. ‘Hayward’) starch were studied. Kiwifruit starch granules were compound, irregular or dome‐shaped with diameters predominantly 4–5 µm or 7–9 µm. Kiwifruit starch exhibited B‐type X‐ray diffraction pattern, an apparent amylose content of 43.1% and absolute amylose content of 18.8%. Kiwifruit amylopectins, relative to other starches, had low weight‐average molecular weight (7.4×10⁷), and gyration radius (200 nm). Average amylopectin branch chain‐length was long (DP 28.6). Onset and peak gelatinization temperatures were 68.9°C and 73.0°C, respectively, and gelatinization enthalpy was high (18.5 J/g). Amylose‐lipid thermal transition was observed. Starch retrograded for 7 d at 4°C had a very high peak melting temperature (60.7°C). Peak (250 RVU), final (238 RVU) and setback (94 RVU) viscosity of 8% kiwifruit starch paste was high relative to other starches and pasting temperature (69.7°C) was marginally higher than onset gelatinization temperature. High paste viscosities and low pasting temperature could give kiwifruit starch some advantages over many cereal starches.     

Optimisation of process conditions for lactose hydrolysis in paneer whey with cold‐active β‐galactosidase from psychrophilic Thalassospira frigidphilosprofundus

The present study was conducted to analyse the physiochemical properties of Indian paneer whey. High concentration of minerals such as potassium, calcium, zinc and sodium, as NaCl, were observed which indicates the suitability of paneer whey in the preparation of beverages. A central composite rotatable design (CCRD) of response surface methodology (RSM) was employed to optimise the hydrolysis of lactose from whey using cold‐active β‐galactosidase of Thalassospira frigidphilosprofundus. Results indicated that 80% of lactose was hydrolysed at pH of 6.5 at 20 °C in 40 min in comparison with 40% at 30 °C. This emphasises the potential use of cold‐active β‐galactosidase in dairy industry.