Crinumin,a chymotrypsin-like but glycosylated serine protease from Crinum asiaticum: Purification and physicochemical characterisation

Plant latex could be a potential source of novel proteases usable in the food and feed industries because of broad substrate specificity with high stability in extreme conditions. Crinumin, a glycosylated serine protease with chymotrypsin-like activity was purified from the latex of Crinumasiaticum using cation-exchange column chromatography. Crinumin shows activity over a wide range of pH (4.5–11.5 and optimum at 8.5), temperature (75 °C and optimum at 70 °C) and is also functional against chaotrophs, organic solvents, and detergents, even after prolonged exposure. The molecular mass (67.7 kDa), extinction coefficient (17.7), isoelectric point (6.9), and numbers of tryptophan (13), tyrosine (24) and cysteine (15 with 7 disulphide bridges) residues were estimated. K_m of the enzyme was 31.7 μM with casein and 5 × 10⁴ μM with N-succinyl-l-phenylalanine-p-nitroanilide. Easy availability of the aqueous latex, simple purification procedure, high yield (33%), stability and activity in adverse conditions makes it applicable for the pharmaceutical and food industries.     

Expression of recombinant Thermomonospora fusca xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it

The mature peptide of Thermomonospora fusca xylanase A (TfxA) was successfully expressed in Pichia pastoris under the control of AOX1 promoter. The activity of recombinant T. fusca xylanase A (reTfxA) in culture supernatant was 117.3 ± 2.4 U/mg, which is 3 times higher than that of the native TfxA. The optimal temperature and pH for reTfxA were 60 °C and 6.0, respectively. When treated at 70 °C and pH 6.0 for 2 min, the residual activity of the reTfxA was 70%. The reTfxA was very stable over a wide pH range (5.0–9.0). After incubation over pH 5.0–9.0 at 25 °C for 1 h, all the residual activity of reTfxA was over 80%. The K_m and k_cat values for reTfxA were 2.45 mg/ml and 139 s⁻¹, respectively. HPLC analysis revealed that xylobiose (X2) was the main hydrolysis product released from birchwood xylan and wheat bran insoluble xylan by reTfxA. Hydrolysis results of xylooligosaccharides showed that reTfxA was an endo-acting xylanase and xylobiose, xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolysed. This is the first report on the expression of reTfxA in yeast and on the determining and quantifying of the hydrolysis products released from xylans and xylooligosaccharides by reTfxA.     

Effect of storage temperature on phenolics stability in hawthorn (Crataegus pinnatifida var. major) fruits and a hawthorn drink

The stability of five major phenolics, namely (−)-epicatechin (EC), procyanidin B₂ (PC-B₂), chlorogenic acid (ChA), hyperoside (HP) and isoquercitrin (IQ), in hawthorn fruits and a canned hawthorn drink were evaluated during 6 months of storage in the dark at three different temperatures (4, 23 and 40 °C). HPLC with a diode-array detector was used to determine the contents of the individual compounds. The results showed that the studied phenolics in the hawthorn fruits and the drink were both stable at 4 °C and relatively unstable at 23 and 40 °C with varied extents of degradation. At room temperature (23 °C), marked degradations of EC and PC-B₂ were observed in both the fruits and the drink with around 50% and 30% decrease after a 6-month storage, respectively. A more significant decrease of the phenolics was observed at 40 °C, especially for EC and PC-B₂, which were almost completely degraded after a 6-month storage. HP, IQ and ChA were relatively stable at 23 °C, but unstable at 40 °C. Therefore, low-temperature storage is recommended for maintaining the quality and efficacy of hawthorn fruits and its preparations.     

Characterisation of a thermostable family 42 β-galactosidase (BgalC) family from Thermotoga maritima showing efficient lactose hydrolysis

A β-galactosidase gene (TM_1195) of Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant β-galactosidase (BgalC), belonging to glycosyl hydrolase (GH) family 42, was purified to homogeneity with 23.4-fold purification and a recovery of 36.6%. Its molecular mass was estimated to be 78 kDa by SDS–PAGE. BgalC exhibited maximum activity at an optimal pH of 5.5 and an optimum temperature of 80 °C. The enzyme displayed important properties, such as stability over a broad pH range of 5.0–9.0 and thermostability up to 75 °C. K_m values of BgalC for p-nitrophenyl-β-galactopyranoside (pNPGal), o-nitrophenyl-β-galactopyranoside (oNPGal) and lactose were 1.21, 7.31 and 6.5 mM, respectively. BgalC was efficient in complete removal of lactose from milk. BgalC is significantly one of the few β-galactosidases from family 42 displaying significant hydrolysis of lactose. These properties make BgalC an ideal candidate for commercial use, in the production of lactose-free milk.     

Mass spectrometric characterization and thermostability of turkey myoglobin

Pink color defect (PCD) is a major quality problem in the turkey industry leading to pink appearance of pre-cooked, uncured turkey. The present study determined the molecular mass of turkey myoglobin (Mb) using mass spectrometry and characterized the thermostability of turkey Mb, in comparison with beef Mb, to elucidate the molecular basis of PCD. Purified turkey and beef myoglobins were analyzed using Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry. The thermostability of turkey and beef oxymyoglobins was examined at pH and Mb concentrations (pH 6.2 and 0.04 mmol/L for turkey; pH 5.6 and 0.15 mmol/L for beef) reflecting inherent conditions in these meats. Turkey and beef oxymyoglobins were incubated at 71, 75, and 80 °C and percentage myoglobin denaturation (PMD) was determined. Molecular mass of turkey Mb (17,295 Da) was 346 Da greater than beef Mb (16,949 Da) and was approximately 300-350 Da greater than those of other red meat myoglobins, suggesting its unique primary structure. PMD was lower (P < 0.05) in turkey Mb than in beef Mb during incubation at 71, 75 and 80 °C, indicating that in-situ turkey Mb is less susceptible to heat-induced denaturation than beef Mb at typical meat cooking conditions. The observed greater thermostability of turkey Mb compared to beef Mb could be, partially, due to inherent greater pH in turkey than in beef. Possible unique primary structure of turkey Mb could have contributed to its greater thermostability, which is one of the reasons for PCD.     

Rapid,simple and sensitive determination of the apparent formation constants of trans-resveratrol complexes with natural cyclodextrins in aqueous medium using HPLC

The study of the complexation of trans-resveratrol with natural cyclodextrins (CDs) in aqueous medium under different physico-chemical conditions of pH or temperature is essential if this antioxidant compound is to be used successfully in the food industry as ingredient of functional foods, due its poor stability, bioavailability and solubility. In this paper, a rapid, simple and sensitive determination of the apparent formation constant of trans-resveratrol/CD complexes by HPLC in aqueous medium has been investigated for first time. It can be observed that trans-resveratrol forms a 1:1 complex with α-, β- and γ-CD. The highest value of the apparent formation constant (K_F = 1922 ± 89 M⁻¹) was found for β-CD and a strong dependence of K_F on pH can be seen in the region where the trans-resveratrol begins the deprotonation of their hydroxyl groups. Moreover, an increase in the system’s temperature produced a decrease in the values of K_F. Finally, to gain information on the mechanism of the trans-resveratrol affinity for CD, the thermodynamic parameters of the complexation were obtained.     

A β-galactosidase from chick pea (Cicer arietinum) seeds: Its purification,biochemical properties and industrial applications

A β-galactosidase from Cicer arietinum seeds has been purified to apparent electrophoretic homogeneity using a combination of various fractionation and chromatographic techniques, giving a final specific activity of 220 units mg⁻¹, with approximately 1840 fold purification. Analysis of the protein by SDS–PAGE revealed two subunits with molecular masses of 48 and 38 kDa, respectively. These bands were further confirmed with LC–MS/MS, indicating that Chick pea β-galactosidase (CpGAL) is a heterodimer. Molecular mass was determined to be 85 kDa by Superose-12 FPLC column, which is in agreement with the molecular mass suggested by mass spectroscopy to be 83 kDa. The optimum pH of the enzyme was 2.8 and it hydrolysed o-nitrophenyl β-d galactopyranoside (ONPG) with a K_m value of 1.73 mM at 37 °C. The energy of activation (E_a) calculated in the range of 35 to 60 °C, using Arrhenius equation, was determined to be 11.32 kcal mol⁻¹. The enzyme could also hydrolyse lactose, with an optimum pH of 4.0 at 40 °C. K_m and E_a for lactose hydrolysis was found to be 10 mM and 10.57 kcal mol⁻¹, respectively. The enzyme was found to be comparatively thermostable showing maximum activity at 60 °C for both ONPG and lactose. Galactose was found to be the competitive inhibitor. β-Galactosidase also exhibited glycoproteineous properties when applied on Con-A Sepharose column. The enzyme was localised in germinated seeds with X-gal activity staining and shown to be expressed prominently at grown radical tip and seed coat. Sequence alignment of CpGAL with other known plant β-galactosidase showed high amino acid sequence homology.     

Stability for olive oil control materials

For the reliability of analytical Quality Control Materials (QCMs), stability studies are essential. In spite of the importance of the stability studies, there is no article relating to QCMs of olive oil. This paper describes an intralaboratory stability study aimed for assuring olive oil QCMs concerning to the physical–chemical characteristics defined in Commission Regulation (EC) No. 1989/2003. The study concluded that, in storage conditions of darkness and low controlled temperature (0–8 °C), critical physical–chemical parameters of the olive oil QCMs, such as acidity level, peroxide value, K-values and total waxes, are stable at least during 24 months (2 years) with, in general, light variability and up trends. Theoretically, according to this study a homogeneous sample olive oil could be used as a QCM during two years if this material were stored in the dark and low temperature. Then, olive oil quality control testing laboratories have an alternative to dispose stable in-house QCMs for the control of their measurements.     

The influence of latitude on the concentration of vitamin D3 and 25-hydroxy-vitamin D3 in Australian red meat

There is little information on the vitamin D content of Australian red meat or on the possible influence of latitude on this content. To determine the content of vitamin D₃ and 25-hydroxy-vitamin D₃ (25OHD₃), lamb and beef were analysed from 34° S with LC–IT-MS. To investigate the possible influence of latitude on vitamin D in meat, the lean meat and fat from five cuts of beef were analysed from 17° S and 41° S. Lamb contained 0.10 μg vitamin D₃/100 g and 0.20 μg 25OHD₃/100 g lean meat, while beef contained 0.12 μg vitamin D₃ and 0.27 μg 25OHD₃/100 g (lean meat). Latitude had no effect on the vitamin D₃ (P = 0.21) or 25OHD₃ (P = 0.29) content of lean beef, but fat from cattle in the 17° S latitude group contained significantly higher (P < 0.01) concentrations of vitamin D₃ than fat from the 41° S group of cattle.     

Partial hydrolysis of soybean oil by phospholipase A1 (Lecitase Ultra)

The partial hydrolysis of soybean oil, as catalysed by phospholipase A₁ (Lecitase Ultra) in a solvent-free system, was investigated in this study. The optimal pH and temperature for the partial hydrolysis of soybean oil by phospholipase A₁ (Lecitase Ultra) were 6.8 and 40 °C, respectively. Phospholipase A₁ (Lecitase Ultra) displayed good stability over a range of pH values from 4.7 to 7.4, and at temperatures below 60 °C. Phospholipase A₁ (Lecitase Ultra) is postulated to possess sn-1,3-position regiospecificity towards triacylglycerols (TAGs), based on the identification of the fatty acids released from TAGs following partial hydrolysis by swine pancreatic lipase (SPL) and phospholipase A₁ (Lecitase Ultra). Alternative TAG hydrolysis routes for phospholipase A₁ (Lecitase Ultra) are postulated, and several reaction equilibrium and rate constants were determined.     

Characterisation of a thermostable family 42 β-galactosidase from Thermotoga maritima

A β-galactosidase gene (TM_0310) of Thermotoga maritima MSB8 was expressed in Escherichia coli. The recombinant β-galactosidase (designated BgalB) was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. BgalB belongs to the glycoside hydrolase family 42. Its molecular mass was estimated to be 78 kDa and 76 kDa by SDS–PAGE and gel filtration, respectively. The enzyme was optimum at pH 5.5, and it was quite stable over the pH range 5.0–11.4 at 70 °C. It was optimally active at 80 °C and was stable up to 75 °C. Besides, BgalB exhibited broad substrate specificity with a preference for p-nitrophenyl-β-galactopyranoside (pNPGal). K_m values of the purified enzyme for pNPGal, o-nitrophenyl-β-galactopyranoside (oNPGal) and pNP-β-fucopyranoside were 2.7 mM, 12.5 mM and 1.4 mM, respectively. These properties make this enzyme an interesting candidate for biotechnological applications. This is the first report of the family 42 β-galactosidases from T. maritima.     

Degradation of curdlan using hydrogen peroxide

Curdlan, a linear glucan interconnected by β-(1 → 3) linkages, is soluble in alkaline solutions but not in water, which limits its wide application, particularly in the food industry. In this study, curdlan was subjected to oxidative degradation using hydrogen peroxide (H₂O₂). The optimal hydrolysis conditions were determined, and the results were as follows: reaction time, 40 min; temperature, 60 °C; H₂O₂ concentration, 1.5% (v/v); and NaOH concentration, 2.5 M. Under these optimised conditions, the maximum dextrose equivalent value (13.49%) was obtained. The composition and the structure of the hydrolysates were characterised by high-performance liquid chromatography and Fourier-transform infra-red spectroscopy, respectively. The hydrolysates were filtered, neutralised with HCl, concentrated to ∼12% (w/v), desalted, and freeze dried to yield a water-soluble, white powder. The (1 → 3)-β-d-glucan oligosaccharide content of the product was 98.6% and the yield was 91.4% (w/w).     

Characterisation of a β-N-acetylhexosaminidase from a commercial papaya latex preparation

A β-N-acetylhexosaminidase (β-NAHA) (EC 3.2.1.52) with molecular mass of 64.1 kDa and isoelectric point of 5.5 was purified from a commercial papaya latex preparation. The optimum pH for p-nitrophenyl-N-acetyl-β-d-glucosaminide (pNP-β-GlcNAc) hydrolysis was five; the optimum temperature was 50 °C; the K_m was 0.18 mM, V_max was 37.6 μmol min⁻¹ mg⁻¹ and activation energy (E_a) was 10.3 kcal/mol. The enzyme was thermally stable after holding at 30–45 °C for 40 min, but its activity decreased significantly when the temperature exceeded 50 °C. Heavy metal ions, Ag⁺ and Hg²⁺, at a concentration of 0.25 mM and Zn²⁺ and Cu²⁺, at a concentration of 0.5 mM, significantly inhibited enzyme activity. The β-NAHA had only one active site for binding both pNP-β-GlcNAc and p-nitrophenyl-N-acetyl-β-d-galactosaminide (pNP-β-GalNAc). A prototropic group with pKa value of about five on the enzyme may be involved in substrate binding and transformation, as examined by Dixon–Webb plots.     

Physicochemical and structural characterisation of protein isolate,globulin and albumin from soapnut seeds (Sapindus mukorossi Gaertn.)

The amino acid (AA) composition and physicochemical and conformational properties of protein isolate (SNPI), globulin (SNG) and albumin (SNA) fractions from soapnut seeds were evaluated. The essential AA of SNG, SNA and SNPI (except sulfur-containing AA) are sufficient for the FAO/WHO suggested requirements for 2–5 year old infants. SNG and SNPI showed similar electrophoresis patterns and AA compositions, the subunit of those proteins consisted of two polypeptides linked by disulfide bonds. In contrast, SNA showed a different AA compositions and SDS–PAGE pattern. Both SNG and SNPI presented a typical U-shape protein solubility (PS)–pH profile, SNA showed a completely different PS–pH profile, especially at pH 2.0–4.0. The near-UV circular dichroism (CD), differential scanning calorimetry (DSC) and tryptophan fluorescence spectra analyses indicated that the flexibility in tertiary conformations decreased in the order: SNA > SNPI > SNG, while soapnut proteins had a similar secondary conformation, with a highly ordered structure (the β-types), as evidenced by far-UV CD spectra.     

Characterisation of an acidic peroxidase from papaya (Carica papaya L. cv Tainung No. 2) latex and its application in the determination of micromolar hydrogen peroxide in milk

An acidic peroxidase isoform, POD-A, with a molecular mass of 69.4 kDa and an isoelectric point of 3.5 was purified from papaya latex. Using o-phenylenediamine (OPD) as a hydrogen donor (citrate–phosphate as pH buffer), the optimum pH for the function of POD-A was 4.6, and the optimum temperature was 50 °C. The peroxidase activity of POD-A toward hydrogen donors was both pH- and concentration-dependent. Under optimal conditions, POD-A catalysed the oxidation of OPD at higher rates than pyrogallol, catechol, quercetin and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The chemical modification reagents N-bromosuccinimide and sodium azide significantly inhibited POD-A activity. The results of kinetic studies indicated that POD-A followed a ping-pong mechanism and had a K_m value of 2.8 mM for OPD. Using CPC silica-immobilised POD-A for the determination of micromolar H₂O₂ in milk, the lower limit of determination was 0.1 μM, and the recoveries of added H₂O₂ were 96–109%.     

Comparative shelf life study of blackberry fruit in bio-based and petroleum-based containers under retail storage conditions

The shelf life of blackberries is relatively short, 2–3 days at 0 °C. Different marketing strategies like packaging can be used to retain blackberry quality during postharvest. This study compares the blackberry retail shelf life performance of different packaging materials, bio-based versus petroleum-based using the same packaging design. ‘Cancaska’ and ‘Chester’ blackberries were packaged in snap-fit closed packages made from oriented poly(lactic acid), OPLA, and oriented poly(styrene), OPS, and stored at 3 °C and 85% RH for three weeks. Both cultivars exhibited an increase in pH, weight loss, SSC to TA ratio, and fungal count, and a reduction in firmness, anthocyanin content, TA, and SSC during storage. The changes in TA, SSC, SSC to TA ratio, and weight loss significantly depended on the packaging material while no such effect was observed on firmness, anthocyanin content, pH and fungal growth. Both cultivars demonstrated better quality in the OPS container with less weight loss, and decrease in SSC and TA. Blackberries in both OPS and OPLA containers met the “US standard No 1” grade for commercialisation for more than 12 days at 3 °C.     

A novel thermostable chitinase (PJC) from pomegranate (Punica granatum) juice

A novel chitinase was isolated and purified to its homogeneity from pomegranate juice by a combination of ammonium sulphate precipitation and ion-exchange chromatography. The pomegranate juice chitinase (PJC) was purified to specific activity of 14.5 U/mg and a recovery of 34%. The monomeric protein migrated on SDS–PAGE at 29 kDa. The enzyme was found to be glycosylated (7.2%). It exhibited optimal activity at pH 4.5 and 70 °C. The enzyme was stable in the pH range 3.0–9.0 and up to 65 °C. The internal peptide sequence results suggest that the purified PJC shared high homology with class III chitinases of other known plant chitinases. The purified enzyme could hydrolyse colloidal chitin to its oligomers. It did not exhibit any antifungal activity.     

Water migration mechanisms in amorphous powder material and related agglomeration propensity

The agglomeration phenomenon of amorphous particulate material is a major problem in the food industry. Currently, the glass transition temperature (T_g) is used as a fundamental parameter to describe and control agglomeration. Models are available that describe the kinetics of the agglomeration process as a function of the distance of the material from T_g (i.e. T − T_g). In practice these models are often not applied because they assume that the powder material is instantly in equilibrium with the humidity conditions of the environment and that solid mobility only occurs at T > T_g. Insights in the kinetics and mechanisms of water transport in powder material can help to better understand and control powder agglomeration. For this purpose, gravimetric step-change water sorption experiments were performed on maltodextrins as a function of the water activity a_w. The maltodextrins vary in dextrose equivalents (DE), particle size and morphology. The experimental results were compared with a Fickian diffusion model in order to understand the dependency of the transport mechanism on water concentration gradient and material relaxation. The water transport kinetics in the maltodextrins with low DE (i.e. 6) were well described by Fickian diffusion for low a_w, independently of particle size and morphology, until relaxation phenomena started to occur at an a_w corresponding to T − T_g = −20 °C. The importance of the matrix relaxation phenomena on the water transport mechanism increased with increasing DE (i.e. 29 and 32), not showing any relationship with the T_g. The results of this study indicate that the water migration mechanism is controlled by relaxation phenomena when the amorphous material is still far from the glass–rubber transition. The T − T_g at which the relaxation phenomena occur depends on the material. On the contrary, the T − T_g parameter could well describe the onset of agglomeration, independently from the material properties. Therefore, it can be concluded that within the conditions of this study, matrix relaxation occurring far below T_g did not affect the onset of agglomeration.     

Extractability and chemical and enzymic degradation of psyllium (Plantago ovata Forsk) seed husk arabinoxylans

To assess the potential of arabinoxylan (AX)-rich psyllium (Plantago ovata Forsk) seed husk (PSH) as a source for production of arabinoxylo-oligosaccharides (AXOS), the parameters determining PSH AX extraction yield and the chemical and enzymic hydrolysis of PSH AX were investigated. The seed husk material had a high content of AX (62.5%) with an arabinose to xylose ratio of 0.41. The water extractability of PSH AX was affected by suspension concentration rather than by temperature. Maximally, 27% of all AX could be extracted, even when using very dilute suspensions (0.1% w/v). When subjected to alkaline conditions, a pH of at least 12 was needed to significantly increase extractability (up to 77% of all AX). The extractability-increasing effect of alkali was, however, reversible, as extractability decreased again when lowering the pH. Acid hydrolysis at high temperature drastically increased AX extraction yields (up to 97% of all AX), released monomeric arabinose (up to 38%), and lowered the average degree of polymerisation (DP_avg) of the AX (down to 31). The presence of substituents (arabinose, xylose, rhamnose and galacturonic acid) on the xylan backbone was an important factor limiting degradation by xylanases. Enzyme preparations containing substituent-removing enzymes were far better for enhancing the extractability of a large portion of the AX population (up to 82%) and for degrading the extracted fragments (down to DP_avg 14) than preparations lacking such activities. The above results show that PSH is a good source for the production of AXOS, since both chemical and enzymic treatments significantly increase the extractability of PSH AX and convert PSH AX molecules into small fragments.