Hydrolysis of Milk Fat By Lipase in Solvent-free Phospholipid Reverse Micellar Media

Butterfat was hydrolyzed with lipases contained in lecithin reverse micelles. The influence of pH, temperature, molar ratio of surfactant to water (R) and surfactant concentration on the hydrolytic reaction indicated linear, quadratic, and interactive effects for reaction systems mediated by R. javanicus and C. rugosa lipases. The initial reaction rate was dependent on reaction parameters; however, the degree of hydrolysis was independent of pH. Both enzymes exhibited high thermal stability. The content and composition of milk fat hydrolysates prepared by R. javanicus lipase were most influenced by reaction temperature and R. The optimum conditions for production of free fatty acids, monoacyl-glycerols, diacylglycerols and specific regio-isomers were defined.     

Preparation Methods for Monodispersed Garlic Oil Microspheres in Water Using the Microemulsion Technique and Their Potential as Antimicrobials

Garlic oil is considered as a natural broad‐spectrum antibiotic because of its well‐known antimicrobial activity. However, the characteristics of easy volatility and poor aqueous solubility limit the application of garlic oil in industry. The purpose of the present work is to develop and evaluate an oil‐free microemulsion by loading garlic oil in microemulsion system. Microemulsions were prepared with ethoxylated hydrogenated castor (Cremophor RH40) as surfactant, n‐butanol (or ethanol) as cosurfactant, oleic acid‐containing garlic oil as oil phase, and ultrapure water as water phase. The effects of the ratio of surfactant to cosurfactant and different oil concentration on the area of oil‐in‐water (O/W) microemulsion region in pseudoternary phase diagrams were investigated. The particle size and garlic oil encapsulation efficiency of the formed microemulsions with different formulations were also investigated. In addition, the antimicrobial activity in vitro against Escherichia coli and Staphylococcus aureus was assessed. The experimental results show that a stable microemulsion region can be obtained when the mass ratio of surfactant to cosurfactant is, respectively, 1:1, 2:1, and 3:1. Especially, when the mixture surfactants of RH40/n‐butanol 2/1 (w/w) is used in the microemulsion formulation, the area of O/W microemulsion region is 0.089 with the particle size 13.29 to 13.85 nm and garlic oil encapsulation efficiency 99.5%. The prepared microemulsion solution exhibits remarkable antibacterial activity against S. aureus.     

Gemini型阳离子表面活性剂反胶束体系萃取纤维素酶的研究

本文研究了Gemini型阳离子酯季铵盐表面活性剂Ⅱ-14-3反胶束萃取纤维素酶的性能,以探索新型表面活性剂在反胶束萃取酶蛋白中的应用。考察了水相pH、离子强度、离子种类、酶浓度、表面活性剂浓度、助溶剂浓度、溶剂比和助表面活性剂(卵磷脂)等因素对萃取率的影响,确定了萃取纤维素酶的最佳条件:[NaCl]=50mmol/L,[Ⅱ-14-3]=0.3mmol/L,pH6.4,C0=0.14,溶剂比S=1.0,萃取率E接近80%,其酶活达到原来的93.38%;若加入适量的卵磷脂([Ⅱ-14-3]/[PC]=36:1)可提高萃取率,萃取率E达90%以上,且酶活达到121.41%。并且从反胶束微观结构给予解释。     

The effect of thermal treatment on β‐glucosidase inactivation in vanilla bean (Vanilla planifolia Andrews)

The conditions for enzyme activity (pH and temperature) and kinetic parameters for the thermal inactivation of β‐glucosidase enzyme in vanilla beans have been investigated. The maximum enzyme activity was detected at pH 6.5 and 38 °C. The values obtained for V_max and K_m were 62.05 units and 2.07 mm, respectively. When hot water treatment (the most practical method of vanilla bean killing) was applied, β‐glucosidase treated at pH 6.0 and 60 °C for 3 min lost 51% of activity, while at 70 °C for 90 s the enzyme lost 60% of activity and at 80 °C for 30 s the enzyme lost 48% of its activity. When vanilla beans were cured in an oven at 60 °C for 36 to 48 h all β‐glucosidase activity was lost.     

EFFECTIVE PURIFICATION PROCEDURE OF ASPERGILLUS ORYZAEα-AMYLASE FROM SOLID STATE FERMENTATION CULTURES INCLUDING CONCANAVALIN A-SEPHAROSE

Solid state fermentation cultures of Aspergillus oryzae NRRL 3485 on moistened wheat bran produced high levels of α-amylase as judged by the specific activity of water extracts (500–700 units/mg protein) and by the enzyme concentration (around 0.15 g/L) in those extracts. The purification of α-amylase by affinity chromatography on Concanavalin A-Sepharose is described. A first DEAE-Sepharose chromatography step (binding of the enzyme contained in the water extract and further elution with 0.2 M NaCl, pH 5) was necessary in order to remove contaminants that hinder the binding of the glycoprotein α-amylase to the lectin. Elution was performed with 7.5 mM α-D-methylmannoside. The enzyme was obtained highly concentrated and purified with a specific activity of 2000 units/mg protein and a recovery of around 60%, free of α-glucosidase, amyloglucosidase and color contaminants. The purity of the enzyme preparation was assessed through nondenaturing gel electrophoresis and sucrose gradient centrifugation. An improvement of the performance of the purification procedure is presented in which the maximum capacities of both the anion exchanger and the lectin matrices were exploited.     

EFFECT OF SURFACTANT GEL AND GUM COMBINATIONS ON DOUGH RHEOLOGICAL CHARACTERISTICS AND QUALITY OF BREAD

Sodium stearoyl‐2‐lactylate (SSL), diacetyl tartaric acid esters of monoglyceride (DATEM), glycerol monostearate (GMS) and distilled glycerol monostearate (DGMS) surfactant gels were made with water. Addition of surfactant gels decreased water absorption by the bread while xanthan, karaya, guar and locust bean gums increased the same. Only DGMS or GMS and gum combinations further improved water absorption. All the gums except for guar along with surfactant gels improved dough stability. Both surfactant gels and gums improved the extensograph dough properties of wheat flour to varying degrees. Alveograph characteristics of wheat flour improved to varying extents with surfactant gels while the gums influenced the viscoelastic properties in differing ways. Different combinations of surfactant gels and gums showed varied influences on rapid visco analyzer characteristics of wheat flour. Both surfactant gels and gums improved the bread making quality. Among surfactants, SSL in combination with gums, and among gums locust bean in combination with surfactant gels improved the bread making quality of wheat flour to a maximum extent.     

BIOCATALYSIS OF CHLOROPHYLLASE IN CANOLA OIL USING ORGANIC SOLVENT SYSTEMS

The hydrolytic activity of a partially purified chlorophyllase, obtained from an algal source, was investigated in a refined-bleached-deodorized (RBD) canola oil model system using various organic solvent media, including water/miscible-organic, biphasic organic, and micellar ternary systems. Although the use of a ternary micellar system containing Span 85 was found to be the most appropriate medium for the hydrolysis of chlorophyll, the hydrolytic activity of chlorophyllase declined as oil content increased. The effects of various parameters, including surfactant concentration, hexane/buffer proportion, enzyme content, reaction time, incubation temperature, shaker speed, and activator concentration, on chloro-phyllase activity in the micellar ternary systems containing 20% RBD oil were also investigated. In addition, phytol displayed a noncompetitive inhibition in reaction media containing polysorbate 80 and Span 85. The plot of 1/v versus oil concentrations revealed that the oil had K _i values of 3.73 and 4.56% in the micellar systems containing polysorbate 80 and Span 85, respectively. Moreover, the presence of 10% oil in the micellar system containing span 85 and polysorbate 80 decreased V _max values of chlorophyllase activity by 6.2 and 9.6-fold, respectively.     

Inactivation of yeast and filamentous fungi by the lactoperoxidase-hydrogen peroxide-thiocyanate-system

The antifungal activity of the lactoperoxidase (LPO) system with glucose oxidase (GOD) as source of hydrogen peroxide was determined in salt solution and in apple juice. The test organisms Rhodutorula rubra and Saccharomyces cerevisiae were cultivated aerobically in apple juice, Mucor rouxii was grown on wort agar adjusted to pH 4.5. Aspergillus niger and Byssochlamys fulva were kept on malt extract agar. Spores of the filamentous fungi were harvested by suspension in salt solution supplemented with Tween 80® and checked microscopically. The antifungal activity of the combined enzyme system was tested with initial counts of approx. 10⁵ cfu · ml^?1 (yeast cells or spores) suspended in salt solution supplemented with 25 mg · l^?1 thiocyanate and 20 g · l^?1 glucose or in apple juice supplemented with the same amount of thiocyanate. The tests were performed with 25 ml of the medium in 100 ml Erlenmeyer flasks shaken at 28 °C under aerobic conditions. Inactivation was achieved for all test organisms in both media. The yeast strains were found to be least stable while B. fulva was most resistant. A combination of 5 U · ml^?1 LPO with 0.5 to 1 U · ml^?1 GOD was sufficient for complete inactivation of this mold in salt solution within 2 h. The enzyme system also showed antifungal activity in apple juice at acid pH (3.2), although its effectiveness was reduced. In this medium, B. fulva was inactivated by 20 U · ml^?1 LPD and 1 U · ml^?1 GOD within 4 h. R. rubra and S. cerevisiae were unable to survive in apple juice at 5 U · ml^?1 LPO combined with 1 U · ml^?1 GOD. For inhibition by GOD alone, higher amounts of this enzyme were needed and even then only M. rouxii and R. rubra have been affected within the concentration range tested (maximum 3 U · ml^?1).     

Chemical Composition and Antidiabetic Activity of Essential Oils Obtained from Two Spices (Syzygium aromaticum and Cuminum cyminum)

The current aim was to evaluate antidiabetic potential of Syzygium aromaticum and Cuminum cyminum essential oils and their emulsions by alpha amylase inhibition assay. Antidiabetic activity of C. cyminum and S. aromaticum was examined in dose dependent mode (1 to 100 µg/mL). The maximum antidiabetic activity for S. aromaticum and C. cyminum essential oils was noted at the highest dose (100 µg/mL). Five emulsions (essential oil + surfactant [tween 80] + co-surfactant [ethanol] + water) of different concentrations for S. aromaticum (A1 to A5) and C. cyminum (B1 to B5) essential oils were formulated. Among different emulsions, A5 of S. aromaticum and B5 of C. cyminum essential oil exhibited a maximum antidiabetic activity with 95.30 and 83.09% inhibition of α-amylase, respectively. Moreover, the analysis of essential oils showed that eugenol (18.7%) and α-pinene (18.8%) were the major components of S. aromaticum and C. cyminum essential oils, respectively.     

Polyphenoloxidase activity from strawberry fruit (Fragaria ananassa,Duch., cv Selva): characterisation and partial purification

In this work, polyphenoloxidase (PPO) from Selva strawberry fruit (Fragaria × ananassa, Duch) was extracted, characterised and partially purified. The activity of PPO was analysed in crude extracts obtained from either fresh fruits or acetone powder. The presence of NaCl and Triton X‐100 in the extraction buffer caused a marked increase in enzyme extractability. The enzyme showed an apparent K_m value of 11.2 mM with pyrocatechol as substrate. The maximum enzyme activity was observed at 50 °C and pH 5.3–6.0 without SDS and pH 7.2 in the presence of SDS. The presence of SDS increased PPO activity at pH 7.2 but diminished it at pH 6.0. The enzyme showed high thermal stability and maintained activities equal to or greater than 50% of its maximum activity in the 2.6–9.3 pH range. One polyphenoloxidase isoenzyme was detected in crude extracts of all ripening stages, showing an isoelectric point of 7.3. The specific activity of PPO decreased continuously through fruit ripening. Maximum specific activities were found at the ‘small green’ and ‘large green’ ripening stages. A total enzyme extract was partially purified by means of (NH₄)₂SO₄ precipitation and cationic exchange chromatography in an FPLC system. The purification grade achieved was near 25. The partially purified enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 135 ± 4 kDa for the native protein. © 2000 Society of Chemical Industry     

RECOVERY OF PROTEINASE FROM PACIFIC WHITING SURIMI WASH WATER

A proteinase from Pacific whiting surimi wash water (SWW) was recovered by ohmic heating, ultrafiltration, and freeze drying with overall yield of 0.83 g protein/L SWW and 78% recovery of activity. Ohmic heating conditions were optimized for the maximum recovery of the enzyme. Different applied voltages (50, 70, 90 V) showed no differences in efficiency for removing protein and retaining cathepsin Lactivity. Cathepsin L activity reached its maximum after ohmic heating to 55C whereas cathepsin B activity decreased constantly with increased temperature. A constant reduction in protein content was observed with the increase in temperatures from 45C to 60C and holding time up to 5 min. The highest retention of both total and specific activity of cathepsin L was obtained with the treatment at 55C for 3 min. Under these conditions, 193% activity was recovered from SWW although a large amount of the activity was lost by the subsequent steps of ultrafiltration and freeze drying.     

Construction of R16F and D19L mutations in the loop I of bile salt hydrolase (BSH) enzyme from Lactobacillus plantarum B14 and structural and functional analysis of the mutant BSHs

Bile salt hydrolase (BSH), found commonly in intestinal species of Lactobacillus and Bifidobacterium, catalyzes the hydrolysis of glycine or taurine-conjugated bile acids into the amino acids and free bile acids. Deconjugated bile acids potentially play an important role in the reduction of blood cholesterol level and formation of some gastrointestinal diseases such as cholestasis, gallstone formation, and colon cancer. Although the crystal and three-dimensional structures of BSH enzyme are known, the working mechanism of catalytic activity of such an important BSH enzyme is not known very well. Previous in silico analysis of multiple BSH has identified that Arginine-16 (R16) and Aspartate-19 (D19) were catalytically important residues in the active site of BSH. To confirm the function of these amino acids, in this study, BSH enzyme from Lactobacillus plantarum B14 strain was cloned into Escherichia coli and strictly conserved polar R16 and D19 amino acids of BSH enzyme were substituted for hydrophobic Phenylalanine-16 (F16) and Leucine-19 (L19) amino acids, respectively, by polymerase chain reaction (PCR)-based site-directed mutagenesis. The effects of the mutations on catalytic activity and structure of the BSH enzymes were detected by ninhidrin assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, respectively. Research results showed that although F16 mutation led to loss of enzyme activity completely, L19 mutation led to abolishment of the synthesis of BSH enzyme. These results indicated that R16 and D19 amino acids located in loop I of the BSH enzyme might be critical for catalytic activity and assembly of the BSH enzyme respectively.     

Modeling Milk Clotting Activity in the Continuous Production of Microbial Rennet from Mucor miehei

Production of microbial rennet, a milk clotting enzyme, from a commercial strain of Mucor miehei (NRRL 3420) was investigated in a continuously fed fermenter system for prolonged periods. The spherical film-wise growth of the culture has been accomplished and the effects of medium pH, mixing and dilution rates, and feed of D-glucose concentration on milk clotting activity was investigated. In model simulation studies, maximum milk clotting activity was generated from a multiple linear function. This was expressed in terms of fermentation medium pH, D-glucose, dissolved oxygen concentrations and dilution rate at the time of maximum milk clotting activity.     

In vitro and in vivo antifungal activity of a water-dilutable cassia oil microemulsion against Geotrichum citri-aurantii

BACKGROUND: Recently, food‐grade microemulsions have been of increasing interest to researchers and have shown great potential in industrial applications. In this study a food‐grade water‐dilutable microemulsion system with cassia oil as oil, ethanol as cosurfactant, Tween 20 as surfactant and water was developed and its antifungal activity in vitro and in vivo against Geotrichum citri‐aurantii was assessed. RESULTS: The phase diagram results confirmed the feasibility of forming a water‐dilutable microemulsion based on cassia oil. One microemulsion formulation, cassia oil/ethanol/Tween 20 = 1:3:6 (w/w/w), was selected with the capability to undergo full dilution with water. The average particle size was 6.3 nm. The in vitro antifungal experiments showed that the microemulsion inhibited fungal growth on solid medium and prevented arthroconidium germination in liquid medium and that cassia oil had stronger activity when encapsulated in the microemulsion. The in vivo antifungal experiments indicated that the water‐dilutable microemulsion was effective in preventing postharvest diseases of citrus fruits caused by G. citri‐aurantii. CONCLUSION: The results of this study suggest a promising utilisation of water‐dilutable microemulsions based on essential oils for the control of postharvest diseases. Copyright © 2012 Society of Chemical Industry     

Effect of Sodium Dodecyl Sulphate on the Physicochemical,Thermal and Pasting Properties of Cassava Starch

The effect of different concentrations of sodium dodecyl sulphate (SDS) on cassava starch properties after complexation and in situ addition were studied. The swelling volume of the starch‐SDS complexes increased to a maximum of 100 mL/g with increase in concentration of SDS (even at 0.004 mol SDS) with a corresponding decrease in solubility. During in situ addition also, a similar but gradual behaviour was observed for swelling volume and solubility. The apparent and total amylose contents showed an irregular trend. The soluble amylose content decreased with increase in concentration of SDS. In vitro enzyme digestibility of starch‐SDS complexes was significantly lowered compared to that of native starch. The water‐binding capacity showed an initial decrease followed by a significant increase at higher concentrations of SDS. DSC studies showed that in addition to the gelatinisation endotherm, a peak corresponding to the melting of the starch‐SDS complex was obtained in the case of in situ addition of the surfactant. The peak viscosity of starch during in situ addition of SDS exhibited a rapid increase except at the highest concentration. The peak viscosity of starch‐SDS complexes underwent an initial reduction followed by a gradual increase at higher concentrations. The breakdown, setback and pasting temperature were also affected.     

Esterolytic and Lipolytic Activities of Lactobacillus Casei-subsp-Casei LLG

The estcrolytic and lipolytic enzymes were produced by cell lysis of Lactobacillus casei-subsp-casei LLG during the late logarithmic growth phase. The enzyme was purified to 67 fold by ion exchange chromatography and gel filtration chromatography using the FPLC system. Polyacrylamide gel electrophoresis and sodium dodecyl sulfate-poly-acrylamide gel electrophoresis using the “Phast” system of the purified enzyme showed a single protein band for butyrate-esterase (3.2 × 10⁵ Dalton), caproate esterase (1.1 × 10⁵ Dalton) and capryate esterase (4.0 × 10⁴ Dalton), respectively. The maximum lipolytic activity was observed at pH 7.2 and 37°C. The enzyme activity was inhibited by silver and mercury ions but magnesium and calcium stimulated lipolytic activity. The Km and Vmax values for esterase-lipase of the strain LLG were 76 μM/min/mg of protein, and 0.57 mM, respectively. This enzyme was stable at room temperature for at least 2 days.     

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).     

POLYPHENOLOXIDASE ACTIVITY, BROWNING POTENTIAL AND PHENOLIC CONTENT OF PEACHES DURING POSTHARVEST RIPENING

Changes in the polyphenoloxidase (PPO) activity and the phenolic content of peaches (Prunus persica cv. Premier) during postharvest ripening were studied. The fruits were stored at 12 or 25C for up to 15 days. The quantity of extractable proteins was maximum at 6–10 days storage at 25 and 12C, coinciding with the onset of the yellowness in the fruits. The PPO activity increased up to the ripening stage, showing a maximum value at 8 days of storage. This was coincident with the maximum degree of browning as evaluated by the absorbance at 440 nm. The amount of total phenolics and chlorogenic acid in the fruits decreased during storage; however, the differences were not significant. The browning potential closely correlated with the enzyme activity, but not with the phenolic content.     

Catalase-based thin-layer enzyme cell used in organic-phase FIA system for determination of moisture in oily foods

The aim of our present work was to study the possibility of constructing a biosensor based on immobilized catalase enzyme (EC 1.11.1.6.) in organic-phase solutions. The catalase enzyme was immobilized by glutaraldehyde on a natural protein membrane in a thin-layer enzyme cell, connected to a stopped-flow injection analyser (SFIA) system with an amperometric detector. Adding FMCA to acetonitrile, the optimal concentration was 7.5 mg l^–1, while with TBATS it was 2.7 mg l^–1. The optimal pH value of the immobilized enzyme in buffer was about 6.0. On studying the role of the buffer solution used in the carrier solvent, the activity of the enzyme changed dramatically. The signal was highest when there was no buffer added to the carrier solution and decreased rapidly when the content increased (0–1.5%). Utilizing these results, a quick analytical method was developed to monitor indirectly the water content (activator) in various butter and margarine samples by maintaining a fixed substrate concentration. The water content of samples was compared with the results obtained by the gravimetric reference method (AOAC Method 920.116); the correlation coefficient (r) was 0.993.     

Flocculation Properties of Poly(��-Glutamic Acid) Produced from Bacillus subtilis Isolate

Bacillus subtilis R 23 produced extracellular biopolymer showing excellent flocculation activity. The biopolymer was confirmed as poly(γ-glutamic acid) (PGA) using high-performance liquid chromatography profile and product characterization. The production, characteristics, and flocculation properties of PGA were studied. PGA produced by B. subtilis R 23 was devoid of any polysaccharides and had a molecular weight of 6.2 × 10⁶ Da. The flocculating activity of PGA in the kaolin suspension was markedly stimulated by the addition of bivalent and trivalent cations in optimum concentration. The pH of reaction mixture also influenced the flocculating activity. Response surface methodology was used to establish the optimum parameters for maximum flocculating activity and to study their interactions. A maximum flocculating activity of 30.32 ± 1.4 1/optical density was obtained with 7.5 mg/L of PGA in combination with 8.0 mM of Ca⁺² at pH 7.5.