Effects of thermal treatments on pectinesterase activity determined in blood oranges juices

The citrus processing industry aims to maintain turbidity and attractive colour of the juice obtained from Sicilian blood oranges. Nevertheless, the presence of pectinesterase (PE, E.C. 3.1.1.11) causes the loss of these peculiar characteristics, due to precipitation of colloids and very fine pieces of pulp in suspension, with negative effects on colour and clarification of the juices. This study compares total PE activity of Sicilian blood oranges (Sanguinello, Moro, Tarocco) with the blonde cultivar Navel, checking enzyme stability with various pasteurisation times and temperatures conditions. Decimal reduction time and temperature (D and z) as well as the kinetic constant (k) were established to optimise and increase the shelf-life of the pasteurised juice. Finally, a heat treatment (85 °C × 3 min) of both microbiological and enzymatic efficacy has been developed that does not compromise anthocyanin stability; this treatment could be used by the citrus fruit processing industry as a valid alternative in the production of blood orange juices.     

Quantification of abdominal and pelvic floor muscle synergies in response to voluntary pelvic floor muscle contractions

The relative levels of pelvic floor muscle (PFM) activation and pressure generated by maximum voluntary PFM contractions were investigated in healthy continent women. The normal sequence of abdominal and PFM activation was determined.Fifteen women performed single and repeated maximum voluntary PFM contractions in supine, sitting and standing. PFM electromyographic (EMG) signals and associated intra-vaginal pressure data were recorded simultaneously. Surface EMG data were recorded from rectus abdominus (RA), external obliques (EO), internal obliques (IO) and transversus abdominus (TA).Abdominal and PFM EMG and intra-vaginal pressure amplitudes generated during voluntary PFM contractions were not different among the positions. Muscle activation sequence differed by position. In supine, EO activation preceded all other muscles by 27 ms (p = 0.043). In sitting, all of the muscles were activated simultaneously. In standing, RA and EO were activated 11 and 17 ms, respectively, prior to the PFMs and TA and IO were activated 10 and 12 ms, respectively, after the PFMs (p ≪ 0.001).The results suggest that women are able to perform equally strong PFM contractions in supine, sitting and standing, however the pattern of abdominal and PFM activation varies by position. These differences may be related to position-dependent urine leakage in women with stress incontinence.     

Does the presence of a vaginal probe alter pelvic floor muscle activation in young,continent women?

Vaginal probes may induce changes in pelvic floor muscle (PFM) recruitment by the very presence of the probes. Fine-wire electrodes allow us to detect muscle activation parameters without altering the natural position and shape of the PFMs. The purpose of this study was to determine whether PFM activation is altered by changes in sensory feedback, muscle length or tissue position caused by two different vaginal probes used to record surface electromyography (EMG). Twelve continent women (30.1 ± 5.4 years), performed PFM maximal voluntary contractions (MVCs) in supine while fine-wire EMG was recorded bilaterally from the PFMs under three conditions: (a) without any probe inserted into the vagina, (b) while a Femiscan? probe was in situ, and (c) while a Periform? vaginal probe was in situ. The reliability of the fine wire EMG data was assessed using intra-class correlation coefficients (ICCs) and coefficients of variation (CV). A repeated measures analysis of variance (ANOVA) model was used to determine if there were differences in EMG amplitude recorded when the different vaginal probes were in situ. For each condition the between-trial reliability was excellent, ICC_(3,1) = 0.93–0.96, (p < 0.001) and CV = 11.2–21.8%. There were no differences in peak EMG amplitude recorded during the MVCs across the three conditions (no probe 63.4 ± 48.4 μV, Femiscan? 55.3 ± 42.4 μV, Periform? 59.4 ± 42.2 μV, p = 0.178). These results suggest that women produce consistent MVCs over multiple contractions, and that PFM muscle activation is not affected by different probes inserted into the vagina.     

Frontal analysis of cell-membrane chromatography for determination of drug-α1D adrenergic receptor affinity

The aim of the present study was to determine drug-α_1D adrenergic receptor (AR) affinity by frontal analysis of cell-membrane chromatography (CMC). The cell-membrane stationary phase (CMSP) was prepared by immobilizing rat aorta cell membranes on porous silica, and the resulting CMSP was used to determine drug binding affinity to α_1D-AR by frontal analysis. The CMSP of rat aorta was stable and reproducible. Relative binding affinities (dissociation constant, K_d) were determined by frontal chromatography for prazosin (166.13 ± 18.36 nmol), BMY7378 (537.40 ± 30.84 nmol), phentolamine (646.92 ± 23.17 nmol), 5-methylurapidil (725.66 ± 25.48 nmol), oxymetazoline (910.56 ± 40.62 nmol) and methoxamine (1299.27 ± 51.73 nmol). These results were consistent with the affinity rank order and showed a good correlation with the affinity of the same compounds for the cloned α_1D-AR subtype obtained from radioligand-binding assay. The study demonstrates that frontal analysis of CMC may be used for direct determination of drug–receptor binding interactions, and that CMC is an alternative reliable method to quantitatively study ligand–receptor interactions.     

Speciation of platinum in blood plasma and urine by micelle-mediated extraction and graphite furnace atomic absorption spectrometry

A highly sensitive and selective technique for the speciation of platinum by cloud point extraction prior to determination by graphite furnace atomic absorption spectrometry (GFAAS) was described. The separation of Pt(II) from Pt(IV) was performed in the presence of 4-(p-chlorophenyl)-1-(pyridin-2-yl)thiosemicarbazide (HCPTS) as chelating agent and Triton X-114 as a non-ionic surfactant. The extraction of Pt(II)–HCPTS complex needs temperature higher than the cloud point temperature of Triton X-114 and pH = 7, while Pt(IV) remains in the aqueous phase. The Pt(II) in the surfactant phase was analyzed by GFAAS, and the concentration of Pt(IV) was calculated by subtraction of Pt(II) from total platinum which was directly determined by GFAAS. The effect of pH, concentration of chelating agent, surfactant, and equilibration temperature were investigated. An enrichment factor of 42 was obtained for the preconcentration of Pt(II) with 50 mL solution. Under the optimum experimental conditions, the calibration curve was linear up to 30 μg L^?1 with detection limit of 0.08 μg L^?1 and the relative standard deviation was 1.8%. No considerable interference was observed due to the presence of coexisting anions and cations. The accuracy of the results was verified by analyzing different spiked samples (tap water, blood plasma and urine). The proposed method was applied to the speciation analysis of Pt in blood plasma and urine with satisfactory results.     

Synthesis,characterization and properties of a new polymerisable surfactant: 12-Methacryloyl dodecylphosphocholine

A new polymerizable surfactant, 12-methacryloyl dodecylphosphocholine (MDPC), has been synthesized using a three-step procedure in moderate yield. Phase transitions were characterized by DSC and phase behavior in water was determined by surface tension and polarizing microscopy. MDPC showed typical surfactant behavior and self-aggregated to micelles above a distinct concentration. The critical micelle concentration (CMC) of MDPC was determined to be 5 × 10^?4 mol/L. MDPC showed mesomorphic properties between 75 and 86 °C as studied by differential scanning calorimetry (DSC). The formation of black lipid membranes was further investigated. The methacrylate functionalized MDPC could form a bilayer membrane (BLM) although it was very unstable (collapsed after 10–30 s). However, it was possible to form stable BLMs in mixture with non-polymerizable two chain phospholipids, i.e. asolectin and diphytanoyl phosphatidylcholine (DPhPC). Stable bilayers could be obtained up to a MDPC content of 50 mol%. Gramicidin A was incorporated into MDPC/DPhPC membranes and exhibited ion-channel activity shown by single channel conductivity measurements.     

Apple juice attenuates genotoxicity and oxidative stress induced by cadmium exposure in multiple organs of rats

The aim of this study was to evaluate the health benefits associated with apple consumption following cadmium exposure. A total of 15 Wistar rats were distributed into three groups (n = 5), as follows: control group (non-treated group, CTRL); cadmium group (Cd) and apple juice group (Cd + AJ). The results showed a decrease in the frequency micronucleated cells in bone marrow and hepatocytes in the group exposed to cadmium and treated with apple juice. Apple juice was also able to reduce the 8OHdG levels and to decrease genetic damage in liver and peripheral blood cells. Catalase (CAT) was decreased following apple juice intake. Taken together, our results demonstrate that apple juice seems to be able to prevent genotoxicity and oxidative stress induced by cadmium exposure in multiple organs of Wistar rats.     

Hollow fiber liquid phase microextraction followed by high performance liquid chromatography for determination of ultra-trace levels of Se(IV) after derivatization in urine,plasma and natural water samples

In the present work, a simple and high sensitive method based on hollow fiber liquid phase microextraction (HF-LPME) was developed followed by high performance liquid chromatography (HPLC) for determination of ultra-trace amounts of Se(IV) after derivatization in biological and natural water samples. Se(IV) was complexed with o-phenylenediamine to form piazselenol. The formed piazselenol was extracted into 20 μL of 1-octanol located in the lumen of a hollow fiber and the solution was injected into HPLC-UV for analysis. Using the Taguchi method, an orthogonal array design (OAD), OA₁₆ (4⁵) was employed to optimize the HF-LPME of piazselenol. The effect of five experimental factors (each factor at four levels) including the volume of the organic phase, extraction time, pH of the solution, stirring rate and ionic strength on the extraction efficiency of piazselenol was studied and optimized. The maximum extraction efficiency of piazselenol was obtained at 20 μL of 1-octanol as the extracting solvent, 30 min extraction time, pH 2, stirring rate of 500 rpm and 30% (w/v) NaCl. Under the optimum conditions, preconcentration factors up to 130 were achieved and the relative standard deviation (%RSD) of the method was <3.7% for different concentrations of Se(IV). The calibration curves were obtained in the ranges of 0.2–100 and 0.05–10 μg L^?1 for the 11 and 50 mL of the sample volumes with reasonable linearity, respectively (r² > 0.995). The limits of detection (LOD) were 0.1 and 0.02 μg L^?1 for the 11 and 50 mL sample volumes, respectively (S/N = 3). Finally, the applicability of the proposed method was evaluated by the extraction and determination of Se(IV) in the plasma, urine and water samples.     

Stimuli responsive polymorphism of C12NO/DOPE/DNA complexes: Effect of pH,temperature and composition

N,N-dimethyldodecylamine-N-oxide (C₁₂NO) is a surfactant that may exist either in a neutral or cationic protonated form depending on the pH of aqueous solutions. Using small angle X-ray diffraction (SAXD) we observe the rich structural polymorphism of pH responsive complexes prepared due to DNA interaction with C₁₂NO/dioleoylphosphatidylethanolamine (DOPE) vesicles and discuss it in view of utilizing the surfactant for the gene delivery vector of a pH sensitive system. In neutral solutions, the DNA uptake is low, and a lamellar L_α phase formed by C₁₂NO/DOPE is prevailing in the complexes at 0.2 ≤ C₁₂NO/DOPE < 0.6 mol/mol. A maximum of ~ 30% of the total DNA volume in the sample is bound in a condensed lamellar phase L_α^C at C₁₂NO/DOPE = 1 mol/mol and pH 7.2. In acidic conditions, a condensed inverted hexagonal phase H_II^C was observed at C₁₂NO/DOPE = 0.2 mol/mol. Commensurate lattice parameters, a_HC ≈ d_LC, were detected at 0.3 ≤ C₁₂NO/DOPE ≤ 0.4 mol/mol and pH = 4.9–6.4 suggesting that L_α^C and H_II^C phases were epitaxially related. While at the same composition but pH ~ 7, the mixture forms a cubic phase (Pn3m) when the complexes were heated to 80 °C and cooled down to 20 °C. Finally, a large portion of the surfactant (C₁₂NO/DOPE > 0.5) stabilizes the L_α^C phase in C₁₂NO/DOPE/DNA complexes and the distance between DNA strands (d_DNA) is modulated by the pH value. Both the composition and pH affect the DNA binding in the complexes reaching up to ~ 95% of the DNA total amount at acidic conditions.     

Nylon capsule method and alfalfa hay crude protein digestibility evaluation

In a trial with two dairy cows we have determined crude protein total tract digestibility (TTDP_NC) by a new nylon capsule method. Nylon capsules were made of nylon cloth with 42 μm apertures. The capsule shape was lenticular, with 10 mm external diameter and 8 mm internal diameter. The capsules were loaded with two stainless steel balls of 149 mg total weight. The average weight of a sample (milled alfalfa hay) in one capsule was 12.9 mg. In each of the two periods 1260 capsules were inserted orally into each of the two cows. Mean capsule passage time (CPT) through the digestive tract was 36.1 h (s.e. 0.49). The total tract dry matter digestibility determined by the nylon capsule method (TTDM_NC) was significantly (P < 0.05) influenced by the time which the capsules spent in the digestive tract. When CPT was 25.4 h, TTDM_NC was 64.8%, while with CPT 50.4 h it was 70.3%. The mean value of TTDP_NC was 93.8%. A similar value, 92.3%, was obtained by calculation based on the values determined by the nylon bag and the mobile bag methods. The results have shown that it is possible to use the nylon capsule method for the determination of total tract digestibility of individual feed components in cattle.     

Effects of maturity status on biochemical content,polyphenol composition and antioxidant capacity of pomegranate fruit arils (cv. ‘Bhagwa’)

Pomegranate fruit (Punica granatum L.) has gained commercial importance in recent years in the food and health industries due to increasing scientific evidence linking its consumption to better health outcomes. In the present study, the evolution of some chemical contents, individual organic acids and sugars, phenolic composition and antioxidant capacity of pomegranate (cv. ‘Bhagwa’) during maturation was investigated. The results showed significant (P < 0.05) increases in sugar content, ascorbic acid and total anthocyanins during fruit maturation, while significant decreases occurred in titratable acidity (TA), organic acids and total phenolic contents (TPCs). The significant increase in TSS/TA ratio, which plays a significant role in juice flavor, peaked at 140 DAFB, while the highest accumulation of anthocyanin content occurred at the full-ripe stage (165 DAFB). Total antioxidant capacity (both DPPH and FRAP) declined during fruit maturation, suggesting a decrease in antioxidant power of fruit juice. Strong correlations between TPC and antioxidant capacity measured by the DPPH (r² = 0.99) and FRAP (r² = 0.96) methods were observed. Fructose and glucose were established to be the major sugars in the fruit cultivar while tartaric acid was the predominant organic acid. Principal component analysis (PCA) showed that harvest maturity of ‘Bhagwa’ pomegranate fruit is dependent on time from full bloom hence could be fixed around 165 DAFB, where fruits were characterized by intense fruit and aril pigmentation as well as high juice quality parameters. This information provided could help pomegranate juice producers to assess and optimize juice quality and antioxidant value of ‘Bhagwa’ pomegranate cultivar through maturity.     

High performance liquid chromatographic determination of plasma free and total tazobactam and piperacillin

A high-pressure liquid chromatography (HPLC) method with ultraviolet detection was developed for the measurement of plasma free and total tazobactam and piperacillin. This method is simple and fast, requiring only 11 min for the HPLC run and a sample preparation of about 11 min for total drugs and 10 min for free drugs. The procedure for the assay involves the treatment of plasma with acetonitrile for total drugs determination, and the use of a centrifugal filter device to deproteinize plasma for free drugs determination. The HPLC column, a Hypersil-ODS, was equilibrated with an eluent mixture composed of acetonitrile–potassium phosphate (pH 2.6). CVs for repeatability of tazobactam and piperacillin measurements ranged from 4.30 to 6.60; CVs for reproducibility ranged from 5.60 to 9.40. Mean analytical recoveries ranged from 100.4 to 103%. A linear relationship was obtained between peak area and drugs concentration in the range studied (0–62.5 mg/L for tazobactam and 0–500 mg/L for piperacillin). The equation for regression line were y = 19x ? 1.4 for tazobactam and y = 1.7x ? 0.9 for piperacillin; correlation coefficients were >0.999. The lower limit of quantitation (LLQ) for standard samples was about 0.12 mg/L for tazobactam and 0.49 mg/L for piperacillin, respectively. The lower limit of detection (LLD) was 0.06 mg/L for tazobactam and 0.24 mg/L for piperacillin. This HPLC assay for tazobactam and piperacillin is sensitive and accurate, and provides a reliable determination of both free and total tazobactam and piperacillin in human plasma, thus allowing the determination of these analytes in patients receiving tazocillin therapy.     

Characteristics of biosurfactant produced by Pseudomonas aeruginosa S6 isolated from oil-containing wastewater

A biosurfactant-producing strain S6 was isolated from oil-containing wastewater and identified as Pseudomonas aeruginosa based on physiological and biochemical tests together with 16S rDNA sequence analysis. Thin layer chromatography (TLC) and high-performance liquid chromatography electrospray ionization mass spectra (HPLC-ESI-MS) worked together to reveal that the strain S6 produced rhamnolipid biosurfactant. Mass spectrometry confirmed the presence of some major components in the rhamnolipid surfactant showing m/z of 675.8, 529.6, 503.3 and 475.4, which corresponded to RhaRhaC₁₀C_12:1, RhaC_12:1C₁₀, RhaC₁₀C₁₀ and RhaC₈C₁₀, respectively. The biosurfactant produced by strain S6 had the ability to decrease the surface tension of water from 72 to 33.9 mN m^?1, with the critical micelle concentration (CMC) of 50 mg L^?1. Emulsification experiment indicated that this biosurfactant effectively emulsified the crude petroleum and the measurements of surface tension demonstrated that the biosurfactant possessed stable surface activity at variable ranges of pH and salinity. The biosurfactant also exhibited good performance of phenanthrene solubilization with about 23 times higher solubility of phenanthrene in water than the control. Thus, this biosurfactant may have a potential for application in bioremediation of crude oil contamination.     

Bifidobacteria in the digestive tract of bumblebees

Bifidobacteria and other bacterial groups (lactobacilli, facultative anaerobes, anaerobes) from the digestive tract of three bumblebee species (Bombus lucorum (34 samples), Bombus pascuorum (18 samples) and Bombus lapidarius (9 samples)) were enumerated and characterised. Counts of facultative anaerobic bacteria and lactobacilli (5.41 ± 2.92 and 2.69 ± 3.02 log CFU/g of digestive tract content) were lower than those of anaerobes (7.66 ± 0.86 log CFU/g). Counts of bifidobacteria were determined using two selective media: MTPY (Modified Trypticase Phytone Yeast extract agar) and a new medium with pollen extract. There was no significant difference between the counts of bifidobacteria from both media, 5.00 ± 2.92 log CFU/g on MTPY and 5.00 ± 2.87 on the pollen medium. Subsequently, 187 bacterial strains of the family Bifidobacteriaceae (fructose-6-phosphate phosphoketolase-positive) were isolated from three different localities and from all three species of bumblebees. Bifidobacteria were found in 42 out of 61 specimens (69%). Twenty-three (38%) specimens had counts of bifidobacteria higher than 7.0 log CFU/g. Bifidobacteria represented the dominant group of anaerobes (>70% of total anaerobes), i.e., the principal group of bacteria in the bumblebee digestive tract, in only fourteen specimens (23% of total). For the first time, bifidobacteria were isolated from the digestive tract of bumblebees. In addition, we suggest, on the basis of biochemical tests (API 50 CHL and RAPID ID 32) and genetic methods (PCR and DGGE), that these bacteria may represent new species within the family of Bifidobacteriaceae.     

Bioassay-guided isolation of a sesquiterpene lactone of deoxyelephantopin from Elephantopus scaber Linn. active on Trypanosome brucei rhodesience

Methanolic extracts of 70 Malaysia plants were screened for their in vitro antitrypanosomal activity using Trypanosome brucei rhodesience, strain STIB 900 and mouse skeletal cell (L-6) in cytotoxicity activity assay. Results indicated that methanol extract from Elephantopus scaber Linn. (E. scaber) possessed the highest value of antitrypanosomal activity with good selectivity index (antitrypanosomal IC₅₀ of 0.22 ± 0.02 μg/ml, SI value of 204.55). Based on these results, E. scaber was chosen for further study by applying bioassay guided fractionation to isolate its antiprotozoal principle. The antiprotozoal principle was isolated from the ethyl acetate partition through solvent fractionation and crystallization process. The isolated active compound 1 was identified as deoxyelephantopin on the basis of its spectral analysis (FTIR, MS, 1D and 2D NMR).     

Digestive capacities allow the Mexican long-nosed bat (Leptonycteris nivalis) to live in cold environments

Digestive capabilities of nectar-feeding vertebrates to assimilate sugars affect their ability to acquire and store energy and could determine the minimal temperatures at which these animals can survive. Here, we described the sugar digestive capability of Leptonycteris nivalis and related it with its capacity to live in cold environments. We measured the enzymatic activity, food intake rate and changes in body mass of bats feeding at four different sucrose concentrations (from 5 to 35% wt./vol.). Additionally, we used a mathematical model to predict food intake and compared it with the food intake of bats. L. nivalis was able to obtain ~ 111.3 kJ of energy regardless of the sugar concentration of their food. Also, bats gained ~ 2.57 g of mass during the experimental trials and this gain was independent of sugar concentration. The affinity (1 / K_m) of sucrase (EC 3.2.1.48) was one order of magnitude higher relative to that reported for its sister species Leptonycteris yerbabuenae (0.250 and 0.0189 mmol^? 1 L, respectively), allowing this species to have a higher energy intake rate. We propose that the high ability to acquire energy conferred L. nivalis the faculty to invade cold environments, avoiding in this way the ecological competition with its sympatric species L. yerbabuenae.     

Structure,properties and applications of rhamnolipids produced by Pseudomonas aeruginosa L2-1 from cassava wastewater

The properties and applications of rhamnolipid surfactants produced by Pseudomonas aeruginosa L2-1 from cassava wastewater added with waste cooking oil (CWO) as low-cost substrate, were investigated and compared with the commercial rhamnolipid mixture JBR599 (Jeneil Biosurfactant Co., Saukville, USA). The rhamnolipids produced by strain L2-1 were characterized by high performance liquid chromatography–mass spectrometry. Sixteen different rhamnolipid congeners were detected, with Rha-C₁₀-C₁₀ and Rha-Rha-C₁₀-C₁₀ being the most abundant. The L2-1 rhamnolipids from CWO showed similar or better tensioactive properties than those from JBR599, with a minimal surface tension of 30 mN/m and a critical micelle concentration (CMC) of 30 mg/l. The L2-1 biosurfactants formed stable emulsions with several hydrocarbons and showed excellent emulsification of soybean oil (100%). These rhamnolipids removed 69% of crude oil present in contaminated sand samples at the CMC and presented antimicrobial activity against Bacillus cereus (32 μg/ml), Micrococcus luteus (32 μg/ml) and Staphylococcus aureus (128 μg/ml). These results demonstrate that the rhamnolipids produced in CWO can be useful for industrial applications, such as the bioremediation of oil spills.     

The 2DX robot: A membrane protein 2D crystallization Swiss Army knife

Among the state-of-the-art techniques that provide experimental information at atomic scale for membrane proteins, electron crystallography, atomic force microscopy and solid state NMR make use of two-dimensional crystals. We present a cyclodextrin-driven method for detergent removal implemented in a fully automated robot. The kinetics of the reconstitution processes is precisely controlled, because the detergent complexation by cyclodextrin is of stoichiometric nature. The method requires smaller volumes and lower protein concentrations than established 2D crystallization methods, making it possible to explore more conditions with the same amount of protein. The method yielded highly ordered 2D crystals diffracting to high resolution from the pore-forming toxin Aeromonas hydrophila aerolysin (2.9 Å), the plant aquaporin SoPIP2;1 (3.1 Å) and the human aquaporin-8 (hAQP8; 3.3 Å). This new method outperforms traditional 2D crystallization approaches in terms of accuracy, flexibility, throughput, and allows the usage of detergents having low critical micelle concentration (CMC), which stabilize the structure of membrane proteins in solution.     

Purification and physicochemical properties of polygalacturonase from Aspergillus niger MTCC 3323

Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was purified. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme was purified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS–PAGE analysis, enzyme was found to be a heterodimer of 34 and 69 kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106 kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45 °C, pH of 4.8, reaction time of 15 min. The enzyme was stable within the pH range of 4.0–5.5 for 1 h. At 4 °C it retained 50% activity after 108 h but at room temperature it lost its 50% activity after 3 h. The addition of Mn²⁺, K⁺, Zn²⁺, Ca²⁺ and Al³⁺ inhibited the enzyme activity; it increased in the presence of Mg²⁺ and Cu²⁺ ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The K_m and V_max values of the enzyme were found to be 0.083 mg/ml and 18.21 μmol/ml/min. The enzyme was used for guava juice extraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%. Addition of purified enzyme increased the %T₆₅₀ from 2.5 to 20.4 and °Brix from 1.9 to 4.8. The pH of the enzyme treated juice decreased from 4.5 to 3.02.     

Improved accumulation of phenylethanoid glycosides by precursor feeding to suspension culture of Cistanche salsa

Effects of some precursors on phenylethanoid glycosides (PeGs) accumulation in Cistanche salsa cell suspension cultures were investigated. Precursors such as tyrosine, phenylalanine, caffeic acid and cucumber juice at proper concentrations could increase the total accumulation of PeGs (echinacoside, acteoside, 2′-acetylacteoside) by 50%, 12%, 12% and 23%, respectively. Under the combined feeding of precursors at proper concentrations, the total production of PeGs in bio-staged culture reached the highest amount of 1358.1 mg l⁻¹ (640.8 mg echinacoside l⁻¹, 689.4 mg acteoside l⁻¹ and 54.9 mg 2′-acetylacteoside l⁻¹), which was about two-fold of that in the control. This study showed promise for obtaining large-scale production of active ingredients in plant cells by the solid–liquid two step culture (SLTSC) technique and also provided for the first time an example for producing PeGs by C. salsa cell culture. The improved production of PeGs was higher than that in previous reports on PeG production by Cistanche deserticola cell culture fed with precursors.