Passage kinetics of C-labeled corn silage components through the gastrointestinal tract of dairy cows

Fractional passage rates form a fundamental element within modern feed evaluation systems for ruminants but knowledge on feed type and feed component specific passage rates are largely lacking. This study describes the use of carbon stable isotopes (¹³C) to assess component-specific passage kinetics of 6 intrinsically ¹³C-labeled corn silages varying in quality (2 cultivars × 3 maturity stages) in a 6 × 6 Latin square design using 6 rumen-fistulated lactating dairy cows. An increase in maturity increased starch and decreased neutral detergent fiber and acid detergent fiber contents of corn silages. Passage kinetics were assessed for an external (chromium mordanted fiber; Cr-NDF) and an internal marker (¹³C isotopes) collected in feces and omasal digesta. The best fit was obtained with a deterministic multicompartmental model compared with stochastic Gn and GnG1 models with increasing order of age dependency (n = 1 to 5) for both sampling sites. The Cr-NDF marker yielded higher rumen fractional passage rates (K₁) than did ¹³C in the dry matter (¹³CDM) in feces (0.042/h vs. 0.023/h). Omasal marker excretion patterns support the conclusions based on conventional fecal marker excretions. Component-specific passage was assessed for acid detergent fiber (¹³CADF) in feces and for starch (¹³CST) in omasal digesta. The fractional passage rate based on fecal ¹³CDM and ¹³CADF did not differ. Omasal ¹³CST provided higher K₁ values (0.042/h) than omasal ¹³CDM (0.034/h) but lower values than omasal Cr-NDF (0.051/h). Fractional passage rates from the proximal colon-cecum (K₂) based on fecal marker concentrations showed trends similar to K₁, with Cr-NDF providing a value (0.425/h) more than twice as high as that of ¹³CDM (0.179/h) and ¹³CADF (0.128/h). Total mean retention time in the gastrointestinal tract was approximately double for ¹³CDM (64.1 h) and ¹³CADF (77.6 h) compared with Cr-NDF (36.4 h). Corn silage quality did not affect any of the estimated passage kinetic parameters. In situ fractional degradation rates did not differ among corn silages, except for a decreased fractional degradation rate of starch with advancing maturity. Results indicate that isotope labeling allows assessment of component-specific passage kinetics of carbohydrate fractions in corn silage.     

Geographical origin of polished rice based on multiple element and stable isotope analyses

We determined carbon and nitrogen contents (C and N contents) and stable carbon, nitrogen, and oxygen isotopic compositions (δ¹³C, δ¹⁵N, and δ¹⁸O) of polished rice in order to develop a simple method to discriminate its geographical origin. As a first attempt, we examined a single cultivar, Koshihikari rice, from 14 different cultivation areas including Australia (n = 1), Japan (n = 12), and USA (n = 1). For all rice samples, C and N contents and the isotopic compositions are consistent with those of general plant materials, being 37.2–40.0% (C content), 0.8–1.4% (N content), −27.1 to −25.4% (δ¹³C), +0.4 to +9.0% (δ¹⁵N), and +18.8 to +22.9% (δ¹⁸O). However, its cultivated area is clearly distinguished by a pentagonal radar plot based on the elemental and isotopic compositions. Thus, the comparison of C and N contents and δ¹³C, δ¹⁵N, and δ¹⁸O values would potentially be useful for rapid and routine discrimination of geographical origin of the polished rice.     

C NMR and electrospray ionization mass spectrometric study of sucrose aqueous solutions at high pH: NMR measurement of sucrose dissociation constant

The ¹³C NMR technique is used for the measurement of the first dissociation constant of sucrose (HL) in highly alkaline solutions. In 1.0 M NaCl/NaOH medium and for 25 °C, the concentration dissociation constant (pK₁) was 13.1 ± 0.3; and, for 60 °C, pK₁ = 12.30 ± 0.05. The β-d-fructofuranosyl ring was found to be responsible for dissociation. The NMR data reveal no clear evidence of the second dissociation step below pH 14, either at 25 °C or at 60 °C. In the solutions with 4–10 mol dm⁻³ NaOH content the ¹³C NMR technique indicated the chemical shift changes, treated as the second dissociation step of sucrose and a sodium complex formation. A very rough estimation, for variable ionic strength, gives the value: pK₂ ∼ 15.8 ± 0.8. The anionic species L⁻ and NaH₋₁L⁻ have been registered by electrospray ionization time-of-flight mass spectrometry (ESI-ToF MS) for 0.01 M sucrose solutions with initial pH 13.     

Evaluation of the performance of osmotic dehydration sheets on freshness parameters in cold-stored beef biceps femoris muscle

This study aimed to evaluate the effects of osmotic dehydration sheet (ODS) packaging on the quality parameters of beef biceps femoris muscle samples stored at 4 °C for 0, 1, 3 and 7 days. Quality indices such as Hunter color values (L^∗, a^∗ and b^∗, the percentage of metmyoglobin (Met-Mb%), K value (freshness index), and the contents of adenosine triphosphate (ATP)-related compounds (ARCs), thiobarbituric acid reactive substances (TBA-RS) and volatile basic nitrogen (VBN) were measured. ODS gave lower a^∗ and b^∗ values and lower Met-Mb% compared with control samples wrapped in polyvinylidene chloride film (PVDCF) (P < 0.01), but had no effect on L^∗ (P < 0.01). As a result, with higher levels of osmotic dehydration produced by the ODS, the percentage of weight loss and the total contents of ARCs and inosine monophosphate of the samples also increased (P < 0.05). The K values of ODS samples were also significantly lower than PVDCF-wrapped samples (P < 0.05). Low performance ODS wrapping reduced the TBA-RS values below those found with PVDCF and high performance ODS processing (P < 0.01). Moreover, the use of ODS had no effect on VBN values. Thus, although the bright red of beef samples changed to a dark purple color and the weights of samples decreased, the ODS approach has potential as a tool for decreasing the deterioration of other quality indices such as Met-Mb%, TBA-RS, ARCs, K values and the VBN content of cold-stored beef.     

Isolation and evaluation of the antioxidant activity of phenolic constituents of the Garcinia brasiliensis epicarp

A new glycosylated biflavonone, morelloflavone-4′″-O-β-d-glycosyl, and the known compounds 1,3,6,7-tetrahydroxyxanthone, morelloflavone (fukugetin) and morelloflavone-7″-O-β-d-glycosyl (fukugeside) were isolated from the epicarp of Garcinia brasiliensis collected in Brazil. The structures of these compounds were established using ¹H and ¹³C NMR, COSY, gHMQC and gHMBC spectroscopy. The compounds exhibited antioxidant activity. The greatest potency was displayed by morelloflavone (2), with IC₅₀ = 49.5 mM against DPPH and absorbance of 0.583 at 400 μg/mL for the reduction of Fe³⁺. The weakest potency was displayed by 1,3,6,7-tetrahydroxyxanthone (1), with IC₅₀ = 148 mM against DPPH and absorbance of 0.194 at 400 μg/mL for the reduction of Fe³⁺.     

Influence of technological processing on apple aroma analysed by high resolution gas chromatography–mass spectrometry and on-line gas chromatography-combustion/pyrolysis-isotope ratio mass spectrometry

Extracts obtained by simultaneous distillation extraction (SDE) from industrial raw materials, namely single strength apple juices, and concentrates and aromas made thereof (each n = 31, from one production line; origin Poland, Germany, Turkey, Romania and China), as well as commercially available juices (n = 27), were analysed by standard controlled capillary gas chromatography–mass spectrometry (HRGC–MS). During the technological processing from juice to the aroma, no qualitative changes in the apple aroma profile were observed. Major constituents of the juices and aromas under study were found to be 1-hexanol (juice, 0.06–5.9 mg/l; aroma, 47–685 mg/l), 1-butanol (juice, 0.1–4.7 mg/l; aroma, 17–370 mg/l); E-2-hexenol (juice, 0.01–3.4 mg/l; aroma, 12–300 mg/l); E-2-hexenal (juice, 0–3.0 mg/l; aroma 0–470 mg/l), and butyl acetate (juice, 0–1.7 mg/l; aroma, 0–165 mg/l). By far the major component of the apple juice concentrates under study was furfural (2.4–56 mg/kg). The observed occurrence of 3-methyl-1-butanol (juice, 0.01–2.1 mg/l; aroma, 1.5–134 mg/l) and, in part, its acetate (juice, 0–0.3 mg/l; aroma, 0–3.3 mg/l), both known not to be genuine apple constituents, was obviously caused by fermentative effects in the course of industrial juice production. In addition, on-line capillary gas chromatography–isotope ratio mass spectrometry was used in the combustion (C) and the pyrolysis (P) modes (HRGC–C/P–IRMS) for the determination of δ¹³C_V-PDB and δ²H_V-SMOW values of selected apple flavour constituents to check potential isotope discrimination during distillative aroma production. As shown by means of the representative examples of E-2-hexenal, 1-hexanol and E-2-hexenol, their δ²H_V-SMOW values were slightly depleted. However, authenticity assessment by stable IRMS will not be influenced by this effect.     

Quality assessment of wild European eel (Anguilla anguilla) stored in ice

The quality assessment of wild European eel (Anguilla anguilla) stored in ice and in boxes without ice (3 ± 1 °C) was investigated by the sensory analysis, levels of nucleotide breakdown products and biogenic amines for up to 19 days. Sensory analysis was assessed using the Tasmanian Food Research Unit Scheme. K and related values (K_i, G, P, H and F_r) were used as freshness indicators. Linear regressions (r²) obtained from K, K_i, G, P, H and F_r were 0.95, 0.96, 0.83, 0.96, 0.99 and 0.96, respectively, for eel stored in ice whereas, for eel kept in boxes without ice, the values were 0.86, 0.86, 0.96, 0.91, 0.98 and 0.86, respectively. When eel stored in ice and in boxes without ice were considered at the limit of acceptability by assessors at ∼12–14 days and ∼5–7 days, respectively, the average K, K_i and P values were ∼70–85%, H values were ∼60% and F_r values were ∼10% for both storage conditions. The level of histamine exceeded the legal limit (5 mg/100 g fish) in eel stored without ice after 6–7 days and, in ice, after 13–14 days of storage, at which time eels were rejected by the sensory panel. The concentrations of biogenic amines were higher in eel stored in boxes without ice than in eel kept in ice. The levels of histamine in the muscle of eel kept in boxes without ice and in ice increased to the maximum levels of 17.9 mg/100 g on day 12 and 12.6 mg/100 g on day 19, respectively.     

Enzymatic synthesis of primeverosides using transfer reaction by Trichoderma longibrachiatum xylanase

Enzymatic synthesis of two phenyl xylopyranosyl glucopyranosides, through transfer reaction by Trichoderma longibrachiatum endoxylanase, was achieved in the presence of n-hexane used as solvent, phenyl glucoside (10 mM) as acceptor and xylan (2 g/l) as donor. Kinetic study showed that only one compound, identified by ¹H and ¹³C NMR and heteronuclear 2D (¹H–¹³C) chemical shift correlation as phenyl primeveroside (phenyl 6-O-β-xylopyranosyl-1-β-d-glucopyranoside), was synthesized when the reaction time was beyond 1 h. Benzyl and hexyl primeverosides were obtained under the same conditions. When several phenyl glucoside concentrations, from 5 to 50 mM, were used with 2 g/l of xylan, a phenyl primeveroside isomer, identified as phenyl 4-O-β-xylopyranosyl-β-d-glucopyranoside, accumulated in the medium whereas the production of phenyl primeveroside decreased. Only phenyl primeveroside was produced when several xylan concentrations from 2 to 10 g/l were used with 10 mM of phenyl glucoside and its concentration in the reaction mixture increased with the increase of xylan concentration.     

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0–11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (K_m) and catalytic constant (K_cat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s⁻¹, respectively. The catalytic efficiency (K_cat/K_m) was 238 s⁻¹ mM⁻¹.     

Chemical composition, in vitro cytotoxic and antioxidant activities of the essential oil and major constituents of Cymbopogon jawarancusa (Kashmir)

The chemical composition of the hydrodistillate of aerial parts of Cymbopogon jawarancusa, a natural grass considered as major forage for animal nutrition, used in food because of the presence of sufficient concentration of minerals like calcium and potassium was analysed by capillary GC–FID, GC–MS and ¹³C NMR. Seventeen constituents representing 97.8% of the total oil with piperitone (58.6%) and elemol (18.6%) as major constituents were identified. In vitro cytotoxicity of the oil and its constituents on human cancer cell lines THP-1 (leukemia), A-549 (lung), HEP-2 (liver) and IGR-OV-1 (ovary) was evaluated by Sulphorhodamine-B assay. The oil was found to be more potent than its components against cancer cell lines tested with IC₅₀ of 6.5 μg/ml (THP-1), 6.3 μg/ml (A-549), 7.2 μg/ml (HEP-2) and 34.4 μg/ml (IGR-OV-1). Antioxidant activity of oil and its constituents was evaluated by DPPH assay. In conclusion, the results demonstrate potent cytotoxic and antioxidant effects of oil, and its components like piperitone, α-pinene, β-caryophyllene and β-elemene.     

Purification and characterization of a novel glucoamylase from Fusarium solani

Thermostable enzymes are currently being investigated to improve industrial processes of starch saccharification. A novel glucoamylase was purified to electrophoretic homogeneity from the culture supernatant of Fusarium solani on a fast protein liquid chromatographic system (FPLC). The recovery of glucoamylase after gel filtration on FPLC was 31.8% with 26.2-fold increase in specific activity. The enzyme had a molecular mass of 40 kDa by SDS-PAGE and 41 kDa by gel filtration. The glucoamylase exhibited optimum activity at pH 4.5. The K_cat and K_m were 441/min and 1.9 mg/ml, respectively, for soluble starch, specificity constant (K_cat/K_m) was 232. The enzyme was thermally stable at 50 °C and retained 79% activity after 60 min at this temperature. The half-life of the enzyme was 26 min at 60°C. The enzyme was slightly stimulated by Cu²⁺ and Mg²⁺ and strongly inhibited by Hg²⁺, Pb²⁺, Zn²⁺, Ni²⁺ and Fe³⁺.     

The production of surfactin during the fermentation of cheonggukjang by potential probiotic Bacillus subtilis CSY191 and the resultant growth suppression of MCF-7 human breast cancer cells

Bacillus subtilis CSY191, the potential probiotics and surfactin-like compound producer, was isolated from doenjang (Korean traditional fermented soybean paste).The survival rate of this strain appeared to be the 58.3% under artificial gastric conditions after 3 h at pH 3.0. Surfactin was purified from the strain CSY191. Three potential surfactin isoforms were detected, with protonated masses of m/z 1030.7, 1044.7, and 1058.71. These different structures were detected in combination with Na⁺, K⁺ and Ca²⁺ ions by MALDI-TOF mass spectrometry. Upon 500 MHz ¹H NMR analysis, the surfactin isoforms had identical amino acids (GLLVDLL) and hydroxy fatty acids (of 13-15 carbons in length). The MTT assay showed that surfactin inhibited growth of MCF-7 human breast cancer cells in a dose-dependent manner, with an IC₅₀ of approximately 10 μg/ml at 24 h. Additionally, the surfactin contents, during cheonggukjang fermentation with strain CSY191, increased from 0.3 to 48.2 mg/kg over 48 h of fermentation, while the level of anticancer activity increased from 2.6- to 5.1-fold.     

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.     

Modelling starch digestion in sweetpotato with biphasic digestograms

The kinetics of starch digestion in 20 sweetpotato samples obtained from Papua New Guinea were investigated using an in vitro procedure based on glucometry. Irrespective of the cultivars (3-mun, Carot kaukau, Wahgi besta, Nillgai, Baiyer kaukau, and 1-mun), provinces, farmers and locations, the samples exhibited biphasic digestograms, possibly due to initial impediments to amylolysis and/or transport of digestion products by non-starch components (e.g. plant cell walls). The biphasic digestograms were segmented into two monophasic digestograms using a second-order polynomial (average r² = 0.723; p < 0.001), and a modified first-order kinetic model adequately described the digestograms (average r² = 0.984, p < 0.001; mean relative deviation modulus, MRDM, =8). Generally, the digestion rate constants (initial, K_i = 3.2-5.2 × 10⁻³ min⁻¹; final, K_f = 3.8-18.4 × 10⁻³ min⁻¹) indicated faster digestion during the final segment after the initial impediments to digestion had possibly been overcome. K_i was significantly (p < 0.05) different among the samples, which did not significantly (p < 0.05) differ in K_f. A logistic model, which treated each digestogram as a single process, also adequately described the biphasic behaviours (average r² = 0.994, p < 0.001; mean relative deviation modulus, MRDM, =15). The digestion parameters from the segment and logistic approaches significantly (p < 0.05) differed among the sweetpotato samples. The rate of digestion from the logistic model was significantly (p < 0.05) related to the initial and final rates of digestion from the segment approach (K = 3.99 K_i + 0.65 K_f; r² = 0.456; p < 0.001). Irrespective of the approach, maximum starch digestion was less than 100% in some samples to indicate the presence of resistant starch type 1, 2 or both. The present study is the first to model biphasic starch digestograms.     

Effects of reaction mixture and other components on the determination of the equilibrium and rate constants of the hydration reactions of anthocyanins

The equilibrium and rate constants of the hydration and deprotonation reactions of anthocyanins show how their color intensity changes with pH. In the cases of several anthocyanins, the constants for each obtained by several methods are different. In an effort to resolve these discrepancies, we have examined the effects of several components of the pH-jump experiments on the values of the constants. Storage of the buffers to be used in pH-jump experiments in Pyrex or borosilicate glass bottles results in increasing Al³⁺ concentration in the buffers over several weeks. When these buffers are used, the anthocyanins with two OH groups on the B ring complex with the Al³⁺ which leads to major changes in their spectra, in the equilibrium position, and in the apparent first-order rate constant. Thus, constants determined on the same anthocyanin using the same buffers stored in glass bottles may be different at different times. During the reduction of the experimental data to the rate and equilibrium constants, two divergences from the expected behavior were found. In the calculation K_a + K_h for the anthocyanin acylated with 4-hydroxy-3,5-dimethoxycinnamic acid (6-O-(4-hydroxy-3,5-dimethoxycinnamoyl)-β-d-glucopyranosyl-(1 → 6)-[β-d-xylopyranosyl-(1 → 2)]-β-d-galactopyranosyl-(1 → O³)-cyanidin), the plotted points appear to fit two straight lines, intersecting at an equilibrium pH near pH 4. In the calculation which leads to the individual constants of both anthocyanins examined here, the points below an equilibrium pH of pH 4 curve upward from the line that describes the points from an equilibrium pH above 4. Differences in the composition of solutions used in pH-jump experiments examined here, include (1) the addition of phosphate to the acetate buffer, (2) the presence of 0.5 M NaCl, and (3) the solution of the anthocyanin in either 0.1 N HCl or 0.1 N HOAc. These changes gave differences that were statistically significant in some of the constants for each of the two anthocyanins examined. The constants were both qualitatively and quantitatively different.     

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.     

Short communication: Comparison of 3 solid digesta passage markers in dairy cows

This study investigated the usefulness of acid-detergent fiber-bound ¹⁵N [acid detergent insoluble (ADI)-¹⁵N] as a solid digesta passage marker in dairy cows compared with chromium (Cr) and ytterbium (Yb) (as labeled fiber or forage, respectively). Intrinsically (ADI-¹⁵N) or extrinsically (Cr, Yb) labeled alfalfa hay was pulse-dosed intraruminally to 7 lactating dairy cows. Following marker administration, spot fecal samples were collected for up to 72 h for marker analyses. Urine and milk samples were also collected and analyzed for Yb and Cr. Fecal marker excretion data were processed using 2-compartment mathematical age-dependent/age-independent (G_n→G₁) models. The rate of passage of the marker in the first, age-dependent compartment tended to be slower for Yb compared with Cr and ADI-¹⁵N, which resulted in a trend for longer mean retention time (MRT) in this compartment when Yb was used as a marker (19.0 h) compared with Cr and ADI-¹⁵N (14.5 and 13.9 h, respectively). The rate constant of marker disappearance for the second or age-independent compartment tended to be greater for Yb compared with Cr and ADI-¹⁵N, which led to a shorter MRT of Yb in this compartment (15.6) versus ADI-¹⁵N (32.1) and Cr (24.8 h). The cumulative MRT was greater for ADI-¹⁵N versus Cr and Yb (46.0, 39.3, and 34.4 h, respectively). Total MRT of marker tended to be greater for ADI-¹⁵N than for Yb (46.6 vs. 36.6 h, respectively). Urine and milk analyses data suggested no measurable losses of Yb along the digestive tract, but about 0.79% of Cr dosed intraruminally was secreted or excreted in milk and urine in the 48-h period following marker administration. Collectively, this study confirmed previous observations that ADI-¹⁵N can be used reliably as a solid digesta passage marker for ruminants, producing pre-duodenal and total-tract retention times similar to that of Cr-labeled fiber. Retention time in the age-independent compartment was underestimated when Yb was used as a marker, emphasizing the need to process forages to isolate fiber before labeling with Yb.     

Milk β-lactoglobulin complexes with tea polyphenols

The effect of milk on the antioxidant capacity of tea polyphenols is not fully understood. The complexation of tea polyphenols with milk proteins can alter the antioxidant activity of tea compounds and the protein secondary structure. This study was designed to examine the interaction of β-lactogolobulin (β-LG) with tea polyphenols (+)-catechin (C), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG) at molecular level, using FTIR, CD and fluorescence spectroscopic methods as well as molecular modelling. The polyphenol binding mode, the binding constant and the effects of polyphenol complexation on β-LG stability and secondary structure were determined. Structural analysis showed that polyphenols bind β-LG via both hydrophilic and hydrophobic interactions with overall binding constants of K_C–β-LG = 2.2 (±0.8) × 10³ M⁻¹, K_EC–β-LG = 3.2 (±1) × 10³ M⁻¹, K_ECG–β-LG = 1.1 (±0.6) × 10⁴ M⁻¹ and K_EGCG–β-LG = 1.3 (±0.8) × 10⁴ M⁻¹. The number of polyphenols bound per protein molecule (n) was 1.1 (C), 0.9 (EC), 0.9 (ECG) and 1.3 (EGCG). Molecular modelling showed the participation of several amino acid residues in polyphenol–protein complexation with extended H-bonding network. The β-LG conformation was altered in the presence of polyphenols with an increase in β-sheet and α-helix suggesting protein structural stabilisation. These data can be used to explain the mechanism by which the antioxidant activity of tea compounds is affected by the addition of milk.     

Purification and characterization of polyphenol oxidase from Ferula sp.

Polyphenol oxidase (PPO) of several Ferula sp. was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis, and gel filtration chromatography. Leaf and stem extracts were used for the determination of enzyme properties. Optimum conditions, for pH, temperature, and ionic strength were determined. The best substrates of PPO were catechol for leaf and (−) epicatechin for stem samples. Optimum pH and temperature were determined. K_M and V_max values were 2.34 × 10⁻³ M and 8541 EU/ml for catechol, and 2.89 × 10⁻³ M and 5308 EU/ml for (−) epicatechin. The most effective inhibitor was sodium diethyl dithiocarbamate for leaf samples and sodium metabisulphite for stem samples. Both inhibitors indicated competitive reactions. PPO showed irreversible denaturation after 40 min at 60 °C.