Solubilization of Cell Wall Bound, Thermostable Pectinesterase from Valencia Orange

Complete solubilization of citrus pectinesterase is not effected by alkaline salt buffers but is partially enhanced after degradation of the cell wall by pectinase and cellulase. After heating at 70°C for 2 min, the pectinesterase fractions solubilized after enzymatic cell wall degradation possessed three times the residual activity of the pectinesterase fractions solubilized by alkaline salt buffer. Isoelectric focusing prints of pectinesterase activity and SDS polyacrylamide gel electrophoresis suggest increased solubilization of a heat stable pectinesterase isozyme after cell wall degradation.     

Cell Wall Monosaccharide Changes During Softening of Brined Cucumber Mesocarp Tissue

Blanched, unheated and fermented cucumber mesocarp tissues stored at pH 3.5 had losses in galacturonic acid, galactose, arabinose, and rhamnose from cell walls isolated by sequential extraction with aqueous sodium dodecyl sulfate (SDS) and phenohacetic acid:water (2:1:1; SDS/PAW). Much smaller amounts of the sugars were solubilized when the cell walls were isolated by extraction with 80% ethanol. The differential solubilization of sugars suggested that a limited degradation of pectic and hemicellulosic substances occurred during cucumber tissue softening. This degradation was readily observed when softened tissue was extracted by SDS/PAW, but not when isolated by the more common ethanol extraction. Similarity in pattern of sugar losses, following low pH storage after three processing treatments, suggested that reactions which resulted in tissue softening and solubilization of cell wall sugars were similar in the three treatments.     

ROLE OF pH IN GEL FORMATION OF WASHED CHICKEN MUSCLE AT LOW IONIC STRENGTH

This work was designed to test the hypothesis that it is not solubilization of the myofibrillar proteins per se that is required to form good gels at low salt concentrations, but the protein‐containing structures must be disorganized. Gels were made from washed minced chicken breast muscle at 0.15, 0.88, and 2.5% sodium chloride. The gels made with varying salt concentrations were evaluated either at pH 6.0–6.5 or pH 7.0–7.4. Strain values, an indicator of protein quality, were high only at neutral pH in the gels containing 0.15 or 0.88% salt. At 2.5% salt, strain values of gels made at acid pH were superior to those at the low salt concentrations at acid pH, but inferior to gels with 2.5% salt at neutral pH. Poor gels were obtained at 0.15% salt and low pH whether or not there was an intermittent adjustment to neutral pH. A neutral salt wash markedly increased the water content of the mince, suggesting that solubility‐inhibiting proteins were removed. Good quality gels were obtained in the absence of any detectable solubilization of myosin and only minimal solubilization of actin.     

Kinetic compensation relations for ascorbic acid degradation and pectinesterase inactivation during orange juice pasteurisations

The thermal degradation kinetics of ascorbic acid and the inactivation kinetics of pectinesterase were studied experimentally with pH-adjusted orange juice. No simple trends were observed in variation of the kinetic parameters with pH, but both kinetic functions agreed with kinetic compensation relations. An isokinetic temperature was observed for ascorbic acid degradation at pH 2.5, 3.0, and 3.5 implying that the same degradation mechanism was valid at these pH values. A large deviation at pH 4.0 from this isokinetic reaction rate implied that the degradation mechanism was different at this pH. There was no isokinetic temperature for pectinesterase inactivation reactions, indicating that pectinesterase was inactivated by different mechanisms at different pH values. The variation of the inactivation mechanism of pectinesterase with pH was attributed to its more complex molecular structure.     

EFFECT OF MgCl2/SODIUM PYROPHOSPHATE ON CHICKEN BREAST MUSCLE MYOSIN SOLUBILIZATION AND GELATION

Sodium chloride (0.29 M) at pH 7 solubilized about 24% of the myosin of washed, minced chicken breast muscle. At a similar pH, 0.2 M sodium chloride in the presence of 10 mM sodiumpyrophosphate and 10 mM magnesium chloride solubilized almost 60% of the myosin. In spite of the greater solubility of myosin under the latter conditions, when gels were prepared with these concentrations of salt at pH 7, the gels without the sodium pyrophosphate and magnesium chloride were slightly superior in both stress (39.3 kPa vs 28.3 kPa) and true strain (2.3 vs 2.0) values. Gels made at a lower pH (6.1–6.5) made much poorer gels. This was true whether the low pH was obtained naturally in the preparation of the sample or re‐adjusted after bringing the mince to a neutral pH. It appears that conditions of pH and salt content that cause solubilization of myosin at more dilute conditions does not contribute to gel quality, but the neutral pH is an important factor for obtaining good gels at ionic strengths <0.3.     

SOLUBILITY OF THE PROTEINS OF MACKEREL LIGHT MUSCLE AT LOW IONIC STRENGTH

Over 90% of the proteins of mackerel light muscle were soluble in solutions of physiological ionic strength or less. To accomplish this solublization, it was necessary to extract certain proteins at moderate ionic strength and neutral pH before extracting the rest of the myofibrillar and cytoskeletal proteins in water. Six proteins were favorably solubilized by sodium chloride solutions of moderate ionic strength at neutral pH under conditions that allowed later dissolution of myofibrillar and cytoskeletal proteins in water. The possibility is suggested that three of these proteins were involved in preventing the solubilization in water of other myofibrillar and cytoskeletal proteins of mackerel light muscle. Based on molecular masses and relative abundance, these proteins could possibly be M-protein (166 kDa), α-actinin (95 kDa) and desmin (56 kDa).     

Degradation of cell wall materials from sweetpotato, cassava, and potato by a bacterial protopectinase and terminal sugar analysis of the resulting solubilized products

Cell wall materials (CWMs) from sweetpotato, cassava, and potato starch residues were degraded using a crude enzyme solution from the culture filtrate of a Bacillus sp. isolated from soil, Bacillus sp. M4. This organism has been found to secrete polygalacturonic acid lyase (PGL) and glycan depolymerase activities, especially arabinanase, but cellulase activity was nearly absent. Sugar analysis of the solubilized product after enzyme treatment at pH 7.0 revealed that it is mainly composed of galacturonic acid, galactose, and arabinose, the sugars found commonly in the pectin fraction. This suggested the presence of a protopectinase (PPase) activity in the culture filtrate. The presence of EDTA completely inhibited PGL but PPase activity was almost retained, suggesting that the PGL is not the primary activity responsible for pectin solubilization. The mode of action of the crude enzyme was determined by terminal sugar analysis using HPAEC-PAD after hydrolysis of the reduced products. Results revealed that galactose is the main neutral sugar at the reducing terminal of the products, although rhamnose was also present in the higher molecular weight component. This suggested that at neutral pH, the primary activity in the culture filtrate of Bacillus sp. M4 is a B-type PPase, which attacked the galactan as well as rhamnogalacturonan moieties of the protopectin, resulting in the release of a soluble pectin fraction.     

Selective Extraction of Thermostable Pectinesterase

Thermostable pectinesterase was estimated to represent 98% of total activity extracted from Marsh grapefruit pulp by 1M NaCl without pH adjustment or 17% when extracted with 1M NaCl, 0.25M Tris-Cl^- pH 8.0. Total units of pectinesterase solubilized were less in 1M NaCl at endogenous pH values than at pH 8.0, but total thermostable pectinesterase units remained constant. No conversion of thermostable to heat sensitive isozymes was observed. Preparative isoelectric focusing indicated that most activity focused at alkaline pH values. The constant specific activity suggested co-migration of basic proteins.     

Effect of extraction conditions on the yield and purity of ulvan extracted from Ulva lactuca

A study of the influence of extraction conditions (pH: 1.5 and 2; temperature: 80 °C and 90 °C; extraction time: 1–3 h), on the yield, chemical composition and purity of the sulphated cell wall polysaccharides ulvan, extracted from the green seaweed Ulva lactuca and precipitated by alcohol is carried out. The alcohol precipitate yields varied from 21.68% to 32.67% (%dw/dw) depending on the pH. At pH 2, the alcohol precipitate yields and the uronic acid recovery from extract juice are higher than those obtained at pH 1.5. Other compounds than ulvan such as cellulose, hemicellulose, proteins and ash are solubilized from the cell walls of Ulva lactuca at both pH, and they are precipitated with alcohol. The alcohol precipitate obtained from different extraction conditions has high uronic acid (20.37%–23.60%) and neutral sugar content (20.09%–29.12%), especially when the conditions (pH, temperature) are drastic. It contains rhamnose (13.35%–15.59%), glucose (2.90%–10.97%), and xylose (2.36%–2.73%). A decrease in the molecular weight of ulvan was observed at acid pH, and for long extraction times. The presence of proteins (1.94%–2.32%) and inorganic material (33.36%–47.15%) in alcohol precipitate prove the lower purity of ulvan extracted and shows that ulvan precipitation with ethanol is not specific.     

Effect of extraction conditions on the yield and purity of apple pomace pectin precipitated but not washed by alcohol

ABSTRACT:  A study of the influence of extraction conditions (pH: 1.5 to 2; temperature: 80 to 90 °C; extraction time: 1 to 3 h), on the yield and purity of apple pomace pectin without elimination of impurities by alcohol washing was carried out. The alcohol precipitate yields varied from 2.9% to 8.9% depending on the pH. At pH 1.5, these yields were higher than those obtained at pH 2 contrary to the galacturonic acid purity (%w/w). Compounds other than pectins were solubilized from the cell walls of apple pomace at pH 1.5, and they were precipitated with alcohol. The apple pectins obtained from the different extraction procedures were highly methylated (54.5% to 79.5%), especially when the conditions (temperature, pH) were drastic. Similar conclusions can be drawn for the neutral sugar content that decreased at pH 1.5 (arabinose, xylose, and galactose) or at the highest temperatures and extraction times (arabinose and galactose). The phenomenon of demethylation and pectin degradation of neutral sugars chains can be observed at acid pH, and for long extraction times. The presence of high quantities of mannose or fructose, glucose, and xylose in the alcohol precipitate showed that pectin precipitation with ethanol was not specific.     

PURIFICATION AND PROPERTIES OF MALT CARBOXYPEPTIDASES ATTACKING HORDEIN

Carboxypeptidases capable of releasing amino acids from barley hordein have been extracted and purified from malted barley. Three enzymes can be distinguished, with similar molecular weights but slightly different ionic characteristics and specificities. The malt carboxypeptidases preferentially attack peptides containing an aromatic amino acid, although other peptides, including those containing proline, are also attacked. The purified carboxypeptidase fractions reduce the viscosity of β-glucan extracted from endosperm cell walls. It is suggested that these enzymes may be instrumental in the initial solubilization of endosperm cell walls, possibly by the hydrolysis of the peptide component of these walls. The pH optima and heat stability data indicate that carboxypeptidases are crucial for the release of amino acids both from hordein, and from peptides derived from hordein during both malting and mashing.     

STABILIZATION OF CLOUD IN PECTINESTERASE ACTIVE ORANGE JUICE BY PECTIC ACID HYDROLYSATES

The cloud of pectinesterase active orange juice was stabilized by inhibiting pectinesterase competitively with pectic acid hydrolysates. Both chemically and enzymatically prepared hydrolysates with an average degree of polymerization of 8 to 15 proved to be suitable, inasmuch as their molecular weight is large enough to inhibit pectinesterase, but too small to cause juice clarification by coagulating themselves. The period of cloud stability of orange juices from various origins could be extended by a factor of 3 to 5 through the additions of 1 mg per ml of hydrolysate with a degree of polymerization of 12. The effect of delayed gelation in orange concentrates was found to be smaller, and higher concentrations of hydrolysates with a somewhat lower degree of plymerization had to be used. Measurement of methanol release proved that pectic acid hydrolysate, added to orange juice, inhibits pectinesterase.     

IRRADIATION OF INDUSTRIAL ENZYME PREPARATIONS II. CHARACTERIZATION OF FUNGAL PECTINASE BY THIN-LAYER ISOELECTRIC FOCUSING AND GEL FILTRATION

Industrial dry fungal pectinase from A. niger was irradiated with doses (up to 1 Mrad) of ⁶⁰ Co-γrays effective in reducing microbial contamination. The pectinase was characterized by thin-layer isoelectric focusing and gel filtration in order to detect possible radiation-induced structural alterations. Thin-layer isoelectric focusing revealed at least fifteen multiple forms with pectin-depolymerizing activity, with isoelectric points in the range pH 4.5–7. Heterogeneity of pectinesterase was also demonstrated, the main band occurring around pH 4. By thin-layer gel filtration the molecular weight of the pectin-depolymerase was estimated as being about 36,000, and that of pectinesterase as about 33,000. Radiation-induced changes of the charge properties or molecular size of the irradiated pectinase preparation were not observed. The feasibility of using ionizing radiation for the reduction of microbial contamination of industrial enzyme preparations looks promising.     

ACID TOLERANCE OF ESCHERICHIA COLI FOLLOWING COLD SHOCK TREATMENT

The effect of an initial cold shock treatment (2 h at 10C), following an abrupt downshift in temperature from 37 to 10C, on the subsequent growth and survival of Escherichia coli strains O157:H7 and MY20 (Biotype 1) in acidified Trypticase soy broth (TSB) and fruit juices (orange, apple) was investigated. Overall, no difference in growth at 37C was observed between each cold shocked and noncold shocked E. coli strain when cultured in TSB adjusted with either acetic acid (pH 6.0)or malic, citric and tartaric acid (each adjusted to: pH 4.5, 5.0, 5.5, 6.0). However, significant (P ± 0.05) differences in survival were observed between cold shocked and noncold shocked populations in TSB acidified with acetic acid (pH 5.0) or citric, malic and tartaric acid (pH 4.0). For strain MY20, survivor levels for cold shocked cells in TSB acidified with acetic acid citric, malic and tartaric acid at 8C were significantly (P ± 0.05) higher than in noncold shocked populations. Also, at 37C survival levels for cold shocked cells were significantly (P ± 0.05) higher than noncold shocked cells in TSB acidified with either malic or tartaric acid (pH 4.0). For the O157:H7 strain, survivor levels were higher (P ± 0.05) for cold shocked cells when maintained in TSB at 37C regardless of acid type. At 8C, cold shock treatment only increased (P ± 0.05) the survival of the O157:H7 strain in TSB adjusted with acetic acid (pH 6.0). Acid cross protection induced by cold shocking, as evidenced by enhanced survival, was not apparent for either E. coil strain in apple (pH 3.5) or orange juice (pH 3.8) maintained at 8C.     

Sugar Composition of Cucumber Cell Walls During Fruit Development

The sugar composition of pickling cucumber cell walls was analyzed in seven cucumber sizes. Relative to total cell wall sugars, glucose did not change, while the other major neutral sugars, galactose, xylose and mannose, underwent relatively small declines during fruit development. The ratios of galacturonic acid/rhamnose and galacturonic acid/arabinose showed large increases during fruit growth, which indicated that pectin structure may change. Pectin methylation in the mesocarp tissue increased from 35 to 64% as cucumber fruit matured. A positive correlation was observed between the firmness of cucumber mesocarp tissue and the total amount of cell wall sugars in the fresh tissue. Cucumber peel tissue contained 4 to 5-fold higher sugar concentrations than either the mesocarp or endocarp tissues.     

EFFECT OF SQUID LIVER EXTRACT ON PROTEINS AND ON THE TEXTURE OF COOKED SQUID MANTLE

ABSTRACT The liver extract of squid Illex argentinus has a proteolytic activity at pH 2–8, with optimum at pH 2.6–3.1. At pH 6 the enzymes retain about 30 and 55% of the maximum proteolytic activity towards hemoglobin and casein, respectively. In squid mantle meat treated with the extract 24 h at 4C and 20C, the sarcoplasmic and myofibrillar proteins were hydrolyzed to low molecular fractions, as was shown by SDS-PAGE. Soaking isolated collagen fibers in the liver extract resulted after 24 h in almost complete solubilization of the fibers in buffered SDS/urea solutions. The solubilized product contained only low molecular weight components. A significant hydrolysis of the proteins in the squid mantle, treated with the liver extract at 20 or 4C, was accompanied by a decrease in toughness of the cooked product by about 65 and 40%, respectively, as compared to the toughness of untreated cooked samples. Cooking of the mantle directly in the liver extract had practically no tenderizing effect.     

A new approach for the purification and characterisation of PA49.5, the main prebiotic of Lactococcus lactis subsp. cremoris

The main autolysin PA49.5, an enzyme that hydrolyzes or destroys the components of a biological endogenous cell or a tissue, was purified 3045 times from the homogenate of a whole cell extract of Lactococcus lactis subsp. cremoris ATCC 9596 (Mc5), with a recovery yield of 52%. The purification of the protein was carried out through a micro-purification technique using SDS-BigCHAP polyacrylamide gel electrophoresis and concentrated with a Microcon-10 filtration system. SDS-polyacrylamide gel electrophoresis of the purified enzyme confirmed the presence of only one band having a molecular weight of 49.5 kDa. In view of its insolubility, PA49.5 contained in the cell extract precipitate was solubilized in the presence of 0.1% (w/v) of BigCHAP, a non-ionic detergent. Higher concentrations of this detergent completely inhibited the activity of solubilized PA49.5 or prevented its solubilization. The optimal pH and temperature for PA49.5 enzymatic activity are 7.5 and 45 degrees C respectively. In addition 0.1% or less of PA49.5 significantly increased Mc5 lysis. We observed 55% more lysis with 0.25 mug of purified PA49.5 compared to the control. Gas chromatography analysis of the components of the crude cell extract, of the precipitate and of the supernatant indicates the presence of at least 6 fatty acids. The long-chained fatty acids (e.g. C18:0 and C18:3) detected represent 81.65% of the precipitate from which PA49.5 was purified. Of these two acids, the C18:0 (stearic acid) alone represents 47.40% of the precipitate. Mc5 releases proteins at the beginning (major peak) and at the end (moderate peak) of the exponential stage of growth. Analysis by denaturing polyacrylamide gel electrophoresis with Mc5 cell walls incorporated as the autolysin's substrate identified a band corresponding to PA49.5 in the second peak of protein secretion.     

THE MODE OF BINDING OF β-GLUCANS AND PENTOSANS IN BARLEY ENDOSPERM CELL WALLS

β-Glucans in barley endosperm cell walls exist as polymers of very high molecular weight (about 4 × 10⁷ daltons) containing firmly linked peptide sequences. This peptidic material is an essential part of the structure of the β-glucan complex as it exists in the cell wall. Rupture of peptide bonds by hydrazinolysis or with the proteolytic enzyme thermolysin gives β-glucans similar in size to those from short-grown green malts (about 10⁶ daltons). This suggests that proteolysis is the first step in β-glucan degradation. Large β-glucans are not all precipitated in 30% (w/v) ammonium sulphate; only 34% of the β-glucan in a hot aqueous extract of cell walls is precipitated. The amount is increased to 63% if the cell walls have been previously dehydrated. Prolonged incubation of cell wall β-glucan at 40°C, mechanical stress, chromatography lasting 8–10 h at or above 65°C, or chromatography in M sodium chloride causes some disassociation of high molecular weight β-glucan to a size of about 10⁷ daltons. Heating a solution for 1 h at 100°C does not disassociate the β-glucan. Pentosans isolated from cell walls are not covalently linked to the β-glucans and can be separated from them by molecular sieve chromatography. They have a higher xylose/arabinose ratio than previously reported for barley pentosans. The pentosan molecules extracted by water are smaller (10⁶ daltons) than those extracted by alkali (5 × 10⁶ daltons). Little difference was observed in the chemical or physical properties of cell wall materials of barley cultivars of different malting qualities.     

A derivative of Lactococcus lactis strain H61 with less interleukin-12 induction has a different cell wall

Lactococcus lactis H61 can increase the cellular immune responses of aged (14-mo-old) senescence-accelerated mice. The aim of this study was to investigate the factors contributing to IL-12 induction by strain H61 by analyzing strains derived from it. Strain H61 derivative no. 13 was obtained by growing the parent strain at 37°C. This derivative induced significantly lower production of IL-12 from J774.1 macrophage cells than did the parent strain H61. The 2 strains differed in the resistance of their whole cells or cell walls to lysozyme, a cell wall-degrading enzyme. Sodium hydroxide treatment to de-O-acetylate muramic acid in the cell walls of the 2 strains reduced the lysozyme resistance, compared with untreated cell walls: at 3h after adding lysozyme, the lysozyme resistance of untreated and NaOH treated cell wall from strain H61 was 55.4% and 11.7%, respectively. The values of untreated and NaOH-treated cell walls from strain no.13 were 73.7 and 42.8%, respectively. The reduction was higher in strain H61, indicating that the cell walls of strain H61 were highly O-acetylated. Trichloroacetic acid treatment to remove wall-associated polymers such as teichoic acids made the lysozyme resistance of the cell walls of both strains similar. The sugar content of cell walls prepared from strain H61 was significantly higher than that of strain no. 13 cell wall. A derivative with less activity for inducing IL-12 by macrophage cells had less O-acetylation and had lower sugar content in the cell wall than did strain H61. Modifying the cell wall of strain H61 may be a useful way to regulate its ability to induce IL-12. Strain H61 has been used as a starter bacterium in the dairy industry. This study could lead to enhancing the value of dairy products made by strain H61 by characterizing the key factor(s) responsible for its stimulation of immunity.     

SOLUBILIZATION OF APPLE POMACE BY EXTRUSION

Apple pomace (AP), a fibrous by-product of apple juice processing, was solubilized by a twin-screw extruder. AP was extruded under 14 different operating conditions, where the process variables are controlled in the following ranges: screw speed, 250–350 rpm; feed rate, 30–40 kg/h; moisture, 20–30%. Water soluble index (WSI) increased linearly with specific mechanical energy (SME). Soluble dietary fiber of extruded AP also increased with SME, which in turn decreased the content of insoluble dietary fiber (IDF). As a result, total dietary fiber (TDF) remained nearly unchanged. Water holding capacity (WHC) remained nearly constant at low SME, but markedly deceased at high SME due to disintegration of cell wall. Gel filtration of WSP exhibited the structural fragmentation of cell wall by extrusion: the higher SME, the higher fragmentation of SME. Ion exchange chromatography of WSP demonstrated that increasing SME resulted in increasing the extractability of free neutral polysaccharides from the cell wall of AP. It was also found that the degradation of AP cell wall by extrusion preferentially occurred at the arabinogalactan sidechains of pectins.