Enzymatic solubilization of a pectinaceous dietary fiber fraction from potato pulp: Optimization of the fiber extraction process

Upgrading of potato pulp, a byproduct stream from industrial manufacture of potato starch, is important for the continued economic competitiveness of the potato starch industry. The major part of potato pulp consists of the tuber plant cell wall material which is particularly rich in galactan branched rhamnogalacturonan I type pectin. In the work reported here, the release of high-molecular weight pectinaceous dietary fiber polysaccharides from starch free potato pulp was accomplished by use of a multicomponent pectinase preparation from Aspergillus aculeatus (Viscozyme^® L). The enzyme reaction conditions for the solubilization were optimized via a surface response design to be addition of 0.27% Viscozyme^® L by weight of potato pulp substrate dry matter, 1 h treatment at pH 3.5, 62.5 °C. Analysis of the molecular size and monomer composition of the enzymatically released fibers showed that they were rich in galactose and uronic acid indicating that the solubilized fibers were mainly made up of galactan branched rhamnogalacturonan I type pectin polymers.     

Definition and characterization of enzymes for maximal biocatalytic solubilization of prebiotic polysaccharides from potato pulp

Potato pulp is a high-volume co-processing product resulting from industrial potato starch manufacturing. Potato pulp is particularly rich in pectin, notably galactan branched rhamnogalacturonan I polysaccharides, which are highly bifidogenic when solubilized. The objective of the present study was to characterize and compare four homogalacturonan degrading enzymes capable of catalyzing the required solubilization of these pectinaceous polysaccharides from potato pulp in a 1 min reaction. An additional purpose was to assess the influence of the pH and the potential buffer chelating effects on the release of these polysaccharides from the potato pulp. The pH and temperature optima of two selected pectin lyases from Emericella nidulans (formerly known as Aspergillus nidulans) and Aspergillus niger were determined to 8.6 and 4.0, respectively, at ≥100 °C within 1 min of reaction. The optima for the two selected polygalacturonases from E. nidulans and Aspergillus aculeatus were determined to pH 4.4 and 46 °C, and pH 3.7 and ≥80 °C, respectively. The polygalacturonase from A. aculeatus was 4-42 times more heat-resistant at 50 °C than the other enzymes. The difference in pH optima of the pectin lyases and the exceptional thermal stabilities of some of the enzymes are proposed to be related to specific amino acid substitutions, stabilizing hydrogen bonding and structural traits of the enzymes. The K_M and V_max values ranged from 0.3-0.6 g/L and 0.5-250.5 U/mg protein, respectively. Phosphate buffer induced release of a higher amount of dry matter than Tris-acetate buffer at pH 6, indicating a chelating effect of the phosphate. Moreover, the phosphate had a higher chelating effect at pH 6 than at pH 4. The optimal conditions for a high yield of polysaccharides from potato pulp were therefore: 1% (w/w) potato pulp treated with 1% (w/w) enzyme/substrate (E/S) pectin lyase from E. nidulans and 1% (w/w) E/S polygalacturonase from A. aculeatus at pH 6.0 and 60 °C for 1 min.     

Statistically designed optimisation of enzyme catalysed starch removal from potato pulp

Potato pulp is a high-volume, low-value byproduct stream resulting from the industrial manufacture of potato starch. The pulp is a rich source of biologically functional dietary fibers, but the targeted valorisation of the fibers requires removal of the residual starch from the pulp. The objective of this study was to release the residual starch, making up 21–22% by weight of the dry matter, from the potato pulp in a rational way employing as few steps, as few enzyme activities, as low enzyme dosages, as low energy input (temperature and time), and as high pulp dry matter as possible. Starch removal to obtain dietary fibers is usually accomplished via a three step, sequential enzymatic treatment procedure using a heat stable α-amylase, protease, and amyloglucosidase. Statistically designed experiments were performed to investigate the influence of enzyme dose, amount of dry matter, incubation time and temperature on the amount of starch released from the potato pulp. The data demonstrated that all the starch could be released from potato pulp in one step when 8% (w/w) dry potato pulp was treated with 0.2% (v/w) (enzyme/substrate (E/S)) of a thermostable Bacillus licheniformis α-amylase (Termamyl^® SC) at 70 °C for at least 65 min. The study also indicated that the amount of other carbohydrates released from the pulp during the release of starch was less than using the AOAC Official Method 985.29 and another recently published starch release method employed as a pretreatment for enzymatic upgrading of a pectinaceous potato pulp fiber.     

Chelating agents improve enzymatic solubilization of pectinaceous co-processing streams

This study investigates the hypothesis that loosening of the egg-box structure by presence of divalent ion chelating agents during enzymatic degradation of homogalacturonan (HG) can improve enzymatic polysaccharide solubilization on pectinaceous, agro-industrial co-processing streams. The influence of different levels of ethylene-diaminetetraacetic acid (EDTA), citric acid, oxalic acid, and phosphate was assessed in relation to enzymatic solubilization of isopropanol precipitatable oligo- and polysaccharides from sugar beet pulp, citrus peel, and two types of potato pulp. The two types of potato pulp were FiberBind 400, a dried commercial potato pulp product, and PUF, a dried calcium reduced product, respectively. The enzymatic treatment consisted of 1% (w/w) of substrate treated with pectin lyase from Aspergillus nidulans and polygalacturonase from A. aculeatus [each dosed at 1.0% (w/w) enzyme/substrate] at 60 °C, pH 6.0 for 1 min. Characterization of the released fractions demonstrated a significantly improved effect of chelating agents for polysaccharide solubilization from FiberBind 400, PUF, and citrus peel, whereas only low amounts of polysaccharides were solubilized from the sugar beet pulp. The results substantiated the importance of chelating agents during enzymatic extraction of pectinaceous polysaccharides. Lower levels of chelating agents were required for the calcium-reduced potato pulp substrate (PUF) indicating the significance of calcium cross-linking in HG in relation to the enzymatic solubilization yields. The effect of the chelating agents correlated to their dissociation constants (pK_a values) and calcium binding constants and citric acid and EDTA exerted highest effects. Maximum polysaccharide yield was obtained for FiberBind 400 where the enzymatic treatment in presence of citric acid yielded 22.5% (w/w) polysaccharides of the initial substrate dry matter.     

Expression and characterization of an endo-1,4-β-galactanase from Emericella nidulans in Pichia pastoris for enzymatic design of potentially prebiotic oligosaccharides from potato galactans

Potato pulp is a high-volume side-stream from industrial potato starch manufacturing. Enzymatically solubilized β-1,4-galactan-rich potato pulp polysaccharides of molecular weights >100 kDa (SPPP) are highly bifidogenic in human fecal sample fermentations in vitro. The objective of the present study was to use potato β-1,4-galactan and the SPPP as substrates for enzymatic production of potentially prebiotic compounds of lower and narrower molecular weight. A novel endo-1,4-β-galactanase from Emericella nidulans (anamorph Aspergillus nidulans), GH family 53, was produced in a recombinant Pichia pastoris strain. The enzyme was purified by Cu(2+) affinity chromatography and its optimal reaction conditions were determined to pH 5 and 49°C via a statistical experimental design. The specific activity of the E. nidulans enzyme expressed in P. pastoris was similar to that of an endo-1,4-β-galactanase from Aspergillus niger used as benchmark. The E. nidulans enzyme expressed in P. pastoris generated a spectrum poly- and oligo-saccharides which were fractionated by membrane filtration. The potential growth promoting properties of each fraction were evaluated by growth of beneficial gut microbes and pathogenic bacteria. All the galactan- and SPPP-derived products promoted the growth of probiotic strains of Bifidobacterium longum and Lactobacillus acidophilus and generally did not support the propagation of Clostridium perfringens in single culture fermentations. Notably the growth of B. longum was significantly higher (p<0.05) or at least as good on galactan- and SPPP-derived products as fructooligosaccharides (FOS). Except in one case these products did not support the growth of the pathogen Cl. perfringens to any significant extent.     

Production of ethanol from potato pulp: Investigation of the role of the enzyme from Acremonium cellulolyticus in conversion of potato pulp into ethanol

In this study, the saccharification and fermentation of the by-product of starch manufacture, potato pulp, were investigated. Analytic results of the components show that the potato pulp contains large amounts of starch, cellulose, and pectin. A commercial enzyme from Acremonium cellulolyticus was found to be highly efficient in the saccharification of potato pulp, since it exhibited high pectinase, α-amylase, and cellulase activities. Hydrothermal treatment of the potato pulp increased the saccharification rate, with a corresponding glucose concentration of 114 g/L and yield of 68% compared to the glucose concentration of 47 g/L and yield of 28% in the untreated case. The hydrolyzate could be used as both nitrogen and carbon sources for ethanol fermentation, showing that bioconversion of potato pulp to ethanol is feasible.     

Polygalacturonate lyase production byThermomonospora curvata on protein-extracted lucerne fiber

Summary Protein-extracted lucerne fiber was used as carbon and energy source for production of extracellular polygalacturonate lyase byThermomonospora curvata. The optimal fiber concentration was 1.5% (w/v); peal lyase activity in culture fluid occurred after 3 days growth at 53°C. During that time, lyase biosynthesis was controlled through induction; production was accelerated by adding small amounts of pectin or by grinding the fiber to 40-mesh particle size to release more inducer. After 3 days growth, lyase activity decreased; inactivation of the enzyme was delayed by the presence of 1 mM Ca or by inhibition of serine proteases with 0.05 mM phenylmethylsulfonyl fluoride. The molecular weight of the lyase produced during growth on the fiber was 35 kDa compared to 56 kDa for the enzyme produced on pure pectin. TheK _m of the 35-kDa form was 0.54% pectin compared to 0.06% for the 56-kDa form. The smaller form was rapidly inactivated at 60°C, the optimal temperature for activity of the larger form.     

Selection of Bacillus species for targeted in situ release of prebiotic galacto-rhamnogalacturonan from potato pulp in piglets

We have previously shown that galacto-rhamnogalacturonan fibers can be enzymatically extracted from potato pulp and that these fibers have potential for exerting a prebiotic effect in piglets. The spore-forming Bacillus species are widely used as probiotics in feed supplements for pigs. In this study, we evaluated the option for further functionalizing Bacillus feed supplements by selecting strains possessing the enzymes required for extraction of the potentially prebiotic fibers. We established that it would require production and secretion of pectin lyase and/or polygalacturonase but no or limited secretion of galactanase and β-galactosidase. By screening a library of 158 Bacillus species isolated from feces and soil, we demonstrated that especially strains of Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus mojavensis have the necessary enzyme profile and thus the capability to degrade polygalacturonan. Using an in vitro porcine gastrointestinal model system, we revealed that specifically strains of B. mojavensis were able to efficiently release galacto-rhamnogalacturonan from potato pulp under simulated gastrointestinal conditions. The work thus demonstrated the feasibility of producing prebiotic fibers via a feed containing Bacillus spores and potato pulp and identified candidates for future in vivo evaluation in piglets.

     

Pectic polysaccharide rhamnogalacturonan II is covalently linked to homogalacturonan.

A borate-containing pectin was solubilized from sugar beet (Beta vulgaris L. ) cell walls by treatment with 0.5 M imidazole, pH 7. The molecular weight of the pectin was reduced when the borate ester was hydrolyzed by treatment with 1 N HCl. Treatment of the acid-treated pectin with boric acid in the presence of Pb(2+) gave a product whose molecular weight distribution was similar to the imidazole-soluble pectin. The imidazole-soluble pectin was saponified and then digested with endo- and exo-polygalacturonases. These treatments shifted the boron peak at the high molecular weight region to the low molecular weight (10 kDa), which corresponds to rhamnogalacturonan II-borate ester cross-linked dimer (dRG-II-B). The treatment also generated rhamnogalacturonan I (RG-I), dRG-II-B, monomeric rhamnogalacturonan II and galacturonic acid. These results show that imidazole solubilizes a high molecular weight borate-containing pectic complex composed of homogalacturonan-rhamnogalacturonan II and RG-I. Our data suggest that borate esters formed between rhamnogalacturonan II molecules cross-link the macromolecular pectin.     

Fructooligosaccharide production by <Emphasis Type="Italic">Penicillium expansum</Emphasis>

Fructooligosaccharides (FOS) production by Penicillium expansum was evaluated. In a first stage, the best conditions for P. expansum growth and sporulation were established with potato/dextrose/agar being the most suitable medium at between 22 and 25°C, giving good growth and good sporulation. The inocula from this medium were used for FOS production using shake-flask cultures, and yielded 0.58 g FOS/g sucrose (3.25 g FOS/l.h), demonstrating the potential of this strain for sucrose conversion to FOS.     

Production and characterization of chitosan obtained from <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> grown on potato chip processing waste

Potato chip processing waste of trimmed potato, potato peel and substandard (low-quality) potato chips, obtained from a potato chip processing plant, were used as substrates for chitosan production from Rhizopus oryzae. It was cultured on each waste product at 30 ± 2°C and 70% moisture content for 21 days. Fermented potato peel had the highest yield after 5 days of fermentation. The cultivation condition of chitosan obtained from R. oryzae was optimum for a peel size of less than 6 mesh, 70% moisture content and a pH of 5. Furthermore, the best extraction condition was using 46% sodium hydroxide at 46°C for 13 h followed by 2% acetic acid at 95°C for 8 h. The maximum chitosan yield obtained by these conditions was 10.8 g/kg substrate. Fungal chitosan properties were found to be 86–90% degree of deacetylation, molecular weight of 80–128 kDa and viscosity of 3.1–6.1 mPa s. Therefore, potato peel could be applied as a low cost substrate for chitosan production from R. oryzae.     

Monoclonal antibodies to rhamnogalacturonan I backbone

Monoclonal antibodies were raised against rhamnogalacturonan I backbone, a pectin domain, using Arabidopsis thaliana seed mucilage-derived rhamnogalacturonan I oligosaccharides—BSA conjugates. Two monoclonal antibodies, designated INRA-RU1 and INRA-RU2, selected for further characterization, were specific for the backbone of rhamnogalacturonan I, displaying no binding activity against the other pectin domains i.e. homogalacturonans, galactans or arabinans. A range of oligosaccharides was prepared by enzymatic digestion of rhamnogalacturonan I isolated from Arabidopsis thaliana seed mucilage and from sugar beet pectin, purified by low-pressure chromatography and characterized by high-performance anion-exchange chromatography and mass spectrometry. These rhamnogalacturonan I oligomers were used to characterize the binding site of the two monoclonal antibodies by competitive inhibition. Both INRA-RU1 and INRA-RU2 showed maximal binding to the [→2)-α-l-rhamnosep-(1→4)-α-d-galacturonic acid p-(1→]₇ structural motif but differed in their minimum binding requirement. INRA-RU2 required at least two disaccharide (rhamnose–galacturonic acid) repeats for the antibody to bind, while INRA-RU1 required a minimum of six disaccharide repeats. Furthermore, the binding capacity of INRA-RU1 decreased steeply as the number of disaccharide repeats go beyond seven. Each of these antibodies reacted with hairy regions isolated from sugar beet pectin. Immunofluorescence microscopy indicated that both antibodies can be readily used to detect rhamnogalacturonan I epitopes in various cell wall samples.     

Optimization of pulp density and particle size in the biooxidation of a pyritic gold concentrate by <Emphasis Type="Italic">Sulfolobus metallicus</Emphasis>

Although increasing pulp densities and decreasing particle sizes have positive effects in the volumetric rate of biooxidation of refractory gold concentrates, a variety of phenomena such as mechanical damage, metabolic stress and inhibition can limit this effect. The objective of this work was to determine pulp density and particle size values that maximize the volumetric solubilization rate of iron from a pyritic gold concentrate. The leaching was carried on in agitated flasks with the thermophilic archaeon Sulfolobus metallicus. The concentrate contained 66.7% pyrite, and the constant operation conditions were 220 rev/min, 68 °C and initial pH of 2.0. Pulp densities were 2.5, 5, 10 and 15% w/v and the size fractions were 150–106, 106–75, 75–38 and <38 m. Total solubilized iron concentrations were in the range of 8–25 g/l. In the 2.5 and 5% pulp density runs, iron extractions were in the range of 80–100%. A complete experimental design of 16 runs allowed the building of response surfaces from which the optimal conditions that maximize the rate of iron solubilization were determined. These conditions are 7.8% pulp density and particle size of 35 m.     

Macerating Activity of Streptomyces fradiae IFO 3439

Streptomyces fradiae IFO 3439 elaborated enzymes with macerating activity toward various plant tissues. The optimum pH of the macerating activity was about 8.0 when the crude enzyme preparation acted on disks of potato tuber or pieces of Ganpi (Wikstroemia sikokiana Fr. et Sav.) bark. Pectolytic activities in this preparation toward free pectin or poly-galacturonic acid were considerably lower than those of fungi or bacteria. However, when the crude enzyme preparation acted on native pectin in Ganpi bark, about 90 per cent of the galacturonic acid residues were recovered as the polygalacturonides having a still high degree of polymerization. These results suggested that the crude enzyme of S. fradiae solubilized Ganpi pectin, degrading it to only a very small extent.     

Bioleaching of cobalt from smelter wastes byThiobacillus ferrooxidans

Summary The bioleaching of cobalt from domestic, industrial smelter wastes was studied.Thiobacillus ferrooxidans solubilized Co from sulfidic dross furnace mattes. At pulp densities of 4% (w/v) up to 600 mg of Co per liter of leaching solution was released from nickel matte, corresponding to removal of about two-thirds of the original amount of Co in the matte. Bioleaching methods may be useful as a component of a process for solubilization and recovery of Co from sulfidic smelter mattes.     

Production of a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> QM9414 xylanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its application in biobleaching of wheat straw pulp

The purpose of this study was to produce a Trichoderma reesei xylanase (XYN2) in Pichia pastoris and to test its potential application for pulp bleaching. The recombinant xylanase was purified by a two-step process of ultrafiltration and gel filtration chromatography. The molecular mass of the recombinant enzyme was 21 and 25 kDa by SDS–PAGE analysis, due to different glycosylation of the native protein. The optimum pH and temperature of the recombinant XYN2 was 5.0 and 50 °C. Enzyme activity was stable at 50 °C and at pH 5.0–7.0. The bleaching ability of the recombinant xylanase was also studied at 50 °C and pH 6.0, using wheat straw pulp. Biobleaching of the xylanase produced chlorine dioxide savings of up to 60%, while retaining brightness at the control level and led to a lower kappa number and small enhancements in tensile, burst and tear strength of pulp fibers.     

Pectinolytic yeasts from cold environments: novel findings of Guehomyces pullulans,Cystofilobasidium infirmominiatum and Cryptococcus adeliensis producing pectinases

One hundred and three yeasts isolated from soil samples from King George Island and Tierra del Fuego province were screened in relation with their capability to produce pectinolytic enzymes. Although all the yeasts showed well-developed colonies at 20 °C, only eight showed a clear halo around the colony, indicative of pectin degradation. A secondary screening demonstrated that only four yeasts were capable to produce pectinases at low temperatures (8 °C). It could be seen that the selected yeasts were able to grow and produce high levels of polygalacturonase activity when submerged fermentations were performed using pectin-containing fruit wastes as substrates. None of the strains produced neither lyase nor rhamnogalacturonan hydrolase activities. Regarding pectin esterase activity, it was only produced in lower amounts by G. pullulans 8E (0.022 U ml⁻¹). A TLC analysis of the substrate cleavage pattern of the pectinolytic systems was consistent with an endo-type activity. The clarification of apple juice was only accomplished by G. pullulans pectinolytic system, with a clarification of 80% (%T₆₅₀) using 4 U/ml of enzyme at 20 °C. As far as we concern this work describes for the first time the production of pectinases by the cold-adapted yeasts species Cystofilobasidium infirmominiatum, Cryptococcus adeliensis and G. pullulans.

     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

Studies on Protopectinase-C Mode of Action: Analysis of the Chemical Structure of the Specific Substrate in Sugar Beet Protopectin and Characterization of the Enzyme Activity

Chemical structures of pectic substances degraded by protopectinase-C (PPase-C) were characterized to identify the releasing mechanism of pectin from sugar beet protopectin by the action of that enzyme. The substrate of PPase-C was a polysaccharide isolated from sugar beet pulp by extraction with NaOH and sequential digestions with rhamnogalacturonase (PPase-T), β-1,4-D-galactanase, and α-L-arabinofuranosidase. The structure of this polysaccharide was analyzed by gas-liquid chromatography (GLC), NMR analysis, and gas chromatography-mass spectrometry (GC-MS), and it was identified as α-1,5-L-arabinan. According to our results, arabinan chains seemed to be connected to rhamnogalacturonan through a chain of β-l,4-D-galactan. PPase-C hydrolyzed both linear α-1,5-L-arabinan and ramified L-arabinan in a random manner, producing L-arabinose. From these results, PPase-C could be classified as arabinan endo-1,5-α-L-arabinase [EC 3.2.1.99]. Moreover, PPase-C seemed to split the L-arabinan of the polysaccharides connecting the rhamnogalacturonan to the other constituents of the plant cell wall in sugar beet pulp, releasing water-soluble pectin.     

The inactivation of pectin depolymerase associated with isolated tomato fruit cell wall: implications for the analysis of pectin solubility and molecular weight

An approach commonly employed to assess the potential role of the enzyme polygalacturonase (PG, EC 3.2.1.15) in tomato fruit cell-wall pectin metabolism includes correlating levels of extractable PG with changes in specific characteristics of cell wall pectins, most notably solubility and molecular weight. Since information on these features of pectins is generally derived from analyses of subfractions of isolated cell wall, assurance of inactivation of the various isoforms of wall-associated PG is imperative. In the present study, cell wall prepared from ripe tomato (Lycopersicon esculentum Mill. cv. Rutgers) fruit was examined for the presence of active PG and for the ability of phenolic solvents to inactivate the enzyme. Using pectin solubility and M_r (relative molecular mass) changes as criteria for the presence of wall-associated PG activity, pectins from phenol-treated and nonphenol-treated (enzymically active) cell wall from ripe fruit incubated in 50 mM Na-acetate, 50 mM cyclohexanetrans-1,2-diamine tetraacetic acid (CDTA), pH 6.5 (outside the catalytic range of PG), were of similar M_r and exhibited no change in size with incubation time. Wall prepared without exposure to the phenolic protein-denaturants exhibited extensive pectin solubilization and depolymerization when incubated in 50 mM Na-acetate, 50 mM CDTA at pH 4.5, indicating the presence of active PG. Based on the changes in the M_r of pectins solubilized in 50 mM Na-acetate, 50 mM CDTA, pH 4.5, active PG was also detected in wall exposed during isolation to phenolacetic acid-water (PAW, 2:1:1, w/v/v), a solvent commonly employed as an enzyme denaturant. Although the depolymerization of pectins in PAW-treated wall was extensive, oligouronides constituted minor reaction products. Interestingly, PAW-treated wall did not exhibit PG-mediated pectin release when incubated under conditions (30 mM Na-acetate, 150 mM NaCl, pH 4.5) in which nonphenol-treated cell wall exhibited high autolytic activity. In an alternative protocol designed to inactivate PG, cell wall was exposed to Tris-buffered phenol (BP). In contrast to pectins released from PAW-treated wall, pectins solubilized from BP-treated wall at pH 4.5 were indistinguishable in M_r from those recovered from BP-treated wall at pH 6.5 Even when incubated at pH 4.5 at 34°C, conditions under which pectins from PAW-treated wall underwent more rapid and extensive depolymerization, pectins from BP-treated wall exhibited no change in M_r, providing evidence that active PG was not present in these wall preparations. The implications of this study in interpreting the solubility and M_r of pectin in cell wall from ripening fruit are discussed.