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Soybean hulls are lignocellulosic residuesgeneratedinthe industrial processing of soybean, representing about 5 % of the mass of the whole bean. This by-product isan importantsource of polymers suchas cellulose(34 %) and hemicellulose (11 %),which could bevalorizedvia biotechnology to improvethe economic returnof the oilseed chain. In the present work,soybean hulls were evaluated as a carbon sourcefor biolipid productionbyLipomycesstarkeyi LPB 53. Initially the hulls were treated physicochemically and enzymatically to obtain fermentable sugars. Subsequently, biomass growth was evaluated using different nitrogen sources andthe lipid production was optimized, reaching a maximum cell biomass concentration of 26.5 g/L with 42.5 % of lipids. Around 65 % of the xylose content was consumed.The obtained oil wasmajorlycomposed of oleic, palmitic, palmitoleic, linoleic and stearic fatty acids in a proportion of 54 %, 32 %, 4 %, 3 % and 2 %, respectively.
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Lipídeos , Lipomyces , Glycine max , FermentaçãoRESUMO
The use of yeasts as starter cultures was boosted with the emergence of large-scale fermentations in the 20th century. Since then, Saccharomyces cerevisiae has been the most common and widely used microorganism in the food industry. However, Candida species have also been used as an adjuvant in cheese production or as starters for coffee, cocoa, vegetable, meat, beer, and wine fermentations. A thorough screening of candidate Candida is sometimes performed to obtain the best performing strains to enhance specific features. Some commonly selected species include C. pulcherrima (teleomorph Metschnikowia pulcherrima) (wine), C. parapsilosis (teleomorph Monilia parapsilosis) (coffee), C. famata (teleomorph Debaryomyces hansenii) (cheese), and C. zeylanoides (teleomorph Kurtzmaniella zeylanoides) and C. norvegensis (teleomorph Pichia norvegensis) (cocoa). These species are associated with the production of key metabolites (food aroma formation) and different enzymes. However, safety-associated selection criteria are often neglected. It is widely known that some Candida species are opportunistic human pathogens, with important clinical relevance. Here, the physiology and metabolism of Candida species are addressed, initially emphasizing their clinical aspects and potential pathogenicity. Then, Candida species used in food fermentations and their functional roles are reported. We recommended that Candida not be used as food cultures if safety assessments are not performed. Some safety features are highlighted to help researchers choose methods and selection criteria.
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In the growing search for therapeutic strategies, there is an interest in foods containing natural antioxidants and other bioactive compounds capable of preventing or reversing pathogenic processes associated with metabolic disease. Fermentation has been used as a potent way of improving the properties of soybean and their components. Microbial metabolism is responsible for producing the ß-glucosidase enzyme that converts glycosidic isoflavones into aglycones with higher biological activity in fermented soy products, in addition to several end-metabolites associated with human health development, including peptides, phenolic acids, fatty acids, vitamins, flavonoids, minerals, and organic acids. Thus, several products have emerged from soybean fermentation by fungi, bacteria, or a combination of both. This review covers the key biological characteristics of soy and fermented soy products, including natto, miso, tofu, douchi, sufu, cheonggukjang, doenjang, kanjang, meju, tempeh, thua-nao, kinema, hawaijar, and tungrymbai. The inclusion of these foods in the diet has been associated with the reduction of chronic diseases, with potential anticancer, anti-obesity, antidiabetic, anticholesterol, anti-inflammatory, and neuroprotective effects. These biological activities and the recently studied potential of fermented soybean molecules against SARS-CoV-2 are discussed. Finally, a patent landscape is presented to provide the state-of-the-art of the transfer of knowledge from the scientific sphere to the industrial application.
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Traditionally, lipid-producing microorganisms have been obtained via conventional bioprospecting based on isolation and screening techniques, demanding time and effort. Thus, high-throughput sequencing combined with conventional microbiological approaches has emerged as an advanced and rapid strategy for recovering novel oleaginous microorganisms from target environments. This review highlights recent developments in lipid-producing microorganism bioprospecting, following (i) from traditional cultivation techniques to state-of-the-art metagenomics approaches; (ii) related topics on workflow, next-generation sequencing platforms, and knowledge bioinformatics; and (iii) biotechnological potential of the production of docosahexaenoic acid (DHA) by Aurantiochytrium limacinum, arachidonic acid (ARA) by Mortierella alpina and biodiesel by Rhodosporidium toruloides. These three species have been shown to be highly promising and studied in research articles, patents and commercialized products. Trends, innovations and future perspectives of these microorganisms are also addressed. Thus, these microbial lipids allow the development of food, feed and biofuels as alternative solutions to animal and vegetable oils.
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Bioprospecção , Metagenômica , Animais , Ácido Araquidônico , Biocombustíveis , MetagenomaRESUMO
The generation of agroindustrial byproducts is rising fast worldwide. The slaughter of animals, the production of bioethanol, and the processing of oil palm, cassava, and milk are industrial activities that, in 2019, generated huge amounts of wastewaters, around 2448, 1650, 256, 85, and 0.143 billion liters, respectively. Thus, it is urgent to reduce the environmental impact of these effluents through new integrated processes applying biorefinery and circular economy concepts to produce energy or new products. This review provides the characteristics of some of the most important agro-industrial wastes, including their physicochemical composition, worldwide average production, and possible environmental impacts. In addition, some alternatives for reusing these materials are addressed, focusing mainly on energy savings and the possibilities of generating value-added products. Finally, this review considers recent research and technological innovations and perspectives for the future.
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Manihot , Águas Residuárias , Animais , Resíduos Industriais , IndústriasRESUMO
Complex carbohydrates, proteins, and other food components require a longer digestion process to be absorbed by the lining of the alimentary canal. In addition to the enzymes of the gastrointestinal tract, gut microbiota, comprising a large range of bacteria and fungi, has complementary action on the production of digestive enzymes. Within this universe of "hidden soldiers", lactobacilli are extensively studied because of their ability to produce lactase, proteases, peptidases, fructanases, amylases, bile salt hydrolases, phytases, and esterases. The administration of living lactobacilli cells has been shown to increase nutrient digestibility. However, it is still little known how these microbial-derived enzymes act in the human body. Enzyme secretion may be affected by variations in temperature, pH, and other extreme conditions faced by the bacterial cells in the human body. Besides, lactobacilli administration cannot itself be considered the only factor interfering with enzyme secretion, human diet (microbial substrate) being determinant in their metabolism. This review highlights the potential of lactobacilli to release functional enzymes associated with the digestive process and how this complex metabolism can be explored to contribute to the human diet. Enzymatic activity of lactobacilli is exerted in a strain-dependent manner, i.e., within the same lactobacilli species, there are different enzyme contents, leading to a large variety of enzymatic activities. Thus, we report current methods to select the most promising lactobacilli strains as sources of bioactive enzymes. Finally, a patent landscape and commercial products are described to provide the state of art of the transfer of knowledge from the scientific sphere to the industrial application.
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6-Fitase , Lactobacillus , Bactérias , Digestão , Trato Gastrointestinal , HumanosRESUMO
The growing global demand for animal products and processed meat has created a challenge for the livestock sector to enhance animal productivity without compromising product quality. The restriction of antibiotics in animal feeds as growth promoters makes the use of probiotics a natural and safe alternative to obtain functional foods that provide animal health and quality and to maintain food safety for consumers. To incorporate these additives into the diet, detailed studies are required, in which in vitro and in vivo assays are used to prove the efficacy and to ensure the safety of probiotic candidate strains. Studies on the use of Bacillus subtilis natto as a spore-forming probiotic bacterium in animal nutrition have shown no hazardous effects and have demonstrated the effectiveness of its use as a probiotic, mainly due to its proven antimicrobial, anti-inflammatory, antioxidant, enzymatic, and immunomodulatory activity. This review summarizes the recent scientific background on the probiotic effects of B. subtilis natto in animal nutrition. It focuses on its safety assessment, host-associated efficacy, and industrial requirements.
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Probióticos , Alimentos de Soja , Ração Animal , Animais , Bacillus subtilis , DietaRESUMO
The sugarcane industry is one of the largest in the world and processes huge volumes of biomass, especially for ethanol and sugar production. These processes also generate several environmentally harmful solid, liquid, and gaseous wastes. Part of these wastes is reused, but with low-added value technologies, while a large unused fraction continues to impact the environment. In this review, the classic waste reuse routes are outlined, and promising green and circular technologies that can positively impact this sector are discussed. To remain competitive and reduce its environmental impact, the sugarcane industry must embrace technologies for bagasse fractionation and pyrolysis, microalgae cultivation for both CO2 recovery and vinasse treatment, CO2 chemical fixation, energy generation through the anaerobic digestion of vinasse, and genetically improved fermentation yeast strains. Considering the technological maturity, the anaerobic digestion of vinasse emerges as an important solution in the short term. However, the greatest environmental opportunity is to use the pure CO2 from fermentation. The other opportunities still require continued research to reach technological maturity. Intensifying the processes, the exploration of driving-change technologies, and the integration of wastes through biorefinery processes can lead to a more sustainable sugarcane processing industry.
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Microalgas , Saccharum , Biomassa , Etanol , GasesRESUMO
OBJECTIVE: This work aimed at evaluating the influence of organic solvents and stationary phases in the extraction with glass beads and chromatographic purification of carotenoids, especially torularhodin, from Sporobolomyces ruberrimus. RESULTS: The combinations of acetone:hexane (1:1 v/v) and acetone:ethyl ether (1:1 v/v) yielded 171.74 and 172.19 µg of total carotenoids.g of cells-1, respectively. The first blend resulted in the highest percent of cell lysis of 57.4%. Among different proportions of acetone:hexane, the 9:1 v/v mixture showed a significant difference (p < 0.05), resulting in a recovery of total carotenoids of 221.88 µg.g of cells-1. The purification of carotenoids was made by preparative chromatography and the yield of the silica-containing stationary phase was higher (24 µg torularhodin.g cells-1). The analyses of the purified fractions in thin layer chromatography and high performance liquid chromatography indicated that the purification of carotenoids, especially of torularhodin, was successfully performed. CONCLUSIONS: The combination of polar (acetone) and non-polar solvents (hexane) and the use of silica as stationary phase was efficient to recover and purify torularhodin from the intracellular pigments of Sporobolomyces ruberrimus.
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Basidiomycota/química , Carotenoides/isolamento & purificação , Solventes/química , Acetona/química , Carotenoides/química , Cromatografia Líquida , Hexanos/químicaRESUMO
Abstract The rapid and accurate diagnosis of tuberculosis (TB), especially considering limited resources, is still a challenge. Development of new methodologies and tests are needed to overcome several disadvantages of the available standard tests. We evaluated the diagnostic potential of two antigens specific for Mycobacterium tuberculosis, the CFP10 and ESAT6 recombinant proteins, and developed stable formulations thereof. Sensitivity and specificity of the delayed-type hypersensitivity (DTH) skin testing and the induction of gamma interferon production (IFN-γ) by lymphocytes, as a non-invasive test, were evaluated using the CFP10 and ESAT6 protein formulations. The recombinant proteins produced by our group presented a high DTH response and the ability to differentiate between tuberculosis infection, BCG vaccination, and the contact with non-tuberculous mycobacteria (NTM). The production of IFN-γ by stimulation with individual and combined proteins was detected in a panel of 40 individuals and showed a specificity of 100% and a sensitivity of 90% when the two proteins were used together. Lyophilized formulations were stable under all conditions, while soluble formulations were stable under freezing at -20 ºC and -80 ºC. The proposed formulations containing the ESAT6 and CFP10 recombinant antigens constitute satisfactory tools for TB testing, suitable to be developed and implemented in a large-scale trial.
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Tuberculose/diagnóstico , Interferon gama , Mycobacterium tuberculosis/isolamento & purificação , Antígenos/químicaRESUMO
SUMMARY This study aimed at evaluating effective methods for breaking the hard and insoluble spores of Ganoderma lucidum to recover functional biomolecules. Rupture techniques were evaluated such as manual maceration (RM), maceration with spheres of various materials (BR), and microwave exposure plus maceration with steel/ chrome spheres (MBR1). Spore rupture was evaluated using UV-Vis spectroscopy, which showed vibrations of 2955, 1642, 1240, 1080 and 1746 cm-1 corresponding to changes in spore walls. The MBR1 extract contained the largest amounts of carbohydrates (19.80 mg.g-1 spores) and polyphenols (2.21 mg.g-1 spores), whereas the BR extract had higher antioxidant activity (57.22%Inb DPPH). The MBR1 and BR extracts contained 62.2 and 73.5% glucose, respectively. Both methods also involved significant extraction of carbohydrates and proteins. The best way to extract biomolecules from spore walls is to perform a microwave heat treatment and break the walls with steel/chrome spheres; this produces large quantities of carbohydrates with antioxidant properties.
RESUMEN El objetivo de este estudio fue evaluar varios métodos de ruptura de las esporas de Ganoderma lucidum y extraer sus propiedades bioactivas. Para este propósito se evaluaron diferentes técnicas de rompimiento como: la maceración manual (RM), la maceración con esferas de diversos materiales (BR) y la exposición a microondas junto la maceración de las esporas con esferas de acero/cromo (MBR1). La ruptura de las esporas fue evaluada por espectroscopia UV-Vis, la cual mostró que las vibraciones 2955, 1642, 1240, 1080 y 1746 cm-1 correspondieron a cambios estructurales en las paredes de las esporas. El extracto MBR1 presento el mayor contenido de carbohidratos (19,80 mg.g-1) y polifenoles (2,21 mg.g-1), mientras que el extracto BR tuvo una mayor actividad antioxidante (57,22% Inb DPPH). Los extractos MBR1 y BR también presentaron en el análisis de monosacáridos un 62,2 y 73,5% de contenido glucosa. Como conclusión la mejor metodología para extraer biomoléculas de las paredes de las esporas de G. lucidum fueron el tratamiento térmico con microondas y la ruptura de las paredes con esferas de acero/cromo, porque este proceso permitió la extracción de una mayor cantidad de carbohidratos con posibles propiedades antioxidantes.
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Lignocellulosic biomass is one of the most abundant organic resources worldwide and is a promising source of renewable energy and bioproducts. It basically consists of three fractions, cellulose, hemicelluloses and lignin, which confer a recalcitrant structure. As such, pretreatment steps are required to make each fraction available for further use, with acidic, alkaline and combined acidic-alkaline treatments being the most common techniques. This review focuses on recent strategies for lignocellulosic biomass pretreatment, with a critical discussion and comparison of their efficiency based on the composition of the materials. Mild pretreatments usually allow the recovery of the three biomass fractions for further transformation and valorisation. An insight is provided of newly developed technologies from recently filed patents on lignocellulosic biomass pretreatment and the transformation of agro-industrial residues into high value-added products, such as biofuels and organic acids.
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Biocombustíveis , Lignina , Biomassa , Celulose , HidróliseRESUMO
Petroleum is an important energy source. Due to its intensive exploration, accidents resulting in oil spills on soil are frequent, which creates consequences to ecosystems and human health. Rhizodegradation is an efficient technique that promotes the decontamination of polluted environments through the selection and use of rhizosphere microorganisms from phytoremediation plants. The aim of this study was to isolate, identify and characterize bacteria capable of degrading petroleum from the rhizosphere of Panicum aquaticum Poir., a plant that grows in petroleum contaminated soils. Three bacteria were isolated and characterized at the morphological (Gram staining), molecular (16S rRNA gene sequence analysis) and biochemical level. These bacteria were identified as new strains of Bacillus thurigiensis, Bacillus pumilus and Rhodococcus hoagii, which have been reported as potential bioremediators in the literature. All three bacteria were able to use petroleum hydrocarbons as the sole carbon source during in vitro degradation assays. Gas chromatography analysis of these assays indicated reductions of petroleum hydrocarbons between 23% and 96% within 48 h. Among the isolated bacteria, Rhodococcus hoagii presented the highest efficiency of petroleum consumption, reaching 87% of degradation after only 24 h of cultivation, which corresponds to a higher and faster degradation than previously reported, confirming the potential use of Rhodococcus hoagii for petroleum biodegradation.
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Biodegradação Ambiental , Panicum/microbiologia , Petróleo/metabolismo , Rizosfera , Rhodococcus equi/metabolismo , DNA Bacteriano/isolamento & purificação , Poluição por Petróleo , RNA Ribossômico 16S/genética , Rhodococcus equi/genética , Rhodococcus equi/isolamento & purificaçãoRESUMO
Coffee can be an ally in the fight against diseases such as type 2 diabetes, cancer, hepatic injury, cirrhosis, depression, suicidal behavior, and neurological and cardiovascular disorders. The properties of coffee also favor gastrointestinal tract and gut microbiota establishment. Coffee bioactive components include phenolic compounds (chlorogenic acids, cafestol and kahweol), alkaloids (caffeine and trigonelin), diterpenes (cafestol and kahweol) and other secondary metabolites. The image of coffee as a super functional food has helped to increase coffee consumption across the globe. This chapter addresses the main health promotion mechanisms associated with coffee consumption. Related topics on coffee production chain, world consumption and reuse of coffee by-products in the production of high-value-adding molecules with potential applications in the food industry are addressed and discussed.
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Doenças Cardiovasculares/prevenção & controle , Café/química , Dieta , Agricultura , Manipulação de Alimentos , Promoção da Saúde , HumanosRESUMO
A new method for CO2 recovery was proposed for cultivation of different microalgae. First, a chemical fixation, where CO2 was injected in alkalinized vinasse to form (bi)carbonate salts, was performed. In addition, biological fixation with CO2-enriched air injection was also accomplished for evaluation of the most promising results. Two bioreactor systems, a stirred-tank reactor and a bubble column reactor, were employed. A higher carbon transfer rate (43.35â¯g.L-1.h-1) was achieved in the bubble column reactor using NaOH-alkalinized vinasse, along with reductions of the chemical oxygen demand (COD), biological oxygen demand (BOD) and turbidity (TD). This allowed the cultivation of microalgae and cyanobacteria at vinasse concentrations between 70 and 100%, reaching a biomass production of 2.25â¯g.L-1 in 15â¯days of culture. The viability of chemical CO2 fixation together with the use of 100% treated vinasse from a bioethanol production unit for microalgae cultivation has been demonstrated in a successfully integrated biorefinery approach.
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Microalgas , Análise da Demanda Biológica de Oxigênio , Biomassa , Carbono , Dióxido de Carbono , GasesRESUMO
The aim of this study was to evaluate the parameters that affect the production of the recombinant 10â¯kDa culture filtrate protein (CFP10), a promising reagent of high specificity for intradermoreaction and other antigen-based methods used in the diagnosis of tuberculosis. Conditions of Escherichia coli growth temperature, induction temperature and IPTG-inducer concentration were evaluated in shake flasks and dissolved O2 concentrations of 15 and 30% were evaluated in a bioreactor. The process parameters defined on small scale were: growth temperature between 30 and 37⯰C, induction temperature of 26⯰C and IPTG concentration of 0.12â¯mM. The process conducted with 15% dissolved O2 presented a recombinant protein yield of 78.6â¯mgâ¯g-1 biomass and a proportion of recombinant protein (insoluble fraction) in relation to total insoluble protein of 72%, at the time of maximum productivity. The operation with 30% dissolved O2 resulted in lower recombinant protein yields of 62.9â¯mgâ¯g-1 biomass and 20% in relation to total insoluble protein, but in higher overall concentration in the culture broth (69.2â¯mgâ¯L-1versus 48.3â¯mgâ¯L-1). The protein identity was confirmed by mass spectrometry, showing high similarity to CFP10, 10â¯kDa of Mycobacterium tuberculosis H37Rv (score 95), and the purified antigen presented reactivity by the Western blotting assay.
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Antígenos de Bactérias , Proteínas de Bactérias , Mycobacterium tuberculosis/genética , Tuberculose/diagnóstico , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
BACKGROUND: Leishmaniasis is caused by parasites of the genus Leishmania, and represents a group of chronic diseases with an epidemiological and clinical diversity. The disease is endemic in tropical regions, being found in 98 countries, affecting around 12 million people, with an estimated increase of 1.5 million per year. METHODS: The present review aims to analyze recent and most important patents regarding development of vaccines to improve immunization against leishmaniasis. For this purpose, the Web of Science - Derwent Innovations Index was consulted. There is also a short description of the licensed vaccines already on the market for commercialization, and a critical opinion on future developments. RESULTS: The data herein presented comprises national and international filings, thus considering the patent's country of origin, and can be used an indicator of a country's technological development regarding a specific field. Several types of vaccines against Leishmania were studied. The main classes comprise: vaccines using live cells (virulent or attenuated); dead cells; containing recombinant protein; using DNA of the parasite. United States (74 patents) leads the ranking of patent applications for vaccines against Leishmania, followed by Brazil (36 patents), which is an endemic region of leishmaniasis with 20,000 human cases of cutaneous leishmaniasis and over 3,000 cases of visceral form. CONCLUSION: This review showed that there is still a lot of space for development regarding the creation of a feasible, effective vaccine against leishmaniasis. The scientific community appears to be taking steps in the right direction, though.
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Invenções/estatística & dados numéricos , Vacinas contra Leishmaniose/biossíntese , Leishmaniose Cutânea/prevenção & controle , Leishmaniose Visceral/prevenção & controle , Vacinas de DNA/biossíntese , DNA de Protozoário/imunologia , Humanos , Leishmania/imunologia , Leishmania/patogenicidade , Vacinas contra Leishmaniose/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Patentes como Assunto , Vacinas de DNA/imunologia , Vacinas Vivas não AtenuadasRESUMO
Laccases are oxidative enzymes related to the degradation of phenolic compounds, including lignin units, with concomitant reduction of oxygen to water. Delignification is a necessary pretreatment step in the process of converting plant biomass into fermentable sugars. The objective of this work was to optimize the production of laccases and to evaluate the delignification of sugarcane bagasse by Pleurotus ostreatus in solid-state fermentation. Among eight variables (pH, water activity, temperature, and concentrations of CuSO4, (NH4)2SO4, KH2PO4, asparagine, and yeast extract), copper sulfate and ammonium sulfate concentrations were demonstrated to significantly influence laccase production. The replacement of ammonium sulfate by yeast extract and the addition of ferulic acid as inducer provided increases of 5.7- and 2.0-fold, respectively, in laccase activity. Optimization of laccase production as a function of yeast extract, copper sulfate, and ferulic acid concentrations was performed by response surface methodology and optimal concentrations were 6.4 g/L, 172.6 µM, and 1.86 mM, respectively. Experimentally, the maximum laccase activity of 151.6 U/g was produced at the 5th day of solid-state fermentation. Lignin content in sugarcane bagasse was reduced from 31.89% to 26.36% after 5 days and to 20.79% after 15 days by the biological treatment of solid-state fermentation.
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Celulose/química , Proteínas Fúngicas/biossíntese , Lacase/biossíntese , Lignina/química , Pleurotus/enzimologia , Saccharum/químicaRESUMO
The valorization of agro-residues by biological routes is a key technology that contributes to the development of sustainable processes and the generation of value-added products. Sugarcane bagasse is an agro-residue generated by the sugar and alcohol industry in Brazil (186 million tons per year), composed essentially of cellulose (32-44%), hemicellulose (27-32%) and lignin (19-24%). The conversion of sugarcane bagasse into fermentable sugars requires essentially two steps: pretreatment and hydrolysis. The aim of the pretreatment is to separate the lignin and break the structure of lignocellulose, and it is one of the most critical steps in the process of converting biomass to fermentable sugars. The aim of this review is to describe different pretreatment strategies to promote the delignification of the sugarcane bagasse by thermo-chemical and biological processes.
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Laccases are oxidative enzymes linked to biological degradation of lignin. The aim of this work was to evaluate the effect of inducers and different concentrations of nitrogen on production level of total laccase activity and pattern of laccase isoforms, produced in solid state fermentation of sugarcane bagasse by a selected strain of Pleurotus ostreatus. The addition of yeast extract 5 g/L, copper sulfate 150 µM and ferulic acid 2 mM provided highest enzymatic activity (167 U/g) and zymograms indicated the presence of six laccase isoforms (POXA1b, POXA3, POXC and three other isoforms). Results of protein identification by mass spectrometry confirmed the presence of POXC and POXA3 as the main isoenzymes, and also identified a glyoxal oxidase and three galactose oxidases. The fact that the isoenzyme POXA1b was not identified in the analyzed samples can be possibly explained by its sensitivity to protease degradation.