Frontiers in Natural Product Chemistry: Volume 6
()
About this ebook
Atta-ur-Rahman
Renowned scientist Dr. Atta-ur-Rahman was appointed as the chairman of United Nations’ committee on Science, Technology and Innovation in March 2016. Formerly Professor Emeritus, International Center for Chemical and Biological Sciences (H. E. J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research), University of Karachi, Pakistan, he was Pakistan Federal Minister for Science and Technology (2000-2002), Federal Minister of Education (2002), and Chairman of the Higher Education Commission with the status of a Federal Minister from 2002-2008. He is a Fellow of the Royal Society of London (FRS) and an UNESCO Science Laureate. A leading scientist, he also has over 930 publications to his name in several fields of organic chemistry
Read more from Atta Ur Rahman
Frontiers in Clinical Drug Research - Anti Infectives: Volume 3 Rating: 0 out of 5 stars0 ratingsFrontiers in Anti-Infective Drug Discovery: Volume 6 Rating: 0 out of 5 stars0 ratingsFrontiers in Clinical Drug Research - Alzheimer Disorders: Volume 6 Rating: 0 out of 5 stars0 ratingsFrontiers in Cardiovascular Drug Discovery: Volume 4 Rating: 0 out of 5 stars0 ratingsGenes in Health and Disease Rating: 0 out of 5 stars0 ratingsAnti-Angiogenesis Drug Discovery and Development: Volume 4 Rating: 0 out of 5 stars0 ratingsApplications of NMR Spectroscopy: Volume 6 Rating: 0 out of 5 stars0 ratingsAdvances in Organic Synthesis: Volume 7 Rating: 0 out of 5 stars0 ratingsTopics in Anti-Cancer Research: Volume 8 Rating: 0 out of 5 stars0 ratingsApplications in Food Sciences Rating: 0 out of 5 stars0 ratingsFrontiers in Drug Design & Discovery: Volume 8 Rating: 0 out of 5 stars0 ratingsFrontiers in Clinical Drug Research - Diabetes and Obesity: Volume 5 Rating: 0 out of 5 stars0 ratingsFrontiers in Clinical Drug Research - Hematology: Volume 5 Rating: 0 out of 5 stars0 ratingsNatural Products in Clinical Trials: Volume 1 Rating: 0 out of 5 stars0 ratingsFrontiers in Drug Design & Discovery: Volume 7 Rating: 0 out of 5 stars0 ratingsFrontiers in Clinical Drug Research - Anti Infectives: Volume 8 Rating: 0 out of 5 stars0 ratingsFrontiers in Stem Cell and Regenerative Medicine Research: Volume 6 Rating: 0 out of 5 stars0 ratingsFrontiers in Clinical Drug Research - Anti-Allergy Agents: Volume 4 Rating: 0 out of 5 stars0 ratingsFrontiers in Stem Cell and Regenerative Medicine Research: Volume 5 Rating: 0 out of 5 stars0 ratingsFrontiers in Clinical Drug Research - Anti Infectives: Volume 6 Rating: 0 out of 5 stars0 ratingsInfectious Diseases Rating: 0 out of 5 stars0 ratingsApplications of NMR Spectroscopy: Volume 8 Rating: 0 out of 5 stars0 ratingsAdvances in Organic Synthesis: Volume 16 Rating: 0 out of 5 stars0 ratingsAdvances in Cancer Drug Targets: Volume 3 Rating: 0 out of 5 stars0 ratingsFrontiers in Clinical Drug Research - Anti-Allergy Agents: Volume 5 Rating: 0 out of 5 stars0 ratingsFrontiers in Clinical Drug Research - Anti-Cancer Agents: Volume 4 Rating: 0 out of 5 stars0 ratingsRecent Advances in Analytical Techniques: Volume 1 Rating: 0 out of 5 stars0 ratingsFrontiers in Clinical Drug Research - Anti Infectives: Volume 2 Rating: 0 out of 5 stars0 ratingsFrontiers in Anti-Cancer Drug Discovery: Volume 10 Rating: 0 out of 5 stars0 ratingsFrontiers in Clinical Drug Research - Anti Infectives: Volume 5 Rating: 0 out of 5 stars0 ratings
Related to Frontiers in Natural Product Chemistry
Related ebooks
Frontiers in Natural Product Chemistry: Volume 6 Rating: 0 out of 5 stars0 ratingsFrontiers in Natural Product Chemistry: Volume 10 Rating: 0 out of 5 stars0 ratingsNew Findings from Natural Substances Rating: 0 out of 5 stars0 ratingsKey Heterocyclic Cores for Smart Anticancer Drug–Design Part II Rating: 0 out of 5 stars0 ratingsFrontiers in Natural Product Chemistry: Volume 11 Rating: 0 out of 5 stars0 ratingsAlkaloids and Other Nitrogen-Containing Derivatives Rating: 0 out of 5 stars0 ratingsScience of Spices and Culinary Herbs - Latest Laboratory, Pre-clinical, and Clinical Studies: Volume 4 Rating: 0 out of 5 stars0 ratingsFrontiers in Natural Product Chemistry: Volume 3 Rating: 0 out of 5 stars0 ratingsPostbiotics: Science, Technology, and Applications Rating: 0 out of 5 stars0 ratingsBiologically Active Natural Products from Asia and Africa: A Selection of Topics Rating: 0 out of 5 stars0 ratingsFree Radical Biology of & Endocrine, Metabolic Immune Disorders Rating: 0 out of 5 stars0 ratingsFrontiers in Computational Chemistry: Volume 4 Rating: 0 out of 5 stars0 ratingsAnti-obesity Drug Discovery and Development: Volume 4 Rating: 0 out of 5 stars0 ratingsThyroid Toxicity Rating: 0 out of 5 stars0 ratingsFrontiers in Drug Design & Discovery: Volume 11 Rating: 0 out of 5 stars0 ratingsNatural Bioactive Compounds from Fruits and Vegetables as Health Promoters: Part 2 Rating: 0 out of 5 stars0 ratingsPlant-derived Hepatoprotective Drugs Rating: 0 out of 5 stars0 ratingsFrontiers in Anti-Cancer Drug Discovery Volume 10 Rating: 0 out of 5 stars0 ratingsCancer Preventive and Therapeutic Compounds: Gift From Mother Nature Rating: 0 out of 5 stars0 ratingsBiopolymers Towards Green and Sustainable Development Rating: 0 out of 5 stars0 ratingsScience of Spices & Culinary Herbs: Volume 5 Rating: 0 out of 5 stars0 ratingsFrontiers in Medicinal Chemistry: Volume 8 Rating: 0 out of 5 stars0 ratingsThe Chemistry Inside Spices & Herbs: Research and Development: Volume 4 Rating: 0 out of 5 stars0 ratingsNatural Products in Clinical Trials: Volume 1 Rating: 0 out of 5 stars0 ratingsFrontiers in Anti-Infective Drug Discovery: Volume 10 Rating: 0 out of 5 stars0 ratingsMushrooms: A Wealth of Nutraceuticals and An Agent of Bioremediation Rating: 0 out of 5 stars0 ratingsFrontiers in Anti-Infective Drug Discovery: Volume 9 Rating: 0 out of 5 stars0 ratingsFrontiers in Clinical Drug Research - Anti Infectives: Volume 3 Rating: 0 out of 5 stars0 ratingsFrontiers in Clinical Drug Research - CNS and Neurological Disorders: Volume 7 Rating: 0 out of 5 stars0 ratingsEmerging Technologies in Agriculture and Food Science Rating: 0 out of 5 stars0 ratings
Medical For You
Brain on Fire: My Month of Madness Rating: 4 out of 5 stars4/5The Vagina Bible: The Vulva and the Vagina: Separating the Myth from the Medicine Rating: 5 out of 5 stars5/5Gut: The Inside Story of Our Body's Most Underrated Organ (Revised Edition) Rating: 4 out of 5 stars4/5Mating in Captivity: Unlocking Erotic Intelligence Rating: 4 out of 5 stars4/5The Little Book of Hygge: Danish Secrets to Happy Living Rating: 4 out of 5 stars4/5The Lost Book of Simple Herbal Remedies: Discover over 100 herbal Medicine for all kinds of Ailment, Inspired By Dr. Barbara O'Neill Rating: 0 out of 5 stars0 ratingsWhat Happened to You?: Conversations on Trauma, Resilience, and Healing Rating: 4 out of 5 stars4/5Mediterranean Diet Meal Prep Cookbook: Easy And Healthy Recipes You Can Meal Prep For The Week Rating: 5 out of 5 stars5/5The Emperor of All Maladies: A Biography of Cancer Rating: 5 out of 5 stars5/5Adult ADHD: How to Succeed as a Hunter in a Farmer's World Rating: 4 out of 5 stars4/5Blitzed: Drugs in the Third Reich Rating: 4 out of 5 stars4/5"Cause Unknown": The Epidemic of Sudden Deaths in 2021 & 2022 Rating: 5 out of 5 stars5/5Peptide Protocols: Volume One Rating: 4 out of 5 stars4/5Women With Attention Deficit Disorder: Embrace Your Differences and Transform Your Life Rating: 5 out of 5 stars5/5The White Coat Investor: A Doctor's Guide to Personal Finance and Investing Rating: 4 out of 5 stars4/5Hidden Lives: True Stories from People Who Live with Mental Illness Rating: 4 out of 5 stars4/5Ghost Boy: The Miraculous Escape of a Misdiagnosed Boy Trapped Inside His Own Body Rating: 4 out of 5 stars4/5The Song of the Cell: An Exploration of Medicine and the New Human Rating: 4 out of 5 stars4/5ATOMIC HABITS:: How to Disagree With Your Brain so You Can Break Bad Habits and End Negative Thinking Rating: 5 out of 5 stars5/5This Is How Your Marriage Ends: A Hopeful Approach to Saving Relationships Rating: 4 out of 5 stars4/5Holistic Herbal: A Safe and Practical Guide to Making and Using Herbal Remedies Rating: 4 out of 5 stars4/5The 40 Day Dopamine Fast Rating: 4 out of 5 stars4/5Woman: An Intimate Geography Rating: 4 out of 5 stars4/5Herbal Healing for Women Rating: 4 out of 5 stars4/5
Reviews for Frontiers in Natural Product Chemistry
0 ratings0 reviews
Book preview
Frontiers in Natural Product Chemistry - Atta-ur-Rahman
Plant Protein Hydrolyzates from Underutilized Agricultural and Agroindustrial Sources: Production, Characterization and Bioactive Properties
María del Mar Contreras¹, *, Minerva Cristina García Vargas¹, ², Antonio Lama-Muñoz¹, Francisco Espínola¹, ³, Manuel Moya¹, ³, Eulogio Castro¹, ³
¹ Department of Chemical, Environmental and Materials Engineering, University of Jaén, Campus Las Lagunillas, 23071Jaén, Spain
² Department of Industrial Engineering of Tecnológico Nacional de México/Instituto Tecnológico de Zitácuaro, Av. Tecnológico, 186 CP 61534, Zitácuaro, Michoacán, Mexico
³ Center for Advanced Studies in Earth Sciences, Energy and Environment, University of Jaén, Spain
Abstract
Today, there is a growing interest in the valorization of agricultural and agroindustrial waste/byproducts, including through obtaining bioactive compounds. Besides the use of plant proteins in animal nutrition, obtaining protein hydrolyzates could give an added value, improving digestibility and exerting functional properties by the generation of bioactive peptides. Bioactive peptides encrypted in plant proteins are latent until released and activated by proteolysis. Generally, to obtain bioactive peptides, enzymatic hydrolysis by peptidases is the most common way, with or without previous solubilization and purification steps of the intact protein. This hydrolysis step can be combined with physical and chemical treatments not only to improve the recovery but also to enhance the bioactivity. Therefore, our chapter presents an overview of different ways of production to obtain bioactive peptides from different underutilized plant sources, including from food, brewing and bioethanol industries. In order to characterize bioactive peptides, the application of conventional methods and more sophisticated methods based on mass spectrometry is also described. Moreover, recent literature on the bioactive properties of those plant peptides and current challenges associated with safety issues are discussed.
Keywords: ACE-inhibitor, Antioxidant, Antihypertensive, Antidiabetic, Byproduct, Bioactive peptide, Carbohydrolase, Hydrolysis, Mass spectrometry, Microwave assisted extraction, Peptidomics, Peptidase, Protein, Sustainability, Valorization.
* Corresponding author María del Mar Contreras: Department of Chemical, Environmental and Materials Engineering, University of Jaén, Campus Las Lagunillas, 23071 Jaén, Spain;
E-mails: [email protected];[email protected];[email protected]
INTRODUCTION
Considering the population factor and the state of natural resources, the need to look for more efficient agroindustry processes is recognized due to demographic growth and the current unsustainable practices. The global population is growing, while our standard of living is increasing; thereby, we have to face environmental challenges. In this sense, it is expected that the world’s population increases by 2 billion people in the next 30 years and could reach around 11 billion in the next century. Based on this prognosis, it is not difficult to understand why the United Nation’s second priority objective for the present century is to End hunger, achieve food security and improve nutrition and promote sustainable agriculture
[1]. Moreover, the current agricultural and food practices also threaten the health of people and the planet: i) 70% of worldwide water use is required by agriculture; ii) it generates huge levels of pollution and waste; iii) risks associated with poor diets are one of the leading causes of death; iv) a double burden of malnutrition exists since millions of people are either eating not enough or eating the wrong types of food. In 2017, this led to one in eight adults (i.e. more than 672 million people) in the world to be obese [2] and forecasts suggest high levels of obesity on the future population [3]. In particular, increased demand for animal-based protein is expected to have a negative environmental impact, generating greenhouse gas emissions, requiring more water and more land [4]. Thereby, plant proteins could be an alternative but sustainable practices are required.
Therefore, against this background, the goal is how to meet the growing global demand for food, including protein and healthy foods, to improve income and employment in rural areas and, at the same time, reduce the environmental impact. This puts pressure on the world’s resources to provide not only more but also different types of food, including more sustainable production of existing sources of protein as well as alternative sources for human consumption [4]. This requires us to move from an oil based economy towards a more sustainable circular bioeconomy model, producing more food and bio-based products from renewable resources, including agricultural and agroindustrial byproducts [5]. In this line, the biorefinery concept has emerged as a sustainable processing of biomass into a portfolio of marketable food and feed ingredients, bio-based products (chemicals, materials, proteins, bioactive compounds, etc.) and energy (fuels, power, heat) [6-8].
When thinking about these resources, plant compounds are usually put forward as their most probable source [9]. This includes macro (cellulose, hemicelluloses, pectins, starch, lignin, proteins, minerals, etc.) and microcomponents (e.g. phytochemicals). In particular, plant byproducts are underutilized sources of proteins and, most of the time are addressed to animal nutrition, but the ruminal degradability of proteins is not high. Nonetheless, proteins can be beneficial not only in terms of nutrition but also from a functional point of view through the generation of bioactive peptides. This means that the breakdown of peptide bonds by enzymatic hydrolysis increases the solubility, digestibility, and functional properties of the precursor proteins and byproduct [6]. Bioactive peptides are known for their high tissue affinity, specificity and efficiency in promoting health [10]. Therefore, apart from the use of plant proteins in animal nutrition, obtaining protein hydrolyzates could give an added value in a biorefinery context, with improved digestibility and exerting functional properties through the generation of bioactive peptides. This could also lead to the formulation of functional ingredients that are in line with the increased consumer awareness towards functional foods, nutraceuticals and personalized diets; the driving force of the functional food and nutraceutical market [10]. Moreover, there is a growing interest in the food industry and among consumers in reducing the use of synthetic additives in food preservation and opting instead for natural ones [11]. All this together connects with the concept of bioeconomy since it can promote a new way to diversify plant byproducts.
Generally, hydrolysis by peptidases, with or without a previous protein extraction step, is the most common way to obtain bioactive peptides with a wide range of biological properties, e.g. antidiabetic, antihypertensive, antimicrobial, antioxidant, and anticancer properties [12-15], but also autolysis and application of microbial suspensions (whole cells) have been applied [13, 16]. Enzymatic hydrolysis can be combined with physical treatments and alkaline extraction not only to improve the recovery but also to enhance the bioactivity [6, 17]. In this context, this book chapter presents an overview of the different ways of production to obtain bioactive protein hydrolyzates from different underutilized plant sources. These sources include byproducts from the cereals industry (wheat germ protein, broken rice by-product), oil industry (olive, and rapeseed/canola byproducts), fruit and vegetable industries (e.g. fruits seeds, potato byproducts, cauliflower leaves), and brewing industry (brewer’s spent grain). Some techniques applied to characterize the hydrolyzates and the peptides are also covered. Moreover, the biological properties of the hydrolyzates have been revised, and the sequence of some bioactive peptides is shown. Finally, some safety issues are also discussed.
WAYS OF PRODUCING PLANT PROTEIN HYDROLYZATES FROM AGRICULTURAL AND AGROINDUSTRIAL SOURCES
There are several ways to produce bioactive hydrolyzates and peptides from natural sources: gastrointestinal digestion, fermentation, enzymatic hydrolysis, and genetic recombination [18]. This can be performed through the use of endogenous and exogenous microorganisms and proteolytic enzymes [19]. In general, microbial alkaline proteases can be produced under culture conditions. Bacillus produces extracellular proteases during post-exponential and stationary phases. Their proteolytic system contributes to the liberation of bioactive peptides [13]. Examples of commercial Bacillus enzymes are Alcalase, Neutrase, Esperase, Thermolysin, etc. Another enzyme employed is Flavourzyme from Aspergillus oryzae. Among them, Alcalase is widely used, probably explained by its high hydrolytic activity [20].
In the case of the agricultural and agroindustrial byproducts/waste the most common way to obtain bioactive peptides is through enzymatic hydrolysis using exogenous proteolytic enzymes. A wide variety of smaller peptides are generated via hydrolysis, depending on the byproduct, enzyme specificity and hydrolysis time [6, 15]. The hydrolysis conditions and enzyme type also affect the degree of hydrolysis and bioactivity due to the generation of peptides with a different amino acid sequences and length [15, 20-23]. Sometimes, gastrointestinal digestion enzymes are applied after enzymatic hydrolysis and ultrafiltration since it can enhance the bioactivity of the hydrolyzates, but it depends on the hydrolysis and ultrafiltration conditions [20, 24, 25]. Other times simulated gastrointestinal conditions are tested to assess the maintenance of the bioactivity as it was in vivo or to find the active peptides [26, 27]. Other studies have revealed that autolysis and fermentation were also plausible ways to recover bioactive peptides from potato and rice byproducts respectively [13, 16]. While the results of autolysis were improved after hydrolysis using Bacillus peptidases [16], the application of suspensions of strains of Bacillus subtilis, Bacillus pumilus and Bacillus licheniformis, which were isolated from different food sources, were successful in generating antioxidant peptides from rice compared to enzymes [13].
Concerning enzymatic hydrolysis, which is the most common way, it can be classified secondarily according to when the hydrolysis step with peptidases is performed; i.e. obtaining proteins and hydrolysis separately (sequential extraction and hydrolysis) (SeEH) or simultaneously (simultaneous extraction and hydrolysis) (SiEH). The proteolysis step can be performed with enzymes and preparations of enzymes from different origins, i.e. microbial enzymes, plant enzymes, and animal enzymes (Table 1). Their effectiveness depends on the method and conditions applied as well as the primary sequence of the proteins. While some enzymes, such as those with subtilisin activity (e.g. preparations like Alcalase) have broad specificity, others cannot work well if they are not favored by the primary sequence of the protein [6]. Other enzyme preparations consist of a mixture of peptidases and other enzymes types: e.g. Flavourzyme from Aspergillus oryzae contains two aminopeptidases, two dipeptidyl peptidases, three endopeptidases, and one α-amylase [28]; pancreatin and Corolase PP contain mainly trypsin and chymotrypsin from animal pancreas, but also other enzymes [29, 30]. The methods applied to generate bioactive peptides from agricultural and agroindustrial byproducts are detailed in the sections below. However, this book chapter is focused on the application of these methods in agricultural and agroindustrial byproducts/waste rather than on the description of the mechanism behind solubilization or their application in other matrices, which has been described in other reviews [6, 19, 31, 32].
Moreover, it should be considered that as a first screening, experimental assays with peptidases can be performed by changing one factor at a time and keeping others fixed, whereas response surface methodology can take several factors into account at the same time (as an example, see [14]). This includes screening tools like factorial designs with a reduced number of assays [33]. Moreover, in silico tools can be applied to choose those enzymes that are able to cut proteins theoretically on the basis of the primary sequence and the extent: e.g. the Peptide-Cutter of ExPASy (https://web.expasy.org/peptide_cutter/; accessed on 17 January 2018) and EnzymePredictor (http://bioware.ucd.ie/~compass/biowar eweb/; accessed on 17 January 2018) [6, 34]. These tools include enzymes like: Arg-C proteinase, caspases, chymotrypsin, pepsin, thermolysin, thrombin, staphylococcal peptidase I, trypsin, enterokinase, glutamyl endopeptidase, proline-endopeptidase, etc.
Table 1 Enzymes reported for the hydrolysis of proteins from agricultural and agroindustrial byproducts/waste. The information about the enzymes was retrieved from the BRENDA database (https://www.brenda-enzymes.org/; accessed 22 January 2020).
aPancreatin and Corolase PP enzyme preparations contain these enzyme activities, among others.
bAlcalase 2.5L, Protex 6L, Esperase contain mainly serine endopeptidase (mainly subtilisin A).
cProtex 14L contains thermolysin activity.
dNeutrase contains bacillolysin activity.
eCorolase L10 and Promod 144MG contain papain activity.
Sequential Extraction and Hydrolysis (SeEH)
If SeEH is selected (Table 2), the main driving force of the protein extraction process should be taken into account, i.e. chemical, biochemical, physical (including, mechanical) and physical-chemical, but a combination of them is also common [6, 31]. In any case, the steps basically consist of: conditioning, extraction/solubilization of the plant protein, concentration (purification or isolation)/drying and hydrolysis by peptidases [6, 35].
Extraction by Chemical Methods and Hydrolysis
In general, these methods normally require a previous grinding of the byproduct, the use of homogenization, shearing and/or thermal treatments to enhance the protein solubilization. Then, the use of neutral solutions, acid solutions, alkaline solutions, organic solvents, salt solutions, surfactants and reducing agents have been applied to recover proteins from plants [31, 36-39]. It also enables the fractionation of cereals proteins, recovering most proteins (98%); e.g. using consecutively, sodium chloride (albumins and globulins), ethanol (prolamins), acetic acid (acid-soluble glutelins), and sodium hydroxide (residual proteins) aqueous solutions [40]. In general, these extraction methods have been reviewed by several publications [31, 37, 38] and, in particular, by our recent review [6], which was focused on plant proteins from agri-food byproducts/residues. Concerning the obtention of protein hydrolyzates, Table 2 exemplifies methods to recover proteins from these sources and the subsequent hydrolysis conditions. In addition, Table 3 shows some of the characteristics of the protein products obtained before and after hydrolysis.
Among these methods, alkaline solutions are widely used as a generally recognized as safe (GRAS) solvent for the extraction of proteins from cereals and seed storage proteins [39], as well as agri-food byproducts [6-8, 17, 41]. In particular, Connolly et al. (2013) [39] reported the use of aqueous-alkaline conditions to recover proteins from pale brewer’s spent grain, which was subjected to protein hydrolysis in subsequent works to generate bioactive peptides [25, 35]. The protein recovery was up to 59%, and hordeins, glutelins and low molecular weight peptides (41% < 10 kDa) were recovered. This value is lower than those reported by Qin et al. [36] (85-95%), who tested acid conditions, either in a single step or two-step treatments (after an alkaline extraction step) [36]. However, proteins were probably obtained in the form of amino acids owing to the strong conditions used. Moreover, alkaline extraction has been also applied to recover proteins from broken rice, canola, palm kernel and walnut cakes after oil extraction [15, 23, 25, 35, 39, 49, 50] with different extraction conditions: pH from 8 to 13.0, from room temperature to 50 ºC, and extraction time from 45 to 180 min, with and without NaCl. Then, hydrolysis with alkaline peptidases, mainly Alcalase, has enabled the obtention of antimicrobial, antioxidants, anticancer and ACE inhibitory (ACEi) peptides, while anticancer peptides were obtained using papain (Table 2).
Other studies have applied buffered extraction at neutral pH, using detergents like sodium dodecyl sulfate (SDS) and reducing agents with, for example, dithiothreitol, to recover proteins from olive seeds [20, 51]. For food appli-cations, these conditions should be adapted to food grade conditions.
Extraction by Enzymatic Methods and Hydrolysis
Adding enzymes during protein extraction increased protein recovery and yield compared to control experiments without enzymes [17]. This included the use of carbohydrolases and peptidases to assist in protein extraction. The first type assists by degrading the cell wall components, while the second type assists through proteolysis. However, only studies focused in the use of carbohydrolases are discussed in this section, while the use of peptidases is the objective of the section below about direct SiEH methods.
Most of studies involving carbohydrolases have been performed at neutral to acidic pH conditions and applied thermal treatments (45-55 ºC) for 2-6 h. They also applied one main enzyme activity like α-amylase and pectinase, but most of them used enzyme preparations and their mixture (Table 2). Examples of the use of carbohydrolases are