Sugar beet leaves for functional ingredients
Tamayo Tenorio, Angelica - \ 2017
Wageningen University. Promotor(en): R.M. Boom, co-promotor(en): A.J. van der Goot. - Wageningen : Wageningen University - ISBN 9789463431378 - 188
sugarbeet - leaves - thylakoids - cellulosic fibres - food - surface proteins - food crops - protein extraction - suikerbieten - bladeren - thylakoïden - cellulosevezels - voedsel - oppervlakte-eiwitten - voedselgewassen - eiwitextractie
Plant leaves are recognised as a potential source for food applications based on their nutritional profile and interesting technological properties of leaf components, and based on the large availability of plant leaves in agricultural waste streams. Besides proteins, leaves have a rich nutritional profile (e.g. dietary fibres, minerals and secondary metabolites) and consist of complex biological structures (e.g. chloroplastic membranes) that can be explored as novel fractions that ultimately broaden the use of leaves. The overall aim of this thesis is to explore green leaves as a food source, with emphasis on neglected leaf fractions. This thesis describes a processing approach that aims at separating/generating enriched- functional fractions rather than pure components, and highlights the implications for value creation out of green leaves. The extraction of leaf membrane proteins is investigated using a proteomics extraction method, while the properties of other valuable leaf components (complexes and fibres) are analysed for techno-functional applications. Furthermore, the feasibility of leaves as a food source is studied at an industrial scale, considering large scale processing and options for leaf stabilisation.
The extraction of proteins from sugar beet leaves is evaluated in Chapter 2 by using a traditional heat coagulation method. The heat treatment is thought to precipitate the insoluble proteins together with fibres, chlorophyll and other components, resulting in a green curd. Therefore, the distribution of soluble and insoluble proteins was followed along the extraction process to discern the effect of the heating step on protein fractionation. This study showed that both soluble and insoluble protein distribute almost evenly over the leaf fractions juice, pulp, supernatant and final pellet. The even distribution of the proteins was attributed to the anatomy of leaves and their biological function, which is predominantly the enzymatic activity related to photosynthesis instead of protein storage, which occurs in other plant tissues. This chapter further concludes that striving for high purity severely compromises the yield, and consequently results in inefficient use of the leave proteins.
Chapter 3 describes the application of proteomic analytical extraction protocols to analyse the fractionation behaviour of leaf proteins. This analysis lead to the translation into food- grade processes based on four fundamental extraction steps: (1) tissue disruption, (2) enzymatic inhibition, (3) removal of interfering compounds, and (4) protein fractionation and purification. Part of these extraction steps can be translated into food-grade alternatives, while the processing conditions determine the potential properties for food of the final products. Nevertheless, it was concluded that harsh and/or non-food grade conditions were required to isolate the leaf membrane proteins with high purity. Those results were explained by the fact that membrane proteins are heterogeneous w.r.t. charge, hydrophobicity, post- translational modification and complexation, leading to non-selective behaviour when compared with a single pool of proteins.
Given the large challenges in isolating membrane proteins from leaves, we studied another approach in which green leaves are considered as a source of naturally structured elements that have relevant techno-functional properties for food products, like the chloroplastic membranes (i.e. thylakoid membranes) and cellulose-rich fibres. Chapter 4 describes the properties of thylakoid membranes and their emulsifying mechanism. These membranes showed surface active properties and their adsorption kinetics were typical for large molecules or soft particles. The thylakoid fragments can effectively stabilise emulsion droplets, even though aggregation was observed already during emulsion preparation and increased with increased thylakoid concentration. Both composition and structure make thylakoid membranes suitable as a biobased material for food and pharma applications.
To continue exploring valuable fractions from leaves, Chapter 5 reports on the interfacial behaviour of cellulose-rich particles obtained from leaf pulp. Cellulosic particles were produced from the pulp obtained after leaf pressing. The particles spontaneous adsorption onto the oil-water interface and interfacial behaviour similar to that of solid particles. Addition of cellulosic particles to oil-in-water emulsions resulted in stable emulsions above a particle concentration of 0.1 w/v%, although phase separation was observed. The particle fines (0.04 – 1.0 µm) stabilised the droplet interface, while large particles formed a network in the continuous phase and rendered a top (green) phase in the emulsions. Finding applications for leaf side streams, like leaf pulp, broadens the options for total leaf processing and contributes to resource use optimisation.
A sustainability assessment of leaf processing is discussed in Chapter 6, considering the challenges that may appear at industrial scale. The seasonal availability of sugar beet plants implies the need of processing large amounts of biomass within a short time due to their high moisture content (85 - 90%) and their sensitivity to spoilage. Processing options were evaluated on their resource use efficiency in terms of energy requirement and exergy indicators. A decentralised process constitutes a good option compared to freezing, since solid side streams can be directly returned the land, leaving nutrients to the soil, and reducing transportation loads. With a decentralised process, freezing of the leaves becomes unnecessary; the leaf juice is transported while chilled, resembling the transportation of fresh milk that is also chill-transported from the farm to a central factory.
Chapter 7 concludes this thesis with a general discussion of the main findings. An integrated process for leaf valorisation is described, which combines the production of functional fractions with the production of bulk products such as protein-rich and fibre-rich fractions. A compilation of data on protein yield and protein purity of fractions obtained from protein crops (e.g. soy, lupine beans, pulses) and from photosynthetic active tissues (e.g., leaves, algae, duckweed) is included. Protein crops reach 50 - 60% protein yield with a protein purity of ~ 90%, whereas leaves and other photosynthetic active tissues reach similar protein purity (60 – 80 w/w% protein) but at much lower yields (10%). We hypothesize that the low yields are due to the small length scale in which protein is structured inside the leaves and the lack of protein storage anatomy in these tissues. Therefore, we conclude that leaf valorisation requires non-conventional approaches that go beyond higher extraction yields but that consider a complete use of the biomass.
Biorefinery of proteins from rubber plantation residues
Widyarani, R. - \ 2016
Wageningen University. Promotor(en): Johan Sanders, co-promotor(en): Marieke Bruins; E. Ratnaningsih. - Wageningen : Wageningen University - ISBN 9789462576643 - 236
biorefinery - biomass conversion - rubber - rubber plants - protein extraction - latex - hydrolysis - hydrophobicity - amino acids - wheat gluten - residual streams - biobased economy - bioraffinage - biomassaconversie - rubber - rubberplanten - eiwitextractie - latex - hydrolyse - hydrofobiciteit - aminozuren - tarwegluten - reststromen - biobased economy
Biorefinery of rubber tree side streams could add economic value and income for farmers, who already grow the trees for latex production. The objective of this research was to design a process for the recovery of proteinaceous fractions from rubber tree. The aimed applications were expected to be suitable for local use, particularly in Indonesia, being one of the world’s largest rubber producers. Rubber seed was selected as a model biomass based on its availability (21-144 kg-protein/ha) and its oil content that enables the combination of protein and biodiesel productions within a biorefinery framework. Experimental works were focused on three parts: separation of protein and oil from rubber seed kernel, enzymatic hydrolysis of rubber seed protein into amino acids, and separation of amino acids from hydrolysate. Using alkaline extraction, up to 80% protein from the total original amount of protein in the kernel could be recovered in the extract, comparable to protein recoveries from other oilseeds and oilseed cakes. Seed type and pre-treatment had the most influence on protein recovery. Following protein extraction, the extracted proteins were recovered via isoelectric precipitation, resulting in rubber seed protein concentrate that can be used as such or can be processed further. Different protease combinations were used to hydrolyse rubber seed protein concentrate. After 24 h hydrolysis of rubber seed protein, up to 53% degree of hydrolysis and 35% protein recovery as free amino acids could be achieved. Combination of Pronase + Peptidase R resulted in the highest recovery and concentration of hydrophobic amino acids (phenylalanine, leucine, isoleucine, tyrosine, tryptophan, valine, methionine, and proline) in the hydrolysate. Some hydrophobic amino acids are essential in human and farm animal diets, therefore they can potentially be applied as a group in food and feed. Ethanol was used as an anti-solvent for selective precipitation of amino acids. Ethanol was able to selectively increase the hydrophobic amino acid fraction in rubber seed protein hydrolysate from 59% (mol/mol) in the starting material to 76% in the supernatant. Leucine and valine contributed most to this increase. The results of this study show that rubber seed proteins can be applied locally as animal feed or in industries for technical applications.
Biomass and its potential for protein and amino acids : valorizing agricultural by-products
Sari, Y.W. - \ 2015
Wageningen University. Promotor(en): Johan Sanders, co-promotor(en): Marieke Bruins. - Wageningen : Wageningen University - ISBN 9789462573185 - 146
landbouwbijproducten - eiwitbronnen - eiwitextractie - bioraffinage - biomassa - proteolyse - economische haalbaarheid - agricultural byproducts - protein sources - protein extraction - biorefinery - biomass - proteolysis - economic viability
The use of biomass for industrial products is not new. Plants have long been used for clothes, shelter, paper, construction, adhesives, tools, and medicine. With the exploitation on fossil fuel usage in the early 20th century and development of petroleum based refinery, the use of biomass for industrial application declined. Since the late 1960s, the petroleum-based products have widely replaced the use of biomass-based products. However, depletion of fossil fuels, rising oil prices, and growing environmental awareness, push the attention and policy towards a transition from fossil into bio-based products. Bio-based products can also be obtained from protein. The amine group (-NH2) in protein shows attractive functionality for nitrogen-containing chemicals production. In petroleum based conversion of crude oil into chemicals, co-reagents such as ammonia have to be used, and various process steps are involved. With the amine in protein, various co-reagent introducing process steps can be by-passed.
Biomass refinery for protein might not only be necessary for supplying feedstock for the chemical industry, before all, it is important to meet the world protein demand for food and feed. Chapter 1 illustrates the protein shortage in 2030 that we will encounter with the current uses of protein in the diet of both humans and animals. The worldwide protein production may provide this demand only if we consider the biomass refinery for protein and use the protein product in an effective and efficient way according the specific need of food, feed, and chemical industry. For this purpose, development in protein extraction technology from various types of biomass is essential. The thesis entitled “Biomass and its potential for protein and amino acids; valorizing agricultural by-products” describes possibilities for using agricultural by-products as protein and or amino acid resources.
An overview on alkaline plant protein extraction was first presented, in Chapter 2, including the potential of addition of different types of enzymes. Protein extraction from common resources such as soybean meal and other oilseed meals were reviewed. Also new protein resources, like microalgae, were discussed on the applicability of alkali based methods for protein extraction. Most of the experimental studies opted for less than 100 min and 50-60°C as extraction time and temperature, respectively. A typical biomass to solvent ratio of 1:10 was selected in some studies. Alkaline pH was selected over acidic pH, because it is far away from the isoelectric point (IEP). Most proteins have the lowest solubility at their IEP, which commonly occurs at pH 4-5. Adding proteases during protein extraction increased protein yield.
Two types of extraction methods were experimentally researched in this study; alkaline and combined alkaline and enzymatic. In Chapter 3, alkaline protein extraction method was used to extract protein from 16 types of biomass, mostly agricultural by-products. Aiming to maximise protein extraction yields, a three step extraction was performed at elevated temperatures; 25, 60, and 120 °C. Protein yield was correlated to biomass chemical composition through Partial Least Square (PLS) regression. The results showed that protein extractability depended crucially on the type of biomass used. Protein from cereals and legumes were highly extracted, compared to other biomass. High protein extractability coincides with the biological function of protein as a storage protein, as opposed to functional protein. Protein extraction was furthermore correlated to the composition of the biomass. Especially cellulose and oil hamper extractability of protein, whereas lignin has no significant influence, suggesting that alkaline treatment removed lignin sufficiently.
In Chapter 4, the effect of proteolysis during protein extraction was studied. Based on their working pHs, both alkaline and acidic proteases tested. Oilseed meals from soybean, rapeseed, and microalgae were considered as protein resource. Proteases that worked at acidic pH assisted protein extraction; but, still, more proteins were extracted using proteases that work at alkaline pH. This finding is in line with the literatures study from Chapter 2 mentioning that more proteins can be extracted at alkaline pH. Protex 40XL, Protex P, and Protex 5L that work at alkaline pH assisted protein extraction, particularly for rapeseed and microalgae meals. To a lesser extent, these proteases also improved protein extraction yield of soybean meal and untreated microalgae.
Having shown that proteolysis aids in protein extraction, proteases were also used to solubilise wheat gluten at alkaline pH. Solubilising wheat gluten is one of the bottle necks for wheat gluten application. In this thesis, wheat gluten was used to represent wheat dried distillers grains with solubles (DDGS). From our perspective, more biomass by-products, such as wheat DDGS derived from ethanol production, will be available, also due to the target to replace 10% fossil fuel with bio-based fuel in 2050. With high glutamic acid content, wheat gluten provides possibilities to serve as an amino acid resource. Glutamic acid, which currently is microbial produced, has potential as feedstock for bulk chemicals production. Large amounts of cheaper glutamic acid can be made available by enabling its production from biomass by-products, such as wheat DDGS. Several methods for producing glutamic acid from wheat gluten were developed and the results were presented in Chapter 5. We found that a combination of enzymatic and mild acid hydrolysis opens up new possibilities for the industrial production of glutamic acid from biomass.
Finally, in Chapter 6, general knowledge obtained from this study is discussed and a perspective on biomass valorization for protein and/or amino acids is presented. It was concluded that biomass, and particularly agricultural residues, are potential resources for protein and/or amino acids. An outlook on protein and/or amino acids production from by-products was also provided in this chapter. For this, economic calculations were provided that focussed on the processing cost. Based on these calculations, overnight alkaline treatment at room temperature was most economical to extract protein from most types of biomass. Residual biomass following protein extraction can be used as animal feed or for energy usage to get to a more integrated biorefinery, thereby reducing protein production cost.
Dry fractionation for sustainable production of plant protein concentrates
Pelgrom, P.J.M. - \ 2015
Wageningen University. Promotor(en): Remko Boom, co-promotor(en): Maarten Schutyser. - Wageningen : Wageningen University - ISBN 9789462572355 - 202
fractionering - peulvruchten (groente) - mechanische eigenschappen - eiwitextractie - voedselverrijking - fractionation - vegetable legumes - mechanical properties - protein extraction - food enrichment
The global demand for protein-rich foods is expected to double in the coming decades due to the increasing prosperity and world population. To keep up with the demand, the transition from an animal to a plant-based protein supply is desirable from long-term economic and environmental perspectives. In particular, legumes such as pea and lupine are of interest due to their nutritional profile and high protein content. Legume proteins are commonly purified by wet fractionation, which consumes large amount of water and energy and alters the native functionality of the proteins. Therefore, this thesis describes a sustainable, dry fractionation method for legumes to obtain functional protein-enriched fractions. Firstly, experiments have been performed to increase understanding of both the material properties of legume seeds and of the process conditions relevant to dry fractionation. Dedicated milling settings were selected for starch-rich and oil-rich legumes based on legume morphology. Milling settings were estimated based on starch granule size in starch-rich legumes, while coarse milling provided better results for oil-rich legumes. Separation of the protein bodies from other cellular components was established by air classification, which consumed ten times less energy and 50 litre water per kg protein less compared to conventional wet fractionation. Secondly, the functionality of the fractions was analysed. The dry-enriched protein fractions provided higher solubility than conventionally produced fractions, making them suitable for high protein drinks. Moreover, pea fractions could also be gelatinized which opens opportunities for preparing solid protein foods such as meat replacers. In conclusion, this thesis contributes to the awareness that the food industry could exploit a more sustainable dry fractionation technique to obtain functional protein fractions rather than focussing on wet extraction of relative pure protein ingredients.
Protein isolation using affinity chromatography
Besselink, T. - \ 2012
Wageningen University. Promotor(en): Remko Boom, co-promotor(en): Anja Janssen. - S.l. : s.n. - ISBN 9789461734266 - 146
eiwitextractie - isolatie - affiniteitschromatografie - harsen - liganden - runderserumalbumine - afvalverwerking - afvalhergebruik - industriële toepassingen - protein extraction - isolation - affinity chromatography - resins - ligands - bovine serum albumin - waste treatment - waste utilization - industrial applications
Many product or even waste streams in the food industry contain components that may have potential for e.g. functional foods. These streams are typically large in volume and the components of interest are only present at low concentrations. A robust and highly selective separation process should be developed for efficient isolation of the components. Affinity chromatography is such a selective method. Ligands immobilized to a stationary phase (e.g., a resin or membrane) are used to bind the component of interest. Affinity chromatography is, however, a costly process, due to the batch-wise operation, the large amount of solvents required and the high costs of the ligands and stationary phases. Therefore, its current use is mainly limited to lab-scale purifications and pharmaceutical applications.
The aim of this research was to investigate the potential of affinity chromatography for the isolation of minor protein in the food industry. The discovery of the VHH ligand, based on the binding domain of a llama antibody, has led to a new class of highly selective ligands, which can be produced on a large scale. We studied the chromatography process to measure productivity, but also to develop a rational protocol for decisions on suitable stationary phases and process configurations. The research presented in this thesis provides insights in the opportunities and challenges for large-scale affinity chromatography.
The isolation of protein using affinity chromatography requires several stages: adsorption, washing, and desorption. In Chapter 2, we studied these stages for the isolation of bovine serum albumin (BSA) from pure BSA solutions with high and low concentration and from actual feedstock, in this case cheese whey. A small-scale packed bed column was used to investigate the yield and productivity. BSA was retrieved in highly pure and concentrated form in the desorption stage. Furthermore, we found that the productivity of the system strongly depended on the point at which the adsorption stage is terminated.
Acids or salts are commonly used to disrupt the bond between ligand and target protein during desorption. This results in the use of large quantities of chemicals, whilst the potential of other methods for desorption, such as an increase in temperature, is not fully explored. In Chapter 3 we measured the thermodynamics of the adsorption reaction between BSA and the VHH ligand with isothermal titration calorimetry (ITC). Temperature and pH were varied to find other conditions for desorption. A buffer with high pH could be used for desorption, and an increase of temperature seemed to weaken the bond between protein and ligand. However, the acidic buffer would in this case still be most effective.
Apart from the bond between ligand and target protein, the stationary phase to which the ligand is immobilised plays a key role in the chromatography process. Many supports are available, of which we investigated a selection of resins for packed bed chromatography in Chapter 4. We found that some resins were unsuitable for our process due to their low adsorption capacity. A ranking and weighing method was presented to determine the optimal resin depending on the requirements of the process.
An important issue we found for all the resins investigated, was the low adsorption capacity compared to other types of adsorptive chromatography processes, such as ion exchange chromatography. Therefore, we studied the immobilization of the ligand to three resins in more detail in Chapter 5. The efficiency of ligand immobilization depended on the ligand concentration used in the immobilization procedure. However, only approximately one out of five immobilized ligands was able to bind to the target. Improvement of ligand immobilization is therefore a potential route to increase the feasibility of affinity chromatography for large-scale processes.
Eventually the lab-scale process has to be scaled-up to industrial scale. The commonly used axial flow column, essentially a cylinder filled with resin through which the feed flows in the axial direction, can have problems at scale-up, because of increased pressure drop as the column is lengthened. Therefore, scale-up usually takes place by widening the column. Another option is to use a radial flow column, in which the resin is confined between two concentric cylinders and liquid flows from the outside inwards or from the inside outwards. The radial flow column can be scaled up in height instead of width. In Chapter 6 we compared axial and radial flow affinity chromatography both experimentally and theoretically. We found that the differences in performance were minimal, because the process was limited by diffusion inside the resin particle. At a small process scale, radial flow columns are impractical in terms of size, but at a larger process scale they may compete with axial flow columns because of their smaller foot print and possibly lower construction costs.
The research in this thesis was focused on a defined ligand-protein system and commercially available resins in packed-bed configuration. The potential of other stationary phases, such as non-porous (magnetic) particles, membranes and monoliths was therefore discussed in Chapter 7. We found that currently the packed bed of porous resin beads still seems to be the most suitable configuration. A radial flow column with porous affinity resin is in theory capable of isolating a low-concentrated protein from a large feed of 10 m3/h. However, the relatively low capacity of the resin, the limited liquid velocity, as well as large buffer usage and the current costs remain important issues to resolve to further expand the opportunities of affinity chromatography for minor protein isolation.
Limiting factors for the enzymatic accessibility of soybean protein
Fischer, M. - \ 2006
Wageningen University. Promotor(en): Harry Gruppen; Fons Voragen. - [S.l.] : S.n. - ISBN 9789085044963 - 139
sojaeiwit - hydrolyse - aggregatie - koolhydraten - eiwitextractie - eiwitvertering - eiwitverteerbaarheid - peptiden - soya protein - hydrolysis - aggregation - carbohydrates - protein extraction - protein digestion - protein digestibility - peptides
Soy is a commonly used ingredient is food and animal feed. With particular focus on the in-soluble fractions, this thesis deals with the effects of proteases and carbohydrate degrading enzymes on different soybean meals subjected to different extent of heating. The primary aim is to improve the understanding of enzymatic hydrolysis of SBM with emphasis on proteins and to identify barriers limiting the efficiency of the process. The results show that aggregation behavior of peptides during enzymatic processing of soy proteins is potentially a limiting factor for efficacy of protein extraction. Surprisingly, it is also demonstrated that aggregation is not limited to in vitro incubations, but is also occurring in vivo in the digestive system of pigs.
|Dry fractionation of peas
Dijkink, B.H. ; Langelaan, H.C. - \ 2000
Industrial Proteins 8 (2000)1. - ISSN 1381-0022 - p. 11 - 13.
erwten - pisum sativum - fractionering - scheiding - groenvoederfractionering - eiwitextractie - agro-industriële sector - postagrarische sector - voedselindustrie - zetmeel - voedselverwerking - kosten - peas - pisum sativum - fractionation - separation - green crop fractionation - protein extraction - agroindustrial sector - postagricultural sector - food industry - starch - food processing - costs
De verschillende factoren die de droge scheiding van erwten in eiwit en zetmeel beinvloeden zijn uiteengezet