Sugar beet leaves: from biorefinery to techno-functionality

Sugar beet leaves (SBL), which are a side stream of the sugar beets cultivation, are currently left unexploited after sugar beets have been harvested. The general aim of this thesis was to study the biorefinery of SBL, with a special focus on the isolation of proteins. To reach this aim the research was divided into three sub-aims: 1) to determine whether there is variability in the chemical composition of the leaves due to pre-harvest conditions (plant age), 2) to evaluate the variability of the techno-functionality of leaf soluble protein concentrate (LSPC) due to system conditions and 3) to extend current product and process synthesis approaches to enable the design of biorefining process. To address the first aim, SBL collected at different time points were used. Despite a small variation in the chemical composition of the leaves of different plant ages, a large effect of the plant age on the quality of LSPC was observed. In particular, LSPC from old plants was brown (indicative of polyphenol oxidase - PPO - activity), whereas LSPC from young plants was yellow. Based on these data, samples extracted with sodium disulfite (to inhibit PPO-mediated browning) were used for further experiments. The obtained LSPC consisted mainly of protein (69.3% w/w db (N∙5.23)) and carbohydrates (5.1% w/w db; half of which was charged carbohydrates). The main protein present in LSPC was Rubisco. The emulsion and foam properties of LSPC were studied as a function of protein concentration (Cp), pH and ionic strength (I). The minimal Cp of LSPC needed to form a stable emulsion (Ccr) was comparable to that of other widely used plant proteins, such as soy protein isolate. A critical ζ-potential (ζcr ~ 11 mV) was identified, below which flocculation occurs. At pH 8.0 and high I (0.5 M) the Ccr was higher than at low I (0.01 M), which relates to a higher protein adsorbed amount at the interface (Γmax). The foam ability (FA) of LSPC increased with Cp at all conditions tested. The FA was related to the soluble and not to the total Cp in the bulk. Interestingly, the minimal Cp; i.e.CcrFA needed to reach highest FA was constant as a function of pH. At high I (0.5 M) LSPC had higher FA than at low I (0.01 M), which was related to the faster adsorption of proteins at the interface. A minimum Cp was required to form stable foams. At pH 3.0 and 5.0 the foam stability of LSPC was higher than at pH 8.0. This was postulated to be due to formation of aggregates (between proteins or between proteins and charged carbohydrates). From these data it was shown that the techno-functional properties of LSPC could be linked to the molecular and interfacial properties of the dominant proteins in the concentrates. Thus, predictions for the techno-functional properties of impure systems, such as LSPC, can be made using only the known molecular properties of the dominant proteins and a small set of experiments. The knowledge acquired through the previous studies was used to adapt an existing methodology; namely the product-driven-process synthesis (PDPS) methodology, to extend its use in biorefinery. The adapted PDPS contained 4 novel steps, which facilitated its use in biorefinery. To illustrate how this new approach can be used in practice, a case study of a sugar beet leaves biorefinery was presented.</p

Towards predicting the emulsion properties of plant protein extracts from sugar beet (Beta vulgaris L.) leaf and soybean (Glycine max)

To apply (novel) proteins such as sugar beet leaf proteins (LSPC) as emulsifier their emulsion properties need to be tested over a wide range of conditions, which is impractical. Recently, a model was proposed to predict the efficiency of proteins to form and stabilize an emulsion -based on the protein molecular properties (e.g. size, charge) and system parameters-. In this model, the critical protein concentration (Ccr), to prevent coalescence during emulsion formation and flocculation induced by changes in system conditions, is the key descriptor. This study investigates whether the model, developed for single protein systems, can be applied to more complex systems containing multiple proteins, i.e. LSPC and soy protein isolate (SPI). Despite the complexity of LSPC and SPI, Ccr for emulsion formation and salt-induced flocculation (at ζ ≥ ζcr) were in close agreement with the predictions. At ζ < ζcr (i.e. pH close to pI), the critical energy barrier of 5 kBT and surface coverage were found to be the most important parameters to predict emulsion stability. As experimental values for Ccr were close to the theoretical Ccr calculated using the model, it was concluded that protein mixtures behave similar as single protein systems. This shows that the model developed to predict the emulsion properties of single protein systems can also be applied, at least to get decent estimations, to more complex (plant) protein systems containing multiple proteins

Using product driven process synthesis in the biorefinery

In this work, we propose the use of the product-driven process synthesis (PDPS) methodology for the product and process design stage in biorefinery. The aim of the biorefinery is to optimize the total use of the whole feedstock - with focus being on various products simultaneously - rather than to maximize the extraction yield of one single product. The challenge is therefore two-fold; first to identify the main compounds of interest, i.e. the products of the biorefinery, and second to design a process scheme that will allow for an optimal quantity and quality of the identified compounds. To illustrate how PDPS can be used in biorefinery a case study based on sugar beet leaves is described. The identification of the main compounds of interest is based on the functionalities that they can deliver in the final applications, rather than on their quantities in the feedstock. To design the process scheme for the extraction of the selected compounds, task networks, currently used for the extraction of the individual compounds of interest, are used after adaptations. These adaptations are done on the basis of the qualitative and/or quantitative changes that certain tasks - used for the extraction of one compound - may cause on another compound of interest. By using the sugar beet leaves biorefinery case, we show that the PDPS methodology can be a useful tool for structured decision making during the product and process design stage in biorefinery

Effect of Plant Age on the Quantity and Quality of Proteins Extracted from Sugar Beet (Beta vulgaris L.) Leaves

Effects of the developmental stage (e.g., young, mature, or senescent) of leaves on their chemical composition have been described in the literature. This study focuses on the variation in chemical composition and quantity and quality of proteins extracted from leaves due to variation in plant age (i.e., harvesting time), using leaves from sugar beets grown in a field (Rhino, Arrival) and in a greenhouse (Isabella). Within the same variety (Rhino, field; Arrival, field; Isabella, greenhouse) the protein content was similar for leaves from young and old plants (22 ± 1, 16 ± 1, and 10 ± 3% w/w db, respectively). Variation in final protein isolation yield was mostly due to variation in nitrogen extractability (28-56%), although no consistent correlation with plant age was found. A significant effect of plant age was observed on the quality (color) of the extracted protein, that is, brown (indicative of polyphenol oxidase activity) and yellow for extracts from old and young plants, respectively

Enzymatic Browning in Sugar Beet Leaves (Beta vulgaris L.) : Influence of Caffeic Acid Derivatives, Oxidative Coupling, and Coupled Oxidation

Sugar beet (Beta vulgaris L.) leaves of 8 month (8m) plants showed more enzymatic browning than those of 3 month (3m). Total phenolic content increased from 4.6 to 9.4 mg/g FW in 3m and 8m, respectively, quantitated by reverse-phase-ultrahigh-performance liquid chromatography-ultraviolet-mass spectrometry (RP-UHPLC-UV-MS). The PPO activity was 6.7 times higher in extracts from 8m than from 3m leaves. Substrate content increased from 0.53 to 2.45 mg/g FW in 3m and 8m, respectively, of which caffeic acid glycosyl esters were most important, increasing 10-fold with age. Caffeic acid glycosides and vitexin derivatives were no substrates. In 3m and 8m, nonsubstrate-to-substrate ratios were 8:1 and 3:1, respectively. A model system showed browning at 3:1 ratio due to formation of products with extensive conjugated systems through oxidative coupling and coupled oxidation. The 8:1 ratio did not turn brown as oxidative coupling occurred without much coupled oxidation. We postulate that differences in nonsubstrate-to-substrate ratio and therewith extent of coupled oxidation explain browning.</p

Minced beef is more rapidly digested and absorbed than beef steak, resulting in greater postprandial protein retention in older men

Older individuals generally experience a reduced food-chewing efficiency. As a consequence, food texture may represent an important factor that modulates dietary protein digestion and absorption kinetics and the subsequent postprandial protein balance

Carbohydrate co-ingestion with protein does not further augment post-prandial muscle protein accretion in older men

Abstract Background A blunted muscle protein synthetic response to protein ingestion may contribute to the age related loss of muscle tissue. We hypothesized that the greater endogenous insulin release following co-ingestion of carbohydrate facilitates post-prandial muscle protein accretion after ingesting a meal-like bolus of protein in older males. Methods Twenty-four healthy older men (75±1 y) were randomly assigned to ingest 20 g intrinsically L-[1-13C] phenylalanine-labeled casein protein with (PRO-CHO) or without (PRO) 40 g carbohydrate. Ingestion of specifically produced intrinsically L-[1-13C] phenylalanine labeled protein allowed us to assess post-prandial incorporation of dietary protein derived amino acids into muscle protein. Blood samples were collected at regular intervals, with muscle biopsies being obtained prior to and 2 and 6 h after protein ingestion. Results Plasma glucose and insulin concentrations showed a greater increase in PRO-CHO compared with PRO (P13C] phenylalanine enrichments tended to increase to a greater extent in PRO-CHO compared with PRO during the first 2 h after protein ingestion (0.0072±0.0013 vs 0.0046±0.010 MPE, respectively; P=0.13). However, 6 h after protein ingestion, differences in muscle protein-bound L-[1-13C] phenylalanine enrichments were no longer observed between experiments (0.0213±0.0024 vs 0.0185±0.0010 MPE, respectively; P=0.30). Conclusions This study shows that carbohydrate ingestion may accelerate, but does not further augment post-prandial incorporation of dietary protein derived amino acids into muscle protein in healthy elderly men.</p

The muscle protein synthetic response to the combined ingestion of protein and carbohydrate is not impaired in healthy older men

Aging is associated with a progressive decline in skeletal muscle mass. It has been hypothesized that an attenuated muscle protein synthetic response to the main anabolic stimuli may contribute to the age-related loss of muscle tissue. The aim of the present study was to compare the muscle protein synthetic response following ingestion of a meal-like amount of dietary protein plus carbohydrate between healthy young and older men. Twelve young ( 21 ± 1 years ) and 12 older ( 75 ± 1 years ) men consumed 20 g of intrinsically l-[1-13C]phenylalanine-labeled protein with 40 g of carbohydrate. Ingestion of specifically produced intrinsically l-[1-13C]phenylalanine-labeled protein allowed us to assess the subsequent incorporation of casein-derived amino acids into muscle protein. Blood samples were collected at regular intervals, with muscle biopsies obtained prior to and 2 and 6 h after protein plus carbohydrate ingestion. The acute post-prandial rise in plasma glucose and insulin concentrations was significantly greater in the older compared with the younger males. Plasma amino acid concentrations increased rapidly following drink ingestion in both groups. However, plasma leucine concentrations were significantly lower at t = 90 min in the older when compared with the young group ( P   0.05 ). We conclude that the use of dietary protein-derived amino acids for muscle protein synthesis is not impaired in healthy older men following intake of protein plus carbohydrate