Protein recovery from bioethanol stillage by liquid hot water treatment

Journal Pre-proof Protein recovery from bioethanol stillage by liquid hot water treatment Anne Lamp, Martin Kaltschmitt, Oliver Ludtke ¨

PII:

S0896-8446(19)30359-6

DOI:

https://doi.org/10.1016/j.supflu.2019.104624

Reference:

SUPFLU 104624

To appear in: Received Date:

21 June 2019

Revised Date:

5 August 2019

Accepted Date:

4 September 2019

Please cite this article as: Lamp A, Kaltschmitt M, Ludtke ¨ O, Protein recovery from bioethanol stillage by liquid hot water treatment, The Journal of Supercritical Fluids (2019), doi: https://doi.org/10.1016/j.supflu.2019.104624

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Protein recovery from bioethanol stillage by liquid hot water treatment Anne Lampa*, Martin Kaltschmitta, Oliver Lüdtkeb a

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Institute of Environmental Technology and Energy Economics, Hamburg University of Technology, Eißendorfer Straße 40, 20357 Hamburg, Germany. b VERBIO Vereinigte Bioenergie AG, Ritterstraße 23, 04109 Leipzig, Germany. *Corresponding author. Tel.: +49 40 428782809. E-mail address: [email protected] (A. Lamp). Graphical abstract

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Highlights     

Liquid hot water is used to extract proteins from bioethanol stillage The effect of temperature, time and pH is investigated Yields and losses of 16 free and protein-bound amino acids are presented Highest total protein recovery in the hydrolysate is 75 % at 170 °C after 20 min A kinetic model for protein solubilization and amino acid degradation is proposed

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Abstract

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Protein recovery from biogenic residues offers a high potential for increasing the added value. This

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paper investigates the protein recovery from bioethanol stillage by liquid hot water treatment at pH 3 - 11 and 110 - 210 °C for 10 - 90 minutes. The results show that protein solubilization starts

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at 89 °C and is limited by temperature. However, all amino acids except glutamic acid start to

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degrade at 170 °C. Especially lysine, arginine and asparagine undergo Maillard reactions, whereby degradation is favoured by bases; acids show a stabilizing effect. The highest protein yield in the hydrolysate is 75 % with a concentration of 53 %-DM at 170 °C and 20 minutes. In addition,

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kinetic modelling of the rate constants for protein solubilization and amino acid degradation is performed. Rate constants for amino acid degradation are about one order of magnitude higher for

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polar than for non-polar amino acids.

Keywords: Bioethanol stillage; protein recovery; liquid hot water; subcritical water; Maillard reactions; kinetic modelling.

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1 Introduction Due to a strongly growing world population and globally rising living standards, the worldwide protein demand is constantly increasing and will raise by almost 50 % until 2050 compared to today’s protein demand [1]. In parallel, globally available arable land for the production of proteins based on animals and plants is limited and clearly decreasing. As a consequence, there is the ne-

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cessity to find new sources of protein for the food and feed markets. One option is the recovery of

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proteins from low value residues provided from processing of agricultural commodities in the context of integrated biorefineries.

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A typical example for such residue streams is whole stillage from grain-based bioethanol pro-

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duction. Approx. 40 % of the initial grain mass remains in the whole stillage after bioethanol fermentation. Typically, whole stillage has a protein content of 20 to 35 %-DM and a dry matter

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content of 8 to 15 % (depending on grain type and process conditions). The total bioethanol stillage production within the EU was 4.7 Mio. t (dry matter) in 2016 [2]. So far, whole stillage is used as

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low quality animal feed (energy feed) whose price is mainly based on its energy content, or in some cases for biogas production. In order to increase the added value, whole stillage could be further processed into high quality protein products with a relative high and clearly defined protein content for use as protein ingredients in food products for human nutrition or as high-protein ani-

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mal feed. For example, high-protein feed from the total bioethanol stillage produced in the EU could substitute 2.3 to 3.7 Mio. t of imported soybean meal, contributing to 12 to 20 % of the total European protein-rich feed demand in 2018 [3]. So far, a commercial recovery of proteins from whole stillage has not been applied [4]. Several different approaches have been proposed for stillage protein extraction, including aqueous ethanol, alkaline-ethanol and enzyme treatments [5–10]. However, protein extraction from whole stillage 4

by liquid hot water (LHW) treatment has not been reported yet (Table 1). Although more energyintensive, protein extraction by LHW treatment has the advantage that no harmful chemicals or expensive enzymes are required. This paper provides a detailed investigation of protein recovery from bioethanol stillage by LHW treatment at temperatures from 110 to 210 °C. By analyzing the amino acid content in the recovered protein fractions, optimal LHW conditions for maximal yield of each of the 16 amino

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acids are obtained. In addition, kinetic modelling of the rate constants for protein solubilization

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and amino acid degradation is performed.

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2 Theoretical Background

Water is called subcritical water above its boiling point and below its critical point at pressures

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high enough to keep it in the liquid state. Subcritical water is often also referred to as liquid hot water (LHW), hot compressed water or pressurized hot water [11]. Liquid hot water has improved

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solvation properties due to changes of its chemical and physical properties. With increasing temperature, the ion product of water increases and the pH decreases to 6.0 at 110 °C and to 5.6 at 210 °C [12,13]. The result is that the reactivity and the catalyzing effect of water increases. In addition, the relative dielectric constant of water decreases with increasing temperature, thus the

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polarity of waters decreases and becomes similar to methanol and ethanol at ambient conditions [12,14–16]. At temperatures above 180 °C, autohydrolysis occurs, where acetyl residues from hemicellulose are liberated in form of acetic acid and further catalyze chemical reactions [11]. Furthermore, increasing the temperature affects the mass transfer properties of LHW by increasing self-diffusivity and decreasing density, viscosity and surface tension [17–19]. That enables faster mass transfer and improved wetting of the sample [12]. 5

2.1 Protein solubilization by LHW treatment Insoluble proteins have a high surface hydrophobicity and / or large molecular sizes. Reduction of the molecular size by partial hydrolysis of the proteins requires a high activation energy that can be reduced by acid catalysts [19,20]. The mechanism of protein solubilization by LHW is, one the one hand, based on the partial hydrolysis of peptide bounds due to the acid-catalyzing effect

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of LHW. On the other hand, hydrophobic surface areas of insoluble proteins become hydrated when the dielectric constant of LHW decreases. By adding acids or bases during LHW treatment,

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the concentrations of H3O+ or OH− ions are increased by several orders of magnitude which further

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increases the hydrolysis reaction rate [21,22]. Consequently, temperature and time required for complete protein solubilization depend on mass transfer limitations (like substrate structure, par-

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2.2 Amino acid degradation

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ticle size, solid-liquid ratio) and the reaction pH.

Besides protein solubilization, also unwanted reactions are catalyzed in LHW. Above 160 °C, the

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α- or ε-amino groups of amino acids can react by nucleophilic addition with the carbonyl groups of reducing carbohydrates (mainly glucose, fructose, maltose, lactose and pentoses) under formation of glycosylamines. Follow-up reactions of glycosylamines produce a complex mixture of Maillard reaction products [23,24]. Especially lysine and arginine have a high tendency to undergo

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Maillard reactions due to their primary ε-amino groups. They can also be cross-linked by a pentose or hexose to form pentosidine [25]. In addition, the α-amino group of asparagine, once hydrolyzed, can react with reducing carbohydrates under the formation of acrylamide [26]. At temperatures above 250 to 300 °C thermal decomposition of amino acids occurs [21,22,27–32]. The consequences of Maillard reactions are often undesired in food products, such as odor and taste changes due to formation of flavors and volatile compounds (especially bitter substances), 6

color changes due to brown pigment formation (melanoidins), decrease in protein digestibility and nutritional quality (loss of essential amino acids) as well as the formation of toxic substances [24]. Measures to inhibit Maillard reactions include lowering the pH, maintaining temperatures as low as possible, avoiding high concentrations of non-reducing sugars and addition of sulfite [15,24]. Sulfurous acid inhibits the Maillard reaction by adding sulfite to carbonyl groups so that these are

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no longer available for subsequent reactions [23].

2.3 Optimal LHW conditions

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The aim of LHW treatment is to maximize protein solubilization while minimizing degradation

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effects. Therefore, it is important to optimize temperature, time and pH. Due to similar effects of temperature and time, it is common to use the severity factor log R0 in LHW experiments, where t

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is the time in minutes and T is the temperature in °C (equation (2.1)) [33]. Although in some cases

in the literature [33,34].

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a combined severity factor is used, where the pH is considered, these correlations are not consistent

𝑇 − 100 )) 14.75

(2.1)

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𝑙𝑜𝑔(𝑅0 ) = 𝑙𝑜𝑔 𝑡 + 𝑙𝑜𝑔 (𝑒𝑥𝑝⁡(

Table 1 summarizes relevant studies on protein extraction from different biomasses by LHW treatment. It is important to note that many studies overestimate the protein yield in the hydrolysate by measuring only the Kjeldahl total nitrogen content (NH4+-N) instead of the total amount of

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undamaged amino acids, as shown by Reisinger et al. [35]. So far, amino acids yields in the hydrolysate were only measured for free amino acids. The yields are generally marginal [28,36–39]. Yields of total (free and bound) amino acids in the hydrolysate, the solid residue and the degraded fraction have not been reported yet. Furthermore, the influence of pH is not reported consistently in the literature. Abdelmoez et al. [32] found that a high pH stabilizes free leucine, isoleucine, phenylalanine, serine, threonine and 7

histidine against degradation and destabilizes free methionine, tyrosine, lysine and arginine. Li et al. [30] showed that rate constants for degradation of free methionine, phenylalanine, proline, serine and threonine are independent of pH in the range of 3.0 to 8.5 and increase at lower pH. Ajandouz et al. [40] found that lysine degradation was reduced by lowering the pH to 4. Kang et al. [21] showed that lowering the pH to 1.4 stabilizes serine against degradation. In general, protein recovery from stillage by LHW treatment has not been reported yet. Lamool-

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phak et al. [39] investigated LHW treatment of baker’s yeast which has similarities with bioethanol

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stillage. However, the amino acid yields were not evaluated individually and the highest protein

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yield was only 33 %.

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3.1 Materials

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3 Experimental

For this study, whole stillage from an ethanol production plant of VERBIO Vereinigte BioEnergie

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AG, Leipzig, Germany, was used. The coarse particles were removed and thin stillage was obtained by a single-stage decantation of the whole stillage. The particle size of the solids in thin stillage were about 10 µm on average. Thin stillage proteins are mostly insoluble, because grains

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contain a high share of insoluble protein (approx. 50 % in rye and corn and almost 80 % in wheat and barley [24]). In addition, stillage proteins are completely denatured which further decreases protein solubility. In order to obtain the insoluble fraction of the thin stillage, a solid-liquid separation was carried out. The insoluble fraction of thin stillage is referred to as TSI in the following. The TSI was diluted with demineralized water to a dry matter content of 4 %. The pH of the diluted TSI was adjusted to 3.2 with sulfuric acid in order to increase the acid-catalyzing effect on the one

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hand and to reduce amino acid degradation on the other hand. The concentration of H3O+ ions was 0.045 mol/L, which is 4 to 5 orders of magnitude higher than the H3O+ concentration of water at 110 to 210 °C.

3.2 Liquid hot water treatment LHW treatment was done according to Kehili et al. [41]. The reaction was carried out in 45 mL

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stainless steel reactors (High Pressure Reactor BR-25, Berghof, Eningen, Germany). The reaction

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temperature was controlled over external electrical heating jackets. In each reactor vessel, 30 g of diluted TSI was weight into a polytetrafluoroethylene (PTFE) cartridge. Nitrogen was used to

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pressurize the reactor to 50 bar in order to maintain the water in its liquid state. The reactions were

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performed at six different temperatures (110, 130, 150, 170, 190 and 210 °C) and for six different reaction times (10, 20, 30, 45, 60 and 90 min). The time was started as soon as 95 % of the set

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temperature was reached. After the treatment time, the reaction was stopped using an iced water bath. For each sample, the entire reactor content was transferred without losses to a centrifugation

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tube and centrifuged for 30 min at 4757 · g. The solid residue was washed with 30 mL demineralized water and centrifuged a second time for 30 min at 4757 · g. The supernatants were united as hydrolysate. Both hydrolysate and residue were freeze dried and analyzed for their amino acid contents. Each experiment was carried out in duplicate.

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The effect of pH was analyzed by adding NaOH to the sample just before closing the reactor. The corresponding experiments were carried out at pH of 3, 5, 7, 9 and 11 for 10 and 40 minutes at 110, 130, 150 and 170 °C.

3.3 Amino acid analysis Free and peptide-bound amino acids were analyzed together. Sample preparation was done by acid 9

hydrolysis with 6 M HCl for 24 h at 110 °C. After cooling, the sample was neutralized with NaOH to pH 1. Then 2 mL of 20 mM internal standard (L-norvaline) were added and the sample was filled up to 200 mL with 0.1 M HCl and filtered (0.45 μm). Analysis of 16 amino acids was carried out with Agilent 1260 HPLC Series. Pre-column derivatization of primary amino acids was done with o-phthaldialdehyde and 3-mercaptopropionic acid (OPA/MPA), while proline was derivatized with 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl). Amino acid separation was achieved

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using a Poroshell LC HPH-18 column (4.6 x 100 mm, 2.7 µm) and a binary gradient system. The

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polar eluent was an aqueous buffer at pH 8.4 and the organic eluent contained methanol, acetonitrile and water (45/45/10, v/v/v). Detection was done by a variable-wavelength FLD-detector. Ex-

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tinction and emission wavelengths were 338 and 454 nm for OPA/MPA derivates and 266 and

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324 nm for FMOC-Cl derivates, respectively. Amino acid losses during sample pretreatment were

3.4 Calculations

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corrected according to [42]. Total protein was expressed as sum of all amino acids.

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Protein yields 𝑦𝑃,𝑖 in states A, B and C are expressed as total protein mass in state i 𝑚𝑃,𝑖 divided by 𝑚𝑃,𝐴0 , the initial TSI protein mass in the reactor at t = 0 (also referred to as A0) (equation (2.1)). Amino acid yields 𝑦𝐴𝐴,𝑖 are calculated accordingly. 𝑚𝑃,𝑖 𝑚𝑃,𝐴0

(3.1)

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𝑦𝑃,𝑖 (%⁡𝑜𝑓⁡𝐴0 ) =

Protein concentrations 𝑐𝑃,𝑖 in states A, B and C are expressed as protein mass in state i 𝑚𝑃,𝑖

divided by the total dry matter in state i 𝑚𝑖 (equation (2.2)). 𝑐𝑃,𝑖 (𝑤𝑡⁡%-𝐷𝑀) =

𝑚𝑃,𝑖 𝑚𝑖

(3.2)

Amino acid shares 𝑥𝑃,𝑖 in states A, B and C are expressed as amino acid mass in state i 𝑚𝐴𝐴,𝑖

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divided by the total protein mass in state i 𝑚𝑃,𝑖 (equation (2.3)). 𝑥𝑃,𝑖 (%) =

𝑚𝐴𝐴,𝑖 𝑚𝑃,𝑖

(3.3)

Yield limits 𝛽 that can be reached for protein release from A at a given temperature and infinite time are calculated by the empirical equation (3.4). T is the temperature in °C and a, b and T0 are constants that are calculated by least square regression. T0 is the temperature in °C where protein

(𝑇 − 𝑇0 )

(3.4)

√𝑏 + (𝑇 − 𝑇0 )2

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𝛽(%⁡𝑜𝑓⁡𝐴0 ) = 𝑎 ∙

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release starts.

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3.5 Kinetic modelling

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The mechanism of protein solubilization and amino acid degradation can be described according to equation (3.5). Amino acids in state A are bound in the insoluble protein fraction, amino acids

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in state B are bound in the soluble protein fraction and amino acids in state C are degraded. Amino acids can be measured in state A (insoluble residue after LHW) and in state B (hydrolysate). Amino

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acids in the combined states C can be calculated by the mass balance. Differential equations for concentrations of A, B and C can be expressed by equations (3.6), (3.7) and (3.8), respectively. A: Insoluble amino acid k1,B (peptide-bound) k1,C

B: Soluble amino acid (free & peptide-bound)

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C: Degraded amino acid (free & peptide-bound)

(3.5)

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C: Degraded amino acid (peptide-bound)

𝑑[𝐴] = −𝑘1,𝐵 [𝐴]𝑛𝐵 − 𝑘1,𝐶 [𝐴]𝑛𝐶 = −𝑘1 [𝐴]𝑛 𝑑𝑡

(3.6)

𝑑[𝐵] = 𝑘1,𝐵 [𝐴]𝐵 − 𝑘2 [𝐵]𝑚 𝑑𝑡

(3.7)

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𝑑[𝐶] = 𝑘1,𝐶 [𝐴]𝑛𝐶 + 𝑘2 [𝐵]𝑚 𝑑𝑡

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= amino acid yield in state A at time t (in wt-% of 𝐴0 ) = amino acid yield in state B at time t (in wt-% of 𝐴0 ) = amino acid yield in state C at time t (in wt-% of 𝐴0 ) = amino acid yield in state A at t = 0 (𝐴0 = 1) = reaction rate of amino acid release (in mol1− n · Ln−1 · s−1) = reaction rate of amino acid solubilization (in mol1− n · Ln−1 · s−1) = reaction rates of amino acid degradation (in mol1− n · Ln−1 · s−1) = reaction order of amino acid release (-) = reaction order of amino acid solubilization (-) = reaction orders of amino acid degradation (-)

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Where 𝐴(𝑡) 𝐵(𝑡) 𝐶(𝑡) 𝐴0 𝑘1 𝑘1,𝐵 𝑘1,𝐶 , 𝑘2 𝑛 𝑛𝐵 𝑛𝐶 , 𝑚

(3.8)

The reaction order 𝑛 for the amino acid release from the insoluble state (A  B + C) in equation

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(3.6) is not obvious due to the multiple effects involved in solubilization (A  B) and degradation

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(A  C). Most publications only consider the reaction rate for the hydrolysis of free amino acids by first order kinetics [19,22,27,28]. However, apart from hydrolysis, other solvation effects con-

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tribute to the solubilization, and degradation of amino acids in state A needs to be considered as well. Furthermore, the addition of acids or bases are to LHW changes the reaction order [15,43].

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So far, kinetic modelling for the release of total (free and bound) amino acids has not been reported. To determine the reaction order n for protein release from state A in this work, first and second order kinetic modelling were compared, while the data were best represented by second order. Regression of the experimental data of protein yields in state A was achieved by equation (3.9).

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Calculation of k1 was done by linearization of equation (3.9). 𝐴(𝑡) = (1 − 𝛽) +

1 (1 + 𝑘1 ⁡𝑡)

(3.9)

The reaction order 𝑚 for the amino acid degradation (B  C) (3.7) is also not obvious due to the complexity of Maillard reactions. In the literature, most papers on kinetic modelling only consider degradation of free amino acids in the hydrolysate and use first order kinetics [21,22,27–32], 12

though zero and second order are also common [44]. In this work, the degradation of amino acid was best represented by first order reaction. Calculation of A0, k1,B and k2 was done with Matlab by least square regression of the data with equation (3.10). 𝐵(𝑡) =

𝐴0 ⁡𝑘1,𝐵 (𝑒 −𝑘2 𝑡 − 𝑒 −𝑘1,𝐵𝑡 ) 𝑘1,𝐵 − 𝑘2

(3.10)

Calculation of activation energies for protein release 𝐸𝐴,1 and amino acid degradation 𝐸𝐴,2 was

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done by linearization of the Arrhenius equation (3.11), where 𝐴 is the pre-exponential factor and 𝑅 is the ideal gas constant.

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𝐸𝐴

(3.11)

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𝑘 = 𝐴⁡𝑒 −𝑅𝑇

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4 Results and Discussion

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The total protein content of untreated thin stillage insolubles (TSI) is 60 %-DM and the share of essential amino acids is 30 %. The residual TSI components are lignin, hemicellulose, starch and

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fats with 14, 10, 8 and 8 %-DM, respectively. In the following sections, the effect of temperature, time and pH during LHW treatment of TSI on the protein and amino acid recoveries are presented. Furthermore, kinetic model results are shown.

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4.1 Total protein recovery

Protein yield. Results for LHW treatment of TSI with varying times and temperatures are shown in Figure 1. Experimental data of protein yields are presented as points in the states A (solid residue), B (hydrolysate) and C (degraded). The results in Figure 1A indicate that the velocity of protein release from TSI is very fast. Most of the protein is released within the first 10 minutes. That shows that the solid to liquid ratio is 13

small enough and mass transfer limitations do not occur. Furthermore, the results confirm that the stillage proteins are not bound anymore to the grain matrix; if the latter would be the case the protein release would be significantly slower (e.g., compared to results found with wheat bran [45]). After ca. 60 minutes, the protein release does not change anymore with time and reaches a constant value. Lamoolphak et al. [39] showed similar results with protein release from baker’s yeast. This temperature limitation of the protein release from TSI can have two reasons. Firstly,

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the protein solubility is influenced by the decreasing polarity of LHW which is constant for each

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temperature. Secondly, activation energies of hydrolysis are not equal for all peptide bounds (see section 4.3). Thus different temperatures are required to crack the various peptide bounds. The

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yield limits for protein release from TSI that can be reached at a given temperature are calculated

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according to equation (3.4). The results are shown in Figure 2. It can be seen that protein release starts at 89 °C and that a complete release can be reached at 230 °C after 90 minutes. The literature

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offers a wide range of results on the temperature and time required for complete protein solubilization. For example, Zetzl et al. [36,45]. reported 48 % and 65 % protein solubilization from wheat

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bran and brewer’s spent grains, respectively, at 210 °C for at 220 minutes. In contrast, 100 % protein solubilization from rice bran at 200 °C for 30 min was reported by Sereewatthanawut et al. [46].

Figure 1B shows that the protein yield in the hydrolysate (B) increases with temperature and

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time until 150 °C. Again, the protein yield reaches a plateau after ca. 30 to 60 minutes. At 170 °C and above, degradation of amino acids can be detected and the protein yield decreases with time. The degradation rate increases with temperature. The highest total protein recovery in the hydrolysate (B) is 75 % at 170 °C and 20 min (severity factor 3.4). This is in good agreement with Watchararuji et al. [47] who reached a maximum total protein yield in the hydrolysate of 75 % after LHW treatment of rice bran at severity factor 5.0. Other authors like Lamoolphak et al. [39], 14

Vegas et al. [48] and Reisinger et al. [35] reached only 33 % at severity factor 5.7 (baker’s yeast), 51 % at severity factor 3.8 (rice husks) and 49 % at severity factor 3.4 (wheat bran), respectively. The reason for the lower yields could be that these proteins were still bound in the cell structure and require a higher severity factor to be released, where amino acid degradation already begins. Figure 1C shows that significant protein losses of more than 10 % of TSI protein start to occur at 190 °C. One reason for this is that the hydrolysis of hemicellulose starts at 180 °C and releases

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free carbonyl groups for the Maillard reaction. The maximum protein loss at 210 °C and 90 min is

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38 % of TSI protein.

By plotting the protein yields against the severity factor (Figure 3), three sections can be ob-

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served:

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1. At severity factors from 1 to 2.2, the protein yield in the hydrolysate (B) is relatively low and increases only slightly with a rising severity factor. The reason might be that the activation

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energy for protein hydrolysis is too high at low severity factors. The slight increase in solubilized protein might only result from the decreasing dielectric constant of water.

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2. In the range of severity factors between 2.2 and 3.3, the protein yield increases fast with rising severity factor. This is probably because in this range the activation energies for hydrolysis are low enough.

3. At severity factor 3.3, the protein yield in the hydrolysate (B) and the yield of released protein

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(1-A) start to differ. That indicates the start of amino acid degradation in the hydrolysate. Thus, that at higher severity factors, the protein yield in the hydrolysate decreases. The yield of released protein (1-A) also shows a reduced increase at severity factors above 3.3. This indicates that the degradation of amino acids also occurs in the insoluble state (A), although not nearly as strong as in the dissolved state (B).

From Figure 3, conclusions for the optimal process conditions can be made. The highest protein 15

yields in the hydrolysate (B) can be achieved at severity factors between 3.3 and 3.8.

Protein concentration. Figure 4 shows the protein concentration in the solid residue (A) and in the hydrolysate (B). The decreasing protein concentration in the solid residue (A) shows that, with increasing severity factor, protein solubilization is favored over the solubilization of other TSI components.

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The protein concentration in the hydrolysate (B) shows the same three sections as the protein

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yield in Figure 3 and increases to about 53 % at a severity factor of 3.0. At higher severity factors (above 3.0), the protein concentration in the hydrolysate stays constant, although the protein yield

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decreases. The reason is that both proteins and other TSI components form volatile degradation

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products entering the vapor phase and thus reducing the total dry mass in the reactor. Figure 5 shows that the total dry mass in the reactor begins to decrease at a severity factor of 3.3 and leads

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to a total mass loss of 25 % at a severity factor of 5.4. Hundreds of volatile products of the Maillard reaction (e.g., pyrroles, pyridines, imidazoles, pyrazines, oxasoles, thiazoles or aldol condensation

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products) have been identified in the literature, as reviewed by [49]. The increase in volatile degradation products could also be smelled during pressure release after LHW treatment producing a typical coffee-like aroma. As a consequence, the optimal severity factor for maximal protein con-

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centration in the hydrolysate is 3.0 to 5.4.

4.2 Amino acid recovery Amino acid yield. In contrast to most studies in the literature, where only free amino acids are considered, the total (free and bound) amino acid contents of the samples were analyzed in this study. That enables the detection of changes in the amino acid profile of the recovered proteins. Figure 6 shows the summarized yields of 16 amino acids in the hydrolysate as a function of the 16

severity factor (individual results for each amino acid for the yields in the solid residue, hydrolysate and degraded fraction can be found in the supplementary material). The amino acids show a similar behavior up to a severity factor of 3.3, where yields begin to differ. 

Significant losses of the polar amino acids histidine, lysine, serine, arginine, threonine and asparagine can be observed. Yield loss rates can be estimated with 34 to 53 wt-% of A0 per severity unit. At the same time, the formation of brown pigments and odorous substances be-

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gins at 170 °C. All polar amino acid side groups are reported to be involved in Maillard reac-

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tions [23,40,44]. These findings are in good agreement with the literature, e. g., Reisinger et al. [35] reported losses of lysine, arginine and asparagine to start at 160 °C at severity factors

For the non-polar amino acids proline, glycine, tyrosine, methionine, leucine, valine, phenyl-

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

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of 3.1, 2.8 and 3.2, respectively.

alanine and isoleucine, only small loss rates of 17 to 21 wt-% of A0 per severity unit were

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detected. Most likely, these losses are less due to reactions of the side groups than to reactions of the released α-amino groups after hydrolysis. The yield of alanine strongly increases at a severity factor of 4.7 and above. It can be assumed

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

that this is caused by the degradation of other amino acids at 210 °C under the formation of alanine. 

The most stable amino acid is glutamic acid; no significant degradation can be detected until

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210 °C (it can be assumed that glutamine is completely converted into glutamic acid during LHW treatment). The reason could be that glutamic acid is the only amino acid with a carboxyl group which has a low reactivity in an acidic aqueous solution.

Table 2 shows the optimal LHW conditions for the highest recovery of the 16 amino acids in the hydrolysate. Process conditions need to be chosen depending on the target application of the recovered protein. 17

In addition, the effect of pH on the amino acid yields in the hydrolysate was investigated from pH 3 to 11 (individual results for each amino acid for the yields in the hydrolysate can be found in the supplementary material). In general, the results indicate that protein solubilization is least effective at pH 7; with increasing acid or base concentrations the amino acid yields in the hydrolysate increase. It can also be seen that at low temperatures, bases are much more effective in protein solubilization than acids. That is evident, since alkaline extraction is a common method for protein

of

solubilization, which is usually carried out at lower temperatures. At higher temperatures and

ro

times, especially lysine, arginine, serine and threonine are stabilized by acids and destabilized in

-p

alkaline conditions. That confirms the findings of Ajandouz et al. [40] and Kang et al. [21].

re

Amino acid share. The distribution of all 16 amino acids in the hydrolysate is shown in Figure 7. Only the relative amount of glutamic acid increases significantly with growing severity factor and

lP

reaches 51 % at a severity factor of 5.2. The proportion of non-polar amino acids remains almost constant, and the summed share of polar amino acids is reduced to less than 5 %. For food and

ur na

feed applications, a high yield and share of essential amino acids is required. The highest yield and share of essential amino acids in the hydrolysate are 69 % and 28 %, respectively, at a severity factor of 3.4. This is almost as much as the share of essential amino acids in TSI. Apart from lysine, the share of all essential amino acids exceed the limits that are recommended for food applications

Jo

by the WHO [50]. The relative lysine content, which is recommended to be 4.5, is 3.0 in the hydrolysate.

4.3 Kinetic modelling Predicted model results for protein release and degradation are shown as lines Figure 1. They are in good correspondence with the actual experimental data points. 18

Protein release. Figure 8 shows the rate constants k1 and the corresponding activation energy EA,1 for the total protein release over time. They are in good agreement with second order kinetics. The corresponding Arrhenius plot has an S-shape. Non-linear Arrhenius plots indicate a change in the reaction order. The reason is that k1 is the sum of protein solubilization (A  B, rate constant k1,B) and protein degradation (A  C, rate constant k1,C). Protein solubilization occurs at all tem-

at 170 °C. That changes the reaction order according to equation (3.6).

of

peratures, whereas degradation starts at 170 °C. Thus, k1,C is zero until 150 °C and starts to increase

ro

Table 3 shows the activation energies EA,1 calculated for each amino acid from the Arrhenius plots of k1. The results show that activation energies for the release of non-polar amino acids are

-p

lower than for polar amino acids. The activation energy for the release of total protein is with

re

61 kJ/mol between the values for polar and non-polar amino acids. Due to the reduced dielectric constant, water interacts more strongly with less polar amino acid side chains, which is why their

lP

peptide bonds are preferentially hydrolyzed with increasing temperature. This is also shown by the

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onset temperatures T0, which are averagely higher for polar amino acids than for nonpolar ones.

Amino acid degradation. For degradation of amino acids, rate constants k2 and activation energies EA,2 that were modelled by first order kinetics according to equations (3.10) and (3.11) are shown in Table 4. The results confirm the findings outlined above. For alanine and glutamic acid,

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k2 is zero. For lysine and arginine, a slight degradation already starts at 150 °C. In general, for the polar amino acids, k2 is about one order of magnitude higher that for the non-polar amino acids.

19

5 Conclusion and Outlook The aim of this study was to investigate liquid hot water treatment of thin stillage in order to extract proteins with high yield, high concentration and high share of essential amino acids. The challenge is to maximize protein solubilization while minimizing degradation effects. The results show that in the severity factor range of 3.3 – 3.8, 75 % of thin stillage proteins can be recovered in the

of

hydrolysate without significant losses of instable amino acids. Further investigations on the down-

ro

stream processes are required. Once separated, the proteins could be used as high-protein feed in or as protein ingredient in food products. In the search for a target application of the protein ob-

re

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tained, other reaction products, browning and the coffee-like odor must be taken into account.

[1]

lP

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26

6 Tables Table 1: Literature review of protein and amino acid recovery by LHW treatment.

Tempera- Time ture (°C) (min)

pH

Analysis

Calculations

Ref.

Free AA

230 – 290

2.5 – 40

2.5 - 10.5

17 free AA

EA and k2 (first order)

[32]

Free AA

250 – 450

0 - 0.5

Natural

Free Ala and Gly

Yield of protein release from A and yields in B; EA, k2 and reaction order

[31]

Free AA

270 – 340

0 – 0.5

1.5 – 8.5

Free His, Phe, Ser, Thr, Pro and Met

EA and k2 (first order)

[30]

Free AA

200 – 340

of

Protein source

Natural

Free Ala, Leu, Phe, Ser and Asp

EA and k2 (first order)

[29]

5 - 30

Natural

Protein (NH4+-N) and total free AA

Yield in B

[39]

Brewer’s spent grain

60 – 210

15 – 220

Natural

Protein (NH4+-N)

BSA

250 – 320

0–3

Natural & Free AA acidified

Corn fiber

160

20

4

Protein (NH4+-N) +-N)

ro

0–3

Baker’s yeast 100 – 250

Yield of protein release from A

[36]

-p

Yield in B; EA, k1 and k2 (first or- [22] der) Concentration in A

[51]

Concentration in A

[52]

160

20

4

Protein (NH4

Fish

180 – 320

5 – 60

Natural

Free AA

EA, k1 and reaction order n

[37]

Fish meat

240 – 270

0 – 60

Natural

Free Ala, Gly and Cys

EA, k1 and k2 (first order)

[28]

Food waste

100 – 200

5 – 30

Natural

Protein (NH4+-N)

Concentration in B

[53]

Hog hair

250

10 – 360

Natural

Free AA

EA, k1 and k2 (zeroth-first order)

[27]

Rice bran

100 – 220

5 – 30

Natural

Protein (NH4+-N), free AA

Yield of protein release from A, free AA yield in B

[46]

Rice bran

120 – 150

1

Natural

Protein (NH4+-N)

lP

re

DDGS

[54]

ur na

Yield and concentration in B

+-N)

Rice bran, 200 – 220 soybean meal

10–30

Natural

Protein (NH4

Yield in B

[47]

Rice husk

180

15 – 40

Natural

Protein (NH4+-N)

Yield in B

[48]

200 – 300

2 - 62

1.4 - 13.2

Free Asp, Ala, Gly and Ser

Yields in B

[21]

160

50 – 190

6.54

Protein (NH4

Yield in B

[55]

140 – 200

10 – 30

Natural

Protein (NH4+-N), total and free AA, degradation products

Yields in B

[35]

2.3 – 3.9

Total Arg, Asp and Lys

Yield in B

[34] [45]

Silk fibroin Waste water sludge

Jo

Wheat bran

+-N)

Wheat bran

120 – 180

5 – 10

Wheat bran

60 – 210

15 – 220

Natural

Protein (NH4

Protein release from A

Thin stillage

110 – 210

10 – 90

3 - 11

Total amount of 16 AA

Yields and concentrations in A, B this and C; EA, k1 and k2 for protein work and 16 AA (first & second order)

+-N)

Abbreviations: Solid residue (A), hydrolysate (B), amino acids (AA), activation energy (EA), rate constant of protein release (k1), rate constant of protein degradation (k2), protein content calculated from total nitrogen by Kjeldahl (NH4+-N).

27

Table 2: Severity factors and corresponding temperatures and times for highest yields of amino acids in the hydrolysate (B).

Max. yield in Severity Tempera- Time hydrolysate factor ture (in wt-% of A0) (log R0) (°C) (min) 82 %

5.2

210

90

Glu

95 %

5.0

210

60

Met*

71 %

4.2

210

10

Tyr

71 %

4.0

170

90

Pro

84 %

3.8

170

60

Leu*

68 %

3.8

170

45

Gly

75 %

3.7

170

45

Phe*

75 %

3.7

170

45

Asp

73 %

3.4

170

20

His

80 %

3.4

170

20

Thr*

71 %

3.4

170

Val*

66 %

3.4

170

Ile*

70 %

3.4

170

Lys*

67 %

3.4

Ser

69 %

3.3

Arg

66 %

2.9

Total*

69 %

Total

75 %

ro

Ala

of

Amino acid

20 20

-p

20 20

150

60

150

30

3.4

170

20

3.4

170

20

re

170

Jo

ur na

lP

*Essential amino acids

28

Table 3: Activation energies EA,1 and pre-exponential factors ln(A) for the protein release, calculated from the rate constants k1,S for each amino acid. T0 is the temperature where protein release starts, calculated from equation (3.4). Values for R2

EA,1 (kJ/mol) Non-polar amino acids Tyr 41

ln A (-)

R2 (-)

T0 (°C)

7

0.72

74.8

44

8

0.84

83.7

Phe

47

9

0.86

88.7

Ile

48

9

0.86

84.6

Leu

49

9

0.87

87.8

Ala

49

10

0.85

88.2

Gly

49

10

0.87

87.7

Pro

56

12

0.94

85.4

Met

59

13

0.94

Polar amino acids Lys 62

14

0.93

65

15

Thr

77

18

Glu

81

20

Ser

86 105 61

84.6

0.89

87.8

0.95

87.3

0.91

89.4

21

0.97

94.9

27

0.99

96.6

14

0.95

88.8

Jo

ur na

Total protein

0.98

re

87

Asp

98.0

21

lP

Arg

85.8

-p

His

ro

Val

of

represent the respective correlation coefficients for the activation energies EA,1.

29

Table 4: Reaction rate constants k2, activation energies EA,2 and pre-exponential factors ln(A) for protein degradation. Values for

Amino acid

Reaction rate constants for amino acid degradation (k2 · 103 min-1) 110 °C

130 °C

Glu

0

0

0

0

0

0

Ala

0

0

0

0

0

0

Tyr

0

0

0

0

0

1.96

Met

0

0

0

0

1.69

4.13

Pro

0

0

0

0

1.73

4.53

Gly

0

0

0

0

2.92

3.03

Leu

0

0

0

0.53

2.60

5.13

101

Phe

0

0

0

0.55

1.86

4.68

96

Val

0

0

0

0.98

2.19

4.15

64

of

R2 represent the respective correlation coefficients for the activation energies EA,2.

Ile

0

0

0

1.20

1.73

3.89

Ser

0

0

0

1.25

12.25

His

0

0

0

1.79

Thr

0

0

0

3.26

Asp

0

0

0

11.70

Lys

0

0

0.76

Arg

0

0

1.33

170 °C

190 °C

EA,2 (kJ/mol)

210 °C

ln(A) (-)

R2 (-)

0.96

18.5

1.00

10.5

1.00

52

7.3

0.94

31.92

145

32.8

0.96

7.34

17.97

103

21.7

0.99

13.48

36.30

108

23.5

0.99

44.36

106.33

98

22.3

0.99

7.23

19.93

29.66

103

22.6

0.91

10.00

33.61

55.41

106

23.9

0.95

re

-p

ro

20.1

Jo

ur na

lP

150 °C

30

of

ro

-p

re

lP

ur na

Jo Figures

31

Figure 1:

Experimental and model results of LHW treatment of TSI at varying times and tem-

peratures. Total protein yields (in wt-% of protein in A0) in the solid residue (A), in the hydrolysate (B) and degraded (C). The experimental data are presented as points and the model results as

Jo

ur na

lP

re

-p

ro

of

lines.

32

of ro -p re lP ur na Jo Figure 2:

Protein yield limit β (in wt-% of A0) that can be released from TSI at a given temper-

ature and infinite time.

33

of ro -p re lP ur na Jo Figure 3:

Protein yields (in wt-% of A0) released from the solid phase (1-A) vs. protein yields

recovered in the hydrolysate (B).

34

Protein concentration (wt-%-DM)

70% 60% 50% 40% 30% 20%

Solid residue (A)

10%

Hydrolysate (B) 0% 1

2

3

4

5

Jo

(B).

Protein concentrations (in wt-%-DM) in the solid residue (A) and in the hydrolysate

ur na

Figure 4:

lP

re

-p

ro

of

Severity factor (log R0)

35

Protein concentration (wt-%-DM)

70% 60% 50% 40% 30% 20%

Solid residue (A)

10%

Hydrolysate (B) 0% 1

2

3

4

5

Jo

verity.

Total mass (in g) in the reactor before and after LHW treatment as a function of se-

ur na

Figure 5:

lP

re

-p

ro

of

Severity factor (log R0)

36

Glu Ala

90%

Pro 80%

Gly

70%

Tyr Met

60%

Phe Ile

50%

Val Leu

40%

His 30%

Lys Ser

20%

Arg 10%

Thr

of

Amino acid yield in hydrolysate (wt-% of A0)

100%

Asp

0% 2

3 4 Severity factor (log R0)

5

6

ur na

Amino acid yields (in wt-% of A0) in the hydrolysate (B).

Jo

Figure 6:

lP

re

-p

ro

1

37

Glu Ala

90%

Pro 80%

Gly

70%

Tyr Met

60%

Phe Ile

50%

Val Leu

40%

His 30%

Lys Ser

20%

Arg 10%

Thr

of

Amino acid yield in hydrolysate (wt-% of A0)

100%

Asp

0% 2

3 4 Severity factor (log R0)

5

6

Amino acid distribution in hydrolysate (B) (in wt-% of total amino acids) as a func-

ur na

Figure 7:

lP

re

-p

ro

1

Jo

tion of severity factor (*essential amino acids).

38

of ro -p re lP ur na Jo Figure 8:

Kinetic modelling of total protein solubilization. Determination of rate constants

(left) and Arrhenius plot (right).

39

Supplementary data of this work can be found in the online version of the paper: 1. Experimental and model results of liquid hot water treatment of thin stillage insolubles (TSI) at varying times and temperatures. Amino acid yields (in wt-% of initial amino acids in TSI) are shown in the solid residue (A), in the hydrolysate (B) and degraded (C). 2. Experimental results of the influence of pH during liquid hot water treatment of thin stil-

Jo

ur na

lP

re

-p

ro

of

lage insolubles (TSI) on the yields of 16 amino acids in the hydrolysate.

40