Internship Report Chemical Engineering - IHOG · 2019-10-08 · Internship Report Chemical...
Transcript of Internship Report Chemical Engineering - IHOG · 2019-10-08 · Internship Report Chemical...
Internship Report Chemical Engineering
The unexpected synergy between potato and hemp
proteins
A study on functional properties of blends from potato and hemp proteins
produced by Avebe and HempFlax
Frederike Gerda Hiltje Klein
9 August 2019
Student number First Assessor
S3241653 Prof. Dr. Ir. H.J. Heeres
Course code Second Assessor
CHTR-10 Dr. P.J. Deuss
Company Company supervisor
Avebe Dr. A.A.C.M Oudhuis
Department
Application Development Centre
Confidential
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Management summary
The aim of this study was to investigate the techno-functional properties of potato/hemp
protein blends, in order to evaluate the commercial potential for combining the protein
streams from Avebe and HempFlax.
Hemp protein meal (HPM) from HempFlax contains 44.7% protein, has low protein solubility
and is not able to form foams, gels or emulsions. Blends of potato protein isolate Solanic 200
(S200) and HPM were not able to form sufficient foams or gels, but they did form stable
emulsions when 60% of oil was used. The emulsions were evaluated on stability, viscosity,
droplet size distribution and emulsifying capacity as a function of the influence of pH, salt
concentration, protein concentration and S200/HPM protein ratio.
From the results it is concluded that up to 85% of the S200 protein can be replaced by protein
from HPM in emulsions with high oil content, while retaining the properties of S200
emulsions.
Since HPM has good nutritional properties and is less expensive then S200 it is recommended
to test S200/HPM blends in emulsion applications such as meat analogues and vegan
mayonnaise.
Abstract
Many food applications use egg protein for its excellent functional properties such as
solubility, foaming, gelling and emulsification. A current challenge in the food industry is to
match these excellent properties with more sustainable plant-based proteins. Two companies
in the province of Groningen, Avebe and HempFlax, produce potato and hemp proteins as a
side stream. In this study the functional properties of both types proteins and blends thereof
were evaluated. A literature research on hemp protein techno-functionality was conducted in
combination with experimental research on the functionality of potato protein isolate Solanic
200 (S200) and hemp protein meal (HPM) blends. S200 contains 93% native potato protein
and has good functional properties in the neutral pH range. For HPM a protein content of
44.7% was determined with Kjeldhal analysis. HPM has low protein solubility and is not able
to form foams, gels or emulsions. Blends of S200 and HPM were not able to form
homogeneous foams or gels, but they did form stable emulsions when 60% of oil was used.
Emulsions with 10% or 30% oil were not stable. The emulsions were evaluated on stability,
viscosity, droplet size distribution and emulsifying capacity. The influence of pH, salt
concentration, protein concentration and S200/HPM protein ratio were tested. The functional
properties improved with the inclusion of NaCl salt in HMP formulations and when proteins
were dissolved at higher pH. The presence of salt and the increased pH can both aid in the
solubilization of the hemp protein. Tests with different S200/HPM protein ratios showed that
in emulsions with 60% oil content up to 85% of the S200 protein can be replaced by protein
from HPM, while obtaining similar properties to S200 emulsions. This synergistic effect
might arise from the presence of lipase in S200, which could hydrolyse the oil in HPM to
fatty acids. This hypothesis was tested with a free fatty acid titration experiment, but the
results were indecisive. Since HPM has good nutritional properties and is less expensive then
S200 the blends can be used to improve food emulsion applications such as vegan sauces and
meat analogues.
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Abbreviations
cP Centipoise (unit of dynamic viscosity)
DIAAS Digestible indispensable amino acid score
FAO Food and Agriculture Organization
HPC Hemp protein concentrate
HPI Hemp protein isolate
HPM Hemp protein meal
kDa kilo Dalton
OHC Oil holding capacity
rpm Rotations per minute
S100 Solanic 100 (potato protein isolate)
S200 Solanic 200 (potato protein isolate)
S200 Solanic 300 (potato protein isolate)
THC Tetrahydrocannabinol
WHC Water holding capacity
WHO World Health Organization
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Table of contents
Management summary ............................................................................................................... 2
Abstract ...................................................................................................................................... 2
Abbreviations ............................................................................................................................. 3
Table of contents ........................................................................................................................ 4
1. Introduction ............................................................................................................................ 6 1.1 The potential of potato proteins ................................................................................................. 6 1.2 The potential of hemp proteins .................................................................................................. 7 1.3 Research scope .............................................................................................................................. 9
2. Literature research on hemp protein techno-functionality ..................................................... 9 2.1 Solubility ....................................................................................................................................... 9 2.2 Foaming ...................................................................................................................................... 10 2.3 Gelling ......................................................................................................................................... 10 2.4 Emulsification ............................................................................................................................. 11 2.5 Water holding capacity and oil holding capacity ........................................................................ 12
3. Experimental ........................................................................................................................ 13 3.1 Materials ..................................................................................................................................... 13 3.2 Protein content of HPM: Kjeldhal protein analysis .................................................................... 13 3.3 Protein solubility of HPM: Kjeldhal protein analysis ................................................................. 13 3.4 Water holding capacity and oil holding capacity ........................................................................ 14 3.5 Preparation of emulsions ............................................................................................................. 14 3.6 Emulsion droplet size distribution .............................................................................................. 14 3.7 Viscosity analysis ........................................................................................................................ 15 3.8 Emulsion stability test with centrifuge ........................................................................................ 15 3.9 Emulsifying capacity measurement ............................................................................................ 15
4. Results and discussion .......................................................................................................... 16 4.1 Kjeldhal protein analysis ............................................................................................................. 16 4.2 Water holding capacity and oil holding capacity ........................................................................ 17 4.3 Emulsion droplet size distribution analysis ................................................................................. 17
4.3.1 Effect of pH ......................................................................................................................... 17 4.3.2 Effect of protein concentration ............................................................................................ 18 4.3.3 Effect of NaCl concentration ............................................................................................... 18 4.3.4 Effect of S200/HPM protein ratio ........................................................................................ 19
4.4 Viscosity analysis ........................................................................................................................ 19 4.4.1 Effect of pH ......................................................................................................................... 20 4.4.2 Effect of protein concentration ............................................................................................ 20 4.4.3 Effect of NaCl concentration ............................................................................................... 20 4.4.4 Effect of S200/HPM protein ratio ........................................................................................ 21
4.5 Emulsion stability test with centrifuge ........................................................................................ 22 4.5.1 Effect of pH ......................................................................................................................... 22 4.5.2 Effect of protein concentration ............................................................................................ 22 4.5.3 Effect of NaCl concentration ............................................................................................... 23 4.5.4 Effect of S200/HPM protein ratio ........................................................................................ 23
4.6 Emulsifying capacity ................................................................................................................... 23
5. Conclusion ............................................................................................................................ 24
6. Recommendations ................................................................................................................ 26
7. References ............................................................................................................................ 27
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Appendix I: Results orientating experiments ........................................................................... 28 Gelling properties HPM .................................................................................................................... 28 Foaming properties HPM .................................................................................................................. 28 Emulsification properties HPM......................................................................................................... 29
Appendix II: Particle size distribution diagrams ...................................................................... 30
Appendix III: Viscosity measurements .................................................................................... 31
Appendix IV: Centrifuge tests .................................................................................................. 33
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1. Introduction
Currently the food industry uses large quantities of egg protein for food applications because
of its excellent solubility, foaming, gelling and emulsification properties. It would be more
sustainable to replace the egg protein with plant-based protein, but it is difficult to match the
excellent properties of egg protein. In the province of Groningen there are two companies,
Avebe and HempFlax, which both produce plant proteins as a side stream. Combining both
streams can possibly expand commercial applications of potato and hemp protein.
Combinations of potato and hemp protein might have additional properties and/or
functionalities, which can be of interest for the food industry.
1.1 The potential of potato proteins
Avebe produces 35.000 MT of protein per year from the tubers of the potato plant (Solanum
tuberosum L.), which is equivalent to 4 billion eggs. This corresponds to approximately 28%
of the eggs annually produced in the Netherlands (FAOSTAT, 2016). There are several
protein isolation methods available such as coagulation, precipitation, adsorption and
filtration. The three main potato protein isolate products at Avebe that are intended for the
food industry are Solanic 100, 200 and 300. Solanic 100 (S100) is a pure potato protein
isolate (80%) and is obtained by heat coagulation (104-108 °C). Due to this heat treatment the
proteins are denatured, resulting in a protein product that has no functionality and is mainly
applied for nutrition. Examples are the protein enrichment in sports nutrition, snacks, cereals
and bakery products. S100 is a high quality protein product with a Digestible Indispensable
Amino Acid Score (DIAAS) of 103%, indicating excellent protein quality. The DIAAS is
based on essential amino acid requirements of humans and their ability to digest them (true
ileal digestibility) as individual nutrients. S100 is nutritionally more valuable than other
vegetable proteins (Figure 1).
Figure 1: Protein qualities assessed by Digestible Indispensable Amino Acid Score (DIAAS). The lines indicate the threshold
levels for protein quality that were established by the FAO (Avebe, 2015)
Solanic 200 (S200) and Solanic 300 (S300) are protein fractions that are isolated by
adsorption (chromatography). These are native proteins that maintain their functionality and
are applied to improve the texture of food products. The main protein in S200 is patatin, a
glycoprotein with a molecular weight from 40 to 45 kDa (Singh & Kaur, 2016). S200 has
good solubility, and therefore functional properties, in the neutral pH range. (Figure 2)
Applications include gluten replacement and vegetarian/vegan meat and cheese substitutes.
S300 is mainly composed of protease inhibitors, which have molecular weights ranging from
Innovation by nature
Protein quality: DIAAS
Values are based on ‘Child’ AA scoring pattern (Table 5) - as recommended by FAO experts - FAO paper No.92 (2013)
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5 to 25 kDa. Protease inhibitors inhibit the function of proteases, which are enzymes that help
breaking down proteins. S300 has good functionality in the acidic pH range. (Figure 2)
Applications include egg replacement in sauces and dressings, gelatine replacement in
confectionery products, gluten replacement and vegan dairy product analogues.
Figure 2: Solubility of S200 and 300 as function of pH
A great advantage of Solanic products is that they do not need allergen labelling like soy and
milk products. In addition they are suitable for vegan and animal-friendly claims, but most
importantly the Solanic proteins are more sustainable then animal proteins. S100 is offered to
the food industry in the same price range as casein. S200 and 300 are more valuable and
offered in the price range of whey proteins.
1.2 The potential of hemp proteins
Hemp proteins are obtained from industrial hemp (Cannabis sativa L.), which is a variety that
is mainly used for the fibres and contains almost no tetrahydrocannabinol (THC) (< 0.2%).
Hemp can grow in similar climates as potato, but the roots of the hemp plant reach much
deeper into the ground, therefore hemp grows better during dry summers compared to potato.
Similar to potato proteins, hemp proteins are not listed as priority allergens that require
labelling. Both hemp seed protein products, meal and isolate, could serve as alternative
protein ingredients to the priority allergens such as soybean and pea proteins, which do
require labelling (Malomo, He, & Aluko, 2014).
Albumin, a globular protein with an estimated molecular weight range from 10 to 42 kDa, and
edestin, a legumin with an estimated molecular weight range between 6 and 35 kDa, are the
two main proteins in hempseed (Malomo & Aluko, 2015a). Edestin accounts for 60-80% of
the total protein content (Galasso, Russo, Mapelli, Ponzoni, Brambilla, Battelli, et al., 2016).
Hempseed protein isolates have been reported to have nutritional values comparable to egg
white and soybean proteins (Callaway, 2004). However, the DIAAS of hemp protein meal
(HPM) is only 59%, with lysine as limiting amino acid (Herreman, Tarazanova, & Wilbrink,
2018). This is relatively low compared to the 103% of Solanic 100, but a combination of 50%
HPM and 50% Solanic 100 would result in a DIAAS of 100%. A mixture could thus result
into a product with excellent protein quality.
Hemp protein isolate (HPI) has superior essential amino acid composition, and most of
essential amino acids are sufficient for the FAO/WHO suggested requirements of infants or
children. However, it shows much poorer protein solubility, emulsifying activities and water
holding capacity compared to soy protein isolate (Tang, Ten, Wang, & Yang, 2006).
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Hemp protein can be isolated from HPM that is obtained by milling and sieving of hemp seed
press cake, which is composed of the biomass, proteins and fibres leftovers from the hemp
seeds cold pressing process (45-48°C) that is used to obtain hemp seed oil. The protein meal
from HempFlax contains approximately 50% protein (Table 1) and is already on the food
market as ingredient for protein shakes (Figure 3). This hemp protein shake retails for
approximately 20 euros per bag of 500 g. The bulk price for HPM from HempFlax is
approximately 10 euros per kg.
Table 1: Composition of Hemp Protein Meal from HempFlax
Protein 49.2%
Fibres 20.8%
Saturated fat 1.2%
Mono unsaturated fat 1.2%
Poly unsaturated fat 8.1%
Total fat 11%
Sugar 7.5%
Starch 2.6%
Total carbohydrates 10%
Salt 0.9%
Calcium 0.140%
Iron 0.018%
Phosphorus 1.420%
Magnesium 0.650%
Potassium 1.28%
Total minerals 3.51 %
Total 94.2%
There are different techniques available for the isolation of hemp protein. The traditional
method of protein isolation is isoelectric precipitation: the proteins are dissolved and the pH
of the protein solution is adjusted to the isoelectric point, whereupon they start to agglomerate
and precipitate. This technique is simple but it damages protein functionality and reduces the
performance of the proteins as an ingredient below the required level for high quality food
products manufacture (Malomo & Aluko, 2015b). More sophisticated methods are ultra- and
diafiltration, where membranes are used to separate the proteins from other compounds.
Optionally enzymes such as cellulase, hemicellulase, xylanase, and phytase can first digest
the non-protein compounds (Malomo & Aluko, 2015b). Next ultrafiltration/diafiltration can
be applied to separate the proteins from the digested fibre and phytate fragments. Target
proteins will remain in the retentate while the digested non-protein compounds are removed
via the permeate (Scheme 1).
Scheme 1: Overview of a diafiltration process
Figure 3: Hemp protein meal obtained
from HempFlax
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Implementing protein isolation is an extra step, which is preferably avoided due to extra
investment costs. Applying the HPM directly would be more efficient and would also broad
the nutritional profile of the final food application due to the presence of fibres, fats and
minerals.
1.3 Research scope
This research comprises two parts: a literature research on hemp protein techno-functionality
and experimental research on the functionality of S200 and HPM blends. The literature
research focuses on solubility, foaming, gelling and emulsification properties of hemp
proteins. The experimental research is mainly focussed on emulsions from S200 and HPM
mixtures. This focus was chosen because from the first orientating experiments it became
clear that HPM is not able to form foams, gels or emulsions, the results are discussed in
Appendix I. Orientating experiments with a mixture of S200 and HPM (3:1) showed that
mixtures of S200 and HPM are not suitable for the formation of homogeneous foams or gels,
but they can form stable emulsions (Appendix I).
2. Literature research on hemp protein techno-functionality
The techno-functional properties of proteins include solubility, viscosity, water binding,
gelation, elasticity, emulsification, foaming and fat and flavour binding (Table 2) (Martin,
2019). The techno-functional properties of main interest for this research will be discussed in
the next sections.
Table 2: Techno-functional properties of proteins
Function Mechanism Food
Solubility Hydrophilicity Beverages
Viscosity Water binding
Hydrodynamic size
Soups, gravies, dressings
Water binding Hydrogen bonding Meat/sausages, cakes, breads
Gelation Network formation Meats, sausages, pasta, baked goods
Elasticity Hydrophobic interactions
Disulphide crosslinks
Meat products, bakery products
Emulsification Interfacial adsorption
Film formation
Sausages, soups, dressings, desserts
Foaming Interfacial adsorption
Film formation
Whipped toppings, cakes, mousse,
nougat
Fat and flavour binding Hydrophobic bonding Bakery products
2.1 Solubility
The solubility of proteins is the lowest at the iso-electric point, where there is no net charge
and therefore no repulsion between the proteins. This results in aggregation of the proteins.
The solubility of proteins is thus strongly dependent on the pH. Other factors that can cause
aggregation are heat, enzymatic hydrolysis and association with non-protein compounds
(Martin, 2019). Studies on the techno-functionality of HPM and HPIs generally report low
solubility. Hadnadev et al. and Malomo and Aluko demonstrated that the solubility of HPI is
dependent on the protein isolation technique (Hadnadev, Dapcevic-Hadnadev, Lazaridou,
Moschakis, Michaelidou, Popovic, et al., 2018; Malomo & Aluko, 2015b). Isoelectric
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precipitation resulted in lower protein solubility compared to isolation by membrane filtration
(Figure 4) (Malomo & Aluko, 2015b). Yin et al. demonstrated that the solubility can be
increased by applying enzymatic hydrolysis (Yin, Tang, Cao, Hu, Wen, & Yang, 2008).
Experiments from Malomo and Aluko showed that albumin from hemp seed has a higher
solubility then edestin from hemp seed (Malomo & Aluko, 2015a). The cold press method
from which HPM is obtained uses a high level of force, and it is likely that the polypeptides
undergo severe protein-protein interactions during this process, which can cause low protein
solubility (Malomo, He, & Aluko, 2014).
Figure 4: Protein solubility profile of hemp seed protein products at different pH values: cHPC, commercial hemp seed
protein concentrate; iHPI, isoelectric pH-precipitated protein isolate; mHPC, membrane ultrafiltration protein concentrate;
HPM, hemp seed protein meal (Malomo & Aluko, 2015b)
2.2 Foaming
A foam is a system where air is dispersed in another phase. A protein can act as foaming
agent if it is easily adsorbed at an air-water interface, where it should reorganize to reduce the
surface tension and form a viscoelastic film (Phillips & Williams, 2011). This can only
happen if the protein has both hydrophilic and hydrophobic areas at its surface. Malomo et al.
demonstrated that higher hemp protein concentrations lead to more stable foams because the
proteins are able to form viscoelastic interfacial membranes through protein–protein
interactions and enhance resistance of air bubbles to destabilization (Malomo, He, & Aluko,
2014). However, lower protein concentrations led to higher foam capacity. HPI showed better
foaming properties compared to HPM. Raikos et al. demonstrated that the foaming capacity
and stability increase with increasing pH for hemp flour (Raikos, Neacsu, Russell, & Duthie,
2014).
2.3 Gelling
Proteins can form a gel when they partially unfold and develop uncoiled polypeptide
segments, which then interact at specific points to form a cross-linked three dimensional
network (Zayas, 1997b). This partial unfolding can be induced by factors such as acids, alkali
and urea, but most commonly heating is used (Phillips & Williams, 2011). A well-known
example is the boiling of an egg (Figure 5). The gelation kinetics, the type of gel and the
strength can be influenced by the heating temperature and time, the protein concentration, the
presence of salts and the pH.
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Figure 5: Schematic representation of the mechanism of gel formation from egg protein, induced by heat (Martin, 2019)
Gels from hemp protein are seldom mentioned in scientific literature. Raikos et al. observed
no gel formation at pH 4, 7 and 10 for hemp flour concentrations between 2-20 w/v%
(Raikos, Neacsu, Russell, & Duthie, 2014). Even at high concentrations (200 g/L) a paste was
produced rather than a cohesive gel, which suggests that the intensity of intermolecular
interactions in hemp flour is too weak to overcome the repulsive forces.
2.4 Emulsification
An emulsion is a thermodynamically unstable system where a liquid phase is dispersed in
another liquid phase, like oil in water (milk, mayonnaise) or water in oil (margarine). There
are different processes that can be involved in the destabilization of emulsions, which are
summarized in Scheme 2. Most proteins have both hydrophobic and hydrophilic parts and can
therefore act as an emulsifier. When proteins unfold at an oil/water interface they decrease the
interfacial tension, and due to their charge they can act as stabilizer by electrostatic repulsion.
The size distributions of the oil droplets that are dispersed in the aqueous phase are an
indication of the quality of an emulsion. Malomo et al. observed smaller oil droplet sizes for
HPM compared to HPI, therefore they suggest that non-protein materials that are present in
the hemp seed press cake could enhance emulsification (Malomo, He, & Aluko, 2014).
Raikos et al. demonstrated that the emulsion activity and stability increase with increasing pH
for hemp flour (Raikos, Neacsu, Russell, & Duthie, 2014).
Scheme 2: Processes involved in destabilization of emulsions (Binks & Horozov, 2006)
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2.5 Water holding capacity and oil holding capacity
Water holding capacity (WHC), sometimes also referred to as water binding capacity or water
retention, is the ability of a food product to physically hold water against gravity (Kinsella,
1979). The WHC is relevant for the juiciness and tenderness of food products, which is
important for meat products and meat analogues. Raikos et al. reported a WHC of 1.5 gr
water/gr sample for hemp flour (Raikos, Neacsu, Russell, & Duthie, 2014). Malomo and
Aluko reported a WHC of 12.3 g/g for a HPM with 37% protein content and a WHC of 12.0
for Hemp protein Isolate (Malomo, He, & Aluko, 2014). Hadnadev et al. reported WHCs of
0.80 for hemp protein isolate prepared by micellization and 1.59 for isolate prepared by
isoelectric precipitation (Hadnadev, et al., 2018). The differences between the results are
likely to arise due to different sample preparation methods (Table 3). The oil holding capacity
(OHC) is the amount of oil, which a protein powder can retain. OHC values from literature
are also not comparable because of the different sample preparation methods, and in addition
the OHC is sometimes expressed in g/g and sometimes in ml/g, while different types of oils
are used.
Table 3: WHC and OHC values of hemp protein products from different studies
Samples WHC g/g OHC g/g or ml/g Source
Hemp protein meal 12.32 ± 0.03 12.54 ± 0.08 g/g (Malomo & Aluko,
2015b; Malomo, He,
& Aluko, 2014)
Hemp protein isolate
(isoelectric precipitate)
12.01 ± 0.08 13.70 ± 0.29 g/g (Malomo & Aluko,
2015b; Malomo, He,
& Aluko, 2014)
Hemp protein isolate
(membrane filtration)
13.18 ± 0.06 13.76 ± 0.19 g/g (Malomo & Aluko,
2015b)
Hemp seed protein concentrate;
commercial
12.05 ± 0.04 12.58 ± 0.06 g/g (Malomo & Aluko,
2015b)
Hemp protein isolate
(micellization)
0.80 ± 0.03 1.62 ± 0.06 g/g (Hadnadev, et al.,
2018)
Hemp protein isolate
(isoelectric precipitate)
1.59 ± 0.05 1.79 ± 0.02 g/g (Hadnadev, et al.,
2018)
Hemp protein isolate
(isoelectric precipitate)
2.6 ± 0.2 3 ml/g (Yin, Tang, Cao, Hu,
Wen, & Yang, 2008)
Hemp seed cake 7.0 ± 0.1 9.0 ± 0.0 ml/g (Teh, Bekhit, Carne,
& Birch, 2014)
Acid soluble hemp protein isolate 8.2 ± 0.1 7.5 ± 0.2 ml/g (Teh, Bekhit, Carne,
& Birch, 2014)
Alkali soluble hemp protein isolate 8.7 ± 0.2 8.0 ± 0.1 ml/g (Teh, Bekhit, Carne,
& Birch, 2014)
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3. Experimental
HPM is a nature product of which the composition and protein content can vary between
batches. It is therefore important to evaluate the protein content of the used HPM before it is
used for experiments. There are different techniques available to evaluate the protein content,
for this study Kjeldhal protein analysis was applied. Furthermore it is essential to test the
solubility of HPM at different conditions, because solubility is a prerequisite for functionality.
The solubility of HPM was also evaluated using Kjeldhal protein analysis. The water and oil
holding capacity of HPM were compared with Solanic 100 and 200. The performance of
HPM as emulsifier was evaluated by preparation of emulsions with mixtures of S200 and
HPM at different conditions. The emulsions were evaluated on the droplet size distribution,
the dynamic viscosity, the relative stability and emulsifying capacity. All the sample
preparation methods and analysis that were used are discussed in the next sections.
3.1 Materials
Hemp (Cannabis sativa L.) protein meal was provided by HempFlax as 500 g bags of
commercial hemp protein shake (batch 0009). Solanic 200 potato protein isolate (batch
20AP8014) and other materials were provided by Avebe.
3.2 Protein content of HPM: Kjeldhal protein analysis
With Kjeldhal analysis the amount of nitrogen in a sample can be determined. The amount of
protein can then be calculated according to Equation 1.
%protein= %N x6.25 (1)
There is however the possibility that there is also non-protein nitrogen present in HPM, such
as free amino acids. The protein content was therefore determined from three types of
samples, which were prepared in duplicate. The first sample was the HPM as is. For the other
samples 10% of HPM was dispersed in demi water and heated in a water bath at 90C for 2
hours to denaturize all the protein. When proteins denaturize they become insoluble in water,
this way they can be easily removed by centrifugation. The samples were then centrifuged at
4000 xg for 30 minutes. The supernatant was transferred into new tubes by decantation. Both
the precipitate with the denatured protein and the supernatant were used as sample for
Kjeldhal analysis to determine if there are free amino acids present in the supernatant.
NutriControl B.V performed the Kjeldhal analysis. The samples were stored in the freezer
until they were sent away for analysis.
3.3 Protein solubility of HPM: Kjeldhal protein analysis
To determine the solubility of the proteins at different pH and salt conditions ten samples
were prepared according to Table 4. The samples were stored in the freezer overnight. The
next day they were briefly vortexed after they had reached room temperature. Next the
samples centrifuged at 4000 g for 30 minutes. The supernatant was transferred into new tubes
by decantation. The supernatants were used as samples for Kjeldhal analysis to determine the
dissolved protein content. NutriControl B.V performed the Kjeldhal analysis. The samples
were stored in the freezer until they were sent away for analysis.
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Table 4: Samples for protein solubility tests
Sample nr. 1 2 3 4 5 6 7 8 9 10
% HPM 5 5 5 5 5 5 5 5 5 5
% NaCl 0 0 0 0 0 1 1 1 1 1
pH 2 4 6 8 10 2 4 6 8 10
3.4 Water holding capacity and oil holding capacity
The water holing capacity (WHC) and oil holing capacity (OHC) were determined for Solanic
100, S200 and HPM according to the method of Hadnadev et al., with slight modifications
(Hadnadev, et al., 2018). First 1 g of protein powder was added to 50 ml centrifuge tubes
whereupon 10 ml of demi water or sunflower oil was added. The suspensions were vortexed
for approximately 1 minute and left for 30 minutes, followed by centrifugation at 3000 g for
20 minutes. The supernatant was discarded and the weight of the precipitate was measured.
The WHC and OHC were calculated according to Equitation 2, were W1 is the mass of the
protein powder sample and W2 is the mass of the precipitate.
WHC or OHC =(W
2-W
1)
W1 (2)
3.5 Preparation of emulsions
To prepare the emulsions first 300 g of protein solution was prepared in a plastic beaker by
gently adding the required amount of S200 to the water while stirring with an overhead stirrer.
To avoid foaming the stirrer was placed near the bottom of the beaker, which results in a
stable vortex in the water while stirring. Once all the S200 was added the required amount of
HPM was also gently added. After all the protein was added the mixture was stirred for at
least 30 additional minutes to ensure complete hydration of the proteins. Next the required
amount of NaCl was added and the pH of the mixture was adjusted to the desired value using
2M HCl or NaOH. Mixtures with HPM cannot reach a stable pH value, as it was observed
that the pH tends to slowly return to the initial value. After the pH was adjusted the mixture
was added to 450 g of sunflower oil form the brand “Reddy”, which was then homogenized
using a Ultra-Turax® homogenizer at max speed for 45 seconds. The obtained emulsion was
then divided over six 125 ml plastic sample pots and three 50 ml centrifuge tubes. The
centrifuge tubes were stored at room temperature for one day until the centrifuge test. Three
of the samples in the sample pots were used for viscosity analysis on the same day. The other
three samples in the sample pots were stored in the fridge for one day until the next viscosity
test.
3.6 Emulsion droplet size distribution
The droplet size distribution of and emulsion can give an indication of the emulsion’s
stability: small droplets indicate a stable emulsion. The droplet size distribution of the
emulsions were analysed using a Sympatec unit with a HELOS laser diffraction sensor and a
QUIXCEL model HD23xx wet dispersion unit at range 2 (0.25/0.45...87.5µm). Each sample
was measured four times with 10 seconds intervals between the measurements.
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3.7 Viscosity analysis
The viscosity of the emulsions was measured in triplicate using a Brookfield DV-II+
viscometer, using helipad spindle nr. 93. The spindle was placed above the emulsion and was
rotated at 10 rpm. Next the spindle was slowly lowered into the emulsion. The viscosity at
T=0 was registered at the moment when the spindle was inserted into the emulsion. At T=30
seconds the viscosity was registered again. Viscosity was analysed both at the day of the
preparation and after one day storage in the fridge at 4C. The samples were taken out of the
fridge 1 hour prior to the measurement.
3.8 Emulsion stability test with centrifuge
Besides the droplet size distribution it is also possible to get an indication of the emulsion
stability with a centrifuge test. A centrifuge test was performed according to the protocol that
was developed by Avebe in 2013 for the comparison of different types of mayonnaise, with
slight modifications (Willemse-Klaassens, 2013). The samples were prepared in triplicate and
two were heated in a water bath at 65C for 30 minutes while the third sample was kept at
room temperature. Next all the samples were centrifuged for 5 minutes at 5000 rpm in a
Hearaeus Megafuge 16R centrifuge. The layer separation of oil, water and solids particles
from the emulsion was recorded.
3.9 Emulsifying capacity measurement
The emulsifying capacity (EC) is defined as the amount of oil that is emulsified by 1 gram of
protein under specific conditions (Zayas, 1997a). The EC for S200 and S200/HPM blends
were determined according to the protocol that was developed by Avebe for the quality
control of Solanic potato proteins, with slight modifications. First 200 g protein solution was
prepared with 2% protein and 1.5% NaCl. The pH was adjusted to pH 8 by addition of 2M
NaOH. From this mixture 150 g was added to 300 g sunflower oil, which was then
homogenized using the Ultra-Turax® at maximum speed for 30 seconds. From the obtained
emulsion 150 g was added to a Hobart bowl of which the weight had been recorded including
the whisk. The emulsion was then whisked in the Hobart mixer while constantly adding
sunflower oil via a plastic tube using a Watson-Marlow static pump at 25 rpm. Oil was added
until the emulsion broke and became a liquid, this was visually assessed. The EC was then
calculated according to Equation 3, were m0 is the weight of the Hobart bowl + whisk, m1 is
the weight of the added emulsion and m2 is the final weight of the Hobart bowl + whisk
including emulsion and added oil. The minimum emulsifying capacity that can be calculated
this way is 100 goil/gprotein.
EC(goil
/ gprotein
) = m2-
m1
3-m
0 (3)
16
4. Results and discussion
4.1 Kjeldhal protein analysis
The results of the Kjeldalh protein analysis, which was performed by NutriControl B.V., are
given in Table 5. The 48.3 % measured protein in the HPM as is close to the 49.2 %, which is
stated on the packaging. The value on the packaging is an average value over different
batches of HPM, and since HPM is a nature product different values between batches are not
unusual. The precipitate contains 44.7 % protein while the supernatant, which should contain
no protein, still showed 4.0 % protein by the Kjeldhal analysis. This result indicates that there
are indeed free amino acids present in the HPM. For the other experiments and analysis in this
research it was therefore assumed that the HPM contains only 44.7 % protein. For S200 a
protein content of 93% was assumed, which is the average value over different batches of
S200 that has been determined by Avebe.
Table 5: Results Kjeldhal protein analysis
Sample HPM Precipitate Supernatant
% Protein 48.30 ± 0.42 44.73 ± 0.40 4.00 ± 0.02
Kjeldhal protein analysis was also used to determine the protein solubility at different
conditions, the results are given in Figure 6. The results are corrected for the 44.7% total
protein content in HPM and show that the solubility of the proteins is in general low. The
results correspond well with the results obtained by Malomo and Aluko, who reported protein
solubility in the range of 5-20% between pH 3 and 9 for HPM (Malomo & Aluko, 2015b).
The HPM from the study of Malomo and Aluko also had very poor foaming properties, but
the emulsifying properties were relatively good, which indicates that the data is indeed
comparable.
Figure 6: Protein solubility of HPM at different pH
The results in Figure 6 show that when 1% of NaCl is added the protein solubility increases,
except at pH 10. This effect can be explained by the Debye-Hückel theory; when salt is
added, the proteins get surrounded by the salt counter ions, which screen the charges on the
protein. Due to this screening the electrostatic free energy of the protein decreases while the
activity of the solvent is increased, which leads to increasing protein solubility.
0
5
10
15
20
25
30
35
40
1 3 5 7 9 11
Pro
tein
so
lub
ilit
y (
%)
pH
0% NaCl
1% NaCl
17
4.2 Water holding capacity and oil holding capacity
The results of the WHC and OHC test are given in Figure 7. For S200 no WHC value could
be obtained because the protein dissolves in the water. The OHC’s are similar for S100 and
S200, while HPM has a relatively low OHC. The results show that S100 has a higher WHC
compared to HPM. The statement from previous internal research at Avebe that HPM has
much higher WHC than S100 seems to be incorrect (Herreman, Tarazanova, & Wilbrink,
2018). In this previous research a WHC of 3-4 g/g for S100 is reported. How this value was
obtained is not mentioned, but this value is compared to the 12-13 g/g for HPM that was
obtained by Malomo and Aluko (Malomo & Aluko, 2015b). Based on these values the
conclusion is drawn that HPM is probably not useful for food applications such as health bars
due to the risk of moisture migration. However, from the literature research described in
section 2.5 it became clear that the literature values of WHC’s couldn’t be directly compared
due to different sample preparation methods; most studies used distilled water to disperse the
samples while some use buffer solutions. The vortex time that is used to disperse the samples
also varies. Furthermore there are differences in centrifugation time and force applied to the
samples. In addition, after decantation the upper phase of the samples is drained in most
studies before measuring the sample weight, but in some studies this step is skipped. Skipping
this step will result in higher WHC values.
Figure 7: Water holding capacity (WHC) and oil holding capacity (OHC) of Solanic proteins and HPM
4.3 Emulsion droplet size distribution analysis
The droplet size distribution can be summarized by three values: the x10, x50 and x90 value.
10% of the particles present in the emulsion have a diameter smaller then the x10 value, 50%
of the particles have a diameter smaller then the x50 value and 90% have a diameter smaller
then the x90 value. The analysis method assumes that the particles are spherically shaped.
4.3.1 Effect of pH
When the droplet size distributions are compared between emulsions that were prepared at
different pH conditions, it can be observed that emulsions with a S200/HPM protein ratio of
1:0 to 1:1 show similar results (Table 6). Emulsions with a 1:3 S200/HPM protein ratio
however, show a clear difference in droplet size distribution at different pH conditions. The
distribution diagram is given in Figure 23 (Appendix II). This could be explained by the fact
that S200 has similar solubility between pH 6-10 while the solubility of protein from HPM
increases at higher pH.
0,0
0,5
1,0
1,5
2,0
2,5
3,0
Solanic 100 Solanic 200 HPM
(g/
g)
WHC
OHC
18
Table 6: Particle size distribution values of S200/HPM emulsions with 60% oil, 1% protein and 0.6 % NaCl at different pH
conditions
Protein ratio S200/HPM pH x10 (µm) x50 (µm) x90 (µm)
1:0 6 1.78 10.81 18.83
1:0 8 1.68 10.20 17.75
1:0 10 1.68 10.46 18.82
3:1 6 1.73 11.20 21.43
3:1 8 1.75 10.75 19.42
3:1 10 1.86 11.21 21.02
1:1 6 1.80 12.83 25.28
1:1 8 1.94 12.51 23.45
1:1 10 2.01 12.36 30.27
1:3 6 6.18 26.09 67.40
1:3 8 2.72 16.57 37.80
1:3 10 1.88 10.76 19.96
4.3.2 Effect of protein concentration
When the protein concentration in the emulsions is increased from 1% to 2% the x10 and x50
values decrease slightly for all different protein ratios (Table 7). The x90 values also decrease
except for the 1:3 S200/HPM protein ratio. The differences are small but adding more protein
has slightly improved the droplet size distribution towards a higher amount of smaller
particles. Such as result is expected since a higher amount of protein can cover a larger
surface area, allowing for smaller particle sizes. In Figure 24 (Appendix II) the droplet size
distribution of emulsions with a 1:1 S200/HPM protein ratio is given as an example.
Table 7: Particle size distribution values of S200/HPM emulsions prepared at pH 8 with 60% oil, 0.6 % NaCl at different
protein concentrations
Protein ratio
S200/HPM
% protein x10 (µm) x50 (µm) x90 (µm)
1:0 1 1.68 10.20 17.75
3:1 1 1.75 10.75 19.42
1:1 1 1.94 12.51 23.45
1:3 1 2.72 16.57 37.80
1:0 2 1.57 9.33 16.63
3:1 2 1.52 9.20 17.68
1:1 2 1.79 11.85 28.14
1:3 2 2.13 15.89 43.46
4.3.3 Effect of NaCl concentration
When no NaCl is added to the emulsions during the preparation the particle size distribution
shows a shift towards larger particle sizes (Table 8). This effect is more clearly visible for
emulsions with less hemp protein, which could be explained by the fact that HPM also
contains 4.4% salts and minerals. In Figure 25 (Appendix II) the droplet size distribution of
emulsions with a 1:1 S200/HPM protein ratio is given as an example.
19
Table 8: Particle size distribution values of S200/HPM emulsions prepared at pH 8 with 60% oil, 1% protein at different
NaCl concentrations
Protein ratio
S200/HPM
% NaCl x10 (µm) x50 (µm) x90 (µm)
1:0 0.6 1.68 10.20 17.75
3:1 0.6 1.75 10.75 19.42
1:1 0.6 1.94 12.51 23.45
1:3 0.6 2.72 16.57 37.80
1:0 0 1.90 11.91 22.46
3:1 0 1.93 12.50 23.30
1:1 0 2.19 14.09 25.88
1:3 0 2.35 15.13 31.81
4.3.4 Effect of S200/HPM protein ratio
When only HPM is used as emulsifier no homogeneous emulsion can be obtained. When part
of the HPM is replaced by S200 homogeneous emulsions can be obtained with similar
properties to S200 emulsions. Emulsions with 1% protein and 0.6% NaCl prepared at pH 8
with small amounts of S200 were tested to investigate to which extend S200 could be
replaced with protein from HPM. From Figure 8 it can be observed that at S200/HPM protein
ratio 10:90 the particle size distribution shows a major shift towards larger particles.
Figure 8: Particle size distribution in S200/HPM emulsions with 60% oil, 1% protein, 0.6% NaCl prepared at pH 8 at
different S200/HPM protein ratios
4.4 Viscosity analysis
In this section the results of the viscosity measurements are discussed. Viscosity was
measured 2 hours after preparation and after one day of storage at 4°C, but mainly the results
after one day storage at 4°C are discussed in this section. The results of the measurements
before storage in the fridge are given in Appendix III. Refrigerated storage tends to increase
the viscosity of the emulsions.
1
10
100
0 20 40 60 80 100
Pa
tric
le s
ize
(µ
m)
% Hemp protein in protein mixture
x10
x50
x90
20
4.4.1 Effect of pH
When prepared at pH 10 the emulsions show the highest viscosity for the ones that contain
HPM (Figure 9). The pH does not influence the viscosity of emulsions with only S200. This
can be explained by the fact that the solubility of S200 is similar in the pH range 6-10, while
the solubility of protein from HPM increases at higher pH values.
Figure 9: Viscosity of S200/HPM elusions prepared at different pH conditions, measured after 1 day storage at 4°C
4.4.2 Effect of protein concentration
In Figure 10 the results are given of the viscosity measurements of emulsions with 1% and
2% protein after 1 day of storage at 4°C. The results of the viscosity measurements on the day
of the preparation are given in Appendix III. Increasing the protein concentration decreases
the viscosity of emulsions with only S200 protein, while the viscosity of emulsions with
S200/HPM protein blends is increased. The trend could be explained by non-protein materials
in the HPM such as polysaccharides, which can increase the viscosity of the emulsions
(Malomo, He, & Aluko, 2014).
Figure 10: Viscosity of S200/HPM elusions prepared with 0.6% NaCl at pH 8 at different protein concentrations, measured
after 1 day storage at 4°C
4.4.3 Effect of NaCl concentration
In Figure 11 the results of the viscosity measurements of emulsions with 0.6% and 0% NaCl
after 1 day of storage at 4°C are given. The results clearly show that without NaCl the
viscosity is much lower. The addition of salt increases the solubility of proteins, which was
0
5000
10000
15000
20000
25000
30000
35000
40000
1:0 3:1 1:1 1:3
Vis
cosi
ty (
cP)
S200/HPM ratio
pH 6
pH 8
pH 10
0
5000
10000
15000
20000
25000
30000
1:0 3:1 1:1 1:3
Vis
cosi
ty (
cP)
Ratio S200/HPM
1% protein
2% protein
21
discussed in section 4.1. When the proteins are better dissolved they also have better
emulsifying properties. If however too much salt will be added all the charges on the protein
will be completely screened and the proteins will no longer have good emulsifying properties.
It is therefore plausible that there is an optimum in NaCl concentration, but the determination
of the optimal NaCl concentration was not part of this study. In the study of Kong the
emulsifying properties of S300 were tested at five different salt concentrations between 0%
and 2% (Kong, 2017). Improvement was observed when NaCl concentration was increased
from 0% to 0.3%, while further increasing to 0.6%, 1% or 2% did not have any significant
effect. For S300 the optimum is thus between 0% and 0.3% NaCl, so it is plausible that for
S200 the optimum concentration is in the same range.
Figure 11: Viscosity of S200/HPM elusions prepared at different NaCl concentrations, measured after 1 day storage at 4°C
4.4.4 Effect of S200/HPM protein ratio
Emulsions were prepared at pH 8 with 1% protein, 0.6% NaCl and small fractions of S200 to
investigate to which extent S200 could be replaced with protein from HPM. When only HPM
is used the viscosity is approximately 2000 cP after 1 day, while emulsions from S200 have a
viscosity of nearly 14000 cP. Similar values are observed when 95% of the S200 protein is
replaced by protein from HPM. So HPM can take over the emulsifying properties of S200 as
long as there is still some S200 present. The highest viscosity was observed for emulsions
with a S200/HPM protein ratio of 1:3.
Figure 12: Viscosity of S200/HPM elusions prepared at different S200/HPM protein ratios, measured after 1 day storage at
4°C
0
5000
10000
15000
20000
25000
1:0 3:1 1:1 1:3
Vis
cosi
ty (
cP)
Ratio S200/HPM
0.6% NaCl
0% NaCl
0
5000
10000
15000
20000
25000
0 10 20 30 40 50 60 70 80 90 100
Vis
cosi
ty (
cP)
% Protien from hemp in protein mixture
Day 2
Day 1
22
4.5 Emulsion stability test with centrifuge
When mayonnaise emulsions are subjected to the centrifuge test they usually show some oil
separation from the emulsion. The emulsions that were prepared in this research however
mainly showed water separation; therefore mainly the water separation is reported and
discussed in this section.
4.5.1 Effect of pH
From Figure 13 can be observed that emulsions with HPM show less water separation when
compared to the emulsions with only S200 protein. This indicates that these emulsions are
more stable, which can be attributed to the water holding capacity of HPM. Figure 13 also
shows that the pH does not have significant influence on the water separation.
Figure 13: Water separation in S200/HPM elusions prepared at different pH, measured after 1 day storage at room
temperature and subsequent centrifuging at 5000 rpm for 5 minutes
4.5.2 Effect of protein concentration
When more protein is present in an emulsion it can be expected that the emulsion is more
stable because there is more protein which can hold on to the water and the oil. Figure 14
demonstrates that emulsions with more protein indeed show less water separation, indicating
a higher stability.
Figure 14: Water separation in S200/HPM elusions prepared at different protein concentrations, measured after 1 day
storage at room temperature and subsequent centrifuging at 5000 rpm for 5 minutes
0
5
10
15
20
25
30
35
1:0 3:1 1:1 1:3
mm
wa
ter
sep
ara
tio
n
Ratio Solanic 200 : HPM
pH 6
pH8
pH10
0
5
10
15
20
25
30
35
1:0 3:1 1:1 1:3
mm
wa
ter
sep
ara
tio
n
Ratio Solanic 200 : HPM
1% protein
2% protein
23
4.5.3 Effect of NaCl concentration
The NaCl concentration has no influence on the water separation from samples that had no
heat treatment (Figure 15). The samples that were heated showed much less water separation
in the 1:0 and 3:1 samples when salt was added (Figure 32, Appendix VI). This finding is
interpreted as heat-induced gelation of the samples, which trapped the water.
Figure 15: Water separation in S200/HPM elusions prepared at different NaCl concentrations, measured after 1 day storage
at room temperature and subsequent centrifuging at 5000 rpm for 5 minutes
4.5.4 Effect of S200/HPM protein ratio
When only S200 protein is used the water separation is the highest for unheated samples and
the lowest for the heated samples. When 25-90% of the S200 protein is replaced by protein
from HPM ± 20 mm water separation is observed, above 90% the water separation increases
slightly to 23-24.5 mm. Samples with only 5% protein form S200 show also 1 mm of oil
separation, while samples without S200 show 50 mm oil separation. Samples with more then
5% protein form S200 show no oil separation after centrifuging. These results show that up to
90% of the S200 can be replaced by protein from HPM without loosing the ability to bind the
oil sufficiently.
Figure 16: Water separation in S200/HPM elusions prepared at different S200/HPM protein ratios, measured after 1 day
storage at room temperature, with and without heating and subsequent centrifuging at 5000 rpm for 5 minutes
4.6 Emulsifying capacity
The EC was evaluated for emulsions with different S200/HPM ratios. The EC for S200 was
measured in duplicate to test the repeatability and was determined at 607 and 614 goil/gprotein
0
5
10
15
20
25
30
35
1:0 3:1 1:1 1:3
mm
wa
ter
sep
ara
tio
n
Ratio Solanic 200 : HPM
0.6% NaCl
0% NaCl
0
5
10
15
20
25
30
35
0 20 40 60 80 100
Wa
ter
sep
ara
tio
n (
mm
)
% protein from hemp in S200/HPM mixture
20°C
65°C
24
(Figure 17). This is also similar to the EC value of 605 goil/gprotein that was obtained by Kong
for S200 at similar conditions (Kong, 2017). It has thus been determined that the emulsifying
capacity test is repeatable. When 25% or 50% of the S200 protein is replaced by protein from
hemp the EC is increased to 662 and 649 goil/gprotein respectively. If 75-90% of the S200
protein is replaced the EC is lower compared to the EC of S200. When 95-100% is replaced
by protein from hemp the EC is below 100 goil/gprotein and could therefore not be determined.
Figure 17: Emulsifying capacity (EC) at different S200/HPM protein ratios. Below 100 goil/gprotein the EC can not be
determined with the method used in this study
These results demonstrate again that HPM can take over the emulsifying properties of S200 as
long as there is still some S200 present. This synergistic effect between S200 and HPM could
arise from the lipase enzymes that are present in S200. This lipase might hydrolyse the fat
from the HPM to fatty acids, which can stabilize the emulsions. This hypothesis was tested
with free fatty acid titration of hemp seed oil after treatment with lipase, but the colour of the
hemp oil interfered too much with this test. The hypothesis could therefore neither be
confirmed nor rejected
5. Conclusion
In this work the functional properties of hemp and potato protein blends were investigated,
with a main focus on emulsifying properties. A literature research was conducted on hemp
protein techno-functionality, and a laboratory study was conducted on the performance of
S200 and HPM in emulsions with 60% oil. The effects of pH, protein concentration, NaCl
concentration and S200/HPM protein ratio on the properties of the emulsions were
investigated.
The scientific literature on HPM reports low solubility of proteins from HPM, and similar
results were obtained in this study from protein solubility experiments with HPM form
HempFlax. The low protein solubility is an indication for low functionality. There are
examples in literature of foams with hemp protein, but the HPM from HempFlax is not
suitable for foam formation because there is still 11% of fat present, which prevents foaming.
Gels from hemp protein are not mentioned in scientific literature and HPM from HempFlax
also showed no gel formation.
100
200
300
400
500
600
700
0 20 40 60 80 100
Em
uls
ify
ing
ca
pa
city
(g
oil
/g
pro
tein
)
% protein from Hemp in S200/HPM emulsion
25
The WHC values for HPM and HPI that are reported in literature vary from 0.80 to 12.32 g/g.
The differences are likely to arise due to different sample preparation methods. OHC values
vary between 1.62 g/g to 13.76 g/g also due to different sample preparation methods. The
HPM from HempFlax had a lower WHC than S100; 1.15 and 2.84 g/g respectively. The OHC
for HPM is lower than for S200, 0.676 and 1.97 g/g respectively.
The literature on emulsions from hemp proteins suggest that HPM forms better emulsions
than HPI because the non-protein materials that are present in the hemp seed press cake
enhance the emulsification. Experiments with emulsions with HPM from HempFlax showed
that no homogeneous emulsions could be obtained. When the HPM is mixed with S200,
homogeneous emulsions can be obtained, but they are not stable when 10% or 30% oil is
used. Emulsions with 60% oil however were stable for longer time.
The replacement of S200 protein with protein from HPM has no major influence on the
particle size distribution up to a S200/HPM protein ratio of 15:85. If more S200 is replaced,
the particle size distribution shows a major shift towards larger particles. Increasing the pH
improves the particle size distributions in emulsions with high HPM content. Increasing the
protein concentration from 1% to 2% has resulted in slightly improved droplet size
distributions towards a higher amount of smaller particles. When no NaCl is added to the
emulsions the particle size distribution shows a shift towards larger particle sizes.
When 95% of the S200 protein is replaced by protein from HPM the viscosity is still similar
to emulsions with only S200 protein. Without NaCl the viscosity is much lower. Increasing
the protein concentration decreases the viscosity of emulsions with only S200 protein, while
the viscosity of emulsions with S200/HPM protein blends is increased due to non-protein
materials in the HPM. The pH does not influence the viscosity of emulsions with only S200,
but increasing the pH results in increased viscosity in emulsions with both S200 and HPM.
Emulsions with HPM showed less water separation compared to emulsions with only S200
protein. The centrifuge test only showed oil separation for emulsions with less then 10%
protein from S200.
A S200/HPM protein blend with 25% or 50% protein from HPM showed higher EC
compared to the 614 goil/gprotein for S200. With 75% or 90% protein from HPM the EC is
lower and with 95% or 100% protein from HPM the EC drops below the measuring limit 100
goil/gprotein.
The inclusion of salt in the HMP formulations improved functional properties. The presence
of salt aids in the solubilization of the hemp protein. From this, it is reasonable to conclude
that the functionality of hemp protein may be improved by increasing its solubility.
The results demonstrate that up to 85% of the S200 protein can be replaced by protein from
HPM in emulsions with high oil content, while obtaining similar properties to S200
emulsions. This synergistic effect might arise from the lipase in S200, which could probably
hydrolyse the oil in HPM to fatty acids, but this hypothesis could not be confirmed nor
rejected in this study.
26
6. Recommendations
Because the hypothesis concerning the synergistic effect between S200 and HPM could not
be confirmed nor rejected in this study, it is recommended to design new experiments to test
if the synergistic effect arises from the lipase in S200. For example HPM could be treated
with lipase before applying it in an emulsion to compare if this has similar effects as the
addition of S200.
Since the functional properties of hemp protein depend on the solubility it is suggested to
investigate hemp oil extraction techniques that can keep the protein solubility more intact. Da
Porto et al. attempted to obtain hemp oil without the cold pressing of the seeds, which
generally results in 65% oil recovery, by extracting the oil out of ground hemp seeds with
supercritical CO2, which resulted in 22 wt.% yield corresponding 72% oil recovery (Da Porto,
Decorti, & Tubaro, 2012). Pre-treating the hemp seeds with ultrasound increased the yield to
24.5 wt.% (Da Porto, Natolino, & Decorti, 2015). The focus of the researchers is on obtaining
the highest yield and quality of oil from the hemp seeds, therefore the quality of the proteins
was not investigated. It would however be very interesting to know how this process
influences the properties of the HPM.
Since HPM has good nutritional properties and is less expensive then S200 it is recommended
to test S200/HPM blends in emulsion applications such as sauces and meat analogues. Sauces
such as mayonnaise contain 70-80% fat, so vegan mayonnaise analogues are a possible option
for further testing. Meat analogues however, such as sausage emulsions, contain much less
fat; 30% (Martin, 2019). Since the emulsions in this research with 30% oil were unstable
additional research is needed on ways to improve the stability of emulsions with lower fat
percentages. As the solubility and the functionality of proteins depend on the salt
concentration it is recommended to test food emulsions at different salt concentrations to find
the optimal concentration.
Applications of S100/HPM blends could also be investigated. Previous research suggested
that a risk of moisture migration in applications with S100 and HPM was present because
HPM had a much higher WHC according to the literature, but this is not the case. HPM could
therefore also have possible applications in fortification of health bars.
Currently the hemp seeds that are produced in the Netherlands cannot be processed into hemp
oil and HPM due to legislations, but if new products can be developed with Solanic and hemp
protein blends this can have a positive influence on the the current lobbying for less strict
legislations. In addition the potato growers that cooperate with Avebe could use hemp for
crop rotation. Especially since the recent potato harvests have declined due to exceptionally
dry weather conditions, which are expected to become more frequent in the future due to
climate change, hemp can be a good alternative crop due to its better drought resistance.
27
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Malomo, S. A., & Aluko, R. E. (2015a). A comparative study of the structural and functional
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28
Appendix I: Results orientating experiments
Gelling properties HPM HPM was not able to form a gel (Figure 18). A mixture of S200 and HPM with protein ratio
3:1 did form a gel, but it was not homogeneous.
Figure 18: From left to right: S200 gel, HPM "gel" and mixed S200/HPM protein (3:1) gel
Foaming properties HPM HPM was not able to form a foam (Figure 19) When a mixture of S200 and HPM (3:1) was
used some foam was obtained, but most of the product remained liquid (Figure 20). The fact
that HPM is unable to form foam can be explained by the fact that HPM contains 11% fat,
which inhibits the foam formation.
Figure 19: No foam could be obtained from HPM
Figure 20: Foam from S200 (left) and liquid with foam on top from S200/HPM (3:1) mixture (right)
29
Emulsification properties HPM Emulsions were prepared with 1% protein. The use of HPM did not result in a homogenous
emulsion when using 60% oil (Figure 21). The use of a S200/ HPM (3:1) mixture resulted in a
homogenous emulsion when using 60% oil, while emulsions with 10% and 30% oil with
HPM were not stable (Figure 22 and Figure 23).
Figure 21: S200 emulsion (left) and HPM emulsion (right)
Figure 22: From left to right: emulsions with 1% protein (S200/HPM = 3:1) with 60, 30 and 10% oil directly after
homogenization
Figure 23: From left to right: emulsions with 1% protein (S200/HPM = 3:1) with 60, 30 and 10% oil 2 hours after
homogenization
30
Appendix II: Particle size distribution diagrams
Figure 24: Particle size distribution of 1% protein 1:3 S200/HPM emulsions with 0.6% NaCl prepared at different pH
conditions. Red = pH 6 Green = pH 8 Pink = pH 10
Figure 25: Particle size distribution of 1:1 S200/HPM protein emulsions with 0.6% NaCl prepared at pH 8 at different
protein concentrations. Red = 1% protein Green = 2% protein Pink = 2% protein (duplicate measurement)
31
Figure 26: Particle size distribution of 1% protein 1:1 S200/HPM prepared at pH 8 at different NaCl concentrations. Red =
0.6% NaCl Green = 0% NaCl
Appendix III: Viscosity measurements
Figure 27: Viscosity of emulsions with 1% protein prepared at pH 6 with 0.6% NaCl
Figure 28: Viscosity of emulsions with 1% protein prepared at pH 8 with 0.6% NaCl
0
5000
10000
15000
20000
25000
30000
35000
1 2
Vis
cosi
ty (
cP)
Day
Ratio 1:0
Ratio 3:1
Ratio 1:1
ratio 1:3
0
5000
10000
15000
20000
25000
30000
1 2
Vis
cosi
ty (
cP)
Day
Ratio 1:0
Ratio 3:1
Ratio 1:1
Ratio 1:3
32
Figure 29: Viscosity of emulsions with 1% protein prepared at pH 10 with 0.6% NaCl
Figure 30: Viscosity of emulsions with 2% protein prepared at pH 8 with 0.6% NaCl
Figure 31: Viscosity of emulsions with 1% protein prepared at pH 8 with 0% NaCl
0
5000
10000
15000
20000
25000
30000
35000
40000
1 2
Vis
cosi
ty (
cP)
Day
Ratio 1:0
Ratio 3:1
Ratio 1:1
Ratio 1:3
0
5000
10000
15000
20000
25000
30000
1 2
Vis
cosi
ty (
cP)
Day
Ratio 1:0
Ratio 3:1
Ratio 1:1
Ratio 1:3
0
5000
10000
15000
20000
25000
30000
1 2
Vis
cosi
ty (
cP)
Day
Ratio 1:0
Ratio 3:1
Ratio 1:1
Ratio 1:3
33
Appendix IV: Centrifuge tests
Figure 32: Water separation of emulsions with 1% protein and 0.6% NaCl prepared at different pH, after heat treatment
(65C, 30 minutes)
Figure 33: Water separation of emulsions with 1% protein prepared at pH 8 with 0.6% and 0% NaCl, after heat treatment
(65C, 30 minutes)
Figure 34: Water separation of emulsions with 0.6%NaCl prepared at pH 8 with 1% and 2% protein, after heat treatment
(65C, 30 minutes)
0
5
10
15
20
25
30
1:0 3:1 1:1 1:3
mm
wa
ter
sep
ara
tio
n
Protein ratio Solanic 200 : HPM
pH 6
pH8
pH10
0
5
10
15
20
25
30
1:0 3:1 1:1 1:3
mm
wa
ter
sep
ara
tio
n
Ratio Solanic 200 : HPM
0.6% NaCl
0% NaCl
0
5
10
15
20
25
30
1:0 3:1 1:1 1:3
mm
wa
ter
sep
ara
tio
n
Ratio Solanic 200 : HPM
1% protein
2% protein