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

2

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.

3

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)



THC Tetrahydrocannabinol

WHC Water holding capacity

WHO World Health Organization

4

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

5

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

6

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)

7

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).

8

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

9

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

10

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.

11

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)

12

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)


(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)


(micellization)

0.80 ± 0.03 1.62 ± 0.06 g/g (Hadnadev, et al.,

2018)



1.59 ± 0.05 1.79 ± 0.02 g/g (Hadnadev, et al.,

2018)



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)

13

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.

14

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


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.

15

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


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


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


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


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


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


1% protein

2% protein

23


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


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


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

7. References Avebe. (2015). Plantaardige eiwitten uit zetmeelaardappelen. Internal communication, Valthermond:

Avebe

Binks, B. P., & Horozov, T. S. (2006). Colloidal particles at liquid interfaces: Cambridge University

Press.

Callaway, J. C. (2004). Hempseed as a nutritional resource: An overview. Euphytica, 140(1-2), 65-72.

Da Porto, C., Decorti, D., & Tubaro, F. (2012). Fatty acid composition and oxidation stability of hemp

(Cannabis sativa L.) seed oil extracted by supercritical carbon dioxide. Industrial Crops and

Products, 36(1), 401-404.

Da Porto, C., Natolino, A., & Decorti, D. (2015). Effect of ultrasound pre-treatment of hemp

(Cannabis sativa L.) seed on supercritical CO2 extraction of oil. Journal of Food Science and

Technology, 52(3), 1748-1753.

FAOSTAT. (2016). Food Supply. Food and Agriculture Organization of the United Nations.

Galasso, I., Russo, R., Mapelli, S., Ponzoni, E., Brambilla, I. M., Battelli, G., & Reggiani, R. (2016).

Variability in Seed Traits in a Collection of Cannabis sativa L. Genotypes. Frontiers in Plant

Science, 7, 9.

Hadnadev, M., Dapcevic-Hadnadev, T., Lazaridou, A., Moschakis, T., Michaelidou, A. M., Popovic,

S., & Biliaderis, C. G. (2018). Hempseed meal protein isolates prepared by different isolation

techniques. Part I. physicochemical properties. Food Hydrocolloids, 79, 526-533.

Herreman, L., Tarazanova, M., & Wilbrink, M. (2018). Hemp seed protein meal characterization.

Internal communication: Avebe.

Kong, L. (2017). Study the physical-chemical properties of potato proteins and food application

development. Internship report. Van Linge, Veendam: Avebe.

Malomo, S. A., & Aluko, R. E. (2015a). A comparative study of the structural and functional

properties of isolated hemp seed (Cannabis sativa L.) albumin and globulin fractions. Food

Hydrocolloids, 43, 743-752.

Malomo, S. A., & Aluko, R. E. (2015b). Conversion of a low protein hemp seed meal into a functional

protein concentrate through enzymatic digestion of fibre coupled with membrane

ultrafiltration. Innovative Food Science & Emerging Technologies, 31, 151-159.

Malomo, S. A., He, R., & Aluko, R. E. (2014). Structural and Functional Properties of Hemp Seed

Protein Products. Journal of Food Science, 79(8), C1512-C1521.

Martin, A. (2019). Proteins - Functionality & Application. Personal communication: TNO.

Phillips, G. O., & Williams, P. A. (2011). Handbook of food proteins: Elsevier.

Raikos, V., Neacsu, M., Russell, W., & Duthie, G. (2014). Comparative study of the functional

properties of lupin, green pea, fava bean, hemp, and buckwheat flours as affected by pH. Food

science & nutrition, 2(6), 802-810.

Singh, J., & Kaur, L. (2016). Advances in potato chemistry and technology. Palmerston North: Nikki

Levy.

Tang, C. H., Ten, Z., Wang, X. S., & Yang, X. Q. (2006). Physicochemical and functional properties

of hemp (Cannabis sativa L.) protein isolate. Journal of Agricultural and Food Chemistry,

54(23), 8945-8950.

Teh, S. S., Bekhit, A. E. D., Carne, A., & Birch, J. (2014). Effect of the defatting process, acid and

alkali extraction on the physicochemical and functional properties of hemp, flax and canola

seed cake protein isolates. Journal of Food Measurement and Characterization, 8(2), 92-104.

Willemse-Klaassens, B. (2013). Emulsion stability test with centrifuge for mayonnaise. Internal

communication: Avebe Commerce Innovation Center Food.

Yin, S. W., Tang, C. H., Cao, J. S., Hu, E. K., Wen, Q. B., & Yang, X. Q. (2008). Effects of limited

enzymatic hydrolysis with trypsin on the functional properties of hemp (Cannabis sativa L.)

protein isolate. Food Chemistry, 106(3), 1004-1013.

Zayas, J. F. (1997a). Emulsifying Properties of Proteins. In Functionality of Proteins in Food, (pp.

134-227). Berlin, Heidelberg: Springer Berlin Heidelberg.

Zayas, J. F. (1997b). Gelling Properties of Proteins. In Functionality of Proteins in Food, (pp. 310-

366). Berlin, Heidelberg: Springer Berlin Heidelberg.

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


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 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


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


1% protein

2% protein

internship report chemical engineering - ihog · 2019-10-08 · internship report chemical...

Documents