The Impact of Species, Roasting and Fermentation on Melanoidin Content

BSC THESIS FOOD TECHNOLOGY Chairgroup Food Quality and Design, WUR Supervisors Vincenzo Fogliano and Xandra Bakker-de Haan September 2016 – May 2017

S. A. Pijnenburg THE IMPACT OF SPECIES, 940819675120 ROASTING AND FERMENTATION ON MELANOIDIN CONTENT AND ANTIOXIDANT CAPACITY IN PROCESSED THEOBROMA BEANS

Abstract The aim of this thesis is to investigate the impact of Theobroma species, fermentation and roasting degree on melanoidin content and antioxidant capacity of Theobroma beans. Beans of T. bicolor, T. grandiflorum and T. cacao were investigated. It was assumed that the water-soluble >20 kiloDalton fraction of the roasted beans consisted nearly purely of water-soluble melanoidins. A higher melanoidin content was found in unfermented cacao than in fermented cacao. A higher melanoidin content was found in mildly roasted cacao than in heavily roasted cacao. Likewise, the antioxidant capacity of melanoidins from unfermented cacao was higher than of melanoidins from fermented cacao. The antioxidant capacity of melanoidins from mildly roasted cacao was higher than the antioxidant capacity of melanoidins from heavily roasted cacao. Mildly roasted unfermented cacao had the highest melanoidin content and antioxidant capacity. With respect to the bicolor and grandiflorum samples, it was found that the estimated total melanoidin content was higher than of the compared cacao samples. However, due to bicolor and grandiflorum’s greater water holding capacity, the melanoidin yield was lower than that of the compared cacao samples.

Acknowledgements Many people at FQD and in my personal life helped me with this thesis, through explanations or moral support. I can’t thank all of them, so hereby; if you talked to me at any point while I was writing this thesis, I probably talked to you about it (I talk a lot). Thank you for listening.

Special thanks to Xandra and Vincenzo; they listened to me talking about cacao and ‘weird beans’ more than anyone else and still managed to see my crazy ideas as creativity and me making things difficult for myself as interesting. Thank you for your support and your patience.

And of course; thank you Jonathan, for the company and fun in the last leg of this thesis! I hope you enjoy your cacao melanoidin trip down the rabbit hole as much as I did.

Contents 1. Introduction ...... 4

1.1 The Theobromae family ...... 4 1.1.1 Cacao ...... 4 1.1.2 Bicolor ...... 5 1.1.3 Grandiflorum ...... 5 1.2 Melanoidins ...... 6 1.3 Cacao processing and melanoidin formation ...... 7 1.4 Research aim ...... 9 2. Materials and methods ...... 10

2.1 Experimental design ...... 10 2.2 Statistical analysis ...... 10 2.3 Materials ...... 11 2.3.1 Theobroma beans ...... 11 2.2.2 Chemicals ...... 11 2.4 Methods ...... 12 2.4.1 Sample preparation ...... 12 2.4.2 Roasting ...... 12 2.4.3 Defatting ...... 14 2.4.4 Extraction ...... 16 2.4.5 Ultrafiltration ...... 17 2.4.6 Antioxidant capacity ...... 17 3. Results ...... 19

3.1 Roasting ...... 19 3.2 Sample characterisation ...... 20 3.2.1 Overview of sample characteristics ...... 20 3.2.2 Colour ...... 21 3.2.3 Foaming capacity ...... 21 3.2.4 pH versus melanoidin content ...... 21 3.3 Bean composition ...... 21 3.3.1 Bean composition ...... 21 3.3.2 Melanoidin content of bean components ...... 22 3.3.3 Antioxidant capacity of bean components ...... 23 3.4 Effects of Theobroma species, fermentation and roasting degree ...... 28 3.4.1 Effect of species and roasting degree ...... 28 3.4.2 Effect of fermentation and roasting degree ...... 30 3.4.3 Effect of roasting degree overall ...... 31 4. Discussion ...... 32

4.1 Measuring error versus found effects/ Reliability ...... 32 4.2 Noise factors/ Variability ...... 32

4.2.1 Reproducibility earlier research ...... 32 4.2.2 Bean origin ...... 32 4.2.3 Roasting degree ...... 32 4.3 Unexpected results ...... 33 4.3.1 Low melanoidin yield ...... 33 4.3.2 Possible contamination of melanoidin fraction...... 33 5. Conclusion ...... 35

5.1 Sample characterisation ...... 35 5.2 Impact of Theobroma species, fermentation and roasting degree on melanoidin yield and antioxidant capacity ...... 35 5.2.1 Species ...... 35 5.2.2 Fermentation ...... 35 5.2.3 Roasting degree ...... 35 5.2.4 Highest melanoidin content and antioxidant capacity ...... 35 5.2.5 In summary; answers to section 1.4 Research aim ...... 36 5.3 Recommendations on further research ...... 36 5.3.1 Melanoidin extraction process ...... 36 5.3.2 Suggestions for further research ...... 37 6. References ...... 38 7 Appendices ...... 40

Ethical appendix ...... 51

1. Introduction Cacao is consumed globally and frequently. It is a current product with a long history going back to Mayan and Aztec culture, when cacao beans were consumed in foamy, invigorating drinks full of spices. Cacao beans are the essential ingredient in chocolate; a much-loved confectionary product. Cacao (Theobroma cacao L.) is part of the Theobromae family, which consists of approximately 20 species. Many of these species were once used in tandem with cacao for food production. Some of them, such as Theobroma grandiflorum and Theobroma bicolor, are still used as such. These botanical relatives are rarely researched for their food/confectionary potential in Western studies, while they may contribute to answering the current threats to the world cacao production such as diseases and global warming. In this introduction the three investigated Theobroma species are introduced, after which the origin and importance of melanoidins are explained. The process from fresh cacao fruit to cacao- and chocolate products is explained, focusing on the steps which are relevant to melanoidin formation. Finally this information is brought together in the aim of this thesis.

1.1 The Theobromae family The Theobroma genus consists of 22 tropical tree species (Pugliese et al, 2003). The research will focus on T. cacao, T. bicolor and T.grandiflorum as these are Theobroma species of which the processed seeds are consumed and/or used for chocolate-like products. Many Theobroma species have a large variety of names for each species, making it difficult to locate and identify useable samples. See Figure 1 below for a side-by-side comparison picture of the pods of these Theobroma species.

FIGURE 1 LEFT TO RIGHT; PODS OF THEOBROMA GRANDIFLORUM, T. BICOLOR AND T. CACAO VAR. FORASTERO

1.1.1 Cacao Theobroma cacao or the cacao tree is the most well-known species of the Theobromae family and used as the source of all cacao beans. These cacao beans are the raw material used to produce cacao (also known as cocoa) powder, cacao butter, and chocolate. Cacao beans are the processed seeds of the Theobroma cacao tree. This process and the resulting impact on melanoidin formation is described in more detail below in section 1.3.

Geographical and genetic research on cacao varieties showed the generally known classification of cacao into varieties Forastero, Criollo and Trinidario is genetically not entirely correct (Motomayor et al., 2008). Forastero cacao was studied in this thesis; the exact genetic variety of Forastero is unknown. According to EU food law (Directive 2000/36/EC), anything containing less than 20% cacao mass (cacao butter + cacao powder) cannot be called chocolate. This complicates matters when talking about chocolate-like products made from non-cacao Theobroma species. Although this is not entirely correct, such products will be referred to as chocolate for ease of explanation.

1.1.2 Bicolor Theobroma bicolor is also known as macambo, pataxte, cacao blanco, cacao do Peru, and jaguar cacao (among others). As the name cacao blanco implies, the fresh beans of this Theobroma species are white (instead of purple like most Theobroma cacao beans). Bicolor beans lack anthocyanins, causing this colour difference and a lack of the astringency that cacao is known for (Aprotosoaie, Luca and Miron, 2015). Bicolor has recently been used in chocolate bars, although these bars are rare. The chocolate making brand Madre chocolate made a ‘jaguar cacao’ bar in the start of 2015. The bar contained T. bicolor and T. cacao bean mass. ChocoSol Traders in Toronto, Canada also makes a ‘jaguar cacao’ bar, similarly using a mix of T. bicolor and T. cacao. Bicolor is mentioned in the Popol’ Vuh, a religious book of the Quiche Mayan people, seeming to share cacao’s importance and high regard (Tedlock, 1996). Fresh bicolor pulp is commonly consumed as fruit and as bicolor pods fall from the tree when ripe whereas cacao pods need to be cut down, bicolor may be simpler to harvest than cacao (Green, 2010). It is said to be used to make cacao powder- and cacao butter-like products in the Mansfeld horticultural encyclopedia stemming from 1959 (Mansfeld and Hanelt, 2001). It was also theorized that bicolor was used in ceremonial foamy cacao drinks consumed in Mesoamerica before it was colonized. In fact, bicolor is said to be the reason for the foaminess of these drinks as creating a stable, long-lasting foam from only cacao was impossible with the tools used (Green, 2010). Bicolor was even found to have a higher aromatic potential for chocolate-like aromas than cacao (Reisdorff et al., 2004). Currently bicolor beans can be found sold as superfoods.

1.1.3 Grandiflorum Theobroma grandiflorum is also known as cupuaçu or cupuassu. Its seeds are used by AMMA, a Brazilian chocolate making company, for their Theobroma grandiflorum/cupuaçu bar. Grandiflorum seeds are also used by the Peruvian chocolate-making company Cocama for their Chocoazú bar. A main issue of grandiflorum bar production is the difficulty in tempering, according to chocolatemakers with AMMA and Cocama. As with bicolor, grandiflorum bars are not widely produced. Unlike bicolor, the grandiflorum bars that can be found are made using only grandiflorum beans as opposed to mixing grandiflorum and cacao. Grandiflorum is mainly used for its pulp and fat; a facial mask containing ‘cupuacu butter’ can be found even at the Dutch drugstore Kruidvat, indicating a global use of grandiflorum fat. The pulp is often consumed fresh as fruit, in juices, or in icecream. The Mansfeld Horticultural Encyclopedia states grandiflorum seeds can be used for ‘good cacao butter’ but leaves out whether the seeds can be used for cocoa or chocolate production (Mansfeld and Hanelt, 2001). In a comparison between grandiflorum mass and cacao mass (processed, ground seeds) it was found that it was possible to produce grandiflorum mass with similar characteristics to cacao mass (Cohen and Jackix, 2005).

In essence cacao, bicolor and grandiflorum have all been used for chocolate production. Bicolor and grandiflorum once had a status equal to cacao in their area of origin but are not well known in the

Western world. Among artisanal chocolatemakers interest in bicolor and grandiflorum seems to be on the rise.

1.2 Melanoidins Not everything about cacao and its nutritional value is understood. One such aspect of cacao is a group of compounds named melanoidins. These compounds behave much like dietary fibres but originate from the roasting step in cacao processing. Melanoidins are a group of heterogeneous, nitrogen- containing, high molecular weight, brown compounds formed by the Maillard reaction. In this thesis melanoidins are defined as having a molecular weight above 20 kiloDalton and the focus is on water- soluble melanoidins. The amount, structure and functionality of melanoidins varies depending on the reaction conditions of their formation. For example, the pH and substrate composition of a model system (Kim and Lee, 2008), temperature during coffee beans roasting (Borrelli et al, 2002) and time of aging of balsamic vinegar (Verzelloni et al, 2010) have an influence on melanoidin formation and structure. Melanoidins are present in many often-consumed foods, such as coffee, bread crust and chocolate/cacao. They can affect the sensory characteristics, such as colour and texture, of foods and may have a biological activity (Morales, Somoza and Fogliano, 2010). Depending on the substrates, melanoidins can have an antioxidant activity (Summa et al, 2008). Melanoidins are not digested and absorbed to a great extent in the gastrointestinal tract, thus arriving mostly intact in the lower gut. In this way, they could be counted among the dietary fibers (Silván et al, 2010). The lack of degradation enables melanoidins to exert an antioxidant activity on the entire GI tract. The relationship between formation conditions and antioxidant capacity of melanoidins in food products is not entirely know yet. Neither is the relationship between formation conditions and melanoidin content or yield.

1.3 Cacao processing and melanoidin formation Below, in Figure 2, the process from cacao tree fruits/seeds to the cacao products we know is visualised. Cacao pods are split open and the pulp-enclosed seeds are separated from the rind. The seeds and pulp then undergo fermentation. During fermentation many of the aroma precursors responsible for ‘chocolate aroma’ are produced. The pulp liquefies during fermentation and is further known as sweatings. Sometimes the inner core around which the seeds grow is co- fermented; sometimes it is not, depending on the producer. The fermented beans are then dried. This can be sun-drying or artifical drying such as fire-drying. Fire-drying can impact the cacao’s aroma by imparting smoky notes. The dried beans are roasted and cracked into nibs; the remainder of the seed coat and pulp surrounding the beans is removed in a process called winnowing. The roasted nibs are ground, producing cacao mass which can be further processed. In some cases the cacao is Dutched. This process involves alkalising the cacao, raising the pH which causes improved water-solubility of the cacao powder and darkens the cacao colour. In the Netherlands, cacao powder sold in supermarkets is nearly always Dutched. Dutching can take place before or after roasting (Moser, 2015). The cacao mass (also known as cacao or cocoa liquor) can be further processed into chocolate or pressed to remove a portion of the cacao fat. The fat is known as cacao butter and used in several applications, such as foods and cosmetics. The pressed FIGURE 2 CACAO AND CHOCOLATE PROCESSING cacao solids can be ground into cacao powder. Cacao solids and fat, can be combined (with additives e.g. sweeteners, vanilla, emulsifiers and milk powder) to create chocolate. The process for making chocolate involves mixing/milling cacao mass, often with such additives, for an

7 extended time. This process is known as conching. Conching serves to remove unwanted flavors and refine particle sizes to a point where individual particles cannot be noticed as grittiness when consuming the chocolate, ideally to roughly 20-30 µm (Beckett, 2003). Conching creates liquid chocolate, which is then tempered to ensure the cacao fat crystallizes into a stable formation. Cacao butter can crystalize in several different crystal types, of which the βV (beta-five) crystal type is ideal for chocolate with a good gloss, snap, shelf life and melting behaviour. Tempering chocolate involves heating and cooling down the liquid chocolate to specific temperatures to seed the chocolate with βV crystals, causing ideally over 90% of cacao fat crystals to have the βV morphology (Windhab, 2008). Incorrectly tempered chocolate has diminished sensorial characteristics and is prone to fat bloom, the recrystalization of cacao fat on the chocolate surface which looks unappealing to consumers.

Significant steps in this process for melanoidin formation are drying, fermentation, Dutching and roasting. These steps are marked with a green oval inFigure 2. Of course, the variety of cacao and pre- and post- harvest conditions and practices are also likely to influence melanoidin formation as these factors can influence cacao bean composition. E.g. varieties which have a higher fat content may be more advantageous for melanoidin formation, as this was found to be favourable for intermediate Maillard reaction product formation and browning in hazelnuts (Fallico, Arena and Zappalà, 2003). The extent to which the beans are dried alters melanoidin formation, as this determines the substrate concentration. As cacao beans are usually dried to the industry standard of 7.5% moisture content, this normally doesn’t affect melanoidin formation much. Fermentation alters the cacao beans’ pH which influences the Maillard reaction and thus the melanoidin formation. Kim and Lee found a higher pH promoted browning and melanoidin formation in model systems (Kim and Lee, 2008). The fermentation process itself differs between producers and may also influence melanoidin formation. For example, Suazo et al showed some differences in browning and antioxidant capacity between unfermented, poorly fermented and properly fermented cacao beans at several roasting time- temperature combinations (Suazo, Davidov-Pardo and Arozarena, 2014). This could indicate the fermentation conditions or dynamics have an impact on melanoidin formation. When Dutching takes place pre-roasting, as the pH is altered, melanoidin formation is affected (Taş and Gökmen, 2016). The roasting step of course also heavily influences melanoidin formation. Further processing of cacao into chocolate, in which the cacao is conched and tempered, is unlikely to cause extensive further Maillard reaction taking place. The cacao mass or chocolate generally does not reach temperatures appropriate for fast Maillard reaction dynamics. Therefore melanoidin formation beyond the roasting step is unlikely to be of great significance.

In summary, melanoidin formation in cacao beans is likely to be influenced by:

- Variety (influences bean composition) - Pre- and post-harvest conditions (influences bean composition) - Fermentation (influences bean structure matrixand pH) - Drying (influences substrate concentration) - Dutching, if applied (influences pH) - Roasting (influences Maillard reaction dynamics)

As various Theobroma species have different compositions, bean species is likely to also influence melanoidin formation.

1.4 Research aim Of the above-mentioned six factors that are likely to impact melanoidin formation, fermentation and roasting were chosen as the focus of this thesis. Furthermore the impact of bean species was examined.

The original aim of this thesis was to investigate the impact of fermentation and roasting degree on cacao melanoidins as a part of ongoing research at FQD. For this four cacao samples (mildly and heavily roasted, fermented and unfermented) were made available.

For reasons of personal interest in the non-cacao Theobroma species, unfermented bicolor and fermented grandiflorum samples were sourced. These beans were roasted mildly and heavily, adding four samples to the research.

Finally the four raw samples (unfermented bicolor and cacao, fermented grandiflorum and cacao) were also added to the sample set. This brought the total amount of samples investigated to 12 instead of the original four.

In summary : the aim of this thesis is to investigate the impact of Theobroma species, fermentation and roasting on melanoidin content and antioxidant capacity of Theobroma beans. It was assumed that the water-soluble >20 kiloDalton fraction of the roasted beans consisted nearly purely of water- soluble melanoidins.

2. Materials and methods In this section the consecutive processing steps to obtain the water-soluble melanoidins are described. The steps followed were sample preparation, roasting, defatting, extraction and ultrafiltration. To characterise the samples, intermediate products were analysed, e.g. the colour and fat content of the samples was measured. To clearly indicate which intermediate products were analysed, methods for these measurements are grouped under the process steps. The antioxidant capacity was assesed on nearly all intermediate products and was thus listed as a group in itself. The entire process is visualised in Figure 3, Figure 4 and Figure 6. These pictures are placed in the methods section at the appropriate process steps.

2.1 Experimental design

The aim of this thesis was to investigate the effect of Theobroma species, fermentation and roasting on the melanoidin yield and antioxidant capacity.

To minimise the amount of samples, a fractional setup with four bean types and three roasting degrees was designed. To study the impact of fermentation on cacao melanoidins, water-soluble melanoidins were extracted from fermented and unfermented Forastero cacao beans. To study the impact of bean species, unfermented bicolor beans were compared to unfermented cacao beans. Fermented grandiflorum beans were compared to fermented cacao beans. To study the impact of roasting degree raw, mildly roasted and heavily roasted samples were compared. By using this experimental setup, it was not necessary to investigate fermented bicolor and unfermented grandiflorum, reducing the amount of samples. 12 samples were investigated, as seen in Table 1

TABLE 1 EXPERIMENTAL DESIGN AND SAMPLE CODES

Fermentation Roasting degree Theobroma Mildly Heavily species Unfermented Fermented Raw roasted roasted

Bicolor X BUR BUM BUH

Grandiflorum X GFR GFM GFH

X CUR CUM CUH Cacao X CFR CFM CFH

2.2 Statistical analysis All statistical analysis was done using Minitab 17 and Minitab Express statistical software. Where possible Anova was used. To identify significantly different samples Games-Howell was used. Main results were anaylsed using main effect plots and interaction plots made using Minitab (both 17 and Express). Tables and graphs were made using Minitab 17, Minitab Express or Microsoft Excel 2010 software.

2.3 Materials

2.3.1 Theobroma beans The T. bicolor beans were sourced from the U.S. company Imlak’esh organics. These beans were wild- harvested, lightly toasted and salted. Due to lack of enzymatic browning and aromas commonly associated with fermentation it was concluded the bicolor beans had not been fermented. The bicolor beans were intact but shelled upon arrival ("Wildcrafted Macambo | Imlak'esh Organics").

The T. cacao beans were already roasted and very roughly ground upon arrival. All cacao was of the Forastero variety. The cacao beans were sourced and prepared by Cinthya Nathaly Quiroz Reyes, who provided the following information on the cacao preparation. The cacao originated from the company CACEP in Villahermosa, Tabasco, Mexico. The cacao was sun-dried to a pre-roasting moisture content of 17-22%. The mildly roasted cacao samples were roasted in a convection oven at 130˚C to a weight loss of 7.5%, which took 1-3 hours. The heavily roasted cacao samples were roasted in a convection oven at 150˚C to a weight loss of 12.5%, which took 3-5 hours. The samples were allowed to cool down at room temperature during 20 minutes before weighing them to determine the weight loss.

The T. grandiflorum beans were sourced from the Brazillian company AMMA. They were grown in an agroforestry system and arrived as fermented and dried raw beans. The beans were intact and unshelled upon arrival ("Theobroma Grandiflorum Cupuaçu | AMMA Chocolate").

2.2.2 Chemicals Cellulose from spruce, Trolox and DPPH were obtained from Sigma-Aldrich. Petroleum ether was obtained from Actu-All Chemicals. Ethanol absolute Ph.Eur /USP was obtained from VWR Chemicals.

2.4 Methods

2.4.1 Sample preparation

The bicolor and grandiflorum beans were processed to nibs before roasting. The bicolor beans were desalted by repeated rinsing with warm running water, then dried overnight in a VWR VENTI-line VL convection oven at 105°C. The grandiflorum beans were hand- shelled.Pre-roasting the bicolor and grandiflorum beans were broken into nibs using a Braun CombiMax 700 food processor. The nibs were sieved using a 12 cm diameter stainless steel spiral wire frying scoop of the brand Handy (Dutch store Blokker ‘Frituurschep’) with 2.5 mm wide gaps to ensure homogeneously-sized nibs and promote even roasting.

2.4.2 Roasting

2.4.2a Bicolor and grandiflorum The nibs were roasted in thin FIGURE 3 MELANOIDIN EXTRACTION PART 1 (maximum 1 cm) layers in open aluminium containers without shaking in a VWR VENTI-line VL 115 oven. The mildly roasted nibs were roasted at 130°C during 30 minutes. The heavily roasted nibs were roasted at 150°C during 210 minutes. Approximately 150g of nibs were roasted for each sample and approximately 50g of nibs were kept raw. This created 6 samples; BR (bicolor raw), BM (bicolor mildly roasted), BH (bicolor heavily roasted), GR (grandiflorum raw), GM (grandiflorum mildly roasted), and GH (grandiflorum heavily roasted). To eliminate variance caused by different ovens the same oven was used for all nib roasting activities. To eliminate variance caused by changes in heat transmission in different containers the same type of aluminium containers were used in all roasting activities.The weight loss during roasting was measured. For this the nibs were weighed pre-roasting, and post-roasting after 20 minutes of cooling down in the FQD Food Safe Laboratory. The nibs were not cooled down in a humidity-free environment as this is unlikely to happen in real-world cacao processing.

2.4.2b Post-roasting nib weight regain; effect of storage environment A nib storage pilot was executed to track weight loss during roasting and weight regain during storage. Bicolor and grandiflorum nibs were roasted as described above at 130˚C. Half of the roasted nibs were stored in a desiccator, the other half were stored next to the desiccator in the FQD Food Safe Laboratory. The humidity, and possibly the temperature, of the environment could influence the nib

12 weight regain rate. Therefore the humidity and temperature in the FQD Food Safe Laboratory, where the samples were stored, were tracked during one week using a TFA HygroTherm digital hygrometer. The humidity and temperature in the FQD Food Safe Laboratory were measured several times per day during a week. The maximum and minimum humidity and temperature during each day and night were also measured using the same hygroscometer. It was assumed that this week represents an average week in this lab.

2.4.2c Correlation between colour changes and weight loss during roasting For this pilot grandiflorum and bicolor nibs were roasted as mentioned above for the BM and BH samples, in the same convection oven and containers at 130°C. 4 containers of nibs were used in this pilot; two containers with each 5g of grandiflorum nibs and two containers with each 5g of bicolor nibs. The containers and nibs were weighed pre-roasting. One container of bicolor nibs and one container of grandiflorum nibs were roasted during 30 minutes, then taken out of the oven and immediately weighed. The colour of the nibs was then measured, which took roughly 30 minutes. For colour measuring method see 2.4.2e Colour measurements. After this 30 minute cooldown the nibs were weighed again. The nibs were then placed back into the oven for another 30 minute roast cycle. This was repeated 4 times; the 3rd roasting cycle continued for 60 minutes instead of 30. Total roasting time was 150 minutes. The other container of bicolor nibs and of grandiflorum nibs was measured in the same manner; for these samples the two roasting cycles were longer to measure the impact of repeated roasting interruptions on nib colour and weight. Two roasting cycles were executed, the first lasting 90 minutes and the second 120 minutes. The measuring/cooldown cycle took 30 minutes. Total roasting time was 210 minutes.

2.4.2d Rough grinding

FIGURE 4 MELANOIDIN EXTRACTION PART 2

Pre-defatting the grandiflorum and bicolor nibs and the cacao powder were roughly ground to obtain a clump-free homogeneous powder. The samples were ground using a Braun CombiMax 700 food processor and sieved through a 1 mm* 1 mm sieve. Particles that did not pass through the sieve were rested shortly and re-ground. The resting step prevented the fat in the particles from melting during grinding, thus avoiding cacao liquor formation.

2.4.2e Colour measurements The colour of roughly ground samples pre-defatting was measured using a HunterLab Colorflex. Colour was measured according to the FQD protocol 36, in CIE L*a*b* colour space coordinates. For 2.4.2c Correlation between colour changes and weight loss during roasting, bicolor and grandiflorum nib colour was measured as well. Samples were packed in glass cylindrical cuvettes to a depth of 1-1.5 cm and covered with white weights to ensure the samples were measured against identical backgrounds. Each sample was measured 8 times, horizontally turning the cuvettes 90 degrees and measuring twice per turned position.

2.4.2f Water activity Water activity of the roughly ground non-defatted samples was measured in triplicate using a Novasina LabMaster-aw according to FQD protocol 32. As the nibs were shown to regain weight due to moisture re-uptake from their environment, water activity is affected by the sample storage environment and duration. Prior to the water activity measurement the samples had spent one week minimum in closed plastic containers in the FQD Food Safe Laboratory. Temperature and humidity of the FQD Food Safe Laboratory were recorded in 2.4.2b Post-roasting nib weight regain; effect of storage environment.

2.4.3 Defatting The samples were defatted by maceration with 40-60 petroleum ether. The roasted samples (CUM, CUH, CFM, CFH, BM, BH, GM, GH) were defatted by, for each sample, filling a 1000mL Duran flask to the 300 mL mark with the roughly ground sample. This amount weighed approximately 150g. Petroleum ether was then added to the 600 mL mark. The flask was closed with a heat-resistant cap and incubated in an electric oven at 30-40˚C during 2 hours. During this maceration, every 15 minutes the flasks were shaken by hand and put back in the oven in a different position, to avoid possible influences of uneven heat distribution inside the oven. This constituted the 2-hour maceration step, after which the flasks were placed inside a fumehood at room temperature overnight to let the sample powder settle. . Fat uptake from the samples caused the petroleum ether to take on a yellow colour. The next day the petroleum ether was carefully poured off, avoiding loss of sample, and new petroleum ether was added to the 600 mL mark.

For the raw samples (BR, GR, CUR, CFR) the same procedure was followed using 35-40 g of sample. A 250 mL Duran flask was used, which the sample filled to the 75 mL mark. The flasks were topped off with petroleum ether to the 150 mL mark. The maceration, settling, pouring off the used petroleum ether and adding new petroleum ether were carried out as with the roasted samples.

This procedure caused the ratio of petroleum ether to sample to change through the maceration repetitions as the samples lose volume and mass due to fat loss, and the petroleum ether was always refilled to the same total volume.

The maceration was repeated until the petroleum ether in a flask was colourless instead of yellow after settling overnight. The sample was then macerated with petroleum ether once more and placed at 5˚C overnight. If there was any fat left in the sample, the fat would solidify and become visible as white

14 solids in the sample. If these white fat solids were found, petroleum ether maceration with overnight settling at 5˚C was repeated until the fat was fully removed.

When the fat was fully removed, the petroleum ether was poured off carefully and the Duran flask containing the sample was placed uncapped in a fumehood at room temperature to evaporate the remaining petroleum ether. The drying samples were stirred occasionally and finely ground when the

FIGURE 5 PARTIALLY DEFATTED CACAO WITH CACAO FAT GLOBULES AND FAT- SATURATED PETROLEUM ETHER (LEFT) AND FULLY DEFATTED CACAO WITH PETROLEUM ETHER (RIGHT) petroleum ether had fully evaporated. Pictures of partially (left) and fully (right) defatted samples are below in Figure 5.

2.4.3a Fine grinding The samples were finely ground using a Retsch Mixer Mill MM 400, then sieved using a Fritsch 250 micron (µm) pore size sieve. The particles that did not pass through the sieve were re-milled until they passed through the sieve.

2.4.3b pH The finely ground defatted samples were mixed with room temperature demineralised water in a 1g of sample: 20 mL of water ratio, and vigorously shaken during the foaming capacity appraisal. The samples were then allowed to settle and pH of the watery top layer, above the settled sample powder, was measured in triplicate using a VWR pHenomenal 1000 L pH meter.

2.4.3c Foaming capacity Foaming capacity was measured by vigorously shaking 1:20 sample: demineralised water mixtures (as used to measure the pH) during 30 seconds. This was repeated 3 times per sample to find the percent volume increase of foam relative to the pre-shaking volume. The 1:20 sample: water ratio was used as in a pilot using Blooker cacao (alkalised cacao powder commercially available in The Netherlands), the highest foaming capacity for defatted cacao was found when using a 1 g of sample : 20 mL of demineralised water ratio.

2.4.4 Extraction

FIGURE 6 MELANOIDIN EXTRACTION PART 3

Pilots were ran to determine optimal extraction sample:water ratio and time. It was found that extraction time had no effect on extraction efficacy, while sample:water ratio was positively correlated with extraction efficacy. Extraction efficacy was determined by calculating the mass percentage loss of extracted cacao powder versus dry, non-extracted cacao powder. A balance between dilute extracts, which had a higher extraction efficiency, and volume and concentration suitable for ultrafiltration was sought in the extraction method.

The defatted, finely ground samples were mixed in 50 mL Greiner centrifuge tubes with miliQ water at 70°C in a 1g sample: 10 mL miliQ ratio. These mixtures were homogenised by vortexing during 15 seconds. The samples were then incubated during 10 minutes in a shaking water bath at 70°C and 170 rpm. After letting the samples cool down to room temperature, they were centrifuged during 5

16 minutes at 3000 rpm and 4°C. The supernatant (extract) was poured off and reserved; the supernatant volume was measured for water holding capacity calculation as described in 2.4.4a Water holding capacity. The extracts were filtered through consecutively Whatman paper filters nr. 4, 44 and 602 consecutively to ensure the absence of solid particles > 2 micron contaminating the extract.The post- Whatman filtering volume was measured to more correctly calculate melanoidin content of the extract.

2.4.4a Water holding capacity Water holding capacity was determined by measuring the volume of sample extract after centrifugation. As the mass of Theobroma sample extracted and the volume of water used was know, this way the water holding capacity could be calculated as millilitres of water retained per gram of defatted, finely ground sample.

2.4.4b Soluble and insoluble content Soluble and insoluble content were determined by extracting 2g of sample with 20 mL of demiwater according to the method described in 2.4.4. The soluble and insoluble fraction (respectively supernatant after Whatman filtering and pellet) were freeze-dried.

2.4.5 Ultrafiltration To obtain the >20 kDa melanoidin fraction, sample extracts were ultrafiltrated using a stirred cell with a maximum of 5 bar nitrogen pressure. The extracts were ultrafiltrated through a 20 kiloDalton cellulose filter in portions of maximum 120 mL. 75% of the extract starting volume was allowed to pass through the 20 kiloDalton filter, after which the remaining 25% of the extract starting volume was washed thrice with 30 mL demineralised water. Each 30 mL was allowed to pass through the filter before the following 30 mL of demineralised water was added. 30 mL more demineralised water was then added to the > 20 kDa melanoidin fraction remaining in the stirred cell above the filter to avoid an overly large concentration gradient and to ensure all of the fraction was collected. This fraction was frozen and freeze-dried to determine the melanoidin yield. The <20 kDa fraction was not further investigated due to time constraints.

2.4.6 Antioxidant capacity Antioxidant capacity was determined using the QUENCHER-DPPH method (Gökmen, Serpen and Fogliano, 2009), according to FQD protocol number 41. Freeze-dried mango, in a 1:6 sample:cellulose dilution, was used as a control. The antioxidant capacity of the defatted finely ground beans, water- soluble compounds, water-insoluble compounds and water-soluble melanoidins was determined for all samples. As the QEUNCHER method works on the solid-liquid interface of the samples, all samples were ground to 250 µm maximum particle size to avoid influence of particle size on the results. For the mango control this fine grinding was not standard operating procedure and therefore omitted. Absorbance measurements were repeated four times per concentration with three different concentrations per sample. The QUENCHER method involves measuring the absorbance at three concentrations for each sample, then calculating one antioxidant capacity from these three points. As this gives only one antioxidant capacity value per sample, no statistical analysis was performed on the final calculated data.

2.4.6a Finely ground defatted beans The finely ground defatted beans were diluted with cellulose from spruce in a 1:200 sample:cellulose ratio. Diluted samples were mixed overnight to ensure homogeneous dispersion of sample particles.

2.4.6b Insoluble The water-insoluble compounds were obtained by freeze-drying the pellet created by the extraction process. These were diluted with cellulose from spruce in a 1:100 sample:cellulose ratio. Diluted samples were mixed overnight to ensure homogeneous dispersion of sample particles.

2.4.6c Soluble The water-soluble compounds were obtained by freeze-drying filtered extract. These were diluted with cellulose from spruce in a 1:100 sample:cellulose ratio. Diluted samples were mixed overnight to ensure homogeneous dispersion of sample particles.

2.4.6c Melanoidins The melanoidin fraction obtained by ultrafiltration was freeze-dried and diluted with cellulose from spruce in a 1:100 sample :cellulose ratio. Diluted samples were mixed during at least 3 hours to ensure homogeneous dispersion of sample particles.

3. Results First the results for the roasting step are presented, then the results for the sample characterisation, followed by the results on bean composition. Lastly the main results on melanoidin content and antioxidant capacity are shown. Detailed results of all measurements can be found in section 7 Appendices.

3.1 Roasting Results were analysed using One-way Anova and samples were compared using the Games-Howell method with 95% confidence. Results that do not share a letter are significantly different.

TABLE 2 ROASTING PARAMETERS AND WEIGHT LOSS DURING ROASTING

Sample Theobroma Fermentation Roasting Roasting Roasting Weight name species degree time temperature loss (hours) (˚C) during roasting (%) BUR Bicolor Unfermented Raw - - - BUM Bicolor Unfermented Mild 0.5 130 4.9a BUH Bicolor Unfermented Heavy 3.5 150 5.7b GFR Grandiflorum Fermented Raw - - - GFM Grandiflorum Fermented Mild 0.5 130 4.2c GFH Grandiflorum Fermented Heavy 3.5 150 5.2d CUR Cacao Unfermented Raw - - - CUM Cacao Unfermented Mild 1-3 130 7.5 CUH Cacao Unfermented Heavy 3-5 150 12.5 CFR Cacao Fermented Raw - - - CFM Cacao Fermented Mild 1-3 130 7.5 CFH Cacao Fermented Heavy 3-5 150 12.5 Weight loss during roasting is shown as percentage of pre-roasting mass.

Cacao samples were already roasted upon arrival according to the sample description (2.3.1 Theobroma beans). For the cacao samples roasting degree was defined by roasting temperature and percentage weight loss during roasting. It was attempted to replicate the roasting degree for the bicolor and grandiflorum samples. This was unsuccesful as the bicolor and grandiflorum nibs could not be roasted beyond 6.5% weight loss without burning. Roasted bicolor and grandiflorum nibs were found to regain weight after roasting. This weight regain was found to be faster in a 23-27% humidity environment than in a dessicator, suggesting moisture reuptake from the environment by the roasted nibs. In addition weight loss and change in nib colour were not found to be strongly correlated for both bicolor and grandiflorum nibs. Detailed results are shown in appendix 7.1 Roasting. Therefore roasting degree was characterised by roasting time as well as roasting temperature and weight loss during roasting. Samples were not moved from the oven until the entire roasting time had passed. To ensure samples were truly different from one another, the samples were extensively characterised, the results of which can be found in section 3.2.1

3.2 Sample characterisation

3.2.1 Overview of sample characteristics Results were analysed using One-way Anova and samples were compared using the Games-Howell method with 95% confidence. Results that do not share a letter are significantly different. An overview of the sample characterisation results is shown in Table 3. Detailed results for the sample characteristics can be found in section 7 Appendices. Colour, foaming capacity and pH are shortly discussed in section 3.2.2 - 3.2.4.

TABLE 3 OVERVIEW OF SAMPLE CHARACTERISTICS

Fat Soluble Insoluble Colour Water FC WHC Sample content pH content content L* a* b* activity (%) (mL/g) (%) (%) (%) BUR 73.49a 2.74k 20.88f 0.459a 54 6.58a 14 3.6a, b 10.9 76.6 BUM 72.16b 3.20j 22.24d 0.100e 54 6.48b 9 3.3c 11.2 79.7 BUH 62.71c 7.48i 28.63a 0.061f 55 6.36c 9 3.8a 8.9 82.2 GFR 41.53f 13.89c 23.19c 0.356b 58 5.82f 2 3.3b, c 12.3 75.8 GFM 35.91h 15.08a 24.07b 0.096e 57 5.57g 5 3.4b, c 11.1 79.1 GFH 21.94i 10.59f 11.74l 0.074e, f 57 5.58g 14 3.6a, b, c 9.2 82.1

CUR 42.40e 10.75f 20.31g 0.434a 43 6.07d 0 2.8e 16.5 70.4 CUM 42.92d 9.26h 16.93j 0.294c 48 6.07d 0 2.4f 15.5 75.0 CUH 42.96d 10.28g 18.93h 0.238d 48 5.98e 0 3.0d 13.8 78.3 CFR 37.93g 14.06b 21.52e 0.343b 50 5.01i 25 2.3g 16.3 71.6 CFM 38.03g 12.94d 18.41i 0.289c 45 4.89j 0 2.3g 12.6 74.6 CFH 35.87h 11.24e 16.01k 0.229d 43 5.06h 14 2.1h 12.8 78.4 Fat content in % mass of non-defatted sample (whole beans) FC = foaming capacity in % volume increase

WHC = water holding capacity in mL water/g defatted finely ground sample Soluble and insoluble content in % mass of defatted sample

3.2.2 Colour

Interval Plot of L* vs sample Results show bicolor was significantly lighter (L*) 95% CI for the Mean 80 in colour than the other samples. In addition the

70 results show lightness was negatively correlated

60 to roasting degree. This was less pronounced in cacao but alignes with the general expectations,

* 50 L as shown in Figure 7. 40

30 3.2.3 Foaming capacity 20 Fermentation was found to increase foaming BUH BUM BUR CFH CFM CFR CUH CUM CUR GFH GFM GFR sample capacity. Foaming capacity of unfermented Individual standard deviations are used to calculate the intervals. bicolor was indeed found to be higher than FIGURE 7 LIGHTNESS MEASUREMENTS OF foaming capacity of unfermented cacao. This is in ROUGHLY GROUND NON-DEFATTED line with literature on bicolor foaming capacity SAMPLES (Green, 2010).

3.2.4 pH versus melanoidin content Fermentation was found to lower pH. Fermented cacao was found to have a lower melanoidin content than unfermented cacao. This is in line with previous findings on the effect of pH on melanoidin foration (Kim and Lee, 2008).

3.3 Bean composition

3.3.1 Bean composition The below Table 4 and Figure 8 show the measured bean composition in mass percentage of the whole beans. The amount of water-soluble molecules larger than 20 kiloDalton actually found is very small relative to the starting material. All fractions and especially the >20 kiloDalton fraction is listed in mass percentage of the whole beans in this table, not as a percentage of the extract or of the defatted beans, because this gives insight into the amount of starting material needed to produce a certain amount of melanoidins.

Theobroma beans consist of fat and solids. The solids in turn consist of soluble and insoluble solids. Part of the solid weight was lost upon watery extraction of the samples; this is indicated simply as extraction loss. This is to say that e.g. sample BUR (unfermented raw bicolor beans) consists of 54.5% fat, 34.8% insoluble solids and 4.9% soluble solids. After ultrafiltration using the 20 kiloDalton filter 0.5% of the starting weight of the whole beans was found to be larger than 20 kiloDalton.

TABLE 4 BEAN COMPOSITION IN MASS PERCENTAGE OF WHOLE (NON-DEFATTED) BEANS

Sample Fat Solids Insoluble Extraction Soluble >20 kDa <20 kDa solids loss solids solids BUR 54.5 45.5 34.8 5.7 4.9 0.5 4.5 BUM 54.0 46.0 36.7 4.2 5.1 1.2 3.9 BUH 55.2 44.8 36.8 4.0 4.0 0.9 3.1 GFR 57.9 42.1 31.9 5.0 5.2 1.6 3.6 GFM 57.1 42.9 33.9 4.2 4.8 1.4 3.4 GFH 57.4 42.6 35.0 3.7 3.9 1.0 2.9 CUR 42.8 57.2 40.3 7.5 9.4 1.7 7.8 CUM 48.1 51.9 38.9 4.9 8.1 1.3 6.7

CUH 47.9 52.1 40.8 4.1 7.2 1.2 6.0 CFR 50.0 50.0 35.8 6.0 8.2 1.0 7.2 CFM 45.4 54.6 40.8 7.0 6.9 1.1 5.8 CFH 43.0 57.0 44.7 5.0 7.3 1.0 6.3

Bean composition in % of whole bean mass 100.0 90.0 80.0 70.0 60.0 extraction loss 50.0 <20 kDa 40.0 >20 kDa 30.0 insoluble 20.0 fat 10.0

0.0 fraction mass % ofwhole bean mass

sample

FIGURE 8 BEAN COMPOSITION IN MASS PERCENTAGE OF WHOLE (NON-DEFATTED) BEANS

3.3.2 Melanoidin content of bean components Below in Table 5 the melanoidin content of various bean fractions is shown. Because the bean composition was not the same amoung samples, melanoidin content of a fraction can’t always be easily translated to melanoidin content of another fraction. To simplify matters for further research in which melanoidin content is compared, melanoidin content of the whole bean, defatted bean solids, soluble matter and extract was listed in Table 5.

This thesis focuses on water-soluble melanoidins. Due to the high water holding capacity of the samples and logistically more convenient single step extraction methodology, part of the water-soluble melanoidins are very likely to have remained trapped in the insoluble matter pellet. The pellet retained part of the extract, as seen in the water holding capacity of the samples. Because of this water holding capacity part of the melanoidins, proportional to the part of the extract retained, was retained by the insoluble fraction of the sample. At other parts of the process, such as Whatman filtering the extract, smaller melanoidin losses could also occur. Melanoidin content is discussed in this thesis as the melanoidin content that was actually found. This is strictly speaking the melanoidin yield; the amount found when following the documented methodology for these samples.The results in sections 3.3.2 to 3.3.4 and conclusions are based on the amount of melanoidins actually found. It was chosen to base the results and conclusion on the melanoidin content actually found as this melanoidin content was emperically proven.

The estimated total melanoidin content was obtained by correcting for the water holding capacity (column WHC in 3.2.1 Overview of sample characteristics) and the loss during Whatman fitration. It is, however, still an estimation and not a verified emperical result. This estimated total melanoidin content is shown below in Table 5, column “C >20 kDa estimated total in defatted bean solids (mg/g)”. The found total melanoidin content, which sections 3.3.2 – 3.3.4 and the conclusions are based on, is shown in Table 5, column “C >20 kDa defatted bean solids (mg/g)”. Column “% extraction yield” in Table 5 shows the mass percentage of melanoidins actually found relative to the estimated total melanoidin content. This shows that the melanoidin extraction efficacy could be improved by mitigating the sample water holding capacity. Column “C >20 kDa in extract (mg/mL)” in Table 5 shows the melanoidin content found in the ultrafiltrated extract. For CUM, CUH and CFH the extract was made in one large portion, then split into four 100 mL portions for ultrafiltration. These four portions were seperately freeze-dried, thus giving an idea of the standard deviation of melanoidin content found in the same extracts. For CUM, CUH and CFM extract melanoidin content is therefore given as miligrams of melanoidins per mililiter of extract ± standard deviation in mg/mL.

TABLE 5 MELANOIDIN CONTENT (C >20 KDA) OF VARIOUS BEAN FRACTIONS

sample C >20 kDa C >20 kDa C >20 kDa C >20 kDa % extraction C >20 kDa in C >20 kDa in of whole whole bean defatted estimated yield soluble extract bean (%) mg/g bean solids total in matter (mg/mL) (mg/g) defatted (mg/g) bean solids (mg/g) BUR 0.5 4.7 10.30 16.0 64.4 94.9 1.60 BUM 1.2 12.0 26.00 52.4 49.6 233.0 5.23 BUH 0.9 8.9 20.97 41.3 50.8 223.6 4.13 GFR 1.6 15.8 37.47 55.5 67.5 304.7 5.40 GFM 1.4 13.8 32.12 57.8 55. 6 290.0 5.80 GFH 1.0 10.1 23.72 45.1 52.6 258.0 4.52 CUR 1.7 16.7 29.23 40.8 71. 7 177.2 4.08 CUM 1.3 13.4 25.76 36.7 70.2 165.8 3.68 ± 0.18 CUH 1.2 11.6 22.34 33.5 66.7 162.4 3.36 ± 0.09 CFR 1.0 9.6 19.19 26.3 73.0 117.5 2.63 CFM 1.1 11.1 20.28 36. 9 55.0 160.4 3.32 CFH 1.0 10.1 17.65 26.5 66.7 137.9 2.66 ± 0.08

3.3.3 Antioxidant capacity of bean components Generally the antioxidant capacity of the defatted beans was higher than that of the individual compounds. For raw fermented and unfermented cacao antioxidant capacity of the soluble fraction was higher than antioxidant capacity of the insoluble fraction. In roasted cacao soluble and insoluble fractions had roughly the same antioxidant capacity. Cacao melanoidins had a higher antioxidant capacity than the insoluble and soluble fraction, but a lower antioxidant capacity than the bean solids (defatted beans). Grandiflorum fractions had a similar antioxidant capacity to fermented cacao. Bicolor had a very low antioxidant capacity which could be explained by its natural lack of anthocyanins.

Antioxidant capacity of the melanoidin fraction is visible in section 3.3.3e and further detailed in section 3.4. Antioxidant capacity of the defatted beans, soluble fraction and insoluble fraction is not detailed further but visible in section 3.3.3a-d.

3.3.3a Comparison of finely ground defatted beans (‘whole) , insoluble compounds, soluble compounds and the soluble >20 kDa fraction.

Antioxidant capacity of Theobroma bean fractions (with 5% error bars) 1.2

1.0

0.8

0.6

0.4

0.2

0.0 BUR BUM BUH GFR GFM GFH CUR CUM CUH CFR CFM CFH

antioxidant capacity (mmol trolox/gsample) sample

whole insoluble soluble >20 kDa

FIGURE 9 ANTIOXIDANT CAPACITY OF THE BEAN SOLIDS ('WHOLE', DEFATTED BEANS); INSOLUBLE AND SOLUBLE COMPOUNDS; AND >20 KDA COMPOUNDS

TABLE 6 ANTIOXIDANT CAPACITY OF THE BEAN SOLIDS ('WHOLE', DEFATTED BEANS); INSOLUBLE AND SOLUBLE COMPOUNDS; AND >20 KDA COMPOUNDS

antioxidant capacity (mmol trolox/g sample sample) whole insoluble soluble >20 kDa BUR 0.153 0.061 0.061 0.029 BUM 0.139 0.046 0.034 0.025 BUH 0.230 0.051 0.054 0.027 GFR 0.607 0.242 0.135 0.228 GFM 0.494 0.237 0.194 0.305 GFH 0.633 0.142 0.203 0.162 CUR 0.908 0.222 0.351 0.546 CUM 0.964 0.425 0.372 0.873 CUH 0.946 0.352 0.322 0.400 CFR 0.754 0.299 0.504 0.543 CFM 0.647 0.272 0.306 0.400

CFH 0.370 0.260 0.216 0.310 mango 0.034 0.026 0.017 0.035 control

3.3.3b “Whole” defatted beans Antioxidant capacity of defatted, finely ground Theobroma beans ('whole') with 5% error bars 1.200

0.908 0.964 0.946 1.000

0.754 0.800 0.647 0.607 0.633 0.600 0.494 0.370 0.400

0.230 0.153 0.200 0.139 0.034

antioxidant capacity (mmol trolox/gsample) 0.000 mango BUR BUM BUH GFR GFM GFH CUR CUM CUH CFR CFM CFH control sample

FIGURE 10 ANTIOXIDANT CAPACITY OF THE DEFATTED BEANS

3.3.3c Insoluble compounds Antioxidant capacity of insoluble Theobroma compounds (with 5% error bars) 0.500 0.425 0.450

0.400 0.352 0.350 0.299 0.272 0.300 0.260 0.242 0.237 0.250 0.222 0.200 0.142 0.150

0.100 0.061 0.026 0.046 0.051 0.050 0.000 mango BUR BUM BUH GFR GFM GFH CUR CUM CUH CFR CFM CFH control

antioxidant capacity (mmol trolox/gsample) sample

FIGURE 11 ANTIOXIDANT CAPACITY OF THE INSOLUBLE COMPOUNDS

3.3.3d Soluble compounds

Antioxidant capacity of water-soluble Theobroma compounds (with 5% error bars) 0.600 0.504 0.500

0.372 0.400 0.351 0.322 0.306 0.300 0.216 0.194 0.203 0.200 0.135

0.100 0.061 0.054 0.034 0.017 0.000 antioxidant capacity (mmol trolox/gsample) mango BUR BUM BUH GFR GFM GFH CUR CUM CUH CFR CFM CFH control sample

FIGURE 12 ANTIOXIDANT CAPACITY OF THE SOLUBLE COMPOUNDS

3.3.3e Melanoidins

Antioxidant capacity of <20 kDa water-soluble Theobroma compounds (with 5% error bars) 1.000 0.873 0.900 0.800 0.700 0.546 0.543 0.600 0.500 0.400 0.400 0.400 0.305 0.310 0.300 0.228 0.162 0.200 0.025 0.100 0.035 0.029 0.027 0.000 mango BUR BUM BUH GFR GFM GFH CUR CUM CUH CFR CFM CFH antioxidant capacity (mmol trolox/gsample) control sample

FIGURE 13 ANTIOXIDANT CAPACITY OF MELANOIDINS (AND >20 KDA COMPOUNDS FOUND IN RAW SAMPLES)

3.4 Effects of Theobroma species, fermentation and roasting degree The research aim was to investigate the effect of 1. Bean species, 2. Fermentation, and 3. Roasting degree on the melanoidin content and on the melanoidin antioxidant capacity. The 12 sample set was intentionally set up to allow balanced comparisons as explained in 2.1, experimental design, and main effect plots ("What Is A Main Effects Plot? - Minitab") and interaction plots ("What Is An Interaction? - Minitab") will be used to separate out the three factors.

3.4.1 Effect of species and roasting degree

3.4.1a Effect of species and roasting degree on melanoidin content To determine the effect of species on melanoidin content, mean melanoidin content of unfermented bicolor over the raw, mildly roasted and heavily roasted samples was compared to mean melanoidin content of unfermented cacao over the raw, mildly roasted and heavily roasted samples. So mean (BUR, BUM, BUH) was compared to mean (CUR, CUM, CUH). And mean (GFR, GFM, GFH) was compared to mean (CFR, CFM, CFH). This way only one factor is varied, in this case species. Therefore if different results are found, these are likely to be caused by the varied factor. This is made visible in the main effects plots. The effect of roasting degree on melanoidin content of the different species is made visible in the interaction plots. If the lines in the interaction plots are not parallel, there may be an interaction. That is to say; roasting degree may not impact melanoidin content of every species in the same way. This is an interaction. Results are shown below inFigure 14.

FIGURE 14 MAIN EFFECTS PLOTS AND INTERACTION PLOTS FOR THE EFFECT OF SPECIES AND ROASTING DEGREE ON MELANOIDIN CONTENT

3.4.1b Effect of species and roasting degree on melanoidin antioxidant capacity Antioxidant capacity is given in trolox equivalents (TE) as mmol trolox/g of melanoidins. Comparisons were made as explained in section 3.2.1a. Results are shown below in Figure 15.

FIGURE 15 MAIN EFFECTS PLOTS AND INTERACTION PLOTS FOR THE EFFECT OF SPECIES AND ROASTING DEGREE ON MELANOIDIN ANTIOXIDANT CAPACITY

3.4.2 Effect of fermentation and roasting degree

3.4.2a Effect of fermentation and roasting degree on melanoidin content To determine the effect of fermentation on melanoidin content, mean >20 kDa content of (CUR, CUM, CUH) was compared to mean >20 kDa content of (CFR, CFM, CFH). This is visible in the main effect plot. The effect of roasting on the melanoidin content of fermented or unfermented cacao is visible in the interaction plot. Results are shown below in Figure 16.

FIGURE 16 MAIN EFFECTS PLOT AND INTERACTION PLOT FOR THE EFFECT OF 3.4.2b Effect of fermentation and roast on melanoidin antioxidant capacity FERMENTATION AND ROASTING DEGREE ON MELANOIDIN CONTENT

3.4.2a Effect of fermentation and roasting degree on melanoidin antioxidant capacity Antioxidant capacity is given in trolox equivalents (TE) as mmol trolox/g of melanoidins. Comparisons were made as explained in section 3.2.2a. Results are shown below in Figure 17.

FIGURE 17 MAIN EFFECTS PLOT AND INTERACTION PLOT FOR THE EFFECT OF FERMENTATION AND ROASTING DEGREE ON MELANOIDIN ANTIOXIDANT CAPACITY

3.4.3 Effect of roasting degree overall

3.4.3a Effect of roasting degree overall on melanoidin content To determine the average effect of roasting on all samples, the mean melanoidin content of all raw samples was compared to the mean melanoidin content of all mildly roasted samples and the mean melanoidin content of all heavily roasted samples. So mean (BUR, GFR, CUR, CFR) was compared with mean (BUM, GFM, CUM, CFM) and with mean (BUH, GFH, CUH, CFH). This is FIGURE 18 MAIN EFFECTS PLOT FOR THE visible in Figure 18. OVERALL EFFECT OF ROASTING DEGREE ON 3.4.3b Effect of roasting degree overall on MELANOIDIN CONTENT melanoidin antioxidant capacity Antioxidant capacity is given in trolox equivalents (TE) as mmol trolox/g of melanoidins. Comparisons were made as explained in section 3.2.3a. Results are shown in Figure 19.

FIGURE 19 MAIN EFFECTS PLOT FOR THE OVERALL EFFECT OF ROASTING DEGREE ON MELANOIDIN ANTIOXIDANT CAPACITY

4. Discussion

4.1 Measuring error versus found effects/ Reliability Several experiments were not repeated and thus could not be statistically analyzed. However, due to the precise and conscientious working method followed during laboratory work and the setup of the experiments, it is estimated that there is a measuring error maximum of 10%. That is to say, when two samples differ from one another by over 10 percent, the samples are assumed to be significally different.

4.2 Noise factors/ Variability

4.2.1 Reproducibility earlier research The exact methods used in earlier research were not always documented to an extent that made the methods easily reproducible. This can lead to variability in methods used by different researchers or students. To mitigate this, methods were very extensively documented in section 2.4 and the non- standard protocols written based on these adapted methods can be requested from the author.

4.2.2 Bean origin The grandiflorum beans and the bicolor beans come from different sources than the cacao beans. The growing conditions and pre- and post-harvest treatment of the bicolor and grandiflorum beans were different from the cacao beans. For the cacao beans the only difference between the fermented and unfermented samples was the fermentation; growing conditions et cetera were the same. Differences between grandiflorum and fermented cacao could be caused by species, but also by the different growing conditions, pre- and post-harvest treatments, or by different fermentation dynamics. Grandiflorum and fermented cacao are both fermented; that does not mean the fermentation process was the exact same. The same applies to bicolor and unfermented cacao; there could be multiple reasons for differences between the samples. Therefore the comparison between fermented cacao and grandiflorum, and unfermented cacao and bicolor, is less precise than the comparison between fermented and unfermented cacao. The two types of cacao investigated simply have fewer uncontrolled reasons for variance. However, as species is likely to have a larger impact on the samples than growing conditions and so on, a comparison can be made between bicolor, cacao and grandiflorum. The variability caused by the different sources of the beans could not be separated from the species variation and therefore was not reported separately.

4.2.3 Roasting degree Weight regain by the roasted nibs and lack of correlation between nib colour change and weight loss during roasting make weight loss during roasting alone a slightly less reliable indicator for roasting degree at a given temperature. Sample placement in the oven and oven type can also impact roasting. To avoid this causing variability, 3 steps were taken:

- Temperature and humidity in the cooldown and storage post-roasting area, the FQD Food Safe Laboratory, were tracked during one week. This week was assumed to be representative and the results can be found in the appendices. Temperature fluctuated between 19.9 and 24.4°C. Humidity fluctuated between 23 and 27%. Detailed results can be found in appendix 7.1.1 Post-roasting nib weight regain; effect of storage environment. - The same convection oven was used for all roasting activities and samples were evenly distributed in the oven. Mildly roasted samples BUM and GFM were roasted simultaneously.

Heavily roasted samples BUH and GFH were roasted simultaneously. These roasting steps took place without interruption (oven door was not opened) for the specified roasting duration. - Samples were extensively characterised and differed quite clearly from one another when looking at sample characteristics.

4.3 Unexpected results

4.3.1 Low melanoidin yield Low melanoidin content was found in general, which was not entirely consistent with the results found previously at FQD (Geerts, 2015). Possible causes for this include the usage of different methods for several steps in the melanoidin extraction. These different methods were adapted from the original methods used by Geerts to increase their suitability for use in the FQD laboratories.

4.3.2 Possible contamination of melanoidin fraction As mentioned before it has been assumed that the >20 kiloDalton fraction of the watery extract of the defatted, roasted beans consists of melanoidins and a negligible amount of contaminations. The melanoidin fraction found in the bicolor samples was very light (see appendix 7.4 Colour bicolor melanoidins). As melanoidins are generally described as being brown in colour, this was slightly unexpected. The melanoidin fraction may have been contaminated. Possible sources of contamination have been investigated.

4.3.2a Plastic compound migration from tubes In defatting pilots it was noticed that the plastic (PPCO , PP) centrifuge tubes available deformed in the presence of petroleum ether. PPCO and PP are not suitable for use with petroleum ether. To avoid contamination by compounds (e.g. plastic softeners) that migrated from centrifuge tubes in the presence of petroleum ether to the sample, the samples were allowed to settle overnight instead in the glass Duran flasks used as defatting vessels, avoiding centrifugation altogether. Thus contamination by migrated plastic compounds during defatting was not possible as the defatting method was adapted to avoid this risk.

4.3.2b Fat The samples were thoroughly defatted, with 6 to 8 maceration repetitions. An excruciatingly small amount of fat may have been left in the samples (<1%) as extracting an absolute 100% of anything isn’t realistically feasible.As the decrease of fat per maceration cycle was not tracked in this thesis, fat contamination remains theoretically possible. However, as the defatting method was adapted to mitigate this risk, it is extremely unlikely.

4.3.2c Microbiological Optical microscopy (40x magnification) showed live micro-organisms in watery extracts that had been stored during 5 days at 5°C. A variety of micro-organisms was seen; these were not fully identified. Amoung them were yeasts, cocci, and rods-shaped organisms of various sized. It is not certain, but definitely possible that the melanoidin fractions were contaminated with micro-organisms that grew in the extract pre- and during ultrafiltration.

4.3.2d Insoluble particles The extract was filtered through Whatman filters 4, 44 and 602 to remove all insoluble particles. Whatman filter 602 was the finest filter used and has a 2 micron pore size. Particles smaller than 2 micron could have passed through this filter and remained in the extract.

4.3.2e Ultrafiltration Contamination by compounds smaller than 20 kiloDalton is assumed to be negligible due to the three washing steps carried out during ultrafiltration.

4.3.2f Proteins and polysaccharides The amound of non-melanoidin compounds >20 kDa fraction has been assumed to be negligible. However, this assumption has not been investigated and is therefore unconfirmed. There may be proteins and/or polysaccharides that did not decompose as expected during roasting present in the melanoidin fraction. This would explain one of the difficulties noticed during ultrafiltration; some samples formed a thin gel-like layer on top of the 20 kDa filter. This was seen in the bicolor samples but whether it occurred in the other samples is unknown. The gel was colourless and could be removed by spraying water on it or gently rubbing the filter under running (demineralised )water. The gel layer was found to slow down ultrafiltra tion drastically. The gel tore easily and could be rolled into a small, dense, colourless ball. It is not known what the gel consisted of, although it is likely to be proteins or polysaccharides. This is in line with the possible contamination of the melanoidin fraction by polysaccharides and/or proteins that did not decompose during roasting.

5. Conclusion

5.1 Sample characterisation All 12 samples were found to have significantly different sample characteristics, caused by the investigated variation on species, fermentation and roasting degree.

5.2 Impact of Theobroma species, fermentation and roasting degree on melanoidin yield and antioxidant capacity

5.2.1 Species The main effects plots in Figure 14 show that unfermented bicolor had a lower melanoidin content than unfermented Forastero cacao, and fermented grandiflorum had a higher melanoidin content than fermented Forastero cacao.

The main effects plots in Figure 15 show that melanoidins from both bicolor and grandiflorum had a lower antioxidant capacity than their cacao counterparts (melanoidins from respectively unfermented and fermented cacao)

The interaction plots in Figure 14 and Figure 15 show that roasting degree and species have an interaction. Roasting degree did not impact all three Theobroma species in the same way .

5.2.2 Fermentation The main effects plots show fermented cacao had a lower melanoidin content (Figure 16) and antioxidant capacity (Figure 17) than unfermented cacao. Fermentation was found to decrease melanoidin content and antioxidant capacity.

Roasting degree had an interaction effect with fermentation of cacao, as shown in Figure 16 and Figure 17. The melanoidin content decreased much more from raw to roasted samples in fermented cacao than in unfermented cacao. This could indicate the polysaccharides and proteins may have been more decomposed with roasting in fermented cacao. It could also indicate that more melanoidin formation took place in unfermented cacao, which is likely as antioxidant capacity for mildly roasted unfermented cacao melanoidins was relatively high. The effect of mildly roasting the cacao versus heavily roasting it mildly was similar in fermented and unfermented cacao.

5.2.3 Roasting degree Roasting did not impact the four bean types (BU, GF, CU, CF) in the same way. Overall mildly roasted samples had a higher melanoidin content (Figure 18) and antioxidant capacity (Figure 19) than heavily roasted samples. The interaction plots inFigure 14, Figure 15, Figure 16 and Figure 17 show that roasting degree has an interaction with the effects of species and fermentation.

5.2.4 Highest melanoidin content and antioxidant capacity As shown in section 3.3.2 and 3.3.3 (Table 5) mildly roasted unfermented bicolor and fermented grandiflorum actually had the highest concentration of melanoidins in the extract. Fermented grandiflorum had a higher melanoidin concentration in the extract than unfermented cacao. Extracts of both mildly and heavily roasted grandiflorum and bicolor (BUM, BUH, GFM, GFH) contained more melanoidins than any roasted cacao extracts (CUM, CUH, CFM, CFH) This even though fermentation lowered melanoidin content in cacao, and mildly roasted samples generally contain more melanoidins than heavily roasted samples. However, as the water holding capacity was higher for bicolor and

35 grandiflorum than for cacao, less of the extract per gram of sample could be ultrafiltrated. This made the melanoidin content actually found in this research (melanoidin yield) for bicolor and grandiflorum lower than of cacao. Antioxidant capacity of cacao was higher than of bicolor and grandiflorum. When looking at the melanoidin content actually found, unfermented mildly roasted cacao (CUM) had the highest melanoidin content. CUM also had the highest melanoidin antioxidant capacity.

5.2.5 In summary; answers to section 1.4 In answer to the original research question; a higher melanoidin content was found in unfermented cacao than in fermented cacao. A higher melanoidin content was found in mildly roasted cacao than in heavily roasted cacao. Likewise, the antioxidant capacity of melanoidins from unfermented cacao was higher than of melanoidins from fermented cacao. The antioxidant capacity of melanoidins from mildly roasted cacao was higher than the antioxidant capacity of melanoidins from heavily roasted cacao. Mildly roasted unfermented cacao had the highest melanoidin content and antioxidant capacity.

With respect to the bicolor and grandiflorum samples, it was found that the estimated total melanoidin content was higher than of the compared cacao samples. However, due to bicolor and grandiflorum’s greater water holding capacity, the amount of melanoidins actually found was lower than that of the compared cacao samples.

As can be seen from the high content of >20 kDa compounds in the raw samples, the melanoidin extraction process using ultrafiltration was not selective for specifically melanoidins. Other large molecules including polysaccharides and proteins are also extracted. The process used was molecular size specific. Because of the heterogeneity of melanoidins an exclusively melanoidin-specific seperation process is currently not available. This is also a possible explaination for the light colour of the found bicolor melanoidins.

5.3 Recommendations on further research

5.3.1 Melanoidin extraction process Perhaps the melanoidin extraction process could be further optimized using the following suggestions:

- To validate the defatting process, I advise tracking the defatting dynamics. This has already been done in follow-up research at FQD by Beuker. - Live micro-organisms were found in stored excess extract. It is unknown whether viable micro- organisms are present in the freeze-dried melanoidins. Beuker’s follow-up research at FQD will focus on melanoidins and lower gut fermentation. To avoid contamination of the microbial assays by micro-organisms from the melanoidins, it is advisable to sterilise or pasteurise the melanoidins before use. - In this thesis controlling the particle size for extraction and sample characterisation was thought to be important. Therefore I finely ground the defatted samples and sieved them. If in further research this controlling of sample particle size is not important, I advise to skip the fine grinding and sieving step as this step can be quite time-consuming. With less finely ground powder, cutting or blending the sample particles into the water is necessary to achieve optimal extraction. An Ultra-Turrax disperser, as it uses blade dispersion, could perhaps be used to homogenise the samples instead of vortexing them in the extraction step (section 2.4.4). - Most importantly, in this thesis single-step extration was used because it was logistically more convenient. Table 3 Overview of sample characteristics shows the samples had a high water

holding capacity; 2.1 to 3.8 mililitres of water were retained per gram of finely ground defatted sample. A sample:water ratio of 1:10 was used. This means that 21-38% of the extract made in the extraction step is retained by the insoluble fraction of the sample. By switching to a multiple-step extraction process, a large part of this held extract can be released. For example say 1/3 of the extract is retained by the insoluble pellet. By re-extracting the pellet once (switching to a two-step extraction), 2/3 of that 1/3 that was retained can be released from the pellet. This drastically increases the amount of extracted compounds; instead of 2/3 of the extracted compounds being found, 8/9 of the extracted compounds would be found. This can also be seen in Table 5 column “estimated extraction yield”. The probable increase in extraction efficacy, due to the high water holding capacity found, by switching to a multiple- step extraction process, is large enough to make a multiple-step extraction process worth the greater logistic difficulcy. A change to a multiple-step extraction process can be considered.

5.3.2 Suggestions for further research For further research on cacao melanoidins, mildly roasted unfermented cacao seems the most suitable.

The intention with the raw samples was to compare the amount of very low molecular weight compounds (<2 kDa) in the raw samples and the roasted samples. This would have indicated whether the small compounds might aggregate or polymerise into the high molecular weight melanoidins. However, due to time constraints, the <20 kDa compounds were not further investigated. The <20 kDa compounds for all samples were frozen and stored; at the time of writing they are available for further research.

Dutching at different points in the cacao processing (see section 1.3) is likely to have an impact cacao melanoidins. The effects of e.g. Dutching pre-roasting versus Dutching post-roasting and not Dutching altogether would be interesting to research.

Research has been done on the impact of incomplete fermentation on cacao, but the impact of fermentation style on melanoidin content and antioxidant capacity is unknown. Fermentation styles such as low temperature long time versus high temperature short time fermentation, or heap fermentation versus box fermentation, could impact melanoidin content and antioxidant capacity and would be interesting to research.

Two Theobroma species, bicolor and grandiflorum, that are almost entirely unknown in the Western world have been studies in this thesis. The Theobromae genus contains over 20 different species. Further research on the properties and especially possible uses of the non-cacao Theobroma species would be very interesting.

6. References (Aprotosoaie, Luca and Miron, 2015) Aprotosoaie, A., Luca, S. and Miron, A. (2015). Flavor Chemistry of Cocoa and Cocoa Products-An Overview. Comprehensive Reviews in Food Science and Food Safety, 15(1), pp.73-91.

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ChocoSol Traders chocolate brand: https://chocosoltraders.com/

(Cohen and Jackix, 2005) Cohen, K. and Jackix, M. (2005). Estudo do liquor de cupuaçu. Ciência e Tecnologia de Alimentos, 25(1), pp.182-190.

(Directive 2000/36/EC) The European Parliament, and The Council of the European Union. "Directive 2000/36/EC Of The European Parliament And Of The Council Of 23 June 2000 Relating To Cocoa And Chocolate Products Intended For Human Consumption". Official Journal of the European Communities (3/8/2000): L 197, page 19- 25. Print.

(Fallico, Arena and Zappalà, 2003) Fallico, B., Arena, E. and Zappalà, M. (2003). Roasting of hazelnuts. Role of oil in colour development and hydroxymethylfurfural formation. Food Chemistry, 81(4), pp.569-573.

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(Geerts, 2015) Geerts, L. (2015). ‘Melanoidins extraction from roasted cocoa using a factorial design 2^3’. BLT thesis. Wageningen University & Research, chairgroup Food Quality and Design. Unpublished

(Gökmen, Serpen and Fogliano, 2009) Gökmen, V., Serpen, A. and Fogliano, V. (2009). Direct measurement of the total antioxidant capacity of foods: the ‘QUENCHER’ approach. Trends in Food Science & Technology, 20(6- 7), pp.278-288.

(Green, 2010) Green, J. (2010). Feasting with Foam: Ceremonial Drinks of Cacao, Maize, and Pataxte Cacao. In: J. Staller and M. Carrasco, ed., Pre-Columbian Foodways: Interdisciplinary Approaches to Food, Culture, and Markets in Ancient Mesoamerica, 1st ed. New York, NY: Springer Science+Business Media, LLC, pp.315-340.

(Kim and Lee, 2008) Kim, J. and Lee, Y. (2008). Effect of reaction pH on enolization and racemization reactions of glucose and fructose on heating with amino acid enantiomers and formation of melanoidins as result of the Maillard reaction. Food Chemistry, 108(2), pp.582-592.

Madre chocolate brand: http://madrechocolate.com/

(Morales, Somoza and Fogliano, 2010) Morales, F., Somoza, V. and Fogliano, V. (2010). Physiological relevance of dietary melanoidins. Amino Acids, 42(4), pp.1097-1109.

(Moser, 2015) Moser, A. (2015). Alkalizing Cocoa and Chocolate. The Manufacturing Confectioner, 95(6), pp.31- 38.

(Motomayor et al., 2008) Motamayor, J., Lachenaud, P., da Silva e Mota, J., Loor, R., Kuhn, D., Brown, J. and Schnell, R. (2008). Geographic and Genetic Population Differentiation of the Amazonian Chocolate Tree (Theobroma cacao L). PLoS ONE, 3(10), p.e3311.

(Oliviero et al., 2009) Oliviero, T., Capuano, E., Cämmerer, B. and Fogliano, V. (2009). Influence of Roasting on the Antioxidant Activity and HMF Formation of a Cocoa Bean Model Systems. Journal of Agricultural and Food Chemistry, 57(1), pp.147-152.

(Pugliese et al., 2013) Pugliese, A., Tomas-Barberan, F., Truchado, P. and Genovese, M. (2013). Flavonoids, Proanthocyanidins, Vitamin C, and Antioxidant Activity of Theobroma grandiflorum (Cupuassu) Pulp and Seeds. Journal of Agricultural and Food Chemistry, 61(11), pp.2720-2728.

(Reisdorff et al., 2004) Reisdorff, C., Rohsius, C., Claret de Souza, A., Gasparotto, L. and Lieberei, R. (2004). Comparative study on the proteolytic activities and storage globulins in seeds of Theobroma grandiflorum (Willd ex Spreng) Schum and Theobroma bicolor Humb Bonpl, in relation to their potential to generate chocolate-like aroma. Journal of the Science of Food and Agriculture, 84(7), pp.693-700.

(Silvan,́ Morales and Saura-Calixto, 2010)Silvan,́ J., Morales, F. and Saura-Calixto, F. (2010). Conceptual Study on Maillardized Dietary Fiber in Coffee. Journal of Agricultural and Food Chemistry, 58(23), pp.12244-12249.

(Suazo, Davidov-Pardo and Arozarena, 2014) Suazo, Y., Davidov-Pardo, G. and Arozarena, I. (2014). Effect of Fermentation and Roasting on the Phenolic Concentration and Antioxidant Activity of Cocoa from Nicaragua. Journal of Food Quality, 37(1), pp.50-56.

(Summa et al., 2008) Summa, C., McCourt, J., Cämmerer, B., Fiala, A., Probst, M., Kun, S., Anklam, E. and Wagner, K. (2008). Radical scavenging activity, anti-bacterial and mutagenic effects of Cocoa bean Maillard Reaction products with degree of roasting. Molecular Nutrition & Food Research, 52(3), pp.342-351.

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(Tedlock, 1996) Tedlock, D. (1996). Popol Vuh. 1st ed. New York [etc.]: Simon & Schuster, pp.111, 146.

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("What Is A Main Effects Plot? - Minitab") "What Is A Main Effects Plot? - Minitab". Support.minitab.com. N.p., 2017. Web. 27 Apr. 2017.

("What Is An Interaction? - Minitab") "What Is An Interaction? - Minitab". Support.minitab.com. N.p., 2017. Web. 27 Apr. 2017.

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(Windhab, 2008) Windhab, E. J. (2008) Tempering, in Industrial Chocolate Manufacture and Use, Fourth Edition (ed S. T. Beckett), Wiley-Blackwell, Oxford, UK. doi: 10.1002/9781444301588.ch13, pp.276-280

Figure 2: Christian, M. (2017). Chocolate Processing Flowchart. [online] the C-spot. Available at: https://www.c- spot.com/editorials/chocolate-processing-flowchart/ [Accessed 29 Apr. 2017].

FQD protocol 32. Bakker, 2014. Use lab Master-AW

FQD protocol 36. Van Twisk & Bakker, 2014. Use HunterLab Colorflex

FQD protocol number 41. Van Twisk, 2014. Anti oxidant measurement quencher procedure

7 Appendices

7.1 Roasting

7.1.1 Post-roasting nib weight regain; effect of storage environment Post-roasting nib weight regain at different humidity 0 0 1000 2000 3000 4000 -1

-2

-3 inside desiccator

-4 outside desiccator

m samples samples m (%) Δ -5

-6

-7 time (min)

temperature in foodsafe lab 8 november 11:06 to 14 november 11.30 30

15 T (˚C) T max (˚C) T min (˚C) temperature (˚C) 10

0 0 1000 2000 3000 4000 5000 6000 7000 t (min)

humidity in foodsafe lab 8 november 11:06 to 14 november 11.30 30

15 humidity (%) humidity max (%) humidity humidity (%) humidity min (%) 10

0 0 2000 4000 6000 8000 t (min)

7.1.2 Correlation between colour, roasting time and weight loss ΔL*vs Δweight 0 0 0.5 1 1.5 2 2.5 3

-5

-10 ΔL* (%) BM3

ΔL* (%) BM2 L* L* (%) Δ ΔL* (%) GM3 -15 ΔL* (%) GM2

-20

-25 Δm (%)

Δa* vs Δweight 50

20 Δa* (%) BM3

Δa* (%) BM2 a* a* (%)

Δ 10 Δa* (%) GM3 Δa* (%) GM2 0 0 0.5 1 1.5 2 2.5 3

-10

-20 Δm (%)

Δb* vs Δweight 20

0 0 0.5 1 1.5 2 2.5 3 Δb* (%) BM3 -5 Δb* (%) BM2

b* (%) b* -10 Δ ΔL* (%) GM3 -15 Δb* (%) GM2 -20

-25

-30

-35 Δm (%)

7.1.3 Colour roughly ground samples L*

Interval Plot of L* vs sample 95% CI for the Mean 80

* 50 L

20 BUH BUM BUR CFH CFM CFR CUH CUM CUR GFH GFM GFR sample Individual standard deviations are used to calculate the intervals.

Games-Howell Pairwise Comparisons

Grouping Information Using the Games-Howell Method and 95% Confidence sample N Mean Grouping

BUR 8 73.4887 A BUM 8 72.1612 B BUH 8 62.7050 C CUH 8 42.9587 D CUM 8 42.9200 D CUR 8 42.3975 E GFR 8 41.528 F CFM 8 38.0337 G CFR 8 37.9337 G GFM 8 35.9100 H CFH 8 35.8712 H GFH 8 21.9387 I Means that do not share a letter are significantly different a*

Interval Plot of a* vs sample 95% CI for the Mean 16

* a 8

2 BUH BUM BUR CFH CFM CFR CUH CUM CUR GFH GFM GFR sample Individual standard deviations are used to calculate the intervals.

Games-Howell Pairwise Comparisons

Grouping Information Using the Games-Howell Method and 95% Confidence sample N Mean Grouping GFM 8 15.0838 A CFR 8 14.0550 B GFR 8 13.8913 C CFM 8 12.9438 D CFH 8 11.2438 E CUR 8 10.7538 F GFH 8 10.5888 F CUH 8 10.2787 G CUM 8 9.2637 H BUH 8 7.4838 I BUM 8 3.1975 J BUR 8 2.74375 K

Means that do not share a letter are significantly different b*

Interval Plot of b* vs sample 95% CI for the Mean 30

* 20 b

10 BUH BUM BUR CFH CFM CFR CUH CUM CUR GFH GFM GFR sample Individual standard deviations are used to calculate the intervals.

Games-Howell Pairwise Comparisons

Grouping Information Using the Games-Howell Method and 95% Confidence sample N Mean Grouping BUH 8 28.6325 A GFM 8 24.0675 B GFR 8 23.1875 C BUM 8 22.2437 D CFR 8 21.5212 E BUR 8 20.8825 F CUR 8 20.3075 G CUH 8 18.9325 H CFM 8 18.4113 I CUM 8 16.9312 J CFH 8 16.0138 K GFH 8 11.7413 L

Means that do not share a letter are significantly different.

7.1.4 Water activity

Interval Plot of water activity vs sample 95% CI for the Mean 0.5

0.4

y t

i 0.3

e t

a 0.2 w

0.1

0.0 BUH BUM BUR CFH CFM CFR CUH CUM CUR GFH GFM GFR sample Individual standard deviations are used to calculate the intervals.

Games-Howell Pairwise Comparisons

Grouping Information Using the Games-Howell Method and 95% Confidence sample N Mean Grouping BUR 3 0.458667 A CUR 3 0.43400 A GFR 3 0.35600 B CFR 3 0.34267 B CUM 3 0.29400 C CFM 3 0.28933 C CUH 3 0.23800 D CFH 3 0.22900 D BUM 3 0.10000 E GFM 3 0.09633 E GFH 3 0.07400 E F BUH 3 0.06067 F

Means that do not share a letter are significantly different.

7.2 Defatting

7.2.1 Fine grinding sample yield fine grinding (%)

CUR 89.4

CUM 93.7

CUH 97.5

CFR 88.7

CFM 83.5

CFH 82.8

BR 90.1

BM 91.0

BH 96.6

GR 98.8

GM 94.8

GH 96.4

7.2.1 pH

Interval Plot of pH vs sample 95% CI for the Mean 6.75

6.50

6.25

6.00 H

p 5.75

5.50

5.25

5.00

BUH BUM BUR CFH CFM CFR CUH CUM CUR GFH GFM GFR sample Individual standard deviations are used to calculate the intervals.

Games-Howell Pairwise Comparisons

Grouping Information Using the Games-Howell Method and 95% Confidence sample N Mean Grouping BUR 3 6.58000 A BUM 3 6.48000 B BUH 3 6.36333 C CUR 3 6.07333 D CUM 3 6.06667 D CUH 3 5.98000 E GFR 3 5.82000 F

GFH 3 5.5833 G GFM 3 5.56667 G CFH 3 5.06000 H CFR 3 5.01333 I CFM 3 4.88667 J

Means that do not share a letter are significantly different.

7.2.3 Foaming capacity Foaming capacity of alkalised Blooker cacao (pilot) vs. sample:water ratio 30

22.5 FC (%) +F FC (%) -F

7.5 foaming capacity foamingcapacity (volume increase) %

0 4 8 12 16 20 24 sample:water ratio (g sample:mL water)

Foaming capacity (%) 30 25 25

15 14 14 14 9 9 10 5 5 2 0 0 0 0 0

% % volumeincrease uponfoaming sample

7.3 Extraction

7.3.1 Water holding capacity

Interval Plot of WHC (mL/g) vs sample 95% CI for the Mean 4.0

3.5

)

/ L

m 3.0

(

H W

2.5

2.0

BUH BUM BUR CFH CFM CFR CUH CUM CUR GFH GFM GFR sample Individual standard deviations are used to calculate the intervals.

Games-Howell Pairwise Comparisons

Grouping Information Using the Games-Howell Method and 95% Confidence sample N Mean Grouping BUH 6 3.8012 A GFH 6 3.5767 A B C BUR 3 3.5591 A B GFM 6 3.3779 B C GFR 4 3.3393 B C BUM 6 3.3133 C CUH 20 2.97242 D CUR 4 2.83098 E CUM 19 2.4326 F CFR 4 2.32753 G CFM 6 2.2512 G CFH 20 2.0553 H

Means that do not share a letter are significantly different.

7.3.2 Soluble and insoluble content

Soluble and insoluble content of defatted beans 100 90 80 70 60 50 insoluble 40 soluble 30

% of dry % ofdry starting mass 20 10 0 BUR BUM BUH GFR GFM GFH CUR CUM CUH CFR CFM CFH sample

sample % % insoluble soluble BUR 76.6 10.9 BUM 79.7 11.2 BUH 82.2 8.9 GFR 75.8 12.3 GFM 79.1 11.1 GFH 82.1 9.2 CUR 70.4 16.5 CUM 75.0 15.5 CUH 78.3 13.8 CFR 71.6 16.3 CFM 74.6 12.6 CFH 78.4 12.8

7.4 Colour bicolor melanoidins

Ethical appendix De ethische aspecten van onderzoek naar cacao-melanoïden

Door: Suzi Pijnenburg Registratienummer: 940819675120 Datum: 9 december 2016 Vakcode: YFS-80824

De morele implicaties van onderzoek naar cacao Oftewel: waarom cadeautjes voor je vader kopen soms veel ingewikkelder blijkt dan je zou denken.

Een redelijk normale dag Ik liep door de supermarkt, zoekende naar een kleinigheidje voor mijn vader. Na wat gedenk besloot ik chocolade voor hem mee te nemen; nu nog kiezen welke. In zo’n chocolade schap is immers vrij veel keuze. Mijn oog viel op een Tony’s Chocolonely-reep (22); deze chocolade is heerlijk én wordt op een verantwoorde manier geproduceerd. Zonder slavernij of kinderarbeid; maar daarover later meer. Ook viel mijn oog op een klein reepje wat heel gezond overkwam. Het was rauwe, veganistische chocolade met probiotica erin van het merk Ombar. Vrij interessant vond ik het, een chocoladeproduct wat in de markt wordt gezet als gezond. Zouden consumenten zoiets snel geloven? Als studente Levensmiddelentechnologie leer je over gezondheidsclaims op voeding, en dat die vaak niet het hele verhaal vertellen. Zo bevat pindakaas met minder vet inderdaad 30% minder vet, maar zit er ineens wel flink wat glucosestroop in (8). Dus zo’n light product hoeft helemaal niet gezonder te zijn dan de originele versie; gezondheidsclaims staan voor exact wat er staat. Niet meer, niet minder dan wat wettelijk gezien nodig is. Ik hoopte dat consumenten nu niet zouden gaan denken dat ze ongelimiteerd Ombar-chocolade konden eten, alleen omdat er wat gezonde aspecten aan verbonden zijn.

Die gezonde aspecten zijn niet het enige interessante aan Ombar-repen; er zit ook cacao in. Maar hoe is die cacao verbouwd?

Een reep slaaf-vrije chocolade Mijn BLT-thesis gaat, ruw gezegd, over cacao. In de cacao-industrie stinkt er wat; een deel van de cacao wordt verbouwd en verkocht voor belachelijk lage prijzen. Dit is mogelijk door uitbuiting van de cacaoboeren.

De meeste chocolade wordt geproduceerd met cacaobonen van allerlei verschillende afkomsten. Het is daardoor moeilijk om die cacao allemaal te traceren. De boeren die de cacaobonen produceren worden vaak onderbetaald of er wordt zelfs gebruik gemaakt van slaven- of kinderarbeid. Dit gebeurt om met de lage marktprijs van cacao alsnog een mooie winst te maken als cacaoproducent. Het Nederlandse televisieprogramma Keuringsdienst van Waarde heeft hier in 2003 een aflevering over uitgezonden (3). In 2005 kwamen er twee vervolgafleveringen (13, 14). In 2010 heeft het programma EenVandaag ook een aflevering over de duurzaamheid en productiemethodes van chocoladeproducten uitgezonden (4).

Er is veel geschreven over deze schrijnende problemen binnen de cacao industrie. Er is sinds 2003 al veel verbeterd. Sinds kort kan zelfs cacaoboter getraceerd/Fair Trade geproduceerd worden op industriële schaal! Dat was hiervoor niet mogelijk. Niet door technische limitatie, maar omdat er niet genoeg interesse was in duurzame cacaoboter. Blijkbaar is die interesse er nu wel (1). Zo merk ik zelf dat in mijn omgeving Tony’s Chocolonely, met hun nadruk op het produceren van slaafvrije cacao, veruit de meest populaire chocolade is. Omdat het je een goed gevoel geeft slaafvrije chocolade te kopen, maar ook omdat het gewoon lekker is.

Er is echter al zoveel geschreven over de hierboven aangestipte problemen in de cacao industrie, dat ik niet het idee heb iets bij te dragen door er ook over te schrijven. Ik kan wel zoor de zoveelste keer de bekende feiten herhalen. Ze bij elkaar zetten en mijn mening erover geven. Maar mijn mening is simpel; ik wil dat cacaoboeren fatsoenlijk kunnen leven van hun werk als cacaoboer. Ik wil dat er geen slavernij gebruikt is bij het produceren van mijn chocolade. Dat hoeft niet nog eens herhaald te worden. Dat betoog is al geschreven; dat bijltje al doorgehakt. Al vele keren zelfs. Om in het dagelijks leven mee te werken aan dat doel voor de chocolade industrie, koop ik voor zover dat mogelijk is op mijn studentenbudget chocolade van traceerbare bonen en afkomsten, waarbij de cacaoboeren fatsoenlijk betaald worden voor hun bonen. Dat kost meer, en die meerprijs betaal ik graag. Dan maar minder chocolade die me beter smaakt. Mijn thesis gaat alleen niet echt over chocolade.

Een thesisonderwerp Het precieze onderwerp van mijn BLT-thesis is de invloed van roosteren en fermentatie op melanoïden in cacao. Daarnaast doe ik onderzoek naar de melanoïden in zaden van Theobroma grandiflorum (cupuaçu) en Theobroma bicolor (pataxte), twee soorten tropische bomen uit hetzelfde geslacht als de cacaoboom. Aangezien de cacaoboom nauw verwant is aan deze andere soorten zouden de melanoïden uit de zaden (bonen) van deze bomen soortgelijke eigenschappen kunnen hebben als de cacao-melanoïden. Melanoïden zijn bruingekleurde moleculen, herkenbaar aan hun hoge moleculaire massa, die ontstaan tijdens de Maillard reactie. (17) Deze reactie is non-enzymatisch en vindt plaats tijdens het roosterproces, waarbij veel van geur- kleur- en smaakstoffen van de cacao worden gevormd. Fogliano

52 en Morales schatten dat de dagelijkse inname van melanoïden rond de 10 gram (6). In verschillende in vitro experimenten is gevonden dat melanoïden onder andere antioxidatieve (10, 11) , antiglycatieve (12) en prebiotische (2)(stimuleert groei van beneficiële darmflora) eigenschappen kunnen hebben. Er is wetenschappelijk bewijs dat melanoïden niet geheel worden afgebroken in het menselijk verteringsstelsel en dus relatief intact in de dikke darm aankomen, waar ze een antioxidatief effect uitoefenen (7, 9). Er zijn ook hypotheses dat melanoïden een preventief effect zouden kunnen hebben op darmkanker (12, 16). Gezien de vrij hoog liggende schatting van de dagelijkse melanoïde-inname is het zeer relevant om de in vivo effecten van melanoïden verder te bestuderen. Mijn thesis gaat over de opbrengst en antioxidatieve werking van melanoïden uit de eerder genoemde cacao, cupuaçu en pataxte zaden. Door te onderzoeken in wat voor zaden de meeste melanoïden te winnen zijn wordt het mogelijk gemaakt om in de toekomst op efficiëntere wijze melanoïden uit deze zaden te verkrijgen voor verder onderzoek.

Het ethische vraagstuk Waarom gezondheidsclaims ook slecht kunnen zijn.

Het dilemma Moge er gevonden worden dat melanoïden inderdaad voordelige effecten hebben op de gezondheid zouden er in de toekomst wel eens gezondheidsclaims over cacao op chocoladeproducten kunnen komen. Dit veroorzaakt voor mij een ethisch dilemma; zijn gezondheidsclaims wel ethisch verantwoord? Is het überhaupt wel ethisch verantwoord voor mij om onderzoek te doen naar stoffen die een effect op de gezondheid lijken te hebben, terwijl ik donders goed weet dat de gevonden informatie zeer makkelijk misbruikt kan worden om consumenten te misleiden zodat ze meer chocolade kopen en consumeren? Zodat de levensmiddelenindustrie meer geld kan verdienen, ten koste van de gezondheid van naïeve consumenten? Aan de ene kant is het natuurlijk goed om consumenten te informeren over de gezondheidsaspecten van voedsel. Aan de andere kant zagen we in het pindakaasvoorbeeld al dat zulke informatie makkelijk verkeerd kan overkomen. Ik denk dat consumenten minder snel light pindakaas zouden kopen als er ‘pindakaas met 30% minder vet maar wel veel meer suiker’ op de pot zou staan. Consumenten kunnen makkelijk misleid worden door gezondheidsclaims. Naar mijns inzicht komt dat door de ongelooflijke complexiteit van de huidige levensmiddelenproductie; zelfs ik, die er al 4 jaar over studeer op universitair niveau, snap nog lang niet alles over ons voedsel. Voor de gemiddelde consument lijkt het me vrijwel onmogelijk alles over wat hij of zij consumeert te bevatten.

Om verder te komen in dit ethische dilemma zal ik in de onderstaande tekst mijn vraagstuk onder andere vanuit het utilisme en de deontologie benaderen.

De deontologie Eerst een korte uitleg; De ethische stroming van de deontologie is gebaseerd op de ethische theorie van Kant. Volgens Kant heeft elk mens bepaalde universele plichten, zoals het niet aantasten van de autonomie (het zelfbeschikkingsrecht) van andere mensen. Het is belangrijk dat de intentie van acties goed was. Wanneer een mens zijn of haar plichten vervult en zijn/haar acties laat leiden door goede intenties, is hij/zij een goed mens (19).

Het utilisme

Het utilisme, daarentegen, stelt niet de wil of intentie maar het resultaat van een actie voorop in de vraag of een actie ethisch goed was. Het utilisme weegt het positieve effect op het genot of gelukkigheid op een persoon of populatie af tegen het negatieve effect op het genot of de gelukkigheid van personen/ populaties. Als er met een actie meer positieve dan negatieve effecten zijn op genot/gelukkigheid, is dit een goede actie. Het utilisme is begonnen in 1789 bij Jeremy Bentham en zijn werk “An Introduction to the Principles of Morals and Legislation” (18). Hierna verfijnde John Stuart Mill de theorie van Betham door te stellen dat er verschillende graden van genot en geluk waren, waarbij bijvoorbeeld intellectueel genot hoger werd gesteld dan fysiek genot. Intellectueel genot weegt volgens Mill dan ook zwaarder mee dan fysiek genot in de optelsom van positieve en negatieve effecten op gelukkigheid/genot (20)

Een voorbeeld Als je door 100 demente hoogbejaarden die niet lang meer te leven hebben te doden, het leven van 1000 doodzieke kleine kinderen kan redden, is het dan ethisch goed om de bejaarden te vermoorden? Als je de bejaarden laat leven, sterven de kinderen. Als je de bejaarden doodt, leven de kinderen een normaal leven uit; dat wil zeggen, ze zijn dan volledig genezen en zoals normale kinderen. Volgens het utilisme zou het vermoorden van de bejaarden een ethisch goede keuze zijn, omdat het de kinderen veel meer gelukkigheid/genot oplevert voor de algehele populatie van het niet vermoorden van de demente hoogbejaarden. Volgens de deontologie mag je de bejaarden niet als middel voor het genezen van de kinderen gebruiken, maar moet je ze als autonome personen en doel op zich zien. Het zou dan niet ethisch goed zijn om de bejaarden te vermoorden; een van je universele plichten is immers geen mensen te vermoorden. De kinderen zouden dan echter sterven.

Mijn oplossing Drie visies op een vraagstuk

De neutraliteit van kennis Ten eerste is kennis neutraal. Het vergaren van kennis is, mits dat op ethisch verantwoorde wijze gebeurt, ethisch neutraal. Je zou zelfs kunnen stellen dat het vergaren van kennis utilistisch-ethisch goed is, omdat daarmee het intellectueel genot wordt vergroot. Ik vergaar kennis; dat is ethisch prima. In principe is vervolgens de marketeer, die de door mij vergaarde kennis gebruikt om consumenten te misleiden, verantwoordelijk voor de ethische last van het misleiden van consumenten. Dit is geruststellend, en zou mijn dilemma oplossen door mij ethisch vrij te stellen van de consequenties van onderzoek naar voedingscomponenten met mogelijke gezondheidsaspecten. Het is echter in mijn ogen niet zo simpel; door mee te werken aan zulk onderzoek stel ik marketeers in staat gezondheidsclaims te maken. Als die gezondheidsclaims vervolgens zo verwoord worden dat er consumentmisleiding plaatsvindt, ben ik op een bepaalde manier toch deels verantwoordelijk voor de misleiding der consumenten. Dit omdat ik de wettelijk verplichte basis lever waardoor de marketeers überhaupt zulke claims op productverpakkingen mogen zetten. Als deze wetenschappelijke basis niet had bestaan hadden zulke claims immers volgens de Europese wetgeving hierover niet op de verpakking mogen staan. Er waren dan geen consumenten misleid geweest. In het geval van light-pindakaas zou men zelfs kunnen stellen dat het product, door de levensmiddelentechnologen die hieraan hebben gewerkt, speciaal is geformuleerd om mensen te misleiden. Pindakaas met 30% minder vet maar meer suiker is ontegenzeggelijk niet zo gezond als het

54 voor een onwetende consument lijkt. De gemiddelde consument zou zich namelijk naar mijn idee niet zo snel beseffen dat iets (suiker) het volume dat verloren ging door de vetreductie moet opvullen. Die heeft over het algemeen teveel andere zaken aan het hoofd om lang en diep na te gaan denken over light-pindakaas. Deze redenering biedt mij dus geen oplossing voor mijn ethische dilemma.

Kennis brengt (intellectueel) genot Ten tweede, ditmaal vanuit een utilistisch perspectief, ben ik van mening dat voor onderzoekers en producenten het intellectueel genot/geluk brengt om meer te weten te komen over die gezondheidsaspecten. Voor de geïnteresseerde consumenten geldt dit ook.

Het brengt consumenten negatief genot/geluk, d.w.z. ze worden er ongelukkiger van, om misleid te worden en daarachter te komen. Mochten de consumenten niet achter de misleiding komen, blijven ze waarschijnlijk in een gelijke staat van gelukkigheid. Ignorance, they say, is bliss.

De geïnteresseerde consument zal zichzelf waarschijnlijk informeren over gezond voedsel en daardoor minder snel misleid worden. Dit kan bijvoorbeeld door de labels van producten te lezen; op het label light-pindakaas staat gewoon in de ingrediëntenlijst dat er meer suiker in zit. Ook energietabellen kunnen veel informatie hierover leveren.

De ongeïnteresseerde consument kan daarentegen wel snel misleid worden. Als die consument er dan achter komt dat hij (of zij, uiteraard) misleid is, zal die consument ongelukkiger worden.

Ik ben er van overtuigd dat het verlies in gelukkigheid van de ongeïnteresseerd consument door misleiding zeer klein is. Dit omdat de consument, aangezien hij misleid werd, op een basaal niveau niet geïnteresseerd is in de gezondheid van voedsel. Het zal de consument dan niet veel ongelukkiger maken om te weten dat het product wat hij heeft geconsumeerd minder gezond was dan hij dacht, omdat de gezondheid van voedsel simpelweg deze consument niet veel boeit.

De toename in (intellectueel) genot van producenten, geïnteresseerde consumenten, en onderzoekers bij het vergroten van de kennis over de gezondheidsaspecten van voedsel is dan groter dan de afname in genot van de misleidde, ongeïnteresseerde consumenten.

Een simpel voorbeeld; Als een consument serieus gelooft dat, omdat er tomaten in de burgers en ketchup zitten, het gezond is om elke dag bij Burger King te eten, is die consument zodanig ongeïnteresseerd in gezond voedsel dat hij zich duidelijk totaal niet erin heeft verdiept. Erachter komen dat Burger King niet gezond is (of nooit erachter komen en vroeg sterven door overmatige consumptie van ongezond fastfood) zal het geluk van deze consument niet genoeg doen afnemen om te kunnen compenseren voor de toename in gelukkigheid van de geïnteresseerde consumenten, Burger King zelf en de onderzoekers door toename van wetenschappelijke kennis over de gezondheidsaspecten van Whoppers (burgers van Burger King). Dus, in de balans, is er een algemene toename van geluk en is onderzoek naar de gezondheidsaspecten van voedsel ethisch verantwoord.

Kennis draagt bij aan onze autonomie Ten derde lijkt het me dat onderzoek doen naar gezondheidsaspecten en het gebruiken van gezondheidsclaims in de marketing van voedsel geen verplichting geeft aan consumenten dit product ook te consumeren.

We zijn namelijk allemaal autonome personen; wij kiezen zelf wat we consumeren, wij kiezen zelf waar we over leren, wij kiezen zelf hoe we ons ontwikkelen.

We kunnen ervoor kiezen te leren over gezond voedsel. We kunnen er ook voor kiezen te leren over auto’s. Of muziek, of sport, medicijnen, er zijn ontelbare mogelijkheden en met de komst van het internet werd meer leren over specifieke onderwerpen ook ineens veel toegankelijker. Iedereen kiest, op basis van hun interesses, referentiekader en mogelijkheden, zelf waar men over leert.

We kunnen ook zelf kiezen wat we eten. We kunnen elke dag friet bakken en eten. We kunnen alleen brood eten en nooit een warme maaltijd. De wereld ligt, qua opties in voedsel, nu meer voor ons open dan ooit. Ook hierin kiest men wat ze eten. Dit is in mijn ogen een deel van wie je bent als persoon; hoe en wat je consumeert. Voedsel (hier valt ook drinken onder, voor de duidelijkheid) wordt gekoppeld aan herinneringen, aan ervaringen. Voor mij is je op gastronomisch gebied ontwikkelen, een deel van je ontplooiing tot volledig persoon; iets wat je hele leven doorgaat en cumulatief is; iets waarmee je aspecten van jezelf en mensen om je heen leert kennen. Ik besef me dat voor veel mensen eten niet zo belangrijk is, voor mij is het dat wel. Voor veel anderen ook.

Door onderzoek te doen (naar gezondheidsaspecten van voedsel) wordt de beschikbare informatie over voedsel vergroot, en krijgen mensen meer mogelijkheden om te leren over wat ze interesseert. Ze behouden de vrije keuze of ze zich willen verdiepen in dat onderzoek of in die gezondheidsclaims. Daarmee is en blijft het de volledig vrije keuze van onderzoekers en producenten om onderzoek te doen naar gezondheidsaspecten van voedsel, en is en blijft het de volledig vrije keuze van consumenten om zich al dan niet te verdiepen in dat onderwerp. Of het al dan niet consumeren van zogezegd ‘gezond’ voedsel.

Daarom vind ik het deontologisch verantwoord om onderzoek te doen naar en/of producten te marketen met gebruik van de gezondheidsaspecten van voedsel. Hiermee stellen we zij die geïnteresseerd zijn in staat zich te verdiepen in gezond voedsel. Consumenten met interesses die elders liggen worden niet gedwongen bepaald voedsel te consumeren. Zo behoudt iedereen zijn of haar zelfbeschikkingsrecht. Terug naar de Whopper van de Burger King als voorbeeld; het is ieders eigen keuze of ze willen leren over de gezondheidsaspecten de fastfood. Door onderzoek naar gezondheidsaspecten van voedselcomponenten te doen maken wetenschappers het mogelijk voor iedereen zich hierin te verdiepen. Dit is ethisch verantwoord mits het op ethisch verantwoorde wijze plaatsvindt (hierover volgt meer uitleg). Het is ook ieders eigen keuze burgers van Burger King te consumeren en men hoort daarin over zelf een keuze te maken; ik ben niet verantwoordelijk voor de keuzes die anderen hierin maken, omdat ik anders hun autonomie zou aantasten. De keuze aan de consument laten is wat dat betreft in mijn ogen deontologisch-ethisch verantwoord.

Het bijltjesmoment Ter conclusie hak ik deze Gordiaanse knoop door; naar mijn mening is onderzoek doen naar gezondheidseigenschappen van voedsel ethisch verantwoord. Daar zet ik graag nog een kanttekening bij. Ik vind onderzoek naar gezondheidsaspecten van voedsel ethisch verantwoord mits dit op een ethisch verantwoorde manier gebeurt. Dit geldt ook voor algemeen onderzoek naar eigenschappen van voedselcomponenten zoals mijn BLT-thesis. Dit onderzoek ondersteunt bij, of geeft aanleiding tot verder onderzoek naar gezondheidsaspecten van voedsel.

Met onderzoek dat op een ethisch verantwoorde manier plaatsvindt bedoel ik e.g. onderzoek met zo min mogelijk dierproeven, als goed wetenschappelijk onderzoek, eerlijk en onafhankelijk.

Ook het marketen van producten met gebruik van gezondheidsclaims die uit deze onderzoeken naar voren komen vind ik ethisch verantwoord, mits;

• Het onderzoek peer-reviewed en wetenschappelijk solide (dus ook e.g. transparant, reproduceerbaar, onafhankelijk etc. ) is. • De claims voldoen aan de Europese wetgeving hierover (ik weet te weinig over de wetgeving over gezondheidsclaims in andere delen van de wereld, zoals de wetgeving van de Verenigde Staten, om daar blind op te vertrouwen. De Europese wetgeving over gezondheidsclaims vind ik rechtvaardig.) (21). • De marketingcampagne als hoofddoel heeft meer producten te verkopen door consumenten te informeren, niet door ze te misleiden. Dit is niet noodzakelijk, maar vind ik persoonlijk prettiger omdat daarbij consumenten als doel an sich worden bekeken, niet als middel om meer geld te verdienen.

Dit vind ik omdat het voor mij zowel deontologisch- als utilistisch-ethisch verantwoord is om onderzoek te doen naar en producten te marketen op basis van de gezondheidsaspecten van voedsel.

Dit gaat overigens op voor consumenten in hoogontwikkelde, westerse landen waarin consumenten genieten van vrije keuze uit en vrij beschikbare kennis over voedsel, zoals Nederland.

Daarmee trek ik de conclusie dat het schrijven van mijn BLT- thesis ethisch goed is.

Referenties

1. Barry Callebaut en Tony’s Chocolonely tekenen strategische samenwerkingsovereenkomst om chocolade te maken van volledig traceerbare duurzame cacao. (2016). 1st ed. [ebook] Zurich, Zwitserland en Amsterdam, Nederland, pp.1-3. Available at: http://www.tonyschocolonely.com/wp-content/uploads/2016/07/160719_NL-Persbericht- traceerbare-cacaoboterDEF.pdf [Accessed 9 Dec. 2016]. 2. Borrelli, R. and Fogliano, V. (2005). Bread crust melanoidins as potential prebiotic ingredients. Molecular Nutrition & Food Research, 49(7), pp.673-678. 3. Cacaoslavernij. (2003). [video] Keuringsdienst van Waarde. 4. EenVandaag, (2010). EenVandaag :: Hoe haalbaar is Fair Trade?. [online] Eenvandaag.nl. Available at: http://www.eenvandaag.nl/buitenland/36557/hoe_haalbaar_is_fair_trade_ [Accessed 9 Dec. 2016]. 5. Faist, V. and Erbersdobler, H. (2001). Metabolic Transit and in vivo Effects of Melanoidins and Precursor Compounds Deriving from the Maillard Reaction. Annals of Nutrition and Metabolism, 45(1), pp.1-12. 6. Fogliano, V. and Morales, F. (2011). Estimation of dietary intake of melanoidins from coffee and bread. Food & Function, 2(2), p.117. 7. Fogliano, V., Corollaro, M., Vitaglione, P., Napolitano, A., Ferracane, R., Travaglia, F., Arlorio, M., Costabile, A., Klinder, A. and Gibson, G. (2011). In vitro bioaccessibility and gut biotransformation of polyphenols present in the water-insoluble cocoa fraction. Molecular Nutrition & Food Research, 55(S1), pp.S44-S55. 8. Gezondergenieten.nl. (2015). Pindakaas: gezond of ongezond?. [online] Available at: http://www.gezondergenieten.nl/pindakaas-gezond-of-ongezond/ [Accessed 9 Dec. 2016]. 9. Morales, F., Somoza, V. and Fogliano, V. (2010). Physiological relevance of dietary melanoidins. Amino Acids, 42(4), pp.1097-1109.

10. Rufián-Henares, J. and Morales, F. (2007). Effect of in Vitro Enzymatic Digestion on Antioxidant Activity of Coffee Melanoidins and Fractions. Journal of Agricultural and Food Chemistry, 55(24), pp.10016-10021. 11. Somoza, V. (2005). Five years of research on health risks and benefits of Maillard reaction products: An update. Molecular Nutrition & Food Research, 49(7), pp.663-672. 12. Tagliazucchi, D. and Bellesia, A. (2015). The gastro-intestinal tract as the major site of biological action of dietary melanoidins. Amino Acids, 47(6), pp.1077-1089. 13. Tony and the chocolate factory part 1. (2005). [film] Keuringsdienst van Waarde. 14. Tony and the chocolate factory part 2. (2005). [video] Keuringsdienst van Waarde. 15. Verzelloni, E., Tagliazucchi, D., Del Rio, D., Calani, L. and Conte, A. (2011). Antiglycative and antioxidative properties of coffee fractions. Food Chemistry, 124(4), pp.1430-1435. 16. Vitaglione, P., Fogliano, V. and Pellegrini, N. (2012). Coffee, colon function and colorectal cancer. Food & Function, 3(9), p.916. 17. Wang, H., Qian, H. and Yao, W. (2011). Melanoidins produced by the Maillard reaction: Structure and biological activity. Food Chemistry, 128(3), pp.573-584. 18. Bentham, J. (1789). An introduction to the principles of morals and legislation. 1st ed. New York: Hafner Pub. Co.

19. Kant, I. and Abbott, T. (1785). Fundamental principles of the metaphysics of morals. 1st ed. Raleigh, N.C.: Alex Catalogue.

20. Mill, J. (1863). Utilitarianism. 1st ed. Peterborough, Ont.: Broadview Press. 21. Eurofoodlaw.com. (2016). Health Claims - EU Food Law. [online] Available at: http://www.eurofoodlaw.com/health-claims/ [Accessed 9 Dec. 2016].

Een (hopelijk) humoristische appendix en referentie 22. Moge u als lezer ondertussen zin heb gekregen in chocolade, raad ik u deze reep zeer aan: verantwoordelijk geproduceerd, een favoriet van mij én van mijn vader, en ook nog eens puur zodat er meer melanoïden in zitten dan in de melk variant. Een welkome afwisseling op de schofterig populaire melk-karamel-zeezout reep van hetzelfde merk en de (vrij lage als je het mij vraagt) prijs meer dan waard. Ik geef ruiterlijk toe dat dit bij deze mijn poging was tot humor over serieuze onderwerpen; het staat dan ook in deze appendix en niet in het serieuze deel. http://webshop.tonyschocolonely.com/chocolade/180-gram/puur-amandel-zeezout-1- stuk