Development of vitamin D gummy supplements and their shelf-life

Kaja Gertin Grétarsdóttir 2019

Thesis for the degree of Master of Science in Food Science

Þróun á D-vítamínbættum gúmmíböngsum og geymsluþol þeirra.

Kaja Gertin Grétarsdóttir

Leiðbeinandi: Prof. Kristberg Kristbergsson

60 einingar

Júní 2019

Ritgerð til meistaragráðu í matvælafræði

Ritgerð þessi er til meistaragráðu í matvælafræði og er óheimilt að afrita ritgerðina á nokkurn hátt nema með leyfi rétthafa.

© Kaja Gertin Grétarsdóttir 2019 Prentun: Háskólaprent ehf. Reykjavík, Ísland 2019 Ágrip

Vegna lítillar sólarljóss hér á landi eru Íslendingar á meðal þeirra þjóða sem að þurfa að innbyrða D vítamín í formi fæðubótar. Samkvæmt nýlegustu könnun ná flestir Íslendingar ekki að uppfylla ráðlagðan dagskammt af vítamíninu þrátt fyrir að taka lýsi og venjulegar D-vítamín töflur. Það væri því hugsanlegt að D-vítamínbættir gúmmíbangsar gætu hjálpað til við það. Enda eru þeir bragðgóðir og gætu einnig verið góð lausn fyrir þá sem eiga erfitt með að kyngja töflum. Markmið þessarar rannsóknar var að þróa D-vítamín gúmmíbangsa og skoða geymsluþol þeirra yfir 14 vikna tímabil. Áhrif fjögurra innihaldsefna gúmmíbangsanna (gelatín, agar, gulrótarsafi og jarðaberjasafi) voru skoðuð með tilliti til áferðar, litar, D-vítamín virknis og skynmats, sem voru jafnframt geymsluþolsþættirnir. Tilraunahögun var notuð til þess að finna hina fullkomnu uppskrift. Lokaútgáfurnar urðu fjórar gerðir af gúmmíböngsum: Gulrótar agar, jarðaberja agar, gulrótar gelatín og jarðaberja gelatín. Mælingar á geymsluþoli hófust svo beint eftir framleiðslu og voru gúmmíbangsarnir geymdir í lokuðum plastílátum við stofuhita. Niðurstöðurnar sýna að D-vítamín virknin í gelatín böngsunum var til staðar í amk fimm vikur á meðan að vítamín virknin í agar böngsunum fór strax að dvína. Þegar að leið á tímann, urðu agar bangsarnir klístraðri, á meðan að gelatín bangsarnir urðu stífari og seigari. Allir gúmmíbangsarnir nema gulróta agar urðu dekkri eftir því sem leið á tímann og rauði og guli liturinn í öllum hlaupunum dofnaði í þeim öllum. Skynmatsdómurunum þótti liturinn á jarðaberja agar böngsunum versna með tímanum. Þegar litið er á geymsluþol hlaupbangsanna er það frekar stutt ef horft er til virknis D- vítamínsins í þeim. Það eru litlar sem engar rannsóknir til um hvernig virkni vítamína gæti varðveist lengur í gúmmíböngsum. Kæling, húðun, breyttar umbúðir og/eða önnur innihaldsefni er allt dæmi um þætti sem gætu hugsanlega varðveitt vítamín í gúmmíböngsum, en þörf er á frekari rannsóknum á því sviði.

iii Abstract

Icelanders are among the world’s inhabitants that need to take vitamin D as a supplement due to lack of sunlight. However, they fail to meet the recommended daily intake of the vitamin in spite of taking cod liver oil or regular vitamin tablets. One of possible solutions to this problem could be gummy supplementation. They are tasty, they help cater “pill fatigue” and could also be a good solution for those who have pill-swallowing problems. The aim of this study was to develop vitamin D gummy supplements and study their shelf life over 14 weeks time period. The effects of four ingredients in the gummies (gelatin, agar, carrot juice and strawberry juice) were examined for texture, color, vitamin D activity and sensory evaluation which were the shelf-life parameters. Experimental designs were used to determine the recipe of the gummies. The final products consisted of four gummies: Carrot agar, strawberry agar, carrot gelatin and strawberry gelatin. Shelf-life measurements began right after production and the gummies were stored in a plastic container at ambient temperature. The results showed that gelatin gummies were able to hold vitamin D for at least five weeks while the vitamin in the agar gummies started to decrease right away. Over time, the agar gummies got stickier, while the gelatin gummies got firmer and tougher. All gummies except carrot agar darkened over time and the red and yellow color in all of the gummies decreased. The color of strawberry agar was less attractive at the end of the time period. The overall shelf-life of the gummies was rather short when looking at the vitamin D activity. There are little or no studies about how vitamin activity in gummy supplements can be improved. Refrigeration, coating, other packaging and/or other ingredients are examples of factors that could possibly preserve vitamins in gummy supplements but further research in this field is needed.

iv Acknowledgements

First of all, I want to thank my supervisor Kristberg Kristbergsson, professor at the Faculty of Food Science and Nutrition for his knowledge and professional guidance through this study. María Guðlaugsdóttir I send my sincere thanks for helping me with the texture analyzer. I would also like to thank Matís for allowing me to access the student’s lab and kitchen and their great staff for their help. The companies Katla matvælaiðja ehf., Kjarnavörur hf., Nói Siríus ehf., Sýni ehf. og Garðyrkjustöðin Kvistar I give my thanks to them for the samples/raw materials. I also send my gratitude to Svana Bára Gerber for loaning me a juicer. Last but not least, I want to thank my parents for the support over the last years and Hrafn Sigurðarson for always believing in me and encouraging me.

v Table of contents

Ágrip ...... iii Abstract ...... iv Acknowledgements ...... v Table of contents ...... vi List of tables ...... viii List of figures ...... x Abbreviations ...... xii 1. Introduction ...... 1 2. Review of the literature ...... 2 2.1 Vitamin D ...... 2 2.2 Gummy supplements ...... 3 2.3 Gums ...... 3 2.3.1 Agar ...... 4 2.3.2 Carrageenan ...... 5 2.3.3 Alginates ...... 6 2.3.4 Gelatin ...... 7 2.3.5 Pectin ...... 8 2.3.6 Gum arabic ...... 10 2.3.7 Guar gum ...... 11 2.3.8 Xanthan gum ...... 11 2.3.9 Health effect of gums ...... 12 2.4 Color types and color measurements ...... 13 2.5 Texture in gummy bears and texture measurements ...... 15 2.6 Sensory evaluation ...... 16 2.7 Food product development ...... 16

3. Objectives ...... 17 4. Materials and methods ...... 18 4.1 Raw materials ...... 19 4.2 Development of the gummy bears ...... 19 4.2.1 Cooking method when developing the gelatin gummy bears ...... 20 4.2.2 Gum testing ...... 20 4.2.3 Moisture ...... 20 4.3 Shelf-life measurements of the gummy bears ...... 21 4.3.1 Cooking method and recipes for the final gummy bear products ...... 21 4.3.2 Texture ...... 24

vi 4.3.3 Color ...... 25 4.3.4 Vitamin D activity ...... 27 4.3.5 Sensory evaluation ...... 28 4.4 Statistics and experimental designs ...... 28 4.4.1 Experimental designs ...... 28 4.4.2 Statistical analysis ...... 29

5. Results and discussion ...... 30 5.1 Development of the gummy bears ...... 30 5.1.1 Screening experiment ...... 30 5.1.2 Optimization experiment ...... 31 5.1.3 Gum testing ...... 34 5.1.4 Moisture ...... 35 5.2 Shelf-life measurements of the gummy bears ...... 36 5.2.1 Texture ...... 36 5.2.2 Color ...... 44 5.2.3 Vitamin D activity ...... 48 5.2.4 Sensory evaluation ...... 50

6. Conclusions ...... 54 7. References ...... 55 8. Appendix ...... 62 8.1 Texture ...... 66 8.2 Color ...... 74 8.3 Sensory evaluation ...... 88

vii List of tables

Table 1. Differences between the main carrageenan types (BeMiller, 2008; Imeson, 2009). ... 5 Table 2. Calculations on how much cholecalciferol was needed for one batch...... 24 Table 3. The recipe for the gelatin- and agar gummies with added vitamin D...... 24 Table 4. Calculated p-values for the effects between the independent and dependent factors in the screening experiment (n=4)...... 30 Table 5. Testing gum types on the recipe...... 34 Table 6. Moisture and dry material in the four gummies...... 35 Table 7. Mean hardness values for each gummy bear (values from the whole time period) and difference between the four gummies...... 38 Table 8. Hardness values for each week (mean ± standard deviation)...... 39 Table 9. Mean toughness values for each gummy bear (values from the whole time period) and difference between the four gummies...... 40 Table 10. Toughness values for each week (mean ± standard deviation)...... 42 Table 11. Mean stickiness values for each gummy bear (values from the whole time period) and difference between the four gummies...... 42 Table 12. Stickiness values for each week (mean ± standard deviation)...... 43 Table 13. Standard deviations from three different color measuring methods...... 44 Table 14. "L values" for each week (mean ± standard deviation)...... 45 Table 15. "a values" for each week (mean ± standard deviation)...... 46 Table 16. "b values" for each week (mean ± standard deviation)...... 47 Table 17. Vitamin D amount in the four gummies (µg cholecalciferol/100g) (result ± expanded measurement uncertainty)...... 49 Table 18. Mean scores for taste for each gummy bear (values from the whole time period) and differences between the four gummies (n=17)...... 50 Table 19. Mean scores for texture for each gummy bear (values from the whole time period) and differences between the four gummies (n=17)...... 50 Table 20. Mean scores for color for each gummy bear (values from the whole time period) and differences between the four gummies (n=17)...... 51 Table 21. Scores from the sensory evaluation in week one and week fourteen (mean ± standard deviation)...... 51 Table 22. Coded levels for the factors of the screening design...... 63 Table 23. A 24 full factorial design for the screening design with four factors and two levels and the mean scores from the sensory evaluation...... 63 Table 24. Coded levels for the factors of the optimization design...... 64 Table 25. Central composite design for the optimization with two factors and five levels and the mean scores from the sensory evaluation...... 64 Table 26. Amount of added water or sugar to each recipe as part of the optimization to adjust the right initial moisture content...... 65 Table 27. Results from all the moisture measurements...... 65 Table 28. Hardness in carrot agar gummy. Comparing means between weeks...... 66 Table 29. Hardness in strawberry agar gummies. Comparing means between weeks...... 67 Table 30. Hardness in carrot gelatin gummies. Comparing means between weeks...... 67

viii Table 31. Hardness in strawberry gelatin gummies. Comparing means between weeks...... 68 Table 32. Toughness in carrot agar gummies. Comparing means between weeks...... 68 Table 33. Toughness in strawberry agar gummies. Comparing means between weeks...... 69 Table 34. Toughness in carrot gelatin gummies. Comparing means between weeks...... 69 Table 35. Toughness in strawberry gelatin gummies. Comparing means between weeks...... 70 Table 36. Stickiness in carrot agar gummies. Comparing means between weeks...... 70 Table 37. Stickiness in strawberry agar gummies. Comparing means between weeks...... 71 Table 38. Stickiness in carrot gelatin gummies. Comparing means between weeks...... 71 Table 39. Stickiness in strawberry gelatin gummies. Comparing means between weeks ...... 72 Table 40. Raw data from the texture measurements...... 73 Table 41. „L values“ comparisons between weeks for carrot agar...... 74 Table 42. „L values“ comparisons between weeks for strawberry agar...... 75 Table 43. „L values“ comparisons between weeks for carrot gelatin...... 76 Table 44. „L values" comparisons between weeks for strawberry gelatin...... 77 Table 45. „a values" comparisons between weeks for carrot agar...... 78 Table 46. „a values" comparisons between weeks for strawberry agar...... 79 Table 47. „a values" comparisons between weeks for carrot gelatin...... 80 Table 48. „a values" comparisons between weeks for strawberry gelatin...... 81 Table 49. „b values" comparisons between weeks for carrot agar...... 82 Table 50. „b values" comparisons between weeks for strawberry agar...... 83 Table 51. „b values" comparisons between weeks for carrot gelatin...... 84 Table 52. „b values" comparisons between weeks for strawberry gelatin...... 85 Table 53. Raw data from the color measurements...... 86 Table 54. Raw data from the color measurements (continued)...... 87 Table 55. Raw data from the sensory evaluation...... 88

ix List of figures

Figure 1. Chemical structure of vitamin D2 and D3 (Teixeira Rabelo, Barroso, & Magalhães, 2017)...... 2 Figure 2. Chemical structure of agarose (Lahaye & Rochas, 1991)...... 4 Figure 3. Chemical structure of agaropectin (Lahaye & Rochas, 1991)...... 5 Figure 4. Chemical structure of kappa-carrageenan (Dul et al., 2015)...... 5 Figure 5. Chemical structure of iota-carrageenan (Dul et al., 2015)...... 6 Figure 6. Chemical structure of lambda-carrageenan (Dul et al., 2015)...... 6 Figure 7. Chemical structure of the G- and M-units of an alginate (Kurt I. Draget & Taylor, 2011)...... 7 Figure 8. "Eggbox" structure formation when alginate is dropped in a calcium solution (Paredes-Juarez et al., 2014)...... 7 Figure 9. The formation of gelatin. Collagen seperates into single coils when heated and forms gel upon cooling where network of chains are held together with hydrogen bonds (Endt & Baker, 2000)...... 8 Figure 10. Chemical structure of pectin monomers. The left monomer is D-galacturonic acid and the right monomer is methylated D-galacturonic acid (Hamman, 2010)...... 9 Figure 11. How HM pectin gels are formed with the help of hydrogen units from a low pH (Hoefler)...... 9 Figure 12. The "eggbox" structure when LM pectin and calcium interact and form a gel (Axelos & Thibault, 1991)...... 9 Figure 13. Gum arabic nodules (Levy, 2018) ...... 10 Figure 14. The carbohydrate/protein structure of gum arabic which causes gum arabic to function as an emulsifier (Williams & Phillips, 2009b)...... 10 Figure 15. Chemical structure of guar gum (Mudgil et al., 2014)...... 11 Figure 16. Chemical structure of xanthan gum (Hamman, 2010)...... 12 Figure 17. Chemical structure of β-carotene ("B-Carotene Molecule," 2016) ...... 13 Figure 18. Chemical structure of the main anthocyanin in strawberries (pelargonidin 3- glucoside) (Lopes-da-Silva et al., 2007)...... 14 Figure 19. The CIELAB color space (Andersen, 2013)...... 15 Figure 20. The strawberries right before they were pressed into juice...... 21 Figure 21. The carrots right before they were pressed into juice...... 22 Figure 22. The sugar-clump right before gelatin-juice mixture was added...... 22 Figure 23. The final gummy bear products standing in the open air for 18 hours...... 23 Figure 24. The final gummy bear products in plastic containers...... 23 Figure 25. A gummy bear before a penetration test in the texture analyzer...... 25 Figure 26. One of three methods tested as a preliminary study: Measuring straight into the plastic container...... 26 Figure 27. One of three methods tested as a preliminary study: Putting two gummybears tight on the measuring head with two fingers (fingers technique not shown)...... 26 Figure 28. One of three methods tested as a preliminary study. Cutting the gummies into small pieces and measuring it in a small glass container...... 27

x Figure 29. All four gummies were sent abroad in a single used plastic containers and labelled appropriately...... 27 Figure 30. The appearance of the recipes from the screening design...... 30 Figure 31. The appearance of the recipes from the optimization design...... 31 Figure 32. Contour plot for the effect of juice and initial moisture on overall likeness...... 31 Figure 33. Contour plot for the effect of juice and initial moisture on color...... 31 Figure 34. Contour plot for the effect of juice and initial moisture on watery mouthfeel...... 32 Figure 35. Response surface method plot for the effect of juice and initial moisture on overall likeness...... 32 Figure 36. Response surface method plot for the effect of juice and initial moisture on color...... 33 Figure 37. Response surface method plot for the effect of juice and initial moisture on watery mouthfeel...... 33 Figure 38. Testing gum arabic on the recipe (heating the gum with the juice and mixing at the same time)...... 35 Figure 39. Results from testing pectin A on the recipe...... 35 Figure 40. A force-time curve from carrot gelatin texture measurement on week three...... 36 Figure 41. A force-time curve from carrot agar texture measurement on week three...... 36 Figure 42. A force-time curve from strawberry agar texture measurement on week seven (many of the samples break)...... 37 Figure 43. A force-time curve from carrot agar texture measurement on week eleven (all of the samples break)...... 37 Figure 44. Hardness changes over the time period in the four gummies...... 39 Figure 45. Toughness changes over the time period in the agar gummies...... 41 Figure 46. Toughness changes over the time period in the gelatin gummies...... 41 Figure 47. Stickiness changes over the time period in the four gummies...... 43 Figure 48. Change in „L value“ in all four gummies over the time period...... 44 Figure 49. Change in „a value“ in all four gummies over the time period...... 45 Figure 50. Change in „b value“ in all four gummies over the time period...... 46 Figure 51. Change in vitamin D amount in all the four gummies over the time period...... 49 Figure 52. Mean scores for taste from the sensory evaluation...... 52 Figure 53. Mean scores for texture from the sensory evaluation...... 52 Figure 54. Mean scores for color from the sensory evaluation...... 53 Figure 55. The gelatin and agar from Sigma Aldrich which was used in the gummies...... 62 Figure 56. The vitamin D from Sigma Aldrich which was used in the gummies...... 62 Figure 57. Temperature of the freezer, where the strawberry and carrot juices were stored. .. 62 Figure 58. Toughness changes over the time period in the four gummies (including week seven)...... 66 Figure 59. The questionnaire used in the sensory evaluation...... 89

xi Abbreviations

Agar Agar-Agar CA Carrot-agar CG Carrot-gelatin SA Strawberry-agar SG Strawberry-gelatin

xii 1. Introduction

People who live in Iceland are one of the world’s inhabitants that need to take vitamin D as a supplement due to lack of sunlight, even though they eat foods that naturally contain vitamin D like fatty fish (Embætti landlæknis, 2017). Vitamin D along with calcium intake plays an important role in maintaining bone health (Holick, 1996). It is most common amongst Icelanders to get their vitamin D source from cod liver oil or regular vitamin D tablets. Even though Icelanders take cod liver oil or vitamin D tablets, they still fail to meet the recommended daily intake of vitamin D. Only 26% of men and 18% of women from Iceland consume cod liver oil daily and only 6% of men and 13% of women take multivitamins daily and the intake of individual vitamins are consumed even less (Bryndís Elfa Gunnarsdóttir et al., 2011). Today, vitamins in the form of gummies have started to gain popularity among adults. They were originally targeted for children as a fun way for them to take their vitamins, but in 2012 a supplement company began marketing them for adults. Consumers find gummy supplements help cater their “pill fatigue” and find them good to hide the “vitamin taste” which often are bad or have a repulsive odor. They might also be a good solution for those who have pill- swallowing problems (Ellin, 2017) since 26-50% of people experience difficulties with swallowing pills (Andersen et al., 1995; Fields et al., 2015). The 7th most popular vitamin D supplement on amazon.com is a gummy supplement. They are even more popular as multivitamins since their top three most sold multivitamins are also gummies (Amazon, 2019). However, gummy supplements usually contain sugar which is well known to increase the risk of obesity and dental decay and should therefore be consumed in a moderate amount (Embætti landlæknis, 2017). There are even indications that chewable vitamins are able to improve nutritional status in children (Kleiman-Weiner et al., 2013; Stewart et al., 2002). A gummy vitamin can consist of different gelling agents, sweeteners, flavorings, colorings and more. When ingredient lists from gummy vitamins which are available now on the market are examined (iHerb.com), it is noticeable that they usually contain either pectin or gelatin as a gelling agent. They also usually contain sugar, syrup, juice concentrates or ready-made color and flavorings and some of them have coatings. Their labelled shelf-life is around 6-18 months. So far, it is unknown for how long vitamins can stay active in a gelling system like this and what factors might preserve the vitamins are also unknown. Consumers today will never really know whether the labelled shelf-life covers the vitamin activity or not.

1 2. Review of the literature

2.1 Vitamin D Vitamin D is a lipid soluble vitamin which we get from diet and from exposure to sunlight. Sources of vitamin D in the diet are mainly fatty fish, cod liver oil, egg yolk, butter as well as vitamin D fortified foods like milk, cereals and more (Insel et al., 2014). When exposed to sunlight, vitamin D is synthesized in the skin. There are two main forms of vitamin D: D3 (cholecalciferol) from the food mentioned above and from exposure to sunlight and D2 (ergocalciferol) from irradiation of plant materials (see figure 1 below) (Lips, 2006).

Figure 1. Chemical structure of vitamin D2 and D3 (Teixeira Rabelo, Barroso, & Magalhães, 2017).

Both vitamin D3 and D2 are converted by the liver and kidneys into the active form of the vitamin: 1,25(OH)2D3. The active form of vitamin D functions like a hormone since it‘s made in one part of the body but regulates activities in other body parts (Insel et al., 2014). Vitamin D along with calcium intake plays an important role in maintaining bone health (Holick, 1996). It has also been shown to reduce the risk of cancer (Garland et al., 2006) and low vitamin D intake has been associated with type 1 diabetes (Hyppönen et al., 2001). In Iceland, the formation of vitamin D in the skin is insufficient during the winter and is therefore essential for Icelandic residents to take vitamin D as a supplement at that time to promote good vitamin D levels (Embætti landlæknis, 2017). A Danish study supports this fact and claims that sunlight- deprived individuals are at risk for vitamin D deficiency and that they need to take vitamin D as supplement (Glerup et al., 2000). The recommended daily dosage of vitamin D today is 15 µg for Icelandic residents (10-70 years old) (Embætti landlæknis, 2016). In 2001-2003 around 60% of Icelanders did take vitamin D supplements (Steingrimsdottir et al., 2005). Around ten years later only 21% of Icelanders consume cod liver oil daily and under 15% take multivitamins and even fewer take individual vitamins like vitamin D daily. The average daily

2 intake of vitamin D in Icelandic residents is 8,1 µg which does not fulfill the recommendations (Bryndís Elfa Gunnarsdóttir et al., 2011).

2.2 Gummy supplements There is no company in Iceland that produces gummy supplements and very few companies import such products into the country. Young Icelandic women get the least amount of vitamin D and should therefore be the main target group for vitamin D gummies. But the target group should also be the ones who don’t consume cod liver oil daily. Because those who do (mostly adults over 60), fulfill the daily recommendations of vitamin D (Bryndís Elfa Gunnarsdóttir et al., 2011). Gummy supplements are gaining popularity all over the world and the three most sold multivitamins on amazon.com are gummies (Amazon, 2019). Gummy supplements have both pros and cons. They usually taste better than conventional vitamin tablets (Sheika, 2017) and might be a good alternative for children and adults that are struggling with swallowing pills (Kannall, 2018). Studies have shown that 26-50% of people experience difficulties with swallowing pills (Andersen et al., 1995; Fields et al., 2015). Consumers also find gummy supplements help cater their “pill fatigue” and that they hide the “vitamin taste” which is often bad or have repulsive odor (Ellin, 2017). One study on children showed that the intake of vitamin A increased when gummy supplementation was used compared to a placebo group (gummies without supplementation) (Stewart et al., 2002). Another study did show that chewable vitamins with high iron content increased the nutritional status in Chinese students more than students that ingested eggs (Kleiman-Weiner et al., 2013). On the other hand, gummy supplements usually contain high quantities of sugar which is well known to increase the risk of obesity and dental decay (Embætti landlæknis, 2017; Sheiham, 1983) as well as having an addictive effect on those consuming it (Ahmed et al., 2013; Avena et al., 2008). Not to mention that eating too much of gummy supplements could end up as vitamin toxicity which could have various negative effects depending on which vitamin is ingested. The main consequence of vitamin D toxicity is hypercalcemia, meaning a high calcium concentration is in the blood. This can for example cause bone loss and kidney stones (Insel et al., 2014).

2.3 Gums What characterizes gums (also called hydrocolloids) is that they’re made of polymers (polysaccharides or proteins) which form complex net structures that can bind large quantities of water. They are commonly used to thicken or gel aqueous solutions and change the viscosity and texture of food products (Williams & Phillips, 2009a). They are used widely in the food

3 industry with different roles in different food products. Thickeners are for example used in sauces, dressings, soups and gravies while gelling agents are mainly used in products like jellies, jams, low calorie gels and candy. In addition to functioning as thickening- and gelling agents, they are also useful as emulsifiers and/or stabilizers (Saha & Bhattacharya, 2010). The origin of gums are extensive, they come from: plants, plant exudates, algal, animal products and microbial fermentation (Williams & Phillips, 2009a). Gums that are mainly used as thickeners are: xanthan gum, locust bean gum, guar gum, gum arabic, gum tragacanth and cellulose deratives. While gums used as gelling agents are mainly: gelatin, alginate, pectin, carrageenan, agar and gellan gum (Saha & Bhattacharya, 2010). When gelling occurs these polymer molecules or bundles of them connect together and junction zones are formed (with hydrogen bonds or cross-linking bonds). Those junction zones then connect with the end of the molecules (or bundles of molecules) that extends outside another junction zone in another area and forms a three-dimensional network (with liquid trapped inside) (BeMiller, 2008; Damodaran et al., 2008). The gums that were used and tested in this project will be described more thoroughly below.

2.3.1 Agar Agar is extracred from red algae (Rhodophyta) (Williams & Phillips, 2009a). It can be divided into two components: agarose and agaropectin (figure 2 and 3). Agarose is a neutral polysaccharide and has greater gelling capability than agaropectin which contains charged polysaccharide components (Labropoulos et al., 2002). Agarose is therefore the preferable component of agar and is used for example in the food-, medicine- and pharmacy industry as well as for bacteria cultures etc. Agar is a linear polysaccharide which consists of alternating 1,3-linked β-D-galactopyranose and 1,4-linked 3,6 anhydro-α-L-galactopyranose units. Agar has a gelling temperature at 40°C and melting temperature close to 90°C (Djabourov et al., 1989).

Figure 2. Chemical structure of agarose (Lahaye & Rochas, 1991).

4 Figure 3. Chemical structure of agaropectin (Lahaye & Rochas, 1991).

2.3.2 Carrageenan Carrageenan is extracted from red algae like agar and they both have a galactose backbone but differ in the location and proportion of side groups (Imeson, 2009). Carrageenan has a variety of functions and is quite sensitive to ionic composition in its environment (Stephen et al., 2006). Three main types of carrageenan are kappa-, iota- and lambda carrageenan (table 1) and they differ in 3,6-anhydrogalactose and ester sulfate content (Imeson, 2009).

Table 1. Differences between the main carrageenan types (BeMiller, 2008; Imeson, 2009). Kappa Iota Lambda Forms gel in presence of: Potassium ions Calcium ions Does not gel Type of gel: Brittle Soft and elastic - Syneresis: Yes No - Freeze-thaw stable: No Yes Yes Ester sulfates content: 22% 32% 37% 3,6-anhydrogalactose 33% 26% ≈ 0% content:

Carrageenan is well known for its use in dairy products where it is used to stabilize and prevent separation for example in chocolate milks, ice creams, milk shakes etc. (Imeson, 2009).

Figure 4. Chemical structure of kappa-carrageenan (Dul et al., 2015).

5 Figure 5. Chemical structure of iota-carrageenan (Dul et al., 2015).

Figure 6. Chemical structure of lambda-carrageenan (Dul et al., 2015).

2.3.3 Alginates Alginates are made from brown algae (Phaeophyceae) and consists of 1,4 linked β-D- mannuronic acid (M) and α-L-guluronic acid (G) residues (figure 7). Also known as G- and M- blocks (Draget & Taylor, 2011). They can also be produced with bacterial fermentation if the formed polysaccharide is prepared by alkaline extraction (Linker & Jones, 1966) but today the most used alginates are the ones from seaweed (Draget, 2009). Alginates, also called alginic acid, forms a gel in the presence of Ca2+ ions and those with the greater G-block content produces stronger gels. G-blocks are segments of the alginic acid which contain only L- guluronopyranosyl units. The holes formed between the G-block chains can hold calcium ions and because of this connection, a so called “eggbox” junction zone is formed, as shown in figure 8 (Damodaran et al., 2008).

6 Figure 7. Chemical structure of the G- and M-units of an alginate (Kurt I. Draget & Taylor, 2011).

Figure 8. "Eggbox" structure formation when alginate is dropped in a calcium solution (Paredes- Juarez et al., 2014).

2.3.4 Gelatin The most used gelling agent in the food industry so far is gelatin. Gelatin is a protein unlike other gum types (which are usually polysaccharides) and is derived from collagen. The collagen most often comes from porcine and bovine tissues like hides, bones and skins (Haug & Draget, 2009). Gelatin gels are very extensible and elastic. The gelations occurs when triple helixes melt when heated but upon cooling, structural rearrangement occurs where net is formed (figure 9) (Damodaran et al., 2008). What characterizes gelatin by contrast with other gum types is that it is classified as food instead of an E number. This is because gelatin is a protein and contains essential amino acids (as well as other amino acids) that are not found in quantity in many foods (Johnston-Banks, 1990). Those are for example: alanine, arginine, glutamine, glycine, hydroxyproline isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, valine etc.

7 (Haug & Draget, 2009). A typical mammalian gelatin melts at 33-34°C and sets at 26-27°C but the temperature can vary between gelatin types, the concentration and cooling- and heating rate (Haug & Draget, 2009).

Figure 9. The formation of gelatin. Collagen seperates into single coils when heated and forms gel upon cooling where network of chains are held together with hydrogen bonds (Endt & Baker, 2000).

2.3.5 Pectin Pectin is found naturally in cell walls of many plants. The main sources of pectin are from citrus peels and apple pomaces (Thakur et al., 1997) . They are a family of a complex polysaccharides which are rich in covalently linked galacturonic acid (pectin monomers shown in figure 10) (Albersheim et al., 1996). Pectin is categorized into high methoxyl (HM) pectins and low methoxyl (LM) pectins. Their gelling ability is based on how many carboxyl groups are esterified with methanol. If more than 50% of the carboxyl groups are methylated they are HM pectin and if the methylation is under 50% they are LM pectin (Damodaran et al., 2008; Sharma et al., 2006). HM pectin forms gel in the presence of sugar and acid where a two-dimensional network is formed (figure 11). LM pectin forms a gel in the presence of Ca2+ ions (or other divalent cations) where a so called “eggbox” structure is formed (figure 12). The lower the methylation the greater gelling ability is in place, however, the presence of acetyl groups prevents gel formation (Sharma et al., 2006). LM pectin is often used in jellies, marmalades and low-calorie jams (Damodaran et al., 2008). Factors like pH, degree of methoxylation,

8 presence of other solutes, molecular size, arrangement of side chains and charge density on the molecule can all affect pectin gelation (Thakur et al., 1997).

Figure 10. Chemical structure of pectin monomers. The left monomer is D-galacturonic acid and the right monomer is methylated D-galacturonic acid (Hamman, 2010).

Figure 11. How HM pectin gels are formed with the help of hydrogen units from a low pH (Hoefler).

Figure 12. The "eggbox" structure when LM pectin and calcium interact and form a gel (Axelos & Thibault, 1991).

9 2.3.6 Gum arabic Gum arabic is a tree gum exudate from Acacia Senegal species. What is special about gum arabic is that it grows only in the Sahelian belt of Africa. The gum substance dries on the branches and forms hard nodules which are then picked by hand and sorted (Phillips & Williams, 2009).

Figure 13. Gum arabic nodules (Levy, 2018) It is the only gum where viscosity is a function of concentration (Williams & Phillips, 2009b). It is a complex of hydrophilic carbohydrate and hydrophobic protein components (Abdullah, 2013). The chemical structure of gum arabic can vary depending on the source, age of the trees, climatic conditions and soil environment. It mainly consist of 1,3 linked β-D- galactopyranosyl units as a backbone and the side chains are composed of two to five units of the same units as the backbone which are linked to the main chain by 1,6 linkages (Ali et al., 2009). Gum arabic works as an emulsifier, thickener, stabilizer, flavoring agent and sometimes as coating agent. It is used in food products like pastilles, gum drops, marshmallows, toffee, soft drinks, chewing gum etc. (Verbeken et al., 2003; Williams & Phillips, 2009b) and it has recently started to gain interest in gluten free bread products (Bourekoua et al., 2018). The concentration of gum arabic depends on how it is used. With increasing concentration of the gum, the viscosity increases. That means it is used at concentration of 40-55% in confectionary products (for example gummy bear candies) (Stephen et al., 2006; Williams & Phillips, 2009b).

Figure 14. The carbohydrate/protein structure of gum arabic which causes gum arabic to function as an emulsifier (Williams & Phillips, 2009b).

10 2.3.7 Guar gum Guar gum is extracted from the seeds of the Cyamopsis tetragonoloba plant which is produced for the most part in India. Guar gum is made from the endosperm and consists of galactomannans which are a linear chain of 1,4 linked β-D-mannopyranosyl units, where 1,6 linked α-D-galactopyranosyl residues are the side chains (figure 15). Guar gum has one of the highest molecular weight of all naturally water soluble polymers. It is used as a thickener, stabilizer and emulsifier (Mudgil et al., 2014). Guar gum is considered as a non-gelling agent (Saha & Bhattacharya, 2010) but it is often used in combination with other gums and it is especially popular in ice creams where its known to prevent growth of ice crystals. It is also used in other dairy products, frozen desserts, prepared meals, bakery products, sauces and pet food (Damodaran et al., 2008).

Figure 15. Chemical structure of guar gum (Mudgil et al., 2014).

2.3.8 Xanthan gum Xanthan gum is secreted from the micro-organism Xanthomonas campestris. It consists of a cellulose backbone 1,4 linked β-D-glucose substituted at C-3 on alternate glucose residues with a trisaccharide side chain, as shown in figure 16 below (Stephen et al., 2006; Sworn, 2009). It is soluble in both hot and cold water which means it doesn’t thicken upon cooling (Damodaran et al., 2008). Xanthan gum is mainly used as a thickener or stabilizer but under certain conditions it can function as a gelling agent. It can form gel when mixed with locust bean gum or tara gum. It can even form a gel when mixed with a enzymatically modified guar gum or with the addition of trivalent cations or borate anions (Stephen et al., 2006). One study has shown that xanthan gum can improve whey protein isolate gels at concentration as low as 0,01% (Bertrand & Turgeon, 2007). Xanthan is used in many types of food products. For example in

11 salad dressings, chocolate syrups, dry mix formulations (sauces, gravies, desserts), doughs and a lot more (Damodaran et al., 2008; Stephen et al., 2006).

Figure 16. Chemical structure of xanthan gum (Hamman, 2010).

2.3.9 Health effect of gums Most available gums can be considered as soluble dietary fiber. However, the definition of dietary fibers has been a debate for a long time (Edwards & Garcia, 2009; Viebke et al., 2014). According to the Panel of the Definition of Dietary Fiber and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes 2001, dietary fiber consists of non-digestible carbohydrates and lignin that are intrinsic and intact in plants (Institute of Medicine, 2001) and that they are carbohydrate polymers with 10 or more monomeric units which are not hydrolyzed in the small intestines of humans (Viebke et al., 2014). Since the majority of gums are considered as soluble dietary fiber, they all have similar health effects on the human body (Edwards & Garcia, 2009). Pectin and guar gum have shown to lower total cholesterol and LDL cholesterol in blood (Jenksins et al., 1975; Rosner er al., 1999) and lower total cholesterol has the benefits of decreasing the risk of coronary heart disease (Gould et al., 2007). Pectin has also the positive effects by lowering serum glucose levels, reduce the risk of cancer and stimulating the immune response (Mohnen, 2008; Sharma et al., 2006). Gum arabic is also considered as a dietary fiber and it acts as a prebiotic at a dose of 10 g/day (Phillips & Phillips, 2011). It increases the growth of lactic acid bacteria which means the colonic pH decreases and small chain fatty acids are produced which inhibits pathogen growth and might lower the risk of colon cancer (Edwards & Garcia, 2009). Xanthan gum has shown to lower serum glucose levels and serum cholesterol (in diabetic subjects) (Osilesi et al., 1985). Agar also behaves like a natural

12 fiber, meaning its digestibility is very low and it can be used as a slow release ingredient for slow absorption of pharmaceutical products (Armisén & Gaiatas, 2009). Alginates have shown to increase satiety after a meal, reduce the desire to eat between meals and reduce blood cholesterol and glucose levels in individuals with BMI over 25 (Dettmar et al., 2011). Carrageenan has shown to lower serum glucose levels and serum cholesterol (Panlasigui et al., 2003). Carrageenan was once considered to cause ulcerative colitis in animals when ingested in high doses but the carrageenan had been degraded (was also called “poligeenan”) and did not function like normal carrageenan (did not have thickening, gelling and stabilizing properties) so that criticism of the safety of carrageenan in food resulted as false (Imeson, 2009). However, gelatin is one of the gums that can’t be considered as a dietary fiber because it is a protein. Proteins in general are critical for good health and play an important role as enzymes, hormones, antibodies, fluid-balancer, acid-base balancer and they transport many key substances like oxygen, vitamins etc. to the right place in the body (Insel et al., 2014). A study has shown that gelatin has antihypertensive activity which is believed to lower the risk of cardiovascular diseases (Möller et al., 2008).

2.4 Color types and color measurements Color is one of the main factors people evaluate food by. Food products can be added with food colorings that are either synthetically or naturally produced. Synthetic colors are widely used in food products since they are cheaper and more stable than natural colors. However, some of the synthetic colors (azo dyes) have shown to have negative effects on human health (Yamjala et al., 2016). The two colors that were used in the gummy bears in this project were naturally occurring colors from carrots-and strawberry juice. Carrots naturally contain the carotenoid pigment and are rich in β-carotene. They are lipid soluble and the β-caroten type is a precursor to vitamin A. They are oxidation sensitive but overall heat resistant (Potter & Hotchkiss, 1998). From different cooking methods, they degrade most by frying (Murador et al., 2014).

Figure 17. Chemical structure of β-carotene ("B-Carotene Molecule," 2016)

13 Strawberries naturally contain anthocyanin, more specifically Pelargonidin-3-Glucoside (Lopes-da-Silva et al., 2007). They are water soluble and the color is sensitive to temperature, pH and light exposure resulting in a more faded color (Cortez et al., 2017). Anthocyanin in strawberries decrease during processing, especially at high temperatures (Cappa et al., 2015; Klopotek et al., 2005) and another study shows it degrades most in pressure steaming (Murador et al., 2014). It has also been shown that sonication can reduce anthocyanin in strawberry juice (Tiwari et al., 2008).

Figure 18. Chemical structure of the main anthocyanin in strawberries (pelargonidin 3-glucoside) (Lopes- da-Silva et al., 2007).

However, there is a way to stabilize the anthocyanin color (copigmentation) and polymers (e.g. gum arabic, pectin etc.) have shown to function as such at pH higher than 3,2 (Cortez et al., 2017). The effect of natural colors on vitamin activity in vitamin added food products have not yet been studied but fruit candies with added grape skin powder (containing anthocyanin) can affect the texture (Cappa et al., 2015). Soft candy with added anthocyanin has shown to improve the sensory properties: more attractive color, more firm and more sweet and having better overall quality compared to candy without the addition of anthocyanin (Adsare et al., 2016). Addition of pitaya fruit puree (where betalain is the natural occurring color) in soft candies have shown a positive effect on color, texture and overall acceptability from sensory analysis test (Hani et al., 2015). Vitamin D is a lipid soluble vitamin which can be damaged because of free radicals. Anthocyanins are antioxidants which means they can prevent oxidation by scavenging free radicals (Martín et al., 2017) and possibly protect the vitamin from being oxidized and therefore, damaged.

14 There is a common method to measure color which is recommended by the Commission International de L’Eclariage, which was used in this project. The color measurement method goes under the name CIELAB color system and is based on the measurement of three color coordinates (L*, a* and b*). L* value analyzes white to black (on a scale 0-100), a* value analyzes red to green and b* value analyzes yellow to blue (on a scale 60 to -60) (Cortez et al., 2017).

Figure 19. The CIELAB color space (Andersen, 2013).

2.5 Texture in gummy bears and texture measurements Gummy confections have usually kind of soft, flexible, firm or brittle texture which is dependent on the gum type and strength (Williams & Phillips, 2009a). For example gelatin gels alone are soft and flexible, but when gellan gel and calcium ions are added to it, its hardness increases and with only addition of calcium the gels tends to be more brittle and less cohesive and springy (Lau et al., 2000). When pectin is added with gelatin it gives a more brittle and less chewy texture as well as the fruity and sweet flavor of the gummies increases (DeMars & Ziegler, 2001). The age of the gummies also affects the texture, since the water content usually changes with time and water is an important factor regarding texture (Ergun et al., 2010; Potter & Hotchkiss, 1998). Texture can both be measured by instrumental tests and by sensory tests (Potter & Hotchkiss, 1998). But it has been shown that sensory analysis obtains greater results compared to instrumental tests (Muñtoz et al., 1986). Textural parameters from a texture analyzer are usually obtained from a force-time curve that’s formed during the compression of the sample (Pang et al., 2014).

15 2.6 Sensory evaluation Sensory evaluation is a method used to measure and analyze products through human senses (sight, smell, touch, taste and hearing). Products are assessed by objective and/or subjective tests. Trained panel is needed for objective testing where sensory attributes are evaluated but for subjective testing, reaction of the consumer of a product is measured. Sensory evaluation is used for product development, quality control (for example shelf-life measurement), marketing and research and some say that it might be the most sensitive and reliable method in some situations (Kemp, 2008).

2.7 Food product development There are many ways to develop a new food product. Experimental design is a common and simple method to systematically look at how different levels (amount) of each variable affects specific characteristic of the product. Variables could be for example sugar, juice, syrup or other ingredients in a gummy bear recipe. Experimental designs are divided into screening- and optimization designs (Lundstedt et al., 1998). The aim of screening experiments is to determine which variables have significant effect on the result. There are a few screening designs to choose between but it is most common to use linear or second order interaction models where variables with the largest influence on the results can be investigated further. The aim of optimization experiments is to find the optimum levels of the variables investigated. There are also a few optimization designs available: response surface methods (RSM) and mixture designs. Mixture designs can only be used for formula factors while RSM may be used to optimize formula and processing factors (Lundstedt et al., 1998).

16 3. Objectives

The aim of this study was twofold: to develop vitamin D gummy supplements and to measure their shelf-life over 14 weeks. The development was performed with experimental designs and the final products consisted of either gelatin or agar as gelling agent and either strawberry- or carrot juice as natural color and flavoring. The effects of those factors on the shelf-life was examined. The shelf-life parameters were: texture, color, vitamin D activity and sensory evaluation. The research question of this study is as following: Do gelatin, agar, strawberry juice or carrot juice affect vitamin D activity over 14 weeks, in vitamin D supplemented gummy bears?

17 4. Materials and methods

The project is divided into two parts; the development of the gummy recipes and shelf life measurements. In the first part, experimental designs were made to invent the recipe of the gummies where gelatin was used as gelling agent. When the final recipe was determined, other gums were tested on the recipe and one gum selected. The final products were made of either gelatin or agar as gelling agent and strawberry- or carrot juice as natural color and flavoring. In the second part, the gummies were made and their shelf-life measured (for 14 weeks). Color and texture were measured with regular intervals, vitamin D activity was measured three times and sensory evaluation was performed at the beginning and the end of the time period.

Development of the

gummy bears

Experimental Sensory evaluation designs (n=4)

Gum testing

Making of the final products

Shelf-life measurements

Instrumental texture Color Vitamin D Sensory evaluation measurements measurements measurements (n=17)

18 4.1 Raw materials When developing the gummies, samples of different gums and other ingredients were donated by several companies in Iceland. Katla matvælaiðja ehf. provided carrageenan, guar gum and xanthan gum. Kjarnavörur hf. provided two types of pectin, powdered gelatin, citric acid and anthocyanin, Nói Siríus ehf. provided gum arabic and glucose syrup (D.E. 37-41), Sýni ehf. provided agar powder and a teacher from the department provided alginate and calcium chloride. Sugar, fresh strawberries and carrots was purchased in a local grocery store by the author. In the final gummy bear products which were used for shelf-life measurements, gelatin (from porcine skin, approximately 250 Bloom), agar and cholecalciferol were obtained from Sigma Aldrich, Steinheim, Germany. Pictures of the Sigma Aldrich products can be found in the appendix in figures 55 and 56. Icelandic strawberries (second grade) were given from the plant nursery Garðyrkjustöðin Kvistar. They were retrieved August 23rd 2018 and pressed the next day. Sugar and Icelandic carrots (from Grafarbakki) were purchased in a local grocery store by the author on September 14th 2018 and the carrots were pressed two days later. Glucose syrup and citric acid were from the same companies as mentioned above, meaning enough of it was left for using it in the final products. The brand of the juicer used for pressing the strawberries and carrots was “Juices Whole Apples” from Solis of Switzerland. The molds used were made from silicon and had a regular gummy bear shape. The molds were sprayed lightly with vegetable oil spray (Vegetable Oil, No-Stick Cooking Spray from Essential Everyday) to prevent the gummy bears from sticking to the molds.

4.2 Development of the gummy bears Recipes from the web, ingredients list on a typical gummy candy packaging and ingredients list on gummy supplements helped the author get an idea of what ingredients could be used and an idea of a recipe. It was decided that the gummy bears would contain the following ingredients: Glucose syrup, sugar, juice, water, gelling agent and citric acid. Experimental designs were used to determine the final recipe of the gummy candies. The designs consisted of a 24 full factorial design (for screening) and central composite design (for optimization). The experimental designs were used to find out which variables affected the results, which were then used for the optimization. Four judges (family members) participated in the sensory evaluation for the experimental designs. The recipes were developed with the use of gelatin as a gelling agent and strawberry juice as flavoring and coloring.

19 4.2.1 Cooking method when developing the gelatin gummy bears It was decided that the gelling agent (gelatin) would be 4% of the total weight and the citric acid would be 1,2 grams per 100 g mixture for each recipe while developing the recipe. Gelatin sheets were allowed to sit in the juice (and water) for about 5-10 minutes at room temperature. Glucose syrup, sugar and citric acid were heated in a pot on a medium heat until a sugar paste was made and then the gelatin-juice mixture was added and mixed gently for a few minutes on a medium heat until everything had dissolved. A plastic syringe was used to put the mixture in the molds.

4.2.2 Gum testing When the final recipe had been invented for gelatin, other gum samples were tested on that same particular recipe. A few tests were done for each gum sample (with a little modification on the recipe) and eventually one gum was chosen, agar. Since the original recipe used on the chosen gum couldn’t properly form a gel, the recipe was modified. The main difference between the gelatin and agar recipe was the amount of gelling agent, added water, cooking temperature and the order of the raw materials. The development of the gummy bears ended in two separate recipes (one for gelatin and one for agar) with two different flavors each, in total four types of gummy bears:

 Carrot agar  Carrot gelatin  Strawberry agar  Strawberry gelatin

4.2.3 Moisture Since the recipes for the gelatin and agar gummy bears were not exactly the same, the moisture content of the gummy bears was measured at the beginning of the time period. This was done in case there were differences between the gummy samples that could be traced to different moisture contents. The moisture was measured using the ISO 6496-1999 method (International Organization for Standardization, 1999). The gummies were cut into small pieces and approximately 5 grams weighed in a dried ceramic container, which had been dried and weighed immediately prior to use. The samples were placed in an oven at 103°C ± 2°C for 4 hours and allowed to cool in a desiccator. The samples were then weighed after drying. The moisture content was calculated as following:

20 W1−W2 Dry material (%) = x 100 W3 Moisture (%) = 100 – dry material (%) where:

W1 = Weight of the container and sample after drying

W2 = Weight of the container before drying

W3 = Weight of the sample

4.3 Shelf-life measurements of the gummy bears Final products of the vitamin D gummy bears were made and subsequently color-, texture- and vitamin D activity was measured with regular intervals as well as sensory evaluation which was performed at the beginning and end of the time period (14 weeks). It was decided to use cholecalciferol in the gummy bears since it has shown to raise the serum 25(OH)D concentrations in blood more than ergocalciferol (Mangoo-Karim et al., 2015; Tripkovic et al., 2012).

4.3.1 Cooking method and recipes for the final gummy bear products The strawberries were gently rinsed with water and dried carefully with a tea towel. The leaves were removed and the berries were pressed into juice. The top and the bottom of the carrots were cut off and then gently rinsed. The carrots were then peeled and pressed into juice. The juices were stored in a freezer (at least -20°C, as shown in figure 57 in the appendix) until the final products were made. Cholecalciferol was added to the juices and mixed by hand in a plastic container right before cooking.

Figure 20. The strawberries right before they were pressed into juice.

21 Figure 21. The carrots right before they were pressed into juice.

Gelatin gummies were prepared by mixing the gelatin with the juice and allowed to sit for 15 minutes. Glucose syrup, sugar and citric acid were heated at a medium heat or until a sugar paste was made (figure 22) then the gelatin-juice mixture was combined with it under a medium heat. The mixture was stirred carefully with a paddle (to prevent foam to form) for about five minutes or until everything was completely dissolved. The foam that came up on top of the mixture was removed before the sugar mixture was put into molds.

Figure 22. The sugar-clump right before gelatin-juice mixture was added.

The agar gummy bears were prepared in a similar way. The agar was allowed to sit in the juice for about 15 minutes. Glucose syrup, water and agar-juice mixture was heated at high heat and boiled for 1 minute while stirring briskly with a whisk. The heat was then reduced and

22 sugar and citric acid added to the mixture and stirred at low heat for approximately one and a half minute. Both the sugar mixtures were deposited in the molds with a plastic syringe and allowed to cool in a refrigerator (<5°C). The gelatin gummies were cooled for 35 minutes and the agar gummies were cooled for 15 minutes. The gummies were then removed from the molds and placed under open air at room temperature for 18 hours (figure 23) and then stored in a plastic container with a volume of one liter each (figure 24).

Figure 23. The final gummy bear products standing in the open air for 18 hours.

Figure 24. The final gummy bear products in plastic containers.

To determine the quantity of cholecalciferol needed for the experiment, the number of gummy bears from an older batch were counted and the total cholecalciferol value calculated. The goal was to have 15 µg of cholecalciferol in a single gummy bear, which is the same amount as the recommended daily dose of vitamin D for icelanders (Embætti landlæknis, 2016). Since there will always be remains of the sugar mixture in the pot and foam removed, the total quantity

23 of cholecalciferol was therefore increased by 5% (table 2). The final recipes for the gummy bears are shown in table 3.

Table 2. Calculations on how much cholecalciferol was needed for one batch. Gelatin Agar Pieces of gummy bears per single batch 60 70 Cholecalciferol per batch (mg) 0,90 1,05 Total cholecalciferol per batch + 5% (mg) 0,95 1,10

Table 3. The recipe for the gelatin- and agar gummies with added vitamin D. Gelatin Agar Glucose syrup (g) 50 50 Sugar (g) 40 40 Juice (g) 33 33 Water (g) 0 30 Gelling agent (g) 5 1,8 Citric acid (g) 1,5 1,5 Cholecalciferol (mg) 0,95 1,1

4.3.2 Texture The texture measurement was performed using TA-HD plus Texture analyzer (Stable Micro Systems, Surrey, UK). The software package Exponent (Stable Micro Systems) was used in combination with the texture analyzer. The samples were compressed once with a flat 26 mm diameter cylinder aluminum probe (which covered the whole gummy bear) at uncontrolled ambient temperature. A single gummy bear was put under the cylinder and pressed at a speed of 1,5 mm/s with a total displacement of five mm. The height of a single gummy bear was eight to nine mm which means, the probe penetrated 56-63% of the sample’s initial height. Five replicates were analyzed for each type (fresh gummy bear every time) and the texture measurements were carried out every two weeks. Since the gummy bears were handmade and not all the exact same size, the gummy bears from the previous measurement were kept and used as a benchmark for the selection of gummy bears to test next time.

24 The following parameters were obtained from the force-time curves:

 Hardness (g), defined as the peak force from 5 mm compression.  Toughness (g/sec), defined as the total force used for the compression.  Stickiness (g), defined as the peak negative force (meaning the force it took for the probe to come off the sample on its way up). The fragileness was also examined by looking at the curves from every measurement.

Figure 25. A gummy bear before a penetration test in the texture analyzer.

4.3.3 Color Color measurements were performed with a Minolta CR-300 Chroma Meter (measuring head) and Minolta data processor DP-301 meter (Minolta Co., Ltd, Osaka, Japan) where the results were expressed as CIELAB values. Before every measurement, the device was calibrated with a white standard. The color measurement was carried out once a week and five replicates were analyzed for each type. The measurement was performed by holding two gummy bears tightly against the measuring head with two fingers. Measurements were done on Wednesdays except for in week 10 the measurement was done on Thursday because of a non-workday that Wednesday. A preliminary study was performed before the weekly measurements started to determine the right method. Tests were performed in three ways; putting the measuring head straight into the plastic container, holding two gummy bears tightly against the measuring head and cutting the gummies in small pieces and measuring it in a small glass container (figures 26 to 28). For this preliminary study, all measurements were repeated six times for each type and the method with the lowest standard deviation was selected.

25

Figure 26. One of three methods tested as a preliminary study: Measuring straight into the plastic container.

Figure 27. One of three methods tested as a preliminary study: Putting two gummybears tight on the measuring head with two fingers (fingers technique not shown).

26 Figure 28. One of three methods tested as a preliminary study. Cutting the gummies into small pieces and measuring it in a small glass container.

4.3.4 Vitamin D activity Vitamin D content in the gummy bears was measured using the EN 12821:2009 method performed by Eurofins in Hamburg, Germany. It was measured in total three times, at the beginning, in the middle and in the end of the time period. Only one measurement was done for each gummy bear type each time due to cost of the analysis.

Figure 29. All four gummies were sent abroad in a single used plastic containers and labelled appropriately.

27 4.3.5 Sensory evaluation Sensory evaluation was performed at the beginning and end of the time period. The judges (non-trained) were both students and family members and answered the same four questions for each gummy bear. The questions were as following:

 How do you like the taste?  How do you like the color?  How do you like the texture?  Do you taste bad taste or aftertaste? All the questions were answered on the scale 0-10 on a hedonic scale where 0 was considered as very bad and 10 as very good. The whole questionnaire can be seen in figure 59 in the appendix. The judges did not know the flavor nor the gelling agent of the gummies so the gummy bear types were named “Red 1 and 2” and “Orange 1 and 2”. The gummies were handed to them in the same order as the order on the questionnaire.

4.4 Statistics and experimental designs

4.4.1 Experimental designs

4.4.1.1 Screening experiment A 24 full factorial experimental design was used with four independent factors (sugar, syrup, juice and water) and two levels, in total 16 recipes. From the combination of each test, the initial moisture content was calculated and used as the “fifth factor”. The characteristics (dependent factors) that were evaluated with sensory evaluation were: watery mouthfeel, overall likeness and color. It was evaluated by four judges (the author himself and family members) in a randomized order on a hedonic scale from 0-10. This was done to find out which factors had the most influence on the taste. The coded levels and the combinations of the recipes and the sensory evaluation ratings are shown in tables 22 and 23 in the appendix. The amount of those ingredients which had no significant impact on the characteristics of the gummies was chosen by the author for the continuing work: 50 g of glucose syrup, 40 g of sugar, 1,5 g of citric acid and 4% of total weight of gelatin.

4.4.1.2 Optimization experiment A central composite design was used for the optimization of the gummies (two factors and five levels). The significant factors from the screening design: juice and initial moisture were optimized. The design was carried out with a 22 full factorial design, four tests at star point and six repeated tests at central point, in total 14 tests which were evaluated with sensory evaluation

28 in the same way as in the screening experiment. The coded levels and the combinations of the recipes and the sensory evaluation ratings are shown in tables 24 and 25 in the appendix. Since initial moisture was a one of the factors which affected the characteristics of the gummies, it was adjusted for each recipe by adding either water or sugar, depending on whether moisture or dryness was needed. The amount of water/sugar added to each recipe to adjust the initial moisture content is listed in table 26 in the appendix. The ideal score for both overall likeness and color were 8,5 and higher but for watery mouthfeel, 2-3 was ideal.

4.4.2 Statistical analysis The data from the screening experiment was analyzed with bivariate analysis (regression analysis) were p<0,05 was considered as significantly different. The data from the optimization experiment was analyzed with contour plot and response surface method. All data in this project was analyzed using the statistic application JMP ® 14.0.0 (SAS Institute Inc.). The texture, color and sensory evaluation changes over the time period, as well as the differences between the gummy bear types were analyzed with one-way anova (regression analysis) where p<0,05 was considered as significantly different.

29 5. Results and discussion

5.1 Development of the gummy bears

5.1.1 Screening experiment

Figure 30. The appearance of the recipes from the screening design.

Table 4. Calculated p-values for the effects between the independent and dependent factors in the screening experiment (n=4). Watery mouthfeel Overall likeness Color Sugar 0,91 0,70 0,91 Glucose syrup 0,12 0,15 0,75 Juice 0,02* 0,24 <0,01* Water 0,07 0,90 0,33 Initial moisture <0,01* 0,41 <0,01* *Significant difference (p<0,05). The amount of juice and the initial moisture content of the gummies has a significant effect on both watery mouthfeel and the color of the gels (table 4). Therefore, it was decided to use those factors for the optimization.

30 5.1.2 Optimization experiment

Figure 31. The appearance of the recipes from the optimization design.

Figure 32. Contour plot for the effect of juice and initial moisture on overall likeness.

Figure 33. Contour plot for the effect of juice and initial moisture on color.

31

Figure 34. Contour plot for the effect of juice and initial moisture on watery mouthfeel. By looking at the contour plots, the results do not really give one optimal result which takes all the sensory factors into account. The best results for overall likeness and color do overlap at the same area (at 32% initial moisture and 35 g juice), but that does not apply to watery mouthfeel. All combinations of the best scores for each sensory factor can be read from the response surface plots below.

Figure 35. Response surface method plot for the effect of juice and initial moisture on overall likeness.

32

Figure 36. Response surface method plot for the effect of juice and initial moisture on color.

Figure 37. Response surface method plot for the effect of juice and initial moisture on watery mouthfeel. After gathering all of the combination that gave the best results and comparing them with each other, there was no one perfect result found. But the most common combinations found were the following:

33  Overall likeness: 32,5% moisture and 33,5 g juice  Watery mouthfeel: 32% moisture and 30 g juice  Color: 32% moisture and 35 g juice From this information, the author determined to use 32% initial moisture and 33 g juice for the final products.

5.1.3 Gum testing After the recipe was completed, it was tested on the different gum samples which resulted in various successes (table 5). The gum that was the first to form a gel was selected for further inspection, in this case agar. When trying out the same recipe with agar as a gelling agent, a little deviation was necessary for the agar to be able to work properly by adding water to the recipe. On the contrary, the cooking temperature for the agar gummies was higher than the cooking temperature for the gelatin gummies.

Table 5. Testing gum types on the recipe. Gum Number of tests How did it go? Did not form gel at first because of insufficient heating and too low water content. Formed gel in the third try with addition of water. Different water Agar 14 amount tested on the recipe and the one with the least amount of water possible was chosen. Tested in two ways:  Sugar mixture with and without added water was let fall in calcium bath. The one with added water formed a very weak and brittle gel. Alginate 3 But too weak to be able to form a gummy bear candy.  Calcium and water were mixed with the recipe. As it cooled it started to clump. After putting it in the molds it resulted in a clumpy, cloudy and weak gel. Did not form a gel but was very close to it. Hard to realize the right Carrageenan 3 cooking method and need for water.

Guar gum + 2 Did not form a gel. Only a thick paste. Xanthan gum Hard to work with by hand. Needed to be stirred under heat for a long Gum Arabic 2 time. Also, hard to put it in the molds by hand because of its thickness.

With and without calcium but only made a thick paste in both cases. Hard 4 Pectin A to realize the right cooking method and need for water.

With added calcium but did not form a gel, only a thick paste like the other Pectin B 2 pectin sample. Hard to realize the right cooking method and need for water.

34 Both of the pectins were LM pectin with different calcium reactivity. The pH of the recipe was not measured and therefore it cannot be determined whether the pH affected the failure of the test.

Figure 38. Testing gum arabic on the recipe (heating the gum with the juice and mixing at the same time).

Figure 39. Results from testing pectin A on the recipe.

5.1.4 Moisture All samples except carrot agar were repeated because of too much difference between the samples. More detailed calculations can be seen in table 27 in the appendix.

Table 6. Moisture and dry material in the four gummies. Strawberry gelatin Strawberry agar Carrot gelatin Carrot agar Dry material (%) 87,0 83,3 86,3 81,7 Moisture (%) 13,0 16,7 13,7 18,3 The moisture at the beginning of the time period is higher in the agar gummies (which holds hand with the fact that there is more water in the agar recipe) and we also see that the carrot juice contains more water than the strawberry juice (table 6).

35 5.2 Shelf-life measurements of the gummy bears To answer the research question in the introduction, it was noticed that gelatin was able to keep vitamin D active for at least five weeks, while the vitamin activity decreased right away from the first measurement in the agar gummies. Strawberry- and carrot juice did not affect the vitamin D activity. Also, in week seven, it was noticed that the agar gummies had more moist around them (got sticky) and that a tiny amount of mold was starting to form on the carrot agar gummies at that time. The agar gummies contained more water than the gelatin gummies, which might explain why their shelf-life was worse.

5.2.1 Texture The force-time curve varied between the two gummies (figure 40 and 41). The tip of the curve of the gelatin gummies was sharper than the agar gummies.

Force (g) 1100

1000

900

800

700

600

500

400

300

200

100

0 0 1 2 3 4 5 Time (sec) -100 Figure 40. A force-time curve from carrot gelatin texture measurement on week three.

Force (g) 4500

4000

3500

3000

2500

2000

1500

1000

500

0 0 1 2 3 4 5 Time (sec)

-500 Figure 41. A force-time curve from carrot agar texture measurement on week three.

36 The agar gummies were more fragile and where they started to break in week seven and the fragileness increased after that (figure 42 and 43) while the gelatin gummies did not break at all over the time period. Agar is defined as a “strong” gel and what characterizes it, is that they rupture and fail at large deformations and do not heal without melting and resetting (Ross- Murphy & Shatwell, 1993) which is in accordance with the texture results above. The probe penetrated more than half of the gummy bears initial height which can be considered as a large deformation.

Force (g) 2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

400

200

0 0 1 2 3 4 5 6 Time (sec) -200 Figure 42. A force-time curve from strawberry agar texture measurement on week seven (many of the samples break).

Force (g) 3500

3250

3000

2750

2500

2250

2000

1750

1500

1250

1000

750

500

250

0 0 1 2 3 4 5 Time (sec) -250 Figure 43. A force-time curve from carrot agar texture measurement on week eleven (all of the samples break).

37 Table 7. Mean hardness values for each gummy bear (values from the whole time period) and difference between the four gummies. P-value P-value P-value P-value Peak force (g) compared to compared to compared to compared to Mean ± Std Dev CA SA CG SG CA 3238 ± 623 - 0,037* <0,01* <0,01*

SA 3017 ± 459 0,037* - <0,01* <0,01*

CG 878 ± 261 <0,01* <0,01* - 0,15 SG 1028 ± 380 <0,01* <0,01* 0,15 - *Significant difference (p<0,05)

The hardness of the agar gummies was approximately threefold higher than the gelatin gummies where the difference was significant. The juices have significant effect on hardness in the agar gummies but not on the gelatin gummies. This is consistent with another study where agar shows to be firmer than gelatin and also that the addition of apple pomace (which probably contains anthocyanin), probiotic and prebiotics reduced the hardness (Lele et al., 2018). The change in hardness over the time period in the four gummies can be seen in figure 44.

38 Figure 44. Hardness changes over the time period in the four gummies.

As mentioned before, the agar gummies did start to break at week seven (figure 42). That means the hardness values for agar after the seventh week cannot really be taken seriously. From week one to week seven is a well-defined gradient for the agar gummies. Where the hardness for both the agar gummies decreases significantly. However, the hardness in the gelatin gummies increases significantly. The definition of the significance (which week is significant) and the mean values for each week can be seen in table 8.

Table 8. Hardness values for each week (mean ± standard deviation). Week 1 Week 3 Week 5 Week 7 Week 9 Week 11 Week 13 CA 3320 ± 633 3890 ± 359* 3521 ± 557 3077 ± 441 2953 ± 787 2557 ± 431* 3313 ± 305

SA 3558 ± 312* 3513 ± 168* 3065 ± 121* 2561 ± 130 2700 ± 186 2755 ± 210 2750 ± 475

CG 535 ± 205* 897 ± 133 1114 ± 248 996 ± 249 792 ± 169 995 ± 179 950 ± 148

SG 741 ± 127 884 ± 182 1018 ± 248 1016 ± 354 971 ± 279 1466 ± 635* 1216 ± 340

*Where more than half of the weeks compared with one week is significant (p<0,05). From the hardness results above it can be concluded that the agar gummies got weaker over time while gelatin gummies got more firm. More detailed information about the significant differences between each week can be seen in tables 28 to 31 in the appendix.

39 The toughness, meaning the total force used for the compression was also observed in the four gummies. The toughness values in the seventh week for both agar and gelatin gummies was taken out of the calculations and graph since they were unusually high (possible reasons are described in the end of chapter 4.2.2.). This is the same timing as when moist (and small amount of mold) was noticed in the agar gummies. The results for toughness are not so different from the hardness results, both when the four gummies are compared with each other as well as when changes over the time period is observed.

Table 9. Mean toughness values for each gummy bear (values from the whole time period) and difference between the four gummies. P-value P-value P-value P-value Peak force (g) compared to compared to compared to compared to Mean ± Std Dev CA SA CG SG

CA 3226 ± 576 - 0,049* <0,01* <0,01*

SA 3038 ± 417 0,049* - <0,01* <0,01*

CG 597 ± 155 <0,01* <0,01* - 0,47 SG 666 ± 204 <0,01* <0,01* 0,47 -

The agar gummies are significantly tougher than the gelatin gummies (approximately five times tougher). The juices have significant effect on toughness in the agar gummies but not on the gelatin gummies. The changes in toughness over the time period in the four gummies can be seen in figures 45 and 46. A graph with the values from week seven can be seen in figure 58 in the appendix.

40 Figure 45. Toughness changes over the time period in the agar gummies.

Figure 46. Toughness changes over the time period in the gelatin gummies.

The toughness decreases over time for the agar gummies and increases a little bit for the gelatin gummies. The decrease in both the agar gummies is not significant but the sudden increase in the CA at the 13th week is significant but uncertain. The toughness in the gelatin

41 gummies increases significantly. The definition of the significance (which week is significant) and the mean values for each week can be seen in table 10.

Table 10. Toughness values for each week (mean ± standard deviation). Week 1 Week 3 Week 5 Week 7 Week 9 Week 11 Week 13

CA 3197 ± 520 3431 ± 406 2843 ± 269 4817 ± 280* 3113 ± 847 2956 ± 451 3827 ± 453*

SA 3128 ± 426 3394 ± 375 2660 ± 446 4290 ± 331* 2862 ± 312 3011 ± 341 3135 ± 350

CG 384 ± 108* 615 ± 56 755 ± 134* 990 ± 211* 574 ± 94 685 ± 95 656 ± 73

SG 501 ± 70* 604 ± 106 666 ± 133 948 ± 269* 624 ± 135 901 ± 315* 768 ± 180

*Where more than half of the weeks compared with one week is significant (p<0,05). It can be concluded that the total force needed to compress the sample does not change for the agar gummies over the time period but the gelatin gummies gets tougher over time. More detailed information about the significant differences between each week can be seen in tables 32 to 35 in the appendix.

Table 11. Mean stickiness values for each gummy bear (values from the whole time period) and difference between the four gummies. P-value P-value P-value P-value Peak force (g) compared to compared to compared to compared to Mean ± Std Dev CA SA CG SG CA -26,9 ± 16,0 - 0,22 <0,01* <0,01* SA -23,8 ± 13,0 0,22 - <0,01* <0,01* CG -7,2 ± 2,1 <0,01* <0,01* - 0,10 SG -11,4 ± 6,2 <0,01* <0,01* 0,10 - *Significant difference (p<0,05). The agar gummies are significantly stickier than the gelatin gummies (table 11). Which holds hands with the fact that in the middle of the time period there was more moist (and small amount of mold) noticed around the agar gummies. However, the juices do not seem to have significant effect on the stickiness. The change in stickiness over the time period in the four gummies can be seen in figure 47.

42 Figure 47. Stickiness changes over the time period in the four gummies. The results for stickiness are easier to interpret than the results for hardness and toughness. There is a clear gradient for the agar gummies, where the stickiness increases significantly over time and the increase was even more for the carrot agar gummies. The stickiness in the gelatin gummies does not decrease over the time period. There are significant changes in the CG where the curve goes up and down alternately while the SG gummies do not change significantly. The definition of the significance (which week is significant) and the mean values for each week can be seen in table 12.

Table 12. Stickiness values for each week (mean ± standard deviation).

Week 1 Week 3 Week 5 Week 7 Week 9 Week 11 Week 13

CA -13,1 ± 3,1* -13,1 ± 2,0* -19,6 ± 4,6 -27,3 ± 8,5 -34,0 ± 5,5 -39,6 ± 13,3* -47,0 ± 24,7*

SA -12,0 ± 1,1* -24,0 ± 21,8 -17,6 ± 3,1 -26,8 ± 4,1 -26,3 ± 10,0 -35,7 ± 18,8 -28,6 ± 7,0

CG -8,6 ± 1,8 -4,5 ± 1,0* -6,9 ± 2,0 -10,0 ± 0,9* -7,5 ± 1,7 -6,2 ± 1,5 -6,2 ± 1,3

SG -11,5 ± 6,8 -8,0 ± 2,6 -11,8 ± 4,6 -12,0 ± 2,2 -9,6 ± 3,6 -16,9 ± 13,0 -10,2 ± 2,3

*Where more than half of the weeks compared with one week is significant (p<0,05). More detailed information about the significant differences between each week can be seen in tables 36 to 39 in the appendix.

43 5.2.2 Color From the preliminary study, the selection stood between two methods (table 13).

Table 13. Standard deviations from three different color measuring methods. Measuring straight into the Two gummies held tightly Small pieces of gummies put

plastic container against the measuring head in a small glass container Standard 2,46 1,90 1,87 deviation Two gummies held tightly against the measuring head ended as the selected method because it was quicker to implement and did not include destroying the gummies. The change in color over the time period in the four gummies can be seen in figures 48 to 50 as well as the mean for each week can be seen in tables 14 to 16. It should be stated that the gummies (especially the strawberry gummies) are pretty much transparent where the background might affect the results.

Figure 48. Change in „L value“ in all four gummies over the time period. The “L value” for the CA gummies do not change significantly over the time period while SA darkens significantly in the first week and lightens back significantly at the end of the time period. Both gelatin gummies darken significantly throughout the time period where the change

44 is most in the first weeks. Both strawberry juice and gelatin causes the samples to darken faster (figure 48). A study showed that gelatin gummies had higher lightness value compared to agar gummies (Lele et al., 2018) which is in accordance to this study where the initial L values of the carrot gummies is higher for gelatin.

Table 14. "L values" for each week (mean ± standard deviation).

Week Week Week Week Week Week Week Week Week Week Week Week Week Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14

35,8 ± 35,5 ± 34,8 ± 34,7 ± 34,5 ± 35,2 ± 35,7 ± 35,2 ± 35,5 ± 34,7 ± 34,6 ± 35,0 ± 33,9 ± 35,9 ± CA 1,5 0,9 0,9 0,7 0,2 1,2 2,0 0,9 1,0 0,2 0,5 1,3 1,6 0,7

32,8 ± 33,3 ± 28,8 ± 28,2 ± 29,1 ± 29,7 ± 28,6 ± 28,8 ± 28,9 ± 29,9 ± 30,5 ± 31,5 ± 29,2 ± 32,6 ± SA 0,8* 1,4* 1,0 1,3* 1,6 0,6 0,7 0,5 1,4 1,0 0,8* 0,9* 1,4 1,0*

38,1 ± 38,6 ± 35,5 ± 34,8 ± 33,0 ± 32,3 ± 35,9 ± 32,5 ± 30,8 ± 31,0 ± 31,5 ± 31,8 ± 31,6 ± 33,0 ± CG 0,7* 0,9* 1,9* 0,9* 1,1* 1,2 2,0 1,1 0,5* 0,7* 1,2 0,5 1,3 1,2*

32,4 ± 34,0 ± 28,8 ± 27,7 ± 28,3 ± 26,4 ± 31,2 ± 27,8 ± 27,3 ± 26,2 ± 26,4 ± 27,4 ± 25,6 ± 26,7 ± SG 0,9* 1,3* 2,2* 1,7 0,9* 0,5 2,4* 1,5 1,5 1,9 0,5 1,1 1,7* 0,5

*Where more than half of the weeks compared with one week is significant (p<0,05). More detailed information about the significant differences between each week can be seen in tables 41 to 44 in the appendix.

Figure 49. Change in „a value“ in all four gummies over the time period.

45 The “a value” for the all of the gummies decreases significantly, meaning their red color decreases over the time period. The red color of the strawberry gummies fades away faster than the carrot gummies (figure 49). The definition of the significance (which week is significant) and the mean values for each week can be seen in table 15.

Table 15. "a values" for each week (mean ± standard deviation).

Week Week Week Week Week Week Week Week Week Week Week Week Week Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14

15,1 ± 13,4 ± 11,1 ± 10,4 ± 9,8 ± 9,5 ± 11,1 ± 9,3 ± 7,6 ± 7,5 ± 7,4 ± 6,5 ± 6,7 ± 7,3 ± CA 3,3* 2,2* 0,6* 0,6* 0,7* 1,0* 2,8* 1,6 0,8 0,2 0,9* 0,7* 0,5* 0,6*

25,9 ± 30,4 ± 18,5 ± 17,4 ± 18,4 ± 17,3 ± 17,0 ± 19,1 ± 15,6 ± 14,2 ± 13,6 ± 14,2 ± 11,7 ± 13,4 ± SA 3,8* 1,9* 2,9* 1,4* 0,9* 1,6* 1,8* 3,2* 1,2 0,6* 2,6* 0,6* 0,9* 0,9*

14,3 ± 15,7 ± 14,7 ± 13,7 ± 13,4 ± 12,2 ± 15,9 ± 12,9 ± 11,1 ± 10,6 ± 9,0 ± 10,2 ± 9,5 ± 9,8 ± CG 0,7* 0,8* 1,4* 0,7* 0,8* 1,3* 1,3* 0,9* 0,5* 0,4* 0,5* 0,2* 0,5* 0,8*

29,0 ± 29,1 ± 23,1 ± 18,2 ± 20,3 ± 18,9 ± 24,3 ± 21,5 ± 18,1 ± 16,5 ± 17,9 ± 14,7 ± 15,9 ± 16,6 ± SG 1,1* 1,6* 3,3* 2,9 1,9* 1,5 3,7* 2,2* 1,8 1,4 1,9 1,6* 2,3* 1,1

*Where more than half of the weeks compared with one week is significant (p<0,05). More detailed information about the significant differences between each week can be seen in tables 45 to 48 in the appendix

Figure 50. Change in „b value“ in all four gummies over the time period.

46 The “b value” for the all of the gummies decreases significantly over the time period. Meaning their yellow color decreases. The decrease of the yellow color in the carrot gummies fades away more significantly throughout the whole time period while the changes in the strawberry gummies are mostly at the beginning. The definition of the significance (which week is significant) and the mean values for each week can be seen in table 16.

Table 16. "b values" for each week (mean ± standard deviation).

Week Week Week Week Week Week Week Week Week Week Week Week Week Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14

18,3 ± 16,2 ± 13,8 ± 12,5 ± 11,8 ± 12,1 ± 12,9 ± 11,3 10,1 ± 9,1 ± 9,5 ± 7,9 ± 8,4 ± 9,0 ± CA 3,6* 2,1* 1,4* 1,0* 1,1 1,4* 3,2* ±1,9 1,9 0,2* 1,1 1,1* 0,5* 0,7*

14,2 ± 18,4 ± 8,3 ± 7,5 ± 8,2 ± 7,7 ± 7,2 ± 9,6 ± 7,0 ± 6,6 ± 6,9 ± 8,2 ± 6,5 ± 9,3 ± SA 4,0* 2,0* 2,8 1,3 0,8 1,6 1,2 3,3 1,2 0,5 2,6 0,6 1,5 0,7

18,2 ± 20,9 ± 17,9 ± 17,3 ± 15,5 ± 14,9 ± 19,1 ± 15,7 ± 13,5 ± 13,4 ± 12,1 ± 13,6 ± 13,0 ± 14,5 ± CG 0,7* 2,7* 2,3* 1,7* 0,9* 1,7* 1,9* 1,7* 1,2* 0,8* 1,0* 0,7* 0,3* 0,8

17,1 ± 17,6 ± 11,6 ± 8,5 ± 9,9 ± 8,9 ± 14,2 ± 11,2 ± 9,0 ± 7,9 ± 9,2 ± 6,2 ± 7,6 ± 8,1 ± SG 1,6* 1,8* 3,1* 2,0 1,8 1,2 3,6* 2,2* 2,0 1,6 1,9 1,3* 2,3 0,8

*Where more than half of the weeks compared with one week is significant (p<0,05). More detailed information about the significant differences between each week can be seen in tables 49 to 52 in the appendix. The red and yellow color decreases significantly in all of the gummies over the time period where both of the colors in the strawberry gummies fades away faster than in the carrot gummies. This is not in accordance with another study, where hard gummies tended to hold anthocyanin better than soft gummies for 4 months of storage (Kordsmeier & Howard, 2011). Agar (the harder gummy) in this study did not seem to hold the red nor yellow color better than gelatin (the softer gummy). However, Kordsmeier & Howard did compare gummies with different gelatin concentration where the hard gummies had high gelatin content. It should also be mentioned that CIELAB values and the amount of natural coloring might not be comparable. In this same study, when gummies stored in a refrigerator were compared to gummies stored at ambient temperature, the anthocyanin amount showed to be higher in the refrigerated gummies after 6 months storage (Kordsmeier & Howard, 2011). From the toughness and color results, it is obvious that something happened in week seven that deviates the results, where a sudden and unexpected increase took place in those measurements. The authors first guess about the sudden increase might be because of temperature fluctuations. It is possible that the temperature of the room where the gummies were kept, got extra warm at some point (because of colder temperature in December, the heat

47 of the radiator could have been increased). It is also possible that when the gummies were being transferred from the authors home to the laboratory that day, they might have stayed in the car for a longer time than only the drive itself and gotten colder than expected. The authors second guess about the sudden increase is that it might have happened because of natural purposes. There is a phenomenon called syneresis, where water separates from the gel, which might have happened to the agar gummies in this project. Syneresis happens because the polymer network shrinks and there is less space to hold water, so the water eventuates on the surface of the gummies (Whytock & Finch, 1991). This happens when the agar gel is stored in a closed container and it can be caused because of the gel strength, gel concentration, ageing, rigidity coefficient, pressure or by the sulphate content (Harris, 1990; Stephen et al., 2006). In this study, it is possible that ageing or the sulphate content caused the syneresis in week seven, since the gummies contained little or no sulphates (high sulphate content prevents the syneresis).

5.2.3 Vitamin D activity The goal was to have 15 µg of cholecalciferol in a single gummy bear. However, a single gummy bear in the final product weighed approximately 1,4 grams which means only around 2-3 µg cholecalciferol was present in a single gummy bear. It can be stated that the 5% addition of vitamin D to the calculated vitamin amount, was not enough since the remains were more than expected. It is possible that part of the vitamin got lost during the production. Also, the first vitamin measurement was performed in the second week (because the samples were sent and analyzed abroad), which means the vitamin amount might have been higher when they were fresh. In the end of the time period, there was only 0,2-1,1 µg of cholecalciferol present in a single gummy bear. It is important to keep in mind that the vitamin D results only represents one measurement per sample so the reliability of the differences over the time period can be debated.

48 Figure 51. Change in vitamin D amount in all the four gummies over the time period.

The vitamin decreases right away in the agar gummies, while the vitamin in the gelatin gummies doesn‘t start to decrease until after five weeks (figure 51). However, the juices do not seem to affect the vitamin activity. Note, that the figure shows the vitamin D amount in 100 grams, not the amount in a single gummy bear.

Table 17. Vitamin D amount in the four gummies (µg cholecalciferol/100g) (result ± expanded measurement uncertainty). Week 2 Week 5 Week 14 CA 189 ± 49,2 105 ± 27,3 11,1 ± 2,9 SA 235 ± 61,2 170 ± 44,2 75,5 ± 19,6 CG 137 ± 35,7 167 ± 43,3 64,3 ± 16,7 SG 135 ± 35,0 153 ± 39,7 51,3 ± 13,3

It is not possible to tell whether the decrease in vitamin D activity is significant, since each sample was only measured once. Vitamin D from week two to week fourteen decreased as following: 1700% in CA, 311% in SA, 213% in CG and 263% in SG. Which shows that the decrease is more in the agar gummies. After a thorough search, there were no studies found on how gelatin, agar, anthocyanin and β-caroten might affect vitamin D, meaning no comparison is possible at this time.

49 5.2.4 Sensory evaluation In total 17 judges (students and family members) participated in the sensory evaluation, thereof 14 in the first week and 12 in the thirteenth week (nine judges participated both times). It was decided to not use the answers from the question about aftertaste/bad taste since the question was not clear enough and a few judges misunderstood it. The carrot agar gummies started to mold in the seventh week so it was decided it would not be tested in the later sensory evaluation.

Table 18. Mean scores for taste for each gummy bear (values from the whole time period) and differences between the four gummies (n=17). P-value P-value P-value P-value Score compared to compared to compared to compared to Mean ± Std Dev CA SA CG SG CA 5,9 ± 2,3 - 0,81 0,50 <0,01* SA 6,1 ± 2,0 0,81 - 0,59 <0,01* CG 6,4 ± 2,1 0,50 0,59 - 0,01* SG 7,8 ± 1,4 <0,01* <0,01* 0,01* - *Significant difference (p<0,05). The taste of the SG gummy is significantly better than all of the other gummies (table 18). This can be confirmed by another study where soft candies with added anthocyanin scored higher in color, texture and taste (Adsare et al., 2016) and judges from another study found gelatin gummies overall better than agar gummies (Lele et al., 2018).

Table 19. Mean scores for texture for each gummy bear (values from the whole time period) and differences between the four gummies (n=17). P-value P-value P-value P-value Score compared to compared to compared to compared to Mean ± Std Dev CA SA CG SG

CA 4,4 ± 2,6 - 0,83 <0,01* <0,01* SA 4,2 ± 2,3 0,83 - <0,01* <0,01* CG 7,6 ± 2,1 <0,01* <0,01* - 0,43 SG 7,1 ± 2,2 <0,01* <0,01* 0,43 - *Significant difference (p<0,05). The texture of the gelatin gummies is significantly better than the agar gummies (table 19).

50 Table 20. Mean scores for color for each gummy bear (values from the whole time period) and differences between the four gummies (n=17).

P-value P-value P-value P-value Score compared to compared to compared to compared to Mean ± Std Dev CA SA CG SG

CA 8,1 ± 2,1 - 0,16 0,57 0,64 SA 7,0 ± 2,4 0,16 - 0,32 0,27 CG 7,7 ± 2,4 0,57 0,32 - 0,90 SG 7,8 ± 2,4 0,64 0,27 0,90 - *Significant difference (p<0,05). There were no significant differences between the four gummies regarding how the judges liked their color (table 20).

Table 21. Scores from the sensory evaluation in week one and week fourteen (mean ± standard deviation). Carrot Strawberry Carrot Strawberry agar agar gelatin gelatin Taste - week 1 (n=14) 5,9 ± 2,3 6,1 ± 1,5 6,3 ± 2,6 7,5 ± 1,6 Taste - week 14 (n=12) NA 6,0 ± 2,6 6,5 ± 1,6 8,1 ± 1,1 P-value NA 0,86 0,84 0,30 Color - week 1 (n=14) 8,1 ± 2,1 8,1 ± 2,1 8,1 ± 2,1 8,3 ± 2,3 Color - week 14 (n=12) NA 5,8 ± 2,2 7,2 ± 2,8 7,2 ± 2,5 P-value NA 0,02* 0,32 0,24 Texture - week 1 (n=14) 4,4 ± 2,6 4,3 ± 2,5 7,1 ± 2,4 6,6 ± 2,6 Texture - week 14 (n=12) NA 4,1 ± 2,2 8,3 ± 1,7 7,8 ± 1,5 P-value NA 0,83 0,17 0,18 *Significant difference between the two tests (p<0,05). The judges found the color of the SA gummies significantly worse after fourteen weeks of storage (table 21). Judges from another study concur with the judges in this study which found agar gels prepared with functional ingredients (probiotic, prebiotics and apple pomace) less acceptable (Lele et al., 2018).

51 Figure 52. Mean scores for taste from the sensory evaluation. There is not so much change in how the judges liked the taste of the gummies. It seems however they like the gelatin gummies a little better after fourteen weeks.

Figure 53. Mean scores for texture from the sensory evaluation. The same can be said about how the judges liked the texture. They seem to find the texture a little better in the gelatin gummies after fourteen weeks (they were stiffer according to the instrumental texture measurements).

52 Figure 54. Mean scores for color from the sensory evaluation. As mentioned before, the color in the strawberry agar gummies was significantly worse in week fourteen compared to the first week. It can also be seen that the judges rate the color in the gelatin gummies lower for that same week.

53 6. Conclusions

The development of vitamin D gummy supplements was done as an idea to improve vitamin D intake of Icelandic residents. The present study shows that gelatin was the only factor (of the four examined) which affected the vitamin D activity in the gummies. The vitamin in the gelatin gummies was active for at least five weeks but faded away instantly in the agar gummies when stored in a plastic container at ambient temperature. However, natural juices did not seem to preserve the vitamin D in the gummies. The agar gummies in this study were more fragile and started to break in week seven while the gelatin gummies did not break at all over the time period when the same force was used. Over time, the agar gummies got less firm but stickier while gelatin gummies got firmer and tougher. Agar gummies were stickier than gelatin gummies which was also noticed by the author in week seven (where also a small amount af mold was noticed on the carrot agar gummy). The color in all the gummies faded away over time, which was also noticed among the sensory evaluation judges for the strawberry agar gummy. Refrigeration has shown to help preserve strawberry anthocyanin in gummies (Kordsmeier & Howard, 2011). It can be argued whether refrigeration might also preserve vitamins in gummy supplements rather than when stored at ambient temperature. Also, what is different between the gummies in this study and regular gummies in a store, is that they do not contain any coating. It would be interesting to see if coating (for example carnauba wax or beeswax) affects vitamin activity in gummy supplements. Overall, there are little or no studies on how vitamin activity in gummy supplements can be improved. Further studies about how refrigeration, coating, different packaging and/or other ingredients might help preserve vitamins in gummy supplements are needed. The author believes that the consumers interest in gummy supplements is growing and further studies on that field are needed. That is, to make sure consumers get a reliable product where the vitamin is active for the whole labelled shelf-life.

54 7. References

Abdullah, A. (2013). Utilization of gum arabic for industries and human health (Vol. 10(10)). Adsare, S. R., Bellary, A. N., Sowbhagya, H. B., Baskaran, R., Prakash, M., & Rastogi, N. K. (2016). Osmotic treatment for the impregnation of anthocyanin in candies from Indian gooseberry (Emblica officinalis). Journal of Food Engineering, 175, 24-32. doi:https://doi.org/10.1016/j.jfoodeng.2015.11.023 Ahmed, S. H., Guillem, K., & Vandaele, Y. (2013). Sugar addiction: pushing the drug-sugar analogy to the limit. Current Opinion in Clinical Nutrition & Metabolic Care, 16(4), 434- 439. doi:10.1097/MCO.0b013e328361c8b8 Albersheim, P., Darvill, A. G., O'Neill, M. A., Schols, H. A., & Voragen, A. G. J. (1996). An hypothesis: The same six polysaccharides are components of the primary cell walls of all higher plants. In J. Visser & A. G. J. Voragen (Eds.), Progress in Biotechnology (Vol. 14, pp. 47-55): Elsevier. Ali, B. H., Ziada, A., & Blunden, G. (2009). Biological effects of gum arabic: A review of some recent research. Food and Chemical Toxicology, 47(1), 1-8. doi:https://doi.org/10.1016/j.fct.2008.07.001 Amazon. (2019). Best Sellers in Vitamin D Supplements. Retrieved 22.05.2019 from https://www.amazon.com/Best-Sellers-Health-Personal-Care-Vitamin- Supplements/zgbs/hpc/3774781/ref=zg_bs_nav_hpc_3_6936790011 Andersen, C. (2013). Daytime Color Appearance of Retroreflective Traffic Control Sign Materials, Publication No. FHWA-HRT-13-018, April 2013. Andersen, O., Zweidorff, O. K., Hjelde, T., & Rødland, E. A. (1995). Problems when swallowing tablets. A questionnaire study from general practice. Tidsskrift for den Norske laegeforening : tidsskrift for praktisk medicin, ny raekke, 115(8), 947-949. Armisén, R., & Gaiatas, F. (2009). 4 - Agar. In G. O. Phillips & P. A. Williams (Eds.), Handbook of Hydrocolloids (Second Edition) (pp. 82-107): Woodhead Publishing. Avena, N. M., Rada, P., & Hoebel, B. G. (2008). Evidence for sugar addiction: Behavioral and neurochemical effects of intermittent, excessive sugar intake. Neuroscience & Biobehavioral Reviews, 32(1), 20-39. doi:https://doi.org/10.1016/j.neubiorev.2007.04.019 Axelos, M. A. V., & Thibault, J. F. (1991). The Chemistry of Low-Methoxyl Pection Gelation. In R. H. Walter (Ed.), The Chemistry and Technology of Pectin (pp. 109-118). San Diego: Academic Press. B-Carotene Molecule. (2016). Retrieved 05.03.2019 from https://www.worldofmolecules.com/colors/bcarotene.htm BeMiller, J. N. (2008). 9 - Hydrocolloids. In E. K. Arendt & F. Dal Bello (Eds.), Gluten-Free Cereal Products and Beverages (pp. 203-215). San Diego: Academic Press. Bertrand, M.-E., & Turgeon, S. L. (2007). Improved gelling properties of whey protein isolate by addition of xanthan gum. Food Hydrocolloids, 21(2), 159-166. doi:https://doi.org/10.1016/j.foodhyd.2006.03.005

55 Bourekoua, H., Różyło, R., Benatallah, L., Wójtowicz, A., Łysiak, G., Zidoune, M. N., & Sujak, A. (2018). Characteristics of gluten-free bread: quality improvement by the addition of starches/hydrocolloids and their combinations using a definitive screening design. European Food Research and Technology, 244(2), 345-354. doi:10.1007/s00217- 017-2960-9 Bryndís Elfa Gunnarsdóttir, Elva Gísladóttir, Hólmfríður Þorgeirsdóttir, Hrund Valgeirsdóttir, Inga Þórsdóttir, Ingibjörg Gunnarsdóttir, Jónína Stefánsdóttir, Laufey Steingrímsdóttir. (2011). Hvað borða íslendingar? Könnun á mataræði Íslendinga 2010-2011. Helstu niðurstöður.: Embætti landlæknis, Matvælastofnun and Rannsóknarstofa í næringarfræði. Cappa, C., Lavelli, V., & Mariotti, M. (2015). Fruit candies enriched with grape skin powders: physicochemical properties. LWT - Food Science and Technology, 62(1, Part 2), 569-575. doi:https://doi.org/10.1016/j.lwt.2014.07.039 Cortez, R., Luna-Vital, D. A., Margulis, D., & Gonzalez de Mejia, E. (2017). Natural Pigments: Stabilization Methods of Anthocyanins for Food Applications. Comprehensive Reviews in Food Science and Food Safety, 16(1), 180-198. doi:doi:10.1111/1541- 4337.12244 Damodaran, S., Parkin, K. L., & Fennema, O. R. (2008). Fennema's Food Chemistry (4 ed.). USA: CRC Press. DeMars, L. L., & Ziegler, G. R. (2001). Texture and structure of gelatin/pectin-based gummy confections. Food Hydrocolloids, 15(4-6), 643-653. doi:10.1016/s0268-005x(01)00044-3 Dettmar, P. W., Strugala, V., & Craig Richardson, J. (2011). The key role alginates play in health. Food Hydrocolloids, 25(2), 263-266. doi:https://doi.org/10.1016/j.foodhyd.2009.09.009 Djabourov, M., Clark, A. H., Rowlands, D. W., & Ross-Murphy, S. B. (1989). Small-angle x- ray scattering characterization of agarose sols and gels. Macromolecules, 22(1), 180-188. doi:10.1021/ma00191a035 Draget, K. I. (2009). 29 - Alginates. In G. O. Phillips & P. A. Williams (Eds.), Handbook of Hydrocolloids (Second Edition) (pp. 807-828): Woodhead Publishing. Draget, K. I., & Taylor, C. (2011). Chemical, physical and biological properties of alginates and their biomedical implications. Food Hydrocolloids, 25(2), 251-256. doi:https://doi.org/10.1016/j.foodhyd.2009.10.007 Dul, M., Paluch, K., Kelly, H., Healy, A., Sasse, A., & Tajber, L. (2015). Self-assembled carrageenan/protamine polyelectrolyte nanoplexes—Investigation of critical parameters governing their formation and characteristics (Vol. 123). Edwards, C. A., & Garcia, A. L. (2009). 3 - The health aspects of hydrocolloids. In G. O. Phillips & P. A. Williams (Eds.), Handbook of Hydrocolloids (Second Edition) (pp. 50- 81): Woodhead Publishing. Ellin, A. (2017). Vitamins Gone Gummy. The New York Times. Retrieved 10.02.2019 from https://www.nytimes.com/2017/02/28/well/eat/vitamins-gone-gummy.html Embætti landlæknis. (2016). Grundvöllur ráðlegginga um mataræði og ráðlagðir dagskammtar næringarefna. Reykjavík: Embætti landlæknis.

56 Embætti landlæknis. (2017). Ráðleggingar um mataræði fyrir fullorðna og börn frá tveggja ára aldri. Reykjavík: Embætti landlæknis. Endt, D. W. v., & Baker, M. T. (2000). The Chemistry of Filled Animal Glue Systems. Retrieved 05.03.2019 from http://albumen.conservation- us.org/library/c20/vonendt1991.html Ergun, R., Lietha, R., & Hartel, R. W. (2010). Moisture and Shelf Life in Sugar Confections. Critical Reviews in Food Science and Nutrition, 50(2), 162-192. doi:10.1080/10408390802248833 Fields, J., Go, J. T., & Schulze, K. S. (2015). Pill Properties that Cause Dysphagia and Treatment Failure. Current therapeutic research, clinical and experimental, 77, 79-82. doi:10.1016/j.curtheres.2015.08.002 Garland, C. F., Garland, F. C., Gorham, E. D., Lipkin, M., Newmark, H., Mohr, S. B., & Holick, M. F. (2006). The Role of Vitamin D in Cancer Prevention. American Journal of Public Health, 96(2), 252-261. doi:10.2105/ajph.2004.045260 Glerup, H., Mikkelsen, K., Poulsen, L., Hass, E., Overbeck, S., Thomsen, J., Charles, P., Eriksen, E. F. (2000). Commonly recommended daily intake of vitamin D is not sufficient if sunlight exposure is limited. Journal of Internal Medicine, 247(2), 260-268. doi:doi:10.1046/j.1365-2796.2000.00595.x Gould, A. L., Davies, G. M., Alemao, E., Yin, D. D., & Cook, J. R. (2007). Cholesterol reduction yields clinical benefits: meta-analysis including recent trials. Clinical Therapeutics, 29(5), 778-794. doi:https://doi.org/10.1016/j.clinthera.2007.05.012 Hamman, J. (2010). Chitosan Based Polyelectrolyte Complexes as Potential Carrier Materials in Drug Delivery Systems (Vol. 8). Hani, N. M., Romli, S. R., & Ahmad, M. (2015). Influences of red pitaya fruit puree and gelling agents on the physico-mechanical properties and quality changes of gummy confections. International Journal of Food Science and Technology, 50(2), 331-339. doi:10.1111/ijfs.12638 Harris, P. (1990). Food Gels. London and New York: Elsevier Science Publishers. Haug, I. J., & Draget, K. I. (2009). 6 - Gelatin. In G. O. Phillips & P. A. Williams (Eds.), Handbook of Hydrocolloids (Second Edition) (pp. 142-163): Woodhead Publishing. Hoefler, A. C. Pectin: Chemistry, Functionality & Applications. Retrieved 05.03.2019 from https://slideplayer.com/slide/14223831/ Holick, M. F. (1996). Vitamin D and Bone Health. The Journal of Nutrition, 126(suppl_4), 1159S-1164S. doi:10.1093/jn/126.suppl_4.1159S Hyppönen, E., Läärä, E., Reunanen, A., Järvelin, M.-R., & Virtanen, S. M. (2001). Intake of vitamin D and risk of type 1 diabetes: a birth-cohort study. The Lancet, 358(9292), 1500- 1503. doi:https://doi.org/10.1016/S0140-6736(01)06580-1 Imeson, A. P. (2009). 7 - Carrageenan and furcellaran. In G. O. Phillips & P. A. Williams (Eds.), Handbook of Hydrocolloids (Second Edition) (pp. 164-185): Woodhead Publishing.

57 Insel, P., Ross, D., McMahon, K., & Bernstein, M. (2014). Nutrition (5 ed.). Burlington: Jones & Bartlett Learning. Institute of Medicine (US) Panel on the Definition of Dietary fiber and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes. (2001). Dietary Reference Intakes Proposed Definition of Dietary Fiber. Washington (DC): National Academies Press (US). International Organization for Standardization. (1999). ISO 6496:1999 (2 ed.): International Organization for Standardization. Jenkins, D. A., Newton, C., Leeds, A., & Cummings, J. (1975). Effect of pectin, guar gum, and wheat fibre on serum-cholesterol. The Lancet, 305(7916), 1116-1117. doi:https://doi.org/10.1016/S0140-6736(75)92503-9 Johnston-Banks, F. A. (1990). Gelatine. In P. Harris (Ed.), Food Gels (pp. 233-289). Dordrecht: Springer Netherlands. Kannall, E. (2018). What are the benefits of chewable vitamins? SF Gate - Healthy Eating. Retrieved 10.02.2019 from https://healthyeating.sfgate.com/benefits-chewable-vitamins- 8933.html Kemp, S. E. (2008). Application of sensory evaluation in food research. International Journal of Food Science & Technology, 43(9), 1507-1511. doi:doi:10.1111/j.1365- 2621.2008.01780.x Kleiman-Weiner, M., Luo, R., Zhang, L., Shi, Y., Medina, A., & Rozelle, S. (2013). Eggs versus chewable vitamins: Which intervention can increase nutrition and test scores in rural China? China Economic Review, 24, 165-176. doi:https://doi.org/10.1016/j.chieco.2012.12.005 Klopotek, Y., Otto, K., & Böhm, V. (2005). Processing Strawberries to Different Products Alters Contents of Vitamin C, Total Phenolics, Total Anthocyanins, and Antioxidant Capacity. Journal of Agricultural and Food Chemistry, 53(14), 5640-5646. doi:10.1021/jf047947v Kordsmeier, M. C., & Howard, L. R. (2011). Storage effects of gel encapsulation on stability of chokeberry monomeric anthocyanins, procyanidins, color density, and percent polymeric color. The Student Journal of Dale Bumpers College of Agricultural, Food and Life Sciences, 12. Labropoulos, K. C., Niesz, D. E., Danforth, S. C., & Kevrekidis, P. G. (2002). Dynamic rheology of agar gels: theory and experiments. Part I. Development of a rheological model. Carbohydrate Polymers, 50(4), 393-406. doi:https://doi.org/10.1016/S0144- 8617(02)00084-X Lahaye, M., & Rochas, C. (1991). Chemical structure and physico-chemical properties of agar. Hydrobiologia, 221(1), 137-148. doi:10.1007/BF00028370 Lau, M. H., Tang, J., & Paulson, A. T. (2000). Texture profile and turbidity of gellan/gelatin mixed gels. Food Research International, 33(8), 665-671. doi:https://doi.org/10.1016/S0963-9969(00)00111-3

58 Lele, V., Ruzauskas, M., Zavistanaviciute, P., Laurusiene, R., Rimene, G., Kiudulaite, D., Tomkeviciute, J., Nemeikstyte, J., Stankevicius, R., Bartkiene, E. (2018). Development and characterization of the gummy–supplements, enriched with probiotics and prebiotics. CyTA - Journal of Food, 16(1), 580-587. doi:10.1080/19476337.2018.1433721 Levy, J. (2018). Is Gum Arabic Harmful or Helpful? Pros & Cons of This Natural Thickening Agent. Retrieved 10.02.2019 from https://draxe.com/gum-arabic/ Linker, A., & Jones, R. S. (1966). A new polysaccharide resembling alginic acid isolated from pseudomonads. J Biol Chem, 241(16), 3845-3851. Lips, P. (2006). Vitamin D physiology. Progress in Biophysics and Molecular Biology, 92(1), 4-8. doi:https://doi.org/10.1016/j.pbiomolbio.2006.02.016 Lopes-da-Silva, F., Escribano-Baion, M. T., & Santos-Buelga, C. (2007). Stability of Pelargonidin 3-glucoside in Model Solutions in the Presence and Absence of Flavanols. American Journal of Food Technology, 2(7), 602-617. doi:10.3923/ajft.2007.602.617 Lundstedt, T., Seifert, E., Abramo, L., Thelin, B., Nyström, Å., Pettersen, J., & Bergman, R. (1998). Experimental design and optimization. Chemometrics and Intelligent Laboratory Systems, 42(1), 3-40. doi:https://doi.org/10.1016/S0169-7439(98)00065-3 Mangoo-Karim, R., Da Silva Abreu, J., Yanev, G. P., Perez, N. N., Stubbs, J. R., & Wetmore, J. B. (2015). Ergocalciferol versus Cholecalciferol for Nutritional Vitamin D Replacement in CKD. Nephron, 130(2), 99-104. doi:10.1159/000430813 Martín, J., Kuskoski, E. M., Navas, M. J., & Asuero, A. G. (2017). Antioxidant Capacity of Anthocyanin Pigments Flavonoids: From Biosynthesis to Human Health: IntechOpen. Mohnen, D. (2008). Pectin structure and biosynthesis. Current Opinion in Plant Biology, 11(3), 266-277. doi:https://doi.org/10.1016/j.pbi.2008.03.006 Mudgil, D., Barak, S., & Khatkar, B. S. (2014). Guar gum: processing, properties and food applications—A Review. Journal of food science and technology, 51(3), 409-418. doi:10.1007/s13197-011-0522-x Muñtoz, A. M., Pangborn, R. M., & Noble, A. (1986). Sensory and mechanical attributes of gel texture. I. Effect of gelatin concentration (Vol. 17). Murador, D. C., da Cunha, D. T., & de Rosso, V. V. (2014). Effects of cooking techniques on vegetable pigments: A meta-analytic approach to carotenoid and anthocyanin levels. Food Research International, 65, 177-183. doi:https://doi.org/10.1016/j.foodres.2014.06.015 Möller, N. P., Scholz-Ahrens, K. E., Roos, N., & Schrezenmeir, J. (2008). Bioactive peptides and proteins from foods: indication for health effects. European Journal of Nutrition, 47(4), 171-182. doi:10.1007/s00394-008-0710-2 Osilesi, O., Trout, D. L., Glover, E. E., Harper, S. M., Koh, E. T., Behall, K. M., O'Dorisio, T. M., Tartt, J. (1985). Use of xanthan gum in dietary management of diabetes mellitus. Am J Clin Nutr, 42(4), 597-603. doi:10.1093/ajcn/42.4.597 Pang, Z., Deeth, H., Sopade, P., Sharma, R., & Bansal, N. (2014). Rheology, texture and microstructure of gelatin gels with and without milk proteins. Food Hydrocolloids, 35, 484-493. doi:https://doi.org/10.1016/j.foodhyd.2013.07.007

59 Panlasigui, L. N., Baello, O. Q., Dimatangal, J. M., & Dumelod, B. D. (2003). Blood cholesterol and lipid-lowering effects of carrageenan on human volunteers. Asia Pac J Clin Nutr, 12(2), 209-214. Paredes-Juarez, G., Spasojevic, M., Faas, M., & de Vos, P. (2014). Immunological and Technical Considerations in Application of Alginate-Based Microencapsulation Systems (Vol. 2). Phillips, A. O., & Phillips, G. O. (2011). Biofunctional behaviour and health benefits of a specific gum arabic. Food Hydrocolloids, 25(2), 165-169. doi:https://doi.org/10.1016/j.foodhyd.2010.03.012 Phillips, G. O., & Williams, P. A. (2009). Handbook of hydrocolloids (2 ed.): Woodhead Publishing Limited. Potter, N. N., & Hotchkiss, J. H. (1998). Food science - Fifth edition (5 ed.): Springer. Rosner, B., Sacks, F. M., Brown, L., & Willett, W. W. (1999). Cholesterol-lowering effects of dietary fiber: a meta-analysis. Am J Clin Nutr, 69(1), 30-42. doi:10.1093/ajcn/69.1.30 Ross-Murphy, S. B., & Shatwell, K. P. (1993). Polysaccharide strong and weak gels. Biorheology, 30(3-4), 217-227. Saha, D., & Bhattacharya, S. (2010). Hydrocolloids as thickening and gelling agents in food: a critical review. Journal of food science and technology, 47(6), 587-597. doi:10.1007/s13197-010-0162-6 Sharma, B. R., Naresh, L., Dhuldhoya, N. C., Merchant, S. U., & Merchant, U. C. (2006). An overview on pectins (Vol. 4). Sheiham, A. (1983). Sugars and dental decay. The Lancet, 321(8319), 282-284. doi:https://doi.org/10.1016/S0140-6736(83)91696-3 Sheika. (2017). Do they work or not? What's the hype about gummy vitamins? Medium. Retrieved 10.02.2019 from https://medium.com/active-beauties/the-pros-and-cons-of- gummy-vitamins-the-big-picture-unveiled-505c352b12b7 Steingrimsdottir, L., Gunnarsson, O., Indridason, O. S., Franzson, L., & Sigurdsson, G. (2005). Relationship Between Serum Parathyroid Hormone Levels, Vitamin D Sufficiency, and Calcium Intake. JAMA, 294(18), 2336-2341. doi:10.1001/jama.294.18.2336 Stephen, A. M., Phillips, G. O., & Williams, P. A. (2006). Food Polysaccharides and Their Applications (2 ed.). Boca Raton: CRC Press. Stewart, R. J., Askew, E. W., McDonald, C. M., Metos, J., Jackson, W. D., Balon, T. W., & Prior, R. L. (2002). Antioxidant Status of Young Children: Response to an Antioxidant Supplement. Journal of the American Dietetic Association, 102(11), 1652-1657. doi:https://doi.org/10.1016/S0002-8223(02)90352-4 Sworn, G. (2009). 8 - Xanthan gum. In G. O. Phillips & P. A. Williams (Eds.), Handbook of Hydrocolloids (Second Edition) (pp. 186-203): Woodhead Publishing.

60 Teixeira Rabelo, H., Barroso, W., & Magalhães, J. (2017). Micronutrients as an Innovation Opportunity for the Pharmaceutical Industry: A Case Study of Vitamin D in Brazil (Vol. 1). Thakur, B. R., Singh, R. K., Handa, A. K., & Rao, M. A. (1997). Chemistry and uses of pectin — A review. Critical Reviews in Food Science and Nutrition, 37(1), 47-73. doi:10.1080/10408399709527767 Tiwari, B. K., OʼDonnell, C. P., Patras, A., & Cullen, P. J. (2008). Anthocyanin and Ascorbic Acid Degradation in Sonicated Strawberry Juice. J Agric Food Chem, 56(21), 10071- 10077. doi:10.1021/jf801824v Tripkovic, L., Lambert, H., Hart, K., Smith, C. P., Bucca, G., Penson, S., Chope, G., Hypponen, E., Berry, J., Vieth, R., Lanham-New, S. (2012). Comparison of vitamin D2 and vitamin D3 supplementation in raising serum 25-hydroxyvitamin D status: a systematic review and meta-analysis. Am J Clin Nutr, 95(6), 1357-1364. doi:10.3945/ajcn.111.031070 Verbeken, D., Dierckx, S., & Dewettinck, K. (2003). Exudate gums: occurrence, production, and applications. Applied Microbiology and Biotechnology, 63(1), 10-21. doi:10.1007/s00253-003-1354-z Viebke, C., Al-Assaf, S., & Phillips, G. O. (2014). Food hydrocolloids and health claims. Bioactive Carbohydrates and Dietary Fibre, 4(2), 101-114. doi:https://doi.org/10.1016/j.bcdf.2014.06.006 Whytock, S., & Finch, J. (1991). The substructure of agarose gels as prepared for electrophoresis. Biopolymers, 31(9), 1025-1028. doi:10.1002/bip.360310902 Williams, P. A., & Phillips, G. O. (2009a). 1 - Introduction to food hydrocolloids. In G. O. Phillips & P. A. Williams (Eds.), Handbook of Hydrocolloids (Second Edition) (pp. 1-22): Woodhead Publishing. Williams, P. A., & Phillips, G. O. (2009b). 11 - Gum arabic. In G. O. Phillips & P. A. Williams (Eds.), Handbook of Hydrocolloids (Second Edition) (pp. 252-273): Woodhead Publishing. Yamjala, K., Nainar, M. S., & Ramisetti, N. R. (2016). Methods for the analysis of azo dyes employed in food industry – A review. Food Chemistry, 192, 813-824. doi:https://doi.org/10.1016/j.foodchem.2015.07.085

61 8. Appendix

Figure 55. The gelatin and agar from Sigma Aldrich which was used in the gummies.

Figure 56. The vitamin D from Sigma Aldrich which was used in the gummies.

Figure 57. Temperature of the freezer, where the strawberry and carrot juices were stored.

62 Table 22. Coded levels for the factors of the screening design. Sugar Glucose syrup Juice Water Coded lower level -1 -1 -1 -1 Coded upper level +1 +1 +1 +1 Real lower level (g) 35 35 15 0 Real upper level (g) 50 50 35 15

Table 23. A 24 full factorial design for the screening design with four factors and two levels and the mean scores from the sensory evaluation. Initial Test Glucose Watery Overall Sugar Juice Water moisture Color no. syrup mouthfeel likeness content* 1 -1 -1 -1 -1 24 1 5 4

2 -1 -1 -1 +1 34 4 4 4

3 -1 -1 +1 -1 37 6 6 9

4 -1 -1 +1 +1 45 5 3 8

5 -1 +1 -1 -1 22 1 4 3

6 -1 +1 -1 +1 32 3 9 6

7 -1 +1 +1 -1 35 2 8 10

8 -1 +1 +1 +1 41 5 6 9

9 +1 -1 -1 -1 20 0 3 4

10 +1 -1 -1 +1 30 5 5 7

11 +1 -1 +1 -1 33 6 7 7

12 +1 -1 +1 +1 40 6 8 8

13 +1 +1 -1 -1 19 0 5 4

14 +1 +1 -1 +1 28 3 7 6

15 +1 +1 +1 -1 31 3 8 7

16 +1 +1 +1 +1 37 3 5 9

*Initial moisture content calculated as the sum of water from the glucose syrup (17% moisture), juice and water. Estimated that the juice is 100% moisture. The dry matter from gelatin and citric acid are also taken into the calculations.

63 Table 24. Coded levels for the factors of the optimization design. -2 -1 0 +1 +2 Juice (g) 15 20 25 30 35 Initial moisture (%) 28 30 32 34 36

Table 25. Central composite design for the optimization with two factors and five levels and the mean scores from the sensory evaluation. Initial Watery Overall Test no. Juice Color moisture mouthfeel likeness 1 +2 0 5 9 10 2 -2 0 4 7,5 8 3 0 +2 5 8 9,5 4 0 -2 3 7,5 8,5 5 0 0 3,5 8,5 9 6 0 0 3 9,5 9 7 0 0 4 8 9 8 +1 +1 2 7 9 9 +1 -1 3 8 9 10 -1 -1 1,5 8,5 8,5 11 -1 +1 5 8 9 12 0 0 3 8 9 13 0 0 3 7,5 9 14 0 0 3,5 8 9

64 Table 26. Amount of added water or sugar to each recipe as part of the optimization to adjust the right initial moisture content. Test no. Juice (g) Initial moisture (%) Water/sugar added 1 35 32 4 g sugar 2 15 32 18 g water 3 25 36 16 g water 4 25 28 1 g water 5 25 32 8 g water 6 25 32 8 g water 7 25 32 8 g water 8 30 34 7 g water 9 30 30 1 g sugar 10 20 30 9 g water 11 20 34 17 g water 12 25 32 8 g water 13 25 32 8 g water 14 25 32 8 g water

To get a better idea of how the calculations were carried out in, calculations for the first two test are shown below.

푊푎푡푒푟 푓푟표푚 푔푙푢푐표푠푒 푠푦푟푢푝+푗푢푖푐푒+푤푎푡푒푟 Initial moisture content(%): 퐺푙푢푐표푠푒 푠푦푟푢푝+푠푢푔푎푟+푗푢푖푐푒+푤푎푡푒푟+푔푒푙푎푡푖푛+푐푖푡푟푖푐 푎푐푖푑

8,5+35 Example, test no. 1: = 32% (extra 4 g sugar for less moisture). 50+44+35+5+1,5

8,5+15+18 Example, test no. 2: = 32% (extra 18 g of water for more moisture). 50+40+15+18+5+1,5

Table 27. Results from all the moisture measurements. First First Second Second Mean measurement measurement measurement measurement value

Strawb. gelatin 87,59 85,71 87,24 87,42 87,0

Strawb. agar 81,46 84,84 83,44 83,34 83,3

Carrot gelatin 86,47 84,81 86,89 87,19 86,3

Carrot agar 81,69 81,71 - - 81,7

65 8.1 Texture

Figure 58. Toughness changes over the time period in the four gummies (including week seven).

Table 28. Hardness in carrot agar gummy. Comparing means between weeks. Week Week Difference p-Value 3 11 1332,738 0,0004* 5 11 963,527 0,0077* 3 9 937,088 0,0093* 3 7 813,650 0,0221* 1 11 762,112 0,0207* 13 11 755,857 0,0325* 3 13 576,881 0,0974 3 1 570,625 0,0775 5 9 567,878 0,1025 7 11 519,088 0,1341 5 7 444,439 0,1974 9 11 395,649 0,2498 3 5 369,211 0,2822 1 9 366,463 0,2496 13 9 360,207 0,2938 1 7 243,025 0,4423 13 7 236,769 0,4879 5 13 207,670 0,5425 5 1 201,414 0,5236 7 9 123,439 0,7168 1 13 6,256 0,9841

66 Table 29. Hardness in strawberry agar gummies. Comparing means between weeks. Week Week Difference p-Value 1 7 997,3061 <,0001* 3 7 952,0398 <,0001* 1 9 858,2545 <,0001* 3 9 812,9882 <,0001* 1 13 808,2089 <,0001* 1 11 803,5187 <,0001* 3 13 762,9426 <,0001* 3 11 758,2524 <,0001* 5 7 504,3130 0,0047* 1 5 492,9931 0,0030* 3 5 447,7268 0,0110* 5 9 365,2614 0,0347* 5 13 315,2158 0,0658 5 11 310,5256 0,0697 11 7 193,7874 0,2497 13 7 189,0972 0,2611 9 7 139,0516 0,4063 11 9 54,7358 0,7425 13 9 50,0456 0,7639 1 3 45,2663 0,7691 11 13 4,6902 0,9775

Table 30. Hardness in carrot gelatin gummies. Comparing means between weeks. Week Week Difference p-Value 5 1 579,5406 <,0001* 7 1 461,4290 0,0004* 11 1 459,9504 0,0004* 13 1 415,1144 0,0011* 3 1 362,4276 0,0035* 5 9 322,0408 0,0142* 9 1 257,4998 0,0321* 5 3 217,1130 0,0894 7 9 203,9292 0,1097 11 9 202,4506 0,1122 5 13 164,4262 0,1938 13 9 157,6146 0,2124 5 11 119,5902 0,3414 5 7 118,1116 0,3473 3 9 104,9278 0,4030 7 3 99,0014 0,4298 11 3 97,5228 0,4367 13 3 52,6868 0,6732 7 13 46,3146 0,7107 11 13 44,8360 0,7196 7 11 1,4786 0,9905

67 Table 31. Hardness in strawberry gelatin gummies. Comparing means between weeks. Week Week Difference p-Value 11 1 725,5823 0,0009* 11 3 582,6098 0,0100* 11 9 495,3124 0,0262* 13 1 475,6947 0,0215* 11 7 450,3024 0,0419* 11 5 448,2914 0,0427* 13 3 332,7222 0,1268 5 1 277,2909 0,1677 7 1 275,2799 0,1707 11 13 249,8876 0,2475 13 9 245,4248 0,2558 9 1 230,2699 0,2496 13 7 200,4148 0,3517 13 5 198,4038 0,3565 3 1 142,9725 0,4717 5 3 134,3184 0,5309 7 3 132,3074 0,5370 9 3 87,2974 0,6832 5 9 47,0210 0,8259 7 9 45,0100 0,8332 5 7 2,0110 0,9925

Table 32. Toughness in carrot agar gummies. Comparing means between weeks. Week Week Difference p-Value 7 5 1974,467 <,0001* 7 11 1860,742 <,0001* 7 9 1703,951 <,0001* 7 1 1619,887 <,0001* 7 3 1385,279 0,0001* 7 13 989,367 0,0037* 13 5 985,099 0,0038* 13 11 871,374 0,0094* 13 9 714,584 0,0302* 13 1 630,520 0,0381* 3 5 589,188 0,0704 3 11 475,463 0,1404 13 3 395,911 0,2171 1 5 354,579 0,2321 3 9 318,673 0,3183 9 5 270,515 0,3958 1 11 240,855 0,4139 3 1 234,609 0,4260 9 11 156,790 0,6212 11 5 113,725 0,7197 1 9 84,064 0,7744

68 Table 33. Toughness in strawberry agar gummies. Comparing means between weeks. Week Week Difference p-Value 7 5 1629,355 <,0001* 7 9 1428,327 <,0001* 7 11 1278,739 <,0001* 7 1 1162,446 <,0001* 7 13 1155,208 <,0001* 7 3 896,219 0,0007* 3 5 733,136 0,0043* 3 9 532,109 0,0326* 13 5 474,146 0,0550 1 5 466,909 0,0420* 3 11 382,520 0,1177 11 5 350,616 0,1502 13 9 273,119 0,2591 3 1 266,227 0,2353 1 9 265,881 0,2359 3 13 258,989 0,2841 9 5 201,027 0,4039 11 9 149,589 0,5335 13 11 123,530 0,6067 1 11 116,293 0,6007 13 1 7,238 0,9740

Table 34. Toughness in carrot gelatin gummies. Comparing means between weeks. Week Week Difference p-Value 7 1 605,9820 <,0001* 7 9 415,5636 <,0001* 7 3 375,0020 <,0001* 5 1 371,3770 <,0001* 7 13 333,5668 0,0001* 7 11 304,5082 0,0003* 11 1 301,4738 0,0002* 13 1 272,4152 0,0005* 7 5 234,6050 0,0040* 3 1 230,9800 0,0024* 9 1 190,4184 0,0104* 5 9 180,9586 0,0225* 5 3 140,3970 0,0717 11 9 111,0554 0,1501 5 13 98,9618 0,1981 13 9 81,9968 0,2842 11 3 70,4938 0,3560 5 11 69,9032 0,3600 13 3 41,4352 0,5857 3 9 40,5616 0,5936 11 13 29,0586 0,7019

69 Table 35. Toughness in strawberry gelatin gummies. Comparing means between weeks. Week Week Difference p-Value 7 1 447,0249 0,0003* 11 1 399,8325 0,0009* 7 3 343,4688 0,0065* 7 9 323,9922 0,0098* 11 3 296,2764 0,0172* 7 5 281,8780 0,0228* 11 9 276,7998 0,0252* 13 1 267,1245 0,0201* 11 5 234,6856 0,0549 7 13 179,9004 0,1362 5 1 165,1469 0,1394 13 3 163,5684 0,1741 13 9 144,0918 0,2296 11 13 132,7080 0,2676 9 1 123,0327 0,2670 3 1 103,5561 0,3487 13 5 101,9776 0,3923 5 3 61,5908 0,6040 7 11 47,1924 0,6908 5 9 42,1142 0,7225 9 3 19,4766 0,8694

Table 36. Stickiness in carrot agar gummies. Comparing means between weeks. Week Week Difference p-Value 3 13 33,87380 <,0001* 1 13 33,84594 <,0001* 5 13 27,34300 0,0005* 3 11 26,46960 0,0007* 1 11 26,44174 0,0003* 3 9 20,88220 0,0058* 1 9 20,85434 0,0032* 5 11 19,93880 0,0081* 7 13 19,69280 0,0088* 5 9 14,35140 0,0500 3 7 14,18100 0,0527 1 7 14,15314 0,0376* 9 13 12,99160 0,0744 7 11 12,28860 0,0906 5 7 7,65020 0,2851 11 13 7,40420 0,3006 7 9 6,70120 0,3480 3 5 6,53080 0,3602 1 5 6,50294 0,3256 9 11 5,58740 0,4329 3 1 0,02786 0,9966

70 Table 37. Stickiness in strawberry agar gummies. Comparing means between weeks. Week Week Difference p-Value 1 11 23,67477 0,0015* 5 11 18,07060 0,0195* 1 13 16,63817 0,0202* 1 7 14,76757 0,0374* 1 9 14,29157 0,0435* 1 3 11,99737 0,0870 3 11 11,67740 0,1213 5 13 11,03400 0,1424 9 11 9,38320 0,2100 5 7 9,16340 0,2205 7 11 8,90720 0,2334 5 9 8,68740 0,2449 13 11 7,03660 0,3444 5 3 6,39320 0,3896 1 5 5,60417 0,4150 3 13 4,64080 0,5311 3 7 2,77020 0,7079 9 13 2,34660 0,7509 3 9 2,29420 0,7563 7 13 1,87060 0,8002 9 7 0,47600 0,9486

Table 38. Stickiness in carrot gelatin gummies. Comparing means between weeks. Week Week Difference p-Value 3 7 5,422200 <,0001* 3 1 4,002743 <,0001* 13 7 3,732000 0,0005* 11 7 3,731400 0,0005* 5 7 3,120000 0,0028* 3 9 2,923600 0,0048* 9 7 2,498600 0,0142* 13 1 2,312543 0,0143* 11 1 2,311943 0,0143* 3 5 2,302200 0,0229* 5 1 1,700543 0,0653 3 11 1,690800 0,0884 3 13 1,690200 0,0885 1 7 1,419457 0,1207 13 9 1,233400 0,2087 11 9 1,232800 0,2089 9 1 1,079143 0,2341 5 9 0,621400 0,5223 13 5 0,612000 0,5286 11 5 0,611400 0,5290 13 11 0,000600 0,9995

71 Table 39. Stickiness in strawberry gelatin gummies. Comparing means between weeks Week Week Difference p-Value 3 11 8,891800 0,0310* 9 11 7,254800 0,0746 13 11 6,694400 0,0986 1 11 5,427057 0,1460 5 11 5,106600 0,2034 7 11 4,845400 0,2268 3 7 4,046400 0,3111 3 5 3,785200 0,3428 3 1 3,464743 0,3482 9 7 2,409400 0,5442 3 13 2,197400 0,5799 9 5 2,148200 0,5884 13 7 1,849000 0,6412 9 1 1,827743 0,6188 3 9 1,637000 0,6798 13 5 1,587800 0,6888 13 1 1,267343 0,7298 1 7 0,581657 0,8740 9 13 0,560400 0,8875 1 5 0,320457 0,9304 5 7 0,261200 0,9474

72 Table 40. Raw data from the texture measurements. Hardness Toughness Stickiness Peak Peak Peak Peak Positive Positive Positive Positive Peak Peak Peak Peak Week force force force force area area area area negative negative negative negative no. (g) (g) (g) (g) (g/sec) (g/sec) (g/sec) (g/sec) force (g) force (g) force (g) force (g) CA SA CG SG CA SA CG SG CA SA CG SG 1 2783,6 3894,2 376,6 715,7 2486,6 3341,4 300,4 491,7 -8,3 -11,9 -6,5 -17,3 1 2810,1 3630,5 834,1 606,9 2913,1 3147,6 556,9 429,4 -17,5 -12,1 -8,0 -14,0 1 3832,6 3276,5 490,7 800,7 3568,5 2761,5 346,2 523,0 -12,5 -14,0 -8,2 -6,0 1 3941,8 3641,6 435,5 926,1 3664,2 3199,9 339,7 586,8 -11,5 -11,2 -8,1 -4,7 1 2620,9 3807,2 436,6 870,3 2696,9 3554,7 339,6 595,7 -14,6 -11,7 -10,7 -9,2 1 3107,8 3657,0 349,7 633,2 3194,9 3517,4 286,8 427,9 -15,9 -10,5 -7,2 -22,8 1 4139,9 3002,4 821,1 632,3 3855,4 2371,6 516,8 451,9 -11,4 -12,6 -11,3 -6,1 3 4479,2 3494,4 797,2 1053,4 4096,5 3869,5 561,3 679,7 -14,8 -62,8 -4,2 -11,2 3 3870,1 3361,0 1072,2 1051,0 3376,0 2966,2 656,8 697,2 -10,8 -12,0 -6,2 -10,4 3 3802,6 3793,1 736,2 743,6 3298,8 3662,3 547,2 525,3 -15,0 -16,8 -4,4 -6,4 3 3502,4 3408,0 954,2 921,5 2990,2 3358,4 664,9 660,8 -11,2 -15,0 -4,4 -6,8 3 3796,5 3509,5 926,8 649,1 3397,0 3113,4 643,4 459,3 -13,6 -13,3 -3,6 -5,3 5 3819,4 2961,9 1062,3 1209,2 2786,9 2071,6 734,1 757,0 -16,7 -14,7 -4,2 -19,9 5 3623,1 3214,6 1008,2 697,9 2569,3 2299,0 716,1 488,6 -17,7 -17,4 -7,0 -9,1 5 3525,5 3170,7 1137,1 819,7 3225,7 2932,0 772,5 556,7 -14,9 -16,3 -6,8 -9,3 5 4045,5 3032,3 1516,0 1265,2 2994,7 3077,9 961,1 771,1 -25,8 -22,9 -9,8 -10,9 5 2591,2 2947,9 848,6 1098,2 2635,9 2923,7 591,9 756,8 -23,1 -16,7 -6,4 -9,6 7 2291,9 2578,9 982,5 1079,8 4343,8 4466,4 1003,3 977,1 -42,1 -28,0 -11,6 -12,6 7 3270,4 2342,4 690,7 1222,1 4806,1 3871,6 713,0 1086,7 -20,8 -23,0 -9,8 -13,8 7 3352,7 2642,1 1332,6 1437,2 4957,4 4118,3 1271,6 1284,1 -23,5 -21,8 -9,6 -14,2 7 3235,0 2566,7 1131,7 812,0 5058,8 4738,0 1085,8 816,8 -24,6 -30,3 -9,3 -10,4 7 3232,5 2675,8 844,2 529,1 4918,7 4256,5 875,0 575,0 -25,4 -30,8 -9,6 -9,2 9 3419,5 2864,4 906,0 1165,2 3545,3 3167,8 648,5 746,6 -30,4 -24,1 -10,1 -15,9 9 2535,3 2520,8 884,8 1329,1 2635,6 2483,8 619,0 761,0 -30,1 -19,4 -6,9 -8,5 9 2426,6 2888,2 575,1 710,8 2471,1 2888,0 455,4 471,6 -30,3 -25,0 -5,3 -7,1 9 2272,4 2733,5 949,6 956,8 2505,3 3159,9 656,0 641,4 -36,6 -43,6 -7,8 -9,0 9 4111,6 2494,2 646,6 693,1 4407,8 2609,7 491,9 499,3 -42,5 -19,3 -7,3 -7,6 11 2108,5 2604,2 835,7 1628,6 2576,6 3010,3 599,5 995,6 -23,9 -37,2 -3,9 -13,3 11 2516,4 2833,6 1117,9 2457,3 2798,0 3364,5 759,5 1394,4 -27,6 -65,4 -5,8 -39,9 11 2451,7 3007,0 1158,3 899,5 2723,7 3275,2 754,8 626,9 -42,6 -36,5 -6,7 -8,6 11 2436,4 2849,9 768,7 1409,7 2957,9 2903,8 565,1 843,2 -53,7 -21,3 -7,3 -10,5 11 3274,1 2479,9 1093,7 936,5 3724,9 2503,3 747,3 643,6 -50,0 -18,0 -7,5 -12,1 13 3218,9 2462,2 1140,3 1255,0 3221,4 2976,8 733,2 799,8 -19,0 -23,8 -6,3 -14,3 13 3100,0 3159,9 779,4 1786,3 3839,6 3465,1 575,5 1067,0 -51,9 -31,4 -4,8 -8,5 13 3822,7 2054,4 955,2 955,4 4392,7 2629,7 655,2 636,7 -43,4 -35,2 -8,3 -9,4 13 3348,9 2994,0 833,6 977,7 3584,2 3452,6 592,9 631,7 -35,2 -34,0 -6,3 -9,3 13 3075,9 3080,8 1041,7 1107,8 4100,1 3150,6 724,2 705,0 -85,4 -18,9 -5,6 -9,6

73 8.2 Color Table 41. „L values“ comparisons between weeks for carrot agar. Week Week Difference p-Value Week Week Difference p-Value 14 13 2,066000 0,0044* 1 6 0,593333 0,3775 1 13 1,939333 0,0052* 6 11 0,568000 0,4184 7 13 1,866000 0,0097* 7 8 0,552000 0,4315 9 13 1,622000 0,0235* 6 10 0,540000 0,4415 2 13 1,610000 0,0245* 8 11 0,536000 0,4449 14 5 1,388000 0,0512 12 5 0,530000 0,4500 6 13 1,346000 0,0584 6 4 0,522000 0,4568 8 13 1,314000 0,0644 7 6 0,520000 0,4586 14 11 1,288000 0,0697 8 10 0,508000 0,4689 1 5 1,261333 0,0637 8 4 0,490000 0,4848 14 10 1,260000 0,0758 14 2 0,456000 0,5155 14 4 1,242000 0,0800 14 9 0,444000 0,5265 12 13 1,208000 0,0884 12 11 0,430000 0,5396 7 5 1,188000 0,0936 9 12 0,414000 0,5548 1 11 1,161333 0,0871 2 12 0,402000 0,5662 1 10 1,133333 0,0948 12 10 0,402000 0,5662 1 4 1,115333 0,1000 12 4 0,384000 0,5837 7 11 1,088000 0,1239 6 3 0,358000 0,6094 14 3 1,078000 0,1274 1 2 0,329333 0,6234 7 10 1,060000 0,1337 8 3 0,326000 0,6417 7 4 1,042000 0,1403 1 9 0,317333 0,6361 3 13 0,988000 0,1616 3 5 0,310000 0,6581 1 3 0,951333 0,1593 9 8 0,308000 0,6601 9 5 0,944000 0,1808 2 8 0,296000 0,6726 2 5 0,932000 0,1863 9 6 0,276000 0,6935 7 3 0,878000 0,2128 2 6 0,264000 0,7062 14 12 0,858000 0,2232 7 2 0,256000 0,7147 9 11 0,844000 0,2308 7 9 0,244000 0,7275 2 11 0,832000 0,2374 12 3 0,220000 0,7534 4 13 0,824000 0,2419 3 11 0,210000 0,7642 9 10 0,816000 0,2464 14 7 0,200000 0,7751 10 13 0,806000 0,2522 3 10 0,182000 0,7949 2 10 0,804000 0,2534 3 4 0,164000 0,8148 9 4 0,798000 0,2569 4 5 0,146000 0,8348 2 4 0,786000 0,2640 6 12 0,138000 0,8437 11 13 0,778000 0,2689 10 5 0,128000 0,8549 14 8 0,752000 0,2850 14 1 0,126667 0,8501 1 12 0,731333 0,2776 8 12 0,106000 0,8796 14 6 0,720000 0,3058 11 5 0,100000 0,8864 5 13 0,678000 0,3346 1 7 0,073333 0,9129 6 5 0,668000 0,3418 4 11 0,046000 0,9476 7 12 0,658000 0,3490 6 8 0,032000 0,9635 8 5 0,636000 0,3652 10 11 0,028000 0,9681 9 3 0,634000 0,3667 4 10 0,018000 0,9795 1 8 0,625333 0,3525 9 2 0,012000 0,9863 2 3 0,622000 0,3758

74 Table 42. „L values“ comparisons between weeks for strawberry agar. Week Week Difference p-Value Week Week Difference p-Value 2 4 5,032000 <,0001* 6 4 1,500000 0,0308* 2 7 4,668000 <,0001* 11 5 1,400000 0,0433* 1 4 4,548333 <,0001* 11 13 1,380000 0,0463* 2 8 4,458000 <,0001* 10 7 1,358000 0,0498* 2 3 4,442000 <,0001* 1 12 1,314333 0,0474* 14 4 4,396000 <,0001* 14 12 1,162000 0,0917 2 9 4,312000 <,0001* 10 8 1,148000 0,0956 1 7 4,184333 <,0001* 6 7 1,136000 0,0991 2 5 4,122000 <,0001* 10 3 1,132000 0,1002 2 13 4,102000 <,0001* 10 9 1,002000 0,1446 14 7 4,032000 <,0001* 13 4 0,930000 0,1752 1 8 3,974333 <,0001* 6 8 0,926000 0,1770 1 3 3,958333 <,0001* 12 11 0,924000 0,1780 1 9 3,828333 <,0001* 5 4 0,910000 0,1845 14 8 3,822000 <,0001* 6 3 0,910000 0,1845 14 3 3,806000 <,0001* 10 5 0,812000 0,2357 14 9 3,676000 <,0001* 11 6 0,810000 0,2368 1 5 3,638333 <,0001* 10 13 0,792000 0,2473 1 13 3,618333 <,0001* 6 9 0,780000 0,2544 2 6 3,532000 <,0001* 9 4 0,720000 0,2924 14 5 3,486000 <,0001* 2 14 0,636000 0,3518 14 13 3,466000 <,0001* 3 4 0,590000 0,3875 2 10 3,310000 <,0001* 6 5 0,590000 0,3875 12 4 3,234000 <,0001* 11 10 0,588000 0,3891 1 6 3,048333 <,0001* 8 4 0,574000 0,4004 14 6 2,896000 <,0001* 6 13 0,570000 0,4037 12 7 2,870000 <,0001* 13 7 0,566000 0,4070 1 10 2,826333 <,0001* 5 7 0,546000 0,4236 2 11 2,722000 0,0002* 2 1 0,483667 0,4589 14 10 2,674000 0,0002* 7 4 0,364000 0,5932 12 8 2,660000 0,0002* 13 8 0,356000 0,6013 12 3 2,644000 0,0003* 9 7 0,356000 0,6013 12 9 2,514000 0,0005* 13 3 0,340000 0,6177 12 5 2,324000 0,0011* 5 8 0,336000 0,6218 11 4 2,310000 0,0012* 5 3 0,320000 0,6385 12 13 2,304000 0,0012* 3 7 0,226000 0,7399 1 11 2,238333 0,0011* 10 6 0,222000 0,7444 14 11 2,086000 0,0032* 13 9 0,210000 0,7577 11 7 1,946000 0,0057* 8 7 0,210000 0,7577 2 12 1,798000 0,0103* 5 9 0,190000 0,7801 11 8 1,736000 0,0131* 1 14 0,152333 0,8152 12 6 1,734000 0,0132* 9 8 0,146000 0,8301 10 4 1,722000 0,0138* 9 3 0,130000 0,8485 11 3 1,720000 0,0139* 13 5 0,020000 0,9766 11 9 1,590000 0,0224* 3 8 0,016000 0,9812 12 10 1,512000 0,0296*

75 Table 43. „L values“ comparisons between weeks for carrot gelatin. Week Week Difference p-Value Week Week Difference p-Value 2 9 7,814000 <,0001* 3 5 2,446000 0,0017* 2 10 7,652000 <,0001* 4 8 2,306000 0,0030* 1 9 7,238333 <,0001* 5 9 2,196000 0,0046* 2 11 7,142000 <,0001* 1 7 2,192333 0,0032* 1 10 7,076333 <,0001* 14 9 2,160000 0,0052* 2 13 7,038000 <,0001* 5 10 2,034000 0,0083* 2 12 6,874000 <,0001* 14 10 1,998000 0,0094* 1 11 6,566333 <,0001* 4 14 1,812000 0,0180* 1 13 6,462333 <,0001* 4 5 1,776000 0,0203* 2 6 6,322000 <,0001* 8 9 1,666000 0,0290* 1 12 6,298333 <,0001* 5 11 1,524000 0,0451* 2 8 6,148000 <,0001* 8 10 1,504000 0,0479* 1 6 5,746333 <,0001* 6 9 1,492000 0,0496* 2 14 5,654000 <,0001* 14 11 1,488000 0,0502 2 5 5,618000 <,0001* 5 13 1,420000 0,0613 1 8 5,572333 <,0001* 14 13 1,384000 0,0679 1 14 5,078333 <,0001* 6 10 1,330000 0,0791 7 9 5,046000 <,0001* 5 12 1,256000 0,0967 1 5 5,042333 <,0001* 14 12 1,220000 0,1065 7 10 4,884000 <,0001* 7 4 1,074000 0,1542 3 9 4,642000 <,0001* 8 11 0,994000 0,1867 3 10 4,480000 <,0001* 12 9 0,940000 0,2114 7 11 4,374000 <,0001* 8 13 0,890000 0,2364 7 13 4,270000 <,0001* 6 11 0,820000 0,2749 7 12 4,106000 <,0001* 12 10 0,778000 0,3000 4 9 3,972000 <,0001* 13 9 0,776000 0,3012 3 11 3,970000 <,0001* 8 12 0,726000 0,3331 3 13 3,866000 <,0001* 6 13 0,716000 0,3398 2 4 3,842000 <,0001* 5 6 0,704000 0,3479 4 10 3,810000 <,0001* 11 9 0,672000 0,3701 3 12 3,702000 <,0001* 3 4 0,670000 0,3715 7 6 3,554000 <,0001* 14 6 0,668000 0,3729 7 8 3,380000 <,0001* 13 10 0,614000 0,4125 4 11 3,300000 <,0001* 2 1 0,575667 0,4222 1 4 3,266333 <,0001* 6 12 0,552000 0,4610 4 13 3,196000 <,0001* 5 8 0,530000 0,4790 2 3 3,172000 <,0001* 11 10 0,510000 0,4957 3 6 3,150000 <,0001* 14 8 0,494000 0,5093 4 12 3,032000 0,0001* 7 3 0,404000 0,5891 3 8 2,976000 0,0002* 12 11 0,268000 0,7199 7 14 2,886000 0,0003* 8 6 0,174000 0,8159 7 5 2,850000 0,0003* 12 13 0,164000 0,8263 2 7 2,768000 0,0005* 10 9 0,162000 0,8284 1 3 2,596333 0,0006* 13 11 0,104000 0,8893 3 14 2,482000 0,0015* 5 14 0,036000 0,9616 4 6 2,480000 0,0015*

76 Table 44. „L values" comparisons between weeks for strawberry gelatin. Week Week Difference p-Value Week Week Difference p-Value 2 13 8,408000 <,0001* 9 13 1,750000 0,0594 2 10 7,774000 <,0001* 5 14 1,646000 0,0756 2 11 7,638000 <,0001* 2 1 1,576667 0,0755 2 6 7,602000 <,0001* 8 10 1,564000 0,0910 2 14 7,338000 <,0001* 3 9 1,514000 0,1015 1 13 6,831333 <,0001* 4 10 1,486000 0,1078 2 9 6,658000 <,0001* 3 12 1,448000 0,1169 2 12 6,592000 <,0001* 8 11 1,428000 0,1220 2 4 6,288000 <,0001* 8 6 1,392000 0,1315 2 8 6,210000 <,0001* 4 11 1,350000 0,1433 1 10 6,197333 <,0001* 4 6 1,314000 0,1541 1 11 6,061333 <,0001* 1 7 1,255333 0,1549 1 6 6,025333 <,0001* 12 10 1,182000 0,1990 1 14 5,761333 <,0001* 3 4 1,144000 0,2136 2 5 5,692000 <,0001* 8 14 1,128000 0,2200 7 13 5,576000 <,0001* 9 10 1,116000 0,2249 2 3 5,144000 <,0001* 14 13 1,070000 0,2444 1 9 5,081333 <,0001* 3 8 1,066000 0,2461 1 12 5,015333 <,0001* 4 14 1,050000 0,2532 7 10 4,942000 <,0001* 12 11 1,046000 0,2550 7 11 4,806000 <,0001* 12 6 1,010000 0,2715 7 6 4,770000 <,0001* 9 11 0,980000 0,2859 1 4 4,711333 <,0001* 5 9 0,966000 0,2927 1 8 4,633333 <,0001* 9 6 0,944000 0,3038 7 14 4,506000 <,0001* 5 12 0,900000 0,3266 1 5 4,115333 <,0001* 6 13 0,806000 0,3793 7 9 3,826000 <,0001* 11 13 0,770000 0,4008 7 12 3,760000 0,0001* 12 14 0,746000 0,4156 1 3 3,567333 0,0001* 9 14 0,680000 0,4578 7 4 3,456000 0,0004* 10 13 0,634000 0,4886 7 8 3,378000 0,0005* 5 4 0,596000 0,5150 3 13 3,264000 0,0007* 3 5 0,548000 0,5493 7 5 2,860000 0,0026* 5 8 0,518000 0,5713 2 7 2,832000 0,0029* 8 9 0,448000 0,6243 5 13 2,716000 0,0042* 14 10 0,436000 0,6335 3 10 2,630000 0,0054* 8 12 0,382000 0,6761 3 11 2,494000 0,0081* 4 9 0,370000 0,6857 3 6 2,458000 0,0091* 4 12 0,304000 0,7395 7 3 2,312000 0,0138* 14 11 0,300000 0,7428 8 13 2,198000 0,0189* 14 6 0,264000 0,7727 3 14 2,194000 0,0191* 6 10 0,172000 0,8507 4 13 2,120000 0,0233* 11 10 0,136000 0,8817 5 10 2,082000 0,0258* 8 4 0,078000 0,9320 5 11 1,946000 0,0367* 12 9 0,066000 0,9424 5 6 1,910000 0,0402* 6 11 0,036000 0,9686 12 13 1,816000 0,0507

77 Table 45. „a values" comparisons between weeks for carrot agar. Week Week Difference p-Value Week Week Difference p-Value 1 12 8,651667 <,0001* 5 14 2,528000 0,0116* 1 13 8,477667 <,0001* 5 11 2,438000 0,0147* 1 14 7,835667 <,0001* 2 3 2,398000 0,0163* 1 11 7,745667 <,0001* 5 10 2,360000 0,0180* 1 10 7,667667 <,0001* 2 7 2,308000 0,0205* 1 9 7,531667 <,0001* 5 9 2,224000 0,0254* 2 12 6,976000 <,0001* 6 14 2,188000 0,0277* 2 13 6,802000 <,0001* 6 11 2,098000 0,0345* 2 14 6,160000 <,0001* 8 14 2,020000 0,0415* 2 11 6,070000 <,0001* 6 10 2,020000 0,0415* 2 10 5,992000 <,0001* 8 11 1,930000 0,0511 2 9 5,856000 <,0001* 6 9 1,884000 0,0567 1 8 5,815667 <,0001* 8 10 1,852000 0,0609 1 6 5,647667 <,0001* 7 8 1,832000 0,0637 1 5 5,307667 <,0001* 3 8 1,742000 0,0774 1 4 4,751667 <,0001* 8 9 1,716000 0,0818 7 12 4,668000 <,0001* 1 2 1,675667 0,0761 3 12 4,578000 <,0001* 7 6 1,664000 0,0913 7 13 4,494000 <,0001* 3 6 1,574000 0,1097 3 13 4,404000 <,0001* 7 5 1,324000 0,1771 2 8 4,140000 <,0001* 3 5 1,234000 0,2079 1 3 4,073667 <,0001* 9 12 1,120000 0,2524 1 7 3,983667 <,0001* 4 8 1,064000 0,2767 2 6 3,972000 0,0001* 10 12 0,984000 0,3140 4 12 3,900000 0,0002* 9 13 0,946000 0,3329 7 14 3,852000 0,0002* 11 12 0,906000 0,3536 7 11 3,762000 0,0003* 4 6 0,896000 0,3589 3 14 3,762000 0,0003* 14 12 0,816000 0,4031 4 13 3,726000 0,0003* 10 13 0,810000 0,4065 7 10 3,684000 0,0004* 7 4 0,768000 0,4312 3 11 3,672000 0,0004* 11 13 0,732000 0,4530 2 5 3,632000 0,0004* 3 4 0,678000 0,4868 3 10 3,594000 0,0005* 14 13 0,642000 0,5102 7 9 3,548000 0,0005* 4 5 0,556000 0,5682 3 9 3,458000 0,0007* 5 8 0,508000 0,6020 5 12 3,344000 0,0011* 5 6 0,340000 0,7269 5 13 3,170000 0,0018* 9 14 0,304000 0,7548 4 14 3,084000 0,0024* 9 11 0,214000 0,8259 2 4 3,076000 0,0024* 13 12 0,174000 0,8581 6 12 3,004000 0,0030* 10 14 0,168000 0,8629 4 11 2,994000 0,0031* 6 8 0,168000 0,8629 4 10 2,916000 0,0039* 9 10 0,136000 0,8888 8 12 2,836000 0,0049* 7 3 0,090000 0,9263 6 13 2,830000 0,0050* 11 14 0,090000 0,9263 4 9 2,780000 0,0058* 10 11 0,078000 0,9361 8 13 2,662000 0,0080*

78 Table 46. „a values" comparisons between weeks for strawberry agar. Week Week Difference p-Value Week Week Difference p-Value 2 13 18,66400 <,0001* 4 11 3,84000 0,0043* 2 14 16,94800 <,0001* 6 11 3,71200 0,0056* 2 11 16,79200 <,0001* 7 14 3,54000 0,0081* 2 10 16,20600 <,0001* 8 9 3,44800 0,0098* 2 12 16,12400 <,0001* 7 11 3,38400 0,0111* 2 9 14,71200 <,0001* 4 10 3,25400 0,0144* 1 13 14,19467 <,0001* 4 12 3,17200 0,0170* 2 7 13,40800 <,0001* 6 10 3,12600 0,0186* 2 6 13,08000 <,0001* 6 12 3,04400 0,0217* 2 4 12,95200 <,0001* 3 9 2,85600 0,0308* 1 14 12,47867 <,0001* 7 10 2,79800 0,0342* 1 11 12,32267 <,0001* 7 12 2,71600 0,0396* 2 5 12,01600 <,0001* 5 9 2,69600 0,0411* 2 3 11,85600 <,0001* 12 13 2,54000 0,0538 1 10 11,73667 <,0001* 10 13 2,45800 0,0617 1 12 11,65467 <,0001* 9 14 2,23600 0,0884 2 8 11,26400 <,0001* 8 7 2,14400 0,1019 1 9 10,24267 <,0001* 9 11 2,08000 0,1123 1 7 8,93867 <,0001* 11 13 1,87200 0,1521 1 6 8,61067 <,0001* 8 6 1,81600 0,1645 1 4 8,48267 <,0001* 4 9 1,76000 0,1777 1 5 7,54667 <,0001* 14 13 1,71600 0,1886 8 13 7,40000 <,0001* 8 4 1,68800 0,1958 1 3 7,38667 <,0001* 6 9 1,63200 0,2109 3 13 6,80800 <,0001* 3 7 1,55200 0,2338 1 8 6,79467 <,0001* 9 10 1,49400 0,2515 5 13 6,64800 <,0001* 9 12 1,41200 0,2782 4 13 5,71200 <,0001* 5 7 1,39200 0,2850 8 14 5,68400 <,0001* 7 9 1,30400 0,3162 6 13 5,58400 <,0001* 3 6 1,22400 0,3466 8 11 5,52800 <,0001* 3 4 1,09600 0,3990 7 13 5,25600 0,0001* 5 6 1,06400 0,4128 3 14 5,09200 0,0002* 5 4 0,93600 0,4710 8 10 4,94200 0,0003* 12 14 0,82400 0,5254 3 11 4,93600 0,0003* 8 5 0,75200 0,5621 5 14 4,93200 0,0003* 10 14 0,74200 0,5673 8 12 4,86000 0,0004* 12 11 0,66800 0,6065 5 11 4,77600 0,0005* 8 3 0,59200 0,6480 2 1 4,46933 0,0006* 10 11 0,58600 0,6513 3 10 4,35000 0,0013* 4 7 0,45600 0,7250 3 12 4,26800 0,0016* 6 7 0,32800 0,8002 5 10 4,19000 0,0019* 3 5 0,16000 0,9017 5 12 4,10800 0,0023* 11 14 0,15600 0,9041 4 14 3,99600 0,0030* 4 6 0,12800 0,9213 9 13 3,95200 0,0033* 12 10 0,08200 0,9495 6 14 3,86800 0,0040*

79 Table 47. „a values" comparisons between weeks for carrot gelatin. Week Week Difference p-Value Week Week Difference p-Value 7 11 6,832000 <,0001* 7 5 2,426000 <,0001* 2 11 6,690000 <,0001* 6 14 2,366000 <,0001* 7 13 6,396000 <,0001* 8 10 2,316000 <,0001* 2 13 6,254000 <,0001* 5 9 2,292000 <,0001* 7 14 6,046000 <,0001* 2 5 2,284000 <,0001* 2 14 5,904000 <,0001* 7 4 2,200000 0,0001* 3 11 5,654000 <,0001* 1 6 2,147333 0,0001* 7 12 5,652000 <,0001* 9 11 2,114000 0,0002* 2 12 5,510000 <,0001* 2 4 2,058000 0,0003* 1 11 5,299333 <,0001* 6 12 1,972000 0,0006* 7 10 5,234000 <,0001* 8 9 1,800000 0,0015* 3 13 5,218000 <,0001* 3 8 1,740000 0,0021* 2 10 5,092000 <,0001* 9 13 1,678000 0,0030* 3 14 4,868000 <,0001* 10 11 1,598000 0,0045* 1 13 4,863333 <,0001* 6 10 1,554000 0,0056* 7 9 4,718000 <,0001* 7 1 1,532667 0,0044* 4 11 4,632000 <,0001* 4 6 1,480000 0,0082* 2 9 4,576000 <,0001* 2 1 1,390667 0,0094* 1 14 4,513333 <,0001* 1 8 1,385333 0,0096* 3 12 4,474000 <,0001* 9 14 1,328000 0,0170* 5 11 4,406000 <,0001* 5 6 1,254000 0,0239* 4 13 4,196000 <,0001* 3 5 1,248000 0,0245* 1 12 4,119333 <,0001* 12 11 1,180000 0,0331* 3 10 4,056000 <,0001* 7 3 1,178000 0,0334* 5 13 3,970000 <,0001* 10 13 1,162000 0,0357* 8 11 3,914000 <,0001* 6 9 1,038000 0,0597 4 14 3,846000 <,0001* 2 3 1,036000 0,0602 1 10 3,701333 <,0001* 3 4 1,022000 0,0636 7 6 3,680000 <,0001* 9 12 0,934000 0,0892 5 14 3,620000 <,0001* 1 5 0,893333 0,0895 3 9 3,540000 <,0001* 10 14 0,812000 0,1383 2 6 3,538000 <,0001* 14 11 0,786000 0,1512 8 13 3,478000 <,0001* 8 6 0,762000 0,1638 4 12 3,452000 <,0001* 12 13 0,744000 0,1738 5 12 3,226000 <,0001* 4 8 0,718000 0,1891 1 9 3,185333 <,0001* 1 4 0,667333 0,2022 6 11 3,152000 <,0001* 9 10 0,516000 0,3435 8 14 3,128000 <,0001* 5 8 0,492000 0,3663 4 10 3,034000 <,0001* 13 11 0,436000 0,4230 7 8 2,918000 <,0001* 10 12 0,418000 0,4423 5 10 2,808000 <,0001* 12 14 0,394000 0,4688 2 8 2,776000 <,0001* 3 1 0,354667 0,4957 8 12 2,734000 <,0001* 14 13 0,350000 0,5197 6 13 2,716000 <,0001* 4 5 0,226000 0,6773 4 9 2,518000 <,0001* 7 2 0,142000 0,7936 3 6 2,502000 <,0001*

80 Table 48. „a values" comparisons between weeks for strawberry gelatin. Week Week Difference p-Value Week Week Difference p-Value 2 12 14,31800 <,0001* 6 12 4,14000 0,0035* 1 12 14,25633 <,0001* 7 5 4,03600 0,0043* 2 13 13,13600 <,0001* 5 10 3,71000 0,0084* 1 13 13,07433 <,0001* 8 11 3,63200 0,0098* 2 10 12,50400 <,0001* 5 14 3,61600 0,0101* 1 10 12,44233 <,0001* 8 9 3,47000 0,0133* 2 14 12,41000 <,0001* 4 12 3,42600 0,0145* 1 14 12,34833 <,0001* 8 4 3,38400 0,0157* 2 11 11,14000 <,0001* 9 12 3,34000 0,0170* 1 11 11,07833 <,0001* 11 12 3,17800 0,0229* 2 9 10,97800 <,0001* 6 13 2,95800 0,0336* 1 9 10,91633 <,0001* 3 5 2,80400 0,0436* 2 4 10,89200 <,0001* 7 8 2,75000 0,0477* 1 4 10,83033 <,0001* 8 6 2,67000 0,0543 2 6 10,17800 <,0001* 5 11 2,34600 0,0896 1 6 10,11633 <,0001* 6 10 2,32600 0,0923 7 12 9,56000 <,0001* 4 13 2,24400 0,1041 2 5 8,79400 <,0001* 6 14 2,23200 0,1059 1 5 8,73233 <,0001* 5 9 2,18400 0,1135 7 13 8,37800 <,0001* 9 13 2,15800 0,1178 3 12 8,32800 <,0001* 5 4 2,09800 0,1281 7 10 7,74600 <,0001* 11 13 1,99600 0,1473 7 14 7,65200 <,0001* 14 12 1,90800 0,1657 2 8 7,50800 <,0001* 10 12 1,81400 0,1871 1 8 7,44633 <,0001* 4 10 1,61200 0,2404 3 13 7,14600 <,0001* 9 10 1,52600 0,2661 8 12 6,81000 <,0001* 3 8 1,51800 0,2686 3 10 6,51400 <,0001* 4 14 1,51800 0,2686 3 14 6,42000 <,0001* 9 14 1,43200 0,2963 7 11 6,38200 <,0001* 5 6 1,38400 0,3127 7 9 6,22000 <,0001* 11 10 1,36400 0,3197 7 4 6,13400 <,0001* 8 5 1,28600 0,3479 2 3 5,99000 <,0001* 11 14 1,27000 0,3539 1 3 5,92833 <,0001* 7 3 1,23200 0,3684 8 13 5,62800 0,0001* 13 12 1,18200 0,3880 5 12 5,52400 0,0001* 6 11 0,96200 0,4818 7 6 5,42000 0,0002* 6 9 0,80000 0,5583 3 11 5,15000 0,0004* 14 13 0,72600 0,5952 8 10 4,99600 0,0005* 6 4 0,71400 0,6013 3 9 4,98800 0,0005* 10 13 0,63200 0,6436 3 4 4,90200 0,0007* 4 11 0,24800 0,8558 8 14 4,90200 0,0007* 9 11 0,16200 0,9055 2 7 4,75800 0,0009* 14 10 0,09400 0,9451 1 7 4,69633 0,0006* 4 9 0,08600 0,9498 5 13 4,34200 0,0023* 2 1 0,06167 0,9624 3 6 4,18800 0,0032*

81 Table 49. „b values" comparisons between weeks for carrot agar. Week Week Difference p-Value Week Week Difference p-Value 1 12 10,34267 <,0001* 6 10 2,90600 0,0158* 1 13 9,89267 <,0001* 5 14 2,78400 0,0205* 1 14 9,28867 <,0001* 7 9 2,74400 0,0223* 1 10 9,14267 <,0001* 5 10 2,63800 0,0278* 1 11 8,82067 <,0001* 6 11 2,58400 0,0310* 2 12 8,24400 <,0001* 3 8 2,43200 0,0419* 1 9 8,17867 <,0001* 2 3 2,41400 0,0434* 2 13 7,79400 <,0001* 4 9 2,36600 0,0475* 2 14 7,19000 <,0001* 8 14 2,34400 0,0496* 2 10 7,04400 <,0001* 5 11 2,31600 0,0523 1 8 6,94467 <,0001* 8 10 2,19800 0,0650 2 11 6,72200 <,0001* 9 12 2,16400 0,0692 1 5 6,50467 <,0001* 1 2 2,09867 0,0657 1 6 6,23667 <,0001* 3 5 1,99200 0,0936 2 9 6,08000 <,0001* 6 9 1,94200 0,1019 3 12 5,83000 <,0001* 8 11 1,87600 0,1138 1 4 5,81267 <,0001* 3 6 1,72400 0,1455 1 7 5,43467 <,0001* 9 13 1,71400 0,1478 3 13 5,38000 <,0001* 5 9 1,67400 0,1574 7 12 4,90800 <,0001* 11 12 1,52200 0,1979 2 8 4,84600 0,0001* 7 8 1,51000 0,2014 3 14 4,77600 0,0001* 3 4 1,30000 0,2705 3 10 4,63000 0,0002* 8 9 1,23400 0,2953 4 12 4,53000 0,0003* 10 12 1,20000 0,3087 1 3 4,51267 0,0002* 4 8 1,13200 0,3367 7 13 4,45800 0,0003* 9 14 1,11000 0,3461 2 5 4,40600 0,0004* 11 13 1,07200 0,3627 3 11 4,30800 0,0005* 7 5 1,07000 0,3636 2 6 4,13800 0,0008* 14 12 1,05400 0,3708 6 12 4,10600 0,0009* 9 10 0,96400 0,4127 4 13 4,08000 0,0009* 3 7 0,92200 0,4333 7 14 3,85400 0,0017* 7 6 0,80200 0,4952 5 12 3,83800 0,0017* 10 13 0,75000 0,5235 2 4 3,71400 0,0024* 6 8 0,70800 0,5469 7 10 3,70800 0,0024* 4 5 0,69200 0,5560 3 9 3,66600 0,0027* 9 11 0,64200 0,5848 6 13 3,65600 0,0028* 14 13 0,60400 0,6072 4 14 3,47600 0,0043* 11 14 0,46800 0,6902 8 12 3,39800 0,0052* 13 12 0,45000 0,7015 5 13 3,38800 0,0053* 5 8 0,44000 0,7079 7 11 3,38600 0,0053* 4 6 0,42400 0,7180 2 7 3,33600 0,0060* 7 4 0,37800 0,7475 4 10 3,33000 0,0061* 11 10 0,32200 0,7838 6 14 3,05200 0,0115* 6 5 0,26800 0,8194 4 11 3,00800 0,0127* 10 14 0,14600 0,9010 8 13 2,94800 0,0144*

82 Table 50. „b values" comparisons between weeks for strawberry agar. Week Week Difference p-Value Week Week Difference p-Value 2 13 11,85600 <,0001* 8 12 1,37000 0,2964 2 10 11,73400 <,0001* 8 5 1,36000 0,2999 2 11 11,46000 <,0001* 5 11 1,32600 0,3120 2 9 11,35000 <,0001* 12 11 1,31600 0,3156 2 7 11,18000 <,0001* 3 9 1,29200 0,3245 2 4 10,82000 <,0001* 8 3 1,28400 0,3274 2 6 10,69000 <,0001* 5 9 1,21600 0,3535 2 12 10,14400 <,0001* 12 9 1,20600 0,3574 2 5 10,13400 <,0001* 6 13 1,16600 0,3735 2 3 10,05800 <,0001* 3 7 1,12200 0,3917 2 14 9,04000 <,0001* 14 12 1,10400 0,3993 2 8 8,77400 <,0001* 14 5 1,09400 0,4035 1 13 7,74267 <,0001* 5 7 1,04600 0,4244 1 10 7,62067 <,0001* 6 10 1,04400 0,4252 1 11 7,34667 <,0001* 12 7 1,03600 0,4288 1 9 7,23667 <,0001* 4 13 1,03600 0,4288 1 7 7,06667 <,0001* 14 3 1,01800 0,4368 1 4 6,70667 <,0001* 4 10 0,91400 0,4848 1 6 6,57667 <,0001* 6 11 0,77000 0,5560 1 12 6,03067 <,0001* 3 4 0,76200 0,5601 1 5 6,02067 <,0001* 5 4 0,68600 0,5997 1 3 5,94467 <,0001* 12 4 0,67600 0,6051 1 14 4,92667 0,0002* 7 13 0,67600 0,6051 1 8 4,66067 0,0004* 6 9 0,66000 0,6136 2 1 4,11333 0,0016* 4 11 0,64000 0,6244 8 13 3,08200 0,0211* 3 6 0,63200 0,6287 8 10 2,96000 0,0266* 5 6 0,55600 0,6705 14 13 2,81600 0,0345* 7 10 0,55400 0,6716 14 10 2,69400 0,0428* 12 6 0,54600 0,6760 8 11 2,68600 0,0434* 4 9 0,53000 0,6850 8 9 2,57600 0,0523 9 13 0,50600 0,6985 14 11 2,42000 0,0678 6 7 0,49000 0,7076 8 7 2,40600 0,0694 11 13 0,39600 0,7618 14 9 2,31000 0,0809 9 10 0,38400 0,7688 14 7 2,14000 0,1052 4 7 0,36000 0,7828 8 4 2,04600 0,1210 7 11 0,28000 0,8302 8 6 1,91600 0,1460 11 10 0,27400 0,8338 3 13 1,79800 0,1720 8 14 0,26600 0,8386 14 4 1,78000 0,1763 7 9 0,17000 0,8964 5 13 1,72200 0,1906 6 4 0,13000 0,9207 12 13 1,71200 0,1931 10 13 0,12200 0,9256 3 10 1,67600 0,2025 9 11 0,11000 0,9329 14 6 1,65000 0,2095 3 12 0,08600 0,9475 5 10 1,60000 0,2234 3 5 0,07600 0,9536 12 10 1,59000 0,2263 5 12 0,01000 0,9939 3 11 1,40200 0,2853

83 Table 51. „b values" comparisons between weeks for carrot gelatin. Week Week Difference p-Value Week Week Difference p-Value 2 11 8,794000 <,0001* 1 8 2,499667 0,0065* 2 13 7,892000 <,0001* 5 13 2,498000 0,0090* 2 10 7,552000 <,0001* 4 6 2,378000 0,0127* 2 9 7,394000 <,0001* 14 11 2,368000 0,0131* 2 12 7,326000 <,0001* 3 5 2,344000 0,0140* 7 11 7,024000 <,0001* 8 10 2,298000 0,0158* 2 14 6,426000 <,0001* 3 8 2,204000 0,0204* 7 13 6,122000 <,0001* 5 10 2,158000 0,0231* 1 11 6,039667 <,0001* 8 9 2,140000 0,0242* 2 6 5,992000 <,0001* 8 12 2,072000 0,0288* 7 10 5,782000 <,0001* 5 9 2,000000 0,0346* 3 11 5,744000 <,0001* 5 12 1,932000 0,0410* 7 9 5,624000 <,0001* 6 13 1,900000 0,0443* 7 12 5,556000 <,0001* 7 4 1,844000 0,0508 2 5 5,394000 <,0001* 4 5 1,780000 0,0590 2 8 5,254000 <,0001* 2 7 1,770000 0,0604 4 11 5,180000 <,0001* 4 8 1,640000 0,0813 1 13 5,137667 <,0001* 6 10 1,560000 0,0968 3 13 4,842000 <,0001* 12 11 1,468000 0,1177 1 10 4,797667 <,0001* 14 13 1,466000 0,1181 7 14 4,656000 <,0001* 6 9 1,402000 0,1347 1 9 4,639667 <,0001* 9 11 1,400000 0,1353 1 12 4,571667 <,0001* 6 12 1,334000 0,1543 3 10 4,502000 <,0001* 7 3 1,280000 0,1714 3 9 4,344000 <,0001* 10 11 1,242000 0,1842 4 13 4,278000 <,0001* 8 14 1,172000 0,2098 3 12 4,276000 <,0001* 14 10 1,126000 0,2280 7 6 4,222000 <,0001* 5 14 1,032000 0,2687 4 10 3,938000 <,0001* 7 1 0,984333 0,2705 4 9 3,780000 0,0001* 14 9 0,968000 0,2992 4 12 3,712000 0,0002* 13 11 0,902000 0,3331 1 14 3,671667 0,0001* 14 12 0,900000 0,3342 7 5 3,624000 0,0002* 1 4 0,859667 0,3353 2 4 3,614000 0,0002* 8 6 0,738000 0,4278 8 11 3,540000 0,0003* 5 6 0,598000 0,5201 7 8 3,484000 0,0004* 12 13 0,566000 0,5426 5 11 3,400000 0,0005* 3 4 0,564000 0,5440 3 14 3,376000 0,0006* 9 13 0,498000 0,5920 1 6 3,237667 0,0006* 6 14 0,434000 0,6404 2 3 3,050000 0,0017* 10 13 0,340000 0,7143 3 6 2,942000 0,0024* 1 3 0,295667 0,7394 4 14 2,812000 0,0035* 12 10 0,226000 0,8077 6 11 2,802000 0,0036* 9 10 0,158000 0,8648 2 1 2,754333 0,0029* 8 5 0,140000 0,8801 1 5 2,639667 0,0042* 12 9 0,068000 0,9416 8 13 2,638000 0,0060*

84 Table 52. „b values" comparisons between weeks for strawberry gelatin. Week Week Difference p-Value Week Week Difference p-Value 2 12 11,34000 <,0001* 3 6 2,68600 0,0445* 1 12 10,91767 <,0001* 6 12 2,68200 0,0448* 2 13 9,92600 <,0001* 8 4 2,67400 0,0454* 2 10 9,61600 <,0001* 3 9 2,60600 0,0510 1 13 9,50367 <,0001* 7 3 2,56200 0,0549 2 14 9,44000 <,0001* 3 11 2,40800 0,0707 1 10 9,19367 <,0001* 4 12 2,29200 0,0849 2 4 9,04800 <,0001* 8 6 2,28400 0,0860 1 14 9,01767 <,0001* 5 13 2,26200 0,0890 2 6 8,65800 <,0001* 8 9 2,20400 0,0973 1 4 8,62567 <,0001* 8 11 2,00600 0,1304 2 9 8,57800 <,0001* 5 10 1,95200 0,1409 2 11 8,38000 <,0001* 14 12 1,90000 0,1516 1 6 8,23567 <,0001* 5 14 1,77600 0,1796 1 9 8,15567 <,0001* 10 12 1,72400 0,1925 1 11 7,95767 <,0001* 3 5 1,69200 0,2008 7 12 7,93000 <,0001* 11 13 1,54600 0,2419 2 5 7,66400 <,0001* 13 12 1,41400 0,2840 1 5 7,24167 <,0001* 5 4 1,38400 0,2942 7 13 6,51600 <,0001* 9 13 1,34800 0,3068 2 8 6,37400 <,0001* 8 5 1,29000 0,3279 7 10 6,20600 <,0001* 6 13 1,26800 0,3362 7 14 6,03000 <,0001* 11 10 1,23600 0,3484 2 3 5,97200 <,0001* 11 14 1,06000 0,4208 1 8 5,95167 <,0001* 9 10 1,03800 0,4305 7 4 5,63800 <,0001* 5 6 0,99400 0,4502 1 3 5,54967 <,0001* 6 10 0,95800 0,4667 3 12 5,36800 0,0001* 5 9 0,91400 0,4873 7 6 5,24800 0,0002* 4 13 0,87800 0,5045 7 9 5,16800 0,0002* 9 14 0,86200 0,5123 7 11 4,97000 0,0004* 6 14 0,78200 0,5521 8 12 4,96600 0,0004* 5 11 0,71600 0,5860 7 5 4,25400 0,0019* 11 4 0,66800 0,6113 3 13 3,95400 0,0037* 4 10 0,56800 0,6656 5 12 3,67600 0,0067* 14 13 0,48600 0,7114 3 10 3,64400 0,0072* 9 4 0,47000 0,7205 8 13 3,55200 0,0087* 2 1 0,42233 0,7370 3 14 3,46800 0,0103* 3 8 0,40200 0,7596 2 7 3,41000 0,0116* 4 14 0,39200 0,7654 8 10 3,24200 0,0161* 6 4 0,39000 0,7665 3 4 3,07600 0,0221* 10 13 0,31000 0,8134 8 14 3,06600 0,0225* 11 6 0,27800 0,8324 1 7 2,98767 0,0203* 11 9 0,19800 0,8801 7 8 2,96400 0,0272* 14 10 0,17600 0,8934 11 12 2,96000 0,0274* 9 6 0,08000 0,9514 9 12 2,76200 0,0390*

85 Table 53. Raw data from the color measurements.

Week L value a value b value L value a value b value L value a value b value L value a value b value no. CA CA CA SA SA SA CG CG CG SG SG SG 1 34,45 15,36 18,27 31,69 20,41 9,72 37,54 15,62 18,79 32,52 30,25 18,48 1 34,36 10,42 13,35 33,59 28,57 16,4 37,38 14,37 18,54 31,11 26,89 14,14 1 36,96 18,85 21,69 33,38 31,22 20,58 37,7 13,86 17,26 33,81 29,55 18,76 1 36,64 16,4 19,52 32,94 26,77 15,22 38,22 13,73 19,05 32,08 29,03 17,17 1 34,53 12,04 14,64 31,75 23,97 12,03 38,36 14,06 17,79 32,94 29,19 17,28 1 37,82 17,72 22,25 33,26 24,44 11,47 39,21 14,3 17,54 32,02 29,02 17,02 2 34,04 11,35 14,5 33,33 31,8 20,01 37,77 16,11 20,69 36 28,36 17,56 2 36,38 16,9 19,71 31,24 27,83 15,76 39,3 14,69 18,39 33,24 29,73 18,01 2 36,02 14,2 16,65 34,98 31,15 18,61 39,3 16,92 25,03 34,54 31,53 20,36 2 35,78 12,02 14,8 33 28,87 16,92 37,52 15,51 18,81 33,4 27,76 15,89 2 35,1 12,81 15,28 33,71 32,18 20,45 39,33 15,34 21,66 32,77 27,87 16 3 35,65 11,77 15,19 29,46 21,89 12,06 37,59 16,38 21,07 30,65 27,84 15,72 3 33,66 11,11 14,97 27,96 20,55 9,67 35,33 15,2 18,67 27,59 20,07 8,73 3 34,14 11,13 13,51 29,79 19,16 8,45 32,91 12,71 14,92 31,43 24,53 13,74 3 35,12 11,19 13,55 27,43 15,82 5,87 34,49 13,74 16,52 28,38 23,08 11,15 3 35,64 10,09 11,65 29,41 15,13 5,41 37,04 15,36 18,15 26,18 19,78 8,62 4 34,07 10,66 12,4 26,21 15,3 5,47 35,62 13,45 17,05 25,37 17,33 7,54 4 35,77 11,06 13,42 28,51 17,79 7,81 34,49 12,63 15,02 29,61 15,86 7,21 4 34,38 10,55 13,48 27,99 16,84 7,09 35,86 14,56 19,72 28,97 15,22 6,69 4 34,34 10,1 12,08 28,77 18,46 8,65 34,01 13,59 18,11 27,5 20,29 9,84 4 34,83 9,53 10,99 29,62 18,68 8,63 34,03 14,05 16,61 27,06 22,09 11,3 5 34,49 9,21 11,38 27,71 19,34 9,14 32,6 13,24 15,38 27,17 19,15 8,91 5 34,56 10,95 13,32 29,4 18,08 8,04 34,93 14,38 16,3 28,89 20,66 10,03 5 34,24 9,98 12,48 31,67 17,78 7,74 33,13 13,66 15,97 29,01 20,13 9,56 5 34,54 9,34 10,51 27,75 17,24 7,15 32,31 13,63 15,88 27,49 18,2 8,12 5 34,83 9,64 11,22 29,12 19,31 9,01 32,16 12,24 14,08 28,93 23,14 12,88 6 33,87 8,3 10,41 29,08 18,94 9,3 33,25 13,35 16,78 26,7 16,38 7,04 6 34,76 9,2 11,47 29,65 18,87 9,41 30,77 10,28 12,74 27,15 19,89 9,8 6 34,91 9,99 12,61 29,49 15,59 6,4 31,29 11,4 14,24 26,17 19,44 9,47 6 36,98 10,81 14,22 30,75 16,09 6,32 33,17 13,01 16,44 26,17 20,21 9,82 6 35,48 9,12 11,54 29,63 16,94 6,87 33,13 12,84 14,42 25,75 18,44 8,4 7 33,9 9,59 11,46 28,14 16,84 6,85 36,23 16,57 18,38 33,89 26,52 17,05 7 35,64 8,57 10,05 28,11 19,82 9,13 38,32 16,18 21,58 32,26 27,52 17,16 7 37,13 13,61 14,04 28,43 16,15 6,39 36,66 17 20,14 31,7 26,89 16,13 7 38,25 14,73 17,97 28,41 15,08 5,93 32,76 13,61 16,5 30,33 20,01 10,24 7 33,68 9,24 10,74 29,83 16,9 7,55 35,41 15,92 19,13 27,61 20,52 10,19 8 34 8,14 9,59 29,19 21,68 12,68 31,07 11,57 13,29 30,05 24,55 14,05 8 36,58 12,06 14,41 28,49 19,41 10,05 32,19 13,44 16,12 28,45 21,8 11,69 8 34,74 8,92 10,92 28,83 22,57 12,7 32,62 14,07 17,22 26,84 19,96 9,07 8 35,32 8,33 10,11 28,12 15 5,34 32,47 12,7 14,68 26,39 18,98 8,92 8 35,2 9,13 11,68 29,34 16,85 7,11 34,13 12,91 17 27,17 22,42 12,22

86 Table 54. Raw data from the color measurements (continued).

Week L value a value B value L value a value b value L value a value b value L value a value b value no. CA CA CA SA SA SA CG CG CG SG SG SG 9 35,45 7,24 9,61 27,47 16,29 7,62 30,12 11,05 13,23 27,72 20,63 11,75 9 34,66 7,14 8,89 27,57 13,64 4,96 31,63 11,45 13,82 26,62 18,64 9,71 9 34,26 6,75 7,93 29,88 16,2 7,48 30,85 11,52 14,14 28,61 18,25 9,22 9 36,37 8,26 11,41 30,5 16,58 8,11 30,71 10,29 11,59 28,7 16,74 7,62 9 36,64 8,61 12,7 29,28 15,56 6,83 30,84 11,38 14,83 25,01 16,1 6,63 10 34,96 7,28 9,11 30,77 13,28 6,24 30,19 10,42 12,79 28,13 15,18 7,28 10 34,73 7,71 9,33 30,03 14,35 6,86 30,43 10,7 13,32 28,19 18,75 10,71 10 34,67 7,29 8,85 30,02 13,83 6,09 31,99 10,56 13,74 24,07 15,48 6,42 10 34,67 7,53 9,01 28,2 14,48 6,67 31,5 11,18 14,46 25,99 16,63 7,36 10 34,27 7,51 9,42 30,69 14,86 7,22 30,85 10,25 12,51 24,7 16,69 7,97 11 35,14 6,23 8,09 31,16 11,4 5,13 32,29 9,64 12,7 25,64 15,44 6,53 11 34,13 7,16 9,37 29,19 12,44 5,14 32,54 9,52 12,89 26,33 18,36 9,27 11 34,86 8,1 10,56 30,77 11,71 5,29 32,36 8,81 12,56 26,44 16,57 8,3 11 34,01 8,38 10,65 30,37 14,67 7,82 30,27 8,37 11,93 26,27 19,17 10,19 11 35,02 7,06 8,66 31,16 17,65 11,07 30,05 8,78 10,53 27,08 20,01 11,63 12 35,34 6,94 9 30,08 14,13 7,6 31,94 10,24 13,8 26,06 14,97 6,34 12 34,96 6,28 7,69 31,21 13,49 8,02 31,45 9,92 12,78 26,52 12,47 4,5 12 34,86 6,27 7,68 32,36 14,76 9,16 32,45 10,01 12,98 27,77 14,35 5,97 12 36,87 5,51 6,36 31,65 14,33 7,96 31,74 10,53 14,42 28,44 16,89 8,23 12 33,28 7,4 8,99 31,97 14,5 8,29 31,27 10,32 13,97 28,2 14,98 6,08 13 32,78 6,1 7,84 30,03 11,95 6,9 31,17 9,85 12,69 27,62 18,08 9,71 13 32,96 6,41 8,73 29,78 11,2 6,52 30,12 9,52 12,94 26,76 18,49 10,46 13 32,41 6,75 8,79 27,59 10,55 4,5 32,11 8,85 12,98 23,92 15,53 6,74 13 35,91 7,42 8,74 27,78 11,79 5,92 33,58 9,06 13,05 23,89 13,97 5,57 13 35,21 6,59 7,87 30,57 13,02 8,63 31,05 10,02 13,46 25,72 13,5 5,71 14 35,42 6,96 9,01 33,82 12,65 8,55 32,97 11,02 15,46 27,33 15,54 7,57 14 35,14 6,41 8,37 33,01 12,38 8,71 32,66 9,06 13,55 26,22 15,79 7,56 14 36,89 7,63 8,82 32,67 13,71 10,21 32,06 9,68 13,9 26,57 17,93 9,43 14 36,16 7,38 8,61 31,17 14,13 9,54 35,11 9,23 14,27 26,26 16,41 7,6 14 35,99 8,1 10,18 32,41 14,22 9,54 32,15 10,06 15,27 26,88 17,53 8,46

87 8.3 Sensory evaluation Table 55. Raw data from the sensory evaluation.

Week Taste Color Texture Taste Color Texture Taste Color Texture Taste Color Texture no. SA SA SA SG SG SG CA CA CA CG CG CG 1 3 4 0 8 4 2 2 8 0 3 8 3 1 5 4 4 6 3 3 5 4 4 5 4 3 1 8 9 5 6 9 4 3 9 5 2 10 5 1 5 9 2 4 9 4 2 6 2 2 6 4 1 8 10 5 8 10 8 8 10 5 9 10 8 1 6 9 5 7 10 7 6 8 5 7 9 7 1 8 10 7 9 10 10 7 10 7 7 10 10 1 5 7 2 6 7 4 7 5 2 7 5 8 1 7 10 6 8 10 9 7 10 6 9 10 10 1 6 10 2 9 10 9 7 10 2 6 10 9 1 7 9 7 7 8 8 8 9 7 8 7 8 1 7 9 4 10 10 8 10 10 6 10 10 8 1 5 7 9 9 9 7 6 9 9 5 9 9 1 6 6 2 8 7 9 5 6 1 8 6 7 14 7 2 6 7 3 7 - - - 5 3 7 14 2 6 3 8 4 8 - - - 5 8 8 14 9 7 4 9 10 7 - - - 10 10 10 14 6 5 4 7 7 8 - - - 6,5 9 8 14 7 9 7 8 9 10 - - - 6 9 10 14 9 8 6 8 10 8 - - - 6 10 10 14 9 8 6 10 10 10 - - - 8 8 10 14 3 4 5 8 7 7 - - - 5 4 8 14 5 3 1 9 4 9 - - - 5 2 5 14 4 7 1 9 8 8 - - - 6 6 6 14 8 7 1 8 8 5 - - - 7 9 9 14 3 4 5 6 6 6 - - - 8 8 8

88 Spurningalisti fyrir skynmat á 4 gúmmíböngsum Vinsamlega merktu við þá tölu sem þér finnst eiga best við

Rauður 1 Hvernig líkar þér bragðið? (Mjög vel) 10 9 8 7 6 5 4 3 2 1 0 (Mjög illa) Hvernig líkar þér liturinn? (Mjög vel) 10 9 8 7 6 5 4 3 2 1 0 (Mjög illa) Hvernig líkar þér áferðin? (Mjög vel) 10 9 8 7 6 5 4 2 1 0 (Mjög illa) Finnur þú eftirbragð eða óbragð? (Já, mikið) 10 9 8 7 6 5 4 3 2 1 0 (Nei, ekkert)

Rauður 2 Hvernig líkar þér bragðið? (Mjög vel) 10 9 8 7 6 5 4 3 2 1 0 (Mjög illa) Hvernig líkar þér liturinn? (Mjög vel) 10 9 8 7 6 5 4 3 2 1 0 (Mjög illa) Hvernig líkar þér áferðin? (Mjög vel) 10 9 8 7 6 5 4 3 2 1 0 (Mjög illa) Finnur þú eftirbragð eða óbragð? (Já, mikið) 10 9 8 7 6 5 4 3 2 1 0 (Nei, ekkert)

Appelsínugulur 1 Hvernig líkar þér bragðið? (Mjög vel) 10 9 8 7 6 5 4 3 2 1 0 (Mjög illa) Hvernig líkar þér liturinn? (Mjög vel) 10 9 8 7 6 5 4 3 2 1 0 (Mjög illa) Hvernig líkar þér áferðin? (Mjög vel) 10 9 8 7 6 5 4 3 2 1 0 (Mjög illa) Finnur þú eftirbragð eða óbragð? (Já, mikið) 10 9 8 7 6 5 4 3 2 1 0 (Nei, ekkert)

Appelsínugulur 2 Hvernig líkar þér bragðið? (Mjög vel) 10 9 8 7 6 5 4 3 2 1 0 (Mjög illa) Hvernig líkar þér liturinn? (Mjög vel) 10 9 8 7 6 5 4 3 2 1 0 (Mjög illa) Hvernig líkar þér áferðin? (Mjög vel) 10 9 8 7 6 5 4 3 2 1 0 (Mjög illa) Finnur þú eftirbragð eða óbragð? (Já, mikið) 10 9 8 7 6 5 4 3 2 1 0 (Nei, ekkert)

Figure 59. The questionnaire used in the sensory evaluation.

89