Danielle Bauer Fall 2018 METHODS TO INCREASE THERMAL

Danielle Bauer Fall 2018 METHODS TO INCREASE THERMAL STABILITY OF ANTHOCYANINS AS NATURAL FOOD COLORANTS

OVERVIEW • Introduction • Challenges • Stabilization Methods • New Research • Discussion • Conclusions and Next Steps

• Colored pigments ‐ red to purple to blue WHAT ARE ANTHOCYANINS? • Water and alcohol soluble • Glycosylated anthocyanidins • Color due to presence of many conjugated double bonds • Structure: • Aglycone – B ring structure • H, OH or OCH 3 substitutions • Glycosidic substitution – positions 3, 5 • Acyl Substitution • Occurs with esterification of organic acids, usually cinnamic or aliphatic • Increases stability Taken from Wrolstad (2004) (Wrolstad 2015) (Cortez and others 2017)

COMMON SOURCES OF ANTHOCYANINS Red cabbage Black Carrot Red Radish Purple Sweet Potato Elderberry Grape Found in many fruits, vegetables, flowers (Wrolstad 2004)

SIX MAIN ANTHOCYANIDINS Cyanidin Pelargonidin Delphinidin Pelargonidin Peonidin Malvidin Petunidin Peonidin Credit: Pixaby Credit: Mali Maeder Malvidin Delphinidin Petunidin More than 600 anthocyanins have been identified Structure of main anthocyanidins Taken from Khoo and others (2017)

CURRENT FOOD & BEVERAGE APPLICATIONS Beverages Fruit products Ice cream Salad Dressing Cereal Candy Sauces Juice beverage colored with purple sweet potato by Carolina Innovative Food Ingredients, Inc. (Barry 2018) (DDWilliamson 2009)

INDUSTRY OUTLOOK Consumer demand for natural colors has long been on the rise 3 in 5 consumers avoid synthetic colors Clean label, natural color Antioxidant health benefits Natural color market projected to hit $2. 5 billion by 2025 7% compound annual growth rate 2016‐ 2025 expected for anthocyanins, specifically (Gelski 2017) (Prince 2017)

CHALLENGES Why are applications limited?

p. H Water activity Temperature Light Oxygen Acylation Enzymes Metal cations FACTORS AFFECTING ANTHOCYANIN STABILITY (Qin and others 2018) (Wrolstad 2004)

Neutral and slightly acidic p. H EFFECT OF p. H Neutral quinoidal bases (purple to violet color) p. H 1‐ 2 p. H > 2 Flavylium cation (red to orange color) p. H 3‐ 6 Carbinol pseudobase (colorless) Anthocyanin structure undergoes reversible changes with changes in p. H Chalcone (colorless) Scheme of the p. H‐dependent structural interconversion between dominant forms of monoglycosylated anthocyanins in aqueous phase (Houbiers et al. 1998) Adapted from He & Guisti 2010. • Flavylium cation is most stable • Red ‐ orange • 100% flavylium p. H 3‐ 6 p. H 6‐ 7 • Carbinol pseudo‐ base & chalcone • Colorless • 50/50 flavylium & carbinol • Quinoidal base • Purple – violet • Mostly carbinol with some quinoidal base and chalcone Most stable < p. H 4 Taken from Wrolstad and Culver (2012)

p. H EXPERIMENT Blueberry anthocyanins 1. 5 3 5 7 9 Taken from Khoo and others (2017) Citric acid Ginger Ale Hydrogen Peroxide Water Baking Soda

EFFECTS OF HEAT • Known to follow first order kinetics • Anthocyanins only thermally stable < p. H 3. 0 (Qin and others 2018) • Enhanced oxidation reactions cause degradation due to heat (Patras and others 2010) • Pigment loss and color fading (Krammer 2016) • Formation of brown compounds and colorless anthocyanin forms known (de Almeida Paula and others 2018) Taken from Patras and others (2010) Possible degradation pathway for two anythocyanins (Patras and others 2010)

HEAT EXPERIMENT Same blueberry anthocyanins heated in microwave 2. 5 MINUTES Graphic from Wrolstad and Culver (2011)

THERMAL STABILITY How can degradation and color loss of anthocyanins be reduced when exposed to heat?

COMMON STABILIZATION TECHNIQUES Copigmentation Self‐Association Encapsulation Metallic ion addition (Qin and others 2018) (Guan and Zhong 2015)

COPIGMENTATION • Non-covalent associations • Conjugated structure allows complexes to form • Anthocyanin complexed with biomacromolecules • Yeast mannoproteins • β‐cyclodextrin • Typically provides protection at a specific p. H • Reduced effect at higher temperatures near boiling point Gum Arabic: significant thermal protection at p. H 5. 0 (Guan and Zhong 2015) (Cortez and others 2017)

COLOR RESULTS Appearance of solutions with 0. 51 mg/m. L anthocyanins at p. H 5. 0 with (A, B, and C) and without (D, E, and F) 10 mg/m. L gum Arabic before (A and D) and after heating at 80 (B and E) and 126 (C and F) C for 30 min. Taken from Guan and Zhong (2015)

METALLIC ION ADDITION • Hydroxyl groups associate with metal ions • Shown to stabilize anthocyanins with more than one free hydroxyl group via chelation • Cyanidin, delphinidin, petunidin • Typical metals used include aluminum, copper, magnesium, potassium, and iron • Alginate and Iron Complex effective at 60°C for 80 minutes at p. H 5. 0 • Enhanced thermal stability of cyanidin-3 glucoside and delphinidin-3 -glucoside Thermal stability of P 3 G, C 3 G, and D 3 G in the presence of Fe 3+ (black bars), Fe 3+ and alginate (gray bars), or in the absence of both cofactors (white bars). • Fe 3+ stabilized color initially, but could not withstand time and also caused aggregation • Alginate stabilized reaction and prevented aggregation (Cortez and others 2017) (Tachibana and others 2014)

ENCAPSULATION • • • Creates a barrier that reduces reactivity and helps maintain functional characteristics Achieved by spray drying or freeze drying Encapsulation agents may be carbohydrates, lipids or proteins • Maltodextrin • Starch • Gum Arabic • Maltodextrin and Gum Arabic: exhibit good stabilization and withstand high temperatures during spray drying of cyanidin‐ 3‐glucoside • Significant impact on increasing half-life at 37°C • Longest half‐life observed at 4°C • No significant effect on L* a* b* values seen at 37°C Half‐life period (days) for roselle pigments encapsulated in different matrices at different temperature. GA, gum Arabic; MD, maltodextrin; SS soluble starch (Idham and others 2010)

• Objective • Can guar gum be used to improve thermal stability? CO‐PIGMENTATION: ADDITI ON OF GUAR G UM & GUAR G UM DOUBLE EMULSION • Previous studies have shown biopolymers can enhance stability by forming non‐covalent complexes • Experimental Design • Commercial anthocyanin extract used • Guar gum concentration ranging from 0. 25‐ 1. 75% in p. H 4. 0 • Samples stored for 10 days at 40°C in presence of light (de Almeida Paula and others 2018)

DEGRADATION RESULTS • First order kinetic degradation • Half‐life increased 2. 4 x at 1. 25% w/v guar gum • 70% retention at 1. 25% vs 41% retention with control Degradation kinetics of anthocyanin presenting in dispersions with different guar gum concentration (0‐ 1. 75%) during 10 days of storage at 40 C. (de Almeida Paula and others 2018) Anthocyanin content in the dispersions with different concentrations of guar gum (0‐ 1. 75%) at time 0 after 10 days.

COLOR RESULTS • Improved stability at concentration > 0. 75% • At < 0. 75% samples had loss of initial color and a yellow‐brown discoloring • Smallest ⍙E was seen at 1. 75% Total color difference (d. E) in relation to time 0, of treatments containing anthocyanins and different guar gum concentrations (0‐ 1. 75%) after storage at 40 C for 10 days. (de Almeida Paula and others 2018)

C ON CLUS IONS • Co‐pigmentation provides stability by slowing the hydration reactions that result in colorless forms • Hydrogen bonding – carbohydrate and anthocyanin • Guar gum is effective in enhancing thermal stability and preventing color fading • 1. 25% Guar gum concentration optimal • Researchers also tested a double emulsion at 1. 25% guar gum that provided increased protective effects (de Almeida Paula and others 2018)

• Objective COPIGMENTATION: M AILLARD REACTION PR ODUCTS FROM W HEY PROTEIN ISOLATE W ITH GLUCOSE • Can Maillard Reaction products stabilize anthocyanins at high temperatures and across p. H ranges? • Experimental Design • Cyanidin‐ 3‐glucoside (C 3 G) • Thermal stability at 80°C, p. H 3. 0‐ 7. 0 • Heated from 0‐ 120 minutes (Qin and others 2018)

DEGRADATION RESULTS • Significantly lower degradation rate when complexed with whey protein isolate and glucose p ≤ 0. 01 • More protective effect at p. H 3. 0 • Largest impact at p. H 6. 0 Thermal degradation analysis of C 3 G under different conditions, WPI‐Glu dispersions containing C 3 G complexes and C 3 G alone were adjusted to p. H 3. 0 and 6. 0, respectively. Subsequently, the solutions were heated for 0, 10, 20, 30, 60, 90, and 120 min at 80 C. Taken from Qin and others (2018)

REACTION KINETICS 1 Taken from Qin and others (2018)

2 3 4 5 6 7 WPI‐Glu only C 3 G only WPI‐Glu + C 3 G COLOR RESULTS • Cyanidin‐ 3‐glucoside alone appears purple from p. H 4‐ 7 • More red appearance with whey protein isolate + glucose • L* decreased as p. H increased, indicating turbidity • a* of C 3 G+WPI‐glu increased p. H 4‐ 7, relative to control • b* higher than that of control Color change exhibited by C 3 G under different conditions (from left to right) p. H 2. 0, 3. 0, 4. 0, 5. 0, 6. 0, and 7. 0; (from top to bottom) WPI‐Glu, C 3 G, and WPI‐Glu+C 3 G (Qin and others 2018)

CONCLUSI ONS • Protein denaturation caused by the Maillard reaction increases reactions with anthocyanins by exposing hydrophobic groups • Whey Protein isolate + glucose was effective in reducing degradation of cyanidin‐ 3‐glucoside and inhibited color change • Limitation: increased turbidity (Qin and others 2018)

DISCUSSION & CONCLUSION What are the next steps?

DISCUSSION What we know now: • Degradation and color fading during thermal processing are a known problem regarding anthocyanins • Due to differences in structure, different anthocyanins inherently have different stability at different conditions of p. H, light, heat • The thermal degradation pathway is largely unknown Limitations: • Many studies have only investigated at lower processing temperatures • Many studies are limited to model systems • Some materials may have unintended side effects such as turbidity

• Copigmentation is a promising method for thermal stability and, thus, color preservation CONCLUSION & FUTURE WORK • More study is needed to find materials/methods that are effective at higher temperatures • Further understanding of the degradation pathway is needed to improve research • Stability at high temperatures could expand applications and increase use as natural colorants • More choices for consumers and potential health benefits

THANK YOU! Questions?

REFERENCES Barry D. 2018. Juice innovation – from ingredients to finished products. Food Business News. [accessed 2018 Oct 10]. https: //www. foodbusinessnews. net/articles/12615‐juice‐innovation‐from‐ingredients‐to‐finished‐products Cortez R, Luna‐Vital DA, Margulis D, Mejia E. 2017. Natural pigments: Stabilization methods of anthocyanins for food applications. Comprehensive Reviews in Food Science and Food Safety. [accessed 2018 Sep 12] 16(1): 180‐ 98. doi: 10. 1111/1541‐ 4337. 12244 DDWilliamson. 2009. Applications of Natural Coloring. DDW The Color House. de Almeida Paula D, Mota Ramos A, Basílio de Oliveira E, Maurício Furtado Martins E, Augusto Ribeiro de Barros F, Cristina Teixeira Ribeiro Vidigal M, de Almeida Costa N, Tatagiba da Rocha C. 2018. Increased thermal stability of anthocyanins at p. H . 0 by guar gum in aqueous dispersions and in double emulsions W/O/W. International Journal of Biological Macromolecules. [accessed 2018 Sep 14]. 117: 665‐ 72. doi: 10. 1016/j. ijbiomac. 2018. 05. 219 Gelski, J. 2017. Demand soars for color from natural sources. Food Business News. [accessed 2018 Sep 14]. https: //www. foodbusinessnews. net/articles/9442‐demand‐soars‐for‐colors‐from‐natural‐sources? page=2 Guan Y, Zhong Q. 2015. The improved thermal stability of anthocyanins at p. H 5. 0 by gum arabic. LWT ‐ Food Science and Technology. [accessed 2018 Oct 23] 64(2): 706‐ 12. doi: http: //dx. doi. org/10. 1016/j. lwt. 2015. 06. 018 Idham Z, Muhamad II, Sarmidi MR. 2012. Degradation kinetics and color stability of spray‐dried encapsulated anthocyanins from hibiscus sabdariffa l. Journal of Food Process Engineering. [accessed 2018 Oct 8]. doi: 10. 1111/j. 1745‐ 4530. 2010. 00605. x

Khoo HE, Azlan A, Tang ST, Lim SM. 2017. Anthocyanidins and anthocyanins: Colored pigments as food, pharmaceutical ingredients, and the potential health benefits. Food & Nutrition Research. [accessed 2018 Oct 8] 61(1): 1361779‐ 21. doi: 10. 1080/16546628. 2017. 1361779 Krammerer D. 2016. Anthocyanins. In: Handbook on natural pigments in food and beverages ‐ industrial applications for improving food color. Elsevier. [accessed 2018 Oct 10]. https: //app. knovel. com/hotlink/pdf/id: kt 010 Y 8 T 1 E/handbook‐ natural‐pigments/susceptibility‐anthocyanins Patras A, Brunton NP, O'Donnell C, Tiwari BK. 2010. Effect of thermal processing on anthocyanin stability in foods; mechanisms and kinetics of degradation. Trends in Food Science & Technology. [accessed 2018 Oct 8]. 21(1): 3‐ 11. doi: //doi‐ org. er. lib. k‐state. edu/10. 1016/j. tifs. 2009. 07. 004 Prince J. 2017. Natural color stability: Challenges and opportunities | nutritional outlook. Nutritional Outlook. [accessed 2018 Oct 10]. http: //www. nutritionaloutlook. com/trends‐business/natural‐color‐stability‐challenges‐and‐opportunities Qin X, Yuan D, Wang Q, Hu Z, Wu Y, Cai J, Huang Q, Li S, Liu G. 2018. Maillard‐reacted whey protein isolates enhance thermal stability of anthocyanins over a wide p. H range. Journal of Agricultural and Food Chemistry. [accessed 2018 Oct 5]. 66(36): 9556‐ 64. doi: 10. 1021/acs. jafc. 8 b 03125 Tachibana N, Kimura Y, Ohno T. 2014. Examination of molecular mechanism for the enhanced thermal stability of anthocyanins by metal cations and polysaccharides. Food Chemistry. [accessed 2018 Sep 30]. 143: 452‐ 8. doi: //doi‐org. er. lib. k ‐state. edu/10. 1016/j. foodchem. 2013. 08. 017 Wrolstad RE. 2004. Symposium 12: Interaction of natural colors with other ingredients. anthocyanin pigments ‐ bioactivity and coloring properties. J Food Sci. 69(5): C 421. [accessed 2018 Sept 12]. doi: //doi‐org. er. lib. k‐state. edu/10. 1111/j. 1365‐ 2621. 2004. tb 10709. x Wrolstad, RE. 2007 Colorants. In: . Food Chemistry: Principles and applications. 2 nd Edition. STS Press. Wrolstad RE. 2012. Alternatives to those artificial FD&C food colorants. Annual Review Food Science Technology. [accessed 2018 Sep 12]. 3(1): 59‐ 77. doi: 10. 1146/annurev‐food‐ 022811‐ 101118; 14

PHOTO CREDITS Radish. Mali Maeder. Pexels. https: //www. pexels. com/photo/close‐up‐colors ‐farm‐produce‐fresh‐ 244393/ Plum. Pixaby. Pexels. https: //www. pexels. com/photo/high‐ angle‐view‐of‐fruit‐bowl‐on‐table‐ 248440/