Anales Científicos THE AMAZING COLORS OF PERUVIAN BIODIVERSITY: SELECT PERUVIAN PLANTS FOR USE AS FOOD COLORANTS

The increasing consumer demand for more nutritious foods, naturally sourced ingredients, and cleaner labels is pushing the food and cosmetic industries to transition from the use of artificial colorants towards naturally sourced alternatives. In this context, the industry is continuously searching for sources of more stable colorants, with special interest in plant sources. The vibrant biodiversity found in Peru represents an exciting economic opportunity. In this review, we highlight select Peruvian crops with excellent potential for use as colorant sources for industrial applications, with -procedures. Huito, an understudied fruit native to the Amazon, is naturally colorless, but it can turn blue when exposed to oxygen or amino acids and can express different hues depending on the source of the primary-amine group. Overall, purple corn, colored-fleshed potatoes, sauco, Berberis species, and huito are promising Peruvian sources of natural colorants for food and cosmetic applications due to their versatility, stability, and attractive color characteristics.

procedures. Huito, an understudied fruit native to the Amazon, is naturally colorless, but it can turn blue when exposed to oxygen or amino acids and can express different hues depending on the source of the primary-amine group. Overall, purple corn, colored-fleshed potatoes, sauco, Berberis species, and huito are promising Peruvian sources of natural colorants for food and cosmetic applications due to their versatility, stability, and attractive color characteristics.

INTRODUCTION
Color is an important aspect of life as it impacts perception and senses (Sigurdson et al., 2017). This influence is epitomized when eating. The color of food and beverages may alter the perceived odor intensity (Zellner & Whitten, 1999), flavor identity (Zampini et al., 2007), sweetness perception (Johnson et al., 1982), and overall acceptability of a product (Johnson et al., 1982;Spence, 2015). Color also indicates that a product is ready for consumption such as with banana peel colors and brown spots being used to indicate ripeness (Mendoza & Aguilera, 2006). The food industry is therefore motivated to deliver food with the proper color-a goal oftentimes achieved by the addition of dyes and pigments. Color additives may be used to standardize natural crops, to compensate for color performance and flavor expectations in products as seen with addition of red colorants to develop a pink hue in strawberry yogurt, or to create new products such as blue raspberry flavor (Sigurdson et al., 2017).
Although safety regulation is robust, consumers are worried about the safety of artificial colorants. One of the most prevalent concerns is a link between childhood hyperactivity and artificial color consumption as reported in the Southampton study (McCann et al., 2007). In a toxicology review by Kobylewski and Jacobson (2012), research was presented showing linkages between the artificial food dyes and allergies, hyperactivity, genotoxicity, and cancer. However, additional research is needed and limitations including bias and study duration exist among many of the current studies (Kobylewski & Jacobson, 2012). Despite continued approval on the safety of artificial colorants by regulatory agencies, consumer concerns and social clean-label trends are leading to the use of naturally derived pigments for coloring food (Sigurdson et al., 2017). These naturally derived colorants generally have favorable consumer perceptions, but it is challenging for food manufacturers to match the hues, stability, and vibrant colors of artificial pigments with the current portfolio of available naturally sourced pigments (Wrolstad & Culver, 2012).
These new consumer trends and the limitations of the existing naturally sourced pigments represent an exciting opportunity for countries with an ample plant diversity such as Peru. In this review, we aim to highlight purple corn, colored-fleshed potatoes, sauco, Berberis species, and huito-pigment sources with potential for industrial applications and the production of naturally sourced colorants.

PURPLE CORN
Purple corn (Zea mays L.), also known as purple maize, is a crop native to the Andes regions of Peru and has been widely cultivated and consumed throughout the Andean region of South America, mainly Peru, Ecuador, Bolivia, and Argentina. Its deep purple shade has led to its pigments being used to color food and beverages (Luna-Vital et al., 2017;Chatham, Howard, and Juvik, 2020). For example, in South America, purple corn extracts are widely applied as colorants in two of the most popular homemade dessert and beverages-mazamorra and chicha morada, respectively (FAO, 2013). Other countries have also shown interest in using this rich source of pigments to color food with purple corn color being recognized by the European Union with the code E-163 and the same code for the Japanese legislation. Imports of port purple corn and its color products are growing. In 2017, Peru exported over 396,000 kilos of purple corn valued at US$645,769, an increase from $602,248 in 2016 (AGAP, 2017).

Purple Corn Pigments
The main class of pigments present in purple corn is the water-soluble anthocyanins. Additionally, the waterinsoluble but alcohol-soluble pholobaphenes have been reported in purple corn (Grotewold et al., 1994;Lee and Harper, 2002). These two pigments partially share the same biosynthetic route in purple corn plants as both are derived from a flavanone intermediate (Figure 1).

Figure 1.
Scheme of the biosynthetic pathway of purple corn phlobaphenes and anthocyanins (adapted from Grotewold, 2005).

Purple Corn Anthocyanins
Anthocyanins are one of the major sources of color which provide the purple reddish hue to purple corn. Anthocyanin content in purple corn ranges from 6.8mg/g fresh weight to 82.3 mg/g fresh weight depending on the section analyzed (Cevallos-Casals and Cisneros-Zevallos, 2003;Wu et al., 2006;Li et al., 2008). This content was higher than most of the known anthocyanin-rich plants based on fresh weight such as blueberries (1.3 to 3.8 mg/g) (Cevallos-Casals and Cisneros-Zevallos, 2003;Wu et al., 2006), strawberries (0.21±0.03 mg/g) (Wang and Lin, 2000; Wu et al., 2006), red cabbage (3.22±0.41 mg/g) (Ahmadiani et al., 2014;Wu et al., 2006), eggplants (8.57 mg/g) (Wu et al., 2006), and chokeberries (14.80 mg/g) (Kulling and Rawel, 2008;Wu et al., 2006). The levels of pigment reported in purple corn have been especially high in the inedible leaf and cob regions (Paulsmeyer, Vermillion, and Juvik 2022;Nankar et al. 2016). The amount of anthocyanin in the cobs ranged from 0.49% to 4.60 % of the dry or fresh weight, respectively, roughly 2 to10 times more than that found in the kernel (Li et al., 2008). Recently, a study on the total anthocyanin content in different purple corn tissues reported that the leaf had the highest average anthocyanin content of 913.56 mg anthocyanins per kg maize, followed by cob (608.65 mg/kg), tassel (588.59 mg/kg), silk (511.82 mg/kg), seeding (500.91 mg/kg), husk (225.78 mg/kg), anther (196.39 mg/kg), and kernels (7.91 mg/kg) containing the lowest concentration of anthocyanins of the tissues (Paulsmeyer, Vermillion, and Juvik 2022). Extraction of purple corn pigments for color usage is usually achieved by soaking the ground purple corn materials into polar solvents, such as water, ethanol, methanol, acetone, and their mixtures (Lao and Giusti, 2018;De Nisi et al., 2021).

COLORED-FLESHED POTATOES
Potato (Solanum tuberosum, belonging to the nightshade family) is a starchy, tuberous crop with cultivars in assorted colors, shapes, and sizes. It was originally domesticated in South America, and a large variety of wild species found in the Andes of Peru and Bolivia were brought into cultivation as a staple food several thousands of years ago (Hawkes, 1992;Salaman et al., 1985). The potato was introduced outside of the Andes region in the 1700s to Europe and later to North America (Ochoa, 1990). Today, it is planted in more than 100 countries and ranks as the world's fourth most important crop following maize, wheat, and rice (FAO, 2009;Hendley, 2006).

Potato varieties and pigmented cultivars
About 5000 potato varieties are recorded worldwide, with vast genetic diversity among cultivars of white-, yellow-, orange-, red-, purple-, and blue-fleshed varieties (Burlingame et al., 2009;Kaspar et al., 2013). The flesh may be partially or solidly pigmented and sometimes only the skin is pigmented. White-and yellow-fleshed potatoes are the most well-known and are rich sources of carotenoids, with yellow-fleshed cultivars containing 10 times more carotenoids than white-fleshed ones (Brown et al., 2005). Other pigmented varieties, such as red-, purple-, and bluefleshed have gained consumer interest due to a higher concentration of anthocyanins (Brown, 2005;Lachman and Hamouz, 2005). Studies have reported higher contents of polyphenols in red-and purplefleshed cultivars than in those with white flesh (Hamouz et al., 2011) (2005) reporting 6.9 to 35 mg/ 100g fresh weight for red potatoes and 5.5 to 17.1 mg/100 g fresh weight for purple varieties. Anthocyanin content of purplefleshed potatoes was higher than that of red-fleshed potatoes (Ezekiel et al., 2013;Lewis et al., 1998) with Lewis et al. (1998) reporting anthocyanin content of purple flesh and red flesh potatoes at 368 mg/100g fresh weight and 22 mg/100g fresh weight, respectively.

Anthocyanins in colored-fleshed potatoes
Pigmented potato cultivars derive their color from anthocyanins, and the type of anthocyanins varies by the color of the potatoes' skin and flesh. Anthocyanins contained in the potatoes with pigmented flesh have been investigated by many researchers due to their antioxidant activity and as alternatives to artificial colorants. In purple and red-fleshed varieties, approximately over 98 % of the total anthocyanins were acylated (Lachman & Hamouz, 2005), an important distinction as acylated anthocyanins generally have better stability than non-acylated anthocyanins (Wrolstad and Culver, 2012).

SAUCO, THE PERUVIAN ELDERBERRY
The genus Sambucus L. (elderberry) belongs to the Adoxaceae family and has been used as a medicinal plant for hundreds of years. The two most known species around the world are the European elderberry (S. nigra L.) and the American elderberry (S. canadensis L. ). Yet, Sambucus peruviana is a Peruvian elderberry species commonly known as "sauco" or "Andean elderberry" (Pangestu et al, 2020). It is recognized at the species level due to its geographical isolation from other Sambucus plants or treated as subspecies of S. nigra L. due to their morphological similarities (Applequist, 2015). This plant is native to Central and South America and can grow in the southern hemisphere between 2800 and 3900 meters of elevation (Porras-Mija et al., 2020). This berry has attracted attention due to its high in vitro antioxidant capacity and rich anthocyanin content that creates its characteristic black-purple color.

Botanical characteristics
Sauco is a large perennial shrub or a small deciduous tree typically around 3-6 meters tall but with the capabilities to grown up to 12 meters in a suitable environment. Its small, bright white flowers bloom between September to February, and its purplishblack, small fruits mature between January and July. The fruits of sauco are between 7 and 12 mm and have a fleshy, juicy, sweet taste. Although its trunk, fruits, and flowers all have high economic values, this species has not been commercially exploited in Peru (other than by small local business) (Mostacero León et al., 2017).
Aligned with their anthocyanin profiles, sauco displayed different color expression patterns and tinctorial capacity compared to S. nigra and S. canadensis. The aqueous extract of sauco displayed orange-red tones at acidic pH salmon pink tones at mildly acidic pH, and brownish purple tones at neutral to alkaline pH (Figure 2). Sauco extract consistently exhibited higher color intensity than the other two elderberry species under acidic pH (pH 1-4) and was capable of resembling the color hue of FD&C No. 40 in a wider pH range (Zhou, 2021). This could be explained by the considerable amount of cyanidin-3lathyroside in sauco as glycosylation with lathyrose is characterized by a lower visual detection threshold as compared to other types of glycosylation (Zhao et al., 2014). Studies have revealed high potential of applying sauco anthocyanin extract as a natural food colorant, especially for dyeing acidic food. Pangestu et al. (2020), demonstrated that sauco extract produced a bright, intense red color to model beverages. The half-life of the chroma of these model beverages colored with sauco was up to 23 weeks, and the addition of copigments such as chlorogenic acid and ferulic acid intensified the color and significantly extended the half-life to 49 weeks in the case with ferulic acid (Pangestu et al., 2020). Overall, anthocyanin extracts of sauco, especially with the addition of ferulic acid, showed great potential on enhancing the color of commercial food and beverage.

BERBERIS SPECIES
The genus Berberidaceae is well studied and characterized, having an extensive distribution around the world.  5-15 mm). The leaves of the long shoots do not participate in photosynthesis but transform into tri-pointed spines and, finally, into short shoots with several leaves (1-10 cm long, simple and entire or with spiny margins) that participate in photosynthesis (Perveen & Qaiser, 2010). In the life cycle of Berberis, there are sexual and asexual reproduction processes that allow the plant to survive in harsh conditions. The reproductive organs of the flower are protected from rain by three inner concave sepals and six petals that completely enclose the anthers and stamens (Peterson et al., 2005).

Berberis commutata Eichler (depicted in
Berberis humbertiana J.F. Macbr (depicted in Figure 4), is an endemic wild berry that grows in central and south Peru between 3000 and 4200 m of elevation (Ulloa and Sagástegui, 2006). This species is also a spiny shrub with sharp thorns and has tiny, dark purple berries of 4.34±0.53 mm in length and 0.040±0.01 grams in weight. The flowers are yellow, and the fruits are available from March until May each year. The anthocyanin content of the berries is high, quantified at approximately 4g/100g of monomeric anthocyanins in seedless berries. Five aglycones (delphinidin, cyanidin, petunidin, peonidin and malvidin) and nine anthocyanins were found in the Berberis humbertiana fruits: delphinidin-3-glucoside (42.7 %), petunidin-3glucoside (19.1 %), malvidin-3-glucoside (16.4 %), cyanidin-3-glucoside (8.7 %), cyanidin-3-rutinoside (3.1 %), petunidin-3-rutinoside (3.1 %), peonidin-3glucoside (2.5 %), delphinidin-3-rutinoside (2.3 %) and malvidin-3-rutinoside (2.2 %)  Wallace and Giusti (2008) incorporated powder from Berberis boliviana Lechler into yogurt samples containing 3 different fat levels. The color of yogurt at 20 mg cyanidin-3-glucoside equivalents/100 g yogurt was similar to commercial blueberry yogurt. The addition of ground B. boliviana berries rich in anthocyanins achieved color characteristics similar to artificially colored commercial brand blueberry yogurt. Due to the remarkably high content of monomeric anthocyanins in B. boliviana dried berries, there was no need for industrial pigment extraction as their addition produced a bright, stable, and acceptable color in yogurt systems. Pigment half-lives were 125 and 104 days for nonacylated anthocyanins at 10 and 20 mg cy-3-glu equivalents/100 g yogurt. In another study, the pH, color, and antioxidant activity of the lyophilized extract of anthocyanins from B. humbertiana and B. boliviana fruits in yogurt were studied. Freeze-dried ethanolic extracts (96 %) acidified with citric acid (pH 3.5) were incorporated into commercial yogurt at concentrations of 80 mg/50 g and 100 mg/50 g of yogurt. The systems maintained an acidic pH, achieved a coloration similar to commercial yogurt, and produced stronger antioxidant activity (Del Carpio-Jiménez, 2021).

HUITO
Genipa americana L., also known commonly as "Huito", "Jagua", "Genipap" or "Genipapo" in addition to many other names , is a tree native to the Amazon region in South America Francis, 1993). It can also be found in areas with

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moist and warm climates in Mexico, Central America, and Jamaica (Francis, 1993). Unripe huito fruits are small and firm with a green skin color, while ripe fruits are larger, softer, and have a more yellow/red skin color (Bentes and Mercadante, 2014), When the fruit is cut and the interior flesh is exposed to oxygen, the fruit becomes blue and continues to get darker and bluer with time .
Traditionally, huito fruit has been used to dye textiles and ceramics (Ramos-De-La-Peña, 2015; Bentes and Mercadante, 2014) and as a skin dye as the blue color can develop through the interaction genipin, an iridoid naturally present in the fruit, with amino acids on the skin (Neri-Numa et al., 2018). Unripe (immature, green) fruits are used most often for dyeing Francis, 1993;Bentes and Mercadante, 2014), while ripe (mature) fruits are used mostly for medicine, such as treating ulcers and wounds (Francis, 1993), and for consumption (Bentes and Mercadante, 2014). In addition to the fruit's many uses, the leaves and trunk of the huito tree are also important resources that can serve many functions.

Genipin and Geniposide
The active pigment in huito that allows for color development is an iridoid in the monoterpenes class called genipin. It is naturally colorless and is found in both Genipa americana L. (genipap fruit) and Gardenia jasminoides Ellis (gardenia fruit) (Neri-Numa et al., 2017;Bentes and Mercadante, 2014). According to Neri-Numa et al. (2017), genipin is characterized by a "cyclopentanoid unit fused with a dihydropyran ring whose hydroxyl group at the C1 position of the genipin pyran ring can be substituted by 1-2 moieties forming the genipin glycosides genipin-1-O-β-glucoside (geniposide) and genipin-1-O-β-D-gentibioside". Both the genipap and gardenia fruits contain genipin and geniposide, with geniposide predominant in the gardenia fruit (Bentes and Mercadante, 2014). However, for the blue pigments to form, geniposide must be hydrolyzed by β-glucosidase which removes the sugar and allows for the genipin aglycone to bind with a primary amine group, as seen in Figure 6 (Bentes and Mercadante, 2014;Neri-Numa et al., 2017;Bellé et al., 2018).

Figure 6.
Conversion of geniposide to genipin through hydrolysis with β-glucosidase followed by blue pigment formation. Adapted from Bentes and Mercadante (2014).

Role of the primary amine group source
The source of the primary amine group that binds with genipin impacts the color expression. However, due to the complexity of the reaction, it is still unclear why some sources are preferable. Pure amino acids have been shown to express different colors, such as blue, green, purple, brown, black, red, yellow, and orange, based on the experimental conditions Cano et al., 2019). For example, glycine produced blue or purple while arginine produced red or brown under specific experimental conditions (i.e. pH, temperature) Cano et al., 2019). Fruit and vegetable juices are a preferable source of amino acids for natural colorants made from huito because they could be considered as natural by consumers. Watermelon juice, lychee juice concentrate, banana puree, and celery juice can all produce blue with fresh huito. The color characteristics of the formed blue pigment include vibrant blue, gray/blue, and purple/blue, with watermelon juice being a preferable amino acid source due to the production of a dark blue color with huito . In addition to the source of primary amine group, the ratio of primary amine to genipin group also impacts the color produced. It has been suggested that there is a maximum limit up to where further addition of the primary amine source will not increase absorbance Brauch, 2016;Lee et al., 2003). However, this ratio may change if βglucosidase is added to the solution which would favor the formation of free genipin to interact with primary amine groups (Brauch, 2016;Cho et al., 2006).

Reaction mechanism
Although the reaction mechanism of pigment formation with genipin is not completely elucidated (Neri-Numa et al., 2017), understanding genipin's crosslinking reaction with primary amine groups can help better predict how pigments may be forming. There are two main stages during the genipinprimary amine group interaction. As seen in Figure  7, the first reaction involves a secondary amide substituting for an ester group on the genipin molecule, and the second reaction is a nucleophilic attack (Ramos-De-La-Peña et al., 2014).  Every primary amine group source has its own molecular structure that produces a unique compound after binding. Additionally, compounds can form monomers, dimers, or polymers (Brauch et al., 2016), increasing the possibilities of binding reactions. Cano et al. (2019) identified polymers of pure amino acids for glycine, lysine, valine, methionine, and proline using chromatography and proposed molecular configurations for genipin with the amino acids.

Huito's pigment stability
Huito is a promising source of blue color due to its enhanced stability to conditions such as pH and temperature. Huito color is stable at acidic pH, and production of the blue color is favored in mildly acidic pH . Blue pigment formation can occur within pH 3-8 but is preferable at pH 4-6 Neri-Numa et al., 2018). It has also been suggested that the pigments formed from genipin are heat stable, with color development increasing with higher temperatures Brauch, 2016). Although promising, studies on the stability of this pigment to light and temperature are limited and show conflicting results (Neri-Numa et al., 2018;. The formation and stability of blue pigments from huito and primary amine groups is largely dependent on the conditions of the experiment and the combination of pH with temperature (Neri-Numa et al., 2018;. More research is needed to elucidate pigment formation mechanisms and their stability in food matrices.

CONCLUSION
Market trends show that naturally sourced colorants are here to stay. Although artificial alternatives will continue to be an attractive option, consumers' demand for clean labels and healthier foods will continue to drive the industry to look for alternatives. Peruvian biodiversity offers exciting new opportunities to the food and cosmetic industry due to the diverse plant materials available. Additional research is needed to fully understand the behavior and capabilities of the pigments obtained from these promising plants.