Article 1

Chocolate bonbon with the addition of astaxanthin oleoresin. 2

Evaluation of texture and antioxidant capacity. 3

Pedro Cerezal-Mezquita 1*, Francisca Salinas-Fuentes 1, Waldo Bugueño-Muñoz 1, Ana Paula Batista 2. 4

1 Laboratorio de Microencapsulación de Compuestos Bioactivos (LAMICBA) del Departamento 5

de Ciencias de los Alimentos y Nutrición, Facultad de Ciencias de la Salud (FACSA), 6

Universidad de Antofagasta. Avda. Universidad de Antofagasta 02800. P.O. Box: 1240000. 7

Antofagasta. Chile. 8

pedro.cerezal@uantof.cl (P.C.M.); francisca.salinas@uantof.cl (F.S.F.); waldo.bugueno@uantof.cl 9

(W.B.M.) 10

2 Joint Research Unit IATE Agropolymers Engineering & Emerging Technology, INRAE, 11

University Montpellier, Institut Agro, CIRAD, 2, Place Pierre Viala, 34060 Montpellier, France. 12

anapaula.batista@gmail.com (A.P.B.) 13
* Correspondence: pedro.cerezal@uantof.cl (P.C.M.). Tel.: +56-55-2637717. 14

Abstract: Astaxanthin is a carotenoid belonging to the family of xanthophylls, its use has spread in 15
the pharmaceutical, cosmetical, and food industries, due to its high antioxidant capacity and the 16
benefit it means for human health. The work aimed to develop a prototype Pigmented Chocolate 17
Bonbon (PCB) with the addition of astaxanthin oleoresin (AO), providing antioxidant properties 18
and being a functional food that is easy to consume. To determine the interaction of astaxanthin in 19
the texture of the chocolates, simple penetration, and cut tests were performed. Micelles’ size formed 20
by AO in the chocolate varied between 0,26 - 3,34 μm2. The determination of antioxidant properties 21
of PCB was determined according to the FRAP (Ferric Reducing Antioxidant Power) method and 22
total polyphenols. Antioxidant capacity in the PCB samples, analyzed at the beginning (freshly pre- 23
pared), presented values of 12044 μmol ET/ 100 g sample. and the total polyphenols reached values 24
of 6.04 mg EAG /100 g sample. In the sensorial evaluation test, 112 consumers participated, where 25
of the totality analyzed, 21.4%, chose the "Very much liked” option, 43.8% favored the "Like a lot" 26
option, and 29.5% selected "Like moderately"; that is, 96.3% of the evaluated population selected the 27
first three preference categories. Only a minor percentage of the population, 3.7% selected the term 28
Citation: To be added by editorial "Neither like nor dislike". 29
staff during production.
Keywords: Chocolate bonbons, astaxanthin oleoresin, antioxidant capacity, phenolic compounds, 30
Academic Editor: Firstname Last-
texture test. 31
name
32
Received: date
Revised: date
1. Introduction 33
Accepted: date
Published: date Astaxanthin (ASTX) is a carotenoid that has great antioxidant potential and has been 34

attributed to an extraordinary role in protecting health [1,2]. This bioactive compound 35

(BAC), is found mainly in Haematococcus pluvialis (HP), a microalga that belongs to the 36
Copyright: © 2023 by the authors.
family of Haematococcaceae and within its metabolism produces proteins and ASTX. The 37
Submitted for possible open access
publication under the terms and latter is responsible for the coloring of some crustaceans, shellfish, and salmonids skin, 38
conditions of the Creative Commons being HP the main and largest source of natural ASTX known [3-5]. 39
Attribution (CC BY) license
(https://creativecommons.org/license
s/by/4.0/).

Processes 2023, 11, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/processes

Processes 2023, 11, x FOR PEER REVIEW 2 of 25

HP has the highest capacity to accumulate ASTX, being 5,02% of cell dry weight, those 40

that contain 36,7 mg/g of trans-astaxanthin (73,1%) and 13,5 mg/g of cis-astaxanthin 41

(26.9%); in the form of monoesters (80%), diesters (15%), and free ASTX (5%) [6-9]. The 42

presence of hydroxyl (OH) and carbonyl (C = O) in the chemical structure of ASTX ex- 43

plains some of its characteristics, such as the ability to be esterified, a more polar nature, 44

and a high antioxidant capacity [10]. The antioxidant activity of ASTX in cells is greater 45

than vitamins C and E, being 11 times more powerful than β-carotene, and 550 times more 46

than α-tocopherol [9,11-12]. Taking advantage of the fat-soluble nature of ASTX [13] and 47

daily doses recommended by the FDA, 4 mg of astaxanthin [10,14-17], some products have 48

been developed that include HP inside or the oleoresin extracted from HP. 49

In recent years, the search for healthier foods and the desire to replace synthetic com- 50

pounds has led to the use of new natural resources, which has allowed the use of new 51

BACs as food additives. Microalgae, and especially HP, has been used in food supple- 52

ments, in the powders form and tablets, within the nutraceutical industry [18]; neverthe- 53

less, ASTX oleoresin has been incorporated into the formulation of bakery products 54

(whole meal biscuits) for flour enrichment, thus taking advantage of properties that re- 55

duce cancer, diabetes, and control of serum cholesterol [19-21]. There are reports of its use 56

as a coloring agent in different types of milk (whole, semi-skimmed, and skim milk) [22] 57

and yogurt (“diet” and “light”) [23]. Moreover, a nutricosmetic formulation elaborated 58

with dark chocolate (72.6% in cocoa) and ASTX has been developed to support the health 59

of the skin and the prevention of aging related to free radicals (Lycotec Ltd, Cambridge, 60

UK) [24]. 61

Chocolate is consumed worldwide, and the largest chocolate manufacturers are based 62

in North America and Europe. The main consumers are the Swiss (8.8 kg/year/per capita), 63

and the lowest level consumers are the Chinese (100 g per year / per capita) [25]). Cocoa 64

beans are known to naturally contain GABA (gamma-aminobutyric acid) a non- 65

proteinogenic amino acid with potent bioactive functions that occurs naturally in plants, 66

animals, and microorganisms [26-27]. GABA plays an important role in the regulation of 67

brain metabolism and has demonstrated physiological functions such as antidepressants, 68

antihypertensive, antioxidant, hypotensive, and insomnia-curing effects [27-28]. The 69

chocolate formulation is in constant development. The changes are the goal of meeting 70

the changing demands of food consumers. There is a growing demand for low-calorie, 71

sugar-free chocolates and vegan product formulations without animal derivatives [29]. 72

The production of chocolate products with attractive sensory characteristics remains a 73

challenge for the food industry. The addition of different vegetable sources has improved 74

the flavor of the chocolate products that are obtained. This is how chocolate bonbons have 75

been designed with the incorporation of carline thistle (Carlina acanthifolia L.) roots, a 76

medicinal plant that has biological activity, with colagogic, diuretic, antibiotic, antioxidant, 77

and cleansing effects [30]. 78

Nevertheless, chocolate products for individual consumption with the addition of 79

ASTX have emerged in some areas of the world, among which stand out the one manu- 80
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factured by "Quality" in Brasilia - Brazil [31]) and by "Carmit Candy" in Israel [32], pre- 81

sented in bonbon and coin format, respectively. Manufacturers report that it may have 82

benefits in cardiovascular health, the immune system, inflammation, and degenerative 83

diseases, as well as aging, specifically the skin, helping to combat the harmful effects of 84

free radicals. Some of these health benefits are reported by Visioli and Artaria [33] and 85

Ling Tan and Norhaizan [34]. 86

Petyaev et al. [35] investigated the effect of highly bioavailable polyphenols of lyco- 87

some-formulated dark chocolate (LF-DC) containing co-crystallized astaxanthin (LF-DC- 88

ASTX) on biological oxidation parameters (BOP). The profound and systemic effect of LF- 89

DC-ASTX on BOP reported may create a rationale for the use of LF-DC-ASTX in the pre- 90

vention and treatment of oxidative disorders far beyond dermatological applications, in- 91

cluding various diseases associated with oxidative stress (cardiovascular disease, type 2 92

diabetes mellitus, and cancer). The highest degree of Oxidized Low-Density Lipoproteins 93

(ox-LDL) and malonic dialdehyde (MDA) decline was observed in the ingestion of the 94

astaxanthin co-crystallized in DC. These results may reflect greater bioavailability and 95

improved pharmacokinetics for cocoa flavanols in conjunction with ASTX [36]. So it was 96

proved that dark chocolate and ASTX were best effective in combined form and it would 97

be a future alternative for the management of oxidative stress-related disorders (cancer, 98

cardiovascular diseases, and diabetes) [35]. 99

Of the existing products on the market, which combine the use of ASTX and chocolate, 100

the ASTX carotenoid is only an antioxidant agent. For this study, the use of natural ASTX 101

seeks the realization of a chocolate-based product, with antioxidant properties, using the 102

characteristic color of ASTX to color the white filling with dark chocolate coating, which 103

we’ll call "Pigmented Chocolate Bonbon" (PCB). 104

Szczesniak [37] has expressed that texture can be defined as the sensory manifestation 105

of the food structure and the manner in which this structure reacts to applied forces, the 106

specific senses involved being vision, kinesthetic, and hearing. Texture profile analysis 107

(TPA) sets up a “bridge” from objective measurement to subjective sensation and makes 108

food texture characteristics more predictable [38]. The instrumental studies of the texture 109

of chocolate are important to research the influence of each ingredient in their composi- 110

tion. One of the aspects that have to be considered is the content and type of fat present in 111

the chocolate since it will bring a characteristic in the texture of a final product, and the 112

modifications that may occur as a result of the subsequent crystallization or recrystalliza- 113

tion of the chocolate during storage [39]. Another important parameter is the size of the 114

chocolate particles, of great value in the sensation of softness of the sample in the mouth 115

[40-41]. Some authors indicate that there exists a great correlation between instrumental 116

measurements and sensory evaluations [40]. 117

This research aimed to make a dark chocolate bonbon for individual consumption, 118

with white chocolate filling and ASTX oleoresin incorporation, obtained by the process of 119

supercritical extraction using CO2 (SFE-CO2). Each chocolate bonbon produced unit will 120

have 20 - 25% of the recommended daily impart antioxidant properties; since recently the 121

European Food Safety Authority (EFSA) concluded that: “the intake of 8 mg/day of natural- 122
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ASTX from food supplements is safe for adults even in combination with an excessive exposure 123

from the background diet”[42]. Texture analysis and its comparison with a commercial choc- 124

olate bonbon will be carried out, to have a pleasant and easy-to-use functional food. 125

2. Materials and Methods 126

2.1. Materials. 127

Two different brands of chocolates were purchased at a local market in the city of 128

Antofagasta, Chile: a) Bitter chocolate coverage, contains milk, Professional Choc 129

Ambrossoli, and b) substitute white chocolate, easy to melt, contains soy lecithin and milk, 130

Puratos (Carat). A commercial chocolate bonbon (CCB) was acquired with dark chocolate 131

coating and stuffed ganache-type praline, to be used as a texture comparison standard, 132

Carezza (Costa). Astaxanthin oleoresin (AO), extracted from Haematococcus pluvialis 133

biomass using supercritical CO2 extraction technology was provided by the company 134

Atacama Bio Naturals Products Inc. (Iquique, Chile), being the content of total carotenoids 135

in AO of 4.41 ± 0.22 %. Of this total, free astaxanthin (FA) corresponded to 1.44 ± 0.45%; 136

astaxanthin esters (AE) 78.21 ± 4.42%; lutein 1.24 ± 0.37%; canthaxanthin 1.16 ± 0.40%; β- 137

carotene 0.90 ± 0.30% and other carotenoids 19.11 ± 0.41%, the sum of FA + (AE) = 79.7 ± 138

4.25%. The reactants: acetone, petroleum ether, n-hexane, dimethyl sulfoxide, and water 139

(HPLC grade) were purchased from Merck. 140

2.2. Elaboration of chocolate bonbon with filling of Astaxanthin oleoresin (AO). 141

The method proposed by Colquichagua [43] was used for the preparation of the 142

bonbons, with some variations in the elaboration of filling, which were: keeping the 143

chocolate tempering machine (model HW-22, Calvac, Chile), with the temperature at 35 ± 144

2°C during the whole process and refrigeration at 10°C for 15 minutes to solidify the 145

chocolate coating and the filling. The amounts of raw materials used for the manufacture 146

of 24 chocolate bonbons, after several preliminary tests, were: 41.6% dark chocolate (DC), 147

used only in the coating, while 58.2% substitute white chocolate (SWC) and 0.2% AO were 148

used in the filling. Each bonbon unit weighed 16 ± 0.5 g. The bonbon filling was made by 149

gradually adding the SWC over the AO and shaking vigorously with a wooden paddle 150

by hand, to achieve a dispersion that allows the color to be distributed in the mixture. 151

Next, the filling mixture was homogenized mechanically using a paddle stirrer (model 152

RW-16 basic, IKA Labortechnik; Germany), for 10 minutes at 736 ± 15 rpm until the AO 153

was fully distributed. The homogeneity of the pigment color throughout the mass was 154

verified by the naked eye. 155

To avoid solidification of the chocolate and possible stratification with fat globules, 156

the paddle stirrer was placed inside the chocolate tempering machine, ensuring stirring 157

and temperature simultaneously (36 ± 2°C). Dimensions of the chocolate bonbon were 40 158

mm x 29 mm x 22 mm (l x w x h) (Figure 1). Each chocolate bonbon produced unit will 159

have 20 - 25% of the recommended daily to impart antioxidant properties, a proposal in 160

the goal of the present research, 1.6 - 2.0 mg natural-ASTX. 161

162
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163
164
165
166
167
168
169
170
171
172

Figure 1. a) Reference sample. Chocolate bonbon (weight ~ 14.2 g ); b) Chocolate bonbon 173

in its ellipsoidal shape. 40 mm x 29 mm x 22 mm (l x w x h). 174

2.3. Observation of ASTX macroparticle in the chocolate bonbon filling. 175

The determination of the size of the macroparticles in the ASTX emulsion blended 176

with SWC was made by sampling a drop and observing in a microscope (model CX31, 177

Olympus Corporation, Japan) with an integrated digital camera (model U-TV0.5XC-3, 178

Olympus, Corporation, Japan), and with the help of the image processing software 179

Micrometrics SE Premium for Windows. To obtain the area of the micelles, the ocular 180

spherical field was divided into 4 quadrants, and one of them was randomly selected for 181

the measurements. The area of the micelles was determined with Equation 1 182

A = π r2 (Equation 1)

2.4. Texture Analysis 183

The texture analysis of the chocolates was carried out using the Texture Analyzer 184

(model TA-XT. plus Stable Micro System, UK) with the help of Exponent software version 185

6.1.5.0 (Stable Micro Systems, Godalming, UK). The samples analyzed were: pigmented 186

chocolate bonbon (PCB), unpigmented chocolate bonbon (UCB), and commercial 187

chocolate bonbon (CCB) with praline filling. 72 chocolate bonbons of the total number of 188

processed chocolates (24 bonbons from each batch produced) were analyzed, and these 189

samples were evaluated in triplicate and subjected to two types of tests: a) Simple 190

Penetration Test and b) Cutting Test (Texture Test), using the conditions indicated in 191

Table 1. Several texture parameters measured, brittleness and hardness were calculated, 192

using the force-deformation relationships with a few modifications (Figure 2) proposed 193

by Cerezal et al. [44] as shown in Equations 2 and 3. 194

Table 1. Parameters Used for Texture Testing 195

Simple Penetration Test Cutting Test

Probe: Aluminium cylindrical 2 mm diameters Probe: Knife Guillotine Blade
Penetration speed: 2 mm/s Penetration speed: 2 mm/s
Penetration depth: 6 mm Penetration depth: 22 mm (bonbon height)
196

197
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198

Force (N)
199
Hardness
200

Brittleness 201

202

203

204

205

206

207

208
d1
d2 209
Deformation in time (s) 210

Figure 2. Typical force–deformation curve of solid according to Cerezal et al. [44]. Where 211

d1 and d2 are the distances in seconds until the first and second maximum peak of the 212

curve, respectively. 213

Brittleness
= 𝑁/𝑠 (Equation 2)
𝑑1
Hardness
= 𝑁/𝑠 (Equation 3)
𝑑2
Where the term on the left-hand side of Equation 2, is known as the first maximum 214

peak in the force–deformation curve (N), and the term on the left-hand side of Equation 215

3, is known as the second maximum peak in the force-deformation curve (N) (Figure. 2). 216

For each sample analyzed, the maximum force in each region corresponded to the 217

hardness of the coating and filling. The area under the curve was also calculated for each 218

region and the total area (coating+filling), which represents the work (force per time) 219

required to penetrate the samples. The negative area associated with the adhesiveness of 220

the samples, i.e., the work required to remove the probe from the sample [45,46] was not 221

considered once the operator needed to hold the bonbon when the probe was removed, 222

which is similar, to a sensory point of view to the degree of compression of the substance 223

between the teeth before breaking [47] and to the force necessary to remove the adherent 224

material from the mouth (palate) during the chewing process performed by the consumer. 225

Other textural tests on the bonbons were carried out by removing the upper part of 226

the coating to measure the hardness of the filling and to interpret how the peripheral and 227

bottom coating contributed to the protection of the filling (Figure 3). 228

The textural properties that allow the structure of a sample to be investigated are 229

firmness (N), the point of maximum force, during penetration; coherence (N s), the 230

positive zone, up to the maximum force during probe descent; cohesion (EN), the 231

maximum peak of the negative region where the probe returns [48,49]. 232

233
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234
235
236
237
238
239
240
241

(B) 242
(A) (C)
Figure 3. (A) Area marked on the bonbon that represents the removal of the coating; (B) 243

View of the penetration of the 2 mm cylindrical probe into the filling without the 244

interference of the coating; (C) Bonbon samples without the coating on top where the hole 245

through which the cylindrical probe has penetrated is observed. 246

2.5. Determination of the stability of the bioactive compound astaxanthin in bonbons using 247

(HPLC). 248

The extraction of the BAC from ASTX was carried out using the alkaline hydrolysis 249

method, for the preparation of the samples the protocol proposed by Cerezal et al. [22], 250

First, a PCB sample was crushed with an analytical mill (model A 11 basic, IKA 251

Labortechnik; Germany), until a powder was obtained, using the quartering method. The 252

portion of the sample to be used was selected 0.5 g PCBs were mixed with 1 mL n-hexane, 253

1 mL DMSO, 2 mL acetone, and 1 mL saline solution in 15 mL glass centrifuge conical 254

tubes. The sample was agitated with a vortex (model VM300, Gemmy Industrial Corp, 255

Taiwan), for 1 min and the tube was centrifuged at 3500 rpm for 3 min. The hexane layer 256

containing ASTX was pipetted into a clean tube containing 1 g of Na 2SO4. Then the hexane 257

layer was transferred to a 50 mL flask, the solvent was removed at 50°C at dryness with a 258

rotary evaporator (model RE 52A. XIan Heb Biotechnology, China). It was then 259

redissolved into 10 mL of acetone, adding 1 mL at a time, and agitating in an ultrasonic 260

bath (model 57H, Neytech, USA). Immediately the total 10 mL of the sample was filtered 261

with a filter millipore Millex-GM Nylon 0.20 µ m the acetone extract was adjusted so that 262

the absorbance was within 0.8-1.2 (analysis in triplicate) 3 mL of sample was used to 263

perform alkaline hydrolysis. 264

Regarding the hydrolysis, the sample with acetone was dried under nitrogen gas flow 265

and the content dissolved in 2 mL of methanol. 0.1 mL of 1% KOH was added and mixed 266

in the vortex (model VM300, Gemmy Industrial Corp, Taiwan). The ASTX esters were 267

hydrolyzed at room temperature under gaseous nitrogen and left in darkness for 18 hours. 268

The methanol phase was extracted with petroleum ether and the organic phase was 269

washed with saline solution. The petroleum ether was washed with Na 2SO4, and the 270

solvent was removed under nitrogen gas flow. This was dissolved again in 3 mL of 271

acetone and analyzed by HPLC. 272

The HPLC was conducted in a liquid chromatograph (model 7100, Hitachi, Japan) 273

equipped with three pumps and a UV-Vis detector. An aliquot of 20 µ L was extracted 274

from the sample using a column RP-18. The mobile phase consisted of a mixture of 275
Processes 2023, 11, x FOR PEER REVIEW 8 of 25

solvents A (acetone), B (methanol), and C (water). For this analysis, the elution procedure 276

used was 60:23:17 A: B: C (v/v) for 2 min; a linear gradient of 60:30:10 A: B: C (v/v) per 4 277

min. The mobile phase was pumped at a flow rate of 1 mL min and detected at 474 nm.
-1 278

The identification of astaxanthin was carried out by comparison of retention times with 279

reference standards through the preparation of standard solutions (1 to 80 ppm) that were 280

injected into the HPLC system for the production of a standard ASTX curve. The 281

calculation for the concentration of ASTX in the samples was performed using the 282

standard curve. 283

2.6. Determination of the moisture of chocolate bonbon 284

The moisture content of chocolate samples was determined using an official 285

gravimetric method [50], with few modifications. A similar method is recommended by 286

the International Office of Cocoa, Chocolate and Sugar Confectionery (IOCCC) [51]). This 287

type of technique is based on the gravimetric determination of the loss of mass of the dried 288

sample to constant mass in an air stove (model Venticell, MMM group, Germany). The 289

sample was weighed between 2 and 5 g, previously crushed and homogenized, and 290

uniformly placed on a Petri glass plate. The sample was introduced in a stove at 103°C ± 291

2, during a time not superior to 24 h to constant weight. 292

2.7. Determination of Antioxidant Activity 293

To determine the antioxidant activity, the FRAP (Ferric Ion Reducing Antioxidant 294

Power) method was used [52,53]; and the Total Polyphenols were determined by the 295

Singleton & Rossi [54] colorimetric method. 296

Using this methodology, solutions of TPTZ, FeCl3- 6H2O (20 mM), and acetate buffer 297

pH 3.6 were prepared daily. For AO samples, 0.5 g (tripled) were weighed (Aux 220, 298

Shimadzu, Japan) and placed in a test tube, then 4 mL of methanol-water (50:50) was 299

added. The test tubes were shaken for 10 minutes in the vortex (VM300, Gemmy Industrial 300

Corp, Taiwan), then exposed to ultrasound (UP100H, Hielscher, Germany) for 10 minutes 301

and centrifuged (5707, Eppendorf, Germany) at 4000 rpm for 15 minutes. The extracted 302

supernatant was reserved, and the pellet was re-extracted, adding 4 mL of acetone:water 303

(70:30 v/v), by repeating the agitation and centrifugation process mentioned above. The 304

supernatant fractions were linked and gauged to 10 mL of distilled water. From the 305

solution obtained, 1 mL was taken and 4 mL of FRAP solvent was added (40% methanol- 306

water: 40% acetone-water: 20% distilled water) and homogenized in the vortex for 2 307

minutes. To finalize the method, 100 μL of the sample from the previous point was added 308

to an Eppendorf tube, with 900 μL of FRAP reagent and the samples were read in the 309

spectrophotometer (model SP 830 UV-VIS, Metertech, Taiwan) at a wavelength of 593 nm. 310

Calculations were obtained based on a Trolox calibration curve 311

For PCB samples, the same FRAP protocol was used, with the modification that 1 g 312

of sample was weighed, which was previously defatted with boiling distilled water and 313

stirring for 1 hour at ambient temperature and the volumes of reagents used were 10 mL. 314

Total phenolic content was determined by the Singleton & Rossi colorimetric method [54], 315
Processes 2023, 11, x FOR PEER REVIEW 9 of 25

based on the reaction of phenols with the Folin-Ciocalteau reagent with some 316

modifications. 317

100 μL of the sample, 800 μL of distilled water, and 100 μL of Folin-Ciocalteau reagent 318

(analytical grade, Merck) were added to an Eppendorf tube. It was shaken and left to rest 319

for 8 minutes. After some time, 50 μL of 20% Na 2CO3 was added. Then the samples were 320

arranged for 1 hour in darkness and then a spectrophotometric reading was made at 760 321

nm. To generate the calibration curve (R = 0.995), gallic acid solutions (Sigma-Aldrich® )
2 322

between 50 - 500 μg·mL -1 were used. The results were expressed as mg gallic acid 323

equivalents (GAE)·g-1 and the values were presented as the mean of the analyses 324

performed in triplicate ± standard deviation. For the preparation of the gallic acid curve 325

(0.1 mg·mL-1), serial volumes from 0 to 160 μL were used, in intervals of 20 μL, completing 326

each volume with 500 μL with distilled water and the measurements were made at 760 327

nm. 328

2.8. Sensory Analysis 329

The sensory evaluation procedures were carried out in two locations: a) The 330

specialized laboratory of Sensory Evaluation that exists in the Department of Food of the 331

Faculty of Health Sciences (FACSA) of the University of Antofagasta, and b) In the 332

Restaurant of a Tourist Center on the outskirts of Antofagasta, Chile. The sensory 333

evaluation analyses carried out focused on two aspects: The first analysis was performed 334

in the Sensory Evaluation Laboratory and consisted of a 7-point category hedonic scale (1 = 335

dislike extremely; 4 = neither like nor dislike; 7 = very much liked) [55]. A second 336

evaluation was performed using an “attribute scale”, which had a maximum value of 10 337

points on a 12 cm long linear scale, limited to 10 cm by anchors, with the highest values 338

of the characteristic at the end right of the scale. In this test, each judge received 3 samples 339

of chocolate bonbons (PCB, UCB, and CCB). 340

The evaluation sheet delivered to each judge is presented in Figure 4 with random 341

three-digit codes for each sample without identifying the type of bonbon chocolate to 342

which they belong. In both evaluations 30 semi-trained judges participated, integrated by 343

students from different university biological careers, these being: food engineering, 344

nutrition, and biotechnology who have already taken the subject of sensory evaluation 345

and handle the basic elements of this discipline. 346

Sample code Evaluated sample result

Code ###
Soft Hard
Code ###
Soft Hard
Code ###
Soft Hard

Figure 4. Sensory evaluation sheet through scale by attributes 347

Processes 2023, 11, x FOR PEER REVIEW 10 of 25

The second sensorial evaluation, performed in the restaurant of the Tourist Center, 348

also consisted of a 7-point hedonic scale test [55]. 112 consumers participated in this test, 349

ranging in age from 65 to 80 years, and whose gender distribution was of the order of 53 % 350

males and 47 % females. 351

2.9. Statistical analysis of the results 352

The analysis of the results was carried out using common statistics and the values 353

were presented in the form of mean values (X) and their respective standard deviations 354

(S). Comparisons between the population mean for two samples were made by the 355

statistician "t of student", in the case of more than two samples, they were done by 356

variance analysis. In those cases where significant differences were found, Duncan's 357

multiple range test was performed [56]. All statistical analyses were performed for a 95% 358

confidence probability 359

3. Results and Discussion 360

3.1. Manufacture of pigmented chocolate bonbons (PCB) in their filling with astaxanthin 361

oleoresin (AO). 362

The Bitter coating of the chocolate was subjected to a "tempering" process at an 363

optimal temperature of 35° C, with the use of this technique it was ensured that the fat 364

crystals present in the chocolate stabilized [57,58], avoiding the phenomenon of "fat 365

blooming", and the difficulty of unmolding [59]. The rapid quenching avoids a critical 366

aspect in the elaboration of chocolates ensuring a fast and adequate crystallization in a 367

stable way achieving a PCB with brightness, stability, and hardness, as suggested by 368

Codini et al. [60]. 369

2.2. Mixing capacity of astaxanthin oleoresin (AO) in the substitute white chocolate 370

(SWC) filling and observation of astaxanthin microparticles in the filling. 371

Figure 5 shows the size of the macroparticles obtained for the measurements made in 372

the middle area of the micelles formed when the AO interacts with the SWC of filling. The 373

filling mix was measured at magnifications from 4X to 100X, achieving a more 374

homogeneous image at this latter value. No notable separations were observed between 375

the micelles due to their very small sizes, resulting in a final mixture of homogeneous 376

orange color without the presence of traces of reddish coloration, which is understood as 377

a good mixture to use as a filling for chocolates. The size of the micelles (areas) in the 378

mixture, with values between 0.26 and 3.34 μm2, observed with an increase of 40X, was 379

determined. With the obtained values, it was possible to classify the micelles within what 380

is established as an acceptable size for the food industry, between 0.1 and 100 µ m [61]. In
2 381

addition, it was observed that the size of the particles in the mixture did not present a 382

uniform size or typical results when the mixtures were made with high-speed 383

homogenization equipment, as occurred in the present investigation. 384

For particle sizes greater than 30 µ m (0.03 mm), the chocolate will produce a gritty 385

tongue feel [62-64]. The range of micelle sizes for this investigation is acceptable, 386
Processes 2023, 11, x FOR PEER REVIEW 11 of 25

considering that the gritty sensation will not occur, since at 37°C (normal temperature in 387

the mouth), the chocolate fat will melt, which adds to the shear that it will suffer in the 388

interaction of teeth and tongue due to chewing. 389

390

391

392

393

394

395

396

397
4X 10X
398

399

400

401

402

403

404

405

40X 100X 406

Figure 5. Particle size in the mixture of the white chocolate filling with the addition of 407

homogenized AO to mechanical mixing 736 ± 15 rpm for 10 min. 408

3.3 Texture analysis of samples of pigmented chocolate bonbons (PCB) with astaxanthin oleoresin 409

(AO). 410

Different authors have investigated the physical-chemical properties of dark 411

chocolate but in particular its textural properties [58,65-69]. It is important to respect the 412

product manufacturing process technologically and in this way the rheological properties 413

of the chocolate are taken care of, thus obtaining a product of high quality and well- 414

defined texture [69]. It is also important to keep the moisture content under control, as 415

this is a factor directly related to the textural properties [70]. Instrumental measurements 416

are known to provide information related to sensory character [71]. Variable texture 417

profiles were observed from the textures obtained from force versus time determinations 418

performed on the PCB samples compared to the same formulation of bonbon but 419

unpigmented in the filling (UCB). This is because both the coating and the filling of the 420

bonbon are hard and brittle, resulting in responses to different penetration forces. 421

Similarly, it is possible to observe a typical texture profile, characterized by presenting 422

two regions with maximum peaks, as reported by Alvis et al. [45], and Cerezal et al. [44], 423

separated by a reversed peak in time 2.0 seconds (Figures 6A and 6B), which is the result 424

of the mean values of 10 replicates of PCB samples. The results of the measurements made 425

on the bonbons are shown in Table 2, it is observed that the only parameters with 426
Processes 2023, 11, x FOR PEER REVIEW 12 of 25

significant differences are the hardness of the coating (p = 0.006) and the total area (p = 427

0.02), present in PCB, with values lower for both parameters, relative to UCB. However, 428

taking into account that the coating must be identical for both bonbons, PCB and UCB and, 429

since this is a minor difference, probably associated with variations in the preparation 430

process, the calculation of the total area represented a minor error (1-2%), considering the 431

areas of coating and filling, individually (5-12%). 432

433

434
435
436
437
438
439
440
441
442
443
444
445
446
447
Figure 6A. Force versus Time Texturogram. Samples of pigmented chocolate bonbon 448
(PCB). Lines of colors indicate different replicates of samples (n = 10). 449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467

Figure 6B. Force versus Time Texturogram. The red color area represents the union of the 468

measurements made (n = 10) in Figure 6A. 469

470
Processes 2023, 11, x FOR PEER REVIEW 13 of 25

Table 2. Texture parameters of whole chocolate bonbons (coating, filling, and coating + filling), subject to simple 471

penetration tests (6 mm) with a 2 mm diameter cylindrical probe. 472

Formulated Chocolates Coating Filling Coating + Filling

Bonbons Fmax (N) A (N.s) Fmax (N) A (N.s) A (N.s)

With Astaxanthin 29.6 ± 1.2 a* 44.9 ± 7.3 a 30.9 ± 1.3 a* 29.4 ± 6.0 a 74.3 ± 3.2a
Without Astaxanthin
31.0 ± 0.5b 44.7 ± 11.5a 31.1 ± 1.5a 34.0 ± 12.1a 78.7 ± 4.6b
(Control)
Note: Different letters in the same column correspond to significant differences (p<0.05) between bonbons with and without
astaxanthin. The presence of an asterisk* in the same line corresponds to significant differences (p<0.05) between the hardness of
the coating in the filling. Force (F) in (N), and A in (N.s) the coherence of the sample.

The analyses performed on the CCB had a completely different texture, this is 473

associated with its "ganache de praliné” filling. A well-defined initial peak (corresponding 474

to the hardness of the coating) and then a threshold (corresponding to the hardness of the 475

filling) were observed (Figures 7A and 7B). For this group of samples, the coating 476

hardness, filling hardness (between anchors 2 and 3), areas under the curve corresponding 477

to coating, and total filling were calculated. Table 3 presents the results, confirming the 478

variability in the data obtained from the coating plus filling analysis of the CCB. 479

480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496

Figure 7A. Force versus Time Texturogram. Samples of commercial chocolate bonbons 497

(CCB) were subjected to simple penetration tests (6 mm) with a 2 mm diameter cylindrical 498

probe. Lines of colors indicate different replicas of samples (n = 10). 499

500
501
Processes 2023, 11, x FOR PEER REVIEW 14 of 25

502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518

Figure 7B. Force versus Time Texturogram. The red color area represents the union of the 519

measurements made (n = 10) in Figure 7A. 520

Table 3. Texture parameters of commercial chocolates (coating, filling, and coating + filling), subjected to simple 521

penetration tests (6 mm) with a cylindrical probe of 2 mm in diameter. 522

Commercial Chocolate Coating Filling Coating + Filling

Bonbons
Fmax (N) A (N.s) Fmax (N) A (N.s) A (N.s)
Coating + filling analysis Group I 17.4 ± 2.6 7.3 ± 2.4 4.6 ± 0.8 10.5 ± 2.4 17.8 ± 2.3
Group II - 6.1 ± 0.8* - 38.2 ± 7.4
Filling analysis - - 3.0 ± 0.4 3.3 ± 0.4 -
* Corresponds to the force at the last point of penetration, that is, for the penetration distance of 6 mm. Force (F) in (N), 523

and A in (N.s) the coherence of the sample. 524

On the other hand, the values of the texture parameters in the fillings of the developed 525

chocolates PCB and UCB presented values of an order of magnitude much higher than in 526

the fillings of commercial chocolates (CCB), in fact, it is evident when the texturograms of 527

the different types of bonbons were compared (Figures 8A and 8B). Nevertheless, the 528

protection of the filling by the coating was observed, which was corroborated when the 529

measurement was made with the cylindrical probe through a hole in the coating (Figure 530

8A) instead of when all the coating was removed, maintaining better the textures on the 531

PCB and UCB than in the CCB commercials (Figure 8B) 532

533
534
Processes 2023, 11, x FOR PEER REVIEW 15 of 25

535
536
537

(A) 538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
(B)
555
556
557
558
559
560
561
562
563
564
565
566
567
568
Figure 8. Comparison of the average texturograms of chocolates. PCB (black), UCB (blue), 569

and CCB (red) were subjected to simple penetration tests with a 2 mm cylindrical probe. 570

A) Measurement of the hardness of the fillings through a hole made in the coating for PCB, 571

UCB, and CCB. B) Measurement of fillings hardness by removing all coating from PCB, 572

UCB, and CCB. 573

The cutting tests performed with the Knife Guillotine Blade probe, on the PCB, UCB, 574

and CCB samples, had erratic results due to the hardness of the dark chocolate coating 575

that prevents the perpendicular penetration of the blade from the beginning of contact 576

with the coating, producing diagonal cuts that were not representative . 577

3.4 Storage stability study of ASTX concentration in PCB 578

Figure 9 shows the behavior of the astaxanthin concentration in the storage time for 579

the PCB samples under environmental conditions, observing linear degradation kinetics, 580

typical of a first-order reaction with a degradation rate constant k = 0.021 days -1 and a 581
Processes 2023, 11, x FOR PEER REVIEW 16 of 25

determination coefficient R2 = 0.884, which resulted in a half-life time of 𝑡½ = 33 days. 582

This result is in correspondence with other studies on the degradation of natural pigments 583

in food matrices, which follow first-order kinetics [72,73]. During the first 7 days, the 584

concentration of astaxanthin remained practically constant in the PCB filling, reaching in 585

42 days a reduction of 17.85% of its initial value. 586

587
588
5.50 589
590
591
5.00 y = - 0.021x + 4.788 592
R² = 0.884 593
Ln (C/Co)

594
4.50
595
596
597
4.00
598
599

3.50 600
601
0 5 10 15 20 25 30 35 40 45
602
Time (days)
603

Figure 9. Graphical representation of Astaxanthin pigment degradation in PCB samples, 604

over a storage period of 42 days, under ambient conditions (T = 20 ± 3°C). 605

3.5 Moisture Analysis of AO Pigmented Chocolate Bonbon (PCB) Samples. 606

For PCB, stored in ambient conditions (T= 20 ± 3°C), their mean values of moisture 607

corresponded to 1.16 ± 0.06%. Moisture contents of all samples were within an acceptable 608

range for chocolate (below 1.5%). Ashkezary et al. [74] reported that the moisture content 609

of the chocolate samples over 1.5 percent would hurt the rheological properties [46], 610

mentioning that, at higher moisture, the bonbon will lose its shine and a whitish layer will 611

appear on the surface known as "fat bloom". This does not affect the nutritional qualities 612

of chocolate, but its attractiveness to the consumer will be less [75]. On the other hand, it 613

is raised that the moisture in the chocolate is a critical factor because an excess causes 614

rheological alterations [74,76]. In addition, high moisture leads to the formation of sugar 615

aggregates (Sugarbloom), which increases friction between particles, also raising viscosity 616

[77]. The fact that the raw materials used for PCBs contain emulsifiers such as soy lecithin, 617

and polyglycerol polyricinoleate (PGPR), provides an advantage as they reduce the 618

negative effect caused by moisture [78]. The hydrophilic bonds, the product of the 619

emulsifiers, retain water that does not affect the fluency of the chocolate [79]. Therefore, 620

the moisture values corresponding to the PCB samples are allowed, since the degradations 621

associated with higher moisture mentioned above will not occur. 622

3.6. Determination of the Antioxidant Capacity and Content of Total Polyphenols of PCB and 623

AO 624
Processes 2023, 11, x FOR PEER REVIEW 17 of 25

In the quantification of the antioxidant capacity, the samples were measured 625

according to their capacity to reduce the ferric iron (Fe ) present in a complex with 2,4,6-
+3 626

tri(2-pyridyl)-s-triazine (TPTZ) up to the ferrous form (Fe ), which had a maximum

+2 627

absorbance at a wavelength of 593 nm [69], for which a Trolox standard curve was made, 628

where a good linear adjustment was obtained (R = 0.997), allowing the quantification of
2 629

the Trolox equivalents (TE). The results were expressed in μmol TE/100 g of the analyzed 630

sample. 631

Figure 10 presents the results of the antioxidant capacity. Freshly prepared PCB 632

samples showed a value of 12044 µ mol ET/100 g, and by the end of the 3-month storage 633

period, a value of 10294 µ mol ET/100 g was obtained, which is equivalent to a 15% 634

reduction in the antioxidant capacity. This may be due to the photoisomerization of 635

astaxanthin. In addition, it must be taken into account that the factors that influence the 636

degradation of carotenoids are not only exposure to light, temperature, presence of 637

oxidants or antioxidants but also the other components of the food product [80-81]. 638

The AO sample presented a value of 30629 μmol ET/100g, proving its high antioxidant 639

capacity mentioned by several authors [82-84]. However, when determining the 640

antioxidant capacity values for the analyzed samples (DC, PCB, and AO) separately 641

(Figure 10), it cannot be ensured that they act synergistically, rather the final product is 642

the result of the interaction of each, factors of process and the food matrix used. 643
644
645
35000
30629 646
30000 647
μmol ET/100 g sample

648
25000 649
650
20000
651

15000 652
12044
10294 653
10000 654
5231 655
5000 656
657
0
658
DC PCB initial PCB final Astaxanthin
659
oleoresin
Samples analysed 660

Figure 10 Antioxidant capacity, by FRAP methodology for the different samples analyzed. 661

The total polyphenols (TPF) determination carried out on the DC sample presented a 662

value of 9.69 mg EAG/100 g, while the initial and at the end of storage PCB sample, 663

reached values of 6.04 and 7.10 mg EAG/100 g, respectively, slightly lower than in DC 664

alone (Figure 11). Chocolate is rich in flavonoids with the structure of catechins and 665

epicatechins, especially the polymeric procyanidins that are formed during the processing 666

of cocoa by joining 2 to 10 epicatechin monomers [85]. For AO samples, the amount of TFP 667

resulted in 20.75 mg EAG/100 g, well above the values presented by DC and PCB alone. 668

It should be borne in mind that the antioxidant effect of polyphenols depends on their 669
Processes 2023, 11, x FOR PEER REVIEW 18 of 25

bioavailability and absorption [86,87]. The content of these compounds in foods is affected 670

by several factors: climate, type of soil, type of crop, and sun exposure, among others [88]. 671
672

25 673
674
20.75
675
20

mg EAG/ 100 g sample

676
677
15
678
9.69 679
10 680
7.1
6.04 681
5 682
683
0 684
DC PCB initialPCB final Astaxanthin 685
oleoresin 686

Samples analysed 687

688

Figure 11. Total polyphenol values for each analyzed sample 689

3.7. Sensory analysis performed on PCB samples 690

The PCB bonbons developed in the present investigation were sensory evaluated to 691

determine their general acceptability regarding the intensity of the characteristics 692

perceived by sight, smell, touch, and taste. The flavor and aroma of chocolate can only be 693

determined sensorially and for these parameters, there is no absolute value, it depends on 694

each process and raw materials of the product, as well as the preference of the consumer 695

[89]. It is important to mention that the consumer, even without previous training, easily 696

detects problems of acidity, moisture, texture, and irregular appearance [90]. 697

In the first sensory evaluation test, the results of each of the sheets issued by the semi- 698

trained judges were analyzed, these being: 18 chose the "Very much liked" option, 6 the 699

"Like a lot" option, 4 the "Like moderately" option, and only 2 selected "Neither like nor 700

dislike" option, which corresponds to 60%, 20%, 13.3%, and 3.7% of the total judgments, 701

respectively. These percentages are represented by the red area in the radial graph of 702

Figure 12. 703

In the second evaluation, carried out with the same 30 semi-trained judges, a 704

comparative hardness test was carried out between the samples, which was evaluated by 705

presenting each judge with a sheet, which showed a linearly structured scale (10 cm) 706

delimited in extremes for all three types of chocolates (PCB, UCB, and CCB) (Figure 13). 707

After analyzing the results and calculating the ANOVA between the samples, it was 708

obtained that the F-ratio = 1.35, (resulting quotient between the intermediate groups and 709

the estimated internal groups). Since the p-value of the F-ratio is greater than or equal to 710

0.05, there was no statistically significant difference. 711

In the third sensorial evaluation test, 112 consumers participated, where of the totality 712

analyzed, 21.4%, chose the "Very much liked” option, 43.8% favored the "Like a lot" option, 713
Processes 2023, 11, x FOR PEER REVIEW 19 of 25

and 29.5% selected the "Like moderately" option; that is, 94.7% of the evaluated 714

population selected the first three preference categories. Only a minor percentage of the 715

population, 5.3% selected the term "Neither like nor dislike" (Figure 8), associating their 716

choice with the fact that they are not recurring consumers of chocolate, whatever its type. 717

These percentages are represented by the orange area in the radial graph of Figure 12. 718
719
Very much liked 720
60.0 721
722
Very much dislike 40.0 Like a lot 723
724
20.0
725
726
0.0
727
Dislike a lot Like moderately 728
729
730
Neither like nor 731
Dislike a little
dislike 732
Consumers Semi-trained judges 733

Figure 12. Sensory Evaluation using a hedonic scale. 30 semi-trained judges (red area) and 734

112 consumers (orange area) participated. 735

736

737

738

739
Hardness (Score)

740

741

742

743

744

745
746
Samples analysed 747

Figure 13. The samples were analyzed by the texture (hardness) sensory panel, but there 748

were no statistically significant differences (p < 0.05) between the CCB, PCB, and UCB 749

samples. 750

4. Conclusions 751
The emulsion made with astaxanthin oleoresin (AO) and substitute white chocolate (SWC) can 752

be classified within the so-called "macro-emulsions", given that the size of the micelles formed var- 753

ied from 0.26 to 3.34 μm2, the acceptable size range for the food industry, considering that the final 754

product will not produce the gritty sensation. In a simple penetration test performed with a 2 mm 755

cylindrical probe, the maximum force exerted for chocolates containing astaxanthin was 35.9 N and 756
Processes 2023, 11, x FOR PEER REVIEW 20 of 25

34.3 N. Instrumental and sensory measurements indicated that astaxanthin did not generate a sig- 757

nificant difference in the texture of the chocolate filling and it was concluded that the simple pene- 758

tration tests performed with a 2 mm diameter cylindrical probe are the most suitable for this type 759

of product resulting in more reproducible texturograms, where the texture parameters can be cal- 760

culated with the lowest error rate. The antioxidant capacity evaluations (FRAP) of the PCB samples, 761

at the beginning of storage (t = 0 days), reached values of 12044 μmol ET/ 100 g and at the end, 10294 762

μmol ET/ 100 g. It is concluded that during the storage period, the PCB sample lost 15% of its initial 763

antioxidant capacity. Regarding the determination of total polyphenols at the beginning and end of 764

storage for the PCB sample, they did not show significant differences. These values are closely re- 765

lated to its antioxidant effect, which depends on its bioavailability and absorption. 766

767

Abbreviations 768

AE: Astaxanthin esters HP: Haematococcus pluvialis

AO: Astaxanthin oleoresin. KOH: Potassium hydroxide.
ASTX: Astaxanthin LF-DC: Lycosome-formulated dark chocolate
BAC: Bioactive compound LF-DC-ASTX: Lycosome-formulated dark chocolate co-
crystallized astaxanthin
BOP: Biological oxidation parameters. MDA: Malonic dialdehyde.
CCB: Commercial chocolate bonbon. Na2SO4: Sodium sulfate.
DC: Dark chocolate. ox-LDL: Oxidized Low-Density Lipoproteins.
DMSO: Dimethyl sulfoxide. PCB: Pigmented Chocolate Bonbon
EN: Cohesion. S: Standard deviations.
FA: Free astaxanthin SWC: Substitute white chocolate.
FDA: Food and Drug Administration TPA: Texture profile analysis
FRAP: Ferric Ion Reducing Antioxidant Power. TPTZ: 2,4,6-Tripyridyl triazine.
GABA: Gamma-aminobutyric acid. UCB: Unpigment Chocolate bonbon.
GAE: Gallic acid equivalents. X: Mean values.

Author Contributions: Conceptualization, P.C.M., and F.S.F.; methodology, P.C.M.; F.S.F.; W.B.M, 769

and A.P.B.; software, P.C.M.; F.S.F., and A.P.B.; validation, P.C.M.; F.S.F. and W.B.M.; formal 770

analysis, F.S.F., and W.B.M. and A.P.B.; investigation, P.C.M.; F.S.F.; and A.P.B.; resources, P.C.M.; 771

F.S.F., and W.B.M.; data curation, P.C.M., and F.S.F.; writing—original draft preparation, P.C.M., 772

and F.S.F.; writing—review and editing, P.C.M., and F.S.F.; visualization, F.S.F.; supervision, P.C.M., 773

and A.P.B.; project administration, P.C.M.; funding acquisition, P.C.M. 774

All authors have read and agreed to the published version of the manuscript. 775

Funding: This research was financed by the public funds of Chile as an undergraduate thesis 776

scholarship in 2016 by the Universidad de Antofagasta. 777

Institutional Review Board Statement: Not applicable. 778

Informed Consent Statement: Not applicable. 779

Data Availability Statement: All data are available in this article. 780
Processes 2023, 11, x FOR PEER REVIEW 21 of 25

Acknowledgments: We thank Atacama Bio Natural Products S.A. for giving the astaxanthin oleo- 781
resin. 782

Conflicts of Interest: The authors declare no conflict of interest. 783

Sample Availability: Not available 784

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