JID: FB

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DOI: 10.1039/D4FB00019F
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10.13039/100018244;Universidad de las Américas Puebla;Unassigned;Mexico

Cladodes of Opuntia ficus indica as a functional ingredient in the

production of cookies: pPhysical, antioxidant and sensory properties

Rocío Aparicio-Ortuñoa, Oscar Jiménez-Gonzáleza, J. Daniel Lozada-Ramírezb, and Ana E. Ortega-Regules* c,ORCID
a . Department of Chemical, Food and Environmental Engineering, Universidad de las Américas Puebla, Sta. Catarina Mártir, C. P. 72820,

Cholula, Puebla, Meéxico; [email protected]; [email protected]

b . Department of Chemical and Biological Sciences, Universidad de las Américas Puebla, Sta. Catarina Mártir, C. P. 72820, Cholula,

Puebla, Meéxico; [email protected]

c . Department of Health Science, Universidad de las Américas Puebla, Sta. Catarina Mártir, C. P. 72820, Cholula, Puebla, Meéxico,*

Correspondence: [email protected]; Tel.: +52-2221650440

Page 1 of 26Page 1 of 26
 Cladodes of Opuntia ficus -indica: Aa Ffunctional Iingredient for Hhealthful Ffoods. Study on Ccookies Ssubstituting Wwheat Fflour

with Ccladode Ppowder.

Opuntia ficus indica cladodes are a rich source of bioactive compounds and dietary fiber; therefore, they could be a functional ingredient in

the production of health-promoting foods. This study aimed to develop cookies by substituting wheat flour with powdered cladodes in

different proportions, 15, 20 and 25%, and analyze them in terms of physical and bioactive compounds and sensory characteristics.

Proximal analysis showed that powdered cladodes was were high in total dietary fiber (54.89%). The analysis of total phenolic compounds

and antioxidant capacity revealed values of 2,050.20 mg GAE/ per 100 g db, and 312.14 mg Trolox/ per 100 g db. Powdered cladodes

supplementation increased hardness and decreased color parameters (L*, a* and b*) compared to control (withiout cladode powder).

Moreover, rising levels of powdered cladodes, contribute to the increased total phenolic compound content and antioxidant capacity

compared to control. Sensory analysis showed that 15% cladode supplementation was adequate for preparing an acceptable functional

cookie. The sorption isotherm showed that it was possible to fit the GAB model to the experimental data and the cookies was were stable at

25 °C. Cladodes of Opuntia ficus indica could be considered as a functional ingredient, and a source of dietary fiber and antioxidants for

the manufacture of foods with benefits to human health and nutrition.

Sustainability sSpotlight statement

Our research group harnessed Opuntia ficus indica cladodes for sustainable and health-conscious food choices, introducing a new

dimension to functional ingredients. In this study, powdered cladodes replaced traditional wheat flour in cookie formulations, revealing

substantial total dietary fiber content and serving as a potent source of bioactive compounds. The cookies exhibited promising stability at

25 °ºC, crucial for sustainability. Opuntia ficus indica cladodes offer a sustainable solution, enabling the food industry to contribute to

health and environmental well-being. This research signifies a step towards integrating sustainable practices in food manufacturing for a

healthier, environmentally conscious future.

1 Introduction

Page 2 of 26Page 2 of 26
 Products made with wheat flour, such as bread, cakes and cookies, are globally consumed in significant quantities; in fact, they are one

of the main components of the human diet, owing to their low cost and easily made formulations, and the world total wheat output in 2023

was nearly at 777 million tonnes.1 The production and consumption of cookies have increased worldwide, and the market reached USD 121

billion by 2021 and is expected to reach USD 164 billion by 2024 (ref. 2). However, the main ingredients in wheat flour-based products

may lack dietary fiber, minerals, and antioxidants, which are relevant to human nutrition and health,; besides, the excessive consumption of

ultra-processed foods contributes to unhealthy diets.3 Conditions such as cardiovascular diseases and obesity limit fat intake in particular

fats rich in saturated fatty acids, such as butter, and the use of unsaturated fatty acids should be recommended to reduce the risk of heart

diseases. 4 According to the Institute of Food Technologists, healthy foods will continue to be a trend in the food market for future years.5

In addition, a healthy diet rich in natural ingredients could act as functional foods and/or nutraceuticals. These types of ingredients have the

potential to improve health and could help in the primary prevention of many chronic diseases, such as dyslipidemia and cardiovascular

diseases. Therefore, consumers have become interested in the food processing, food ingredients and how they interact with health;5 also,

food markets are interested in local, organic foods or products which incorporate these ingredients.

On the other hand, epidemiological studies6,7 have shown that regular consumption of plant-based foods is associated to with reduced the

risk of chronic diseases related to oxidative stress. Therefore, functional ingredients rich in antioxidants, dietary fibers, minerals, and

vitamins, low in calories and fat, and free of synthetic additives used in the formulation of conventional foods can enhance their

nutraceutical potential. In this context, the composition of Opuntia ficus indica cladodes makes them a potential ingredient to produce

functional foods that promote health. In Mexico, in addition to being consumed as a vegetable, Opuntia ficus indica cladodes are used in

traditional medicine to treat diabetes, hyperlipidemia, obesity, and gastrointestinal disorders. 8,9 Cladodes contain high amounts of fibers,

including mucilage, pectin, lignin, cellulose, and hemicellulose, which influence the metabolism of lipids and carbohydrates. 10–12 However,

fresh cladodes have limited acceptance among many consumers including kids, due to their mucilaginous texture, acidity, astringency, and

herbal taste.13 Consequently, an alternative to increase their use and consumption is to transform them into powder to and use them as a

functional ingredient that maintains the nutritional, medicinal, and nutraceutical properties. Therefore, the incorporation of these powders in

the formulation of flour-based products could represent a promising strategy, to acting as a good carrier for functional ingredients and

Page 3 of 26Page 3 of 26
offering the possibility of introducing substances with health benefits through the diet to increase their nutritional profile and confer

additional functional properties.7,14–16

In this context, the aim of the present study was to evaluate the effect of replacing wheat flour with powdered cladodes of Opuntia ficus

indica var. Atlixco and replacing butter with vegetable oil in the preparation of cookies and how this substitution influences its their

physical properties, polyphenols content, antioxidants capacity, and sensory properties. Likewise, for the best evaluated sensorial cookie,

the sorption isotherm was used as an instrument to predict the shelf life and acceptability of the products.

2 Materials and mMethods

2.1. . Preparation of cladodes powder

Opuntia ficus indica variety Atlixco cladodes with more than 60 days of maturation and a length greater than 30 cm were collected in

May 2023 in Atlixco, Puebla, Mexico (18° 54′' 4.935″” N 98° 27′' 11.346″” W), which and were free of pesticides and chemical

fertilizers. Manual depinning was performed with a stainless-steel knife, and they were washed with distilled water, and disinfected with a

solution of 10% sodium hypochlorite. Then, cladodes were sliced into strips of 2 cm lengthwise, dried in a dehydrator at 45 °C/30 h

(Excalibur® EXC10EL, Sacramento, California, USA), and ground at 22 rpm/4 min (WARING, blender 7011 HS, USA). The powder

obtained was stored at 25 °C hermetically in sealed bags until further use.

2.2. . Proximal chemical analysis

Cladode powder analysis was carried out according to the methods of A. O. A. C. (2000).17 The moisture content (950.46) and ash

content (31.012) were determined by from the weight difference after dehydration and calcination, respectively. The crude protein content

was determined using the micro-Kjeldahl method (2001.11), and the total nitrogen content was used for protein content calculation using a

factor of 6.25. The crude fat content (920.3) was determined by solvent extraction using a Fat Extractor E-500 (BUCHI, Fat Extractor E-

500, Flawil, Switzerland). Total dietary fiber, insoluble dietary fiber, and soluble dietary fiber were determined using an enzymatic-

Page 4 of 26Page 4 of 26
gravimetric method 991.43 (Merck enzymes, 112979, Germany) and the procedure of Prosky et al.18 The Ccarbohydrate content was

determined by from the difference in the content of the other components.

2.3. Physical properties

2.3.1. . Bulk density measurement

The assay was carried out according to the technique described by Chandra et al.19 with slight modifications. Briefly, 10 g of sample

was placed in a 25 mL graduated test tube, it was hit on a wooden table several times, until a visible decrease in the level of the graduated

test tube was no longer noticed, and depending on the weight and volume, the bulk density (g /mL−1) was calculated.

2.3.2. . Water and oil holding capacity (WHC and OHC)

Water and oil holding capabilities were determined using the procedure described by Chandra et al.19 with minor modifications. 1 g of

sample was mixed with 50 mL of distilled water or 50 mL of soybean oil, stirred for 1 min at high speed (Vortex-Gene 2 G560, Scientific

Industries, USA), kept at rest at room temperature (25 °C) for 30 min,. Tthen centrifuged at 2,000 × g × g during for 30 min, and the

volume of water or oil in the supernatant was measured. Water and oil holding capabilities were expressed as a percentage of bound water

or oil per gram of powdered cladodes.

2.4. . Bioactive properties

2.4.1. . Extract obtention

The extract was obtained with the Santiago et al.20 methodology with slight modifications. A mixture of 2 g of sample and 25 mLl of

methanol/water was added (50 : :50) and placed in an ultrasound bath (Branson, M1800H-E, USA) for 30 min. Then, the mixture was

centrifuged at 2200 × g × g for 10 min (Centrifuge Z 366 KK, HERMLE Labortechnik, Germany). A second extraction was performed

Page 5 of 26Page 5 of 26
with 25 mLl of acetone/water (70 : :30). Both supernatants were mixed and filtered through a Whatman No. 1 (Sigma-Aldrich) filter paper

and concentrated in a rotary evaporator (R-300, Buchi, Flawil, Switzerland) at 51 °C and 235 mm Hg until half of the initial volume was

evaporated.

2.4.2. . Determination of total phenolic compounds

Total phenolics quantification was made through the Folin-–Ciocalteu colorimetric method.21,22 A solution of the 0.1 M Folin-–

Ciocalteu reagent (Sigma-Aldrich) was mixed with 100 μL of extract. The Mmixture was incubated for 6 min, then 1 mL of Na2CO3 at 5%

was added and incubated in the dark at room temperature (25 °C) for 30 min. Solutions were measured with a spectrophotometer at 765 nm

(UV-Vis Spectrophotometer 1900i, Shimadzu, Japan). A calibration curve was prepared with gallic acid (Sigma-Aldrich, Mexico) at

different concentrations: y = 0.0042x (0–-200 ppm) + 0.0592, (RR2 = 0.991). The results were expressed as mg gallic acid equivalents

(GAEs) per 100 g dry weight and calculated using eqn (1).

d4fb00019f-t1 1

wWhere Abs is the absorbance of each solution (sample/standard) and DF is the dilution factor.

2.4.3. . In vitro antioxidant capacity by DPPH (2,2-diphenyl-1-picrylhydrazyl) assay

Antioxidant capacity was determined by DPPH assay developed by Brand-Williams et al.23 with some modifications. A mixture of 200

μL of extract was added to 2 mL of DPPH ethanolic solution (0.1 mM). The Rresulting mixture was incubated for 30 minutes at room

temperature (25 °C). Absorption was read at 517 nm in on a UV-Vis Sspectrophotometer (UV-Vis Spectrophotometer 1900i, Shimadzu,

Japan), and used to calculate the inhibition percentage (I%) of the antioxidant capacity (eqn (2)). A calibration curve was prepared using

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; Sigma-Aldrich, Mexico) as the antioxidant at different concentrations.

Page 6 of 26Page 6 of 26
The concentration of Trolox was plotted against the percentage of inhibition to obtain the calibration curve ( I(%) = =0.872 × x [0–−300

ppm] − 0.418 , RR2 = =0.962). The Rresults were expressed as mg Trolox equivalents (TEs) per 100 g dry weight (eqn (3)).

d4fb00019f-t2 2

d4fb00019f-t3 3

wWhere ADPPH is the absorbance of the blank without sample or standard, AASs is the absorbance of the sample after 30 min of reaction, and,

DF is the dilution factor.

2.5. . Preparation of functional cookies

2.5.1. . Cookies formulation

For the preparation of cookies, a full factorial design was used, and different relations proportions of canola oil/butter and cladode

powder/wheat fluorflour were used for cookies elaboration following the values shown in Table 1tbl1. The Bbasic formulation consisted of

canola oil/butter creamed with 33 g of brown sugar in a Kitchen aid blender (Whirlpool Corporation, USA). Subsequently, 18 g of eggs

and 2 mL of vanilla were added. Finally, 100 g of a mixture of cladode powder and wheat flour was added until a homogeneous and

smooth dough was obtained. The resulting dough was molded with a roller and the cookies were cut and cooked at 180 °C for 15 min in a

convection oven (Zucchelli Forni, Italy). The cookies were allowed to cool (20 min) and were stored in airtight bags for further analysis.

Table 1 Experimental design

<!--Col Count:3-->Formulation % Cladodes/Wwheat flour Ffffffflour % Vegetable oil/Bbutter

Page 7 of 26Page 7 of 26
 powder/Wwheat flour
Control 0/100 0/100
F1 15/85 70/30
F2 25/75 70/30
F3 25/75 50/50
F4 25/75 30/70
F5 15/85 30/70
F6 20/80 70/30
F7 20/80 30/70
F8 20/80 50/50
F9 15/85 50/50

2.5.2. . Characteristics of cookies

After elaboration, cookies were evaluated in terms of physical properties, water activity, color (L*, a*, b*), and hardness. In addition,

biochemical compounds (total polyphenol content and antioxidant capacity by DPPH assay) were also determined. Finally, the sensory

evaluation (color, smell, texture, taste, and general acceptance) was performed.

2.5.2.1. . Water activity

The determination of water activity (aaw) was made performed with an Aqualab 4TEV equipment, and a proper amount of cookie

sample powder was placed on the tray for the determination.

2.5.2.2. . Color

Determination was made on the surface of the cookies with the help of a colorimeter (Konica Minolta CR-410, Tokyo, Japan) in the

transmittance mode with a D65 standard illuminant, and a 2° standard observer. The parameters L * (Lluminosity), a * (trend towards

green -−, or red +), and b * (trend towards blue -−, or yellow +) values were recorded.

2.5.2.3. . Hardness

Page 8 of 26Page 8 of 26
 The texture of the cookies was measured with an EZ-SX texturometer (Shimadzu Corporation, Kyoto, Japan). The maximum force

(hardness) for cookie brake break was measured with the a cutting device in the middle of the cookie, and the upper blade was moved down

at a speed of 1 mm /s−1 with a penetration of 5 mm. The hardness value is expressed in Nnewton (N).

2.5.2.4. . Bioactive compounds of cookies

Total phenolic compounds and antioxidant capacity were determined following the methodology previously mentioned.

2.5.2.5. . Sensory evaluation

The preferences or acceptance of cookies was were evaluated with potential consumers. At the beginning of the test, each judge was

informed about the test they were going to perform. Only judges who agreed with the evaluation were allowed to participate. Judges were

also informed that “all products were safe for consumption” and that they could leave the test at any time without a reason. The evaluation

panel consisted of 30 untrained men and women with ages between 22 and -59 years from the Universidad de las Americas Puebla

(UDLAP). Evaluators tasted the cookies and rated the color, smell, texture, taste, and general acceptance, using a hedonic scale of 9-point

where 1 = I dislike it very much and 9 = I like it very much. The samples were placed on white plates identified with by random three-digit

numbers. One sample was given at a time.

2.6. . Moisture sorption isotherms

Moisture sorption isotherms was were performance obtained for the to control cookie and the cookie best ranged in sensory attribute of

general acceptance. The evaluation of moisture sorption isotherms was carried out, as a quality parameter during storage under at different

humidity levels. The methodology of Jimenez, et al.24 with slight modifications was used. Saturated solutions of salts used to control

relative humidity were, LiCl (11.3%), CH3COOK (22.5%), K2CO3 (44.3%), Mg(NO3)2 (53.6%), NaBr (56.6%), NaCl (75.3%), BaCl2

(90.3%), and, K2SO4 (97%). Then, 0.5 g of cookie for in triplicate was placed inside the hermetically sealed glass jars. The complete set of

solutions were was placed in an environment with a controlled temperature of 25 °C. The weight of the samples was registered recorded

Page 9 of 26Page 9 of 26
every three days for four weeks. The experimental data of moisture sorption were adjusted to the GAB (Guggenheim, Anderson, and De

Boer) model. The moisture content was carried outobtained by the AOAC 950.46 method.

2.7. . Statistical analysis

All experiments were made performed by in triplicate except for texture analysis where five samples was were used for the test. The

Rresults were expressed as mean ± standard deviation. The comparison of the means was analyzed by analysis of variance (ANOVA)

followed by the Tukey test to identify significant differences (PP ≤ 0.05) between the samples. Data were analyzed using Minitab software

(version 19, Minitab Inc., USA).

3 Results and discussion

3.1. . Chemical composition of cladode powder

The results obtained for cladode powder composition were are presented in Table 2tbl2. The protein content (11.5%) obtained was

similar to the those reported from other studies.25,26 Differences in protein content values depend to on the cultivar variety. Specifically, for

Atlixco variety, the values round range from 9.6% 25 to 12.6%.26 On the other hand, the main component of cladode powder was Ttotal

dDietary Ffiber (TDF) (54.89%). Specifically, Iinsoluble Ddietary Ffiber (IDF) (cellulose, hemicellulose, and lignin) was higher to than

Ssoluble Ddietary Ffiber (SDF) (pectin, gums, and mucilage) which is typical from of this type of products. The Vvalues correspond to the

those previously reported by other authors, ; TDF values in a the range of 45 to 66%, FDI IDF in a the range of 32 to 56%, and SDF with in

the ranges of 5.6 to 25.22%.27–29 However, the relation proportion of IDF FDI to SDF changes with the maturation stage, IDFFDI

increases with maturation.29,30 High values of TDF indicates that cladode powder could be considered as a high source of fiber and a key

ingredient to in food fortification. Different studies have demonstrated the beneficial effect of cladodes; Uebelhack et al.31 showed that

cladode fiber helps reduce body weight by binding to dietary lipids and increasing their excretion. Moran et al.8 tested the effect of

powdered cladodes in diet of obese rats diet, and a decrease of total cholesterol serum levels was observed. Also, the authors suggest an

Page 10 of 26Page 10 of 26
effect on liver function by decreasing triglyceride accumulation and fatty acid peroxidation, thereby reducing hepatic oxidative stress.

Padilla et al.32 studied changes in cholesterol levels with in mice and prevented hyperlipidemia. Finally, the ash content agrees with

previous studies.26,33 In addition, the authors also mention that the main minerals found in cladodes are potassium, calcium and

magnesium;, however, mineral content varied with the maturation stage.34

3.2. . Physical properties

The Pphysical characteristics of cladode powder were are presented in Table 2. The bulk density was 0.77 g /mL−1, (Table 2), which is

consistent with the value reported by Ayadi et al.16 of 0.647 g /mL−1 for powdered cladodes. Water and oil retention capacities are important

parameters that affect the texture, mouthfeel and consistency of food products.6 Regarding the value of WHC (Wwater Hholding

Ccapacity), powdered cladode showed around 34.67% which was higher than the OHC (Ooil Hholding Ccapacity) with by 8%;, this is

related to the high fiber content (54.89 ± 3.76 g per /100 g db), as well as the protein content (11.50 ± 2.74 g per /100 db), since its

hydrophilic and hydrophobic nature allows them it to interact with water and/or oil in food.15 Cladode powder, due to its high water

retention capacity and swelling properties, may be appropriate to improve the texture and stability of a variety of foods such as bakery

products;, likewise, hydrophobic components, such as insoluble fiber (35.25 ± 2.74 g per /100 gg db, Table 2), may improve fat retention

and flavor in some foods, as it in the case of cookies.12

Table 2 Characterization of cladode powder from Opuntia ficus indica var. Atlixco

<!--Col Count:2-->Parameters Value a

Moisture (g per /100 g db) 8.52 ± 0. 88
Proteins (g per /100 g db) 11.50 ± 0.56
Lipids (g per /100 g db) 1.83 ± 0. 17
Carbohydrates b 62.77
Total dDietary fFiber (g per /100 g
54.89 ± 3. 76
db)
Insoluble dDietary fFiber (g per
35.25 ± 2. 74
/100 g db)
Soluble dDietary fFiber (g per /100 19.64 ± 1. 49

Page 11 of 26Page 11 of 26
<!--Col Count:2-->Parameters Value a
g db)
Ash (g per /100 g db) 15.38 ± 0. 10
−1
Bulk density (g /mL ) 0.77 ± 0.01
Water hHolding cCapacity % 34.67 ± 1.15
Oil hHolding cCapacity % 8.0 ± 1.5
Total polyphenols (mg GAE per 2,050.20 ±
/100 g db) 71.5
Antioxidant capacity (DPPH; mg 312.14 ±
Trolox per /100 g db) 55.64
a
Values are the mean ± standard deviation.,

b
Calculated by from the equation: Ccarbohydrates = 100 -− (Mmoisture + Pprotein + Llipids + Aash), db: dry basebasis.

3.3. . Bioactive properties

Phenolic compounds are an important component contributing to the Aantioxidant Ccapacity (AC) of foods and there is a synergy with

vitamins, carotenoids and other components.35 The total content of polyphenols (Table 2) in powdered cladodes was 2,050.20 mg GAE per

/100 g db, and are is similar with to those obtained by Msaddak et al.,12 2,485 mg GAE per /100 g db, and Bensadón et al.,35 2,690 mg

GAE per /100 g db. The small difference in total phenolic compounds, could be related to the variety, maturity stage, and the extraction

method/solvent. Regarding to the AC, a value of 312.14 mg Trolox per /100 g db was foundobtained. The combination of the high

percentage of dietary fiber and the content of phenolic compounds present in powdered cladodes can result in an antioxidant dietary fiber

with beneficial properties for use as a functional ingredient in food processing.36

3.4. . Physical characteristics of formulated cookies with powdered cladode

3.4.1. . Water activity

Water activity values (aw) were in a the range of 0.20 to 0.29; the control showed a value of 0.14 (Table 3tbl3). Values lower than 0.3

indicate that the cookies are chemically and microbiologically stables.37 The difference in the aw of the control in relation to cookies

Page 12 of 26Page 12 of 26
formulated with powdered cladodes may be due to the content of dietary fiber present since it has a greater water holding capacity. The aw

values, showed significant differences (pp < 0.05) according to the formulation. The highest aw (0.290 ± 0.003) belongs to the formula

with the highest content of powdered cladodes (25%). Similarly, high values of aw were presented observed for formulaes with 15 and 20%

of cladodes powder but had the same percentage of vegetable oil/butter (30/70),; therefore, a higher butter content, also influenced the aw of

the cookies due to the presences of milk solids in butter such as proteins and lactose. The lowest values were for cookies with a higher

percentage of vegetable oil/butter (70/30). The different ingredients, such as powdered cladodes, wheat flour, vegetable oil and butter,

given their chemical structure and water retention capacity, will influence the final aw. Similar aw values (0.25 to 0.41) were reported by

Uriarte et al.37 for cookies made with amaranth, mushrooms and powdered cladodes.

3.4.2. . Color

The addition of cladode powder changes the color characteristics (L*, a*, and b*) of cookies with yellow/brown color to different

shades of green. The three-color coordinates present significant differences (pp < 0.05) according to the cookie composition (Table 3).

Specifically, the color parameter L* decreases in relation to the control (67.52), and the lowest value was for cookies with 25% powdered

cladodes. The values of a* and b* also decreased in relation to the control (5.83 and 29.36, respectively). Values for a* were near to czero

or negative, and b* values were greater than 19 indicating that the predominance of green shades color. This may be due to the content of

chlorophyll and other phenolic compounds present in the powdered cladodes and the nonenzymatic reaction (Maillard reaction) that takes

place between reducing sugars and amino acids causing a browning effect in theduring cooking.6

Table 3 Physical characteristics of cookies made with cladodes powder/wheat flour and vegetable

oil/buttera

<!--Col
CP/WF Hardness
Count:8-- O/B% aw L* a* b*
% (N)
>Formulation
Control 0/100 0/100 0.14 ± 67.52 5.83 29.36 5.56 ± 1.
0.03e ± ± ± 0.94c 62d

Page 13 of 26Page 13 of 26
<!--Col
CP/WF Hardness
Count:8-- O/B% aw L* a* b*
% (N)
>Formulation
2.24d 0.95d
1.18
49.76 24.60
0.20 ± ± 12.32 ±
F1 15/85 70/30 ± 1.81 ± 0.89
0.004 d a, b 0.43 a 4.93 a, b
a, b

43.55 2.38 19.42

0.20 ± 8.92 ±
F2 25/75 70/30 d ± 1.43 ± ± 2.90
0.005 c 1.01 a, b, c, d
0.84 a b
47.41 1.03 23.09
0.20 ± 7.88 ±
F3 25/75 50/50 ± 1.08 ± ± 0.56
0.001 d b 1.10 b, c, d
0.48 b a, b
−-
47.79 23.49
0.29 ± 0.43 12.50 ±
F4 25/75 30/70 a ± 0.37 ± 1.20
0.003 b ± a 1.64 a
c
0.32
−-
0.28 ± 52.71 26.79
a, 0.62 9.13 ±
F5 15/85 30/70 0.014 ± 0.25 ± 1.21
b a ± a 1.65 a, b, c, d
0.41 c
−-
48.06 23.96
0.23 ± 0.26 8.36 ±
F6 20/80 70/30 ± 1.08 ± 0.71
0.001 c b ± a 1.82 a, b, c, d
c
0.34
−-
50.03 24.91
0.26 ± 0.69 10.03 ±
F7 20/80 30/70 b ± 1.30 ± 1.94
0.008 a, b ± a 2.47 a, b, c
0.28 c
−-
48.63 23.12
0.26 ± 0.72 6.37 ±
F8 20/80 50/50 ± 0.52 ± 0.77
0.003 b b ± a 1.65 c, d
c
0.40
−-
0.22 ± 51.93 25.33
0.50 5.63 ±
F9 15/85 50/50 0.001 c, ± 0.13 ± 0.30
d a ± a 2.14 d
c
0.14
a
CP/WF: Ccladodes Ppowder/Wwheat Fflour, O/B: Vvegetable oil/Bbutter, aw: water activity, color parameter: (L*, a*, and b*). Values

are the mean ± standard deviation. Different letters in the same column means significantly different differences (pp < 0.05).

3.4.3. . Hardness

Page 14 of 26Page 14 of 26
 The hardness values (Table 3) showed a significant difference (pp < 0.05), between all samples. The control cookie (5.56) and the

formula with 25% powdered cladodes and an O/B percentage of 30/70 (F4) has have the highest increase in hardness. Hardness increases

with the substitution of oil at 70%, and 30%, in contraston the contrary, have a slight decrease at 50% of oil substitution is observed.

Similar trends were observed in at the three levels of cladode addition. This could be related with to the amount of air entrapped into the

cookie dough during its elaboration, but this cannot be retained into the cookie made making them hard.38 In general, all formulaes with

powdered cladodes caused an increase in the hardness of the cookies. Similar trends were reported by Msaddak et al.15 in cookies made

with powdered cladodes. As the percentage of cladode increased, the hardness of the cookies also increased. This could be attributed to the

spreadability of dough during baking,; the internal microstructure of cookies changes because of the particle size of cladode powder, by

which preventing prevents the formation of air cells and a foamy texture.

Fig. 1 (a) Total Pphenolics tTotal Ccompounds (TPCs), and (b) Aantioxidant capacity (AC), in of the

different cookies'’ formulations. Different letters between formulations means significant differences

(pp < 0.05).

3.4.4. . Bioactive compounds of cookies

The Rresults for Ttotal Pphenolic Ccompounds (TPC) and Aantioxidant cCapacity (AC) of cookies are shown in Fig. 1fig1. The TPC

content for control was 75.74 mg GAE per /100 g db and was significantly lower (pp < 0.05) than those of the rest of the formulations.

According to Fig. 1a), the increase in the concentration of powdered cladodes increases the TPC. In contrarycontrast, the oil/butter (O/B)

ratio did not influence the TPC of the cookies. No significant difference (pp < 0.05) was observed between sample 5, 9 and 1 (values near

to 180 mg GAE per /100 g db); 4, 3 and 2 (values near to 230 mg GAE per /100 g db); and 7, 6 and 8 (values near to 340 mg GAE per /100

g db). Different studies suggest that TPC values varied in the range of 100 to 500 mg GAE per /100 g db,6,7,13,15 based for on foods such as

cookies, bread, cake, and cladode flakes in different percentages with supplementation of powdered cladodes around 10 to 25%.

Page 15 of 26Page 15 of 26
 For the AC, similar behaviors to TPC were observed, (a correlation between both values were was RR2 = 0.8741);, the control cookie

had a value of 43.28 mg TE per /100 g db showing a significant difference (pp < 0.05) with respect to the other cookies. In all cases, by

increasing the content of powdered cladodes, the CA was higher, with a good capacity to eliminate free radicals by the DPPH method. The

resulting values relate are in agreement with those found by other authors,7,15,39 in similar foods with data ranging from 53.29 to 120 mg TE

per /100 g db. The results obtained show that cookies added with powdered cladodes have a greater antioxidant potential than control

cookies, which reinforces their nutritional value.

3.4.5. . Sensory evaluation

The sensory analysis of the different formulations of cookies, as well as the control, was carried out for color, smell, texture, taste and

general acceptance, and the results are presented in Table 4tbl4. Fig. 2fig2 shows the cookies elaborated for sensory analysis. Control

cookies were the better rated compareding to the cladode added cookies, most of the values were up to 8 score (Fig. 3fig3). Specifically,

lower values were obtained in color for formulations F2, F3, F4, F6, F7 and F8, with 20 and 25% (6.58 to 7.10), and as was expected the

control (8.5 score) and the lower concentration cladode powder (7.13 score) cookies present the greatest evaluation (pp < 0.05), ); this

difference could be related to the green color of the cookies with a great high amount of cladode powder (Fig. 2, and Table 3). With respect

to smell, no significant difference was observed (pp > 0.05) except for the F8 sample;, neither the percentage of cladode nor the ratio of

oil/butter influences in acceptability. In terms of texture, the increase in hardness due to the addition of the cladode powder influences the

preference of cookies. However, as the oil content increases, this could influence result in hard texture as was discussed previously, and this

could affect the acceptability. However, F6 have has most of the values near to nine (Fig. 3).

Table 4 Results of the sensory evaluation for different cookies made with cladodes powder/wheat flour

and vegetable oil/buttera

Page 16 of 26Page 16 of 26
<!--Col
CP/WF O/B General
Count:8-- Color Smell Texture Taste
% % acceptance
>Formulation
0/10 8.50 ± 8.0 ± 8.30 ± 8.55 ± 8.50 ± 0.61
Control 0/100
0 0.84 a 1.52 a 0.81 a 0.61 a a
7.19 ±
70/3 6.58 ± a, 6.71 ± 7.35 ± 7.03 ± 1.22
F1 15/85 b 1.42
0 1.54 b 1.53 c, d 1.45 b c, d
6.84 ±
70/3 6.58 ± a, 6.29 ± 7.10 ± 6.61 ± 1.38
F2 25/75 b 1.21 d
0 1.61 b 1.60 1.35 b d
7.39 ± 7.71 ±
50/5 7.10 ± a, 6.68 ± 7.06 ± 1.15
F3 25/75 1.15 1.13 a, b, c, d
0 1.56 b b 1.70 c, d b
7.03 ±
30/7 6.61 ± 6.58 ± 7.32 ± 6.97 ± 1.05
F4 25/75 1.40 a,
0 1.63 b b 1.86 c, d 1.30 b c, d
7.45 ± 7.39 ± 8.00 ±
30/7 a, a, 8.00 ± a, 7.97 ± 0.84
F5 15/85 1.31 1.31 a, b 1.00 a, b
0 b b 0.77 b

7.53 ± 7.47 ±
70/3 6.67 ± a, 6.70 ± 7.07 ± 1.41
F6 20/80 1.11 1.28 a, b, c, d
0 1.38 b b 1.62 c, d b
7.07 ± 7.23 ± 7.70 ±
30/7 6.80 ± a, 7.40 ± 1.33
F7 20/80 b 1.36 1.38 a, b, 1.15 a, b, c, d
0 1.56 b c, d b

6.87 ±
50/5 6.70 ± 6.70 ± b, c, 7.37 ± 7.07 ± 1.08
F8 20/85 b b 1.57
0 1.64 1.76 d 1.33 b b, c, d
7.13 ± 7.27 ± 7.63 ± 7.77 ±
50/5 7.77 ± 1.10
F9 15/85 1.68 a, 1.53 a, 1.27 a, b, 1.43 a, a, b, c
0 b b c b

a
CP/WF: Ccladodes powder/Wwheat flour, O/B: Vvegetable oil/Bbutter. The values are the mean ± standard deviation (n n= 30). Values

that do not share letters (a, b, c, and d) in the same column are significantly different (pp < 0.05).

Fig. 2 Images of the cookies obtained for the different formulations (Table 1).

The scores for the taste characteristic, show a significant difference (p p< 0.05) between the control (8.55) and formulaes 1, 2, 4 and 8

with scores of 7.10 to 7.37, and these formulaes have percentages of powdered cladodes of 15, 20 and 25%. Therefore, the acceptance of

Page 17 of 26Page 17 of 26
the flavor, is also influenced by the content of vegetable oil/butter since higher scores generally have a higher butter content or a 50/50

ratio. Finally, for the general acceptance of the cookies, there was no significant difference between the control cookie, with a score of 8.5

and F5 and F9 (15% of powdered cladodes, O/B 30/70 and 50/50) with values of 7.97 and 7.77, respectively. The results obtained show

that in general the best evaluated sensory attributes, after the control cookie scores, are for cookies with an addition of 15% powdered

cladodes and a vegetable oil/butter ratio of 30/70.

3.5. . Moisture sorption isotherms

Sorption isotherms (Fig. 4fig4) were performed recorded for the control cookie and the cookie with the highest score in the general

acceptance of the sensory corresponding to 15% CP and 30% oil/70% butter (F5). The cookies had aAn initial humidity of 1.87% was

observed for control cookies and 3.24% for the cookies with 15% CP. The isotherms showed an increase in equilibrium moisture content as

with increasing aw at the studied temperature of 25 °C. The water adsorbed for each sample corresponds to 0.11 to 0.99 g water per /g dry

solids for control and 0.17 to 1.15 g water per /g dry solids for the selected cookie (F5). The shape of the isotherms was sigmoidal type III,

which corresponds to products rich in sugar.40 The moisture sorption data were adjusted to the GAB mathematical model, and the results

obtained for the model parameter are shown in Table 5tbl5. The value of the monolayer moisture content (XM) indicates the amount of

water that is strongly adsorbed at specific sites on the surface and is considered as the desirable value for food stability. 41,42 In this study, the

XM values were 0.27 and 0.31 gg water per /g dry solids for the control and selected cookie (15% CP), respectively, and the difference may

be due to the soluble dietary fiber content (mucilage) present in the powdered cladodes, which causes greater water sorption.42. The

correlation coefficients RR2 of the isotherms were greater than 0.9, and the error less than 0.01, indicating that the GAB model can be used

to predict the behavior of the experimental data. The water sorption properties of food are of great importance in selecting the right

packaging materials and being able to predict changes in stability and humidity during storage. An increase in moisture content can directly

affect the texture of cookies, which is a key factor in their acceptance by consumers.43.

Page 18 of 26Page 18 of 26
Fig. 3 Effect of the % cladode powder (CP) and % vegetable oil (Ooil) on the sensory attributes. u1

F1, u2 F2, u3 F3, u4 F4, u5 F5, u6 F6, u7 F7, u8 F8, u9 F9, u10 Ccontrol.

Fig. 4 Sorption isotherms for the control cookie and the cookie with 15% Ccladodes Ppowder (CP) at

25 °C, adjusted to the GAB model. The values are the mean ± standard deviation.

Table 5 GAB model parameter values and statistical coefficients for cookie isotherms evaluated at 25

°Ca

<!--Col
Count:3-- Control cookie Cookie 15% CP
>Parameter
XM 0.2723 0.3169
C 0.8148 0.9817
K 0.8155 0.810
2
R 0.9980 0.997
E 0.006 0.002
ERMS 0.0313 0.0161
a
XM : Mmoisture content of the monolayer. C and K are interaction energy constants for the monolayer and the other water molecules. R2:

linearl regression coefficient, E: Eerror, ERMS: Rroot Mmean Ssquare error.

4Conclusion

In summary, the proximal chemical analysis of the powdered cladodes of Opuntia ficus indica var. Atlixco showed a high content of

dietary fiber and phenolic compounds with antioxidant capacity. In the formulation of the cookies, the substitution of wheat flour for

powdered cladodes significantly improved the content of total phenolic compounds and the antioxidant capacity in all cases compared to

cookies made only with wheat flour, being higher for formulations with 25% powdered cladodes. For the sensory evaluation, cookies with

Page 19 of 26Page 19 of 26
15% powdered cladodes and 30% vegetable oil content showed the best taste and overall acceptability score, relative to the control. The

sorption isotherm showed that it was possible to fit the GAB model to the experimental data and the cookies are stable at 25 °C. Based on

the results obtained, powdered cladodes can be considered as a functional ingredient, and a source of dietary fiber and antioxidants for the

manufacture of foods with benefits to human health and nutrition.

Author cContributions

Aparicio-Ortuño, Rocío: conceptualization, methodology, investigation, data curation, writing-original draft preparation, writing-review

and editing, and visualization; Jiménez-González, Oscar: data curation, formal analysis, and methodology; Lozada-Ramírez, J. Daniel:

conceptualization, methodology, formal analysis, draft preparation, writing -review and editing, and supervision; Ortega-Regules, Ana E:

conceptualization, methodology, validation, formal analysis, investigation, resources, writing-original draft preparation, writing-review and

editing, supervision, project administration, and, funding acquisition.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

Aparicio-Ortuño acknowledges to Universidad de las Américas PueblaUniversidad de las Américas Puebla (UDLAP) and Consejo

Nacional de Humanidades, Ciencias y Tecnología (CONAHCyTCONAHCyT) for the scholarship granted to complete her doctoral

degree.

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