Minimally processed yam beam roots fortified with

Original Article

probiotics and phenolic compound from microencapsulated green coffee

Miguel Abud-Archila1

and Claudia Mendoza-Avendaño1*

1 Tecnológico Nacional de México/IT de Tuxtla Gutiérrez, División de Estudios de Posgrado e Investigación. Carretera Panamericana km 1080, Tuxtla Gutiérrez, Chiapas, México. C.P. 29050.

Jícama mínimamente procesada fortificada con probióticos y compuestos fenólicos de café verde microencapsulado



ABSTRACT

Yam beam (Pachyrhizus erosus L.) root, commonly known as jicama, is widely consumed by health-conscious individuals due to its low caloric content, However, its nutritional value is relatively low. To enhance these nutritional properties, jicama can be supplemented with probiotics and antioxidant compounds. In this study, the jicama pieces were coated with an edible layer containing microencapsulated Lactobacillus acidophilus, Bifidobacterium spp. and phenolic compounds derived from green coffee, which were microencapsulated using a double spray drying technique. The probiotics and phenolic compounds were dried using double spray drying with chitosan at 120 and 140 °C. The results showed that the inlet air temperature did not have a statistically significant effect (p ≥ 0.05) on the encapsulation efficiency of probiotics, chlorogenic acid and caffeine content, or antioxidant activity expressed as IC50 value (110 - 116 µg/mL). After 6 d of storage at 4 °C, the jicama supplemented with the microcapsules containing Lactobacillus acidophilus and Bifidobacterium spp. exhibited a reduction in microbial viability by 1 and 2 log CFU/g, respectively. However, the addition of microcapsules allowed a higher concentration of phenolic compounds than the control group. Minimally processed jicama containing microcapsules with probiotics and phenolic compounds could be a functional food, and the reported procedure could be applied for industrial purposes.

Keywords: edible coating; antioxidant compounds; spray- dried.


RESUMEN

La raíz de ñame (Pachyrhizus erosus L.) comúnmente llamada jícama, es consumida por personas preocupadas por su salud ya que tiene bajo contenido calórico y, a pesar de que contie- ne algunas vitaminas, su contenido nutrimental es bajo. Para mejorar estas propiedades nutricionales, la jícama puede ser suplementada con probióticos y compuestos antioxidantes. En este estudio, la jícama fresca en trozos fue recubierta con microcápsulas que contenían Lactobacillus acidophilus, Bifidobacterium spp. y compuestos fenólicos de café verde, los cuales fueron microencapsulados mediante secado por aspersión doble. Los probióticos y compuestos fenólicos se secaron mediante doble secado por aspersión con quitosano a 120 y 140 °C. Los resultados indicaron que la temperatura

*Author for correspondence: Claudia Mendoza-Avendaño e-mail: claudia.ma@tuxtla.tecnm.mx

Received: June 10, 2024

Accepted: September 7, 2024

Published: October 8, 2024

de entrada del aire no afectó estadísticamente (p ≥ 0.05) la eficiencia de encapsulación de los microorganismos, el contenido de ácido clorogénico y cafeína, ni la actividad anti- oxidante expresada como IC50 (110 - 116 µg/mL). Después de 6 d de almacenamiento a 4 °C, en la jícama adicionada con las microcápsulas, la viabilidad de Lactobacillus acidophilus y Bifidobacterium spp. tuvieron una reducción de 1 y 2 log UFC/g, respectivamente. Sin embargo, la concentración de compuestos fenólicos fue superior que en la jícama del grupo control. La jícama que contiene las microcápsulas con probióticos y compuestos fenólicos podría ser un alimento funcional, y el procedimiento desarrollado podría aplicarse con fines industriales.

Palabras clave: recubrimiento comestible; compuestos anti- oxidantes; secado por aspersión


INTRODUCTION

In 2022, around 7100 Ha in México were planted with Pachyrhizus erosus L. (jicama) with an average production of 24 tons/Ha (SIAP, 2022). Jicama is a legume; the edible struc- tural organ of this plant is the root, which is consumed fresh. The root is low in calories (40 cal) and contains vitamins, minerals and starch (Ramírez-Balboa et al., 2023). Although jicama contains most essential amino acids, vitamins and minerals (Duke, 1992), these nutritional values are relatively low. Therefore, jicama is a product that can be supplemented with other bioactive compounds. In this sense, antioxidant compounds and probiotics have been used to fortify other products (Granato et al., 2020).


1

Phenolic compounds exhibit significant biological activ- ities, including antimicrobial, anti-inflammatory (Albuquer- que et al., 2021), and antitumoral (Heleno et al., 2015), among others. Granato et al. (2020) reported that an increased intake of natural phenolic compound antioxidants is associ- ated with a reduced risk of coronary disease. While phenolic compounds are present in many fresh foods, they can also be incorporated into foods during processing. In that sense, coffee is widely recognized as a functional food with antiox- idant properties, primarily due to its phenolic compounds, as noted by Jeszka-Skowron et al. (2016). Although coffee is mostly consumed processed, green coffee has been reported to be a rich source of phenolic compounds, such as chloro- genic acids, hydroxycinnamic acids, caffeine, and caffeic acid


Volume XXVI

DOI: 10.18633/biotecnia.v26.2350

Journal of biological and health sciences http://biotecnia.unison.mx

Universidad de Sonora

ISSN: 1665-1456

(Macheiner et al., 2019), among others. However, these com- pounds are easily oxidized during processing and storage, which reduces their effectiveness. Due to the importance of these compounds for human health, researchers are interest- ed in developing systems that protect bioactive compounds during co-administration (Bednarska and Janiszewska-Turak, 2020).

In the context of improving people’s health, researchers are looking for new strains of probiotics, as these have been shown to provide health benefits to the host, as demonstrat- ed by Ramírez-Pérez et al. (2023) in in vivo studies using Wistar rats. Probiotics are live microorganisms that, when adminis- tered in adequate amounts, promote a benefit in the health of the host (Ramírez-Pérez et al., 2022). Probiotics can help treat gastrointestinal diseases (Fragnant et al., 2023), reduce serum cholesterol and heart disease (Oniszczuk et al., 2021), regulate glycemic indexes (Rezazadeh et al., 2019), control urogenital tract infections (Nader-Macías and Juárez, 2015) and stimulate the immune system (Salami et al., 2019). Two common probiotic microorganisms are the bacteria from the genera Bifidobacterium and Lactobacillus (Ramos-Clamont et al., 2013; Yao et al., 2019). To provide these benefits, probi- otic bacteria must be present with a minimum of 106 to 107 colony-forming units (CFU) per g or mL of food (FAO/WHO, 2006). However, factors such as stress produced during their management, storage and transit through gastrointestinal tract can decrease their viability (López-Fernández et al., 2019; Pupa et al., 2021).

To avoid the loss of probiotic viability and to protect phe- nolic compounds against adverse environmental conditions such as light, moisture, and oxygen, microencapsulation pro- cesses are often employed. Several microencapsulation pro- cesses have been developed for this purpose, which involve trapping the bioactive compound within a coating material (Reque and Brandelli, 2021). Spray drying is the most widely used technique for protecting probiotics (Russo et al., 2022) and phenolic compounds. Because it is difficult for a single encapsulant matrix to have all the required characteristics, it is common to use carbohydrates, proteins and polysac- charides at different ratios (Navarro-Flores et al., 2020) or multilayers of these (Abrahão et al., 2019; Pupa et al., 2021). Chitosan is one of the most promising coating materials used for microencapsulation to improve the stability of phenolic compounds and probiotics (Pupa et al., 2021). Microcapsules obtained by spray drying with chitosan are very stable in storage and demonstrate controlled release characteristics because of their low solubility at neutral pH (Flores-Belmont et al., 2015).

Although the separate microencapsulation of phenolic compounds and probiotic has been reported in several studies, few have evaluated the effect of double microen- capsulation by spray drying on cell viability, phenolic com- pounds content and the properties of the microcapsules. Additionally, only a limited number of papers have explored the used of these microcapsules in the development of functional foods. Given that jicama has limited attractive nutritional characteristics but is consumed for its low caloric

content, this research aimed to determine the effect of inlet air temperature on the properties of microcapsules obtained by single and double spray drying. Moreover, the effect of the addition of probiotics and phenolic compounds from green coffee microencapsulated in coating form on the microbiological, physicochemical, and sensory properties of minimally processed jicama roots was tested.

MATERIAL AND METHODS

Microorganisms and conditions of cultivation Lactobacillus acidophilus and Bifidobacterium spp. (Vivolac, Mexico) were reactivated in Man Rogosa Sharpe (MRS) broth supplemented with 0.05 % (w/v) cysteine, and incubated at 38 °C for 20 h. Subsequently, cells were centrifuged at 4500 rpm for 15 min, at 4 °C. The pellet containing the cells was washed twice with 0.9 % (w/v) saline solution and centri- fuged using the same conditions (Odila et al., 2016).

Preparation of green coffee extract

The green coffee beans were ground and then passed in a sieve 40 (0.420 mm) to produce green coffee powder. To obtain the green coffee extract, the methodology of Budryn et al. (2013) was employed with some modifications. Briefly, the green coffee powder was mixed with distilled water at a 1:5 (w/v) ratio and heated at 90 °C for 1 h. Subsequently, the solution was filtered using filter paper (0.16 mm pore size). Finally, the green coffee extract (GCE) was stored in amber jars at 4 °C until use.

Encapsulation of probiotics and phenolic compounds The double microencapsulation of microorganisms and coffee extract was performed following the methodology proposed by Flores-Belmont et al. (2015), with some modifi- cations. In the first step, an aqueous of gelatin-maltodextrin (1:25) solution, at 26 % (w/w), was prepared in an ascorbic acid solution at 1 % (w/v). The GCE was added to a final concentration of 4.2 mg gallic acid equivalent/mL. Lacto- bacillus acidophilus and Bifidobacterium spp. were added to a final concentration of 109 and 108 CFU/mL, respectively. Subsequently, the mixture was homogenized using an Ultra Turrax T-25 Basic Homogenizer at 4500 rpm for 5 min. The mixture was fed into a spray dryer (BUCHI Mini B-290, Flawil, Switzerland) at a constant flow of 14 mL/min, and two inlet air temperatures, 120 °C and 140 °C, were evaluated, with an outlet air temperature of 50 °C. The microcapsules obtained in the first spray-dried process were hydrated and dried by spray drying in a second step. For this, 10 g of the microcap- sules were added to 100 mL of a 0.5 % (w/v) chitosan solution prepared in 1 % (v/v) acetic acid, and the mixture was sub- jected to the drying process following the same conditions as the first step. Finally, the microcapsules were stored in vacuum-sealed metal bags until use.

Efficiency of probiotic microencapsulation

The microencapsulation efficiency of probiotic microorgan- isms (MEP) was evaluated using one gram of the suspension before drying or one gram of the microcapsules which were

mixed with 9 mL of sterile peptone water (0.1 %, w/v). Viable cell counts were determined in triplicate by plate seeding us- ing MRS agar supplemented with L-cysteine (0.05 % w/v) and incubated at 37 °C (72 h). Previous results showed that the morphology of colonies was different for Lactobacillus aci- dophilus and Bifidobacterium spp. The results were expressed as a log CFU/g sample as suggested by Pupa et al. (2021). The MEP was calculated by equation1:

(1)


where No and N represented the log of the number of via- ble cells (CFU) before and after the encapsulation process, respectively.

Powder properties

After simple and double spray drying, microcapsules were characterized in terms of water solubility index (WSI), water absorption rate (WAR), swelling capacity (SC), morphology, microencapsulation efficiency of phenolic compounds (MYp) and antioxidant activity (AA).

Water solubility index (WSI), water absorption rate (WAR) and swelling capacity (SC)

The WSI was determined according to Paini et al. (2015). One gram of the microcapsules was mixed with 12 mL of distilled water, mixed and incubated at 30 °C for 30 min. The sample was then centrifuged at 3500 rpm for 10 min. The superna- tant was transferred to a capsule and dried at 105 °C until it reached a constant weight. The WSI, WAR, and SC were calculated using equations 2, 3, and 4, respectively:


(2)


(3)


(4)


Microcapsule morphology

The morphology of the microcapsules was examined by scanning electron microscopy (SEM) using a high-resolution, high-vacuum microscope (SM-71480 JEOL, Massachusetts, USA). The microcapsules were attached to the sample holder with double-sided adhesive tape. SEM images were taken at room temperature and examined using an acceleration voltage of 15 kV according Navarro-Flores et al. (2020).


Microencapsulation efficiency of phenolic compounds (MYp)

The microencapsulation efficiency of phenolic compounds (MYp) was calculated by using the total and superficial phenolic compounds in microcapsules, following the methodology described by Navarro-Flores et al. (2020). Briefly, to measure the total phenol content, 200 mg of the microcapsules were mixed with 2 mL of methanol:acetic

acid:water solution (50:8:42 v/v/v). The mixture was shaken for 1 min, sonicated twice in a Cole-Palmer ultrasonic bath model 08855-00 (Cole-Palmer, Vernon Hills, IL, USA) at 25 °C for 20 min, and finally centrifuged at 4000 rpm for 5 min. The supernatant was used for quantifying the total phenolic con- tent. For determination of superficial phenolic compounds content, 200 mg of the microcapsules were mixed with 2 mL of ethanol:methanol solution (1:1), agitated for 1 min, and then centrifuged at 4,000 rpm for 5 min, and the content of phenolic compounds was determined according to Navar- ro-Flores et al. (2020). The content of the total and superficial phenolic compounds was determined with Folin-Ciocalteu reagent, with the method described by Singleton et al. (1999) using gallic acid as the standard. The results were expressed as milligrams of gallic acid equivalents (GAE) per gram of powder. The efficiency of the microencapsulation of phenolic compounds was determined by Eq. 5:


(5)


where PCtotal is the total phenolic compound (mg GAE/g) and PCsup is the superficial phenolic compound (mg GAE/g).

Antioxidant activity (AA)

The AA was determined by measuring the inhibitory effect against the DPPH radical, following the method described by Shekhar and Anju (2014), with some modifications. Briefly, several microcapsules´s solutions (25, 50, 100, 150, and 200 µg/mL) were prepared. Three milliliters of each solution were mixed with 1 mL of DPPH (0.1 mM). After 30 min of incuba- tion, the absorbance of the solution was measured at 517 nm. The AA was calculated using equation 6:


(6)


where Abs control is the absorbance of the control and Abs sample is the absorbance of the sample.

The EC50 value of the sample, which represents con- centration required to inhibit 50 % of the DPPH radical, was calculated using the inhibition curve.

Finally, microcapsules with the best properties, such as low solubility index, higher antioxidant activity, and greater encapsulation efficiency of phenolic compounds and probi- otics, were selected for the next stage of this research. Once selected, the effect of applying these microcapsules to fresh pieces of jicama was studied.

Coating of minimally processed jicama roots

To determine the effect of the addition of the edible coating on the physicochemical and microbiological properties of the minimally processed jicama roots, five types of coatings were evaluated: (1) gelatin-maltodextrin (1:25) 26 % (w/w) aque- ous solution (referred as “C”); (2) GCE at a final concentration of 4.2 mg GAE/mL and Lactobacillus acidophilus and Bifido- bacterium spp. at a final concentration of 109 and 108 CFU/ mL, respectively, added to a gelatin-maltodextrin (1:25) at 26

% (w/w) aqueous solution (referred as “EP”); (3) GCE at a final concentration of 4.2 mg GAE/mL added to gelatin-maltodex- trin (1:25) 26 % (w/w) aqueous solution (referred as “E”); (4) Lactobacillus acidophilus and Bifidobacterium spp. at a final concentration of 109 and 108 CFU/mL, respectively, added to gelatin-maltodextrin (1:25) 26 % (w/w) aqueous solution (referred as “P”), and (5) microcapsules obtained after double spray drying process (referred as “MC”).

The jicama roots were washed, disinfected, and cut into cubes (2 x 2 x 1 cm, 5±0.5 g) using a sterile knife. For coatings C, EP, E, and P, the jicama roots were immersed in the respec- tive mixtures for one minute. For treatment MC, the jicama was coated with a thin layer of powder (approximately 0.3 grams of microcapsules per piece). Previous results indicated that this method produced a uniform layer of the microcap- sules on the surface of each jicama piece. The samples coated were stored in polypropylene containers (4 x 6 x 6 cm) at 4 °C for six days. Microbiological and physicochemical properties (weight loss, pH, color, total phenolic compounds, caffeine and chlorogenic acid) of the samples were analyzed at 0, 1, 3, and 6 days.


Determination of shelf-life characteristics of jicama roots To measure the weight loss, the jicama was weighed on the sampling days. The total weight loss was calculated by equation 7:


(7)


The surface color of the jicama was determined using a portable colorimeter (ColorTec, Clinton, NJ, USA). The values of L* (luminosity), a* (-green a + red), and b* (-blue a + yellow) were recorded, and the chromaticity (C*) was then calculated using equation 8:


(8)


To measure the pH, the samples were blended, and the juices were centrifuged at 4500 rpm for 3 min at 4 °C before measuring pH using a pH meter (Hanna Instruments HI981031, Woonsocket, RI, USA).

Quantification of total phenolic compounds (TPC), caffeine (CF) and chlorogenic acid (CL) in the jicama roots The extracts used for TPC, CF and CL determinations were obtained according to the methodology described by Desai et al. (2019). For that, jicama samples were freeze-dried using a lyophilizer (Labconco FreeZone 4.5 L, Kansas City, USA) at - 40 °C and 0.250 mbar for 48 h. Then, 0.5 g of the lyophilized sample was mixed with 2.5 mL of a methanol:acetic acid:wa- ter solution (50:8:42 v/v/v). The mixture was shaken for 1 min, sonicated twice in a Cole-Palmer ultrasonic bath model 08855-00 (Cole-Palmer, Vernon Hills, IL, USA) at 25 °C for 20 min, and finally centrifuged at 4000 rpm for 5 min. The su-

pernatant was used for quantifying the phenolic compound content according to Joya-Dávila et al. (2023).

Caffeine and chlorogenic acid were quantified by high-performance liquid chromatography (HPLC) with a Kromasil 100-5-C18 column (4.6 x 150 mm, 5 µm, 100 A- Supelco, Bellefonte, CA, USA), using a diode array detector (PerkinElmer Series 200 HPLC Systems, Shelton, CT, USA). The samples were filtered with a 0.22 µm millipore membrane. The mobile phase was acetonitrile/formic acid at 0.1 % (80:20, v:v) (Phase A) and formic acid at 1 % (v/v) (Phase B) at a 10:90 ratio with a constant flow of 1 mL per min in isocratic mode. Quantification was performed at 280 nm for CF and 320 nm for CL, and 10 µL of the sample were injected into the HPLC. In addition, standard solutions of the analytes to be quantified (50, 100, 200, 300, 400, 500, and 700 mg/L) were prepared for elution times and respective calibration curves. Metabolites were expressed in milligram GAE per gram of jicama root in dry basis.


Experimental design and statistical analysis

A completely randomized experimental design with three replicates was employed for two evaluations. The results were analyzed using an analysis of variance (ANOVA) to determine significant differences between treatments (p

≤ 0.05). Honestly-significant-difference (HSD) or Tukey test were used for mean comparisons. Statistical analyses were carried out using Statgraphics Centurion XVI software.


RESULTS AND DISCUSSION

Survival of probiotics during the spray drying process The results indicated that the encapsulation efficiency of Lactobacillus acidophilus and Bifidobacterium spp. was not significantly affected by the inlet air temperature, regardless of whether single or double spray drying was used. After the single drying, cell viability ranged from 9.39 to 9.12 log CFU/g for Lactobacillus acidophilus and 7.91 to 7.85 log CFU/g for Bifidobacterium spp. The encapsulation efficiency of both microorganisms at 120 or 140 °C after the single drying was around 90 % (Table 1). However, the encapsulation efficiency for both microorganisms after the double drying process was around 73- 77 %. Despite this reduction, cell viability remained at 7.2 log CFU/g for Lactobacillus acidophilus and

6.2 log CFU/g for Bifidobacterium spp. Despite the decrease in

cell viability, the use of chitosan as a coating material allowed obtaining powders with a probiotic content higher than 106 CFU/g of microcapsules. Similar results were reported by Pupa et al. (2021) and Flores-Belmont et al. (2015), who encapsulated different species of lactic acid bacteria with chitosan through a double spray drying process, with encap- sulation efficiencies of approximately 70 %.


Microcapsules properties

Micrographs, water solubility index (WSI), water absorp- tion rate (WAR) and swelling capacity (SC) of microcap- sules after single and double spray drying

Micrographs show that the microcapsules had a spherical

Table 1. Inlet air temperature effect on microencapsulation efficiencies of Lactobacillus acidophilus and Bifidobacterium spp. after the spray drying process.

Tabla 1. Efecto de la temperatura del aire de entrada sobre la eficiencia

de microencapsulación de Lactobacillus acidophilus y Bifidobacterium spp. después del proceso de secado por aspersión.

Spray drying

Inlet air

temperature (°C)

Lactobacillus

acidophilus (%)

Bifidobacterium

spp. (%)


120

92.13±1.28 a*

92.55±1.34 a

Single

140

92.28±1.49 a

91.31±2.83 a


120

76.24±0.54 b

77.18±0.11 b

Double

140

73.30±1.07 b

73.48±2.23 b

HSD

4.68

7.81

* Means followed by different lowercase letters in a column are significantly different according to the Tukey HSD test (p ≤ 0.05).

Medias seguidas con diferentes letras minúsculas en una columna son significativamente diferentes de acuerdo a la prueba de Tukey (p ≤ 0.05).

shape with dents and free of cracks on the surface, and an approximate average diameter of 15 µm (Figure 1). For mi- crocapsules obtained by single and double spray drying at 120 and 140 °C, the size did not differ. The solubility of the mi- crocapsules obtained by single spray drying ranged from 88 to 89 % (Table 2). These results are similar to those reported by Navarro-Flores et al. (2020), who encapsulated phenolic compounds using maltodextrin and other unconventional agents. These high solubility index could be attributed to the high solubility of the encapsulant agents (Fazaeli et al., 2012). Additionally, gelatin and maltodextrin contain hydrophilic sections, so they could interact and create a more soluble particle in aqueous environments (Semenova et al., 2002). Moreover, the water solubility index decreased for the micro- capsules obtained by double microencapsulation through spray drying with chitosan (Table 2), compared with micro- capsules obtained by single spray drying. This reduction can be attributed to the low solubility of chitosan at pH values above 6.5 (Aranaz et al., 2021). These results are similar to those reported by Flores-Belmont et al. (2015), who indicated that double microencapsulation with chitosan resulted in less insoluble powders in water (pH 7).

Water absorption rate values of microcapsules ranged from 0.11 to 0.34 g/g (Table 2). These values are similar to those reported by other authors (Da Costa et al., 2018; Na- varro-Flores et al., 2020). It has been reported that variations in WAR may be due to the different degrees of participation of hydroxyl groups present in encapsulant agents in the formation of hydrogen bonds with water (Ahmed et al., 2010; Da Costa et al., 2018). The WAR of microcapsules obtained by single spray drying were lower than those by double spray drying with chitosan. This reduction can be attributed to the low solubility of chitosan at pH values above 6.5 as men- tioned previously. At basic pH, the amino groups of chitosan are in their deprotonated form (-NH2), which reduces their ability to interact with water and decreases the polymer´s solubility (Aranaz et al., 2021). SC values ranged from 0.037 to

0.092 g/g (Table 2). The SC values decreased significantly (p ≤ 0.05) for the microcapsules obtained by double spray drying


Figure 1. SEM micrographs of spray-dried powder particle; simple spray- dried process (a) and the double spray-dried process (b).

Figura 1. Micrografías SEM de partículas de polvo secado por aspersión; se-

cado por aspersión simple (a) y secado por aspersión doble (b).

compared with microcapsules obtained by single spray dry- ing, probably due to the presence of chitosan. Ahmed et al. (2010) reported that a low swelling capacity is related to the greater stability of microcapsules, which reduces their ability to swell.


Encapsulation efficiency of phenolic compounds and antioxidant activity of microcapsules after single and double spray drying

The inlet air temperature and the double encapsulation process with chitosan did not have a statistically significant effect (p ≥ 0.05) on the encapsulation efficiency of phenolic compounds, with percentages ranges of 91.60 - 93.16 % (Table 3). The total phenol content in microcapsules ranged from 8.06 to 11.89 mg GAE/g of powder (Table 3). These values are similar to those reported by Desai et al. (2019) for green coffee extract encapsulated with maltodextrin, with a TPC of 11.98 mg GAE/g of powder.

The results indicated that increasing the inlet air tem- perature to 140 °C or the double spray drying process, the total phenol concentration of the microcapsules decreased significantly (p ≤ 0.05). Despite the decrease in phenolic com- pounds, the antioxidant activity, measured as IC50, remained unchanged. During spray drying, some phenolic compounds may degrade; however, new derivative compounds that are highly effective at inhibiting free radicals can be formed, as noted by Abrahão et al. (2019). Additionally, during thermal

Table 2. Water solubility index, water absorption, swelling capacity, phenolic compounds, chlorogenic acid content, caffeine content, IC50, and the microen- capsulation efficiency of phenolic compounds of microcapsules by simple and double spray drying.

Tabla 2. Índice de solubilidad en agua, absorción de agua, capacidad de hinchamiento, compuestos fenólicos, contenido de ácido clorogénico, contenido de

cafeína, IC50 y eficiencia de microencapsulación de compuestos fenólicos de microcápsulas mediante secado por aspersión simple o doble.

Water solubility Water Swelling Phenolic Chlorogenic Microencapsulation

Treatment index absorption rate capacity compound acid Caffeine efficiency of phenolic IC50

(%) (g/g) (g/g) (mg EAG/g) (mg/g) (mg/g) compound (%) (µg/mL)

Single 120 °C

88.53±1.47 a*

0.11±0.04

b

0.078±0.011

a

11.89±0.01 a

4.57±0.02

a

2.74±0.38

a

92.14±0.11

a

110.21±2.61 a

Single 140 °C

89.93±1.95 a

0.12±0.02

b

0.092±0.022

a

8.93±0.66 b

4.43±0.22

a

2.70±0.04

a

93.16±0.60

a

114.43±1.65 a

Double 120 °C

78.90±0.76 b

0.34±0.05

a

0.037±0.002

b

9.62±0.12 b

4.21±0.31

a

2.47±0.05

a

91.60±0.40

a

111.42±3.37 a

Double 140 °C

78.39±0.15 b

0.30±0.08

a

0.037±0.002

b

8.06±0.44 b

4.28±0.01

a

2.22±0.12

a

92.19±0.23

a

116.28±0.97 a

HSD

3.34

0.14


0.03


1.64

0.77


0.83


1.56


9.51

*Means followed by different lowercase letters in the same column are significantly different according to the Tukey HSD test (p ≤ 0.05).

Medias seguidas con diferentes letras minúsculas en la misma columna son significativamente diferentes de acuerdo a la prueba de Tukey (p ≤ 0.05).

Table 3. Total phenolic compounds content in jicama roots during storage at 4 °C for six days.

Tabla 3. Contenido total de compuestos fenólicos en jícama durante el almacenamiento a 4 °C durante seis días.

Phenolic compounds (mg GAE/g of jicama roots in dry base)

Time (days)

Treatment                                                                                                                                      HSD

0 1 3 6

C

0.99±0.01 cA*

0.99±0.01 cA

0.96±0.01 cA

0.99d±0.02 cA

0.06

EP

1.50±0.02 bA

1.50±0.04 bA

1.50±0.01 bA

1.49.44±0.01 bA

0.10

E

1.50±02 bA

1.50±0.03 bA

1.50±0.03 bA

1.49±0.01 bA

0.07

P

0.97±0.01 cA

0.98±0.01 cA

0.98±0.1 cA

0.98±0.01 cA

0.03

MC

4.31±0.12 aA

3.93±0.02 aAB

3.38±0.21 aBC

3.00±0.12 aC

0.57

HSD

0.22

0.09

0.40

0.22


C (Control), EP (green coffee extract/probiotics), E (green coffee extract), P (probiotics), MC (microcapsules with chitosan obtained by double spray drying). *Means followed by different lowercase letters in the same column are significantly different according to the Tukey HSD test (p ≤ 0.05). Means followed by different uppercase letters in the same row are significantly different according to the Tukey HSD test (p ≤ 0.05).

C (Control), EP (extracto de café verde/probiótico), E (extracto de café verde), P (probióticos), M (microcápsulas con quitosano obtenidas mediante doble secado por aspersión). * Medias seguidas con diferentes letras minúsculas en la misma columna son significativamente diferentes de acuerdo a la prueba de Tukey (p ≤ 0.05). Medias seguidas con diferentes letras mayúsculas en la misma linea son significativamente diferentes de acuerdo a la prueba de Tukey (p≤ 0.05).


process, the Maillard reaction can occur, producing complex- es with varying degrees of antioxidant activity (Liang et al., 2016).

The results also showed that around 110 - 116 µg/ mL of microcapsules was needed to inhibit 50 % of DPPH radical (IC50), and the double encapsulation process did not significantly affect this IC50 value. In addition, neither the inlet air temperature and double encapsulation caused sig- nificant differences in the concentrations of chlorogenic acid and caffeine. This suggests that the double encapsulation process using chitosan effectively preserves both phenolic compounds and their antioxidant activity.

Based on these results, only the microcapsules obtained by double spray drying with chitosan at an inlet air tempera- ture of 120 °C were used for the subsequent jicama coating experiments.

Coating of minimally processed jicama roots Determination of shelf-life characteristics

Weight loss results (Figure 2a) showed that jicama coated with microcapsules obtained by double encapsulation with chitosan (MC) showed the highest weight loss at the end of a 6 days of storage. Moreover, EP, E, and P treatments provided a significant additional protection (p ≥ 0.05) against weight loss compared to treatment MC. Wong et al. (2021) reported that weight loss in food during storage is mainly due to water migration from plant tissues to the outdoor environment through transpiration. In addition, the moisture difference between the jicama and the environment was probably the driving force for weight loss.

The pH of the jicama decreased significantly during storage (p ≤ 0.05), with the lowest pH values observed in treatments containing probiotic microorganisms (MC, EP and P) compared to the treatments without probiotics


Figure 2. Effect of different types of coatings on weight loss (a), pH (b), luminosity (c) and chromaticity (d) of jicama during storage at 4 °C for six days. C (Control); EP (green coffee extract/probiotics); E (green coffee extract); P (probiotics); MC (mi- crocapsules with chitosan obtained by double spray drying).

Figura 2. Efecto de diferentes tipos de recubrimientos sobre la pérdida de peso (a), pH (b), luminosidad (c) y cromaticidad

(d) de jícama mínimamente procesada durante el almacenamiento a 4 °C durante seis días. C (Control); EP (extracto de café verde/probióticos); E (extracto de café verde); P (probióticos); MC (microcápsulas con quitosano obtenidas por secado por aspersión doble).

(Figure 2a). This could be attributed to the fact that during storage of jicama, even under refrigeration, probiotics could use the nutrients present in the coating and/or jicama piec- es, producing organic acids, such as lactic acid, which could cause a decrease in pH. Wong et al. (2021) reported similar pH decreases in fresh-cut apple slices coated with Lactoba- cillus plantarum. The decrease in pH during the storage of minimally processed foods can be attributed to the activity of endogenous enzymes, which can produce acids and con- tribute to pH reduction. In treatments containing probiotics, the pH decrease can be attributed to the microbiological activity of the added microorganisms (Varoquaux and Wiley, 2017). Among the treatments with probiotics, the most pronounce pH reduction in jicama was observed in those with free microorganisms (treatments EP and P) (Figure 2a). This pH reduction could suggest a likely decrease in product acceptance. Otherwise, the pH of the jicama coated with the microcapsules (treatment MC) remained almost constant during storage. This could be attributed to the fact that spray drying decreased the cell metabolic activity of the bacterial cells (Behboudi-Jobbehdar et al., 2013).

Color is another important attribute of minimally pro- cessed foods, as affects the appearance and consumer’s ac- ceptance of the product. On the cutting surface, cell rupture

can occur, allowing substrates and oxidizers to come into contact. Therefore, one of the main objectives during the minimum processing of fruit and vegetables is to preserve the original color. Changes in the color of the samples were expressed through chromaticity (value C*) and luminosity (L*) with respect to time (Figure 2 c). In general, the results indicated that at the beginning of storage, the samples that had the coating with the unencapsulated green coffee ex- tract (E and EP) were significantly opaquer than the control, whereas samples coated with powder obtained from double spray drying showed L* values similar to the control treat- ment. This behavior could be attributed to the color masking effect of the encapsulant agents on the color of the green coffee extract (Piedrahíta et al., 2018). After 6 days of storage, however, there was no significant statistical difference (p ≥ 0.05) in the luminosity of the samples. Color changes in the samples were expressed through chromaticity (C* value) over time; after 6 days of storage, there were no significant changes in the color of the samples.


Viability of probiotic microorganisms in jicama

The viability of Lactobacillus acidophilus and Bifidobacterium spp. in different types of coatings for minimally processed of jicama´s pieces during storage at 4 °C are shown in Figures

3a and 3b, respectively. As can be seen, the number of Lacto- bacillus acidophilus and Bifidobacterium spp. cells remained constant during the 6 days of storage in EP and P treatments. This could have occurred because microorganisms remain metabolically active, probably by using impregnated carbo- hydrates and/or nutrients from the jicama as a carbon source (Wong et al., 2021). For the MC treatment (jicama coated with microcapsules) after 3 days of storage, the viability of microorganisms decreased significantly (p ≤ 0.05) (Figures 3a and 3b), probably because double spray drying with chitosan caused damage to the cell membrane of the probiotics. Therefore, when the microcapsules were applied to the jica- ma, they were hydrated, and the probiotics were reactivated. However, the microorganisms, having been damaged in the double drying, began their death phase.


Total phenol, caffeine, and chlorogenic acid content during the storage of minimally processed jicama roots The results of the total phenol content (mg/g of jicama roots in dry basis) during storage are presented in Table 3. The con-


Figure 3. Effect of different types of coatings on cell viability of Lactobaci- llus acidophilus (a) and Bifidobacterium spp. (b) during the storage of jicama roots at 4 °C for six days. EP (green coffee extract/probiotics); P (probiotics); MC (microencapsulated with chitosan obtained by double spray drying). The other treatments are omitted because they were not inoculated.

Figura 3. Efecto de diferentes tipos de recubrimientos sobre la viabilidad

celular de Lactobacillus acidophilus (a) y Bifidobacterium spp. (b) durante el almacenamiento de jícama a 4 °C durante seis días. EP (extracto de café ver- de/probióticos); P (probióticos); MC (microencapsulado con quitosano obte- nido por secado por doble pulverización). Los demás tratamientos se omiten por no ser inoculados.

trol treatment had a total phenol concentration of 0.99 mg GAE/g. Treatments E, EP, and MC exhibit higher total phenol content than the control (C). These results could be attributed to the green coffee extract present in these treatments, which were reported as an excellent source of phenolic compounds (Desai et al., 2019). The main phenolic compounds present in the green coffee extract are caffeine and chlorogenic acid, so these metabolites were quantified (Supplementary Fig. S1). Treatments C and P did not show detectable levels of caffeine or chlorogenic acid. In contrast, treatments E and EP resulted in caffeine and chlorogenic acid content to remain in the range of 0.43 - 0.38 mg/g and 0.42- 0.41 mg/g, respectively, throughout storage.

In the MC treatment, the content of caffeine (0.72 - 0.48 mg/g) and chlorogenic acid (1.11- 0.55 mg/g) were signifi- cantly higher (p ≤ 0.05) during the entire storage compared with the other treatments. During storage, the concentration of total phenols, caffeine, and chlorogenic acid decreased




Figure S1. Effect of different types of coating on the caffeine (a) and chlo- rogenic acid (b) content in jicama roots during storage at 4 °C for six days. E: green coffee extract; EP: green coffee extract/probiotics; MC: Microencapsu- lated with chitosan obtained by double spray drying.

Figura S1. Efecto de diferentes tipos de recubrimiento sobre el contenido de cafeína (a) y ácido clorogénico (b) de raíces de jícama durante el alma- cenamiento a 4 °C durante seis días. E: extracto de café verde; EP: extracto de café verde/probióticos; MC: Microencapsulado con quitosano obtenido mediante secado por aspersión doble.

significantly (p ≤ 0.05) in the samples of the MC treatment (jicama coated with the microcapsules obtained by double spray drying), compared with the other treatments. It has been reported that incorporating probiotics into vegetables matrices using the immersion technique allows microor- ganisms to enter the interior of the food through capillarity, promoting their adherence and protecting them from exter- nal conditions (De Oliveira et al., 2017). Additionally, refriger- ation helps maintain the stability of probiotics, as reported by Wong et al. (2021). However, the MC treatment presents a higher content of these phenolic compounds (caffeine and chlorogenic acid) throughout the complete jicama storage. The reduction of these compounds concentration could be originated for the microcapsule’s hydration and their partial hydration during the storage of the food, which causes the re- lease of phenolic compounds into the outside environment. Franҫa et al. (2018) reported that in chitosan microcapsules the active compound is trapped in the nucleus and covered by a chitosan layer, which, depending on storage conditions, can swell and then release the nutrient.

CONCLUSIONS

In this study, microcapsules containing probiotics and phe- nolic compounds were obtained through single and double spray drying at 120 and 140 °C. Double spray drying with chitosan allowed microcapsules with a microbial popula- tion higher than 106 CFU/g and 9.62 mg GAE/g of phenolic compounds. Moreover, these microcapsules were used as an edible coating containing probiotics and phenolic com- pounds from green coffee, applied to minimally processed jicama root. Jicama pieces with encapsulated probiotics and phenolic compounds can be considered functional food due to their enhanced nutritional quality. To the best of the au- thors’ knowledge, this is the first investigation incorporating a chitosan coating, obtained through a double spray drying process, into a minimally processed food. The microcapsules developed with the encapsulated probiotics and phenolic compounds allowed preserved the viability of probiotics and the concentration of phenolic compounds, such as caffeine and chlorogenic acid, in jicama during storage. This study reports the formulation and production of a functional food with high nutritional value and that can be used as a healthy snack. However, further research is recommended to extend the shelf life of coated jicama to enhance its appeal to indus- trial manufacturers.

ACKNOWLEDGMENTS

Mendoza-Avendaño thanks the Consejo Nacional de Hu- manidades, Ciencia y Tecnología (CONAHCyT, Mexico) for the postdoctoral scholarship (2731630). The authors thank Edith Ponce-Recinos (Universidad Politécnica de Chiapas, México) for support with the SEM technique.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

REFERENCES

Abrahão, F.R., Rocha, L.C.R, Santos, T.A., Carmo, E.L., Pereira,

L.A.S., Borges, S.V., Alvarenga, R.G.F.P. and Botrel, D.A. 2018. Microencapsulation of bioactive compounds from espresso spent coffee by spray drying. LWT-Food Science and Technology. 103: 116-124. Doi: 10.10.1016/j.lwt.2018.12.061

Albuquerque, B., Heleno, S.A., Oliveira, B.P.P., Barros, L. and Ferreira, C.F.R. 2021. Phenolic compounds: current industrial applications, limitations and future challenges. Food & Function. 12: 14-29. Doi:10.1039/d0fo02324h

Ahmed, M., Akter, M.S., Lee, J.C. and Eun, J.B. 2010. Encapsulation by spray drying of bioactive components, physicochemical and morphological properties from purple sweet potato. LWT-Food Science and Technology. 43(9): 1307-1312. Doi: 10.1016/j.lwt.2010.05.014

Aranaz, I., Alacántara, A.R., Civera, M.C., Arias, C. Elorza, B., Caballero, A.H. and Acosta, N. 2021. Chitosan: An overview of its properties and applications. Polymers. 13(19): 1-27. Doi: 3390/polym13193256

Bednarska, M.A. and Janiszewska-Turak, E. 2020. The influence of spray drying parameters and carrier material on the physico-chemical properties and quality of chokeberry juice powder. Journal of Food Science and Technology. 57(2): 564-

577. Doi: 10.1007/s13197-019-04088-8

Behboudi-Jobbehdar, S., Soukoulis, C., Yonekura, L. and Fisk, I. 2013. Optimization of spray-drying process conditions for the production of maximally viable microencapsulated Lactobacillus acidophilus NCIMB 701748. Drying Technology. 31(11): 1274-1283. Doi: 10. 1080/07373937.2013.788509

Budryn, G., Żyżelewicz, D., Nebesny, E., Oracz, J. and Krysiak, W. 2013. Influence of addition of green tea and green coffee extracts on the properties of fine yeast pastry fried products. Food Research International. 50(1): 149-160. Doi: 10.1016/j. foodres.2012.10.006

Da Costa, R.S., Texteira, B.C., Gabbay, A.T.V., Ribeiro-Costa, R.M., Casazza, A.A., Aliakbarian, B., Converti, A., Silva, J.J.O.C. and Perego, P. 2018. Optimization of spray drying conditions to microencapsulate cupuassu (Theobroma grandiflorum) seed by-product extract. Natural Product Research. 33(18): 1-9. Doi: 10.1080/14786419.2018.1462178

De Oliveira, P.M., Ramos, A.M., Martins, E.M.F., Vieira, É.N.R., Soares, A.S. and de Noronha, M.C. 2017. Comparison of vacuum impregnation and soaking techniques for addition of the probiotic Lactobacillus acidophilus to minimally processed melon. International Journal of Food Science & Technology. 52(12): 2547-2554. Doi: 10.1111/ijfs.13540

Desai, M.N., Haware, D.J., Basavaraj, K. and Murthy, P.S. 2019. Microencapsulation of antioxidant phenolic compounds from green coffee. Preparative Biochemistry Biotechnology. 49(4): 400-406. Doi: 10.1080/10826068.2019.1575858

Duke, J.A. 1992. Dr. Duke´s phytochemical and ethnobotanical databases. Green Farmacy Garden 8210 Murphy Rood. Fulton, MD 20759, USA. Dataset.

FAO/WHO. 2006. Probiotic in food. Health and nutritional properties and guidelines for evaluation. Rome, Italy. FAO: Food and Nutrition. 85.

Fazaeli, M., Emam-Djomeh, Z., Ashtari, A.K. and Omid, M. 2012. Effect of spray drying conditions and feed composition on the physical properties of black mulberry juice powder. Food and Bioproducts Processing. 90(4): 667-675. Doi: 10.1016/j. fbp.2012.04.006

Franҫa, D., Medina, F.A., Messa, L.L., Souza, C.F. and Faez, R. 2018. Chitosan spray-dried microcapsule and microcapsule and microsphere as fertilizer host for swellable-controlled release materials. Carbohydrate Polymers. 196(15): 47-55. Doi: 10.1016/j.carbpol.2018.05.014

Fragnant, H.S., Isidean, S.D., Wilson, L., Bukhari, A.S., Allen, J.T.,

Agans, R.T., Lee, D.M., Hatch-McChesney, A., Whitney, C.C., Sullo, E., Porter, C.K. and Karl, P.K. 2023. Orally ingested probiotic, prebiotic, and symbiotic interventions as countermeasures for gastrointestinal tract infections in nonelderly adults: A systematic review and meta-analysis. Advances in Nutrition. 14(3): 539-554. Doi: 10.1016/j. advnut.2023.02.002

Flores-Belmont, I.A., Palou, E., López-Malo, A. and Jiménez- Munguía, T. 2015. Simple and double microencapsulation of Lactobacillus acidophilus with chitosan using spray drying. International Journal of Food Studies. 4: 188-200. Doi: 10.7455/ijfs/4.2.2015.a7

Granato, D., Barba, F.J., Kovačević, D.B., Lorenzo, J.M., Cruz, A.G. and Putnik, P. 2020. Functional foods: product development, technological trends, efficacy testing, and safety. Annual Review of Food Science and Technology. 11: 93-118. Doi: 10.1146/annurev-food-032519-051708

Heleno, S.A, Martins, A., Queiroz, M.J.R.P. and Ferreira, I.C.F.R. 2015. Bioactivity of phenolic acids: metabolites versus parent compounds: a review. Food Chemistry. 173: 501-513. Doi: 10. 1016/j.foodchem.2014.10.057

Jeszka-Skowron, M., Sentkowska, A., Pyrzynska, K. and Paz De Peña, M. 2016. Chlorogenic acids, caffeine content and antioxidant properties of green coffee extracts: influence of green coffee bean preparation. European Food Research Technology. 242: 1403-1409. Doi: 10.1007/s00217-016- 2643-y

Joya-Dávila, J.G., Gutiérrez-Miceli, F.A., Luján-Hidalgo, M.C., Serrano-Gómez, L. and Ruíz-Sesma, B. 2023. Cambios bioquímicos y morfométricos en Coffea arabica posterior a un tratamiento con metanosulfonato de etilo. Biotecnia. 25(3): 36-41. Doi: 10.18633/biotecnia.v25i3.1969

Liang, N., Xue, W., Kennepohl, P. and Kitts, D.D. 2016. Interactions between major chlorogenic acid isomers and chemical changes in coffee brew that affect antioxidant activities. Food Chemistry. 213: 251-259. Doi: 10.1016/j. foodchem.2016.06.041

López-Fernández, S.G., Fernández, M.S.G., Armenta, C.R.I., García-Galaz, A., Moreno-Rivas, S.C., Vázquez-Moreno, L. and Ramos-Clamont, M.G. 2019. Caracterización del co- encapsulamiento de Lactobacillus plantarum y ácidos grasos omega-3 en una matriz de alginato-pectina. Biotecnia. 21(2): 38-46. Doi: 10.18633/biotecnia.v21i2.904

Macheiner, L., Schmidt, A., Scheriner, M. and Mayer, H.K. 2019. Green coffee infusion as a source of caffeine and chlorogenic acid. Journal of Food Composition and Analysis. 84: 1-8. Doi: 10.1016/j.jfca.2019.103307

Nader-Macías, M.E.F. and Juárez, T.M.S. 2015. Profiles and technological requirements of urogenital probiotics. Advanced Drug Delivery Reviews. 92(15): 84-104. Doi: 10.1016/j.addr.2015.03.016

Navarro-Flores, M.J., Ventura-Canseco, L.M.C., Meza-Gordillo, R., Ayora-Talavera, T.R. and Abud-Archila, M. 2020. Spray drying encapsulation of native plant extract rich in phenolic compounds with combinations of maltodextrin and non- conventional wall. Journal of Food Science and Technology. 57: 4111-4122. Doi: 10.1007/s13197-020-04447-w

Odila, P.J., Soares, J., Sousa, S., Madureira, A.R., Gomes, A. and Pintado, M. 2016. Edible films as carrier for lactic acid bacteria. LWT-Food Science and Technology. 73: 543-550. Doi: 10. 1016/j.lwt.2016.06.060

Oniszczuk, A., Oniszczuk, T., Gancarz, M. and Szymańska, J. 2021. Role of gut microbiota, probiotics and prebiotics in the cardiovascular diseases. Molecules. 26(4): 1-15. Doi: 10.3390/ molecules26041172

Paini, M., Aliakbarian, B., Casazza, A.A., Lagazzo, A., Botter, R. and Perego, P. 2015. Microencapsulation of phenolic compounds from olive pomace using spray drying: a study of operative parameters. LWT-Food Science and Technology. 62(1): 177-

186. Doi: 10.1016/j.lwt.2015.01.022

Piedrahíta, M.D.G., Fuenmayot, C.A. and Suarez, M.H. 2018. Effect of chitosan-propolis edible coatings in stability of refrigerated cachama (Piaractus brachypomus) vacuum- packed fish fillets. Packaging Technology and Science. 32(3): 1-11. Doi: 10.1002/pts.2422

Pupa, P., Apiwatsiri, P., Sirichokchatchawan, W., Pirarat, N., Muangsin, N., Ali, S.A. and Prapasarakul, N. 2021. The efficacy of three double microencapsulation methods for preservation of probiotic bacteria. Scientific Reports. 11(13753): 1-9. Doi: 10.1038/s41598-021-93263-z

Ramírez-Balboa, G., Balois-Morales, R., León-Fernández, A.E., Bautista-Rosales, P.U., Jiménez-Zurita, J.O. and Montalvo- González, E. 2023. Physicochemical and proximal characterization of starch and flour of jicama (Pachyrhizus erosus L.). Revista Bio Ciencias, 10 (e1427): 1-17. Doi: 10. 15741/revbio.10.e1427

Ramírez-Pérez, J.I., Álvarez Gutiérrez, P.E., Luján Hidalgo, M.C., Ovando Chacón, S.L., Soria Guerra, R.E., Ruiz Cabrera, M.A., Grajales Lagunes, A. and Abud Archila, M. 2022. Effect of linear and branched fructans on growth and probiotic characteristics of seven Lactobacillus spp. isolated from an autochthonous beverage from Chiapas, Mexico. Archives of Microbiology. 204 (7) 364. Doi: 10.1007/s00203-022- 02984-w

Ramírez-Pérez, J.I., Abud-Archila, M., Ovando-Chacón, S.L., Soria- Guerra, R.E., Ruiz-Cabrera, M.A., Godínez-Hernández, C.I., and Grajales-Lagunes, A. 2023. Effect of coadministration of Lactiplantibacillus fabifermentans and linear/branched fructans mixtures on the intestinal health of Wistar rats. International Journal of Biological Macromolecules. 247, 125748. Doi: 10.1016/j.ijbiomac.2023.125748

Ramos-Clamont, G., Hernández-González, L.E., Fernández- Michel, S.G., Froto-Madariaga, L. and Vázquez-Moreno,

L. 2013. Estrategias para mejorar la sobrevivencia de probióticos en helados. Biotecnia. 15(2): 31-38. Doi: 10.18633/bt.v15i2.147

Reque, P.M. and Brandelli, A. 2021. Encapsulation of probiotics and nutraceuticals: Applications in functional food industry. Trends in Food Science & Technology. 114: 1-10. Doi: 10. 1016/j.tifs.2021.05.022

Rezazadeh, L., Gargari, B.P., Jafarabadi, M.A. and Alipour, B. 2019. Effects of probiotic yogurt on glycemic indexes and endothelial dysfunction markers in patients with metabolic syndrome. Nutrition. 62: 162-168. Doi: 10.016/j. nut.2018.12.011

Russo, M.I., Abeijón-Mukdsi, M.C., Santacruz, A., Ross, R., López, M.A., Gauffin-Cano, P. and Medina, R.B. 2022. Spray dried lactobacilli maintain viability and ferulotyl esterease activity

during prolonged storage and under gastrointestinal tract conditions. Journal of Food Science and Technology. 59: 1202-1210. Doi: 10.1007/s13197-021-05125-1

Salami, M., Kouchaki, E., Asemi, Z. and Tamtaji, O.R. 2019. How probiotic bacteria influence the motor and mental behaviors as well as immunological and oxidative biomarkers in multiple sclerosis? A double-blind clinical trial. Journal of Functional Foods, 52: 8-13. Doi: 10.1016/j.jff.2018.10.023

Semenova, M.G., Antipova, A.S. and Belyakova, L.E. 2002. Food protein interactions in sugar solutions. Current Opinion in Colloid & Interface Science. 7(5-6): 438-444. Doi: 10.1016/ S1359-0294(02)00079-1

Servicio de Información Agroalimentaria y Pesquera (SIAP). 2022. Estadística de Producción Agrícola. Datos Abiertos (siap.gob.mx).

Shekhar, T.C. and Anju, G. 2014. Antioxidant activity by DPPH radical scavenging method of Ageratum conyzoides Linn. Leaves. American Journal of Ethnomedicine. 1(4): 244-249.

Singleton, V.L., Orthofer, R., Lamuela-Raventós, R.M. 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods in Enzymology. 299: 152-178. Doi: 10.1016/S0076- 6879(99)99017-1

Varoquaux, P. and Wiley, R.C. 2017. Biological and biochemical changes in minimally processed refrigerated fruits and vegetables. In Minimally Processed Refrigerated Fruits and Vegetables. F. Yildiz and R. Wiley (ed.), pp 153-186. Springer. Boston. Doi: 10.1007/978-1-4939-7018-6_5

Wong, C.H., Mak, I.E.K. and Li, D. 2021. Bilayer edible coating with stabilized Lactobacillus plantarum 299v improved the shelf life and safety quality of fresh-cut apple slices. Food Package Shelf Life. 30: 1-6. Doi: 10.1016/j.fpsl.2021.100746

Yao, M., Xie, J., Du, H., McClements, D.J., Xiao, H. and Li, L. 2019. Progress in microencapsulation of probiotics: A review. Comprehensive Reviews in Food Science and Food Safety. 19(2): 857-874. Doi: 10.1111/1541-4337.12532