The viability of probiotic Lactobacillus paracasei IMPC2.1 coating on apple slices during dehydration and simulated gastro-intestinal digestion

Food Bioscience 34 (2020) 100533

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The viability of probiotic Lactobacillus paracasei IMPC2.1 coating on apple slices during dehydration and simulated gastro-intestinal digestion

T

Francesca Valerioa, Maria Grazia Volpeb, Gabriella Santagatac, Floriana Boscainob, Costantina Barbarisib, Mariaelena Di Biasea, Anna Rita Bavaroa, Stella Lisa Lonigroa, Paola Lavermicoccaa,∗ a

Institute of Sciences of Food Production, National Council of Research of Italy, 70126, Bari, Italy Institute of Food Science, National Council of Research of Italy, 83100, Avellino, Italy c Institute for Polymers, Composites and Biomaterials, National Council of Research of Italy, 80078, Pozzuoli, Naples, Italy b

A R T I C LE I N FO

A B S T R A C T

Keywords: Dried apple Lactobacillus paracasei Probiotic survival Citrus pectin Edible coating Gastro-intestinal digestion

A pectin coated dehydrated apple snack containing ≥9 log colony forming units/20 g portion of the probiotic Lactobacillus paracasei IMPC2.1, was developed. The strain was incorporated into the apple slices using vacuum impregnation (IP1) or soaking/stirring (IP2); apple slices were then coated with citrus pectin and dehydrated at 4 °C for 14 days. Microbial application led to colonization of the apple surface, as seen using scanning electron microscope images, and all probiotic products showed comparable radical scavenging activity and total phenol content. IP2 led to better bacterial survival during 30 days storage at 4 °C and color parameters comparable to the fresh apple. The simulated gastro-intestinal (GI) digestion suggested the ability of the strain to survive the digestive process. This inclusion/coating/dehydration process led to a probiotic snack, which could successfully deliver bacterial cells in a viable form to the GI tract.

1. Introduction The manipulation of microorganisms inhabiting the gastro-intestinal (GI) tract is a suitable approach for treating or preventing alteration of GI microbiota (dysbiosis) thus maintaining health (de Almada, de Almada, Martinez, & de Souza Sant’Ana, 2015). The GI microbiota can be modulated by introducing fibers, prebiotics and probiotics in the diet. Particularly, probiotic bacteria are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002). Many commercial products, including fermented foods, have probiotic strains belonging to Bifidobacterium and Lactobacillus species. The introduction of probiotic populations in the diet (≥109 cfu/day) can beneficially modulate GI symptoms linked to some intestinal illnesses (Bron et al., 2017; FAO/WHO, 2002; Riezzo et al., 2012). The probiotic strain has to survive the GI transit and reach the small intestine in adequate amounts to have a beneficial role. Among the probiotic strains, Lactobacillus paracasei IMPC2.1 was shown to have functional properties, i.e., immunomodulatory, anti-proliferative and pro-apoptotic effects (D'Arienzo et al., 2011; Orlando et al., 2012; Sisto et al., 2016). The probiotic features of this strain were also highlighted in clinical studies

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(Riezzo et al., 2012; Valerio et al., 2010) and human feeding trials (Lavermicocca et al., 2005; Valerio et al., 2006; 2015), when carried by vegetable or fish ready-to-eat products. These studies showed the ability of the strain to reach and transiently colonize the human small intestine. Moreover, the strain was able to maintain a mild fermentation of vegetables (table olives, artichokes and cabbage) or fish (swordfish fillets) products, while preserving the nutritional and functional quality of the food (Lavermicocca et al., 2005; Sarvan et al., 2013; Valerio et al., 2006; 2015). Food and their components (i.e., prebiotic molecules) have an important role in modulating the activity of the strain during food processing, helping to buffer the probiotics during GI transit and contributed to an efficient implantation of bacterial cells regulating the intestinal colonization and having probiotic features (Ranadheera, Baines, & Adams, 2010). Non-dairy alternative food matrices are fruits and their products (do Espírito Santo, Perego, Converti, & Oliveira, 2011; Flach, van der Waal, van den Nieuwboer, Claassen, & Larsen, 2017; Moreira, Cassani, Martín-Belloso, & Soliva-Fortuny, 2015; Ribeiro et al., 2014). Among fruits, apples have been studied as a suitable carrier for probiotic cells, because of their nutritional and functional properties (Betoret et al., 2003; Emser, Barbosa, Teixeira, & de Morais, 2017; Tapia et al., 2007). Apples are relatively inexpensive,

Corresponding author. Via Amendola122/O, 70126, Bari, Italy. E-mail address: [email protected] (P. Lavermicocca).

https://doi.org/10.1016/j.fbio.2020.100533 Received 29 October 2018; Received in revised form 14 January 2020; Accepted 15 January 2020 Available online 20 January 2020 2212-4292/ © 2020 Elsevier Ltd. All rights reserved.

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List of abbreviations ANOVA aw cfu DPPH GAE GI IP IP1 IP1U IP2 IP2U

LAB MRD MRS NA PCA RSA% SE SEM SGF SIF SM SSF TBC TPC YM

analysis of variance water activity colony forming units 1,1-diphenyl-2-picrylhydrazyl gallic acid equivalents gastro-intestinal inclusion procedure inclusion procedure using a vacuum impregnation method inclusion procedure using a vacuum impregnation method uncoated inclusion procedure using a soaking/stirring method inclusion procedure using a soaking/stirring method uncoated

available, and have polyphenols, such as dihydrochalcones, flavonols, hydroxycinnamates and flavanol; and fibers, which include cellulose, hemicellulose and pectin. The beneficial effect of apple components on gut microbiota composition and on the metabolic pattern was shown in an in vitro batch culture colonic model (Koutsos et al., 2017). Apple non-digestible polysaccharides (prebiotics) and polyphenols reaching the colon acted as substrates for colonic fermentation, producing phenolic acids and short chain fatty acids, whose health benefits are known (Koutsos et al., 2017). Moreover, the parenchyma of the apple matrix has a large volume of intercellular spaces (20–25% of total volume) filled with gas and liquid that can be replaced by bioactive compounds or microorganisms (Noorbakhsh, Yaghmaee, & Durance, 2013). Therefore, apples can be considered a suitable functional matrix to carry live probiotic cells into the human gut. Several studies have been done to select the appropriate method for including probiotic cells into fruit matrices. Vacuum impregnation was the most effective way to obtain fruit fortified with a large amount of bioactive compounds (Betoret, Betoret, Rocculi, & Dalla Rosa, 2015). This technique is based on the mass transfer between a liquid medium and a solid porous food: the pressure promotes the replacement of gas and liquid contained in the matrix spaces with the bacterial cells or bioactive molecules. However, vacuum impregnation can result in visual defects such as browning of fruit and bacterial cellular damage, which could compromise their survival with stress conditions (de Oliveira et al., 2017; Puente, Betoret, & Cortés, 2009; Sunny-Roberts, 2009). Alternatively, the soaking technique, a traditional, mild impregnation procedure, has the benefit of not interfering with the physiological state of bacterial

lactic acid bacteria maximum recovery diluent de Man-Rogosa-Sharpe nutrient agar plate count agar radical scavenging activity standard error of the mean scanning electron microscopy simulated gastric fluid simulated intestinal fluid skim milk simulated salivary fluid total aerobic bacterial count total polyphenol content yeast and molds

cells. Probiotic cells can also be incorporated into the fruit matrix using edible coating and films (Espitia, Batista, Azeredo, & Otoni, 2016). However, to ensure the bacterial viability during processing and GI digestion, the microencapsulation of bacterial cells before incorporation into the edible coating was also investigated (Betoret et al., 2015; Espitia et al., 2016; Pavli, Tassou, Nychas, & Chorianopoulos, 2018; Tripathi & Giri, 2014). In addition, the edible coatings may act as physical protective systems prolonging shelf life and preserving nutritional and functional properties, and such coatings have also been developed to reduce the use of non-biodegradable packaging materials (Debeaufort, Quezada-Gallo, & Voilley, 1998). Dehydration is a traditional technology used with fruits and vegetables (Akbarian, Ghasemkhani, & Moayedi, 2014). Recently, a pectinhoney edible coating for cut fruits was developed as a bioactive agent that provided functional characteristics to dried fruits including apple slices (Santagata et al., 2018). Dehydrated apples have become a snack food recommended by nutritionists (Morais et al., 2018). Preserving the intrinsic nutritional quality of fruit while adding substances such as probiotics would increase the benefits of eating fruit (Sarkar et al., 2016). However, no studies were found on the development of a probiotic coating for fruits, especially determining that the organism reached the GI tract. Dehydrated fruits and vegetables can be obtained using several processes that differ primarily with the drying method used, whose choice depends on the structural and nutritional characteristics of the food matrix (Khan, 2012). The success of dehydrated fruits and vegetables is due both to the nutritional content and to the greater consumer

Fig. 1. Process to obtain probiotic pectin coated dehydrated apple slices. Two different probiotic inclusion procedures (IP), vacuum impregnation (IP1) and soaking/ stirring (IP2), were used. After the probiotic inclusion, drained apple slices were coated or not (uncoated IP1U and IP2U) with citrus pectin and placed on a grid at 4 °C for 14 days for dehydration. 2

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2. Materials and methods

was maintained at 45 °C until its use as a coating material. Drained apple slices were dipped in the pectin solution for 30 s and placed on the grid at 4 °C for 14 days for dehydration. After coating and/or dehydration, L. paracasei IMPC2.1 was measured (section 2.3.2). Dehydrated pectin coated apple slices, without the probiotic strain, were used as a control. All probiotic samples after dehydration (IP1 and IP2) were stored for 30 days at 4 °C in sealed polyethylene bags (Seward Ltd., Worthing, West Sussex, UK) and L. paracasei numbers measured again.

2.1. Probiotic strain

2.3. Sample analyses

The probiotic strain used was Lactobacillus paracasei IMPC2.1 (from human intestine) belonging to the Institute of Sciences of Food Production (ITEM 17146, ISPA Collection, Bari, Italy) and deposited with number LMG P-22043 in the Belgian Coordinated Collections of Microorganisms (Ghent, Belgium). The strain was obtained as a freeze dried powder (log 2 ± 0.5 × 1011 cfu/g).

2.3.1. Microbiological analyses Microbiological analyses were done to evaluate the microbiological quality of all dehydrated samples and to determine the presence of the probiotic strain only in IP samples. The microbiological quality of coated and uncoated dehydrated apples and of the control was done after the dehydration process, as reported by Santagata et al. (2018), with slight modifications. Briefly, apple samples (5 g) were homogenized for 2 min in sterile maximum recovery diluent (MRD) prepared mixing 0.1% Bactopeptone (Difco, Becton Dickinson Co., Sparks, MD, USA) with 0.85% NaCl, pH 7.0 (1:5 dilution) using a Stomacher (Seward, London, UK). After 30 min at room temperature, the suspension was directly spread plated (1 mL) on three agar plates, and an aliquot was decimally diluted and spread plated (100 μL) on plate count agar (PCA, Difco) supplemented with cycloheximide (Sigma-Aldrich Div.) (0.17 g/L, 30 °C, 48 h) for total aerobic bacterial count (TBC); Pseudomonas agar with CFC supplement (Oxoid Ltd., Basintoke Hampshire, UK) (25 °C, 48 h) for Pseudomonas spp.; de Man-Rogosa-Sharpe agar (MRS, Oxoid) (37 °C, 48 h) for lactic acid bacteria (LAB); Sabouraud dextrose agar (Difco) supplemented with chloramphenicol and chlortetracycline (both 0.05 g/L) (25 °C, 5–7 days) for yeast and molds (YM). Total counts of Enterobacteriaceae were obtained using pourplating dilutions (1 mL) in violet red bile glucose agar (VRBGA, Difco), incubated at 37 °C for 24 h. Another aliquot of the food suspension was heat treated (80 °C for 10 min) to determine spore forming bacteria. Therefore, the suspension was directly spread plated (1 mL) on three nutrient agar (NA) (Oxoid) plates, and an aliquot was decimally diluted and spread plated (100 μL) and plates incubated at 37 °C for 24 h. The presence of the probiotic strain was ascertained as described in paragraph 2.3.2. During the product preparation and storage, 5 g portions of apple slices were homogenized in 50 mL sterile MRD for 2 min in the Stomacher and decimal dilutions (100 μL) were plated on MRS agar plates incubated at 37 °C for 48 h. After the simulated digestion, an aliquot of digested sample was removed, decimally diluted with MRD and plated on MRS agar plates, incubated at 37 °C for 48 h. Microbial counts were measured as cfu/g of apple and the resulting averaged data were transformed to log cfu/g.

attention towards ready-to-eat foods (Thienhirun & Chung, 2018). To obtain a functional coated dehydrated apple snack containing the optimal amount of probiotic Lactobacillus paracasei IMPC2.1 cells, two different impregnation techniques were tested on apple slices. The resulting products were microbiologically and physico-chemically characterized, while strain survival was monitored during processing and after simulated GI digestion.

2.2. Sample preparation 2.2.1. Apple samples The "Golden Delicious" apple variety, cultivated in Italy and sold as "Mela Alto Adige IGP", according to the European Commission, 28AD that also establish a minimum diameter of 65 mm to include apples in the commercial category "Extra". Apples were purchased from a local fruit retailer and immediately stored at 4 °C. Uniform sized, defect-free fruits were selected. Apples (1 Kg to obtain ~100 apple slices) were hand-peeled with a knife, cored and cut into slices with uniform thickness of ~2 cm and subjected to probiotic inclusion and pectin coating. The pH of fresh apple was 3.5 while the acidity was 0.53%. The pH was measured using a portable pH meter (type 110, Eutech Instruments Ltd., Singapore) supplied with a Double Pore D electrode (Hamilton, Bonaduz, Switzerland), while the acidity was determined using titration with 0.1 N NaOH and expressed as % malic acid. 2.2.2. Probiotic inclusion and dehydration To obtain probiotic pectin coated or uncoated dehydrated apple slices, two different inclusion procedures (IP), vacuum impregnation (IP1) and soaking/stirring (IP2), were used (Fig. 1). A probiotic suspension was prepared mixing 7.5 g of freeze-dried culture with 1.5 L 0.85% NaCl to obtain a final concentration of 1.5 × 109 cfu/g of apple. For each procedure, 1 Kg of apple slices and 1.5 L of the probiotic suspension were used. For IP1, the vacuum impregnation method of Betoret et al. (2003), with slight modifications, was used. In brief, apple slices were dipped in the bacterial suspension at room temperature (22 ± 1.0 °C) for 20 min at atmospheric pressure; then, the vacuum was used for 10 min using a vacuum pump (KNF Laboport, Trenton, NJ, USA) and then atmospheric pressure was restored while leaving the apple slices in the liquid for an additional 10 min, closed in the flask. For IP2, the apple slices were soaked in the bacterial suspension (soaking) and stirred (150 rev/min) on an orbital shaker Certomat U (B. Braun Biotech International, Goettingen, Germany) for 1 h at room temperature. After the IP, apple slices were allowed to drain on polyethylene grids for food contact (hole dimension: 16 × 16 mm; Italian Food Technology, Mantova, Italy) for 10 min at room temperature and microbiological analyses were done to determine the presence of the probiotic strain, as described in paragraph 2.3.2. After the probiotic inclusion, some apple slices were directly placed on the grid, left to dehydrate at 4 °C for 14 days and used as controls (IP1U and IP2U) for each IP. The coating and the dehydration processes were done as described by Santagata et al. (2018) using citrus pectin (low degree of esterification) obtained from Herbstreith & Fox KG, Pektin-Fabriken (CAS No: 9000-69-5; Neuenbürg, Germany). Briefly, citrus pectin (2% w/v) was dissolved in demineralized water (Sigma-Aldrich Div., Milan, Italy) at 100 °C with stirring. After pectin solubilization, the solution

2.3.2. Genetic identification of L. paracasei IMPC2.1 The identification of L. paracasei IMPC2.1 was done as reported by Valerio et al. (2015). Briefly, 20% of total presumptive LAB colonies randomly picked from countable MRS agar plates, were isolated and checked for purity by streaking each colony on MRS agar plates. Purified colonies were grown overnight in MRS broth at 37 °C and bacterial DNA was extracted using a Clonesaver Card Kit (Whatman, Maidstone, UK) according to manufacturer's recommendations. Briefly, a 5 μL aliquot of each bacterial culture was applied to the center of a printed circle on the card and allowed to air-dry for 1 h. Discs from the center of each dried sample spot were removed (Harris Uni-Core disposable 5.0 mm punch, Whatman), placed in a microcentrifuge tube and, after three washing steps with sterile TE−1 extraction buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0), the DNA was eluted from each disc by adding 5 μL sterile TE−1 buffer in the tube (50 min at room temperature). DNA samples were amplified using REP-PCR using two degenerate primers, REP-1R-Dt (5′-IIINCGNCGNCATCNGGC-3′) (where N is A, T, C, or G 3

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centrifuge tubes (29 × 115 mm) in a centrifuge (Model 4235, A.C.L. International S.r.l., Milano, Italy). The TPC and antioxidant capacity were measured for the supernatant. The Folin-Ciocalteu procedure was used to determine the TPC using the method reported by Soong and Barlow (2004). Briefly, 50 μL samples were mixed with 2.5 mL Folin–Ciocalteu reagent previously diluted with distilled water (1:10). After 5 min, 2 mL 30% sodium carbonate solution was added. The absorbance was measured at 760 nm using a UV–visible spectrophotometer (Model DU 730, Beckman Coulter, Brea, CA, USA), after incubation for 90 min at room temperature. Gallic acid (GA) was used as a reference standard, and the results were expressed as mg GA equivalents (GAE)/100 g of dehydrated apple. Folin-Ciocalteu, GA and ethanol were obtained from Sigma-Aldrich.

and I is inosine) and REP-2R-Dt (5′-NCGNCTTATCNGGCCTAC-3′), as previously described by De Bellis, Valerio, Sisto, Lonigro, and Lavermicocca (2010). The amplifications were done in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). The amplification fragments were resolved using a microfluidic Lab-on-a-Chip (LoaC) electrophoresis carried out on a 2100 Bioanalyzer using the DNA 7500 LabChip kit (Agilent Technologies, Waldbronn, Germany). The chip loading was done according to the manufacturer's instruction (Agilent Technologies, 2016). REP-PCR profiles were automatically obtained within 30 min and data evaluated using 2100 Expert Software provided by the company, including generating the peaks of the electropherogram and the bands for the gel images assuming Beer-Lambert's law. Genetic identification of L. paracasei IMPC2.1 was based on the comparison of the REP-PCR profile of each isolate with the specific pattern obtained from the pure culture of the strain. The concentration of L. paracasei IMPC2.1 (cfu/g) was calculated on the basis of the number of identified colonies.

2.3.6. DPPH free radical scavenging assay The antioxidant activity was measured in terms of hydrogen-donating or radical scavenging ability of the extracts. The ability to counteract the DPPH radical (1,1-diphenyl-2-picrylhydrazyl) was investigated using the method of Yeo, Jeong, and Lee (2010). Briefly, 2.4 mL 0.1 mM solution of DPPH (Sigma-Aldrich) in methanol was added to 100 μL of sample. After shaking, the mixture was kept at room temperature for 30 min. Then the absorbance (A) was measured at 515 nm. The radical scavenging activity percentage (RSA%) was calculated as:

2.3.3. pH and water activity determination The pH and water activity (aw) were monitored after the dehydration process. The aw value was measured using an AquaLab Series 3 (Decagon Devices, Pullman, WA, USA). 2.3.4. Color analysis Parameters considered for color were L* (lightness), a* (redness), b* (yellowness), measured on 4 random points on the sample surface (3 slices for each sample replicate). A colorimeter (CR-400, Konica Minolta, Osaka, Japan) equipped with a D65 illuminant in the reflectance mode and in the CIE L* a* b* color scale (CIE, 1986), was used. The colorimeter was calibrated with a standard reference having values of L*, a*, b* corresponding to 97.55, 1.32 and 1.41, respectively.

RSA% = [(Acontrol-Asample)/Acontrol × 100]

(1)

2.3.7. Scanning electron microscopy (SEM) Morphological analyses of all dehydrated apple slice surfaces were done using a Quanta 200 FEG (FEI, Eindhoven, The Netherlands). SEM observations were done in low vacuum mode (PH2O: 0.7 torr), using a large field detector (LFD) and an acceleration voltage of 5–20 kV. Prior to the observation, the sample surfaces were coated with a homogeneous layer (18 ± 0.2 nm) of Au–Pd alloy using a sputtering device (MED 020, BAL-TEC AG, Balzers, Liechtenstein). Three different specimens, each one investigated in 5 different areas, were observed at several magnifications (800-5000x).

2.3.5. Total polyphenol content (TPC) The TPC was measured in probiotic coated and uncoated dehydrated apple samples and in the control as reported by Santagata et al. (2018). Briefly, dehydrated samples were minced with a laboratory mixer (Ultra Turrax T8, Ika-Werke, Staufen, Germany) at 12,000 rpm for 10 min and aliquots of 0.5 g were treated with 10 mL of ethanol:water mixture (50:50 v/v) for 30 min with stirring at room temperature. The samples were centrifuged for 30 min at 2930×g using

Table 1 Preparation of stock solutions of simulated digestion fluids as reported by Minekus et al. (2014). The volumes are calculated for a final volume of 500 mL for each simulated fluid. The electrolyte stock solutions simulated salivary fluid (SSF), simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), were prepared as 1.25X concentrate solutions and used as reported in the material and method section. The addition of enzymes, bile salts, Ca2+ solution, etc. and water will result in the correct electrolyte concentration in the final digestion mixture. CaCl2 (H2O)2 has to be added to the final mixture of simulated digestion fluid and sample to avoid salt precipitation.

Constituent

KCl KH2PO4 NaHCO3 NaCl MgCl2(H2O)6 (NH4)2CO3 For pH adjustment

Stock conc.

SGF

SIF

pH 7

pH 3

pH 7

Vol. of stock

Conc. in SSF

Vol. of stock

Conc. in SGF

Vol. of stock

Conc. in SIF

g L−1

mol L−1

mL

mmol L−1

mL

mmol L−1

mL

mmol L−1

37.3 68 84 117 30.5 48

0.05 0.05 1 2 0.15 0.5

15.1 3.7 6.8 – 0.5 0.06

15.1 3.7 13.6 – 0.15 0.06

6.9 0.9 12.5 11.8 0.4 0.5

6.9 0.9 25 47.2 0.1 0.5

6.8 0.8 42.5 9.6 1.1 –

6.8 0.8 85 38.4 0.33 –

mmol L−1 – 15.6

mL – 0.7

mmol L−1 – 8.4

mol L−1 NaOH 1 HCl 6 CaCl2(H2O)2 is not added to the simulated digestion g L−1 mol L−1 CaCl2(H2O)2 44.1 0.3 a

SSF

mL mmol L−1 mL – – – 0.09 1.1 1.3 fluids until the end, see details in legend −1 mmol L 1.5 (0.75a)

The Ca2+ concentration in the final digestion mixture is reported in parenthesis. 4

mmol L−1 0.15 (0.075a)

mmol L−1 0.6 (0.3a)

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followed using Fisher LSD test. Statistical significance was measured at a level of 5% (P < 0.05). The STATISTICA data analysis software system, version 10 (StatSoft, Inc., Tulsa, OK, USA) was used.

2.4. Simulated GI digestion Samples were subjected to simulated GI digestion as reported by Minekus et al. (2014) with slight modifications. A control suspension of the L. paracasei strain in skim milk (SM, Oxoid), prepared by adding 0.25 g freeze dried culture in 50 mL sterilized medium was used to obtain a final load of 1 × 109 cfu/mL. The simulated GI digestion consisted of oral, gastric and intestinal phases and simulated digestion fluids were prepared as reported in Table 1. All solutions used for the simulated digestion were sterilized by filtration through 0.45 μm filters (Millipore, Millipore Corp., Bedford, MA, USA). All digestion phases were done at 37 °C and a preliminary experiment was done to determine the amount of HCl or NaOH needed to maintain the correct pH value in each phase. Measurement of pH was done as reported in paragraph 2.2.1 and the electrode was periodically sterilized with ethanol. Briefly, mastication was simulated by homogenizing 5 g apples or 5 mL SM sample with 4 mL of simulated salivary fluid (SSF) stock solution using a blender (LB20EG, Waring Laboratory Science, Torrington, CT, USA) for 1 min; after that 56 mg α-amylase from porcine pancreas (EC 3.2.1.1) (Sigma-Aldrich), 25 μL 0.3 M CaCl2 and 975 μL water were added to a final ratio of sample:SSF of 1:1 (w/v or v/ v). The reaction time was 2 min. To simulate the gastric phase, the oral bolus was mixed with 9.1 mL of simulated gastric fluid (SGF) stock solution, 55 mg pepsin from porcine gastric mucosa (EC 3.4.23.1) (Sigma-Aldrich), 5 μL 0.3 M CaCl2, 0.2 mL 1 M HCl and 695 μL water to obtain a final ratio of sample:SGF of 1:1 (v/v). Sterile HCl was added to reduce the pH to 3.0 as described in Emser et al. (2017). The time of reaction was 2 h at 37 °C. The pH was re-adjusted during digestion with 1 M HCl. After the gastric phase, the chime was mixed with 11 mL simulated intestinal fluid (SIF) stock solution, 5 mL pancreatin, from porcine pancreas (4 × USP, Sigma-Aldrich), solution (0.072 mg/mL) in SIF, 2.16 g bovine bile salts (Sigma-Aldrich) to obtain a bile:pancreatin ratio of 6:1 as reported by Hollebeeck, Borlon, Schneider, Larondelle, and Rogez (2013), 2.5 mL water, 40 μL 0.3 M CaCl2, 1 M NaOH sterile solution was added to neutralize the mixture to pH 7.0 and, finally, to obtain a final ratio of sample:SIF of 1:1 (v/v), an adequate volume of water was added. The time of reaction was 2 h. The pH was re-adjusted during digestion. At the end of simulated digestion, the survival of the L. paracasei IMPC2.1 strain in each sample was determined as in section 2.3.2. The survival of the strain was expressed as logarithmic reduction: log (N/ N0), where N is the cfu/g (or cfu/mL for SM) mean value after digestion, N0 is the cfu/g mean value before digestion.

3. Results and discussion 3.1. Viability of L. paracasei IMPC2.1 during dehydrated apple slices preparation Both IP procedures led to a dehydrated product with viable bacterial cells ≥7 log cfu/g (≥9 log cfu of cells/20 g portion) (Table 2) as required for probiotic foods by the Italian Ministry of Health (Guidelines on probiotics and prebiotics, 2018) and by FAO (FAO/WHO, 2002). A portion of ~20 g of dehydrated apples can be considered an acceptable amount to be introduced as a snack in the daily diet, as suggested by the increased commercial availability of small packages of dehydrated fruits. As shown in Table 2, at each process step no differences (P ≥ 0.05) in the probiotic concentration were observed between samples; however, a slight reduction was observed after the dehydration step without affecting the desired probiotic load (≥7 log cfu/g). The coating step did not influence the probiotic adhesion to the apple matrix. As a result, both uncoated samples had the needed bacterial concentration. The vacuum impregnation favored the adsorption of probiotic cells to a fruit matrix (Morais et al., 2018), even if it resulted in some disadvantages such as lower strain viability and lower brightness (Puente et al., 2009; Rêgo, Freixo, Silva, Gibbs, & Teixeira, 2013). In particular, Rêgo et al. (2013) reported that the application of a pressure of 50 mbar for 1.2 s affected the viability of probiotic strains using this method. In this study, no differences in the probiotic concentrations were observed between the two IP, suggesting that the time used for vacuum impregnation (10 min) favored the absorption of the bacterial suspension. Nevertheless, the two impregnation methods influenced the distribution of probiotic cells differently on apple slices, as shown by morphological observations (see section 3.2). 3.2. SEM observations SEM images showed the heavy colonization of the apple surface by the L. paracasei IMPC2.1. In Fig. 2 (a and b), as an example, two SEM micrographs of IP1U and IP2U samples are shown. The micrographs showed a high number of rod-shaped probiotic cells adhering well to both dehydrated apple slice substrates. The adhesion of probiotic microorganisms to fruit tissue could be due to the internal structure of plant tissues, able to harbor bacteria in places that allow their survival. In particular, from Fig. 2 (a and b) the grip of L. paracasei IMPC2.1 to apple mainly occurs into the intercellular spaces of the parenchymal tissue of the fruit, even if some cells could also be observed on the fruit surface. The apple parenchymal cells, ranging between 50 and 500 μm in diameter, had an important role in microorganism allocation and adhesion. Indeed, bacterial cells, whose size ranged in 0.5 μm in diameter and ~2.0 μm in length, could penetrate inside the cellular compartments of the apples and give rise to some agglomerations,

2.5. Statistical analysis Experiments were done twice with two replicates each. The mean and standard error of the mean were calculated for each experimental parameter. Values of pH (-log [H+]) and of microbial counts (cfu/mL or cfu/g) were first averaged and then expressed as log mean values ± standard error of the mean (SE). Differences among the experimental samples were determined using one-way analysis of variance (ANOVA)

Table 2 Viability of L. paracasei IMPC2.1 in dehydrated apple slices coated (IP1 and IP2) or not (IP1U or IP2U) with citrus pectin obtained using vacuum impregnation (IP1) and soaking/stirring (IP2) procedures. Process step

IP1

IP2

IP1U

IP2U

8.1 ± 0.1 aA 8.0 ± 0.1 aA 7.8 ± 0.1 aA 7.98 ± 0.02 aB

8.5 ± 0.1 aA – 7.8 ± 0.1bA 6.9 ± 0.3 cA

8.2 ± 0.1 aA – 8.0 ± 0.1 aA 6.9 ± 0.1bA

log cfu/g ± SE Probiotic inclusion Coating Dehydration Storage (after 30 days)

8.15 ± 0.03 aA 8.4 ± 0.1 aA 7.9 ± 0.2 aA 6.8 ± 0.3bA

Values (mean ± standard error of the mean) with different lower case (column) or capital (row) letters are significantly different (P < 0.05). 5

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Fig. 2. SEM surface pictures of dehydrated apple slices containing or not the probiotic strain Lactobacillus paracasei IMPC2.1: uncoated probiotic samples IP1U (a) and IP2U (b), coated probiotic samples IP1 (c) and IP2 (d), coated apple slice not including probiotic cells (control, e). Enlarged images (red boxes) show the adhesion of the rod-shaped probiotic cells on coated (c, d) and uncoated (a, b) dehydrated apple slices while no cells are visible on the control sample (e).

of the pectin coating resulted in the formation of a regular and smoothed layer covering the active cells. Nevertheless, some differences concerning the coating thickness could be observed. In particular, the IP1 sample was characterized by a lower thickness range (0.5–2 mm) compared to IP2 sample, which seemed to have a thicker layer of 1.5–2.5 mm (micrographs not shown). In the control sample, the distribution of the coating on the surface of apples not including probiotic cells was responsible for a structural shrinking, as previously observed by Santagata et al. (2018), likely due to the strong physical interactions occurring between polar groups of apple and pectin solution, as a consequence of some permeation of the pectin-water solution inside the

particularly visible in IP1 samples. While in the IP2U sample, except with high concentration of probiotic cells, it was possible to discern the bright domains and the boundary regions of the single shaped rods. In the IP1U sample the smoother topography of the cell distribution suggested a possible sort of mutual cell gluing, and, except for some sporadic discrete rods, it was difficult to distinguish the single cells. In Fig. 2 (c, d, e) examples of IP1, IP2 and coated apple slice (control), respectively, are shown. It was possible to observe the embedding of the probiotic cells under the pectin coating (Fig. 2 c, d). In both cases, the application of the edible film did not modify the main structural aspects of the probiotic cell distribution. Hence, the addition 6

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porous apple structure (Rojas-Graü, Soliva-Fortuny, & Martín-Belloso, 2009). This outcome led to diffuse macroscopic buckling of the fruit slices.

Table 4 Color parameters (L*, a*, b* ± SE) relevant to probiotic, coated or uncoated, dehydrated apple slices, control (coated and dehydrated but not containing the probiotic L. paracasei IMPC2.1 strain) in comparison to fresh apple slices.

3.3. Microbiological and physical qualities of dehydrated apple slices

sample

L*

a*

b*

All dehydrated samples showed comparable (P ≥ 0.05) aw and pH values (Table 3). Enterobacteriaceae and Pseudomonas spp. were not detected, while a higher (P < 0.05) TBC in the probiotic samples compared to the control was observed. Spore forming bacteria were detected only in the control sample, while no significant differences were observed for the remaining populations. The LAB count was representative of the probiotic population. The color parameters were measured on all samples, including fresh apple slices and control. Results shown in Table 4 indicate a lighter color (higher L* and lower a*) for IP2 apple samples compared to IP1 samples, suggesting that the vacuum impregnation method strongly influenced the final color of apples. As reported by several authors (de Oliveira et al., 2017; Puente et al., 2009), the vacuum-method led to the formation of voids and pores able to absorb the bacterial suspension replacing the air bubbles, leading to a higher light reflectance and consequent lower brightness. Both IP2 and IP2U samples showed a brightness (L*) and redness (a*) comparable to the fresh apple, suggesting that this procedure allowed the preservation of the visual quality of the apples, a fundamental sensory characteristic for this food. The IP1 sample became as brown as the control not containing the probiotic strain.

Fresh apple Control IP1 IP2 IP1U IP2U

79 ± 1a 67 ± 2bd 62 ± 2b 83 ± 1ac 68 ± 2d 86 ± 1c

−0.4 ± 0.1 ab 10 ± 1c 8.1 ± 0.3c 1.5 ± 1.2a 5 ± 1d 0.8 ± 0.1b

28 ± 1a 40 ± 1b 38 ± 2bc 39 ± 1bc 36 ± 1cd 34 ± 1d

For a given determination (column) values (mean ± standard error of the mean) with different lower case letters are significantly different (P < 0.05). Table 5 Total polyphenol content (TPC ± SE) and radical scavenging activity percentage (RSA% ± SE) of probiotic, pectin coated (IP1 and IP2) or not (IP1U and IP2U), dehydrated apple samples and of the coated control not containing the probiotic strain. Sample Control IP1 IP2 IP1U IP2U

TPC (mg GAE/Kg apple) 2

11.2 ± 0.4 × 10 a 13.6 ± 0.4 × 102b 139 ± 0.2 × 101b 133 ± 0.3 × 101b 13.7 ± 0.1 × 102b

RSA (%) 80 ± 1a 82 ± 3b 82 ± 2b 81 ± 0.4b 85 ± 1b

For a given determination (column) values (mean ± standard error of the mean) with different lower case letters are significantly different (P < 0.05).

3.4. TPC and RSA%

form into the gut since after digestion, a good survival rate was observed confirming its ability to adhere to the apple matrix (Fig. 2) and to tolerate the harsh GI conditions (Lavermicocca et al., 2005; Valerio et al., 2006; 2015). Both IP led to a good survival of the strain with ~2 log reduction for both coated and uncoated samples (Table 6). Therefore, a 20 g portion of dehydrated apples containing 9 log cfu of live cells, would supply the GI tract with ~7 log cfu of probiotic cells. This amount was considerably higher (P < 0.05) than that obtained with SM. However, when samples were stored for about 30 days at 4 °C and subjected to simulated digestion, the survival of the probiotic strain was affected only in IP1 samples (data not shown), suggesting an effect of sub-atmospheric pressure on the physiological state of bacterial cells making them more susceptible to the GI conditions. As previously reported, bacterial cells can be irreversibly damaged during processes such as lyophilization, vacuum or spray–drying (Sunny-Roberts, 2009). As shown in Fig. 2, bacterial cells adhering to the IP1 apple surface created a smooth layer of unshaped cells suggesting damage of these cells and a consequent lower cell viability after GI digestion. The previous hypotheses were consistent with the morphological analysis of IP1 apple surfaces, as seen by the development of overlapped joined cell clusters, probably affected by the vacuum impregnation method.

All probiotic samples showed comparable TPC values (Table 5), while the control was lower, suggesting a protective action of the probiotic cells covering the apple surface, that slowed the passage of oxygen and the consequent oxidation of polyphenols. No differences were observed for the antioxidant activity between probiotic samples, while the control was non-significantly lower. These results were consistent with the observed lighter color of IP2 samples because of the mild IP and the uniform bacterial layer of cells contributed to maintaining a good visual and nutritional quality of the dried product. 3.5. Evaluation of the survival of L. paracasei IMPC2.1 on coated and uncoated dehydrated apples slices exposed to simulated GI digestion To obtain a probiotic product, a bacterial load ≥7 log cfu/g and ≥9 log cfu/portion, is needed. Live probiotic cells should be delivered into the human gut in an adequate amount to exert a beneficial effect (FAO/ WHO, 2002). Therefore, the food matrix should have compositional and structural properties protecting the probiotic viability during processing, storage and passage during the GI tract (Flach et al., 2017; Ranadheera et al., 2010). Dehydrated apple slices successfully delivered L. paracasei in viable

Table 3 Microbiological quality, water activity and pH values of probiotic, coated (IP1 and IP2) or uncoated (IP1U and IP2U), dehydrated apple slices, and of the control (coated and dehydrated but not containing the probiotic L. paracasei IMPC2.1 strain). Sample

TBC

LAB

2.7 ± 0.1a 3.09 ± 0.01a 2.6 ± 0.4a 2.8 ± 0.6a 3.6 ± 0.9a

-a 7.9 7.8 7.8 7.9

YM

Spore forming bacteria

aw ± SE

2.6 ± 0.1 – – – –

0.55 0.56 0.54 0.53 0.51

pH ± SE

log cfu/g ± SE Control IP1 IP2 IP1U IP2U

± ± ± ±

0.2a 0.1a 0.1a 0.1a

2.6 2.9 1.9 2.0 1.3

± ± ± ± ±

0.1a 0.1a 0.1a 0.2a 0.3b

± ± ± ± ±

0.01a 0.01a 0.003a 0.02a 0.03a

Values (mean ± standard error of the mean) in each column with different lower case letters are significantly different (P < 0.05). a For LAB population the detection limit (DL) was 2 log cfu/g; for the other populations DL was 1 log cfu/g. 7

3.9 3.9 4.0 3.8 3.7

± ± ± ± ±

0.1a 0.2a 0.2a 0.1a 0.1a

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References

Table 6 Survival of L. paracasei IMPC2.1 tested in skim milk (SM) and in coated (IP1 and IP2) and uncoated (IP1U and IP2U) dehydrated apple samples, after the simulated GI digestion. Sample

log (N/N0) ± SE

SM IP1 IP2 IP1U IP2U

−3.9 −2.7 −2.3 −2.4 −2.3

± ± ± ± ±

Agilent Technologies. Agilent DNA 7500 and DNA 12000, kit guide. (2016). Available online at: https://www.agilent.com/cs/library/usermanuals/public/G2938-90024_ DNA7500-12000_KG.pdf Accessed 2nd July 2019 . Akbarian, M., Ghasemkhani, N., & Moayedi, F. (2014). Osmotic dehydration of fruits in food industrial: A review. International Journal of Biosciences, 4(1), 42–57. de Almada, C. N., de Almada, C. N., Martinez, R. C. R., & de Souza Sant'Ana, A. (2015). Characterization of the intestinal microbiota and its interaction with probiotics and health impacts. Applied Microbiology and Biotechnology, 99(10), 4175–4199. Betoret, E., Betoret, N., Rocculi, P., & Dalla Rosa, M. (2015). Strategies to improve food functionality: Structure–property relationships on high pressures homogenization, vacuum impregnation and drying technologies. Trends in Food Science & Technology, 46(1), 1–12. Betoret, N., Puente, L., Dıaz, M. J., Pagán, M. J., Garcıa, M. J., Gras, M. L., et al. (2003). Development of probiotic-enriched dried fruits by vacuum impregnation. Journal of Food Engineering, 56(2–3), 273–277. Bron, P. A., Kleerebezem, M., Brummer, R. J., Cani, P. D., Mercenier, A., MacDonald, T. T., et al. (2017). Can probiotics modulate human disease by impacting intestinal barrier function? British Journal of Nutrition, 117(1), 93–107. CIE (1986). Publication No. 15.2. Colorimetry (2nd ed.). Vienna, Austria: CIE Commission International de L'eclairage. De Bellis, P., Valerio, F., Sisto, A., Lonigro, S. L., & Lavermicocca, P. (2010). Probiotic table olives: Microbial populations adhering on olive surface in fermentation sets inoculated with the probiotic strain Lactobacillus paracasei IMPC2.1 in an industrial plant. International Journal of Food Microbiology, 140(1), 6–13. Debeaufort, F., Quezada-Gallo, J. A., & Voilley, A. (1998). Edible films and coatings: Tomorrow's packagings: A review. Critical Reviews in Food Science, 38(4), 299–313. D'Arienzo, R., Stefanile, R., Maurano, F., Mazzarella, G., Ricca, E., Troncone, R., et al. (2011). Immunomodulatory effects of Lactobacillus casei administration in a mouse model of gliadin‐sensitive enteropathy. Scandinavian Journal of Immunology, 74(4), 335–341. Emser, K., Barbosa, J., Teixeira, P., & de Morais, A. M. M. B. (2017). Lactobacillus plantarum survival during the osmotic dehydration and storage of probiotic cut apple. Journal of Functional Foods, 38, 519–528. do Espírito Santo, A. P., Perego, P., Converti, A., & Oliveira, M. N. (2011). Influence of food matrices on probiotic viability – a review focusing on the fruity bases. Trends in Food Science & Technology, 22(7), 377–385. Espitia, P. J., Batista, R. A., Azeredo, H. M., & Otoni, C. G. (2016). Probiotics and their potential applications in active edible films and coatings. Food Research International, 90, 42–52. European Commission (28AD). Council regulation amendment application in accordance with article 9 “mela Alto Adige”/“Südtiroler apfel” EC No: IT-PGI-0105-020709.09.2011. Official Journal of the European Union, 55 C 125/05. FAO/WHO (2002). Guidelines for the evaluation of probiotics in food. Report of a joint FAO/ WHO expert consultation on guidelines for the evaluation of probiotics in foodLondon Ontario, Canada: World Health Organization and Food and Agriculture Organization of the United Nations. Flach, J., van der Waal, M. B., van den Nieuwboer, M., Claassen, E., & Larsen, O. F. (2017). The underexposed role of food matrices in probiotic products: Reviewing the relationship between carrier matrices and product parameters. Critical Reviews in Food Science and Nutrition, 1–15. Hollebeeck, S., Borlon, F., Schneider, Y. J., Larondelle, Y., & Rogez, H. (2013). Development of a standardised human in vitro digestion protocol based on macronutrient digestion using response surface methodology. Food Chemistry, 138(2–3), 1936–1944. Italian Ministry of Health. Guidelines on probiotics and prebiotics. (2018). Revised in March 2018. Available online at: http://www.salute.gov.it/imgs/C_17_pubblicazioni_1016_ ulterioriallegati_ulterioreallegato_0_alleg.pdf Accessed 20th March 2019 . Khan, M. R. (2012). Osmotic dehydration technique for fruits preservation - a review. Pakistan Journal of Food Sciences, 22(2), 71–85. Koutsos, A., Lima, M., Conterno, L., Gasperotti, M., Bianchi, M., Fava, F., et al. (2017). Effects of commercial apple varieties on human gut microbiota composition and metabolic output using an in vitro colonic model. Nutrients, 9(6), 533. Lavermicocca, P., Valerio, F., Lonigro, S. L., De Angelis, M., Morelli, L., Callegari, M. L., et al. (2005). Study of adhesion and survival of lactobacilli and bifidobacteria on table olives with the aim of formulating a new probiotic food. Applied and Environmental Microbiology, 71(8), 4233–4240. Minekus, M., Alminger, M., Alvito, P., Ballance, S., Bohn, T., Bourlieu, C., et al. (2014). A standardised static in vitro digestion method suitable for food – an international consensus. Food & Function, 5(6), 1113–1124. Morais, R. M. S. C., Morais, A. M. M. B., Dammak, I., Bonilla, J., Sobral, P. J. A., Laguerre, J. C., et al. (2018). Functional dehydrated foods for health preservation. Journal of Food Quality, 2018, 1739636 29 Pages. Moreira, M. R., Cassani, L., Martín-Belloso, O., & Soliva-Fortuny, R. (2015). Effects of polysaccharide-based edible coatings enriched with dietary fiber on quality attributes of fresh-cut apples. Journal of Food Science & Technology, 52(12), 7795–7805. Noorbakhsh, R., Yaghmaee, P., & Durance, T. (2013). Radiant energy under vacuum (REV) technology: A novel approach for producing probiotic enriched apple snacks. Journal of Functional Foods, 5(3), 1049–1056. de Oliveira, P. M., Ramos, A. M., Martins, E. M. F., Vieira, É. N. R., Soares, A. D. S., & 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 and Technology, 52(12), 2547–2554. Orlando, A., Refolo, M. G., Messa, C., Amati, L., Lavermicocca, P., Guerra, V., et al.

a

0.1a 0.2b 0.2b 0.1b 0.2b

Values with different lower case letters are significantly different (P < 0.05). a Survival is represented as the logarithmic reduction: log (N/N0) ± standard error of the mean, N is the cfu/g mean value after digestion, N0 is the cfu/ g mean value before digestion.

4. Conclusions The inclusion/coating/dehydration process led to a snack containing > 9 log cfu/portion of the probiotic L. paracasei IMPC2.1 strain, which survived during fruit processing and simulated GI digestion. The procedure including a mild probiotic inclusion step (soaking and stirring, IP2) can be considered an easy method to obtain an attractive snack containing adequate amounts of probiotic populations, that would survive the GI tract so that they can have a beneficial effect. This IP led to a uniform layer on the apple surface and a good visual and nutritional quality of the dried product which could be maintained for 30 days and did not affect the survival of the probiotic strain during a 30-day storage period. Further efforts will be focused on transferring the technology to a food enterprise. CRediT authorship contribution statement Francesca Valerio: Conceptualization, Methodology, Writing - review & editing. Maria Grazia Volpe: Conceptualization, Methodology, Writing - review & editing. Gabriella Santagata: Investigation, Data curation, Writing - review & editing. Floriana Boscaino: Investigation, Data curation, Writing - original draft. Costantina Barbarisi: Investigation, Data curation, Writing - original draft. Mariaelena Di Biase: Investigation, Data curation, Writing - original draft. Anna Rita Bavaro: Investigation, Data curation, Writing - original draft. Stella Lisa Lonigro: Investigation, Data curation, Writing - original draft. Paola Lavermicocca: Supervision. Declaration of competing interest The authors confirm that they have no conflicts of interest with respect to the study described in this manuscript. Acknowledgements The authors thank the project “SOstenibilità della FIliera Agroalimentare” (SO.FI.A), in the framework of the National Technological Cluster of Ministry of Education, University and Research (MIUR), for their financial support. The authors thank the technician Maria Cristina Del Barone, for her support in performing morphological analysis at the LAMeST Laboratory of Scanning and Transmission Electron Microscopy (IPCB-CNR, Pozzuoli (NA), Italy). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fbio.2020.100533. 8

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by the probiotic strain Lactobacillus paracasei LMG-P22043. Food Research International, 54, 706–710. Sisto, A., Luongo, D., Treppiccione, L., De Bellis, P., Di Venere, D., Lavermicocca, P., et al. (2016). Effect of Lactobacillus paracasei culture filtrates and artichoke polyphenols on cytokine production by dendritic cells. Nutrients, 8(10), 635. Soong, Y. Y., & Barlow, P. J. (2004). Antioxidant activity and phenolic content of selected fruit seeds. Food Chemistry, 88(3), 411–417. Sunny-Roberts, E. O. (2009). Application of disaccarides pre-treatment in improving tolerances of Lactobacillus rhamnosus strains to environmental stresses or during vacuum- and spray-drying processesDoctoral dissertation. Berlin, Germany: Berlin University of Technology. Tapia, M. S., Rojas‐Graü, M. A., Rodríguez, F. J., Ramírez, J., Carmona, A., & Martin‐Belloso, O. (2007). Alginate‐and gellan‐based edible films for probiotic coatings on fresh‐cut fruits. Journal of Food Science, 72(4), E190–E196. Thienhirun, S., & Chung, S. (2018). Consumer attitudes and preferences toward crosscultural ready-to-eat (RTE) food. Journal of Food Products Marketing, 24(1), 56–79. Tripathi, M. K., & Giri, S. K. (2014). Probiotic functional foods: Survival of probiotics during processing and storage. Journal of Functional Foods, 9, 225–241. Valerio, F., De Bellis, P., Lonigro, S. L., Morelli, L., Visconti, A., & Lavermicocca, P. (2006). In vitro and in vivo survival and transit tolerance of potentially probiotic strains carried by artichokes in the gastrointestinal tract. Applied and Environmental Microbiology, 72(4), 3042–3045. Valerio, F., Lonigro, S. L., Giribaldi, M., Di Biase, M., De Bellis, P., Cavallarin, L., et al. (2015). Probiotic Lactobacillus paracasei IMPC 2.1 strain delivered by ready-to-eat swordfish fillets colonizes the human gut after alternate-day supplementation. Journal of Functional Foods, 17, 468–475. Valerio, F., Russo, F., de Candia, S., Riezzo, G., Orlando, A., Lonigro, S. L., et al. (2010). Effects of probiotic Lactobacillus paracasei-enriched artichokes on constipated patients: A pilot study. Journal of Clinical Gastroenterology, 44, S49–S53. Yeo, J. D., Jeong, M. K., & Lee, J. H. (2010). Application of DPPH absorbance method to monitor the degree of oxidation in thermally-oxidized oil model system with antioxidants. Food Science and Biotechnology, 19(1), 253–256.

(2012). Antiproliferative and proapoptotic effects of viable or heat-killed Lactobacillus paracasei IMPC2. 1 and Lactobacillus rhamnosus GG in HGC-27 gastric and DLD-1 colon cell lines. Nutrition and Cancer, 64(7), 1103–1111. Pavli, F., Tassou, C., Nychas, G. J. E., & Chorianopoulos, N. (2018). Probiotic incorporation in edible films and coatings: Bioactive solution for functional foods. International Journal of Molecular Sciences, 19(1), 150. Puente, D. L., Betoret, V. N., & Cortés, R. M. (2009). Evolution of probiotic content and color of apples impregnated with lactic acid bacteria. Vitae, 16(3), 297–303. Ranadheera, R. D. C. S., Baines, S. K., & Adams, M. C. (2010). Importance of food in probiotic efficacy. Food Research International, 43(1), 1–7. Rêgo, A., Freixo, R., Silva, J., Gibbs, P., & Teixeira, P. (2013). A functional dried fruit matrix incorporated with probiotic strains: Lactobacillus plantarum and Lactobacillus kefir. Focusing on Modern Food Industry, 2(3), 138–143. Ribeiro, C., Freixo, R., Silva, J., Gibbs, P., Morais, A. M., & Teixeira, P. (2014). Dried fruit matrices incorporated with a probiotic strain of Lactobacillus plantarum. International Journal of Food Studies, 3, 69–73. Riezzo, G., Orlando, A., D'attoma, B., Guerra, V., Valerio, F., Lavermicocca, P., et al. (2012). Randomised clinical trial: Efficacy of Lactobacillus paracasei‐enriched artichokes in the treatment of patients with functional constipation–a double‐blind, controlled, crossover study. Alimentary Pharmacology & Therapeutics, 35(4), 441–450. Rojas-Graü, M. A., Soliva-Fortuny, R. C., & Martín-Belloso, O. (2009). Edible coatings to incorporate active ingredients to fresh-cut fruits: A review. Trends in Food Science & Technology, 20(10), 438–447. Santagata, G., Mallardo, S., Fasulo, G., Lavermicocca, P., Valerio, F., Di Biase, M., et al. (2018). Pectin-honey coating as novel dehydrating bioactive agent for cut fruit: Enhancements of the functional properties of coated dried fruits. Food Chemistry, 258, 104–110. Sarkar, S., Sur, A., Sarkar, K., Majhi, R., Basu, S., Chatterjee, K., et al. (2016). Probiotics: A way of value addition in functional food. International Journal of Food Science, Nutrition and Dietetics, 5, 290–293. Sarvan, I., Valerio, F., Lonigro, S. L., De Candia, S., Verkerk, R., Dekker, M., et al. (2013). Glucosinolate content of blanched cabbage (Brassica oleracea var. capitata) fermented

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