Accepted Manuscript

Novel functional blueberries: Fructo-oligosaccharides and

probiotic lactobacilli incorporated into alginate edible coatings

María Florencia Bambace, María Victoria Alvarez, María del

Rosario Moreira

PII: S0963-9969(19)30040-7
DOI: https://doi.org/10.1016/j.foodres.2019.01.040
Reference: FRIN 8230
To appear in: Food Research International
Received date: 31 August 2018
Revised date: 26 December 2018
Accepted date: 16 January 2019

Please cite this article as: María Florencia Bambace, María Victoria Alvarez, María del
Rosario Moreira , Novel functional blueberries: Fructo-oligosaccharides and probiotic
lactobacilli incorporated into alginate edible coatings. Frin (2019), https://doi.org/10.1016/
j.foodres.2019.01.040

This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.
 ACCEPTED MANUSCRIPT

Novel functional blueberries: fructo-oligosaccharides and probiotic lactobacilli

incorporated into alginate edible coatings

María Florencia Bambacea,b, María Victoria Alvareza,b*, María del Rosario Moreira a,b

a
Grupo de Investigación en Ingeniería en Alimentos, Facultad de Ingeniería, Universidad

Nacional de Mar del Plata. Argentina

b
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Argentina

*
Corresponding author E-mail address: mvalvarez@fi.mdp.edu.ar

Juan B. Justo 4302, Mar del Plata. Buenos Aires. Argentina.

Tel.: +54 - (0223) 481-6600.

Short title: Functional ready-to-eat fruits

Abstract

Minimally processed fruits are an alternative to dairy products to deliver probiotics. Bio-

protection against several factors that affect their viability has been proposed in the food

industry. In this study, probiotic Lactobacillus rhamnosus CECT 8361 was added to

alginate-based coatings enriched with inulin and oligofructose and applied on fresh-

blueberries. Probiotic viability, microbiological, physicochemical and sensory quality

parameters of blueberries were monitored during 21 days of refrigerated storage. Also, L.

rhamnosus CECT 8361 was tested for its antagonistic effect against inoculated Listeria

innocua and E. coli O157:H7.

1
 ACCEPTED MANUSCRIPT

Advantageously, prebiotic compounds allowed improving probiotic viability with counts

above 6.2 log CFU/g for the entire period. Native microbiota counts remained under safe

levels. Overall visual quality, odor and flavor were acceptable up to day 14 of storage.

Regarding antimicrobial activity, L. rhamnosus CECT 8361 was able to reduce L. innocua

counts by 1.7 log in inoculated blueberries. These results encourage further implementation

of new fruit-based foods with multifunctional properties.

Keywords: functional foods, lactobacilli, antimicrobial effect, sensory acceptability, ready-

to-eat blueberries, probiotic survival.

1. Introduction

The consumption of probiotic foods has increased because of consumer concerns

regarding healthy diets and wellness. Non-dairy probiotic products have drawn attention

due to the growing interest in veganism, as well as to the higher number of consumers with

diet restrictions such as lactose intolerance and allergies to milk proteins (Furtado Martins

et al., 2013). In this way, FAO/WHO (2002) defined probiotics as “live microorganisms

which, when administered in adequate amounts, confer a health benefit on the host”.

Lactobacillus and Bifidobacterium are common genera of the endogenous intestinal tracts

of mammals. They have been widely used as probiotics and are able to stabilise the

intestinal microbiome, inducing host immunomodulation and reducing the symptoms of a

wide range of gastrointestinal disorders (Valcarce et al., 2017). With the objective of

expanding the spectrum of available probiotic products, intensive research efforts have

focused on caring for the viability of probiotic cultures during processing, storage and

2
 ACCEPTED MANUSCRIPT

gastrointestinal transit (Tripathi & Giri, 2014). It has been indicated that supplementing

probiotic foods with non-digestible food ingredients known as prebiotic compounds could

help in the passage through the digestive tract maintaining the viability of bacteria and, in

this way, exert a beneficial effect on human health (Romano et al., 2014). Probiotics and

prebiotic fibers may be carried within edible polymer matrices used in the food packaging

industry. However, the use of films and edible coatings added with probiotics and

prebiotics destined to synbiotic active food packaging has been little explored by

researchers, with limited available information (Espitia, Batista, Azeredo & Otoni, 2016).

Probiotic fortification is a well-established approach to produce dairy-foods with

functional properties. Thereby, probiotic vegetables and fruits are considered a promising

alternative to probiotic dairy products (Gupta & Abu-Ghannam, 2012). In the last years,

several products of plant origin have been suggested for the consumption of probiotic

bacteria including purees, table olives, kimchi, and fermented juices (Di Cagno, Coda, De

Angelis, & Gobbetti, 2013). However, the use of minimally processed fruits as carriers of

beneficial microbes was restricted to a limited range of products including probiotic-

enriched fresh-cut papaya, apple, and pineapple (Alegre, Viñas, Usall, Anguera, & Abadias,

2011; Rößle, Auty, Brunton, Gormley, & Butler, 2010; Russo et al., 2014). Furthermore, to

the best of our knowledge, literature on the applications of probiotics and their interaction

with prebiotic fibers in active food packaging applied on minimally processed berries is not

available. Previous studies reported the probiotic and functional properties of several strains

of Lactobacillus rhamnosus (Grompone et al., 2012; Muñoz-Quezada et al., 2013; Plaza-

Diaz et al., 2013). Particularly, the probiotc strain L. rhamnosus CECT 8361 was studied by

Valcarce et al. (2017) and a significant antioxidant activity was reported. Moreover, these

3
 ACCEPTED MANUSCRIPT

authors analysed the effect of the ingestion of two selected antioxidant probiotics strains

(L.rhamnosus CECT 8361 and Bifidobacterium longum CECT 7347) and reported

significant improvements on male fertility (Robles et al., 2018; Valcarce et al., 2017).

The objective of this work was to analyze the impact of L. rhamnosus CECT 8361

and inulin/oligofructose incorporated into alginate-edible coating on the microbiological,

physicochemical and sensory quality of fresh blueberries. A potential protective effect of

prebiotic fibers on survival of L. rhamnosus CECT 8361 was also evaluated. Also,

probiotic bacteria may be used as an alternative strategy for controlling pathogenic

microorganisms due to their antimicrobial capacity, through bacteriocins, or by competition

in situ (Messaoudi et al., 2013). For these reasons, the incorporation of probiotic bacteria

into alginate-prebiotic fiber coating applied on blueberries and their potential antagonistic

effect on E. coli O157:H7 and L. innocua inoculated in the product was evaluated.

2. Materials and methods

2.1. Plant material

Fresh blueberries (Vaccinium corymbosum L.) were acquired in a wholesale

distributor of local produce in General Pueyrredon, Buenos Aires province (Argentina), and

immediately stored at 5 ± 1 °C for a few hours until processing. A selection based on

uniformity of size and color was made. Fresh fruits at commercial maturity (pH 3.32 ±

0.11; 12.6 ± 0.1 °Brix) were washed using tap water with citric acid at 5 g /L for 30 s,

drained and air-dried for 30 min prior to coating application.

2.2. Preparation of coating solutions

4
 ACCEPTED MANUSCRIPT

Food-grade sodium alginate (No. 18047, Sigma-Aldrich, St Luis, MO, USA) was

used for coating formulation. Glycerol was added to the coatings as a plasticizer. Inulin and

oligofructose were supplied by Saporiti S.A. (Buenos Aires, Argentina). An aqueous

calcium chloride solution (20 g/kg) was used to crosslink biopolymers. To prepare the

coating solutions, sodium alginate (A) at 20 g/kg was dissolved into distilled water by

gently stirring at 70 °C until the solution became clear. Glycerol was used at a

concentration of 15 g/kg. The enrichment with inulin and oligofructose was made in equal

proportion (1:1) using 80 g/kg of each prebiotic fiber in the coating solutions (AF). These

concentrations were selected based on preliminary assays, where the same fibers at

concentrations of 40 and 80 g/kg of each one (1:1) were tested (data not shown). Both

concentrations allowed a good retention of probiotic viability and filled in the requirements

of The Code of Federal Regulations (Title 21, Part 101.54), but the highest concentration

was selected in order to enhance the nutritional value of the proposed food product.

2.3. Incorporation of viable L. rhamnosus CECT 8361 into alginate-coating solution

The freeze-dried Lactobacillus rhamnosus BPL015 (CECT 8361) culture (Biopolis

S.L., Valencia, Spain), commercialized as probiotic strain, was activated (48 h) and then

propagated (24 h) in Man, Rogosa and Sharpe (MRS) broth with L-cysteine·HCl at 0.5 g/L

at 37 °C under anaerobic incubation, using Oxoid gas jars (HP11) and anaerobic gas packs

(Oxoid, Unipath Ltd., U.K.). Bacterial cells were harvested by centrifugation at 6000 g for

15 min at 5 °C as described by Pereira et al. (2016). To formulate alginate-probiotic (AP)

and alginate-prebiotic fiber-probiotic (AFP) coatings, A and AF were mixed aseptically

with the biomass sediment of L. rhamnosus CECT 8361 at 50 g/kg to obtain suspensions of

about 109 colony forming units (CFU) per mL.

5
 ACCEPTED MANUSCRIPT

2.4. Pathogenic culture and inoculation

The bacterial strains used in this work were non-toxigenic Escherichia coli

O157:H7 FP605/03 provided by Malbran Institute (Buenos Aires, Argentina) and Listeria

innocua CIP 8011 provided by Faculty of Pharmacy and Biochemistry (University of

Buenos Aires, Argentina). Particularly, L. innocua was selected as a biological indicator

instead of pathogenic L. monocytogenes due to its similar susceptibility to physical,

chemical or thermal procedures. A stock culture was maintained on tryptic soy broth

(Britania, Buenos Aires, Argentina) at 4 °C. Strains were grown at 35-37 °C in Brain–Heart

Infusion (BHI, Britania, Buenos Aires, Argentina) during 24 h. Before each assay, 100 µL

of each culture was added to 9.9 mL of BHI and incubated 24 h at 35-37 °C. This

procedure was repeated twice consecutively in order to achieve cells in stationary growth

phase. To evaluate the antagonistic effect of L. rhamnosus against pathogens, 100 L of E.

coli O157:H7 and L. innocua bacterial suspension were added to AFP solution (final

pathogen concentration of approximately 4-5 log CFU/g), obtaining AFPEc and AFPLi

treatments, respectively.

2.5. Fruit coating

Fruits were coated using AF, AP, AFP, AFPEc and AFPLi dipping solutions. For

this, blueberries were immersed into the chilled (5 °C) solutions for 2 min. The excess of

coating material was allowed to drip off for 1 min before dipping the fruit again for 2 min

in the calcium chloride cross-linking solution. An uncoated sample was dipped into

6
 ACCEPTED MANUSCRIPT

distilled water as a fresh control (C). Treated blueberries were air dried at room temperature

for 30 min. All handling was performed in a biosecurity cabin class II (Bioquell, England)

under laminar airflow to avoid any external contamination.

After being treated, 90 grams of blueberries were packed into 290-mL

polypropylene containers with hinged lid (Boulevares SRL, Cordoba, Argentina) and stored

in darkness at 5 ± 1°C for 21 days. At each sampling day (0, 7, 14 and 21) three containers

of each treatment were removed from storage and used for experimental determinations.

2.6. Microbial quality and fruit decay

The evolution of each microbial population was monitored through 21 days of

refrigerated storage. For this, samples of 10 g of fruit were separated from each container,

placed in a sterile bag, diluted with 90 mL of 1 g/L aqueous solution of peptone and treated

in a stomacher blender during 2 min. Serial dilutions (1:10) of homogenized samples were

made and surface spread. Native microflora was evaluated as follows: mesophilic (MES)

and psychotropic bacteria (PSI) were enumerated on plate count agar (PCA) incubated at

35 °C for 48 h and at 7 °C for 5 days, respectively and yeasts and molds (YM) on yeast–

glucose–chloramphenicol (YGC) medium incubated at 25 °C for 5 days. Also, the

enumeration of E. coli O157:H7 and L. innocua populations was performed on Eosin

Methylene Blue (EMB) agar and Oxford Agar with Oxford Selective Supplement

(CCCFA), respectively. The colonies were counted after incubation at 37 °C for 24-48 h.

All culture medium were from Britania, Buenos Aires, Argentina. Microbial counts were

expressed as log CFU/g of treated blueberries. Determinations were done in duplicate at

each sampling day from three randomly sampled containers of each treatment.

7
 ACCEPTED MANUSCRIPT

The evaluation of fungal decay was performed by visual inspection, removing the

fruits with visible mold growth from containers at each sampling day. Results were

expressed as percentage of berries presenting decay signs in each container, according to

Alvarez, Ponce & Moreira (2018).

2.7. Determination of viable L. rhamnosus CECT 8361

Viable probiotic bacteria in coated berries (with and without fiber) were determined

by sampling three containers of each treatment, preparing appropriate dilutions as explained

in the previous section and plating them in MRS agar supplemented with 0.5 g/L of L-

cysteine·HCl (Iglesias, Abadias, Anguera, Sabata, & Viñas, 2017). Plates were incubated at

37 °C for 24-48 h under anaerobic conditions. Counts were determined in duplicate and

expressed as log CFU/g.

2.8. Surface color and firmness

A colorimeter (CR-400, Minolta, Tokyo, Japan) was used to determine the surface

color of treated blueberries. Color was recorded using the CIE L*a*b* uniform color space.

A white standard plate (Y =94.00, x =0.3158, y =0.3322) was used for calibration. The

color was evaluated in fifteen berries corresponding to three containers per treatment and

sampling date (n=15). Color modification was evaluated through changes in lightness (L*)

and hue angle (h°). The latter was calculated from a* (green/red) and b* (blue/yellow)

chromatic values as h° =tan−1(b*a*-1).

Fruit firmness was measured with a digital penetrometer (FHT-801; Test Equipment

Depot, Melrose, MA, USA) with a 3.5 mm plunger diameter. Maximum strength required

to penetrate the fruit on the calyx side was recorded and expressed as N/cm2. The

8
 ACCEPTED MANUSCRIPT

measurements were made on fifteen fruits corresponding to three containers per treatment

and sampling date (n=15).

2.9. Sensory evaluation

The samples were subjected to a quantitative descriptive analysis. The methodology

was performed according to Alvarez et al. (2018). Briefly, nine panelists of the UNMdP

Food Engineering Group aged 25–50 years old with experience in fruit and vegetable

sensory quality assessment carried out the sensory analysis. An unstructured five-point

scale was used to score the following attributes: overall visual quality (OVQ, from

0=highly spoiled appearance to 5=fresh appearance), odd-odor (from 0=intense odd-odors

to 5=fresh-like odor) and odd-flavor (from 0=intense odd flavor to 5=typical-no odd

flavor). These attributes were defined in previous sessions where judges identified and

discussed them and the final selection was based on the most important defects presented in

blueberries at refrigerated storage. Samples were provided to judges with 3-digit code

numbers in random order to ensure independent evaluations. The sensory attributes were

judged at 0, 7, 14 and 21 days of storage. The limit of acceptance was established on 50%

of the scale (2.5 points).

2.10. Statistical analysis

The experimental design used in this study was completely randomized with two

factors: treatment and storage time. Assays were performed at least in duplicate on three

independent experimental runs. Analysis of variance ANOVA (P <0.05) was performed

and differences between means were determined using the LSD (least significant

9
 ACCEPTED MANUSCRIPT

difference) test with a 95% confidence level. To analyze data, the statistical software

InfoStat (v2013) was used (Universidad de Córdoba, Córdoba, Argentina).

3. Results and Discussion

The main physicochemical features of fresh-blueberries were monitored in order to

determine the impact of the probiotic and prebiotic enrichment on microbial, nutritional and

sensorial quality attributes of the fruit. Also, the protective effect of inulin/oligofructose

(IN/OL) on L. rhamnosus viability was analyzed. Finally, this probiotic strain was tested

for its antagonistic effect against L. innocua and E. coli O157:H7.

3.1. Protective effect of IN/OL enrichment on L. rhamnosus CECT 8361 viability during

storage

In this work we evaluated the putative protective effect of IN/OL on L. rhamnosus viability,

using an alginate-edible coating as vehicle for both components. These mixtures of

probiotics and prebiotic compounds represent a synbiotic that improves the survival and

implantation of probiotic bacteria and, therefore benefits the host. Thus, L. rhamnosus

viability in blueberries with and without IN/OL is presented in Figure 1. The obtained

results showed that there were no significant differences in the initial L. rhamnosus counts

between treatments (AFP and AP) with values in the range of 7.1-7.6 CFU/g. However, up

to the end of storage, viable L. rhamnosus population in blueberries without prebiotic fibers

was in the range of 5 log cycles CFU/g, while berries coated with AFP presented probiotic

counts significantly (p<0.05) higher (6.2 log CFU/g). This effect on L. rhamnosus counts in

10
 ACCEPTED MANUSCRIPT

the final product represents a great advantage since berries with prebiotic fibers could be

considered as the therapeutic minimum to attain health benefits. Several authors reported

encouraging results about the enhancement of probiotics viability due to prebiotic fibers

enrichment on food products. Tang et al. (2015) assessed the incorporation of inulin,

chitosan oligosaccharide (COS), galactooligosaccharide (GOS) and FOS into maize starch-

based edible films for prebiotic purposes and achieved a promoter effect of the growth of

the probiotic bacteria Bifidobacterium infantis ATCC 15697 and Lactobacillus fermentum

ATCC 9398 by in vitro assays. Soukoulis, Behboudi-Jobbehdar, Yonekura, Parmenter &

Fisk (2014) studied gelatin films containing L. rhamnosus GG, E-96666, VTT culture

collection, Espoo, Finland and four selected prebiotic components: inulin, polydextrose,

GOS, and wheat dextrin. These authors reported that inulin was the most effective prebiotic

agent to protect the probiotic during storage. Tavera-Quiroz et al. (2015) developed snacks

of baked green apples coated with methylcellulose containing Lactobacillus plantarum

CIDCA 83114 and FOS (30 g/L) obtaining a good retention after 90 days of storage at 20

°C and 60% RH. Related to juices and another non-dairy beverages, Santos, Andrade &

Gouveia (2017) used commercial pectin and pectin extracted from passion fruit peel as

carriers of L. rhamnosus ATCC 7469 in a fruit beverage and verified that the incorporation

of sucrose increased survival of L. rhamnosus during refrigerated storage of the product.

Also, Krasaekoopt & Watcharapoka (2014) achieved 0.5 and 0.4 log counts higher for L.

acidophilus (LA 5) and L. casei (LC 01) in orange juice added with 15 g/L of GOS

compared to juice without prebiotic fiber at the end of four-week storage.

Benefits of probiotic consumption can be obtained from foods containing at least 6–

7 log CFU/g of viable microorganisms at the end of storage (Mamede da Costa et al., 2017;

Romano et al., 2014; Russo et al., 2014; Russo et al., 2015). In this way, the viability of L.

11
 ACCEPTED MANUSCRIPT

rhamnosus was monitored during 21 days of storage at refrigeration temperatures (5 ± 1

°C). As explained above, the enrichment with IN/OL prebiotic fibers allowed keeping the

L. rhamnosus counts above the required level. Therefore, the coated blueberries (AFP)

could be considered a probiotic food in the proposed storage time (21 days). However,

blueberries without prebiotic addition (AP) contained less viable probiotics than the

minimum level recommended to provide health benefits. Previous works were carried out

by several authors analyzing the viability of probiotic strains on fresh-fruits and juices

along different conditions of storage. Russo et al. (2014) worked with L. plantarum B2

(CECT 8328) and L. fermentum PBCC11.5 (CECT 8448) as probiotic bacteria inoculated

on fresh-cut pineapples in order to obtain a new functional food. With a similar approach,

the same LAB strains were suggested for the preparation of probiotic fresh-cut cantaloupes

(Russo et al., 2015). Alegre et al. (2011) studied fresh-cut apple as carrier of probiotic

strain and reported that the probiotic viability was maintained above recommended levels

until 14 days of storage at 5 °C. In addition, Rößle et al. (2010) applied L. rhamnosus GG

to fresh-cut apple and reported that it contained approximately 108 CFU/g of inoculum over

a 10 day period at 2-4 °C. Mamede da Costa et al. (2017) assessed the viability of L.

paracasei ssp. (L. casei-01, Hansen) in orange juice supplemented with oligofructose (20

g/L). These authors reported that the cell viability remained higher than 10 7 CFU/mL after

28 days of storage, indicating that the orange juices manufactured with probiotic culture

and/or prebiotics may be considered a potential functional food.

The challenge of maintaining the recommended levels of probiotic throughout the

shelf life of a potential fruit-origin product has been taken on by many authors. A study

conducted by Kim, Chae & In (2010) reported that fermented pear puree by Leuconostoc

mesenteroides 51-3 strain isolated from Kimchi showed high viable cell counts (109

12
 ACCEPTED MANUSCRIPT

CFU/g) for two weeks of storage at 4°C. Furthermore, three commercial probiotics (L.

plantarum ATCC 20174, L. casei ATCC 393 and L. rhamnosus ATCC 7469) and an

Iranian native probiotic strain of L. casei (TD4 and T4) were used for the manufacture of

cornelian cherry juice (Nematollahi, Sohrabvandi, Mohammad, & Jazaeri, 2016). Authors

adjusted the pH (from 2.6 to 3.5) to increase the cell viability of the probiotic strain during

cold storage at 4 °C for 28 days. The viability of industrial strains L. rhamnosus and L.

plantarum decreased from the initial number of 8.00 log CFU/mL to 4.24 and 4.20 log

CFU/mL respectively, after 7 days, but the viability of L. casei had a slight increase after

28 days of storage, remaining higher than 8 log CFU/mL. On the contrary, Mousavi,

Mousavi, Razavi, Eman-Djomeh & Kiani (2011), did not obtain viable cells of

Lactobacillus plantarum DSMZ 20174, L. acidophilus DSMZ 20079, L. paracasei DSMZ

15996, and L. delbrueckii DSMZ 20006 after 21 days of storage at 4 °C in pomegranate

juice.

3.2. Microbial quality of coated blueberries

From a microbiological point of view, the addition of probiotic bacteria

significantly affects the dynamic populations of mesophilic, psychrophilic and yeasts and

molds microorganisms (Fig. 2). The initial mesophilic counts observed in samples

inoculated with L. rhamnosus were approximately 3 orders log higher with respect to

samples without probiotic strain (Fig. 2A). These differences could be explained by the

growth of the probiotic in PCA. In accordance with our results, Panigrahi et al. (2004)

reported no significant differences on total counts and LAB counts in water samples during

an aquaculture study using L. rhamnosus JCM 1136. In addition, aerobic growth of L.

rhamnosus species was reported in some studies (Lavari et al.,2015; Zotta et al., 2014).

13
 ACCEPTED MANUSCRIPT

During 21 days of storage, fruits with probiotic strain (AP and AFP) showed

mesophilic and psychrotrophic bacteria counts values significantly (p<0.05) higher than

fruits without probiotic (Figs. 2 A-B). For yeast and molds, counts did not show significant

differences in the initial values obtained for samples with and without probiotic strain.

Nevertheless, during storage, the evolution of these population counts was similar to that

observed for the rest of native microflora, with final values significantly (p<0.05) higher for

blueberries supplemented with probiotic strain (Fig. 2C). This fact could be attributed to the

presence of maltodextrins used as cell viability protector and support material in the

formulation of the commercial freeze-dried probiotic strain. Despite this, after 21 days of

storage, none of native microbiota populations (MES, PSI and YM) exceeded the

maximum limit of microorganisms of 7.00 log CFU/g in minimally processed foods set by

the Spanish Regulation (BOE, 2001). Fungal decay evolution (Fig. 3) may be perfectly

related to the yeast and molds growth observed during storage. None of samples exhibited

decay until day 7, when uncoated blueberries (C) presented a 7% of decay. This inhibition

of coating treatments regarding C on fruit decay may be due to moisture and semi-

permeable gas barrier formed by the coating, resulting in control of microbial growth

(Kumar & Sethi, 2018). Other authors have also reported improvements on these attributes

when they evaluated different coatings on blueberries (Alvarez et al., 2018; Vieira et al.,

2016). Until day 14, all samples showed low levels of fungal decay, while during the third

week of storage those samples with probiotic significantly (p<0.05) increased their visible

mold growth. Fungal decay rate did not present significant differences on behavior for C y

AF samples at day 14 and 21 of storage (Fig. 3). For this reason, the responsibility of the

increment on decay rate of AP and AFP could be attributed to the support material of

freeze-dried probiotic.

14
 ACCEPTED MANUSCRIPT

3.3. Color and texture of coated blueberries

The color parameters and firmness results obtained are presented in Table 1.

Regarding firmness, the application of coatings did not introduce any significant changes

on this attribute, compared to control. In this sense, neither the fiber nor the probiotic were

able to enhance or reduce coated blueberries firmness. Also, no significant (p>0.05)

differences were detected throughout storage. The results obtained for this parameter could

be explained by the fact that variability within a fruit sample may make differences

between treatments more difficult to perceive. The problem of within-batch variability and

even within each fruit is often treated to be minimized as much as possible (Bavay et al.,

2013), but this variability is an accepted fact in these food matrices. Color parameters were

more influenced by storage time than by type of treatment. For both luminosity and hue

angle parameters, the application of coatings had no significant (p>0.05) effect. A

significant (p<0.05) increase was observed on L* on average throughout 21 days of storage,

regardless of the applied treatments. Concerning °Hue, a significant (p<0.05) increase was

shown between day 7 and 21 of storage. The increase of L* is related with changes in the

surface reflection properties of coatings and, in this case, it indicates that there was not loss

of brightness in average on storage. The increase on °Hue from about 279 to 300 shows the

variation of berries color from blue to violet.

In agreement with our results, Russo et al. (2015) reported a minimal and non-

significant color variation when they evaluated the inoculation of Lactobacillus plantarum

B2 (CECT 8328) and L. fermentum PBCC11.5 (CECT 8448) on fresh-cut cantaloupe

during 11 days of storage. In terms of firmness, Martins, Ramos, Martins & Leite Junior

15
 ACCEPTED MANUSCRIPT

(2016) found that the addition of L. rhamnosus did not produce significant changes in fruit

salad texture. Despite this, Rößle et al. (2010) observed a significant firmness loss in both

fresh-cut apples enriched with Lactobacillus rhamnosus GG and control without probiotic,

after the second day of storage.

3.4. Impact of functional coatings on sensory quality of blueberries

The taste and aroma of the food product may be altered by addition of probiotics

strain due to production of different metabolic components. The presence of the probiotic

culture in food product, therefore, should not adversely affect product quality or sensory

properties. Although the microbiological characterization was made, in order for a non-

dairy probiotic product to be able to be proposed as a new functional food, it should firstly

have to be well characterized from a sensory point of view. Thus, Figure 4 shows the

results obtained from the quantitative descriptive analysis of blueberries samples at 0, 7, 14

and 21 days of storage. Initially, the addition of prebiotic fibers and probiotic strain into the

edible coating did not exert changes on any of the sensory attributes studied. At day 7, all

samples maintained high scores for flavor and OVQ and moderate values for odor. After

the second week of storage, only OVQ and odd-odor were assessed. Thus, after 14 days of

storage, fruits with probiotic strain showed good appearance and higher scores of odor and

OVQ than those without probiotic (Fig. 4). Finally, on the last day of sampling (21)

blueberries coated with probiotics were scored below the established limit (2.5 points of

scale). This result can be related to higher fungal decay presented by AP and AFP,

compared to C and AF samples.

In agreement with our results, Nematollahi et al. (2016) reported that the product

containing L. casei TD4 had no off-flavor in the sensory analysis of probiotic cherry juice.

16
 ACCEPTED MANUSCRIPT

Mamede da Costa et al. (2017) studied the sensory acceptance of unfermented orange juice

and found that the addition of prebiotic fiber and probiotic bacteria did not affect the

acceptance (appearance, aroma, flavor, texture and overall impression) of the juice. In a

study carried out by Alegre et al. (2011), the panelists did not observe any off-flavor or off-

odor development in any of the samples of functional apples at the end of storage (14 days),

demonstrating that high concentrations of probiotic bacteria had no effect on the

degradation rate of the sensory properties of the product. Finally, several researchers

proved that probiotic cultures did not affect the overall acceptability of non-dairy products.

Still, these products are yet to come to the market (Panghal et al., 2018).

3.5. Effect of L. rhamnosus CECT 8361 on the survival of E. coli O157:H7 and L. innocua

Apart from presenting beneficial health effects, probiotic microorganisms have also

been related to effects against other undesirable microorganisms when they were inoculated

into foods. Therefore, the antagonistic effect of L. rhamnosus against L. innocua and E. coli

O157:H7 strains were tested to enhance the safety of minimally processed blueberries and

the obtained results are shown in Table 2. L. innocua counts evolution along storage was

different for AFLi and AFPLi. In samples without probiotic, L. innocua counts remained

constant for the entire period, while in co-inoculated samples (pathogen-probiotic) the

viability of this strain was affected by the presence of L. rhamnosus. Hence, a significant

bactericidal effect of L. rhamnosus against L. innocua was observed with reductions up to

1.7 log units during the storage period, but no antimicrobial effect was showed immediately

after coatings application. Contrarily, non-antagonistic effect of L. rhamnosus on the

viability of E. coli was exhibited. Non-significant (p>0.05) differences were observed in E.

coli counts between AFEc and AFPEc samples during storage. Lastly, the impact of E. coli

17
 ACCEPTED MANUSCRIPT

and L. innocua on L. rhamnosus viability was also monitored at each sampling time. As a

remarkable result, co-inoculation probiotic-pathogen did not influence in any case the

growth of L. rhamnosus (data not shown).

Alegre et al. (2011) tested the antimicrobial activity of a probiotic culture (L.

rhamnosus GG ATCC 53103) against the representatives of two potentially pathogenic

species: Salmonella and L. monocytogenes. Results reported by these authors showed that

Salmonella was not affected by L. rhamnosus GG, but the population of L. monocytogenes

was 1 log cycle lower in the presence of the probiotic. With a similar approach, Russo et al.

(2014) studied the effect of L. plantarum B2 (CECT 8328) and L. fermentum PBCC11.5

(CECT 8448) against E. coli O157:H7 and L. monocytogenes on fresh-cut pineapples.

Interestingly, L. plantarum was able to reduce the dynamic populations of both E. coli

O157:H7 and L. monocytogenes, while L. fermentum was effective only against L.

monocytogenes, supporting that antagonism could be a strain or species-depending feature.

The capability of LAB to inhibit pathogen strains was confirmed to be related to the

amounts of metabolites produced by probiotic, such as lactic acid or antimicrobial peptides

(Iglesias et al., 2017). Some authors reported that Gram-positive bacteria are more affected

by lactobacilli than Gram-negative ones (Coman et al., 2014). In addition, bacteriocins have

specific inhibitory activity against Gram-positive bacteria, whereas organic acids are more

effective against Gram-negative pathogens (Marianelli, Cifani, & Pasquali, 2010).

4. Conclusions

In this work, a microbial approach was proposed to produce multifunctional ready-

to-eat blueberries. In this way, our results showed that the addition of L. rhamnosus CECT

8361 strain into alginate-prebiotic fibers solution could be a valuable strategy to improve

18
 ACCEPTED MANUSCRIPT

the quality of fresh blueberries for different reasons. For the first time, minimally processed

blueberries may be suggested as carrier of beneficial microorganisms for the preparation of

a new functional food. Secondly, the prebiotic fibers added to alginate were able to improve

the probiotic viability retention during refrigerated storage. Finally, L. rhamnosus CECT

8361 could enhance the safety of fresh berries due to its antagonistic effect against

pathogens potentially present in refrigerated fruits. As an advantage, the addition of this L.

rhamnosus strain did not affect the sensory acceptability of the coated berries stored for 14

days under refrigerated conditions. We can conclude that, probably, fresh fruits such as

blueberries would be the next category of non-dairy foods where healthy probiotic bacteria

and prebiotic compounds will make their mark.

Acknowledgments

This work was supported by Consejo Nacional de Investigaciones Científicas y

Técnicas, Argentina (CONICET), Agencia Nacional de Promoción Científica y

Tecnológica (ANPCyT) and Universidad Nacional de Mar del Plata, Argentina (UNMDP).

Dr. Empar Chenoll from the Department of Food Biotechnology of Biopolis S.L.

(Valencia, Spain) is acknowledged for providing the probiotic strain used in this study.

19
 ACCEPTED MANUSCRIPT

References

Alegre, I., Viñas, I., Usall, J., Anguera, M., & Abadias, M. (2011). Microbiological and

physicochemical quality of fresh-cut apple enriched with the probiotic strain

Lactobacillus rhamnosus GG. Food Microbiology, 28, 59–66.

Alvarez, M. V, Ponce, A. G., & Moreira, M. R. (2018). Influence of polysaccharide-based

edible coatings as carriers of prebiotic fibers on quality attributes of ready-to-eat fresh

blueberries. Journal of the Science of Food and Agriculture, 98, 2587–2597.

Bavay, C., Symoneaux, R., Maître, I., Kuznetsova, A., Brockhoff, P. B., & Mehinagic, E.

(2013). Importance of fruit variability in the assessment of apple quality by sensory

evaluation. Postharvest Biology and Technology, 77, 67–74.

Coman, M. M., Verdenelli, M. C., Cecchini, C., Silvi, S., Orpianesi, C., Boyko, N., &

Cresci, A. (2014). In vitro evaluation of antimicrobial activity of Lactobacillus

rhamnosus IMC 501®, Lactobacillus paracasei IMC 502® and SYNBIO® against

pathogens. Journal of Applied Microbiology, 117, 518–527.

Di Cagno, R., Coda, R., De Angelis, M., & Gobbetti, M. (2013). Exploitation of vegetables

and fruits through lactic acid fermentation. Food Microbiology, 33, 1–10.

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.

FAO/WHO. (2002). Guidelines for the evaluation of probiotics in food, 1–11.

Furtado Martins, E. M., Mota Ramos, A., Silva Lago Vanzela, E., Stringheta, P. C., de

Oliveira Pinto, C. L., & Martins, J. M. (2013). Products of vegetable origin: A new

alternative for the consumption of probiotic bacteria. Food Research International, 51,

20
 ACCEPTED MANUSCRIPT

764–770.

Grompone, G., Martorell, P., Llopis, S., González, N., Genovés, S., Mulet, A. P., ... &

Foligné, B. (2012). Anti-inflammatory Lactobacillus rhamnosus CNCM I-3690 strain

protects against oxidative stress and increases lifespan in Caenorhabditis elegans.

PloS one, 7, e52493.

Gupta, S., & Abu-Ghannam, N. (2012). Probiotic fermentation of plant based products:

Possibilities and opportunities. Critical Reviews in Food Science and Nutrition, 52,

183–199.

Iglesias, M. B., Abadias, M., Anguera, M., Sabata, J., & Viñas, I. (2017). Antagonistic

effect of probiotic bacteria against foodborne pathogens on fresh-cut pear. LWT - Food

Science and Technology, 81, 243–249.

Kim, D. C., Chae, H. J., & In, M. (2010). Fermentation characteristics of Korean pear

(Pyrus pyrifolia Nakai) puree by the Leuconostoc mesenteroides 51-3 strain isolated

from Kimchi. African Journal of Biotechnology, 9, 5735–5738.

Krasaekoopt, W., & Watcharapoka, S. (2014). Effect of addition of inulin and

galactooligosaccharide on the survival of microencapsulated probiotics in alginate

beads coated with chitosan in simulated digestive system, yogurt and fruit juice. LWT -

Food Science and Technology, 57, 761–766.

Kumar, P., & Sethi, S. (2018). Edible coating for fresh fruit: a review. International

Journal of Current Microbiology and Applied Sciences, 7, 2619–2626.

Lavari, L., Ianniello, R., Páez, R., Zotta, T., Cuatrin, A., Reinheimer, J., ... & Vinderola, G.

(2015). Growth of Lactobacillus rhamnosus 64 in whey permeate and study of the

effect of mild stresses on survival to spray drying. LWT-Food Science and

Technology, 63(1), 322-330.

21
 ACCEPTED MANUSCRIPT

Mamede da Costa, G., De Carvalho Silva, J. V., Dias Mingotti, J., Barao, C. E., Jensen

Klososki, S., & Colombo Pimentel, T. (2017). Effect of ascorbic acid or oligofructose

supplementation on L. paracasei viability, physicochemical characteristics and

acceptance of probiotic orange juice. LWT - Food Science and Technology, 75, 195–

201.

Marianelli, C., Cifani, N., & Pasquali, P. (2010). Evaluation of antimicrobial activity of

probiotic bacteria against Salmonella enterica subsp. enterica serovar typhimurium

1344 in a common medium under different environmental conditions. Research in

Microbiology, 161, 673–680.

Martins, E. M. F., Ramos, A. M., Martins, M. L., & Leite Junior, B. R. de C. (2016). Fruit

salad as a new vehicle for probiotic bacteria. Food Science and Technology

(Campinas), 36, 540–548.

Messaoudi, S., Manai, M., Kergourlay, G., Prévost, H., Connil, N., Chobert, J. M., &

Dousset, X. (2013). Lactobacillus salivarius: Bacteriocin and probiotic activity. Food

Microbiology, 36, 296–304.

Mousavi, Z. E., Mousavi, S. M., Razavi, S. H., Eman-Djomeh, Z., & Kiani, H. (2011).

Fermentation of pomegranate juice by probiotic lactic acid bacteria. World Journal of

Microbiology and Biotechnology, 27, 123–128.

Muñoz-Quezada, S., Chenoll, E., Vieites, J. M., Genovés, S., Maldonado, J., Bermúdez-

Brito, M., ... & Suárez, A. (2013). Isolation, identification and characterisation of three

novel probiotic strains (Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve

CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036) from the faeces of

exclusively breast-fed infants. British Journal of Nutrition, 109, S51-S62.

Nematollahi, A., Sohrabvandi, S., Mohammad, A., & Jazaeri, S. (2016). Viability of

22
 ACCEPTED MANUSCRIPT

probiotic bacteria and some chemical and sensory characteristics in cornelian cherry

juice during cold storage. Electronic Journal of Biotechnology, 21, 49–53.

Panghal, A., Janghu, S., Virkar, K., Gat, Y., Kumar, V., & Chhikara, N. (2018). Potential

non-dairy probiotic products – A healthy approach. Food Bioscience, 21, 80–89.

Panigrahi, A., Kiron, V., Kobayashi, T., Puangkaew, J., Satoh, S., & Sugita, H. (2004).

Immune responses in rainbow trout Oncorhynchus mykiss induced by a potential

probiotic bacteria Lactobacillus rhamnosus JCM 1136. Veterinary Immunology and

Immunopathology,102(4), 379-388.

Pereira, J. O., Soares, J., Sousa, S., Madureira, A. R., Gomes, A., & Pintado, M. (2016).

Edible films as carrier for lactic acid bacteria. LWT - Food Science and Technology,

73, 543–550.

Plaza-Diaz, J., Gomez-Llorente, C., Campaña-Martin, L., Matencio, E., Ortuño, I.,

Martínez-Silla, R., ... & Genovés, S. (2013). Safety and immunomodulatory effects of

three probiotic strains isolated from the feces of breast-fed infants in healthy adults:

SETOPROB study. PLoS One, 8, e78111.

Robles Rodríguez, V., García, V. D., Ramón, V. D., Genovés, M. S., Martorell Guerola, P.,

& Chenoll Cuadros, M. (2018). U.S. Patent Application No. 15/539,542.

Romano, N., Tavera-Quiroz, M. J., Bertola, N., Mobili, P., Pinotti, A., & Gómez-Zavaglia,

A. (2014). Edible methylcellulose-based films containing fructo-oligosaccharides as

vehicles for lactic acid bacteria. Food Research International, 64, 560–566.

Rößle, C., Auty, M. A. E., Brunton, N., Gormley, R. T., & Butler, F. (2010). Evaluation of

fresh-cut apple slices enriched with probiotic bacteria. Innovative Food Science and

Emerging Technologies, 11, 203–209.

23
 ACCEPTED MANUSCRIPT

Russo, P., Chiara, M. L. V., Vernile, A., Amodio, M. L., Arena, M. P., Capozzi, V., …

Spano, G. (2014). Fresh-cut pineapple as a new carrier of probiotic lactic acid bacteria.

BioMed Research International, 2014, 1–9.

Russo, P., Peña, N., de Chiara, M. L. V., Amodio, M. L., Colelli, G., & Spano, G. (2015).

Probiotic lactic acid bacteria for the production of multifunctional fresh-cut

cantaloupe. Food Research International, 77, 762–772.

Santos, E., Andrade, R., & Gouveia, E. (2017). Utilization of the pectin and pulp of the

passion fruit from Caatinga as probiotic food carriers. Food Bioscience, 20, 56–61.

Soukoulis, C., Behboudi-Jobbehdar, S., Yonekura, L., Parmenter, C., & Fisk, I. D. (2014).

Stability of Lactobacillus rhamnosus GG in prebiotic edible films. Food Chemistry,

159, 302–308.

Tang, Y., Xie, F., Zhang, D., Zhu, M., Liu, L., Liu, P., & Gu, C. (2015). Physical properties

and prebiotic activity of maize starch-based functional films. Starch, 67, 124–131.

Tavera-Quiroz, M. J., Romano, N., Mobili, P., Pinotti, A., Gomez-Zavaglia, A., & Bertola,

N. (2015). Green apple baked snacks functionalized with edible coatings of

methylcellulose containing Lactobacillus plantarum. Journal of Functional Foods, 16,

164–173.

Tripathi, M. K., & Giri, S. K. (2014). Probiotic functional foods: Survival of probiotics

during processing and storage. Journal of Functional Foods, 9, 225–241.

Valcarce, D. G., Genovés, S., Riesco, M. F., Martorell, P., Herráez, M. P., Ramón, D., &

Robles, V. (2017). Probiotic administration improves sperm quality in

asthenozoospermic human donors. Beneficial microbes, 8(2), 193-206.

Vieira, J. M., Flores-lópez, M. L., Jasso, D., De Rodríguez, D. J., Sousa, M. C., Vicente, A.

24
 ACCEPTED MANUSCRIPT

A., & Martins, J. T. (2016). Effect of chitosan–Aloe vera coating on postharvest

quality of blueberry (Vaccinium corymbosum) fruit. Postharvest Biology and

Technology, 116, 88–97.

Zotta, T., Ricciardi, A., Ianniello, R. G., Parente, E., Reale, A., Rossi, F., ... & Coppola, R.

(2014). Assessment of aerobic and respiratory growth in the Lactobacillus casei

group. PLOS one, 9(6), e99189.

25
 ACCEPTED MANUSCRIPT

Figure captions

Figure 1. Effect of inulin/oligofructose addition on L. rhamnosus CECT 8361 viability in

alginate-coated blueberries throughout 21 days of refrigerated storage. AP: alginate-

probiotic coated blueberries, AFP: alginate-prebiotic fiber-probiotic coated blueberries.

Different lowercase letters indicate significant differences (p<0.05) between treatments at

each sampling time. Different capital letters indicate significant differences (p<0.05)

through storage time for each treatment.

Figure 2. Evolution of native microbiota of coated blueberries during 21 days of

refrigerated storage.(A) Aerobic mesophilic microorganisms; (B) psychrotrophic bacteria;

(C) yeasts and molds. Treatments: C: uncoated control, AF: alginate-prebiotic fiber coated

blueberries, AP: alginate-probiotic coated blueberries, AFP: alginate-prebiotic fiber-

probiotic coated blueberries.

Figure 3. Fungal decay evolution of coated blueberries throughout 21 days of refrigerated

storage. Treatments: C: uncoated control, AF: alginate-prebiotic fiber coated blueberries,

AP: alginate-probiotic coated blueberries, AFP: alginate-prebiotic fiber-probiotic coated

blueberries.

Figure 4. Changes in sensory attributes of coated blueberries. (A) Overall Visual Quality

(OVQ); (B) odd odor; (C) odd flavor. Treatments: C: uncoated control, AF: alginate-

prebiotic fiber coated blueberries, AP: alginate-probiotic coated blueberries, AFP: alginate-

prebiotic fiber-probiotic coated blueberries.

26
 ACCEPTED MANUSCRIPT

Table 1. Impact of different coatings on blueberries firmness (F) and color parameters (lightness L*,

hue angle h°) throughout 21 days of storage.

Time (days)
Parameter Treatment 0 7 14 21

C 23.3 ± 8.1 23.5 ± 8.7 24.9 ± 5.5 19.6 ± 7.7

AF 17.3 ± 4.4 25.6 ± 9.8 18.4 ± 5.6 19.4 ± 8.5

F NS’
(N/cm2)
AP 19.1 ± 7.2 22.9 ± 8.2 26.0 ± 13.9 25.4 ± 10.7

AFP 18.7 ± 6.1 21.2 ± 6.9 24.3 ± 6.8 15.6 ± 8.1

C 24.69 ± 1.08 26.86 ± 0.75 24.80 ± 1.41 27.75 ± 1.80

AF 26.53 ± 2.25 28.92 ± 2.28 25.02 ± 1.48 27.93 ± 2.70

L*
AP 24.90 ± 1.46 29.62 ± 2.70 27.99 ± 3.09 30.82 ± 3.98 NS

AFP 25.33 ± 2.38 29.46 ± 2.54 27.60 ± 1.74 30.84 ± 1.14

Mean 25.36 ± 0.82a 28.72 ± 1.28bc 26.35 ± 1.68ab 29.33 ± 1.73c

C 300.00 ± 35.40 279.07 ± 22.44 302.96 ± 38.40 268.61 ± 15.46

AF 280.53 ± 20.88 286.47 ± 35.37 280.00 ± 30.82 294.39 ± 45.61

h°
AP 293.48 ± 28.76 269.39 ± 21.40 300.02 ± 45.07 315.62 ± 79.45 NS

AFP 285.53 ± 29.12 284.22 ± 35.63 287.65 ± 33.37 322.89 ± 51.04

Mean 289.88 ± 8.60ab 279.79 ± 7.59a 292.66 ± 10.73ab 300.38 ± 24.39b

NS: treatment was a non-significant factor.

NS’: treatment and time were non-significant factors.
C: uncoated control, AF: alginate-prebiotic fiber enriched coated blueberries, AP: alginate-probiotic
coated blueberries, AFP: alginate-prebiotic fiber-probiotic coated blueberries.

27
 ACCEPTED MANUSCRIPT

Different lowercase letters indicate significant differences between treatments at each sampling time.

28
 ACCEPTED MANUSCRIPT

Table 2: Evolution of L. innocua and E. coli O157:H7 populations alone (AFLi and AFEc)

and co-inoculated with L. rhamnosus CECT 8361 (AFPLi and AFPEc) on alginate-

prebiotic fiber coatings applied on blueberries through 21 days of storage (log CFU/g).

Days of
AFLi AFPLi
storage

0 4.61 ± 0.29ªA 4.46 ± 0.60aA

7 4.63 ± 0.28ªA 3.15 ± 0.21bBC

14 4.45 ± 0.17ªA 2.76 ± 0.03bC

21 4.74 ± 0.03ªA 3.79 ± 0.07bAB

AFEc AFPEc Means

0 4.19 ± 0.35 NS 4.16 ± 0.20 4.18A

7 4.71 ± 0.13 5.46 ± 0.38 5.09BC

14 4.90 ± 0.09 5.47 ± 0.20 5.18C

21 4.53 ± 0.13 4.71 ± 0.28 4.62AB

Different lowercase letters in the same raw indicate significant
differences (p<0.05) between treatments at each sampling time.
Different capital letters in the same column indicate significant
differences (p<0.05) trough storage time, for each treatment.

NS: treatment was a non-significant factor.

29
 ACCEPTED MANUSCRIPT

Highlights

- Functional blueberries were developed by probiotic coating application.

- Prebiotic addition allows maintaining L. rhamnosus counts above 6 log CFU/g until

21 days.

- The coating enriched with L. rhamnosus showed a significative antilisterial effect.

- Sensory and quality attributes remained acceptable until the end of storage.

30
Figure 1
Figure 2
Figure 3
Figure 4