Viability of Lactobacillus fermentum microencapsulated in flavoured alginate beads and added to a gelatine dessert

Journal of Functional Foods 38 (2017) 447–453

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Viability of Lactobacillus fermentum microencapsulated in flavoured alginate beads and added to a gelatine dessert Emma Mani-López, Enrique Jiménez-Hernández, Enrique Palou, Aurelio López-Malo ⇑ Departamento de Ingeniería Química y Alimentos, Universidad de las Américas Puebla, San Andrés Cholula, Puebla 72810, Mexico

a r t i c l e

i n f o

Article history: Received 20 November 2016 Received in revised form 12 September 2017 Accepted 14 September 2017

Keywords: Lactobacillus fermentum Encapsulation Flavoured alginate beads Gelatine dessert

a b s t r a c t The aim of this work was to formulate flavoured alginate beads focussed on maximum viability of Lactobacillus fermentum and evaluate its viability in flavoured beads when added to a mango gelatine dessert during 28 days of storage at 5 °C. Flavoured alginate beads were developed from mango nectar at different pHs and soluble solid contents. Gelatine desserts’ pH, soluble solids, hardness, and adhesiveness were determined while their sensory evaluation was also performed. Maximum encapsulation yield was obtained at pH 4.5 and 13.5 °Brix of mango nectar. Encapsulated L. fermentum maintained higher (p < 0.05) counts than free cells during storage. Gelatine desserts’ pH incremented when free cells were added. Gelatine desserts’ hardness increased (p < 0.05) when flavoured alginate beads were incorporated, meanwhile their adhesiveness increased (p < 0.05) by addition of free cells. Gelatine desserts containing flavoured alginate beads were well rated by sensory panellists. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Human microbiota is a complex habitat where dominant species are mainly influenced by diet. For example, Lactobacillus acidophilus is commonly present in the Western’s people gut whereas these species are not present in Eastern people; Lactobacillus fermentum was the predominant species isolated from healthy elderly Korean man from a longevity village (Park et al., 2015). L. fermentum is a heterofermentative lactic acid bacterium commonly found in some fermented foods. On the other hand, some strains of L. fermentum have shown functional properties similar to probiotics, benefits such as lowering cholesterol (strain F1, Zeng, Pan, & Zhou, 2011), oligosaccharide synthesis (strain CM33, Sriphannam et al., 2012), anti-oxidative and antiinflammatory protecting effects on host cells (strain Lf1, Chauhan et al., 2014), pathogen inhibitory activity and immune-enhancing activity (strain PL9988, Park et al., 2015), as well as reducing severity of symptoms and diminishing respiratory illness in male athÒ letes (strain PCC , West et al., 2011) have been reported. FAO/ WHO. (2002) declared that regular consumption of probiotics in sufficient quantities (106 to 107 CFU/g) contributed to health benefits of the host. Most research about beneficial bacteria has focused on evidencing health benefits of L. fermentum on humans;

⇑ Corresponding author at: Departamento de Ingeniería Química, Alimentos y Ambiental, Universidad de las Américas Puebla, Cholula, Puebla 72810, Mexico. E-mail address: [email protected] (A. López-Malo). https://doi.org/10.1016/j.jff.2017.09.026 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.

however, very few studies are available with regards to developing new food carriers from this lactic acid bacterium. Pharmaceutical and food supplements are the main available products for intake of probiotics; besides, some foods are being developed with this purpose. Ingesting probiotics in foods could be a pleasant way to consume them. The main challenge when probiotics are added to foods is to guarantee probiotic viability until product consumption. Encapsulation can protect probiotics from food components and improve their viability during food manufacturing and storage. Alginate, one of the most studied biopolymers, has shown effective protection of probiotics against food environmental factors. Counts approximately of 5.2 log10 CFU/mL were reported for 5 lactobacilli (Lactobacillus rahmnosus, Lactobacillus salivarus, Lactobacillus plantarum, L. acidophilus, Lactobacillus paracasei) encapsulated in alginate and incorporated to orange juice after 6 weeks of refrigerated storage (4 °C), while their free cells had the same counts after 3 weeks; thus alginate encapsulation provided protection to tested probiotics (Ding & Shah, 2008). Lactobacillus reuteri had better viability when encapsulated in alginate (7.2 log10 CFU/ g) than when L. reuteri free cells (5.8 log10 CFU/g) were added to blackberry jam and stored for 30 days at 5 °C (García-Ceja, ManiLópez, Palou, & López-Malo, 2015). Up to date, few authors suggested that L. fermentum should be added to foods; Martin, LaraVilloslada, Ruiz, and Morales (2013) reported beneficial effects of microencapsulating L. fermentum in alginate or in alginate-starch. Yogurts and fermented milks are main dairy products added with probiotics (as free or encapsulated cells) due to their buffering

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capacity that promotes cell viability in these products until their consumption. However, lactose intolerance and allergy to milk proteins restrict yogurt and fermented milk consumption and in consequence these types of probiotic food products. Other foods have been studied as probiotic carriers such as: fruit and vegetable juices, purees and beverages; soy drinks or desserts; cereal-based desserts; meat products; cassava-flour products; and starchsaccharified drinks (Granato, Branco, Nazzaro, Cruz, & Faria, 2010). In the last decade, some studies proposed adding probiotics (as free cells) to desserts as ice creams (Di Criscio et al., 2010), chocolate mousses (Aragon-Alegro, Alegro, Cardarelli, Chiu, & Saad, 2007; Cardarelli, Aragon-Alegro, Alegro, Castro, & Saad, 2008), coconut flans (Corrêa, Castro, & Saad, 2008), cacaopuddings (Irkin & Guldas, 2011), and chocolate flans (Silva et al., 2012). Very few studies have included encapsulated probiotics; Shah and Ravula (2004) proposed adding lyophilized alginatevegetal oil-calcium capsules to ice cream in order to incorporate probiotics and maintaining them alive until consumption. Gelatine desserts are well accepted by consumers in Mexico and usually marketed under refrigeration conditions, thus they could be good options to include probiotics. Therefore, the aim of this work was to formulate flavoured alginate beads focussed on maximum viability of L. fermentum as well as to evaluate L. fermentum viability in flavoured beads when added to a mango gelatine dessert during storage at 5 °C for 28 days. Selected physicochemical and sensory properties of studied gelatine desserts were also determined. 2. Materials and methods 2.1. Strains, growth conditions and materials L. fermentum NRRL B-1917 was acquired in lyophilised form from the USDA (Agricultural Research Service, Peoria, Illinois, USA), which was activated and routinely sub-cultured in de-Man Rogosa and Sharpe (MRS, DifcoTM BD, Sparks, Maryland, USA) broth at 37 °C for 24 h. Biomass production was obtained from L. fermentum activated and grown in MRS broth (500 mL) at 37 °C for 20 h under anaerobic conditions. Cells were harvested by centrifugation at 8000g for 10 min at 5 °C and washed twice with phosphate buffer (1 mol/L, pH 7.0). Cell pellet was suspended in commercial pasteurized mango nectar (pH 3.5, 13.5 °Brix) and subjected to microencapsulation as described below. Sodium alginate was acquired from FMC Biopolymer (Haugesund, Rogaland, Norway), and calcium chloride from RBM (Puebla, Puebla, Mexico) while mango nectar and gelatine powder (mango flavour) were acquired from a local supermarket.

at 5 cm from a 150 mL of sterile calcium chloride (0.1 mol/L) solution using a sterile syringe (needle size 0.70
0.32 mm) and left for curing (1 h) at 5 °C. Alginate beads were then drained and washed with sterile distilled water and then placed on sterile filter paper to remove water excess. Beads were kept in refrigeration until being used but no more than two hours. Thirty beads were taken in order to determine their size with a digital micrometre (Mitutoyo Corporation, Kanagawa, Japan). Flavoured alginate beads were measured only before they were added to the gelatine dessert. 2.3. Preparation of studied gelatine desserts A commercial gelatine dessert powder (35 g, mango flavour) was dissolved in boiling water (1 L) following the manufacturer’s instructions. The commercial gelatine dessert powder ingredients were gelatine, acidulant, non-caloric sweeteners (aspartame and potassium acesulfame), artificial and natural flavours, sodium chloride, ascorbic acid, as well as artificial and natural dyes (sunset yellow and tartrazine). The homogeneous blend was then cooled to 30 °C. Two grams of flavoured alginate beads and 18 g of gelatine solution were placed in polystyrene cups (20 mL); mixtures were refrigerated for 5 min, and then stirred for complete dispersion of beads into gelatine. Cups containing the mixture were closed with a polystyrene cover. Gelatine cups were stored and maintained at 5 °C for 28 days and every 7 days, 8 gelatine cups were removed for analyses. Fig. 1 exhibits a gelatine dessert added with flavoured alginate beads containing L. fermentum. Furthermore, in order to evaluate the viability of free cells in studied gelatine desserts and their effects on gelatine desserts’ properties, gelatine desserts containing L. fermentum free cells were also prepared. Biomass was prepared as previously described, cell pellet was suspended in mango nectar and 2 g of cell suspension were placed into cups while 18 g of gelatine were poured, mixed, closed with polystyrene cover, and refrigerated as previously described. Eight of these cups were removed for analysis every 7 days for 28 days. 2.4. Determination of microbial viability To determine L. fermentum’s viability before and after the encapsulation process, one gram of dispersion (free cells + alginate + mango nectar) was taken or one gram of washed beads were grounded, and diluted in 9 mL of sterile peptone (Merck, Darmstadt, Germany) water (0.1 g/100 mL). Appropriate dilutions were platted on MRS agar (DifcoTM BD, Sparks, Maryland, USA). Plates were incubated anaerobically at 37 °C for 48 h. Encapsulation yield (EY) was determined using the following formula:



2.2. Formulation of flavoured alginate beads with L. fermentum

%EY ¼ Flavoured alginate beads were developed in order to provide an adequate taste to beads and therefore enhance the gelatine dessert taste where beads will be incorporated. Thus, commercial mango nectar was used as alginate solvent as well as cell pellet suspending medium. Adjustments in pH and soluble solids of mango nectar were investigated in order to locate the highest viability of L. fermentum during the encapsulation process. The pH of mango nectar was adjusted with citric acid (20 g/100 mL) or NaOH (10 mol/L) solutions up to 3.0 and 4.5. Soluble solids were adjusted with sucrose at 20 °Brix. In addition, mango nectar without pH and soluble solids’ adjustments was also tested. One gram of alginate was dissolved in 50 mL of mango nectar at 75 °C under stirring, when alginate was completely dissolved, the blend was sterilized and cooled to 35 °C and combined with 50 mL of mango nectar containing suspended cell pellet, and then mixed for 10 min (alginate final concentration was 1 g/100 g of nectar). This mixture was dropped

N N0



 100

Fig. 1. Gelatine dessert contained flavoured alginate beads.

ð1Þ

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where N is the number of viable encapsulated cells released from beads and N0 is the number of free cells in the alginate dispersion using mango nectar as solvent, during production of the beads. To determine L. fermentum viability in studied gelatine desserts, three gelatine cups containing flavoured alginate beads and three gelatine cups containing free cells were removed from the refrigerator and heated on a water bath up to 25 °C; then one gram of beads was separated from gelatine and washed with sterile water. Beads were grounded and diluted in 9 mL of sterile peptone water (0.1 g/100 mL). For gelatine containing free cells, one gram of liquid gelatine was taken. Appropriate dilutions were platted on MRS agar. Plates were incubated anaerobically at 37 °C for 48 h. 2.5. pH, soluble solids and texture analysis of gelatine desserts These tests were carried out for gelatine desserts added with free or microencapsulated cells of L. fermentum as well as for gelatine desserts without free cells or beads. After gelatine desserts were melted in a water bath, pH was measured (by triplicate) using a pH-meter by electrode immersion (Oakton Instruments pH 700, Vernon Hills, IL, USA). Soluble solids were determined (by triplicate) using a digital refractometer (AR200, Reichert Inc., Depew, NY, USA). For the texture tests, a texture analyser (Stable Micro Systems, Godalming, UK) was utilized to determine texture profile analysis (TPA) by triplicate. Solid gelatine desserts were removed from cups and cylinders of 20 mm in diameter and 10 mm in height were cut and then refrigerated for one hour. A plate of stainless steel (35 mm in diameter) was moved at a test speed of 1 mm/s from the gelatine surface until a compression of 25% was achieved. TPA parameters were obtained with the Texture Expert Software for the TA.TX2 Texture Analyser. 2.6. Sensory evaluation of gelatine desserts Sensory tests were carried out with gelatine desserts after 24 h of refrigeration. Twenty untrained panellists evaluated flavour, texture, colour and overall acceptability. Consumer panellists were selected from university staff and students, who regularly consumed gelatine desserts. A 1–9 hedonic scale was used to evaluate studied samples in which a score of 1 represents the attributes most disliked and a score of 9 represents the attributes most liked. Gelatine desserts with or without beads were evaluated and asked for preference in the hedonic scale. Before the tests were performed, panellists were informed about the gelatine dessert added with flavoured alginate beads, as well as possible benefits of ingestion of lactic acid bacteria. Scores around 6 were considered acceptable; furthermore, comments from panellists about the products were encouraged. 2.7. Statistical analysis Statistical analysis of the data was performed by ANOVA and Tukey’s mean comparison tests (p < 0.05) using MINITAB statistical package (ver. 16), to identify significant differences in lactobacilli viability, pH, °Brix and TPA and sensory evaluation parameters. 3. Results and discussion 3.1. Flavoured alginate beads formulation The pH and soluble solids of assessed mango nectar were 3.5 and 13.5 °Brix, respectively. Beads were well-formed at every examined condition, so tested acidic media and soluble solids did not affect formation of alginate beads. No published reports

regarding juice or nectar based alginate beads were found. However, Marcotte, Taherian, and Ramaswamy (2000) formulated alginate particles using carrot puree; they reported that the critical formation step of particles was the alginate/calcium chloride ratio; since high alginate concentrations and low CaCl2 concentrations resulted in no gel network formation (no particles were formed). Table 1 presents encapsulation yield for L. fermentum from different mango nectar-alginate pHs and soluble solid contents. Encapsulation yield was among 68.7 and 91.9% for tested conditions. The best encapsulation yield was obtained from mango nectar at pH 4.5 and 13.5 °Brix. Low pH and 20 °Brix reduced microbial viability during tested encapsulation process. L. fermentum’s viability was strongly affected by low pH, especially when the pH was equal or lower than 3.5. Close to 30% reductions were observed due to low pHs. Acid tolerance depends on the lactobacilli strain; a previous report observed unchanged counts of L. fermentum KC5 b when cells were maintained in acid conditions at pH 2.0 after 2 h (Pereira & Gibson, 2002). While, other report showed poor viability in acidic media (pH=3.5, such as fruit juices) of some beneficial Lactobacillus (Ding & Shah, 2008). Our encapsulation yields were similar to those reported for L. fermentum CECT5716 microencapsulated in alginate (74.41 ± 1.76%) or in alginate-starch (97.26 ± 0.33%) by Martin et al. (2013). Alginate dispersed in mango nectar at 4.5 and 13.5 °Brix (92% of encapsulation yield) was used as the formulation for beads that were added to studied gelatine desserts. Flavoured alginate beads were spherical in shape with an average diameter of 1.63±0.03 mm. Some studies reported larger alginate beads’ diameters; Muthukumarasamy, Allan-Wojtas, and Holley (2006) observed diameters of 2.37 mm for selected alginate concentrations; García-Ceja et al. (2015) reported diameters of 2.06– 2.10 mm for alginate (3 g/100 mL) beads containing L. acidophilus or L. reuteri. Alginate (1 g/100 mL) beads seasoned with 1.0% mackerel tuna-based flavouring solution contained Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis DN-173 010 had an average size of 2.8 ± 0.05 mm (Guimarães, Vendramini, Santos, Leite, & Miguel, 2013); alginate concentrations and needle size influenced bead size.

3.2. Microbial viability on studied gelatine desserts Fig. 2 shows viable counts of L. fermentum when added to gelatine desserts as free or encapsulated cells, during refrigerated storage (5 °C). Microencapsulated cells maintained 1.7 log10 CFU of L. fermentum/g more than free cells when they were added into gelatine desserts; furthermore, this difference was still significant (p < 0.05) after 28 days of storage. L. fermentum counts above 106 CFU/g in the gelatine desserts were maintained for 14 or 21 days, in the gelatine desserts with free or encapsulated cells, respectively. The observed cell viability reductions could be attributed to scarce nutrients, acid pH, low temperature, and tested strain; since fermentable sugars were not available in studied gelatines, pH were 4.5, and L. fermentum does not grow at 15 °C (Sneath, Table 1 Encapsulation yield of Lactobacillus fermentum at different pHs and soluble solid contents for flavoured alginate beads. Initial count was 10.41 ± 0.29 log10 CFU/mL. pH

Soluble solids (°Brix)

% Encapsulation yield

3.0 3.0 3.5 3.5 4.5 4.5

13.5 20.0 13.5 20.0 13.5 20.0

68.7 ± 2.04d 70.9 ± 1.19 cd 75.6 ± 0.39c 69.5 ± 3.24 cd 91.9 ± 0.40a 83.4 ± 1.20b

Data followed by different letters are significantly different (p < 0.05).

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10

a, A

Beads Free-cells

a, A

3.3. Physicochemical properties of studied gelatine desserts

b, A c, A

8

b, B d, A

-1

Log N (CFU g )

b, B

c, B

6

e, A

d, B 4

2

0 0

had poor acid tolerance and did not growth at 5 °C; as a result, loss of viability was observed.

7

14

21

28

Time (day) Fig. 2. Viability of Lactobacillus fermentum (in flavoured alginate beads or as free cells) added to a gelatine dessert (mango flavour) during refrigerated storage (5 °C). Data followed by different lowercase letters are significantly different (p < 0.05) when comparing viability during storage. Data followed by different capital letters are significantly different (p < 0.05) when comparing viability of encapsulated (beads) or free cells.

Mair, Sharpe, & Holt, 1986). Very few published reports regarding L. fermentum’s encapsulation or entrapment were found; likewise, no published reports concerning addition of encapsulated L. fermentum to foods were found. Martin et al. (2013) entrapped cells of L. fermentum in alginate-starch micro-particles that were stored 45 days at 4 °C; they observed constant counts (around 9.3 log10 CFU/g) during storage and refer that alginate-starch microparticles maintained better viability than alginate ones. Differences between their results and the ones from our study could be due to strain differences and tested entrapment methods. Viability of other beneficial lactic acid bacteria encapsulated in alginate added to a dessert have been reported by Borges, Ferreira, and Costa (2004); after 20 days of storage they observed 3 or 2 log10 CFU/g reductions for free or encapsulated cells (respectively) of L. acidophilus in a calcium-alginate matrix added to chocolate mousses. Most published studies with reference to lactic acid bacteria incorporated to desserts (coconut flan, cacao pudding, chocolate mousse, etc.) reported addition of free cells; thus very few data are available to compare our results with encapsulated cells. L. paracasei LBC 82 maintained constant counts of 7.27 log10 CFU/g after 28 days at 4 °C when was added as free cells to a chocolate mousse (Cardarelli et al., 2008). Increments of 2 log10 CFU/g in population of L. paracasei LBC 82 was reported when free cells were added to a coconut flan and stored at 5 °C for 28 days (Corrêa et al., 2008). After 15 days at 4 °C, Lactobacillus casei decreased 1.2 log10 when incorporated as free cells to a chocolate flan (Silva et al., 2012). Reductions around 2.7 log10 CFU/g were observed for B. animalis subsp. lactis LAFTI 94 or L. casei LAFTI L26 as free cells incorporated in cacao puddings, after their storage for 25 days at 4 °C (Irkin & Guldas, 2011). In our study, reductions were higher than those reported, despite encapsulation of L. fermentum. Certainly, chocolate mousse, cacao pudding and coconut/chocolate flans are milk-based desserts and rich in sugars; favouring the viability or growth of cells due to milk’s buffering capacity and available nutrients. Also, these milk-based desserts had a pH between 5.6 and 6.1; thus, tested lactic acid bacteria maintained good viability at this pH range. In the present study, L. fermentum strain

3.3.1. pH Table 2 presents the pH values of gelatine desserts during refrigerated storage. The original gelatine dessert had a pH of 4.42, which was 4.41 when added with flavoured alginate beads, while free cells addition increased its pH to 4.55, due to cells and mango nectar pH (4.5). In general, pH remained constant during refrigerated storage up to 21 days for the gelatine dessert alone or with flavoured alginate beads; while after 28 days, the pH of the gelatine dessert without beads slightly decreased. Regarding the gelatine dessert with free cells, it increased its pH during storage, probably due to partial hydrolysis of gelatine’s protein by L. fermentum. Lactic acid bacteria are recognized by their proteolytic ability (Sneath et al., 1986); hence low quantities of ammonium could be released. Therefore, the best option to add L. fermentum into the studied gelatine dessert is in beads. Furthermore, an unpleasant appearance (turbid gel referred by panellists as ‘‘milky”) when free cells were added was observed. Other authors observed minimal or null pH changes when adding probiotic Lactobacillus to selected desserts; Corrêa et al. (2008) observed a decrement of pH from 6.58 to 6.05 in coconut flan added with L. paracasei after 28 days of refrigerated storage, authors attributed this reduction in pH to L. paracasei metabolic activity. A pH reduction (6.6 to 5.6) was also reported for cacao puddings added with free cells of L. casei but not with free cells of L. acidophilus when they were stored for 25 days at 4 °C; L. casei grew, thus pH of puddings diminished (Irkin & Guldas, 2011). Therefore, pH values during storage of desserts depend on the tested microorganism’s metabolic activity and its ability to use sugars and proteins of the corresponding food matrix. 3.3.2. Soluble solids Soluble solids of studied gelatine desserts during refrigerated storage are presented in Table 2. Soluble solids’ content was constant for the gelatine dessert alone and gelatine dessert added with flavoured beads during their storage (p > 0.05). Soluble solids of gelatine dessert containing free cells slightly decreased during storage, maybe due to a scarce metabolic activity that consumed some sugars. There were not significant differences (p > 0.05) in soluble solids among the three studied gelatine desserts. Previous studies with desserts added with Lactobacillus did not include soluble solids analysis of food matrices. However, it is important to evaluate this property in order to know the effect of the tested bacteria in the studied food. 3.3.3. Hardness and adhesiveness Texture is a complex parameter that involves structural organization of micro- and macro-molecular interactions. Gelatine protein usually forms gels with cross-linked flexible macromolecules (by a continuous colloidal tri-dimensional network), and it is the interaction between the colloidal network and the medium that determines the mechanical behaviour and stability of the system and consequently its properties (Rosenthal, 1999). Gel analyses by TPA are useful in order to understand gel texture based on the force/deformation curve generated by the compression test (Rosenthal, 1999). In this study, hardness and adhesiveness were selected to understand studied gelatine desserts’ changes due to addition of free cells or beads. Hardness is an important property of foods that represents the ‘‘force required for compressing a material between tongue and palate”, and is defined as the ‘‘maximum force required to compress the sample” (Bourne, 1978). Hardness of studied gelatine desserts is displayed in Fig. 3.

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E. Mani-López et al. / Journal of Functional Foods 38 (2017) 447–453 Table 2 Changes in pH and soluble solid contents of studied gelatine desserts during storage at 5 °C. Storage (day)

pH

0 7 14 21 28

Soluble solids (°Brix)

Gelatine dessert

Gelatine + Lactobacillus fermentum free cells

Gelatine + flavoured alginate beads containing L. fermentum

Gelatine dessert

Gelatine + L. fermentum free cells

Gelatine + flavoured alginate beads containing L. fermentum

4.42 ± 0.01a,B 4.39 ± 0.01a,B 4.43 ± 0.06a,B 4.38 ± 0.01a,B 4.25 ± 0.02b,C

4.55 ± 0.02c,A 4.70 ± 0.03ab,A 4.69 ± 0.02ab,A 4.68 ± 0.05b,A 4.76 ± 0.02a,A

4.41 ± 0.02ab,B 4.39 ± 0.02b,B 4.41 ± 0.02ab,B 4.39 ± 0.01b,B 4.44 ± 0.02a,B

4.00 ± 0.20a,A 4.13 ± 0.15a,A 4.17 ± 0.06a,A 4.07 ± 0.21a,A 4.03 ± 0.06a,A

4.27 ± 0.23a,A 4.07 ± 0.12b,A 4.17 ± 0.15ab,A 4.07 ± 0.12b,A 4.03 ± 0.06b,A

4.07 ± 0.15a,A 4.10 ± 0.17a,A 4.10 ± 0.20a,A 4.13 ± 0.06a,A 4.13 ± 0.06a,A

Data followed by different lowercase letters in the same column are significantly different (p < 0.05) when comparing pHs or soluble solid contents during storage. Data followed by different capital letters in the same row are significantly different (p < 0.05) when comparing studied gelatine desserts.

Gelatine dessert Gelatine dessert + free-cells Gelatine dessert + flavoured alginate beads a,A a,A a,A a,A

8

a,A

Hardness (N)

6 a,AB

a,B

a,B

4

a,A a,B

a,A b,A

a,B a,A 2

a,C

0 0

7

14

21

28

Tiime (day) Fig. 3. Hardness values during refrigerated storage (5 °C) of studied gelatine desserts. Data followed by different lowercase letters are significantly different (p < 0.05) when comparing different times of storage. Data followed by different capital letters are significantly different (p < 0.05) when comparing gelatine dessert, gelatine dessert + Lactobacillus fermentum free cells, or gelatine dessert + flavoured alginate beads containing L. fermentum.

Hardness values were 1.47–3.17 N for the gelatine dessert alone, 3.76–4.40 N for gelatine dessert added with L. fermentum free cells, and 6.80–1.95 N for gelatine dessert added with flavoured alginate beads containing L. fermentum. Storage time had no significant (p > 0.05) effect on hardness of gelatine desserts without L. fermentum. Hardness was strongly affected (p < 0.05) by free cells (76% more) and flavoured alginate beads (176% more); thus, higher hardness values were observed when they were incorporated to gelatine desserts. We expected important changes in textural parameters because cell and beads modified the cross-linked pattern of gelatine protein gel. Typical gelatine gels are soft and vibrating, but incorporated cells and beads seem to toughen gels, probably due to a reduction in the amount of porous in network and an increment of solid structures (cells and beads). Even more, alginate beads are harder than gelatine; therefore, gelatine desserts added with alginate beads increased the force necessary to compress them. Gelatine desserts’ hardness values of our study were similar to the ones of gelatine gels with 2 g of gelatine protein/100 mL (5.0 N) reported by Brewer, Peterson, Carr, McCusker, and Novakofski (2005) and to the ones of 6.5 g of fish gelatine protein/100 mL (5.88 N) reported by Boran, Lawless, and Regenstein (2010). A commercial gelatine dessert has approximately 2–3 g/100 mL of gelatine protein in order to form the typical gel. On the other hand, gelatine protein – alginate have

shown good interactions and biocompatibilities for different applications such as films from combined biopolymers (Xiao, Liu, Lu, & Zhang, 2001) and for gelatine beads coated with alginate (DíazBandera, Villanueva-Carvajal, Dublán-García, Quintero-Salazar, & Dominguez-Lopez, 2013). Hardness during storage was higher for gelatine desserts added with beads than for gelatine desserts alone from the beginning of storage and up to 21 days; later, a brittle gel structure was observed. On the other hand, gelatine desserts added with L. fermentum free cells presented an unclear trend during storage; as mentioned above, increases in pH values suggested probably the partial hydrolysis of proteins; however, this trend cannot be related to desserts’ softening. Probiotic cells (L. paracasei) increased hardness of chocolate mousse up 12% when free cells were added, and 36% when free cells plus inulin were added to mousse (Cardarelli et al., 2008). Few published studies are available with regards to textural effects when free cells or beads are incorporated into foods. However, they are very important due to their effect in several food product quality parameters. Adhesiveness represents the ‘‘force required removing the material that adheres to the mouth, generally the palate, during the normal eating process”, and during the TPA test it is ‘‘the negative area of the curve obtained when the plate returns to its original position” (Bourne, 1978). Fig. 4 presents adhesiveness of studied gelatine desserts during storage. At the beginning of storage, free cells addition increased the absolute value of adhesiveness up to 4 times, from 1.2 to 4.5 N s but during storage unclear trends were observed. Meanwhile, incorporation of alginate beads decreased these values during gelatine desserts’ storage 2 times. In general, gelatine desserts added with free cells maintained adhesiveness values close to the ones of gelatine desserts alone. Published reports suggest that Lactobacillus cells interaction with milk proteins are strong and specific, and factors such as tested strain, pH, and nature of the proteins influenced their interactions (Burgain et al., 2013). It is possible that gelatine’s proteins also had strong interactions with L. fermentum cells and allowed to form a sticky gel with similar adhesiveness to the gelatine gel; however, further analyses and specific studies about gelatine – L. fermentum interactions are still necessary. Meanwhile, studied flavoured beads formed greater discontinuities in the gelatine network due to their size and resulted in low adhesiveness of corresponding gelatine desserts. Our gelatine desserts had higher values of adhesiveness compared with those reported (1.10 Ns) for gelatine (7 g/100 mL) – modified corn starch (3 g/100 mL) gels by Marfil, Anhê, and Telis (2012); in addition, these authors observed that increments in gelatine concentration decreased gels’ adhesiveness. Increased adhesiveness was reported for chocolate mousses added with free cells of L. paracasei (3.6%) or L. paracasei and inulin (10.7%) by Cardarelli et al. (2008). 3.3.4. Sensory evaluation Table 3 presents sensory average scores of studied gelatine desserts. No significant differences (p > 0.05) were observed in the

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the sensory profile was not changed in studied gelatine desserts when flavoured alginate beads where added, probably due to the information provided before the sensory test and the age of our panellists.

1 Gelatin dessert Gelatin dessert + free cells Gelatin dessert + flavoured alginate beads 0

Adhesiveness (N·s)

4. Conclusions

a,A

-1

a,A

a,A

a,A a,A

-2 b,B a,A b,AB

-3

a,B

a,C -4

-5

a,B 0

c,B 7

a,B 14

a,B

c,B

21

28

Time (day) Fig. 4. Adhesiveness values during refrigerated storage (5 °C) of studied gelatine desserts. Data followed by different lowercase letters are significantly different (p < 0.05) when comparing different times of storage. Data followed by different capital letters are significantly different (p < 0.05) when comparing gelatine dessert, gelatine dessert + Lactobacillus fermentum free cells, or gelatine dessert + flavoured alginate beads containing L. fermentum.

Flavoured alginate beads were successfully developed and high counts of L. fermentum were encapsulated. Favourable conditions for mango nectar-alginate dispersion were pH 4.50 and 13.5 °Brix for maximum encapsulation yield of L. fermentum. Microencapsulation of L. fermentum in flavoured alginate beads maintained its viability after 21 days at 6.7 log10 CFU/g when added to the studied gelatine dessert and stored at 5 °C; viable counts were higher than when free cells of L. fermentum were added to the gelatine dessert. Gelatine desserts’ pH added with L. fermentum free cells slightly increased, possibly due to L. fermentum proteolytic activity on gelatine protein. Soluble solids content of studied gelatine desserts was unchanged during storage. Hardness and adhesiveness of the studied gelatine dessert were modified by addition of either L. fermentum free cells or flavoured beads containing L. fermentum. Gelatine desserts with flavoured alginate beads were well accepted by sensory panellists. The studied gelatine dessert could be a good carrier to incorporate flavoured alginate beads containing L. fermentum. Acknowledgments

Table 3 Sensory scores of studied gelatine desserts after 24 h of refrigeration. Attribute

Gelatine dessert

Gelatine dessert + flavoured alginate beads containing Lactobacillus fermentum

Flavour Texture Colour Overall acceptability

7.89 ± 0.89a 7.55 ± 0.88a 7.70 ± 0.92a 7.80 ± 0.69a

7.52 ± 0.96a 7.80 ± 0.89a 7.55 ± 0.88a 7.95 ± 0.82a

Data followed by different letters in the same row are significantly different (p < 0.05).

preference of the evaluated attributes of gelatine desserts. These unexpected results could be due to the panellists were informed about gelatine desserts’ characteristics before evaluating them. Young panellists (less than 25 years old) gave higher scores to the gelatine dessert with beads than older panellists. These findings are in agreement with Krasaekoopt and Kitsawad (2010) whom found that young people are willing to experience new flavours and textures. Some thought-provoking comments about the gelatine dessert added with flavoured alginate beads include ‘‘interesting and different texture, intensely flavoured, beads’ fermented taste, and beads ensure lactic acid bacteria content”. Different results have been previously reported; microencapsulated (in alginate) L. reuteri added to dry sausages did not change the sensory profile of sausages (Muthukumarasamy & Holley, 2006); however, other authors reported sensory rejection of milk, peach nectar (García-Ceja et al., 2015) or yogurt (Ortakci & Sert, 2012) containing alginate beads (with L. acidophilus); furthermore, an improved sensory profile (including textural attributes) of mayonnaise was reported when alginate capsules with Bifidobacterium bifidum were added to it (Khalil & Mansour, 1998). The addition of ‘‘large” beads to foods is a concern due to possible rejection by consumers; however, sensory evaluation results will depend on type of food and the nature of the sensory panel (e.g., panellists’ age, expectations, etc.) among other factors. Moreover, an informed consumption renders more objective food evaluations. In our case,

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