or milk-based synbiotic apple ice creams

    In vitro gastrointestinal resistance of Lactobacillus acidophilus La-5 and Bifidobacterium animalis bb-12 in soy and/or milk-based synbiotic apple ice creams Natalia Silva Matias, Marina Padilha, Raquel Bedani, Susana Marta Isay Saad PII: DOI: Reference:

S0168-1605(16)30330-0 doi: 10.1016/j.ijfoodmicro.2016.06.037 FOOD 7284

To appear in:

International Journal of Food Microbiology

Received date: Revised date: Accepted date:

15 December 2015 17 June 2016 26 June 2016

Please cite this article as: Matias, Natalia Silva, Padilha, Marina, Bedani, Raquel, Saad, Susana Marta Isay, In vitro gastrointestinal resistance of Lactobacillus acidophilus La-5 and Bifidobacterium animalis bb-12 in soy and/or milk-based synbiotic apple ice creams, International Journal of Food Microbiology (2016), doi: 10.1016/j.ijfoodmicro.2016.06.037

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 In vitro gastrointestinal resistance of Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 in soy and/or milk-based synbiotic apple ice

SC RI

PT

creams

Natalia Silva Matiasa, Marina Padilhaa, Raquel Bedania, Susana Marta Isay

a

Department

of

Biochemical

and

NU

Saada*

Pharmaceutical

Technology,

School

of

PT

ED

05508-000 São Paulo, SP, Brazil

MA

Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 580, B16,

CE

*Corresponding author at: Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, Av. Prof.

AC

Lineu Prestes, 580, B16, 05508-000 São Paulo, SP, Brazil. Phone: +55 11 3091-2378; Fax: +55 11 3815-6386. e-mail adress: [email protected] (S.M.I. Saad).

1

ACCEPTED MANUSCRIPT Abstract The viability and resistance to simulated gastrointestinal (GI) conditions of

T

Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 in synbiotic ice

IP

creams, in which milk was replaced by soy extract and/or whey protein isolate (WPI)

SC R

with inulin, were investigated. The ice creams were showed to be satisfactory vehicles for La-5 and Bb-12 (populations around 7.5 log cfu/g), even after the whole storage

NU

period (84 days/-18°C). In all formulations, the propidium monoazide qPCR (PMAqPCR) analysis demonstrated that probiotics could resist the in vitro GI assay, with

MA

significant survival levels, achieving survival rates exceeding 50%. Additionally, scanning electron microscopy images evidenced cells with morphological differences,

D

suggesting physiological changes in response to the induced stress during the in vitro

TE

assay. Although all formulations provided resistance to the probiotic strains under GI

CE P

stress, the variation found in probiotic survival suggests that GI tolerance is indeed

AC

affected by the choice of the food matrix.

Keywords: probiotic, prebiotic, in vitro gastrointestinal survival, propidium monoazide qPCR, functional food, SEM.

2

ACCEPTED MANUSCRIPT 1

Introduction Ice cream is an excellent source of dietary energy and nutritive compounds (Goff

T

and Hartel, 2013). Moreover, the addition of probiotic cultures in ice creams provides

IP

functionality, besides adding value to the product (Cruz et al., 2009). Probiotics are

SC R

defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 2014), and studies have shown that ice cream is

NU

an excellent vehicle for probiotic bacteria (Di Criscio et al., 2010; Ferraz et al., 2012; Senaka Ranadheera et al., 2013; Silva et al., 2015).

MA

Inulin and oligofructose are the best-known prebiotics, and have several functional and nutritional properties that make them suitable to be used to formulate innovative

D

healthy foods. These fibres have been shown to increase the population of

TE

Bifidobacterium spp. and Lactobacillus spp. (Cardarelli et al., 2007). Strains belonging

CE P

to these genera are widely used as probiotic microorganisms and extensively incorporated into various fermented products, either dairy products (Meira et al., 2015; Taverniti et al., 2014; Verruck et al., 2015) or soy products (Bedani, Rossi and Isay

AC

Saad, 2013; Bedani et al., 2014; Matias et al., 2014). Lactobacillus. acidophilus La-5 and B. animalis Bb-12 are two strains with demonstrated beneficial health effects such as recolonization of the intestinal microbiota (Nord et al., 1997), reduction of the duration of stomach upset (de Vrese et al., 2011), and intestinal microbiota modulation (Savard et al., 2011). The wide diversity of assays and conditions used to simulate in vitro digestion makes it somewhat difficult to compare the survival data described in the scientific literature (Villarreal et al., 2013). However, they may be helpful for selecting a suitable combination between the food matrix and probiotic strains, since the survival of probiotics can be positively or negatively influenced by the presence of ingredients like 3

ACCEPTED MANUSCRIPT prebiotics, fruits, fats, and whey protein isolates or concentrates (Bedani, Rossi and Saad, 2013; Bedani et al., 2014; Hernandez-Hernandez et al., 2012)

T

In response to the stress of processing and food composition, a part of the living

IP

probiotic microorganisms might enter a viable but not cultivable state (VBNC). In this

SC R

condition, they are in a dormant state, even though they are metabolically active. However, they may not be detectable using the traditional plate culture method, leading to an underestimation of the colony forming units numbers (Davis, 2014). Due to

NU

expected reductions in probiotic populations, it is necessary to reliably assess cell

MA

viability under the phases of the simulated in vitro gastrointestinal conditions. The quantitative PCR (qPCR) method used alone is able to determine the whole DNA extracted from the sample, including non-viable cells and, therefore, does not

TE

D

distinguish live from dead cells, which can overestimate the number of intact cells. Several studies have suggested the application of the qPCR method combined with a

CE P

propidium monoazide (PMA) sample pre-treatment, which allows the exclusion of the dead cells in PCR amplification (García-Cayuela et al., 2009; Gensberger et al., 2014;

AC

Nocker et al., 2009; Zhang et al., 2015). The qPCR enables a more rapid and specific detection of the targeted organisms and PMA treatment facilitates the exclusion of false positive results caused by DNA from dead cells (Gensberger et al., 2014). This occurs because PMA binds to DNA of cells in which the membrane is damaged and can be covalently crosslinked to it by light exposure. Thus, the PCR amplification of such modified DNA is strongly inhibited (Nocker et al., 2009). Soymilk and whey protein isolate (WPI, less than 0.5% lactose) may be alternatives for lactose-intolerant individuals, since WPI can be safely consumed by these individuals, even though the WPI is a milk-based product (Geiser, 2003). Among the benefits of WPI, its high nutritional value due to the content of essential amino-acids 4

ACCEPTED MANUSCRIPT and of high quality proteins is remarkable, besides vitamin B and immunoglobulin contents, in addition to its antioxidant, antihypertensive, antitumor, and antiviral

T

properties (Geiser, 2003; Krissansen, 2007; Yalcin, 2006)

IP

The challenge is to develop different ice cream formulations that maintain the

SC R

viability of the probiotic microorganisms L. acidophilus La-5 and B. animalis Bb-12 in an environment with reduced lactose content, using soybean extract and WPI. In fact, obtaining apple ice-cream matrices supplemented with probiotic microorganisms and

NU

prebiotic ingredients may result in a product with excellent functional potential. Thus,

MA

besides the good market prospects, this product could supply lactose intolerant consumers in varying degrees and of all ages, offering the benefits of probiotics and prebiotics. Therefore, the aim of this study was to investigate the effect of the total or

TE

D

partial replacement of milk by soy extract and/or WPI combined with inulin on the viability and resistance to in vitro simulated GI conditions of L. acidophilus La-5 and B.

CE P

animalis Bb-12 incorporated into synbiotic ice cream formulations and to monitor La-5 and Bb-12 morphological changes during gastrointestinal stress.

2.1

Material and methods

AC

2

Ingredients

The following ingredients were used in this work: skimmed milk powder (Molico, Nestlé, Araçatuba, SP, Brazil); sucrose (União, Coopersucar-União, Limeira, SP, Brazil); glucose powder (Nutre, São Paulo, SP, Brazil); palm oil 370 SE (Agropalma, Belém, PA, Brazil); soy extract (Provesol PSA Olvebra, Eldorado do Sul, RS, Brazil); whey protein isolate (WPI) (Alibra, Campinas, SP, Brazil); inulin (Beneo GR®, Orafti, Oreye, Belgium); nature-identical flavouring apple 8500120 (Duas Rodas, Jaraguá do Sul, SC, Brazil); Grindsted guar 250 (DuPont Danisco, Cotia, SP, Brazil); Grindsted 5

ACCEPTED MANUSCRIPT xanthan 80 (DuPont Danisco); Grindsted carrageenan CY-500 (DuPont Danisco); pasteurized and frozen concentrated apple juice (Malus domestica Borkhausen, Yakult,

T

São Joaquim, SC, Brazil); emulsifier (Emustab, Duas Rodas); citric acid (Doce Aroma,

IP

São Paulo, SP, Brazil); fructo-oligosaccharides (FOS) (Beneo P95, Orafti), and maltose

2.2

SC R

(Difco, Le Pont de Claix, France). Cultures employed

NU

The freeze-dried probiotic cultures L. acidophilus La-5 and B. animalis subsp. lactis Bb-12 were purchased from Christian Hansen (Hørsholm, Denmark). These probiotic

MA

cultures were previously activated, as follows: each culture was weighed and transferred aseptically to vials containing 40 mL of reconstituted skimmed milk powder or

D

reconstituted soy extract (both prepared at concentrations of 10 g/100 mL distilled water

TE

and sterilized). The vials were kept at 37 °C for 90 min prior to addition to the mix

CE P

(blend of the ice cream ingredients prior to freezing) at the end of its preparation. The cultures, previously activated in milk, were used in ice creams containing milk powder

AC

and when activated in soy extract they were employed in the formulations without milk powder, with the aim of not adding lactose. 2.3

Experimental design and synbiotic ice cream manufacture Eight pilot-scale apple ice cream-making trials were prepared in duplicate (two

different batches of the same formulation) according to Table 1, including an axial point randomly chosen, using a simplex-centroid design, from which part of the milk (x1) or the whole amount of it was replaced by soy extract (x2) and/or WPI combined with inulin (x3). The complete list of ingredients used for the production of the different ice creams is described in Table 2. Different combinations of the ingredients: skimmed milk powder, soy extract, and whey protein isolate combined with inulin (3 g of WPI for each 6

ACCEPTED MANUSCRIPT 1 g inulin) were used. All trials were performed using the probiotic cultures of L. acidophilus La-5 and of B. animalis Bb-12 and the prebiotic fibre FOS. The proportion

T

of 6% FOS in all trials was chosen, according to the amounts of fructans needed to

IP

confer prebiotic benefits and to compose a synbiotic food.

SC R

Ice creams were produced in batches of 5 kg with commercial pasteurized and frozen concentrated apple juice, which did not contain any added preservatives that might reduce probiotic viability (since it was natural, pasteurized, and frozen), and an

NU

emulsifier. In addition, to produce the mix, the skimmed milk powder (or soy and/ or

MA

WPI + inulin - see Table 2), sucrose, glucose powder, palm oil, and the gums were mixed and heated for 10 min, in order to achieve 85 °C, during 25 s in a pilot-scale universal mixer machine (Geiger UMMSK-12, Geiger, Pinhais, PR, Brazil). Next, the

TE

D

temperature was reduced to 40 °C for the addition of the emulsifier, pasteurized apple juice, flavour, citric acid, and the pre-activated L. acidophilus and B. animalis cultures.

CE P

Subsequently, the mix was transferred to a container and kept refrigerated at 4 ± 1 °C for 20 h for maturation. After the maturation period, the mix was transferred to the ice

AC

cream producer (Skymsen BSK-16, Metalúrgica Skymsen, Brusque, SC, Brazil), in which the refrigerating fluid was kept at -20 °C for simultaneous beating and freezing. When the product reached the temperature of -3 to -4 °C, the ice creams were packed in appropriate polypropylene plastic pots for food products (68 mm in diameter, 32 mm in height, 55 mL in total volume, Tries Aditivos Plásticos, São Paulo, SP, Brazil) and covered with a plastic cover. The ice creams were stored frozen (-18 ± 3 °C) for up to 84 days. Ice cream samples from each formulation were analysed after 0 (before dynamic freezing), 1, 7, 14, 28, 42, and 84 days of frozen storage regarding probiotic viability

7

ACCEPTED MANUSCRIPT and pH, and after 7, 42, and 84 days of storage to evaluate probiotic resistance to simulated gastrointestinal conditions. Determination of pH

IP

T

2.4

SC R

The pH values were determined in duplicate ice cream samples (two different pots of the same batch, totalling 4 pots for each formulation at each storage period) using a Orion pH meter (model Three Stars, Thermo Fisher Scientific, Waltham, MA, USA)

NU

equipped with a penetration electrode model 2AO4-GF (Analyser, São Paulo, SP,

2.5

MA

Brazil). Probiotic viability determination

D

To determine the probiotic viability in the products during storage, portions of 10

TE

g of each ice cream in duplicate samples (two different pots of the same batch, totalling

CE P

four pots for each formulation at each storage period) were blended with 90 mL of 1 g/L peptone water in a Bag Mixer 400 (Interscience, St. Nom, France) and serial dilutions were performed in the same diluent. Lactobacillus acidophilus La-5 was counted by

AC

pour-plating 1 mL of each dilution of the samples in modified DeMan-Rogosa-Sharpe (MRS) agar by the substitution of glucose for maltose (Difco, Le Pont de Claix, France) as the main carbohydrate source (InternationalDairyFederation, 1995), followed by 72 h of aerobic incubation at 37 ºC (Vinderola and Reinheimer, 1999). Bifidobacterium animalis Bb-12 was counted by pour-plating each dilution (1mL) in MRS agar (Oxoid), and the following solutions were added: lithium chloride (2 g/L) (Merck, Darmstadt, Germany) and sodium propionate (3 g/L) (Sigma-Aldrich, St. Louis, MO, USA) in LP-MRS agar, followed by anaerobic incubation for 72 h (Anaerobic System Anaerogen, Oxoid) at 37 ºC as previously described (Vinderola and

8

ACCEPTED MANUSCRIPT Reinheimer, 1999). The selectivity of each medium was checked by a random selection of colonies for Gram test and microscopy analysis. The results obtained by plate count

T

were presented as log cfu/g. Coliforms and Escherichia coli, and yeasts and moulds

IP

were analysed using Petrifilm EC™ Count Plates (37 °C for 24 h) and Petrifilm YM™

2.6

SC R

Count Plates (3M Microbiology, St Paul, MN, USA) (25 °C for 5 days), respectively. Survival of L. acidophilus and B. animalis under simulated GI conditions

NU

2.6.1 In vitro simulated GI conditions assay

MA

The evaluation of probiotic survival in the ice cream samples submitted to simulated gastric and enteric conditions followed the procedure elsewhere described

D

(Buriti et al., 2010; Liserre et al., 2007) with some adaptations, as described next.

TE

Samples of 10 mL of each ice cream triplicate dilution in 0.85 g/100 mL NaCl solution were transferred to 9 sterile flasks, composing a total of 9 flasks containing the samples

CE P

(3 dilutions carried out with 3 different samples of the same batch in the same period of storage). The pH was adjusted to 2.3-2.6 with 1 mol equi/L HCl (Merck). In addition,

AC

pepsin (from porcine stomach mucosa, Sigma-Aldrich) and lipase (Amano lipase G, from Penicillium camemberti, Sigma-Aldrich) were added to samples reaching a final concentration of 3 g/L and 0.9 mg/L, respectively. Flasks were incubated at 37 ºC for 2 h in a shaker with agitation of 150 rpm (Metabolic Water Bath Dubnoff MA-095, Marconi, Piracicaba, SP, Brazil), leading to the simulated gastric phase. Then, the pH of samples was adjusted to 5.4-5.7 using an alkaline solution [150 mL of 1 mol equi/L NaOH (Synth, Diadema, SP, Brazil) and 14 g of PO4H2Na.2H2O (Synth) and distilled water up to 1 L]. Bile (bovine bile, Sigma-Aldrich) and pancreatin (pancreatin from porcine pancreas, Sigma-Aldrich) were added to a final concentration of 10 g/L and of 1 g/L, respectively. Next, samples were incubated again at 37 °C for 2 h under agitation to 9

ACCEPTED MANUSCRIPT simulate enteric phase I. Finally, the pH was adjusted to 6.8-7.2 using the same alkaline solution described above, containing bile and pancreatin adjusted to maintain the

T

concentration of 10 g/L and 1 g/L, respectively, and the samples were incubated again at

IP

37 °C for 2 h under agitation to stimulate enteric phase 2 and reaching 6 h of assay.

SC R

2.6.2 Determination of probiotic survival by PMA-qPCR

To quantify the viability of La-5 and Bb-12 cells before (0 h) and after each phase

NU

of the in vitro assay (2 h, 4 h, and 6 h) under simulated GI conditions PMA-qPCR was

slight modifications as follows.

MA

employed. The procedures were carried out according to (Villarreal et al., 2013), with

Total DNA was isolated from the product and from the in vitro assay samples.

D

Briefly, at each sampling day of storage (7, 42, and 84), portions of 2 g from the product

TE

or of 3 mL from the in vitro samples were homogenized with, respectively, 18 or 27 mL

CE P

of trisodium citrate dehydrate solution (2%, w/v), and incubated (45 ºC for 30 min). The resulting suspensions were centrifuged (9,000 x g for 10 min at 4 ºC) and the pellet was

AC

washed and resuspended in 500 µL of Tris-EDTA (10 mM Tris-HCl, 1 mM EDTA, [pH 8]) buffer. The samples were stored at -20 ºC prior to proceed DNA isolation. The PMA treatment was performed according to Nocker et al. (2006) and Villarreal et al. (2013). The cells suspensions (500 µL) were treated with PMA (phenanthridium,

3-amino-8-azido-5-[3-(diethylmethylammonio)

propyl]-6-phenyl

dichloride; Biotium, Inc., Hayward, CA, USA) to a final concentration of 50 mM. After a 5 min incubation in the dark, the samples were exposed to light for 15 min using a 650-W halogen light device (DWE, 650W, 120V, GE Lighting, East Cleveland, OH, USA) at a distance of 20 cm from the light. The samples tubes were mixed occasionally and placed horizontally on ice to avoid excessive heating. The samples were subsequently centrifuged at 10,000 x g, for 10 min at 4 ºC. The resulting pellet was 10

ACCEPTED MANUSCRIPT washed with PBS buffer with the purpose to eliminate the inactivated PMA. Afterwards, another centrifugation was carried out and the cell pellet was resuspended

T

in Tris-EDTA buffer (500 µL).

IP

For DNA isolation, cells suspensions of PMA-treated samples were transferred

SC R

to 2 mL tube containing 0.3 g of 0.1 mm zirconia beads (Biospec, Bartlesville, OK, USA) and 150 µL of buffer-saturated phenol (Invitrogen, Carlsbad, CA, USA). A mechanical cell disruption was performed, using a FastPrep-24 bead-beater (MP

NU

Biomedical, Solon, OH, USA), followed by subsequent phenol-chloroform:isoamyl

MA

alcohol extractions, until obtaining a clear interface. The DNA was precipitated through ethanol, collected by centrifugation, and resuspended in 30 µL of TE buffer. The determination of DNA quality and concentrations were carried out using a Nanodrop

TE

D

ND-1000 (Thermo Scientific, Waltham, MA, USA). Lactobacillus acidophilus La-5 and B. animalis Bb-12 quantification were

CE P

performed using the ABI-PRISM 7500 sequencing detection system (Applied Biosystems, Bridgewater, NJ, USA). For bacteria analysis, each 25 µL reaction volume

AC

contained 5 µL of nucleic acid isolated was diluted from 10 to 1000-fold in sterilized nuclease-free water and 20 µL of PCR Master Mix. For B. animalis Bb-12 quantification, 1X TaqMan Master Mix (Applied Biosystems), 200 nM of F_Bifid 09c forward and R_Bifid 06 reverse primers, and 250 nM of P_Bifid probe were used. The primers were the same as those previously described (Furet et al., 2009). The amplification conditions used for Bb-12 were 50 ºC for 2 min, 95 ºC for 10 min, and 40 cycles of 95 ºC for 30 s and 60 ºC for 1 min. For L. acidophilus La-5 quantification, 1X Power SYBR Green PCR Master Mix (Applied Biosystems) and 400 nM of each primer was used, according to Tabasco et al. (2007). The amplification program used was 50 ºC for 2 min, 95 ºC for 10 min, and 40 cycles of

11

ACCEPTED MANUSCRIPT 95 ºC for 10 s and 60 ºC for 30 s. For SYBR Green qPCR, the melting curve analysis was carried out after amplification reaction, in order to discriminate the target from the

T

non-target PCR products.

IP

To quantify the microorganisms, standard curves were constructed for each

SC R

strain by purified genomic DNA isolated from pure cultures serially diluted with sterilized nuclease-free water for obtaining a range of DNA concentrations equivalent to approximately 5 x 108 to 100 genomes copies of each microorganism per amplification

NU

reaction mixture. The number of copies was estimated considering the total genome size

MA

of L. acidophilus (NCBI-ID1099) and B. animalis (NCBI-ID844). Finally, the quantification was estimated by comparing the samples threshold cycle (Ct) and a standard curve Ct.

TE

D

The coefficients of efficiency were from 91 to 103% and the correlation coefficients (r2) were between 0.991 and 0.999. Non-template controls (NTC) samples

CE P

were added in all PCR runs and tested negative. All assays were performed at least in triplicates and the average values were used for analysis. The results of probiotic

AC

survival were expressed in log cfu equivalents/g of synbiotic ice cream. In order to evaluate La-5 and Bb-12 survival under simulated GI conditions in different storage periods, the survival rate was calculated according to the following equation (Guo et al., 2009):

Survival rate % = (log cfu N1/ log cfu N0) x 100

Eq. (1)

N1, the total viable count of probiotic strains after exposure to in vitro simulated GI conditions (6 h); N0, the total viable count of probiotic strains before exposure to in vitro simulated GI conditions (0 h).

12

ACCEPTED MANUSCRIPT 2.7

Scanning electron microscopy (SEM) The SEM analysis was carried out according to Villarreal et al. (2013). Briefly, the

T

samples, obtained from the in vitro simulated GI conditions assay (section 2.6.1), were

IP

centrifuged (5,000 x g for 10 min) to remove the supernatant. The resulting pellets were

SC R

resuspended in NaCl solution (0.9%, w/v) at a final concentration of around 5 log cfu/mL. The cell suspensions (1 mL) were filtered through 0.2 mm-pore size membrane

NU

filters (Isopore, Millipore, Billerica, MA, USA) and fixed in a 2% (w/v) glutaraldehyde solution for 2 h. Afterwards, the membranes were washed using Milli-Q water

MA

(Millipore). The washing procedure was repeated two more times, after which the membranes were dehydrated in ethanol solutions in the following sequence: 25, 50, 75,

D

90, and 95%, and finally with 100% ethanol (three times), and dried using the critical-

TE

point CO2 method. The dried membranes were placed on aluminum stubs, sputter

CE P

coated with gold and their analyses were performed using a field emission scanning electron microscope (JEOL JSM-7401F; JEOL, Tokyo, Japan) at 2.5 kV. Sample images

2.8

AC

were acquired at different magnifications (x5,000, x15,000 and x30,000). Statistical analysis The experiment constituted a simplex-centroid design with three factors. Initially, data were checked regarding homogeneity of variances and normality. Differences between ice cream samples for the same day and between different days of storage for the same ice cream were statistically analysed using repeated measures ANOVA, followed by the post hoc Tukey test (p<0.05). For data showing non-homogenous variance, the nonparametric Kruskall-Wallis test and Dunn’s multiple comparison procedure (p<0.05) were employed to compare the formulations and Friedman test with Bonferroni correction were used for comparison between days. According to data of this 13

ACCEPTED MANUSCRIPT study, it was not possible to obtain significant regression models from the experimental design; therefore the results were expressed as mean ± SD. Statistical analysis was

3.1

IP

Results and discussion

SC R

3

T

performed using STATISTICA 12 (StatSoft Inc. Tulsa, OK, USA).

pH values and microbiological parameters

NU

The pH mean values of the apple ice creams ranged from 4.63 to 5.32 during frozen storage (Table 3). Significant variations were not observed for ice creams prepared with

MA

milk (M), milk and WPI + inulin (MPI), milk and soy and WPI + inulin (MSPI), and random ice cream (R) during the storage period studied, suggesting that the presence of

D

milk in combination with whey protein isolate and inulin may influence the

TE

maintenance of pH. On the other hand, the blend process of soy and milk or soy and whey protein isolate with inulin apparently contributed to raise the pH, since there was a

CE P

slight but significant increase (p<0.05) in pH values of ice creams made with soy (S), milk and soy (MS), and soy and WPI + inulin (SPI). A distinct result was observed for

AC

ice cream containing only WPI + inulin (PI), which presented the lowest pH values when compared to the other ice cream formulations and a slight reduction during storage (p<0.05). The low pH buffering capacity of soy proteins could explain, at least in part, the pH increase observed in S, MS, and MSPI formulations (Wang et al., 2009). Coliforms, E. coli, and yeasts and moulds were not detected in any of the ice cream samples, indicating they were microbiologically safe for human consumption (data not shown). All the ice cream formulations produced showed to be feasible as food matrices for the probiotic microorganisms studied, since the average viability of L. acidophilus La-5 and B. animalis Bb-12 over the storage period was always above 7.5 log cfu/g (Table 14

ACCEPTED MANUSCRIPT 4). Although no general agreement has been reached on the recommended levels of probiotic strains, it is suggested they should be present in a food to a minimum level of

T

8 log cfu/day, with the aim of balancing the possible reduction in the population of the

IP

probiotic microorganisms during the passage through the gut. Considering a serving

SC R

portion of 60 g of ice cream, probiotic populations of 7 to 8 log cfu/g would result in a daily intake of 8 to 9 log cfu of these microorganisms. In the present study, throughout the whole storage, L. acidophilus La-5 as well as B. animalis Bb-12 maintained the

NU

minimum viability currently recommended by Brazilian legislation (Anvisa, 2008) of 8

MA

to 9 log cfu per daily serving portions of a probiotic product. Populations of L. acidophilus La-5 obtained by the plate count method have shown slight but significant changes (p<0.05) during storage (Table 4). However these

TE

D

changes can be considered of little microbiological significance, since they did not exceed 0.5 log cfu/g in all of the ice cream formulations and periods analysed.

CE P

Populations of B. animalis Bb-12 (Table 4) decreased significantly (p<0.05), notably in the ice cream samples PI and MPI, in which there was a variation of 1 and of 0.6 log

AC

cycle during the whole storage, respectively. In general, the viability of Bb-12 was lower (p<0.05) in PI when compared to the other ice cream samples. Additionally, we observed that after the dynamic freezing (day 1) there was a slight, but significant reduction (p<0.05) of the populations of La-5 and Bb-12 in most of the ice creams. However, this reduction in the viability had microbiological significance only in PI and MSPI for Bb-12. The dynamic freezing process may cause damage to the probiotic microorganisms and may reduce their viability, possibly due to stress caused by the incorporation of air and freezing, which may damage the cell wall or promote a plasmatic membrane rupture given the eventually formation of ice crystals inside and outside the cell (Cruz et al., 2009). Moreover, low freezing rates associated with slow

15

ACCEPTED MANUSCRIPT supercooling and low ice nucleation have also been reported to be associated with plasmolysis (Soukoulis et al., 2014). Our results of overrun do not seem to have

T

influenced the viability. On the other hand, no significant reduction in L. rhamnosus

IP

DSM 20021 and L. casei DSM 20011 was observed after freezing and during 112 days

SC R

of probiotic and synbiotic ice cream storage (Di Criscio et al., 2010). The addition of FOS in all ice cream formulations may have contributed to the maintenance of La-5 and Bb-12 high viability during storage. Even though bacteria

NU

metabolism is limited at low temperatures, the prebiotic ingredient may have the role of

MA

balancing the osmotic pressure of probiotics, avoiding cell damage and maintaining high viability. Moreover, since the ice cream has a low eutectic point, a portion of the

D

water containing the nutrients and the microorganisms is probably present in the liquid

TE

form at -18C, and this may have supported the growth of certain number of bacteria. Since some of these bacteria were probably present in the viable but not cultivable state,

CE P

due to freezing injury, the populations were more or less constant, when determined by the classic plate-count method.

AC

A protective effect of inulin on probiotics is commonly observed in frozen synbiotic products, resulting in the maintenance of the viability throughout their shelf life or even in increased viability compared to products that do not contain this prebiotic ingredient (Akın et al., 2007; Di Criscio et al., 2010; Vasconcelos et al., 2014). However, in our study, a distinct behaviour was observed in the ice cream formulations with the highest proportion of inulin (PI), which had the lowest viability of Bb-12 in most of the sampling periods. Nevertheless, this behaviour might be associated with the higher levels of the whey protein isolate (WPI) present, since WPI may have interfered with the adaptation of the probiotic strain in this food matrix. In fact, according to Soukoulis et al. (2014) the sub-lethal impact of each factor may vary from insignificant 16

ACCEPTED MANUSCRIPT to very considerable, and their combination might result in severe lethality of probiotic cells. Akalin and Erisir (2008) found a significant decrease in the viability of La-5 e Bb-

T

12 in low-fat ice creams supplemented with oligofructose or inulin. Although the

IP

populations of these probiotics were significantly enhanced in ice cream with

SC R

oligofructose, at 90 days of storage, only Bb-12 was found in amounts slightly exceeding 6 log cfu/g.

In the preliminary assays performed to adjust the ingredient proportions of the

NU

ice cream formulations produced in this study, we observed a coagulation process of the

MA

proteins during mix pasteurization and a reduction of 2 log cycles on the viability of Bb12 at the end of 84 days (data not shown). We believe that the type of processing (such as pasteurization time and temperature) used during the manufacturing of the ice cream

TE

D

may influence the probiotic ability to adapt to the matrix. Besides, the survival of bacteria when faced with adverse factors during processing and product development is

3.2

CE P

strain dependent (Tripathi and Giri, 2014). Probiotic resistance and morphological changes during the in vitro simulated

AC

GI conditions

In general, the survival of L. acidophilus La-5 and B. animalis Bb-12, obtained by the PMA-qPCR method, revealed an average reduction of 4.2 log cycles after 6 h of assay under simulated GI conditions (Fig. 1 and Fig. 2). In the gastric phase (after 2 h of the in vitro assay) there was a decline in the viability of La-5 (p<0.05), except for SPI at 42 days and S at 84 days (Fig. 1). At the end of the 6 h of incubation in the enteric phase II, the greatest variation in relation to the initial population occurred in SPI (7 days), S (42 days), and M (84 days), which had a reduction of, respectively, 3.6, 4.2, and 4.2 log.

17

ACCEPTED MANUSCRIPT A decline in the viability of Bb-12 in the gastric phase was observed in all the ice cream formulations (Fig. 2). The greatest variation in relation to the initial amount of

T

Bb-12 occurred in MS (7 days), PI (42 days), and PI (84 days) with, respectively, 4.2,

IP

3.0, and 4.1 log after 6 h assay.

SC R

At the 7th day of storage, the ice cream formulation MSPI showed the highest survival (p<0.05) of La-5 after the 6 h assay (5.78 log cfu equivalents/g). At 42 days, MS, MPI, and MSPI had a surviving population of 5.99 log cfu equivalents/g after the 6

NU

h assay, indicating that the combination of milk with soy or whey protein, or both had a

MA

greater protective effect on La-5 during the simulated GI conditions. After 84 days of storage, the greatest survival was observed for MPI (5.75 log cfu equivalents/g), MSPI (5.65 log cfu equivalents/g), and R (5.84 log cfu equivalents/g).

TE

D

Considering the survival of Bb-12, after 6 h of the in vitro assay on the 7th day of storage, the counts by PMA-qPCR were 5.90 log cfu equivalents/g and 6.02 log cfu

CE P

equivalents/g, respectively, in SPI and MSPI. The same ice cream formulations had the highest survival (p<0.05) on the 84th day of storage under the simulated GI conditions,

AC

leading to 7.27 log cfu equivalents/g (SPI) and to 7.42 log cfu equivalents/g (MSPI) after 6 h (Fig. 2). These observations showed a favourable interaction between soy extract, whey protein, and inulin on the probiotic resistance to the stress conditions assayed. García-Cayuela et al. (2009) evaluated the viability of La-5 and Bb-12 in a synbiotic commercial product by selective agar methods and qPCR combined with PMA treatment and the results were similar between both methods, even 60 days after the expiration date. We also employed the traditional qPCR method (without using the PMA treatment) in this study (data not shown) to evaluate the in vitro survival, but the results were higher than those obtained by plate count and by the PMA-qPCR methods.

18

ACCEPTED MANUSCRIPT Moreover, results were maintained on a plateau and relevant variations between the phases of the in vitro assay were not observed. On the 84th day, for example, after 6 h of

T

incubation under GI simulated conditions, the variation of viability obtained was 1.2 log

IP

unit and 1.5 log unit, respectively, for La-5 and Bb-12 (data not shown). These results

SC R

suggest that the qPCR method combined with PMA was effective in reducing the interference of dead cells, evidencing the differences between the phases of the test, whereas the qPCR method was not able to demonstrate these differences.

NU

Similarly, according to Villarreal et al. (2013), the traditional qPCR was not a

MA

reliable enumeration method for the quantification of intact probiotic populations in petit-suisse cheeses and the use of PMA overcame the qPCR inability to differentiate between dead and alive cells. Additionally, bacteria plate counts were much lower than

TE

D

with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms, therefore unable to form colonies.

CE P

Studies have shown that cellular components of probiotic bacteria, like their genomic DNA, or their inactivated and dead cells promote beneficial effects in the host

AC

(Ghadimi et al., 2008; Taverniti and Guglielmetti, 2011). However, the current consensus is that probiotics should be alive to exert their beneficial effects (Hill et al., 2014).

The survival rate of La-5 and Bb-12 during the in vitro assays was always above 50% even after 84 days of storage at -18 °C (Table 5). Generally, the survival rate of La-5 decreased (p<0.05) in 3 ice cream formulations - M, S, and MS throughout the storage period, while Bb-12 survival rates decreased in formulations M, and PI. In the MPI, MSPI, and R formulations, the survival rate of La-5 was higher than in the other formulations at the end of the storage period. The same happened with Bb-12 for MPI, SPI, MSPI, and R formulations. Additionally, at 84 days, formulations M and PI

19

ACCEPTED MANUSCRIPT presented a greater amount of injured cells of Bb-12 after 6 h of the in vitro assay, when compared to S, MPI, SPI, MSPI, and R (Fig. 2).

T

The use of prebiotic ingredients may be an alternative to support the growth of

IP

probiotic strains and improve their survival through the GIT (Hernandez-Hernandez et

SC R

al., 2012). However, contrary to our expectations, the mix of WPI and inulin did not offer additional protection to the strains tested during simulated GI environment in ice cream formulation PI, but when combined with milk or soy, an increased survival rate

NU

of the probiotics was observed in the ice creams at the end of storage.

MA

Scanning electron micrographs of La-5 and Bb-12 strains showed morphological alterations in the cells surface. Untreated probiotic cells have normal morphological structure, evidenced by a smooth continuous surface along the cell structure, adhered

TE

D

through an intact ice cream matrix (Fig. 3A). Thus, during subsequent incubation periods under simulated GI stress conditions, we observed a reduction of the cell

CE P

amount in the ice cream samples, which may be related to the digestion process of the ice cream by the added enzymes (Fig. 3A-C). In addition, some of the microorganisms

AC

showed a number of protrusions at the cells’ surface, similar to wrinkled structures, together with a fine shrunken phenotype, as indicated by white arrows in Fig. 3B-C. Finally, at the end of the digestion period, we observed a more intense stress phenotype in some particular cells, such as cell membrane clumping and damage events, evidenced by black arrows (Fig. 3Db-c). The long term exposure of a microorganism to different environment conditions, varying from the gastric phase to the enteric phases I and II in the presence of bile and pancreatin, as the pH increases, could result in physiological changes in the microorganisms. These changes affected its surface structure integrity (plasmatic membrane, cell wall or capsule), which in turn attempted to adapt to this dynamic

20

ACCEPTED MANUSCRIPT environment. In fact, previous studies have reported that lactobacilli are known to modify their surface properties in response to environmental changes (Fozo et al., 2004;

T

Taranto et al., 2003; Villarreal et al., 2013). After the first 2 hours of incubation in the

IP

gastric phase, under low pH (2.4-2.9) and in the presence of pepsin and lipase, we

SC R

observed the first morphological changes. Previous studies have reported the effect of the pH in the morphology and viability of different Lactobacillus strains (Hossein Nezhad et al., 2010; Norton et al., 1993; Sanhueza et al., 2015). Moreover, the presence

NU

of proteases such as pepsin and trypsin have also been reported to influence not only the

MA

viability of different strains of lactobacilli, but also their ability to adherence (Darilmaz et al., 2011).

The survival ability at the aggressive conditions of the GI, overcoming the

TE

D

presence of a mix solution with bile salts and other digestive factors, is crucial for the host colonization by probiotics (Koskenniemi et al., 2011). However, these digestive

CE P

agents present in the gastric and pancreatic secretions (as well as other physiological factors, such as peristalsis and competition with host microbiota) could affect probiotic

AC

functions (Darilmaz et al., 2011) In the present study, the probiotic viability results obtained by the PMA-qPCR assay could be hereby confirmed by the SEM qualitative assay, which shows a stress intensification process induced throughout the stages of the in vitro simulated digestion, evidenced by different morphological aspects acquired by the cells, culminating in loss of cell integrity and consequentially, their viability. A portion of the microorganisms showed their surface structure gradually wrinkled and even shrunken. This event might have occurred in response to the effect of the digestion process, such as gradually increasing of wrinkled structures in the time point 2 to 4 h. This finally resulted in some cell lysis events observed at the end of the

21

ACCEPTED MANUSCRIPT incubation period. A previous study has linked the denatured conditions of the cell surface to increased cell death process, including aberrant protrusions on the cell

T

surface, together with morphological denaturation, without causing extensive damage to

IP

the cell architecture (Adams et al., 2000).

SC R

Loss of cell viability observed in the final stages of the digestion assay of the present work supports the hypothesis that these cells gradually lose their viability. However, this loss is still low since many other cells still remain morphologically

NU

stable, as confirmed by quantitation assays with PMA-qPCR, indicating the

MA

effectiveness of the formulations tested in maintenance of the cell viability and physiological function between 4 h and 6 h (enteric phase II). Conclusions

D

4

TE

The present study showed that the viability of L. acidophilus La-5 and B.

CE P

animalis Bb-12 in all synbiotic apple ice cream formulations tested was satisfactory until the 84th day of frozen storage, with populations of around 7.5 to 8.5 log cfu/g,

AC

showing that the ice cream formulations are good matrices for carrying and delivering the probiotic microorganisms. Using the PMA-qPCR technique, we demonstrated that La-5 and Bb-12 survived pretty well in GI conditions simulated in vitro, with survival rates exceeding 50% after 6 h of the in vitro assay even at 84 days of frozen storage. The SEM results confirmed that there are cells in different physiological and morphological stages during the in vitro assay. The variation found in La-5 and Bb-12 in vitro survival among the ice cream formulations suggests that the gastrointestinal tolerance of probiotic strains can potentially be affected by the choice of the food matrix. However, the use of soymilk and whey protein isolate + inulin instead of milk showed to be feasible in order to obtain a food matrix that may provide protection to the probiotic strains under GI stress conditions when compared to the milk-based 22

ACCEPTED MANUSCRIPT counterpart. Most importantly, the ice cream mixtures containing soy extract and/or WPI + inulin may expand the range of synbiotic products for individuals with varying

T

degrees of lactose intolerance or lactose sensitiveness. It is necessary to extend this

IP

study to clinical trials, and thus a forthcoming study to evaluate the survival of these

SC R

microorganisms in the faeces of healthy subjects consuming the formulations assessed, since all of the ice cream matrices maintained the minimum probiotic levels currently recommended of 9 daily log cfu in an ice cream portion of 60 g throughout the 84 days

AC

CE P

TE

D

MA

NU

of storage.

23

ACCEPTED MANUSCRIPT Table 1. Simplex-centroid experimental design employed in the present study. Proportion of each

Amount of each ingredient (g) in

ingredient

Skimmed (X1, X2, X3)

milk powder

M3

(1, 0, 0)

10

S4

(0, 1, 0)

0

PI 5

(0, 0, 1)

0

MS

(1/2, 1/2, 0)

5

MPI

(1/2, 0, 1/2)

5

SPI

(0, 1/2, 1/2)

MSPI

(1/3, 1/3, 1/3)

R6

(1/10, 6/10, 3/10)

Soy extract

WPI1 + inulin 2

X2

X3

SC R

X1

T

100 g of ice cream

in the mixture

IP

Trials

0

10

0

0

10

5

0

0

5

0

5

5

10/3

10/3

10/3

1

6

3

D

MA

NU

0

WPI=whey protein isolate. 2 Proportion of the mixture of WPI + inulin: 3g of WPI for each 1 g inulin. 3 M=milk. 4 S=soy extract. 5 PI=protein+inulin. 6 Random formulation containing M, S, and PI in different proportion from the other ice creams.

AC

CE P

TE

1

24

ACCEPTED MANUSCRIPT

Table 2. Ingredients used for the production of the ice cream formulations, according to the experimental design described in Table 1. Ice cream Ingredients S

PI

MS

Variable ingredients powder

MSPI

R

0.00

0.00

5.00

5.00

0.00

3.33

1.00

Soy extract (S)

0.00

10.00

0.00

5.00

0.00

5.00

3.33

6.00

WPI1 + inulin2 (PI)

0.00

0.00

10.00

0.00

5.00

5.00

3.33

3.00

Water

54.52

54.52

54.52

54.52

54.52

54.52

54.52

54.52

Concentrated apple juice

19.40

19.40

19.40

19.40

19.40

19.40

19.40

19.40

FOS

6.00

6.00

6.00

6.00

6.00

6.00

6.00

6.00

Sucrose

4.00

4.00

4.00

4.00

4.00

4.00

4.00

4.00

Glucose powder

3.00

3.00

3.00

3.00

3.00

3.00

3.00

3.00

Palm oil

2.00

2.00

2.00

2.00

2.00

2.00

2.00

2.00

Emulsifier

0.40

0.40

0.40

0.40

0.40

0.40

0.40

0.40

Citric acid

0.30

0.30

0.30

0.30

0.30

0.30

0.30

0.30

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.04

0.04

0.04

0.04

0.04

0.04

0.04

0.04

0.05

0.05

0.05

0.05

0.05

0.05

0.05

0.05

0.05

0.05

0.05

0.05

0.05

0.05

0.05

0.05

(M)

Natural apple flavour

Xanthan gum

AC

Guar gum

CE P

TE

Carrageenan gum

Lactobacillus acidophilus La-5

Bifidobacterium animalis Bb-12 TOTAL

MA

Fixed ingredients

SC R

10.00

NU

milk

SPI

D

Skimmed

MPI

T

M

IP

(g/100 mL of mix)

100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00

1

WPI= whey protein isolate. 2Proportion of the mixture of WPI + inulin: 3g of WPI for each 1 g inulin.

25

ACCEPTED MANUSCRIPT

MSPI

R

5.21 ± 0.12Aa 4.80 ± 0.05CDabc 4.72 ± 0.06Dab

4.99 ± 0.03Bb

5.00 ± 0.07Ba

4.70 ± 0.13Db

4.92 ± 0.13BCa

4.75 ± 0.10CDa

1

5.27 ± 0.11Aa 4.75 ± 0.06Cbc

4.71 ± 0.06Cabc

4.94 ± 0.04Bb

4.97 ± 0.04Ba

4.84 ± 0.09BCa

4.93 ± 0.12Ba

4.86 ± 0.16BCa

2

5.25 ± 0.06Aa 4.76 ± 0.16DEc

4.67 ± 0.02Eabcd 4.99 ± 0.09Bb

4.92 ± 0.03BCa 4.81 ± 0.08CDEa 4.91 ± 0.07BCDa 4.81 ± 0.08BCDEa

7

5.32 ± 0.03Aa 4.78 ± 0.10Cbc

4.75 ± 0.06Ca

4.98 ± 0.04Bb

4.90 ± 0.01BCa 4.79 ± 0.07Cab

14

5.28 ± 0.03Aa 4.82 ± 0.13CDbc

4.71 ± 0.05Dab

5.03 ± 0.06Bab 4.92 ± 0.16BCa 4.84 ± 0.06BCDa 4.93 ± 0.12BCa

28

5.26 ± 0.10Aa 4.80 ± 0.06BCabc 4.63 ± 0.01Ccd

4.96 ± 0.03Bb

4.92 ± 0.18Ba

4.87 ± 0.14Ba

4.93 ± 0.16Ba

4.88 ± 0.15Ba

42

5.31 ± 0.02Aa 4.86 ± 0.11Bab

4.65 ± 0.07Cc

5.02 ± 0.02Bab 4.96 ± 0.13Ba

4.88 ± 0.13Ba

4.93 ± 0.06Ba

4.98 ± 0.26BCa

84

5.28 ± 0.04Aa 4.94 ± 0.05CDa

4.66 ± 0.05Ebcd

5.10 ± 0.04Ba

4.84 ± 0.05Da

4.97 ± 0.09Ca

5.05 ± 0.07BCa

CE P

TE D

MA N

US

0

4.97 ± 0.11Ca

4.86 ± 0.06BCa

4.82 ± 0.20Ca 4.83 ± 0.17BCDa

Means of six replicates. 2See Table 2 for the description of ice creams. A,B Different superscript capital letters in a row denote significant differences (p<0.05) between different ice cream formulations for the same day of storage. a,b Different superscript lower-case letters in a column denote significant differences (p<0.05) between different days of storage for the same ice cream formulation.

AC

1

CR

IP

T

Table 3. Mean pH values (mean ± SD) of synbiotic apple ice cream formulations during frozen storage (-18 ± 3 °C). pH1 Storage Ice cream2 period (days) M S PI MS MPI SPI

26

ACCEPTED MANUSCRIPT

T

Table 4. Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12 viability (mean ± SD) in synbiotic apple ice cream formulations during frozen storage (−18 ± 3 °C) obtained by plate counting.

B. animalis Bb-12

IP

Ice cream MS MPI ABa 8.02 ± 0.06 7.99 ± 0.07ABa Aab 7.91 ± 0.07 7.74 ± 0.11BCb 7.90 ± 0.10ABab 7.76 ± 0.12BCb 8.03 ± 0.05ABa 7.96 ± 0.04BCDa 7.93 ± 0.09ABab 7.84 ± 0.10BCab 7.87 ± 0.14Aab 7.74 ± 0.10ABb 8.00 ± 0.08Aa 7.91 ± 0.05ABab Eb 7.79 ± 0.04 7.84 ± 0.01DEab 8.55 ± 0.11Aa 8.54 ± 0.08Aa 8.43 ± 0.13ABabc 8.04 ± 0.02Cbc 8.25 ± 0.08ABcde 8.13 ± 0.07BCb 8.35 ± 0.03Babcd 8.12 ± 0.02Cbc 8.23 ± 0.10ABCde 8.11 ± 0.08BCbc 8.28 ± 0.10Bbcde 8.06 ± 0.08BCbc 8.10 ± 0.09BCe 8.14 ± 0.10ABCb 8.46 ± 0.09ABCab 7.98 ± 0.05Ec

CR

PI 8.00 ± 0.09ABa 7.77 ± 0.18BCb 7.67 ± 0.13Cb 7.74 ± 0.05Eb 7.78 ± 0.10BCab 7.57 ± 0.16Bb 7.67 ± 0.04Cb 7.56 ± 0.06Fb 8.32 ± 0.16Ba 7.84 ± 0.09Db 8.19 ± 0.23BCa 7.64 ± 0.05Dbc 7.72 ± 0.12Dbc 7.54 ± 0.17Dcd 7.60 ± 0.13Dbcd 7.31 ± 0.04Fd

MA N

S 7.94 ± 0.04ABCab 7.81 ± 0.10ABCcd 7.87 ± 0.07ABbcd 8.00 ± 0.03ABCa 7.86 ± 0.05BCbcd 7.77 ± 0.07Ad 7.93 ± 0.02ABabc 8.00 ± 0.07Ba 8.62 ± 0.06Aa 8.50 ± 0.11Aab 8.53 ± 0.17Aab 8.62 ± 0.05Aa 8.43 ± 0.14Aab 8.57 ± 0.09Aab 8.35 ± 0.05Ab 8.58 ± 0.13Aab

TE D

L. acidophilus La-5

M 7.90 ± 0.03BCa 7.68 ± 0.07Ce 7.80 ± 0.05ABbcd 7.86 ± 0.03Dabcd 7.76 ± 0.02BCde 7.78 ± 0.08Acd 7.88 ± 0.05Babc 7.89 ± 0.04CDEab 8.59 ± 0.06Aa 8.19 ± 0.10Cd 8.46 ± 0.13Aab 8.42 ± 0.06Babc 8.36 ± 0.11ABbcd 8.25 ± 0.08Bcd 8.34 ± 0.09Abcd 8.37 ± 0.06BCbcd

US

Microorganism

CE P

0 1 2 7 14 28 42 84 0 1 2 7 14 28 42 84

Populations of probiotic microorganisms (log cfu g-1)

AC

Storage period (days)

SPI 7.83 ± 0.09Cabc 7.65 ± 0.11Ccd 7.78 ± 0.10ABCabcd 7.90 ± 0.03CDab 7.73 ± 0.18Cbcd 7.58 ± 0.11ABd 7.82 ± 0.07Babc 7.97 ± 0.03BCa 8.55 ± 0.08Aa 8.11 ± 0.13Cbc 8.01 ± 0.20Cc 8.33 ± 0.09Bab 8.04 ± 0.18Cc 7.92 ± 0.15Cc 8.02 ± 0.11Cc 8.12 ± 0.02DEbc

MSPI 7.95 ± 0.07BCbc 7.94 ± 0.05Ac 7.97 ± 0.09Abc 8.10 ± 0.02Aab 7.92 ± 0.10ABCc 7.88 ± 0.08Ac 7.94 ± 0.04ABc 8.20 ± 0.03Aa 8.54 ± 0.04Aa 8.20 ± 0.16Ccd 8.08 ± 0.14BCd 8.36 ± 0.04Babc 8.09 ± 0.13Cd 8.11 ± 0.10BCd 8.27 ± 0.08ABbcd 8.49 ± 0.12ABab

R 8.05 ± 0.04Aab 7.86 ± 0.07ABcd 7.91 ± 0.11Abcd 8.01 ± 0.09ABCabc 8.08 ± 0.11Aa 7.82 ± 0.09Ad 7.91 ± 0.03ABabcd 7.94 ± 0.05BCDabcd 8.54 ± 0.07Aa 8.23 ± 0.14BCb 8.32 ± 0.19ABab 8.36 ± 0.07Bab 8.10 ± 0.17Cb 8.16 ± 0.15Bb 8.07 ± 0.06BCb 8.29 ± 0.04CDab

See Table 2 for the description of ice creams; n = 4. For the same microorganism: A,B Different superscript capital letters in a row denote significant differences (p<0.05) between different ice cream formulations for the same day of storage. a,b Different superscript lower-case letters in a column denote significant differences (p<0.05) between different days of storage for the same ice cream formulation.

27

ACCEPTED MANUSCRIPT

Table 5. Survival rate (%) of L. acidophilus La-5 and B. animalis subsp. lactis Bb-12 in ice cream formulations assessed by PMA-qPCR.

7

M L. acidophilus La-5

71.1 ± 1.2

S Aa

65.0 ± 1.4

PI BCa

62.7 ± 2.7

T

period (days)

Ice cream MS CDab

IP

Microorganism

71.3 ± 0.9

Aa

CR

Storage

MPI

71.0 ± 1.5

SPI Aa

59.1 ± 0.6

MSPI Dab

68.1 ± 1.5

R ABa

65.5 ± 0.5BCb

64.0 ± 0.8Aa

55.3 ± 0.4Bb

71.9 ± 3.2 Aa

65.1 ± 1.5Aa

69.0 ± 1.5Aa

67.5 ± 3.3 Aa

67.5 ± 1.0Aa

54.8 ± 0.3Bc

84

52.2 ± 0.5Cb

55.2 ± 0.8Cb

54.6 ± 1.2Cb

55.2 ± 3.7Cb

70.3 ± 1.3ABa

52.7 ± 1.6Cb

64.1 ± 3.0Ba

72.7 ± 0.5Aa

60.8 ± 0.6Cb

65.5 ± 0.9Bb

59.7 ± 0.4Ca

56.7 ± 1.4Db

57.1 ± 0.8Dc

69.7 ± 0.3Ab

61.8 ± 1.1Cc

68.5 ± 0.5Ab

42

71.2 ± 2.4BCa

75.8 ± 0.6Aa

62.0 ± 0.5Ea

68.4 ± 0.7CDa

70.6 ± 1.2BCb

65.5 ± 0.1Dc

69.7 ± 1.1BCb

72.0 ± 1.3Ba

84

54.6 ± 2.4Dc

66.9 ± 1.1Cb

53.0 ± 1.5Db

66.7 ± 0.4Ca

74.4 ± 1.4ABa

74.0 ± 0.5ABa

75.7 ± 0.4Aa

72.2 ± 0.6Ba

B. animalis Bb-12

MA N

7

US

42

AC

CE P

TE D

See Table 2 for the description of ice creams; n = 3. For the same microorganism: A, B Different superscript capital letters denote significant differences (p<0.05) between different ice cream formulations for the same day of storage. a,b Different superscript lower-case letters denote significant differences (p<0.05) between different days of storage for the same ice cream formulation.

28

ACCEPTED MANUSCRIPT Acknowledgements The authors wish to thank Fundação de Amparo à Pesquisa do Estado de São Paulo

T

(FAPESP) (Projects 2011/12981-0 and 2013/50506-8), Coordenação de Aperfeiçoamento de

IP

Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e

SC R

Tecnológico (CNPq) for financial support and fellowships. Thanks are due to Marina Gomes Rodrigues and Fernanda Perlis Ferreira for their clever technical assistance. The authors

NU

would also like to thank Yakult, Duas Rodas, Agropalma, and DuPont Danisco companies for

AC

CE P

TE

D

MA

providing part of material resources employed in the present study.

29

ACCEPTED MANUSCRIPT References

AC

CE P

TE

D

MA

NU

SC R

IP

T

Adams, L.B., Soileau, N.A., Battista, J.R., Krahenbuhl, J.L., 2000. Inhibition of metabolism and growth of Mycobacterium leprae by gamma irradiation. Int. J. Lepr. Other Mycobact. Dis. 68, 1-10. Akalin, A.S., Erisir, D., 2008. Effects of inulin and oligofructose on the rheological characteristics and probiotic culture survival in low-fat probiotic ice cream. J. Food Sci. 73, M184-188. Akın, M.B., Akın, M.S., Kırmacı, Z., 2007. Effects of inulin and sugar levels on the viability of yogurt and probiotic bacteria and the physical and sensory characteristics in probiotic icecream. Food Chem. 104, 93-99. Anvisa, 2008. Brazilian Agency of Sanitary Surveillance. Food with health claims, new foods/ingredients, bioactive compounds and probiotics. Bedani, R., Rossi, E.A., Isay Saad, S.M., 2013. Impact of inulin and okara on Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 viability in a fermented soy product and probiotic survival under in vitro simulated gastrointestinal conditions. Food Microbiol. 34, 382-389. Bedani, R., Rossi, E.A., Saad, S.M.I., 2013. Impact of inulin and okara on Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 viability in a fermented soy product and probiotic survival under in vitro simulated gastrointestinal conditions. Food Microbiol. 34, 382-389. Bedani, R., Vieira, A.D.S., Rossi, E.A., Saad, S.M.I., 2014. Tropical fruit pulps decreased probiotic survival to in vitro gastrointestinal stress in synbiotic soy yoghurt with okara during storage. LWT - Food Sci. Technol. 55, 436-443. Buriti, F.C., Castro, I.A., Saad, S.M., 2010. Viability of Lactobacillus acidophilus in synbiotic guava mousses and its survival under in vitro simulated gastrointestinal conditions. Int. J. Food Microbiol. 137, 121-129. Cardarelli, H.R., Saad, S.M.I., Gibson, G.R., Vulevic, J., 2007. Functional petit-suisse cheese: measure of the prebiotic effect. Anaerobe 13, 200-207. Cruz, A.G., Antunes, A.E.C., Sousa, A.L.O.P., Faria, J.A.F., Saad, S.M.I., 2009. Ice-cream as a probiotic food carrier. Food Res. Int. 42, 1233-1239. Darilmaz, D.O., Aslım, B., Suludere, Z., Akca, G., 2011. Influence of gastrointestinal system conditions on adhesion of exopolysaccharide-producing Lactobacillus delbrueckii subsp. bulgaricus strains to caco-2 cells. Braz. Arch. Biol. Technol. 54, 917-926. Davis, C., 2014. Enumeration of probiotic strains: review of culture-dependent and alternative techniques to quantify viable bacteria. J. Microbiol. Methods 103, 9-17. de Vrese, M., Kristen, H., Rautenberg, P., Laue, C., Schrezenmeir, J., 2011. Probiotic lactobacilli and bifidobacteria in a fermented milk product with added fruit preparation reduce antibiotic associated diarrhea and Helicobacter pylori activity. J. Dairy Res. 78, 396-403. Di Criscio, T., Fratianni, A., Mignogna, R., Cinquanta, L., Coppola, R., Sorrentino, E., Panfili, G., 2010. Production of functional probiotic, prebiotic, and synbiotic ice creams. J. Dairy Sci. 93, 4555-4564. Ferraz, J.L., Cruz, A.G., Cadena, R.S., Freitas, M.Q., Pinto, U.M., Carvalho, C.C., Faria, J.A., Bolini, H.M., 2012. Sensory acceptance and survival of probiotic bacteria in ice cream produced with different overrun levels. J. Food Sci. 77, S24-28. Fozo, E.M., Kajfasz, J.K., Quivey, R.G., 2004. Low pH-induced membrane fatty acid alterations in oral bacteria. FEMS Microbiol. Lett. 238, 291-295. Furet, J.P., Firmesse, O., Gourmelon, M., Bridonneau, C., Tap, J., Mondot, S., Dore, J., Corthier, G., 2009. Comparative assessment of human and farm animal faecal microbiota using real-time quantitative PCR. FEMS Microbiol. Ecol. 68, 351-362. 30

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

García-Cayuela, T., Tabasco, R., Peláez, C., Requena, T., 2009. Simultaneous detection and enumeration of viable lactic acid bacteria and bifidobacteria in fermented milk by using propidium monoazide and real-time PCR. Int. Dairy J. 19, 405-409. Geiser, M., 2003. The wonders of whey protein. NSCA’s Performance Training Journal 2, 1315. Gensberger, E.T., Polt, M., Konrad-Köszler, M., Kinner, P., Sessitsch, A., Kostić, T., 2014. Evaluation of quantitative PCR combined with PMA treatment for molecular assessment of microbial water quality. Water Res. 67, 367-376. Ghadimi, D., Folster-Holst, R., de Vrese, M., Winkler, P., Heller, K.J., Schrezenmeir, J., 2008. Effects of probiotic bacteria and their genomic DNA on TH1/TH2-cytokine production by peripheral blood mononuclear cells (PBMCs) of healthy and allergic subjects. Immunobiology 213, 677-692. Goff, H.D., Hartel, R.W., 2013. Ice Cream. Springer US, New York. Guo, Z., Wang, J., Yan, L., Chen, W., Liu, X.M., Zhang, H.P., 2009. In vitro comparison of probiotic properties of Lactobacillus casei Zhang, a potential new probiotic, with selected probiotic strains. LWT - Food Sci. Technol. 42, 1640-1646. Hernandez-Hernandez, O., Muthaiyan, A., Moreno, F.J., Montilla, A., Sanz, M.L., Ricke, S.C., 2012. Effect of prebiotic carbohydrates on the growth and tolerance of Lactobacillus. Food Microbiol. 30, 355-361. Hill, C., Guarner, F., Reid, G., Gibson, G.R., Merenstein, D.J., Pot, B., Morelli, L., Canani, R.B., Flint, H.J., Salminen, S., Calder, P.C., Sanders, M.E., 2014. Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat. Rev. Gastroenterol. Hepatol. 11, 506-514. Hossein Nezhad, M., Stenzel, D., Britz, M., 2010. Effect of growth at low pH on the cell surface properties of a typical strain of Lactobacillus casei group. Iran J. Microbiol. 2, 147154. International Dairy Federation, 1995. Fermented and non-fermented milk products: detection and enumeration of Lactobacillus acidophilus. Bulletin of IDF 306, 23-33. Koskenniemi, K., Laakso, K., Koponen, J., Kankainen, M., Greco, D., Auvinen, P., Savijoki, K., Nyman, T.A., Surakka, A., Salusjarvi, T., de Vos, W.M., Tynkkynen, S., Kalkkinen, N., Varmanen, P., 2011. Proteomics and transcriptomics characterization of bile stress response in probiotic Lactobacillus rhamnosus GG. Mol. Cell. Proteomics 10, M110 002741. Krissansen, G.W., 2007. Emerging health properties of whey proteins and their clinical implications. J. Am. Coll. Nutr. 26, 713S-723S. Liserre, A.M., Ré, M.I., Franco, B.D.G.M., 2007. Microencapsulation of Bifidobacterium animalis subsp. lactis in modified alginate-chitosan beads and evaluation of survival in simulated gastrointestinal conditions. Food Biotechnol. 21, 1-16. Matias, N.S., Bedani, R., Castro, I.A., Saad, S.M.I., 2014. A probiotic soy-based innovative product as an alternative to petit-suisse cheese. LWT - Food Sci. Technol. 59, 411-417. Meira, Q.G.S., Magnani, M., de Medeiros Júnior, F.C., Queiroga, R.C.R.E., Madruga, M.S., Gullón, B., Gomes, A.M.P., Pintado, M.M.E., de Souza, E.L., 2015. Effects of added Lactobacillus acidophilus and Bifidobacterium lactis probiotics on the quality characteristics of goat ricotta and their survival under simulated gastrointestinal conditions. Food Res. Int. 76, 828-838. Nocker, A., Cheung, C.-Y., Camper, A.K., 2006. Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J. Microbiol. Methods 67, 310-320.

31

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Nocker, A., Mazza, A., Masson, L., Camper, A.K., Brousseau, R., 2009. Selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology. J. Microbiol. Methods 76, 253-261. Nord, C.E., Lidbeck, A., Orrhage, K., Sjöstedt, S., 1997. Oral supplementation with lactic acid-producing bacteria during intake of clindamycin. Clin. Microbiol. Infect. 3, 124-132. Norton, S., Lacroix, C., Vuillemard, J.C., 1993. Effect of pH on the morphology of Lactobacillus helveticus in free‐cell batch and immobilized‐cell continuous fermentation. Food Biotechnol. 7, 235-251. Ranadheera, C.S., Evans, C.A., Adams, M.C., Baines, S.K., 2013. Production of probiotic ice cream from goat's milk and effect of packaging materials on product quality. Small Rumin. Res. 112, 174-180. Sanhueza, E., Paredes-Osses, E., González, C.L., García, A., 2015. Effect of pH in the survival of Lactobacillus salivarius strain UCO_979C wild type and the pH acid acclimated variant. Electron. J. Biotechnol. 18, 343-346. Savard, P., Lamarche, B., Paradis, M.E., Thiboutot, H., Laurin, E., Roy, D., 2011. Impact of Bifidobacterium animalis subsp. lactis BB-12 and, Lactobacillus acidophilus LA-5containing yoghurt, on fecal bacterial counts of healthy adults. Int. J. Food Microbiol. 149, 50-57. Silva, P.D.L.d., Bezerra, M.d.F., Santos, K.M.O.d., Correia, R.T.P., 2015. Potentially probiotic ice cream from goat's milk: Characterization and cell viability during processing, storage and simulated gastrointestinal conditions. LWT - Food Sci. Technol. 62, 452-457. Soukoulis, C., Fisk, I.D., Bohn, T., 2014. Ice cream as a vehicle for incorporating healthpromoting ingredients: conceptualization and overview of quality and storage stability. Compr. Rev. Food Sci. Food Saf. 13, 627-655. Tabasco, R., Paarup, T., Janer, C., Peláez, C., Requena, T., 2007. Selective enumeration and identification of mixed cultures of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. acidophilus, L. paracasei subsp. paracasei and Bifidobacterium lactis in fermented milk. Int. Dairy J. 17, 1107-1114. Taranto, M.P., Fernandez Murga, M.L., Lorca, G., de Valdez, G.F., 2003. Bile salts and cholesterol induce changes in the lipid cell membrane of Lactobacillus reuteri. J. Appl. Microbiol. 95, 86-91. Taverniti, V., Guglielmetti, S., 2011. The immunomodulatory properties of probiotic microorganisms beyond their viability (ghost probiotics: proposal of paraprobiotic concept). Genes Nutr. 6, 261-274. Taverniti, V., Scabiosi, C., Arioli, S., Mora, D., Guglielmetti, S., 2014. Short-term daily intake of 6 billion live probiotic cells can be insufficient in healthy adults to modulate the intestinal bifidobacteria and lactobacilli. J. Funct. Foods 6, 482-491. Tripathi, M.K., Giri, S.K., 2014. Probiotic functional foods: Survival of probiotics during processing and storage. J. Funct. Foods 9, 225-241. Vasconcelos, B.G., Martinez, R.C.R., de Castro, I.A., Saad, S.M.I., 2014. Innovative açaí (Euterpe oleracea, Mart., Arecaceae) functional frozen dessert exhibits high probiotic viability throughout shelf-life and supplementation with inulin improves sensory acceptance. Food Sci. Biotechnol. 23, 1843-1849. Verruck, S., Prudêncio, E.S., Vieira, C.R.W., Amante, E.R., Amboni, R.D.d.M.C., 2015. The buffalo Minas Frescal cheese as a protective matrix of Bifidobacterium BB-12 under in vitro simulated gastrointestinal conditions. LWT - Food Sci. Technol. 63, 1179-1183. Villarreal, M.L.M., Padilha, M., Vieira, A.D.S., Franco, B.D.G.M., Martinez, R.C.R., Saad, S.M.I., 2013. Advantageous direct quantification of viable closely related probiotics in petitsuisse cheeses under in vitro gastrointestinal conditions by propidium monoazide - qPCR. PLoS ONE 8, e82102. 32

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Vinderola, C.G., Reinheimer, J.A., 1999. Culture media for the enumeration of Bifidobacterium bifidum and Lactobacillus acidophilus in the presence of yoghurt bacteria. Int. Dairy J. 9, 497-505. Wang, J., Guo, Z., Zhang, Q., Yan, L., Chen, W., Liu, X.M., Zhang, H.P., 2009. Fermentation characteristics and transit tolerance of probiotic Lactobacillus casei Zhang in soymilk and bovine milk during storage. J. Dairy Sci. 92, 2468-2476. Yalcin, A.S., 2006. Emerging therapeutic potential of whey proteins and peptides. Curr. Pharm. Des. 12, 1637-1643. Zhang, Z., Liu, W., Xu, H., Aguilar, Z.P., Shah, N.P., Wei, H., 2015. Propidium monoazide combined with real-time PCR for selective detection of viable Staphylococcus aureus in milk powder and meat products. J. Dairy Sci. 98, 1625-1633.

33

ACCEPTED MANUSCRIPT Fig. 1. Survival of L. acidophilus La-5 obtained by PMA-qPCR in ice cream samples before (0 h) and during exposition to simulated gastrointestinal conditions for 2 h (gastric phase), 4 h (enteric phase 1), and 6 h (enteric phase 2) at 7, 42, and 84 days (i, ii, and iii, respectively) of A,B

different superscript capital letters denote significant

T

storage. For the same day of storage,

IP

differences among ice cream formulations for the same phase of the in vitro assay; a,b different lowercase superscript letters denote significant differences among different phases of the in

SC R

vitro assay for the same ice cream. Mean of triplicates (log cfu equivalents/g) as calculated

NU

from Ct values.

Fig. 2. Survival of B. animalis subsp. lactis Bb-12 obtained by PMA-qPCR in ice cream

MA

formulations before (0 h) and during exposition to simulated gastrointestinal conditions for 2 h (gastric phase), 4 h (enteric phase 1), and 6 h (enteric phase 2) at 7, 42, and 84 days (i, ii, and iii, respectively) of storage. For the same day of storage,

A,B

different superscript capital

a,b

different lowercase superscript letters denote significant differences among

TE

in vitro assay;

D

letters denote significant differences among ice cream formulations for the same phase of the

different phases of the in vitro assay for the same ice cream formulation. Mean of triplicates

CE P

(log cfu equivalents/g) as calculated from Ct values.

Fig. 3. Scanning electron microscopy showing morphological changes in L. acidophilus La-5

AC

and B. animalis Bb-12 cells throughout the in vitro simulated digestion of ice cream samples. Representative photographs were obtained at different magnifications a, b, and c (x5000, x15000, and 30000, respectively). Images were acquired in the following order, A: Initial phase with no enzymes addition; B: 2 h of incubation in the gastric phase (pH 2.4-2.9 in the presence of pepsin and lipase); C: 4 h of incubation in the enteric phase I (pH 4.8-5.6 in the presence of bile and pancreatin; and D: after 6 h of incubation in the enteric phase II (pH 6.46.9 in the presence of bile and pancreatin). White arrows indicate surface protrusions of the cell wall/capsule; black arrows indicate cell membrane clumping and damage.

34

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

Fig. 1

35

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

Fig. 2

36

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

Fig. 3

37

ACCEPTED MANUSCRIPT

In vitro gastrointestinal resistance of Lactobacillus acidophilus La-5 and Bifidobacterium

IP

T

animalis Bb-12 in soy and/or milk-based synbiotic apple ice creams

NU

HIGHLIGHTS

SC R

Natalia Silva Matias, Marina Padilha, Raquel Bedani, Susana Marta Isay Saad

Synbiotic ice creams developed with milk, soy, and whey protein isolate/inulin



Viability, in vitro GI resistance, and morphological changes were assessed



Counts were above 7.5 log cfu/g both by culture-dependent methods and PMA-qPCR



Survival rates were above 50% in all the ice creams after the in vitro assay



Scanning electron microscopy confirmed the intensification of the in vitro stress

AC

CE P

TE

D

MA



38