Effect of full-fat goat's milk and prebiotics use on Bifidobacterium BB-12 survival and on the physical properties of spray-dried powders under storage conditions

Accepted Manuscript Effect of full-fat goat's milk and prebiotics use on Bifidobacterium BB-12 survival and on the physical properties of spray-dried powders under storage conditions

Silvani Verruck, Gabriela Rodrigues de Liz, Carolinne Odebrech Dias, Renata Dias de Mello Castanho Amboni, Elane Schwinden Prudencio PII: DOI: Reference:

S0963-9969(18)30828-7 doi:10.1016/j.foodres.2018.10.042 FRIN 8012

To appear in:

Food Research International

Received date: Revised date: Accepted date:

29 June 2018 28 August 2018 11 October 2018

Please cite this article as: Silvani Verruck, Gabriela Rodrigues de Liz, Carolinne Odebrech Dias, Renata Dias de Mello Castanho Amboni, Elane Schwinden Prudencio , Effect of fullfat goat's milk and prebiotics use on Bifidobacterium BB-12 survival and on the physical properties of spray-dried powders under storage conditions. Frin (2018), doi:10.1016/ j.foodres.2018.10.042

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ACCEPTED MANUSCRIPT Effect of full-fat goat’s milk and prebiotics use on Bifidobacterium BB-12 survival and on the physical properties of spray-dried powders under storage conditions

Silvani Verrucka, Gabriela Rodrigues de Liza, Carolinne Odebrech Diasa, Renata Dias

Department of Food Science and Technology, Agricultural Sciences Center, Federal

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a

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de Mello Castanho Ambonia, Elane Schwinden Prudencioa,*

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University of Santa Catarina, Rod. Admar Gonzaga, 1346, Itacorubi, 88034-001,

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Florianópolis, SC, Brazil.

* Corresponding author: Elane Schwinden Prudencio, Department of Food Science and Technology, Agricultural Sciences Center, Federal University of Santa Catarina, Rod. Admar Gonzaga, 1346, Itacorubi, 88034-001, Florianópolis, SC, Brazil. +55 48 3721 5366, e-mail adress: [email protected]

ACCEPTED MANUSCRIPT Abstract The effects of full-fat goat’s milk and/or inulin and/or oligofructose, as carrier agents, were investigated to improve the survival rates of Bifidobacterium BB-12, and the physical properties of the microcapsules under storage conditions. On the day of their manufacture, the microcapsules were evaluated for morphology, particle size, and

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distribution of fat and bifidobacteria. The viability of the bifidobacteria, moisture and

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fat content, water activity, solubility, bulk and tapped density, flowability, cohesiveness

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and color properties were evaluated for 120 days at 4 ºC and 25 ºC. The full-fat goat’s milk powder with or without inulin as encapsulating agents showed the highest survival

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rates of Bifidobacterium BB-12 after spray drying and storage. Considering the bifidobacteria survival, both of these spray-dried powders showed the most desirable

properties

are

highlighted

for

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physical properties, i.e., lowest water activity and solubility, respectively. Both better

stability

of

spray-dried

powders,

with

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microcapsules, during storage time. These results are credited to full-fat goat’s milk (200 g L-1 ) and the presence of inulin (100 g L-1 ), besides the fat content showing a high

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correlation with the solubility values. The lowest volume occupied by the spray-dried powders was noted when oligofructose was used as the carrier agent. The samples that

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showed the presence of cracks influenced negatively on the bifidobacteria viability. These cracks were responsible by the greater water escape, resulting in powders with more desirable lower water activity. In relation to the color parameters, lower stability was noted when oligofructose was used, while the best stability was also noted for the powders

with full-fat

goat’s

milk

and/or inulin. During storage time,

the best

performance was achieved by the microencapsulation process that used only full-fat goat’s milk and/or inulin and storage at 4 °C. Keywords: goat’s milk; inulin; oligofructose; bifidobacteria; microcapsule.

ACCEPTED MANUSCRIPT 1. Introduction It is well known that the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) have defined that probiotics are “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (Hill et al., 2014). Therefore, probiotics are one of the key elements

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that are being recognized as a driving force of the functional food market because of

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their now well-documented health benefits. Ribeiro et al. (2014) reported that studies

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performed with these microorganisms show the benefits probiotics provide to humans, such as the reduction of lactose intolerance, lower cholesterol levels, stimulation of the

anti-mutagenic,

anti-carcinogenic,

and

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immune system, relief from constipation, increased absorption of minerals, as well as anti-hypertensive

effects.

In

this

respect,

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probiotics must be metabolically stable and active in the food product, and the Bifidobacterium strains are the probiotic bacteria that are mostly used (Shori, 2017).

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Unfortunately, many conditions of manufacture and storage of food products lead to losses in the viability of this probiotic bacterium. Champagne, Raymond, Guertin, and

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Bélanger (2015) stated that means to improve the stability of probiotics in foods are needed. Therefore, different approaches to increase the resistance of these sensitive

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bacteria against adverse conditions, such as microencapsulation by spray drying, have been proposed.

Microencapsulation is one of the most efficient methods of protecting probiotics and has been under especial consideration and investigation (Martín et al., 2015). This method is defined as the process in which cells are retained within an encapsulating membrane in order to reduce or minimize injuries and loss of the probiotic bacteria (Chen, Chen, & Liu, 2006). Ribeiro et al. (2014) stated that the incorporation of encapsulated probiotic bacteria in a food ensures a better survival of this microorganism

ACCEPTED MANUSCRIPT when compared to the free microorganism. The carrier agents used as probiotic encapsulating material have different compositions; however, in the past few years, the use of prebiotics has shown many advantages. Darjani, Hosseini Nezhad, Kadkhodaee, and Milani (2016) reported that prebiotics are non-digestible food compounds that are selectively fermented by the gut

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microbiota, and thus improve the host’s health by stimulating the growth and activity of

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the probiotics. Moreover, they can also serve as a nutrient for the encapsulated probiotic

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cells once they are released. Many of the prebiotics, such as inulin and oligofructose, have been investigated for their biological importance in promoting human health.

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Yang, Zhang, Mao, You, and Chen (2016) stated that inulin is a type of fructose polymer whose units are linked by (2-1) glycosidic bonds and that the degree of

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polymerization of these fructans can reach up to 60 units. These authors also reported that inulin can be hydrolyzed into oligofructose by inulinase. It is noteworthy that these

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prebiotic fibers have been classified by the Food and Drug Administration (FDA) as safe for human consumption. Apart from that, inulin and oligofructose have been

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applied, usually in association with other materials, as encapsulating agents for some probiotic strains (Freire et al., 2012; Pinto et al., 2015a). Currently, there are a lot of

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other encapsulation agents that are being used and have advantages of their own, such as cow’s milk. However, the use of full-fat goat’s milk with/without the addition of prebiotics as encapsulating agents is the novelty employed by our research group in this study. Albenzio et al. (2015) reported that goat’s milk is considered an excellent matrix for developing a large variety of innovative products, such as the microcapsules. Apart from that, Clark and García (2017) cited that that greatest proportion of goat’s milk fat globules were < 4 μm in diameter, while for the cow milk the fat globules were > 4 μm

ACCEPTED MANUSCRIPT in diameter. Amigo and Fontecha (2011) reported that the fat in goat’s milk naturally does not form aggregate because they lack agglutinin, responsible for the aggregation of fat globules in cow’s milk. When the goat’s milk is used as carrier agent generates smaller microcapsules, preventing the coalescence and agglomeration of the spray-dried powder. Thereby, the goat’s milk fat and the agglutinin lack could collaborate for the

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higher retention and survival of the bacteria entrapped in the microcapsules.

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The characterization and the monitoring of the microcapsules during storage at

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different temperatures are also important approaches to improve and determine the optimal conditions when adding them to food products. Given the importance of this

inulin

and/or

oligofructose

as

carrier

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topic, the highlight of our work is the use of full-fat goat’s milk either with or without agents

for

the

microencapsulation

of

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Bifidobacterium BB-12. In order to ensure the success of the spray-dried powders, we evaluated the stability of the properties of the feed solutions, the microcapsules, and the

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spray-dried powders. The properties of the feed solutions as well as morphology, particle size, and confocal laser scanning microscopy of the microcapsules were

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evaluated only on the day that the spray-dried powder was manufactured. Meanwhile, the effect of full-fat goat’s milk, inulin and/or oligofructose on the microcapsules of

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Bifidobacterium BB-12 and the properties of the spray-dried powders were determined during the 120 days of storage at 4 °C and at 25 °C.

2. Materials and methods 2.1 Materials A probiotic culture composed of Bifidobacterium BB-12 (BB-12®, Chr. Hansen, Hønsholm, Denmark) was used as the active material for the microcapsules, while UHT (ultra-high temperature) goat’s milk (Caprilat®, Rio de Janeiro, Brazil, was employed

ACCEPTED MANUSCRIPT for the preparation of the bacterial suspension. Full-fat goat’s milk powder (Caprilat®, Rio de Janeiro, Brazil) and prebiotics agents like inulin (Orafti® Gr, Tienen, Belgium), with degree of polymerization (DP) ≥ 10, and oligofructose (Orafti® P95, Tienen, Belgium), with DP ranging from 2 to 8, were used as carrier agents for bifidobacteria. For the viable cell count of bifidobacteria, it was employed MRS Agar (Difco, Sparks,

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USA), lithium chloride (Vetec, Rio de Janeiro, Brazil), sodium propionate (Fluka, Neu-

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Ulm, Germany) and AnaeroGen® (Oxoid, Hampshire, UK). For confocal laser scanning

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microscopy were used the Nile red (9-(diethyl amino) benzo [a]phenoxazin-5(5H)-one) and the DAPI (4',6-Diamidino-2-phenylindole) dyes from Sigma–Aldrich (St. Louis,

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MO, USA). All other chemicals were of analytical grade or equivalent purity.

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2.2 Bifidobacterium BB-12 microencapsulation by spray drying For Bifidobacterium BB-12 microencapsulation by spray drying, four feed

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solutions were prepared employing 1 liter of distilled water for each one, as diluting medium, and according to the carrier agents denoted as: GM: 200 grams of full-fat

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goat’s milk powder; GMI: 100 grams of full-fat goat’s milk powder and 100 grams of inulin; GMO: 100 grams of full-fat goat’s milk powder and 100 grams of oligofructose;

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and GMIO: 100 grams of full-fat goat’s milk powder, 50 grams of inulin and 50 grams of oligofructose (Verruck et al., 2017). All the feed solutions were homogenized and heated at 80 ± 1ºC for 30 min and left to cool down to room temperature. The bacterial suspension was prepared according to the procedures described by Fritzen-Freire et al. (2012), with modifications. The Bifidobacterium BB-12 freezedried cells were rehydrated at 25 g L−1 using 1 liter of UHT goat’s milk and frozen as a stock solution at −18 ± 2°C into sterile glass bottles. This stock solution was incubated

ACCEPTED MANUSCRIPT at 37 ± 1ºC for 2 h and either inoculated (100 mL L-1 ) into the four feed solutions for microencapsulation by spray drying. The microencapsulation process was carried out with a laboratory scale spray dryer (B-290 mini spray dryer, Buchi, Flawil, Switzerland), operating at constant air inlet temperature of 150 ± 1ºC and an outlet temperature of 50 ± 3ºC. The four feed

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solutions containing Bifidobacterium BB-12 were kept under magnetic agitation at

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room temperature and fed into the main chamber through a peristaltic pump, with feed

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flow of 20 mL min−1 , drying airflow rate of 35 m3 h−1 , and compressor air pressure of 0.7 MPa. From each feed solutions were obtained the following spray dried powder

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samples, which were designated as GM1 from GM; GMI2 from GMI; GMO3 from GMO; and GMIO4 from GMIO. The entire process for the production of the probiotic

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microcapsules was performed in triplicate. Each spray dried powder sample type was collected from the base of the cyclone, pooled and placed in sterile plastic vials and

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stored.

As the spray dried powder properties are significantly affected by feed solutions

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composition, it was analyzed their total solids content and density. With the exception of product yield, morphology, particle size, and confocal laser scanning microscopy

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analyzes, which were evaluated only on the day of spray dried powder manufacture; the effect of goat’s milk, inulin and/or oligofructose on the Bifidobacterium BB-12 microcapsules properties

were

also

determined

during 120

days with storage

temperature equal to 4 ± 1ºC and 25 ± 1ºC, respectively. All analyzes were performed in triplicate, except those for morphology and confocal laser scanning microscopy (CSLM), which were evaluated from multiple images once.

2.3 Viable Bifidobacterium BB-12 cells count

ACCEPTED MANUSCRIPT Aiming to ensure the survival of Bifidobacterium BB-12 after spray drying process, the viable cells count was conducted for the four feed solutions (GM, GMI, GMO, GMIO) and for their spray dried powders (GM1, GMI2, GMO3, GMIO4), respectively. For the spray-dried powders, the viable cells count was also determined during 120 days using storage temperatures equal to 4 ± 1ºC and 25 ± 1ºC. The viable

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Bifidobacterium BB-12 cells count was expressed as log colony-forming units per gram

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of dry matter in the four feed solution, and the four spray dried powders (log CFU g-1 of

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dry matter) according to Verruck et al. (2017).

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2.4 Microcapsules and spray dried powders properties 2.4.1 Morphology and particle size

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The morphology and particle size of the microcapsules were evaluated using a Jeol scanning electron microscope, model JSM 6390 LV (Jeol, Tokyo, Japan), at an

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accelerating voltage of 10 kV. Firstly, the samples were coated with gold with a vacuum sputtering coater (Leica, model EM SCD 500, Wetzlar, Germany) (Lian, Hsiao, &

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Chou, 2002). The microcapsules diameter was measured from the Scanning electron microscopy (SEM) micrographs in their original magnification using ImageJ (version

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1.47; http://rsb.info.nih.gov/ij/). The diameter of the particles was measured for each spray-dried powder (GM1, GMI2, GMO3, GMIO4) considering the mean of 300 particles, as described by Pinto et al. (2015a).

2.4.2 Confocal laser scanning microscopy (CLSM) For the localization and visualization of the fat globules and Bifidobacterium BB-12 disposition into the microcapsules, a Leica TCS-SP5 confocal laser scanning microscope (CSLM) (Wetzlar, Germany) was used. The fat globules were stained with

ACCEPTED MANUSCRIPT Nile red at a concentration of 0.1 g L-1 in 1,2-propanediol (Auty, Twomey, Guinee, & Mulvihill, 2001) and the Bifidobacterium BB-12 were stained with DAPI at a concentration of 0.1 g L-1 in phosphate buffered saline (PBS buffer). Fat globules and Bifidobacterium BB-12 were imaged simultaneously at 514 nm and 405 nm laser excitation, respectively. Images were acquired on CLSM using a 100 x objective (HCX

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PL APO CS with a numerical aperture of 1.44 – oil immersion objective) with Leica

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Type F immersion oil. Images were taken with a Leica microsystem camera. The count

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of fat globules per microcapsule and the measure of size range of fat globules were determined from 10 microcapsules of each spray dried powder sample (GM1, GMI2,

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GMO3, GMIO4), while the measure of the pixels intensity were performed from around 100 microcapsules, considering the same acquisition configurations and same area for

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all samples. The acquisition and processing of data were carried out using the LAS AF

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Lite software.

2.4.3 Moisture, water activity, and fat content

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The moisture content was determined gravimetrically by oven drying at 102 °C until reaching constant weight, as described by IDF (IDF, 1993). Water activity was

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measured at 25°C using an Aqualab 4TE analyzer (Decagon Devices, USA) after the samples had been stabilized for 15 min. The fat content was analyzed according to AOAC (AOAC, 2005) by Gerber butyrometer method.

2.4.4 Solubility in water The solubility in water was evaluated by adding 1 g of each spray dried powder samples (GM1, GMI2, GMO3, GMIO4) in 25 mL of distilled water and stirred for 5 minutes using a magnetic stirrer. The solution was centrifuged at 760 × g for 10

ACCEPTED MANUSCRIPT minutes, and a 20 mL aliquot of the supernatant was transferred to a pre-weighed Petri dish and subjected to drying in an oven at 80°C overnight, as described by Eastman and Moore (1984). The solubility in water (%) was calculated as the percentage of dried supernatant compared to the initial amount of powder (1 g).

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2.4.5 Bulk and tapped densities

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Bulk (ρbulk ) and tapped (ρtapped) densities were calculated as proposed by

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Jinapong, Suphantharika, and Jamnong (2008), with modifications. For ρbulk calculation, 2 g of each spray dried powder sample (GM1, GMI2, GMO3, GMIO4) was gently

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loaded into 10 mL tared graduated cylinders, and the filled volume was read. The same procedure was realized for the tapped density (ρtapped), however, before reading the

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volume, the cylinder was tapped vigorously until no further change in volume occurred. Both bulk and tapped density were calculated by dividing the mass of the spray-dried

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powder by the volume occupied in the graduated cylinder.

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2.4.6 Flowability and cohesiveness

The flowability and cohesiveness of all spray dried powders were carried out

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regarding Carr’s index (CI) and Hausner ratio (HR), as realized by methodology proposed by Carr (Carr, 1965) and Hausner (Hausner, 1967), respectively. Both CI and HR ratio were calculated from the bulk (ρbulk ) and tapped (ρtapped) densities.

2.4.7 Color analysis The color analysis of spray dried powders was determined using a colorimeter Minolta Chroma Meter CR-400 (Konica Minolta, Osaka, Japan), adjusted to operate with D65 lightning and 10º of observation angle. The total difference of color (ΔE*)

ACCEPTED MANUSCRIPT between the measured values in the final storage time (day 120) and initial storage time (day 1) was calculated as described by Verruck et al. (2014). Hue angle (h*) and Chroma (C*) values were determined according to Jha (2010).

2.6 Statistical analysis

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To determine significant differences (P < 0.05) between the results, one-way

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analysis of variance (ANOVA) and Tukey studentized range test was used. When

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appropriate, the data also were submitted to linear correlation (R) from the regression analysis. All statistical analyses were performed using STATISTICA 13.0 software

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(StatSoft Inc., Tulsa, USA). The data were expressed as mean ± standard deviation.

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3. Results and discussion

3.1 Viable Bifidobacterium BB-12 cell count

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After the microencapsulation process, the viable Bifidobacterium BB-12 cell count reduced in 0.52, 0.53, 1.20 and 1.12 log cycles, for GM1, GMI2, GMO3, and

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GMIO4, respectively (Table 1).

All spray-dried powders showed viable cell counts

above 7.98 log CFU g-1 of the product. According to Hill et al. (2014) for a product be

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considered with potential benefits to the human health, it must have a viable cell counts equal to or greater than 6 log CFU g-1 . However, the results showed that full-fat goat’s milk with/without inulin were the best carrier agents for the microencapsulation of bifidobacteria by spray drying. Picot and Lacroix (2004) reported that probiotic cell survival during the microencapsulation process could be credited to the presence of milk fat, such as our initial supposition. Liu et al. (2015) hypothesized that the melting of the fat would absorb part of the heat energy, decreasing the internal temperature of the microcapsules, and thus preventing rupture of the probiotics’ cell membrane. Fritzen-

ACCEPTED MANUSCRIPT Freire et al. (2012) also observed that inulin had a positive effect on the protection of bifidobacteria during the microencapsulation process and attributed this effect to a possible thermoprotection provided by this carrier agent to the cells undergoing the drying process. According to Ananta, Volkert, and Knorr (2005), some carrier agents with DP = 2 – 8 interact directly with polar head groups, reducing the protection of the

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membranes during the drying process. On the other hand, these authors also state that

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steric hindrance of the polymers due to their large size prevented them from interacting

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with dried proteins or membrane lipids. Therefore, the total maintenance of the structural and functional integrity of the bacterial cell membrane during the spray drying

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process could be affected.

The viable Bifidobacterium BB-12 cell counts throughout the storage time of

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120 days at 4 ± 1°C and 25 ± 1°C are shown in Fig. 1(a) and (b), respectively. In relation to storage at 4°C, our findings suggest that full-fat goat’s milk or its partial

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replacement with inulin or oligofructose in the spray drying process caused no negative effect on the survival of Bifidobacterium BB-12. According to Pinto et al. (2015a),

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these changes were of little microbiological significance since they were always of values lower than 0.5 log CFU g−1 and the viable cell count of the microcapsules

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remained higher than 6 log CFU g-1 . Pedroso, Thomazini, Heinemann, and FavaroTrindade (2012) reported that this result was obtained probably because at this temperature the cells have their chemical interactions reduced. Regarding the samples stored at 25ºC, distinct behaviors on the viable bifidobacteria cell count were noted for all the spray-dried powders (GM1, GMI2, GMO3, and GMIO4). On all the days of storage, the spray-dried powders containing microencapsulated bifidobacteria showed a decrease (P < 0.05) in viable cell count. It is noteworthy

that

the

protection

performance

during

storage

was lower when

ACCEPTED MANUSCRIPT oligofructose was used as the carrier agent. Ananta, Volkert, and Knorr (2005) also noted an adverse impact on the stability of Lactobacillus rhamnosus GG during prolonged storage when part of the milk was replaced with oligosaccharides with a degree of polymerization between 2 and 8. These authors attributed the reduction in viable cell count to the net quantity of directly interacting molecules in the drying

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medium. On the other hand, the decrease in the viable cell count during storage at 25°C

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might be correlated with the natural mechanism that involves the degradation of lifeessential macromolecules. At this temperature, the cell counts remained higher than 6

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log CFU g-1 for GM1 and GMI2 for 40 days, while for GMIO4 they remained higher

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than 6 log CFU g-1 only for 20 days. However, it is known that the use of different

possibly affect their functionality.

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carrier agents can result in microcapsules with different physical properties, and thus

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3.2 Properties of the microcapsules and the spray-dried powders Fig. 2 shows the scanning electron microscopy (SEM) micrographs of the

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Bifidobacterium BB-12 microcapsules from the spray-dried powders using full-fat goat’s milk powder, inulin and/or oligofructose as carrier agents. These SEM images

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indicate that the Bifidobacterium BB-12 cells were trapped inside the microcapsules because it was possible to note the absence of cells either outside or on the surface of the microcapsules. It was also possible to note that all the microcapsules from the spraydried powders showed particles that were spherical and of different sizes. However, the presence of concavities was verified only for the microcapsules from GM1 (black arrows, Fig. 2a, 2b). Pinto et al. (2015a) stated that these behaviors are typical characteristics of spray-dried products.

ACCEPTED MANUSCRIPT Some cracks were noted in the external surface of the microcapsules from GMI2, GMO3, and GMIO4 (white arrows, Fig. 2d, 2f, 2h). These cracks occur when there is slow crust formation during the drying of atomized droplets. Duongthingoc, George, Katopo, Gorczyca, and Kasapis (2013) highlighted that rapid crust formation, and therefore less presence of cracks potentially shields the core material sooner by

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reducing the extent of the thermal shock transmitted through the interior of the high-

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solid matrix. Therefore, the absence of cracks noted in the surface of the microcapsules

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from GM1 could be related with the best survival rate of Bifidobacterium BB-12 during the storage time evaluated (Fig. 1b). The absence of cracks on GM1 could be related

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with the milk-rich formulation, containing a higher lactose and protein presence, providing the highest degree of cell protection during drying. Despite this behavior,

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Huang et al. (2017) hypothesized that this mechanism is due to the stabilizing effect of low molar mass carbohydrate and proteins on cell membranes, by replacing the water

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around polar residues within these macromolecular structures, decreasing the membrane phase transition temperature.

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In relation to the morphology, Parthasarathi and Anandharamakrishnan (2016) stated that the particle size of the microcapsules is affected by the type of carrier agent

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used. These authors also stated that particle size is an important property for the microcapsules because of its strong influence on product solubility, appearance, and acceptability. Duongthingoc et al. (2013) recommended that a population with an average particle size below 40 μm in spray-dried particles is expected to provide acceptable mouthfeel of food products. Our findings (Table 2) indicate that the sizes of the particles are in accordance with those recommended. The influence of fat milk distribution and the presence of Bifidobacterium BB12 in the microcapsules were evaluated by confocal laser scanning microscopy (CLSM)

ACCEPTED MANUSCRIPT (Table 2 and Fig. 3). These images show the presence of the bifidobacteria inside all the microcapsules produced. It was possible to note that fat globules and the bacterial cells were evenly dispersed throughout the entire matrix of the microcapsules. Moreover, it was possible to confirm the spherical shape of the microcapsules. However, the images also indicate that the microcapsules from GM1 showed less affinity with the dyes that

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were used and, consequently, a lower breaking down of the primary microcapsule

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structure was noted. This behavior could also be associated with the absence of cracks

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in the external surface of these microcapsules previously detected by SEM. Due to the composition of the microcapsules, higher (P < 0.05) values for the

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number, size, and the occupied area of the fat globules were noted for the GM1 microcapsules. As well as the thermoprotective effect already established for inulin,

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these results lead us to believe that the fat globules also contributed to the protection of Bifidobacterium BB-12. The affinity of bacteria to milk fat was noticed by Burgain et

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al. (2014). According to these authors, components such as mucins, phospholipids, proteins, glycophospholipids, and gangliosides located in the membrane of milk fat

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globules are known to show affinity to the bacterial cell surface. According to Ly, Vo, Le, Belin, and Waché (2006), bacteria possess surface properties that are related with

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their charge and with their Lewis acid/base and hydrophobicity characteristics, which are involved in the attachment of microorganisms to the surface of fat globules. Despite the spray-dried powders (GM1, GMI2, GMO3, GMIO4) showing an increase in moisture content and water activity during the storage time at the different temperatures used, their moisture content remained below 4 g 100 g−1 (Table 3). These values are in accordance with those obtained by Schuck (2011), who reported that this is usually the recommended value for adequate maintenance of the viability of the probiotics in the microcapsules and prevention of caking during storage. This maximum

ACCEPTED MANUSCRIPT content needs to be taken into consideration because, as was reported by Broeckx, Vandenheuvel, Claes, Lebeer, and Kiekens (2016), high molecular mobility destabilizes the carrier agents and changes the characteristics of the powder, leading to loss of the probiotics’ viability. Also, there may be a decrease in the shelf life of the stored powder. The use of inulin followed by the use of oligofructose or oligofructose and inulin

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contributed to produce spray-dried powders that showed water activity values lower (P

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< 0.05) than those for GM1; even so, all these values remained below 0.3. Tonon,

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Brabet, Pallet, Brat, and Hubinger (2009) reported that this maximum value is recommended for the stability of the microcapsules during storage. The higher (P <

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0.05) water activity values for GM1 could be associated with the double of full fat used in the GM feed solution. As mentioned before, fat is able to absorb part of the heat

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during the spray drying process, and thus decrease the internal temperature of the particles, contributing to water retention inside the microcapsules. This behavior can be

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seen in Fig. 2 by the presence of cracks in the surface of the microcapsules from GMI2, GMO3, and GMIO4, which facilitate the outflow of water.

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The fat content showed high correlation with the solubility values (R= - 0.974, P < 0.05). This finding corroborates those obtained by Kim, Chen, and Pearce (2009) for

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industrial spray-dried milk powders. These authors concluded that when fat is present on the surface of the matrix of the powder, the solubility of the powder decreases because of fat’s hydrophobic nature. Regarding the solubility values, it was also possible to verify that the use of both prebiotics resulted in microcapsules that are more soluble (P < 0.05) than those produced only with full-fat goat’s milk (GM1) as a carrier agent. Karimi, Azizi, Ghasemlou, and Vaziri

(2015) stated that the solubility of

prebiotics is related with the hydroxyl groups in the molecular structure because of their higher ability to interact with water than with other molecular parts. Therefore, the

ACCEPTED MANUSCRIPT lowest solubility rates contributed to the longest microcapsule dissolution time. Pinto et al. (2015b) emphasized that longer dissolution times ensure good control of the release of the probiotic cells when in contact with an aqueous solution, which is expected when adding microcapsules in food products. Other physical properties of the microcapsules are represented by bulk and

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tapped densities. It was possible to note (Table 4) that the carrier agents employed in

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this study were that factor that mostly caused the differences between the values

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obtained for the bulk and the tapped densities of the powders. In general, the use of oligofructose as the carrier agent contributed to the lowest volume occupied. Rajam and

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Anandharamakrishnan (2015) noted that the fructooligosaccharide ratio contributed to the decrease of the volume. These authors suggest that the fructooligosaccharide

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encapsulates showed higher bulk density because of the higher particle aggregation and fewer interspaces between the particles. Kosasih, Bhandari, Prakash, Bansal, and Gaiani

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(2016) stated that bulk and tapped densities are related to the amount of air entrapped in the microcapsules. Therefore, the cracks in the surface of the microcapsules, as shown

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in Fig. 2, may have affected the bulk density values by releasing more of the entrapped air from the samples produced with inulin and oligofructose as carrier agents.

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The values for flowability and cohesiveness (Table 4) obtained for all the spraydried powders were high. Parthasarathi and Anandharamakrishnan (2016) stated that flowability results above 38% are classified as “very, very poor” flowability. These same authors state that a Hausner ratio that is greater than or equal to 1.25 indicates a powder with “poor” flowability. Our spray-dried powders showed a Carr’s index and a Hausner ratio > 36% and ≥ 1.51, respectively, which indicate “very, very poor” flow properties. However, for both measures, lower values were obtained with the spraydried powders that had oligofructose or inulin and oligofructose as carrier agents than

ACCEPTED MANUSCRIPT those produced only with full-fat goat’s milk. According to Parthasarathi and Anandharamakrishnan (2016), a “poor” flowability might be attributed to the cracks in the surface of the spray-dried powders. Moreover, Xinde, Shanjin, Ning, and Bin (2007) reported that besides the surface properties of powders, other factors, such as their interaction forces and their particle sizes, are also important for the flow function. It is

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noteworthy that all the spray-dried powders produced in our study showed low moisture

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contents (Table 3) and no liquid bridging was observed in the SEM micrographs (Fig.

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2). In this case, Xinde, Shanjin, Ning, and Bin (2007) stated that the cohesion is mainly affected by van der Waals force and/or chemical bonds. Besides, the smaller

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microcapsules size by SEM (Table 2) would result in a more cohesive flow. These authors stated that a smaller particle size would probably provide a greater surface area

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for interparticle attractive forces to interact, and thus would probably result in a more cohesive behavior, which in turn is determined by the ingredient of the wall material.

spray dried powders.

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These results are desirable for the economic point of view, and the industry handling of

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Table 5 shows the color parameters for the spray-dried powders (GM1, GMI2, GMO3, GMIO4) during storage time.

Martínez-Cervera, Salvador, Muguerza, Moulay,

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and Fiszman (2011) reported that total color difference (ΔE*) values lower than 3 cannot be visually perceived by the human eye. Therefore, it was possible to verify that only GMO3 and GMIO4, i.e., with the addition of oligofructose and storage at 25°C for 120 days, were affected. According to Mensink, Frijlink, Maarschalk, and Hinrichs (2015), the hydrolysis of inulin into oligofructose results in a product that contains high amounts of reducing groups which are able to participate in Maillard reactions. As was noted in our study (Fig. 1), the formation of these compounds was reported by Kurtmann, Skibsted, and Carlsen (2009) to cause a decrease in the viability of freeze-

ACCEPTED MANUSCRIPT dried Lactobacillus acidophilus throughout storage time. These findings can enhance the information about the decrease in bifidobacteria viability during the formation of browning compounds. Therefore, the presence of browning reactions needs to be taken into consideration when choosing carrier agents for stabilizing probiotics. In the near future, these results will further facilitate decision making and may

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be useful to elucidate risks associated with the utilization of these microcapsules and/or

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spray-dried powders. Finally, according to our study in the field of microencapsulation

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of probiotic bacteria, it is expected in future fabricating novel microcapsules with the combination of different carries agents, such as the milk, with the different fat content

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ratio, and different prebiotics.

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4. Conclusion

The use of full-fat goat’s milk with or without inulin as microcapsule carrier

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agents produced the best results for the viability of Bifidobacterium BB-12 after the spray drying process. The results for storage at 4°C showed that full-fat goat’s milk or

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its partial replacement with inulin or oligofructose did not reduce the viable cell count of Bifidobacterium BB-12, whereas for storage at 25°C the all results showed a However,

this protection performance was lower with the use of

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reduction.

oligofructose. The best Bifidobacterium BB-12 survival results may be related to the absence of cracks in microcapsules, which can be verified in the morphology analysis. As for the morphology, the sizes of the particle of the microcapsules were affected by the type of carrier agent used. In general, the use of oligofructose contributed to the lowest volume occupied by the spray-dried powders. Besides, among the results obtained for microcapsules physical properties, it was also possible to verify that the inulin addition contributed to obtain spray-dried powders with higher product yield and

ACCEPTED MANUSCRIPT density, and therefore with lower occupied volume. Therefore, during the storage time and temperature evaluated, the inulin addition also generated spray dried powders with better stability of the color parameters. However, all the microcapsules showed a tendency toward a slightly greenish yellow.

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Conflict of interest statement

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Authors declare no conflicts of interest.

Acknowledgments

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The authors thank the LCME-UFSC facilities for technical support during electron microscopy and confocal laser scanning microscopy analysis. This work was

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supported by the National Council of Technological and Scientific Development [CNPq, 405965/2016-8] and the Coordination of Improvement of Higher Education

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Personnel [CAPES, scholarship]. The authors are thankful to BENEO Latinoamerica for

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ACCEPTED MANUSCRIPT Fig. 1. Bifidobacterium BB-12 viable cells count from spray dried powder during 120 days of storage at (a) 4 ± 1°C and (b) 25 ± 1°C. (□) GM1, (○) GMI2, (Δ) GMO3 and (◊) GMIO4 are the spray dried powders from feed solutions prepared employing 1 L of distilled water, and with the following carrier agents 100 g of full-fat goat’s milk powder, 100 g of full-fat goat’s milk powder and 100 g of inulin, 100 g of full-fat goat’s

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milk powder and 100 g of oligofructose, and with 100 g of full-fat goat’s milk powder,

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50 g of inulin and 50 g of oligofructose, respectively. Error bars represent standard

2.

Scanning electron microscopy micrographs of Bifidobacterium BB-12

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Fig.

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deviations of the mean of the experiment.

microcapsules of the spray dried powders from feed solutions prepared employing 1 L

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of distilled water, and with the following carrier agents: (a.b) 100 g of full-fat goat’s milk powder (GM1); (c,d) 100 g of full-fat goat’s milk powder and 100 g of inulin

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(GMI2); (e,f) 100 g of full-fat goat’s milk powder and 100 g of oligofructose (GMO3); and (g,h) 100 g of full-fat goat’s milk powder, 50 g of inulin and 50 g of oligofructose

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(GMIO4). Black arrows indicate concavities on the microcapsules surfaces. White

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arrows indicate cracks in the microcapsules surfaces.

Fig. 3. Confocal scanning laser micrographs of Bifidobacterium BB-12 microcapsules of the spray dried powders from feed solutions prepared employing 1 L of distilled water, and with the following carrier agents: (a)

100 g of full-fat goat’s milk powder

(GM1); (b) 100 g of full-fat goat’s milk powder and 100 g of inulin (GMI2); (c) 100 g of full-fat goat’s milk powder and 100 g of oligofructose (GMO3); and (d) 100 g of fullfat goat’s milk powder, 50 g of inulin and 50 g of oligofructose (GMIO4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

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ACCEPTED MANUSCRIPT Table 1 Bifidobacterium BB-12 viable cells count for the feed solutions and for the spray dried powders. Viable cells count (log CFU g−1 ) 9.22 ± 0.06a

GMI

9.11 ± 0.20a

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Feed solutions

GM

9.33 ± 0.13a

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GMO

9.10 ± 0.13a

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GMIO

GMI2

8.58 ± 0.13b 8.13 ± 0.16c

GMIO4

7.98 ± 0.05c

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Means ± standard deviation with different superscript lowercase letters indicate

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a–c

8.69 ± 0.02b

GMO3

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Spray dried powders

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GM1

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significant differences (P < 0.05) between samples.

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ACCEPTED MANUSCRIPT Table 2 SEM and CLSM morphological properties of Bifidobacterium BB-12 microcapsules. Microcapsule

Microcapsule

Number of fat

Size range of

Area occupied by

size (μm) by

size range

globules per

fat globules

fat globules per

SEM

(μm) by

microcapsule by

(μm) by

particle by CLSM

SEM

CLSM

CLSM

(pixel intensity)

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Samples

6.37 ± 4.17a

2.01 – 19.10

279.67 ± 76.51a

0.08 – 7.02

38.85 ± 7.66a

GMI2

6.26 ± 4.05a

2.01 – 19.50

133.33 ± 23.71b

0.14 – 4.00

18.25 ± 5.09b

GMO3

6.00 ± 3.47a

1.99 – 18.93

114.50 ± 33.00b

0.04 – 4.95

16.76 ± 4.02b

GMIO4

5.74 ± 3.54a

2.02 – 18.81

136.75 ± 27.61b

0.06 – 5.80

22.34 ± 2.26b

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a–b

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GM1

Means ± standard deviation with different superscript letters in the same column indicate significant

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differences (P < 0.05) between the microcapsules. SEM: Scanning electron microscopy; CLSM: Confocal

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Table 3 Moisture content, water activity, fat content, and solubility Bifidobacterium BB-12 microcapsules during the storage period.

properties

Storage day

Temperature (ºC)

Moisture (g 100 g −1 )

Water activity

Fat (g 100 g −1 )

Solubility (%)

GM1

0

4

3.68 ± 0.15bA

0.208 ± 0.004cA

27.63 ± 0.81aA

60.82 ± 2.21aB

20

4

3.62 ± 0.12bA

0.215 ± 0.001bA

27.59 ± 0.79aA

62.12 ± 2.33aB

40

4

3.75 ± 0.16abA

0.218 ± 0.003bA

28.47 ± 0.20aA

60.14 ± 0.55aB

60

4

3.88 ± 0.14abA

0.225 ± 0.010abA

28.03 ± 0.60aA

59.44 ± 0.57aB

120

4

3.93 ± 0.09aA

0.237 ± 0.002aA

28.75 ± 0.59aA

59.88 ± 0.72aB

0

25

3.68 ± 0.15bA

0.208 ± 0.004cA

27.63 ± 0.81aA

60.82 ± 2.21aB

20

25

3.53 ± 0.17bA

0.217 ± 0.004bA

27.61 ± 0.79aA

60.13 ± 0.93aB

40

25

3.79 ± 0.12aA

0.216 ± 0.001bA

27.90 ± 0.61aA

62.57 ± 0.21aB

60

25

3.95 ± 0.07aA

0.227 ± 0.013abA

27.65 ± 0.95aA

63.05 ± 0.59aB

25

3.98 ± 0.08aA

0.235 ± 0.004aA

28.60 ± 0.39aA

61.33 ± 0.78aB

4

3.48 ± 0.13cA

0.148 ± 0.007bC

10.78 ± 0.35aB

71.40 ± 1.77aA

0

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120

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Samples

20

4

3.50 ± 0.14cA

0.155 ± 0.007abC

10.70 ± 0.77aB

71.97 ± 1.00aA

40

4

3.71 ± 0.11bcA

0.158 ± 0.004abC

10.76 ± 0.65aB

71.24 ± 1.05aA

60

4

3.82 ± 0.07abA

0.153 ± 0.003abC

10.71 ± 0.72aB

69.42 ± 0.49aA

120

4

3.93 ± 0.09aA

0.157 ± 0.001aC

10.71 ± 0.72aB

70.90 ± 0.30aA

0

25

3.48 ± 0.13bA

0.148 ± 0.007bC

10.78 ± 0.35aB

71.40 ± 1.77aA

of

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0.157 ± 0.003abC

10.69 ± 0.19aB

71.04 ± 0.79aA

40

25

3.56 ± 0.15bA

0.155 ± 0.007abC

11.28 ± 0.67aB

72.15 ± 0.26aA

60

25

3.76 ± 0.14abA

0.153 ± 0.004abC

10.79 ± 0.53aB

70.02 ± 2.33aA

120

25

3.87 ± 0.13aA

0.160 ± 0.002aC

11.24 ± 0.88aB

69.93 ± 2.15aA

0

4

3.46 ± 0.15bA

0.179 ± 0.002bB

20

4

3.71 ± 0.08bA

0.181 ± 0.001bB

40

4

3.68 ± 0.12bA

60

4

3.66 ± 0.14bA

120

4

3.97 ± 0.15aA

0

25

20

25

40

25

70.15 ± 1.71aA

10.76 ± 0.22aB

71.24 ± 0.28aA

0.180 ± 0.001bB

10.77 ± 0.95aB

70.31 ± 1.28aA

0.184 ± 0.001aB

10.82 ± 0.88aB

69.42 ± 0.72aA

0.186 ± 0.003aB

10.68 ± 0.91aB

69.88 ± 0.95aA

3.46 ± 0.15bA

0.179 ± 0.002bB

10.79 ± 0.59aB

70.15 ± 1.71aA

3.54 ± 0.17bA

0.180 ± 0.001bB

11.28 ± 0.67aB

72.15 ± 2.26aA

3.80 ± 0.19abA

0.182 ± 0.001abB

10.71 ± 0.87aB

72.41 ± 0.58aA

25

3.91 ± 0.09aA

0.185 ± 0.002aB

10.66 ± 0.79aB

70.35 ± 2.16aA

25

3.83 ± 0.07aA

0.187 ± 0.002aB

10.27 ± 0.18aB

70.37 ± 2.19aA

0

4

3.42 ± 0.19bA

0.179 ± 0.001bB

11.29 ± 0.61aB

70.23 ± 2.41aA

20

4

3.65 ± 0.13bA

0.180 ± 0.001bB

10.84 ± 0.80aB

70.26 ± 1.86aA

40

4

3.63 ± 0.10bA

0.179 ± 0.001bB

11.33 ± 0.60aB

69.45 ± 1.67aA

60

4

3.68 ± 0.16bA

0.180 ± 0.002abB

11.27 ± 0.52aB

69.51 ± 1.19aA

120

4

3.98 ± 0.11aA

0.184 ± 0.002aB

10.72 ± 0.87aB

70.26 ± 0.69aA

AC C

120

ED

MA

NU

SC

RI

10.79 ± 0.59aB

60

GMIO4

PT

25

EP T

GMO3

20

ACCEPTED MANUSCRIPT 36 3.42 ± 0.19bA

0.179 ± 0.001bB

11.29 ± 0.61aB

70.23 ± 2.41aA

20

25

3.61 ± 0.11abA

0.180 ± 0.002bB

11.39 ± 0.24aB

70.14 ± 0.40aA

40

25

3.75 ± 0.16aA

0.180 ± 0.001bB

10.40 ± 0.34aB

72.09 ± 1.93aA

60

25

3.79 ± 0.09aA

0.186 ± 0.001aB

10.50 ± 0.56aB

69.28 ± 1.24aA

120

25

3.81 ± 0.15aA

0.185 ± 0.001aB

PT

25

10.66 ± 0.79aB

69.67 ± 0.21aA

Means ± standard deviation with different superscript lowercase letters in the same column indicate

RI

a–b

0

significant differences (P < 0.05) on the same temperature among the different days of storage for each A-D

Means ± standard deviation with different superscript uppercase letters in the same column

SC

sample.

indicate significant differences (P < 0.05) among the samples on the same storage day and at the same

AC C

EP T

ED

MA

NU

temperature.

Table 4

ACCEPTED MANUSCRIPT 37

Bulk

density,

tapped

density,

flowability

and

cohesiveness

properties

of

Bifidobacterium BB-12 microcapsules during the storage period. Storage day

Temperature (ºC)

Bulk density (g cm−3 )

Tapped density (g cm−3 )

Flowability (%)

Cohesiveness

GM1

0

4

0.27 ± 0.02bC

0.50 ± 0.03aC

46.77 ± 1.62aA

1.88 ± 0.06aA

20

4

0.25 ± 0.01bC

0.48 ± 0.05aC

46.97 ± 3.00aA

1.89 ± 0.11aA

40

4

0.27 ± 0.03abC

0.52 ± 0.02aC

47.93 ± 6.44aA

1.94 ± 0.23aA

60

4

0.31 ± 0.01aC

0.57 ± 0.04aC

45.87 ± 4.63aA

1.86 ± 0.16aA

120

4

0.32 ± 0.01aC

0.57 ± 0.04aC

43.15 ± 6.29aA

1.77 ± 0.20aA

0

25

0.27 ± 0.02bC

46.77 ± 1.62aA

1.88 ± 0.06aA

20

25

0.26 ± 0.01bC

0.49 ± 0.03aC

44.24 ± 3.91aA

1.80 ± 0.12aA

40

25

0.29 ± 0.02abC

0.53 ± 0.04aC

44.22 ± 4.49aA

1.80 ± 0.14aA

60

25

0.29 ± 0.02abC

0.55 ± 0.02aC

46.75 ± 3.18aA

1.88 ± 0.11aA

25

0.31 ± 0.01aC

0.56 ± 0.04aC

45.34 ± 4.37aA

1.84 ± 0.14aA

4

0.36 ± 0.03bB

0.65 ± 0.04aB

44.37 ± 3.01aA

1.83 ± 0.10aA

0

AC C

GMI2

RI

SC

NU

0.50 ± 0.03aC

MA

ED

EP T

120

PT

Samples

20

4

0.34 ± 0.01bB

0.61 ± 0.02aB

44.01 ± 3.17aA

1.79 ± 0.10aA

40

4

0.36 ± 0.01bB

0.65 ± 0.03aB

44.58 ± 2.41aA

1.81 ± 0.08aA

60

4

0.40 ± 0.01aB

0.68 ± 0.05aB

40.27 ± 3.77aA

1.68 ± 0.10aA

120

4

0.42 ± 0.02aB

0.66 ± 0.03aB

36.80 ± 5.76aA

1.59 ± 0.14aA

0

25

0.36 ± 0.03bB

0.65 ± 0.04aB

44.37 ± 3.01aA

1.83 ± 0.10aA

ACCEPTED MANUSCRIPT 0.34 ± 0.02bB

0.60 ± 0.04aB

41.75 ± 1.96aA

1.72 ± 0.06aA

40

25

0.38 ± 0.02bB

0.61 ± 0.03aB

37.91 ± 3.14aAB

1.59 ± 0.18aAB

60

25

0.39 ± 0.03abB

0.67 ± 0.03aB

44.07 ± 2.88aA

1.79 ± 0.09aAB

120

25

0.42 ± 0.01aB

0.68 ± 0.04aB

38.46 ± 2.05aB

1.63 ± 0.13aA

0

4

0.50 ± 0.03aA

0.78 ± 0.03aA

36.31 ± 3.82aB

1.57 ± 0.14aB

20

4

0.47 ± 0.02aA

0.75 ± 0.05aA

36.99 ± 3.56aB

1.58 ± 0.08aB

40

4

0.48 ± 0.04aA

0.80 ± 0.03aA

40.72 ± 3.44aA

1.66 ± 0.10aA

60

4

0.48 ± 0.02aA

0.82 ± 0.02aA

41.11 ± 2.98aA

1.70 ± 0.13aA

120

4

0.46 ± 0.01aA

43.83 ± 3.82aA

1.78 ± 0.16aA

0

25

0.50 ± 0.03aA

0.78 ± 0.03aA

36.31 ± 1.82aB

1.57 ± 0.14aB

20

25

0.48 ± 0.02aA

0.75 ± 0.01aA

36.59 ± 1.18aB

1.51 ± 0.13aB

40

25

0.47 ± 0.01aA

0.74 ± 0.03aA

36.23 ± 1.20aB

1.57 ± 0.11aB

25

0.47 ± 0.03aA

0.79 ± 0.05aA

40.62 ± 3.07aA

1.68 ± 0.13aB

SC

NU

ED

MA

0.82 ± 0.02aA

120

25

0.47 ± 0.03aA

0.79 ± 0.05aA

40.62 ± 3.07aAB

1.68 ± 0.13aA

0

4

0.40 ± 0.02aB

0.70 ± 0.05aAB

42.53 ± 5.07aAB

1.75 ± 0.20aAB

20

4

0.35 ± 0.03aB

0.63 ± 0.05aB

44.77 ± 5.36aAB

1.82 ± 0.17aAB

40

4

0.37 ± 0.02aB

0.70 ± 0.04aB

47.03 ± 5.58aA

1.90 ± 0.21aA

60

4

0.42 ± 0.01aB

0.71 ± 0.03aB

40.98 ± 2.49aA

1.70 ± 0.04aA

120

4

0.42 ± 0.02aB

0.71 ± 0.03aB

40.09 ± 3.73aA

1.67 ± 0.07aA

AC C

60

GMIO4

RI

25

EP T

GMO3

20

PT

38

ACCEPTED MANUSCRIPT 25

0.40 ± 0.02aB

0.70 ± 0.05aAB

42.53 ± 5.07aAB

1.75 ± 0.20aAB

20

25

0.36 ± 0.05aB

0.65 ± 0.03aB

44.00 ± 2.70aA

1.79 ± 0.09aA

40

25

0.40 ± 0.02aB

0.68 ± 0.04aAB

41.73 ± 1.04aA

1.72 ± 0.03aA

60

25

0.41 ± 0.01aB

0.71 ± 0.03aAB

45.17 ± 3.22aA

1.83 ± 0.11aAB

120

25

0.42 ± 0.01aB

0.70 ± 0.04aAB

43.33 ± 2.52aA

1.77 ± 0.08aA

Means ± standard deviation with different superscript lowercase letters in the same column indicate

RI

a–b

0

PT

39

significant differences (P < 0.05) on the same temperature among the different days of storage for each A-D

Means ± standard deviation with different superscript uppercase let ters in the same column

SC

sample.

indicate significant differences (P < 0.05) among the samples on the same storage day and at the same

AC C

EP T

ED

MA

NU

temperature.

Table 5

ACCEPTED MANUSCRIPT 40

Color parameters (L*, a*, b*, C*, h*, ∆E*) of the spray dried powders during the storage period. Storage day

Temperature (°C)

L*

a*

b*

C*

h*

∆E*

GM1

0

4

95.17 ± 0.36bB

-1.06 ± 0.04cA

8.35 ± 0.05bB

8.41 ± 0.05bB

97.22 ± 0.33cA

1.65

20

4

96.15 ± 0.69abA

-1.36 ± 0.08bA

8.97 ± 0.21aAB

9.07 ± 0.21aAB

98.61 ± 0.53bA

40

4

96.17 ± 0.33aA

-1.56 ± 0.15aA

8.73 ± 0.13aB

8.87 ± 0.15aB

100.14 ± 0.86aA

60

4

95.47 ± 0.77abAB

-1.44 ± 0.07abA

8.55 ± 0.24abB

8.67 ± 0.23abB

99.59 ± 0.60abA

120

4

96.71 ± 0.85aAB

-1.58 ± 0.08aA

0

25

95.17 ± 0.36bB

-1.06 ± 0.04bA

20

25

95.84 ± 0.32bA

40

25

95.15 ± 0.65bB

60

25

120

25

0

4

SC

100.42 ± 0.20aA

8.35 ± 0.05cB

8.41 ± 0.05cB

97.22 ± 0.33bA

-1.38 ± 0.06aA

9.06 ± 0.26bB

9.16 ± 0.27bB

98.66 ± 0.21aA

-1.33 ± 0.09aA

9.37 ± 0.03bC

9.48 ± 0.03bC

98.83 ± 0.27aA

ED

RI 8.71 ± 0.18aB

96.88 ± 0.27aA

-1.46 ± 0.04aA

9.25 ± 0.12bC

9.35 ± 0.11bC

98.17 ± 0.55aB

96.83 ± 0.06aA

-1.47 ± 0.06aA

10.14 ± 0.30aC

10.25 ± 0.30aC

98.26 ± 0.20aB

96.04 ± 0.45aA

-0.76 ± 0.04cB

6.70 ± 0.06aC

6.75 ± 0.06aC

96.45 ± 0.29cB

MA

NU

8.57 ± 0.18abB

EP T

AC C

GMI2

PT

Samples

20

4

96.46 ± 0.74aA

-0.93 ± 0.08bB

7.57 ± 1.44aB

7.62 ± 1.42aB

97.23 ± 1.61bcA

40

4

96.72 ± 0.52aA

-1.22 ± 0.11aB

6.73 ± 0.14aC

6.84 ± 0.12aC

100.28 ± 1.10aA

60

4

96.01 ± 0.60aAB

-1.05 ± 0.09abB

6.52 ± 0.16aC

6.60 ± 0.17aC

99.13 ± 0.61abA

120

4

96.85 ± 0.54aA

-1.15 ± 0.13aB

6.86 ± 0.24aC

6.96 ± 0.25aC

99.53 ± 1.07abA

0

25

96.04 ± 0.45aA

-0.76 ± 0.04cB

6.70 ± 0.06dC

6.75 ± 0.06dC

96.45 ± 0.29bB

20

25

96.15 ± 0.42aA

-1.14 ± 0.08bB

7.35 ± 0.17cC

7.44 ± 0.17cC

98.82 ± 0.53aA

2.49

0.98

1.73

ACCEPTED MANUSCRIPT 7.80 ± 0.11bD

7.91 ± 0.10bD

99.47 ± 0.49aA

60

25

96.41 ± 0.54aA

-1.23 ± 0.03abB

7.71 ± 0.07bD

7.81 ± 0.07bD

99.07 ± 0.24aA

120

25

96.70 ± 0.48aA

-1.30 ± 0.07aB

8.16 ± 0.17aD

8.26 ± 0.17aD

99.06 ± 0.46aA

0

4

96.20 ± 0.29aA

-0.72 ± 0.05cB

10.46 ± 0.12aA

10.49 ± 0.12aA

93.91 ± 0.25cC

20

4

96.23 ± 0.41aA

-0.86 ± 0.04bBC

9.56 ± 0.25bA

9.59 ± 0.25bA

95.11 ± 0.14bB

40

4

95.99 ± 0.26aA

-1.11 ± 0.11aB

9.75 ± 0.28bA

9.78 ± 0.29bA

96.55 ± 0.81aB

60

4

96.29 ± 0.40aA

-1.03 ± 0.06aB

9.56 ± 0.24bA

9.61 ± 0.24bA

96.13 ± 0.20aB

120

4

96.06 ± 0.51aAB

-1.04 ± 0.06aB

9.51 ± 0.35bA

9.77 ± 0.35bA

96.27 ± 0.59aB

0

25

96.20 ± 0.29aA

-0.72 ± 0.05bB

10.46 ± 0.12eA

10.49 ± 0.12eA

93.91 ± 0.25bC

20

25

95.74 ± 0.30aA

-0.67 ± 0.21bC

12.48 ± 0.41dA

12.51 ± 0.41dA

93.07 ± 1.92abB

40

25

94.45 ± 0.31bBC

-1.06 ± 0.04aB

13.43 ± 0.12cB

13.47 ± 0.15cB

94.50 ± 0.22aB

60

25

94.35 ± 0.44bcB

-1.11 ± 0.03aC

13.71 ± 0.07bB

13.75 ± 0.07bB

94.63 ± 0.10aC

120

25

93.93 ± 0.67cB

-1.03 ± 0.05aC

14.33 ± 0.14aB

14.37 ± 0.14aB

94.11 ± 0.17abC

0

4

95.19 ± 0.23bB

-0.70 ± 0.04bB

10.60 ± 0.17aA

10.63 ± 0.17aA

94.32 ± 0.19bC

MA

ED

1.37

RI

PT

-1.30 ± 0.05aA

NU

96.89 ± 0.40aA

EP T

GMIO4

25

AC C

GMO3

40

SC

41

20

4

95.33 ± 0.77abA

-0.72 ± 0.10abC

9.42 ± 0.34bA

9.45 ± 0.33bA

94.36 ± 0.66abB

40

4

95.82 ± 0.42abA

-0.78 ± 0.20abC

9.71 ± 0.25bA

9.75 ± 0.25bA

94.58 ± 1.18abB

60

4

95.35 ± 0.25abB

-0.76 ± 0.07abC

9.70 ± 0.16bA

9.74 ± 0.16cA

94.96 ± 0.51abC

120

4

95.96 ± 0.20aB

-0.84 ± 0.03aC

9.83 ± 0.45bA

9.87 ± 0.45bA

94.87 ± 0.32aC

0

25

95.19 ± 0.23aB

-0.70 ± 0.04abB

10.60 ± 0.17dA

10.63 ± 0.17dA

94.32 ± 0.19aC

4.55

1.16

5.28

ACCEPTED MANUSCRIPT 42

25

94.22 ± 0.25bB

-0.80 ± 0.07abC

12.98 ± 0.24cA

13.00 ± 0.25cA

93.54 ± 0.27bB

40

25

93.96 ± 0.18bcC

-0.87 ± 0.13aC

14.16 ± 0.42bA

14.19 ± 0.41bA

93.53 ± 0.55bC

60

25

93.53 ± 0.38cB

-0.70 ± 0.17abD

14.23 ± 0.17bA

14.25 ± 0.17bA

92.82 ± 0.67bcD

120

25

93.61 ± 0.37bcB

-0.64 ± 0.07bD

15.63 ± 0.42aA

15.64 ± 0.42aA

92.33 ± 0.29cD

Means ± standard deviation with different superscript lowercase letters in the same column indicate

PT

a–e

20

significant differences (P < 0.05) on the same temperature among the different days of storage for each A-D

Means ± standard deviation with different superscript uppercase letters in the same column

RI

sample.

indicate significant differences (P < 0.05) among the samples on the same storage day and at the same

AC C

EP T

ED

MA

NU

SC

temperature.

ACCEPTED MANUSCRIPT 43

- Bifidobacteria survival was higher using goat’s milk and inulin, as carrier agents. - Highest survival rates after storage were noted using goat’s milk and/or inulin. - Spray-dried powder produced with goat’s milk showed the better solubility properties.

PT

- Spray-dried powder produced with goat’s milk and inulin has lowest water activity.

AC C

EP T

ED

MA

NU

SC

RI

- These data contribute to an innovative approach to the goat’s milk industry.

Figure 1

Figure 2

Figure 3