Thermal Aggregation of Calcium-Fortified Skim Milk Enhances

Jul 15, 2016 - Probiotic Protection during Convective Droplet Drying ... ABSTRACT: Probiotic bacteria have been reported to confer benefits on hosts w...
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Thermal aggregation of calcium-fortified skim milk enhances probiotic protection during convective droplet drying Juan Wang, Song Huang, Nan Fu, Romain Jeantet, and Xiao Dong Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02205 • Publication Date (Web): 15 Jul 2016 Downloaded from http://pubs.acs.org on July 18, 2016

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Thermal aggregation of calcium-fortified skim milk enhances probiotic protection during

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convective droplet drying Juan Wanga, Song Huanga,b*, Nan Fua, Romain Jeantetb, Xiao Dong Chena*

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4

5

a

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Environmental Engineering, College of Chemistry, Chemical Engineering and Material Science,

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Soochow University, Suzhou 215123, Jiangsu Province, China

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b

Suzhou Key Laboratory of Green Chemical Engineering, School of Chemical and

STLO, Agrocampus Ouest, INRA, 35000, Rennes, France

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*Corresponding authors:

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Xiao Dong Chen, 911 Building, Soochow University, 199 Ren’ai road, Suzhou 215123, Jiangsu

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Province, China.

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Email: [email protected]; Tel: 0512-65882767; Fax: 0512-65882750

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Song Huang, 65 rue de Saint Brieuc, UMR1253 STLO, 35000, Rennes, France

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Email : [email protected]; Tel: +33 (0) 223485342

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Abstract

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Probiotic bacteria have been reported to confer benefits on hosts when delivered in adequate dose.

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Spray drying is expected to produce dried and microencapsulated probiotic products due to its

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low production cost and high-energy efficiency. The bottleneck in probiotic application addresses

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the thermal and dehydration inactivation of bacteria during process. A protective drying matrix

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was designed by modifying skim milk with the principle of calcium-induced protein thermal

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aggregation. The well-defined single droplet drying technique was used to monitor the droplet-

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particle conversion and protective effect of this modified Ca-aggregated milk on Lactobacillus

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rhamnosus GG. The Ca-aggregated milk exhibited a higher drying efficiency and a superior

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protection on L. rhamnosus GG during thermal convective drying. The mechanism was explained

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by the aggregation in milk, causing the lower binding of water in serum phase and conversely

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local concentrated milk aggregates involved in bacteria entrapment in the course of drying. This

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work may open new avenues for development of probiotic products with high bacterial viability

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and calcium enrichment.

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Key words

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Probiotics; Protein aggregation; Calcium; Single droplet drying; Particle formation; Spray drying

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Introduction

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Probiotics consist in viable microorganisms that confer a health benefit to the host. Increasing

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number of studies have evidenced the benefit of probiotics in the improvement of health and

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prevention of therapeutic interventions linked to various diseases 1. However, these probiotic

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effects were mostly dose-dependent 2. The probiotic products containing stable and high level of

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active bacterial counts are therefore increasingly demanded in the current era.

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The technical bottlenecks of probiotic production address the maintenance of probiotic viability

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during production process, storage and digestion. In this sense, the design of food grade

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microparticles to encapsulate and protect probiotic bacteria is now gaining interest 3. Drying is a

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common method to stabilize foods containing microorganisms such as bacteria and yeast for a

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prolonged period of time 4. Drying matrixes may also work as encapsulated wall materials or

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delivery vehicles to protect and release probiotic bacteria at the required site of action in the

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digestive tract 5.

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Freeze drying is still the most often used technique in drying live microorganisms. However, its

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high production cost, batch processing mode and low energy efficiency turn to be a stumbling

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block when facing the increasing demand of probiotic products 6. Spray drying is therefore

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expected as a promising alternative technique to produce dried probiotics due to its low cost

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(6~10 times cheaper than freeze drying) and high productivity 7. However, the challenge consists

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in the considerable inactivation of bacteria caused by high temperature, dehydration and high

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shear during process 8. Besides, during real spray drying process, billions of droplets are sprayed

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into a relatively large tower, resulting in the complexity in understanding the ‘in-process’ droplet-

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particle conversion and the related protective mechanism of the matrix on bacteria. To 3

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circumvent these difficulties, single droplet drying is therefore established to map the drying

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behavior of bacteria in the droplet level 9. Several studies have been done based on single droplet

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drying technique, to design or optimize the drying matrixes for probiotic bacteria 10–12.

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Dairy products, such as skim milk or milk proteins, were widely applied to encapsulate probiotics

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due to its compatibility with probiotics and possibility in promoting probiotic efficacy

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Calcium-fortified milk is of value from both nutrition and marketing perspectives. The

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combination of calcium supplement with probiotics can improve either the absorption of calcium

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and the efficacy of probiotics

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protein aggregation upon heat treatment. The characteristics of thermal aggregation of milk

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proteins induced by calcium supplementation has been extensively studied

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principles of milk protein aggregation, encapsulation of probiotics via emulsion or gelation

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technique has recently gained attention due to its potential effects on protecting probiotics under

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digestion stress

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microencapsulate probiotics by drying has rarely been reported. In the present work, we

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employed the single droplet drying technique to map the drying behavior of the calcium-induced

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aggregated skim milk, of which the protection capacity on probiotic Lactobacillus rhamnosus GG

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was also studied during droplet-particle conversion.

19,20

15–17

13,14

.

. Supplementation of calcium in milk can cause the milk

18

. Based on the

. However, the feasibility of applying calcium-fortified milk to

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Materials and methods

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Strains and growth conditions

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Lactobacillus rhamnosus GG (ATCC 53101, LGG in brief) was routinely cultivated in MRS

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broth as described before 21. Briefly, LGG strain was retrieved by pouring one stick of Culturelle

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powder into 10 mL sterilized water. The resulting LGG suspension was streaked on MRS agar. A

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single colony was aseptically transferred to 10 mL MRS broths and then grew statically at 37 ºC

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for overnight as a pre-culture. Subsequently, a 1% (v/v) inoculum of LGG was made from the

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pre-culture to MRS broth, to grow at 37 oC for 24 h without agitation.

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Preparation of bacteria-suspended milk matrixes

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The Ca-aggregated milk was prepared as described previously

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(Devondale, Australia) was added by 10 mM CaCl2 (Sinopharm, Shanghai, China) and then

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heated at 90 ºC for 30 min to cause the protein aggregation. Apart from the untreated UHT skim

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milk, the 10 mM CaCl2 added milk without heating was also used in this work as a control to

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exclude the effects of both CaCl2 addition and higher dry matter (Ca-added milk in brief). All the

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milk matrixes were full vortexed and then placed at 25 ºC for 30 min by a thermal mixer

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(Eppendorf, Germany) before mixing with LGG cells.

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LGG cells were harvested in the 2 mL Eppendorf tube from the 24 h culture by centrifuging at

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8000 g for 15 min (Heal Force, Hong Kong, China). The cell pellets were washed by 0.5% (w/v)

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peptone water in the same centrifuging condition and then suspended in 1:1 volume of the above

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milk matrix. All the bacteria suspended matrixes were fully vortexed and placed at 4 ºC before

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further use.

21

. Briefly, UHT skim milk

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Single droplet drying

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Single droplet drying was carried out with the SDPA MARK II equipment (Dong-Concept New

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Material Technology Co. Ltd, China). The working principles of this equipment have been

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described in detail elsewhere

22,23

. In brief, the system consists of an air supply unit to provide

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laminar air flow with controllable temperature, humidity and velocity, and a droplet drying unit

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where an isolated droplet can be suspended at the tip of a specially made glass filament for drying

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in the conditioned air stream. Three droplet suspension modules were equipped in the droplet

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drying unit for measuring the changes of droplet temperature, mass and diameter respectively. A

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high-resolution video camera was coupled with the droplet drying unit to monitor and record the

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change of droplet morphology during drying. Droplet moisture content on dry basis (Xd, kg kg-1)

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was calculated with the following equation:

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Xd=(Md−Ms)/Ms

(1)

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where Md is the mass of droplet and Ms is the total solids mass of the matrix (mg).

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The single droplet was generated from the above bacteria suspended milk matrixes with a gas

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chromatograph micro-syringe (SGE Analytical Science Pty Ltd, Australia). Control consisted of

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the drying of sterilized Milli-Q water droplets. For each drying run, the syringe was rinsed by

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Milli-Q water, 75% (v/v) ethanol, and sterilized Milli-Q water subsequently. The remaining LGG

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viability within droplets was measured as described before

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stopped at certain time by blocking the air flow with a cold humid barrier. The droplet was then

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immediately rehydrated in 1 mL of 0.5% peptone water, and subsequently serially diluted for

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measuring the viable bacteria population by pouring on MRS agar plate. The agar plates were

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incubated at 37 ºC for 48 h. The initial bacteria population in three milk matrixes were also

10,24

. Briefly, the drying process was

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measured before drying (~ 109 CFU mL-1 for all three matrixes). The drying conditions used in

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this work were set as following: air temperature 65 ± 0.2 ºC, air velocity 0.91 ± 0.02 m s-1, air

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moisture content 1 ± 0.01 g kg-1 and droplet volume 2 ± 0.05 µL.

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Particle size analysis

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The size distribution of dispersed elements in milk matrixes were measured by laser light

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diffraction (Horiba LA-960, Japan).

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Viscosity measurement

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The viscosity of milk matrixes was measured using an oscillatory rheometer (Kinexus Pro+,

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Malvern, UK). The samples were first equilibrated at 25 ºC and then quickly pipetted onto the

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bottom plate of the rheometer. A 40 mm Cone (4º) geometry was used with the sheer rate at 50 s-1

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and the operation temperature was controlled at 25 ± 0.5 ºC.

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One-dimensional polyacrylamide gel electrophoresis

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The content of milk whey proteins in the serum phase of different milk matrixes was compared

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by electrophoresis SDS-PAGE. Milk matrixes were firstly centrifuged at 8000 g for 15 min. The

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supernatant was collected and then mixed with the LDS sample buffer (NuPGE, ThemoFisher

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Scientific, United States) at the volume ratio of 1:1 prior to heat denaturation at 75 °C for 10 min.

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One-dimensional polyacrylamide gel electrophoresis was conducted on the XCell SureLock

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Mini-Cell system (ThermoFisher Scientific, United States) with 12% Bis-Tris Precast gel

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(NuPAGE, ThermoFisher Scientific, United States). The gel was stained by Bio-Safe Coomassie

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blue reagent (Biorad, United States) prior to photograph with the Imaging System (ChemiDoc

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XRS+, Biorad, United States). 7

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Nuclear magnetic resonance (NMR) measurement

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To characterize the mobility of water in bacteria-suspended milk matrixes, the transverse

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relaxation time (T2) was measured using a 23 MHz 1H-NMR spectrometer (Suzhou Niumai

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Electronic Technology Co., Ltd., China) at 32 ± 0.1 ºC. The equilibrated samples were placed in

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the 15 mm-diameter tube. The tube was sealed with parafilm to prevent the water loss during

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measurement. The T2 distributions were measured using the Carr-Purcell-Meiboom-Gill (CPMG)

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pulse sequence with the 90-180º pulse spacing varied between 25 and 50 µs. The echo number

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was set to 4000, the echo time was 0.5 s, the waiting time was 2 s, and 16 scans were acquired for

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each measurement. The data were collected and analyzed by the instrument MultiExp Invfit-T

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Analysis software.

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Scanning electron microscopy

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The particles after drying at 65 °C for 420 s were collected and cut by a surgical knife blade. The

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particle pieces were placed on the carbon tape and then sputter-coated by gold-palladium. These

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samples were observed by scanning electron microscopy (Hitachi Ltd, Japan).

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Statistical analysis

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All the experiments were repeated independently for three times. The features reported were

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repeatedly observed in three independent experiments. The results of bacteria population and

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distrubution of T2 relaxation time were expressed as mean value ± standard deviation. Statistical

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analysis was performed using one-way ANOVA and Student t-Test (Excel, Microsoft, USA).

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Differences were considered significant at p < 0.05.

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Results and Discussion

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Physical and chemical properties of liquid milk matrixes

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The size distribution of dispersed elements in the three bacteria-suspended milk matrixes is

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shown in Figure 1A. The dispersed elements in untreated skim milk and Ca-added milk showed

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very similar size distribution, consisting mostly in elements ranging approximately from 70 to

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500 nm, and conversely in a less proportion of elements with large diameter from 3 to 10 µm.

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These later might correspond respectively to the casein micelles and LGG cells in the milk

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Therefore, the addition of 10 mM CaCl2 had no influence on the solid size distribution in skim

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milk. By contrast, most of dispersed elements in the Ca-aggregated milk had diameters ranging

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from 5 to 50 µm. In addition, the Ca-aggregated milk contained a portion of elements with

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diameter larger than 100 µm. The protein aggregation and increasing of the casein micelle size

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emerged in the skim milk with additional Ca2+ upon the heat treatment taken place.

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Besides, the content of proteins in the serum phase of Ca-aggregated milk was largely decreased

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(Figure 1B) due to the formation of whey protein - casein micelle aggregates during the heat

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treatment in the presence of added Ca2+ 18,27,28. The Ca-aggregated milk displayed a considerably

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higher viscosity when compared to the skim milk and Ca-added milk (Table 1). The higher

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viscosity of Ca-aggregated milk was caused by the heat treatment, leading to denaturation of

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whey proteins and further aggregation either in the soluble milk phase or with the casein micelles

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29

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distribution, as shown in Table 1. However, the Ca-aggregated milk displayed a left shift of the

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T2 distribution with a smaller area under the peak, which indicated a decrease of T2 30. It can be

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concluded that water had a lower mobility in the Ca-aggregated milk than that in untreated and

25,26

.

. In addition, the untreated and Ca-added skim milk showed a similar T2 relaxation times

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Ca-added skim milk. It could result from the aggregation process leading to the entrapping of

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water into the protein network 31,32.

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Drying kinetics of milk matrixes

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The drying kinetics of three different bacteria-suspended milk matrixes during convective drying

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are shown in Figure 2 through description of temperature and mass changes. The features of

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droplet temperature (Td) and mass (Md) were coherent with previous studies

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temperature curve, four stages can be identified during drying: 1st stage, the Td was rapidly pre-

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heated to the wet-bulb range (~ 23 °C); 2nd stage, the Td maintained nearly constant for tens of

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seconds; 3rd stage, the Td curve experienced a progressive increase with time and tended toward a

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final plateau with a sigmoid shape; 4th and last stage, the Td approached to the air temperature at

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around 65 °C. In the same time, the curve of droplet mass Md decreased progressively with time.

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Considering the temperature curve stages previously mentioned, the decrease of Md was almost

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linear with time at the 1st and 2nd stages. The mass loss rate slowed down gradually from the 3rd

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stage until approaching zero at the 4th stage. The untreated skim milk and Ca-added milk showed

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similar Td and Md behaviors during drying. The addition of 10 mM CaCl2 without further thermal

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aggregation had no significant influence on the drying kinetics of skim milk. However, Ca-

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aggregated milk displayed a distinct drying kinetics, with a longer 2nd stage and a shorter time to

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reach the final 4th stage plateau.

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The features of Ca-aggregated milk were closer to that of pure water droplet under the same

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drying condition. It is known that the evaporation of pure water can be considered as diffusion-

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controlled and has the highest dehydration rate under the same condition compared to other

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materials. Thus, the Ca-aggregated milk experienced longer time of diffusion-controlled

24,33

. For the

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evaporation in comparison with untreated and Ca-added skim milk. The overall drying efficiency

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was thus the highest among the three milk matrixes tested in this work. It can be assumed that the

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water in the serum phase of the Ca-aggregated milk was bound to a lesser extent, due to its lower

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protein content (Figure 1B). It may therefore lead to the more water-like drying behaviour

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previously observed, with a longer 2nd stage and faster drying rate during final convective drying.

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Droplet morphology evolution during drying

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The evolution of surface morphology of droplet during drying was recorded. The droplet

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morphology with the corresponding drying time for each milk matrix is shown in Figure 3.

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Regardless of the milk matrixes, the droplets had a similar shape and size at the beginning of

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drying. They shrank homogeneously from the beginning of drying to the end of 2nd stage, which

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can be assumed as diffusion controlled 34. By the end of 2nd stage, the spherical shape of droplets

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were maintained but the droplet size of Ca-aggregated milk was smaller than that of untreated

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and Ca-added milk. When the drying proceeded to the 3rd stage, the droplet began to distort due

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to the skin formation on the surface and progressively lost its spherical shape

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droplet-particle conversion stage, the water diffused through the droplet surface because of the

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porous properties of the skin. However, as the sol-gel transition took place over the free surface

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of droplet, the droplet skin developed to a thickened shell which fixed the shape of droplet from

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further change and progressively hindered the evaporation

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progressively formed inside the particle, as indicated by the transparent feature of droplets. This

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could be explained by the pressure change across the droplet skin, which made it possible for the

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air to enter the droplet at the same time as water diffused through the shell 33,37. This transparent

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feature was particularly apparent on the final skim milk and Ca-added milk particles.

33,37

35,36

. During this

. In this stage, a vacuole

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Interestingly, the particle formed by the Ca-aggregated milk was hemispherical (lower height)

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compared to the skim milk and Ca-added milk during the droplet-particle conversion. The

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morphology observed by the SEM displayed an even more remarkable different structure of the

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Ca-aggregated milk dried particle (Figure 4). Specifically, this latter presented a wrinkled surface

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while the surface of skim milk and Ca-added milk particles were relatively smooth (Figure 3). It

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has been reported in the convective droplet drying that the particles formed by the whey proteins

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displayed a relatively smooth surface and spherical shape, while the casein micelles would form

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preferentially wrinkled particles

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of the untreated skim milk and Ca-added milk could be attributed to the predominant

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participation of whey proteins in the shell formation due to their small size and thereby faster

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diffusion rate compared to the casein micelles. As discussed above, the whey proteins in Ca-

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aggregated milk were associated with the casein micelles to form the large size of aggregates.

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Hence, the wrinkled particle surface of the Ca-aggregated milk was due to the main participation

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of casein micelles in the shell formation.

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Last, the inner-morphology of dried particles is also shown in Figure 4. The vacuole structure

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was indeed formed inside the particles regardless of the matrix type. However, the vacuole size of

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the Ca-aggregated milk particles was smaller than that of untreated and Ca-added skim milk

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particles. When comparing the particle shell section, the skim milk and Ca-added milk showed a

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similar compact and thin shell, while the Ca-aggregated milk displayed a dense shell with the

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bubble-like structure. It supports our above hypothesis that the casein micelles played the main

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role in the shell formation of the particles dried from Ca-aggregated milk 38,39. In the figures with

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10000 magnification, the network-like structure in the shell was clearly observed in the particle

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of Ca-aggregated milk. It was significantly different from that of matrix-filled structure displayed

38–40

. We suggest that the smooth and spherical particle surface

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in the particle shell of untreated and Ca-added skim milk. This porous properties of shell may

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also explain the distinct drying kinetics of the Ca-aggregated milk. As an evidence, it should be

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easier for water to diffuse across a porous shell, leading thereby to the longer 2nd stage and faster

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drying rate observed.

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Bacteria viability during single droplet drying

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The inactivation kinetics of LGG is shown in Figure 5A. The initial bacterial population was

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around 1.0×109 CFU g-1 before drying, whatever the milk matrix. The viability of LGG in the

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three milk matrixes maintained constant within the first 200 s of drying, corresponding to the

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period from 1st stage to the end of 3rd stage in Figure 1. The inactivation of bacteria increased

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when the drying proceeded to the 4th stage (~210 s): the viable bacterial population in skim milk

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and Ca-added milk decreased dramatically to a level lower than 106 CFU g-1 within 240 s. In

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comparison, the Ca-aggregated milk showed a remarkable protection on LGG. Specifically, the

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LGG population in Ca-aggregated milk decreased significantly slower than that in the skim milk

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and Ca-added milk from 210 s to 420 s. At the end of drying, the remaining bacterial population

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in the particle of Ca-aggregated milk was still higher than the level of 107 CFU g-1, which made

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the final viability around 2 logs higher than that of in the skim milk or Ca-added milk.

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Inactivation of LGG in the droplets was also plotted as a function of droplet temperature (Figure

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5B). It was found that the viable LGG population in the three milk matrixes all remained at the

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initial level of 109 CFU g-1 for temperature lower than 50 °C. However, as the droplet

268

temperature increased continually, the bacterial population in the skim milk and Ca-added milk

269

droplets decreased by approximately 1 log CFU g-1 from around 53 to 60 °C, and then dropped

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sharply when the temperature overcame 60 °C. By contrast, the Ca-aggregated milk matrix better 13

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protected LGG bacteria in the temperature range from 53 to 62 °C. For this matrix, a significant

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loss of bacterial population was only observed during the 4th stage, when the droplet temperature

273

approached the air temperature (~63 °C). In addition, the bacterial inactivation as a function of

274

droplet moisture content is shown in Figure 5C. The reduction of bacterial population cropped up

275

when the droplet moisture content decreased to approximately 0.5 (kg kg-1). Similarly as in

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Figure 5B, the Ca-aggregated milk exhibited a better LGG protection at the end of drying, i.e.

277

when the droplet moisture content was lower than 0.3 kg kg-1.

278

The inactivation of LGG during single droplet drying mainly rose when the droplet moisture

279

content was lower than 0.5 kg kg--1 and the temperature reached above 60 ºC. The Ca-aggregated

280

milk also exerted its protective effects on LGG during this period. It is known that the

281

inactivation of bacteria during single droplet drying is due to the thermal and dehydration stress 9.

282

Previously, the Ca-aggregated milk has been found to protect LGG under thermal stress due to

283

the better interaction between milk solids and bacterial cells, and more stable extracellular

284

environment during heat treatment

285

bacteria by Ca-aggregated milk observed here at the end of thermal convective drying. In our

286

previous work, we reported that the 10 mM Ca2+ could improve the heat resistance of lactic acid

287

bacteria

288

LGG in the present work. It should be reminded that the Ca2+ added in milk was subjected to salt

289

equilibrium between the soluble phase and casein micelles, ruled by HPO4Ca solubility; it was

290

then partially transferred to the casein micelles calcium phosphate nanoclusters, instead of acting

291

on the bacterial cells 42,43.

292

The improved survival of LGG in the Ca-aggregated milk may also be attributed to its

293

physicochemical properties and distinct drying behavior. The Ca-aggregated milk matrix before

41

21

. Such mechanism could explain the protection of LGG

. However, the skim milk with 10 mM Ca2+ addition did not increase the survival of

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drying presented a higher viscosity compared to the untreated and Ca-added skim milk (Table 1).

295

It has been assumed that the high viscosity of media could retard molecular mobility and reaction

296

rate, hence stabilizing the biological system

297

inhomogeneous and consisted of a network of large and insoluble aggregates that incorporated

298

denatured whey proteins and entrapped part of water. This matrix may provide a local protective

299

environment for the embedded bacterial cells under the dehydration and thermal stress

300

addition, the droplet of Ca-aggregated milk had the longer 2nd stage during evaporation (Figure 2),

301

that is to say was maintained at lower temperature; conversely, it was faster to reach the final 4th

302

stage to form the dried particle. It has been reported that the fast drying may induce instant

303

fixation of the cells and thereby prevent the dehydration inactivation of bacteria 12. The bacteria

304

cells hence accumulated less dehydration inactivation within the droplet of Ca-aggregated milk in

305

comparison to that of untreated and Ca-added skim milk. Besides, it has been observed that the

306

bacteria cells are more heat-resistant in the dry state than in a hydrated environment

307

the temperature reached the harmful range (mostly the upper limit of the growth temperature, i.e.

308

~40 ºC for lactobacillis), the Ca-aggregated milk provided a drier extracellular environment for

309

LGG, likely to explain the better survival of LGG at the high temperature range. Besides and

310

according to

311

(Figure 4) should be soft and ductile, which may indicate that the bacteria has experienced less

312

mechanical stress during the sol-gel transition and shrinkage of droplet. This remains a

313

hypothesis for further research.

314

To conclude, the protein aggregation occurring in the Ca-aggregated milk led to lower protein

315

concentration in the milk serum phase and conversely protein-enriched casein micelle phase. This

316

microstructure resulted in a more water-like drying kinetics, different feature of droplet-particle

39,49

44

. The Ca-aggregated milk was also relatively

45,46

47,48

. In

. When

, the mechanical properties of the porous structure in the Ca-aggregated particle

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conversion especially regarding the porosity of the shell formed, and a superior protective

318

capacity on preservation of L. rhamnosus GG during single droplet drying. This work may open

319

new avenues for development of probiotic products with high bacterial viability and calcium

320

enrichment. It may also provide a novel strategy for the optimization of drying efficiency and

321

powder morphology in the dairy industry. However, the feasibility of scaling-up this application

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and its applicability on other probiotic strains should now be explored on the real spray drying

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scale.

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Acknowledgements

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Xiao Dong Chen thanks the startup grant provided by Soochow University for him to setup the

327

new school of chemical and environmental engineering; The authors also like to acknowledge the

328

financial support from the “Jiangsu Innovation and Entrepreneurship (Shuang Chuang)” Program,

329

and the joint PhD program between Soochow University and Agrocampus Ouest.

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Tables Table 1. Viscocity and distribution of T2 relaxation time of the three bacteria-suspended milk matrixes

T2 distribution Viscocity

Area

Milk matrixes (mPa·s)

Start (ms)

Peak (ms)

End (ms)

Area

proportion (%)

Untreated skim milk

1.4±0.1a

75.6±0.1a

141.0±0.0a

265.6±0.1a

5440±110a

98.3±0.6a

Ca-added milk

1.3±0.1a

75.6±0.0a

141.0±0.1a

265.6±0.0a

5339±27ab

98.5±0.2a

Ca-aggregated milk

28.9±0.5b

65.8±0.1b

131.2±0.1b

231.0±0.2b

5187±100b

98.2±1.0a

Different superscript letters (by column) refer to significant difference (p