Thermal Aggregation of Calcium-Fortified Skim Milk Enhances

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36

Journal of Agricultural and Food Chemistry

1

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

2

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

3

4

5

a

6

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

7

Soochow University, Suzhou 215123, Jiangsu Province, China

8

b

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

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

9

10

*Corresponding authors:

11

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

12

Province, China.

13

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

14

15

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

16

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

17

1

ACS Paragon Plus Environment


Page 2 of 36

18

Abstract

19

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

20

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

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

and calcium enrichment.

32

33

Key words

34

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

35

2


Page 3 of 36


36

Introduction

37

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

38

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

39

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

40

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

41

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

42

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

43

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

44

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

45

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

46

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

47

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

48

digestive tract 5.

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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



Page 4 of 36

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

and the efficacy of probiotics

66

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

67

proteins induced by calcium supplementation has been extensively studied

68

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

69

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

70

digestion stress

71

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

72

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

73

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

74

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

4


Page 5 of 36


75

Materials and methods

76

Strains and growth conditions

77

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

78

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

79

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

80

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

81

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

82

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

83

Preparation of bacteria-suspended milk matrixes

84

The Ca-aggregated milk was prepared as described previously

85

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

86

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

87

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

88

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

89

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

90

(Eppendorf, Germany) before mixing with LGG cells.

91

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

92

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

93

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

94

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

95

further use.

21

. Briefly, UHT skim milk

5



Page 6 of 36

96

Single droplet drying

97

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

98

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

99

described in detail elsewhere

22,23

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

100

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

101

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

102

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

103

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

104

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

105

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

106

was calculated with the following equation:

107

Xd=(Md−Ms)/Ms

(1)

108

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

109

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

110

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

111

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

112

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

113

viability within droplets was measured as described before

114

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

115

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

116

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

117

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

10,24

. Briefly, the drying process was

6


Page 7 of 36


118

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

119

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

120

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

121

Particle size analysis

122

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

123

diffraction (Horiba LA-960, Japan).

124

Viscosity measurement

125

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

126

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

127

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

128

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

129

One-dimensional polyacrylamide gel electrophoresis

130

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

131

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

132

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

133

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

134

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

135

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

136

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

137

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

138

XRS+, Biorad, United States). 7



Page 8 of 36

139

Nuclear magnetic resonance (NMR) measurement

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

Analysis software.

149

Scanning electron microscopy

150

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

151

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

152

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

153

Statistical analysis

154

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

155

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

156

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

157

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

158

Differences were considered significant at p < 0.05.

159 8


Page 9 of 36


160

Results and Discussion

161

Physical and chemical properties of liquid milk matrixes

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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

178

29

179

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

180

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

181

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

9



Page 10 of 36

182

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

183

water into the protein network 31,32.

184

Drying kinetics of milk matrixes

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

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

199

reach the final 4th stage plateau.

200

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

201

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

202

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

203

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

24,33

. For the

10


Page 11 of 36


204

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

205

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

206

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

207

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

208

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

209

Droplet morphology evolution during drying

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

further change and progressively hindered the evaporation

222

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

223

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

224

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

225

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

11



Page 12 of 36

226

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

227

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

228

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

229

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

230

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

231

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

232

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

233

preferentially wrinkled particles

234

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

235

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

236

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

237

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

238

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

239

of casein micelles in the shell formation.

240

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

241

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

242

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

243

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

244

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

245

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

246

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

247

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

248

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

12


Page 13 of 36


249

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

250

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

251

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

252

drying rate observed.

253

Bacteria viability during single droplet drying

254

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

255

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

256

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

257

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

258

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

259

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

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

270

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



Page 14 of 36

271

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

272

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

276

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

14


Page 15 of 36


294

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

15



Page 16 of 36

317

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

322

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

323

scale.

324

325

Acknowledgements

326

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.

330

16


Page 17 of 36


331

Reference

332

(1)

333 334

Klaenhammer, T. R.; Kleerebezem, M.; Kopp, M. V.; Rescigno, M. The impact of probiotics and prebiotics on the immune system. Nat. Rev. Immunol. 2012, 12 (10), 728–734.

(2)

Johansson, M. E. V.; Jakobsson, H. E.; Holmén-Larsson, J.; Schütte, A.; Ermund, A.; Rodríguez-

335

Piñeiro, A. M.; Arike, L.; Wising, C.; Svensson, F.; Bäckhed, F.; et al. Normalization of Host

336

Intestinal Mucus Layers Requires Long-Term Microbial Colonization. Cell Host Microbe 2015, 18

337

(5), 582–592.

338

(3)

339 340

Nutr. 2015, 0 (ja), 00–00. (4)

341 342

Sarao, L. K.; M, A. Probiotics, Prebiotics and Microencapsulation - A Review. Crit. Rev. Food Sci.

Fu, N.; Chen, X. D. Towards a maximal cell survival in convective thermal drying processes. Food Res. Int. 2011, 44 (5), 1127–1149.

(5)

Martín, M. J.; Lara-Villoslada, F.; Ruiz, M. A.; Morales, M. E. Microencapsulation of bacteria: A

343

review of different technologies and their impact on the probiotic effects. Innov. Food Sci. Emerg.

344

Technol. 2015, 27, 15–25.

345

(6)

Barbosa, J.; Borges, S.; Amorim, M.; Pereira, M. J.; Oliveira, A.; Pintado, M. E.; Teixeira, P.

346

Comparison of spray drying, freeze drying and convective hot air drying for the production of a

347

probiotic orange powder. J. Funct. Foods 2015, 17, 340–351.

348

(7)

349 350

opportunities to improve the viability of bacteria powders. Int. Dairy J. 2013, 31 (1), 12–17. (8)

351 352 353

Schuck, P.; Dolivet, A.; Méjean, S.; Hervé, C.; Jeantet, R. Spray drying of dairy bacteria: New

Peighambardoust, S. H.; Golshan Tafti, A.; Hesari, J. Application of spray drying for preservation of lactic acid starter cultures: a review. Trends Food Sci. Technol. 2011, 22 (5), 215–224.

(9)

Schutyser, M. A. I.; Perdana, J.; Boom, R. M. Single droplet drying for optimal spray drying of enzymes and probiotics. Trends Food Sci. Technol. 2012, 27 (2), 73–82.

17



354

(10)

355 356

Fu, N.; Woo, M. W.; Selomulya, C.; Chen, X. D. Inactivation of Lactococcus lactis ssp. cremoris cells in a droplet during convective drying. Biochem. Eng. J. 2013, 79, 46–56.

(11)

357 358

Khem, S.; Woo, M. W.; Small, D. M.; Chen, X. D.; May, B. K. Agent selection and protective effects during single droplet drying of bacteria. Food Chem. 2015, 166, 206–214.

(12)

Perdana, J.; Bereschenko, L.; Fox, M. B.; Kuperus, J. H.; Kleerebezem, M.; Boom, R. M.; Schutyser,

359

M. A. I. Dehydration and thermal inactivation of Lactobacillus plantarum WCFS1: Comparing

360

single droplet drying to spray and freeze drying. Food Res. Int. 2013, 54 (2), 1351–1359.

361

(13)

Burgain, J.; Scher, J.; Lebeer, S.; Vanderleyden, J.; Cailliez-Grimal, C.; Corgneau, M.; Francius, G.;

362

Gaiani, C. Significance of bacterial surface molecules interactions with milk proteins to enhance

363

microencapsulation of Lactobacillus rhamnosus GG. Food Hydrocoll. 2014, 41, 60–70.

364

(14)

365 366

Lee, B.; Yin, X.; Griffey, S. M.; Marco, M. L. Attenuation of colitis by Lactobacillus casei BL23 is dependent on the dairy delivery matrix. Appl. Environ. Microbiol. 2015, AEM.01360-15.

(15)

Pérez, A. V.; Picotto, G.; Carpentieri, A. R.; Rivoira, M. A.; Peralta López, M. E.;

367

Tolosa de Talamoni, N. G. Minireview on Regulation of Intestinal Calcium Absorption. Digestion

368

2008, 77 (1), 22–34.

369

Page 18 of 36

(16)

Scholz-Ahrens, K. E.; Ade, P.; Marten, B.; Weber, P.; Timm, W.; Açil, Y.; Glüer, C.-C.; Schrezenmeir,

370

J. Prebiotics, probiotics, and synbiotics affect mineral absorption, bone mineral content, and bone

371

structure. J. Nutr. 2007, 137 (3 Suppl 2), 838S–46S.

372

(17)

Trautvetter, U.; Ditscheid, B.; Kiehntopf, M.; Jahreis, G. A combination of calcium phosphate and

373

probiotics beneficially influences intestinal lactobacilli and cholesterol metabolism in humans. Clin.

374

Nutr. 2012, 31 (2), 230–237.

375

(18)

Gaucheron, F. The minerals of milk. Reprod. Nutr. Dev. 2005, 45 (4), 473–483.

18


Page 19 of 36

376

(19)


Doherty, S. B.; Gee, V. L.; Ross, R. P.; Stanton, C.; Fitzgerald, G. F.; Brodkorb, A. Development and

377

characterisation of whey protein micro-beads as potential matrices for probiotic protection. Food

378

Hydrocoll. 2011, 25 (6), 1604–1617.

379

(20)

380 381

Heidebach, T.; Först, P.; Kulozik, U. Microencapsulation of probiotic cells by means of rennetgelation of milk proteins. Food Hydrocoll. 2009, 23 (7), 1670–1677.

(21)

Huang, S.; Yang, Y.; Fu, N.; Qin, Q.; Zhang, L.; Chen, X. D. Calcium-Aggregated Milk: a Potential

382

New Option for Improving the Viability of Lactic Acid Bacteria Under Heat Stress. Food Bioprocess

383

Technol. 2014, 7 (11), 3147–3155.

384

(22)

Fu, N.; Wai Woo, M.; Qi Lin, S. X.; Zhou, Z.; Dong Chen, X. Reaction Engineering Approach (REA) to

385

model the drying kinetics of droplets with different initial sizes—experiments and analyses. Chem.

386

Eng. Sci. 2011, 66 (8), 1738–1747.

387

(23)

388 389

QI Lin, S. X.; Chen, X. D. Improving the Glass-Filament Method for Accurate Measurement of Drying Kinetics of Liquid Droplets. Chem. Eng. Res. Des. 2002, 80 (4), 401–410.

(24)

Zheng, X.; Fu, N.; Duan, M.; Woo, M. W.; Selomulya, C.; Chen, X. D. The mechanisms of the

390

protective effects of reconstituted skim milk during convective droplet drying of lactic acid

391

bacteria. Food Res. Int. 2015, 76, 478–488.

392

(25)

393 394

Kim, H.; Park, K.; Oh, S.; Chang, I. S. Rapid detection of lactobacillus and yeast concentrations using a particle size distribution analyser. J. Appl. Microbiol. 2009, 107 (5), 1499–1504.

(26)

McGann, T. C. A.; Donnelly, W. J.; Kearney, R. D.; Buchhemm, W. Composition and size

395

distribution of bovine casein micelles. Biochim. Biophys. Acta BBA - Gen. Subj. 1980, 630 (2), 261–

396

270.

397 398

(27)

Anema, S. G.; Li, Y. Association of denatured whey proteins with casein micelles in heated reconstituted skim milk and its effect on casein micelle size. J. Dairy Res. 2003, 70 (1), 73–83.

19



399

(28)

400 401

(29)

(30)

(31)

Belloque, J.; Ramos, M. Application of NMR spectroscopy to milk and dairy products. Trends Food Sci. Technol. 1999, 10 (10), 313–320.

(32)

408 409

Li, X.; Ma, L. Z.; Tao, Y.; Kong, B. H.; Li, P. J. Low Field-NMR in Measuring Water Mobility and Distribution in Beef Granules during Drying Process. Adv. Mater. Res. 2012, 550–553, 3406–3410.

406 407

Jeurnink, T. J. M.; De Kruif, K. G. Changes in milk on heating: viscosity measurements. J. Dairy Res. 1993, 60 (2), 139–150.

404 405

Dalgleish, D. G. Denaturation and aggregation of serum proteins and caseins in heated milk. J. Agric. Food Chem. 1990, 38 (11), 1995–1999.

402 403

Lambelet, P.; Berrocal, R.; Renevey, F. Low-field nuclear magnetic resonance relaxation study of thermal effects on milk proteins. J. Dairy Res. 1992, 59 (4), 517–526.

(33) Sadek, C.; Tabuteau, H.; Schuck, P.; Fallourd, Y.; Pradeau, N.; Le Floch-Fouéré, C.; Jeantet, R. Shape,

410

Shell, and Vacuole Formation during the Drying of a Single Concentrated Whey Protein Droplet.

411

Langmuir 2013, 29 (50), 15606–15613.

412

(34)

Arcamone, J.; Dujardin, E.; Rius, G.; Pérez-Murano, F.; Ondarçuhu, T. Evaporation of Femtoliter

413

Sessile Droplets Monitored with Nanomechanical Mass Sensors. J. Phys. Chem. B 2007, 111 (45),

414

13020–13027.

415

(35)

416 417

420

Fu, N.; Woo, M. W.; Selomulya, C.; Chen, X. D. Shrinkage behaviour of skim milk droplets during air drying. J. Food Eng. 2013, 116 (1), 37–44.

(36)

418 419

Page 20 of 36

Pauchard, L.; Allain, C. Buckling instability induced by polymer solution drying. Europhys. Lett. EPL 2003, 62 (6), 897–903.

(37)

Arai, S.; Doi, M. Skin formation and bubble growth during drying process of polymer solution. Eur. Phys. J. E 2012, 35 (7).

20


Page 21 of 36

421

(38)


Sadek, C.; Li, H.; Schuck, P.; Fallourd, Y.; Pradeau, N.; Le Floch-Fouéré, C.; Jeantet, R. To What

422

Extent Do Whey and Casein Micelle Proteins Influence the Morphology and Properties of the

423

Resulting Powder? Dry. Technol. 2014, 32 (13), 1540–1551.

424

(39)

Sadek, C.; Pauchard, L.; Schuck, P.; Fallourd, Y.; Pradeau, N.; Le Floch-Fouéré, C.; Jeantet, R.

425

Mechanical properties of milk protein skin layers after drying: Understanding the mechanisms of

426

particle formation from whey protein isolate and native phosphocaseinate. Food Hydrocoll. 2015,

427

48, 8–16.

428

(40)

Sadek, C.; Schuck, P.; Fallourd, Y.; Pradeau, N.; Jeantet, R.; Le Floch-Fouéré, C. Buckling and

429

collapse during drying of a single aqueous dispersion of casein micelle droplet. Food Hydrocoll.

430

2016, 52, 161–166.

431

(41)

432 433

bacteria. FEMS Microbiol. Lett. 2013, 344 (1), 31–38. (42)

434 435

Huang, S.; Chen, X. D. Significant effect of Ca2+ on improving the heat resistance of lactic acid

Mekmene, O.; Le Graët, Y.; Gaucheron, F. A model for predicting salt equilibria in milk and mineral-enriched milks. Food Chem. 2009, 116 (1), 233–239.

(43)

Arifin, M.; Swedlund, P. J.; Hemar, Y.; McKinnon, I. R. Calcium Phosphates in Ca 2+ -Fortified Milk:

436

Phase Identification and Quantification by Raman Spectroscopy. J. Agric. Food Chem. 2014, 62

437

(50), 12223–12228.

438

(44)

Buitink, J.; van den Dries, I. J.; Hoekstra, F. A.; Alberda, M.; Hemminga, M. A. High Critical

439

Temperature above Tg May Contribute to the Stability of Biological Systems. Biophys. J. 2000, 79

440

(2), 1119–1128.

441

(45)

442 443

Hansen, N.-H.; Riemann, H. Factors Affecting the Heat Resistance of Nonsporing Organisms. J. Appl. Bacteriol. 1963, 26 (3), 314–333.

(46)

Potts, M. Desiccation tolerance of prokaryotes. Microbiol. Rev. 1994, 58 (4), 755–805.

21



444

(47)

Page 22 of 36

Xing, Y.; Li, A.; Felker, D. L.; Burggraf, L. W. Nanoscale Structural and Mechanical Analysis of

445

Bacillus anthracis Spores Inactivated with Rapid Dry Heating. Appl. Environ. Microbiol. 2014, 80 (5),

446

1739–1749.

447

(48)

448 449 450

Zhang, L.; Huang, S.; Ananingsih, V. K.; Zhou, W.; Chen, X. D. A study on Bifidobacterium lactis Bb12 viability in bread during baking. J. Food Eng. 2014, 122, 33–37.

(49)

Mezzenga, R.; Schurtenberger, P.; Burbidge, A.; Michel, M. Understanding foods as soft materials. Nat. Mater. 2005, 4 (10), 729–740.

451

22


Page 23 of 36


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

Thermal Aggregation of Calcium-Fortified Skim Milk Enhances

Recommend Documents