Role of Ca-alginate and mannitol in protecting Bifidobacterium

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Role of Ca-alginate and mannitol in protecting Bifidobacterium 3 the running title (not to exceed 54 characters and spaces)?? 4 5

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Dianawati Dianawatia,b, Vijay Mishraa and Nagendra P. Shaha,c* 9

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aSchool of Biomedical and Health Sciences, Victoria University, Werribee campus, P.O. Box 13 14428, Melbourne, Victoria 8001, Australia. 14

bTribhuwana Tunggadewi University, Jalan Telaga Warna, Malang 65145, East Java, Indonesia. 15

cFood and Nutritional Science - School of Biological Sciences, The University of Hong Kong, 16

Pokfulam Road, Hong Kong 17

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*Corresponding author: Prof. Dr. Nagendra P. Shah; [email protected] 24

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01724-12 AEM Accepts, published online ahead of print on 27 July 2012

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Abstract 26

Fourier Transform Infrared Spectroscopy (FTIR) was carried out to ascertain the protection 27

mechanism of Ca-alginate and mannitol on cell envelope components and secondary proteins of 28

Bifidobacterium animalis ssp. lactis Bb12 after freeze drying and after 10 week of storage at 29

room temperature (25 oC) at low water activities (aw) of 0.07, 0.1 and 0.2. Preparation of Ca-30

alginate and Ca-alginate-mannitol as microencapsulant was carried out by dropping alginate or 31

alginate-mannitol emulsion containing bacteria using a burette into CaCl2 solution to obtain Ca-32

alginate beads or Ca-alginate-mannitol beads, respectively. The wet beads were then freeze 33

dried. The aw of freeze dried beads was then adjusted to 0.07, 0.1 and 0.2 using saturated salt 34

solutions; control was prepared by keeping Ca-alginate and Ca-alginate-mannitol in an 35

aluminium foil without aw adjustment. Mannitol in Ca-alginate system interacted with cell 36

envelopes during freeze drying and during storage at low aw. In contrast, Ca-alginate protected 37

cell envelopes after freeze drying but not during 10 week storage. Unlike Ca-alginate, Ca-38

alginate-mannitol was effective in retarding the changes in secondary proteins during freeze 39

drying and during 10 week of storage at low aw. It appears that Ca-alginate-mannitol was more 40

effective than Ca-alginate in preserving cell envelopes and proteins after freeze drying and after 41

10 weeks of storage at room temperature (25oC). 42

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Words: 211 44

Keywords: alginate, mannitol, freeze drying, storage, low aw, cell envelopes, secondary proteins 45

Abbreviations: CA = calcium alginate without bacteria; CAM = calcium alginate and mannitol without 46

bacteria; CAB = calcium alginate containing bacteria; CAMB = calcium alginate and mannitol containing 47

bacteria; SA = sodium alginate in powder form 48

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Introduction 52

Microencapsulation of probiotic bacteria using alginate or alginate fortified with mono-, 53

di- or polysaccharides or proteins has been widely studied (1, 9, 29, 31, 39, 46, 54). Fortification 54

of sugars or proteins to alginate was effective in improving bacterial survival; however, 55

Krasaekoopt and others (31) reported that alginate combined with chitosan was not successful in 56

protecting Bifidobacterium in very low pH environment. Similarly, Zohar-Perez and 57

Nussinovitch (61) found that alginate was not effective as bacterial encapsulant. Stabilization of 58

alginate-based microencapsulated bacteria was influenced by many factors such as type and 59

concentration of microencapsulants, and storage conditions such as temperature, water activity 60

and the presence of oxygen. The use of mannitol was found effective in maintaining bacterial 61

viability during drying and during storage at room temperature (15, 28, 40) and also in 62

stabilizing protein during freeze drying (27) and during storage (8). Mannitol protected probiotic 63

bacteria during storage likely due to its characteristic as hydroxyl radical scavenger (15, 51). 64

Water activity (aw) is a critical factor for bacterial stabilization during storage at room 65

temperature. Storage at low aw at room temperature maintained the glassy state and minimized 66

chemical reactions, hence the survival of bacteria improved (24). Storage at low aw (0.07 and 67

0.1) maintained high viability of bacteria during long term storage at room temperature; 68

however, this phenomenon depended on the type of sugars as a protectant incorporation of 69

mannitol in Ca-alginate improved the survival of B. animalis ssp. lactis Bb12 and preserved 70

some glycolitic enzymes during long term storage at room temperature at low aw (11, 12). 71

Even though Ca-alginate is known as an encapsulant for probiotic bacteria (26), its 72

protection mechanism on probiotic bacteria has not been established. Changes in functional 73

groups of polypeptides, lipids and secondary proteins can be observed by FTIR spectroscopy 74


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(53). FTIR spectroscopy has been successfully applied in identifying microorganisms such as 75

probiotic bacteria and fungi (14, 16, 58, 59). Characterization of lactic acid bacteria (LAB) 76

encapsulated with alginate using FTIR spectroscopy has been carried out by Le-Tien and others 77

(34). The effect of drying and as well as of sugars on bacterial membrane structure and on 78

secondary proteins was studied by Izutsu and others (27); Sharma and Kalonia (49); Oldenhof 79

and others (42); Santivarangkna and others (48); Santivarangkna and others (47). Trehalose and 80

sorbitol were effective in protecting cell envelopes and secondary proteins of probiotic bacteria 81

during drying (35, 42, 47). However, polysaccharides such as maltodextrin acts as an inert 82

bulking substance instead of bacterial protectant (35). Lyophilization causes reversible changes 83

to secondary proteins (21) as well as affected the stability of phophatidylcholine and liposomes 84

(56). However, freeze drying is a common method for dehydration of probiotic bacteria. In this 85

study, FTIR was used to establish the protection mechanism provided by encapsulants such as 86

Ca-alginate with or without mannitol on cell envelope components and secondary proteins of 87

probiotic bacteria during freeze drying and after 10 week storage at low aw. Interaction between 88

Ca-alginate and mannitol was also observed in order to understand the effectiveness of mannitol 89

as a protectant in Ca-alginate matrix. 90

Materials and Methods 91

B. animalis ssp. lactis Bb12 and their cultivation 92

Cultivation of B. animalis ssp. lactis Bb12 was carried out according to Ding and Shah 93

(13). The freeze dried pure culture of B. animalis ssp. lactis Bb12 was received from Chr. 94

Hansen (Bayswater, Victoria, Australia). The organism was grown in MRS broth (Oxoid Ltd., 95

Hampshire, U.K.) supplemented with filter sterilized 0.05% w/v L-cysteine-hydrochloride 96


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(Sigma Chemical Co., Castle Hill, Australia) at 37 oC using 1% inoculum. The organism was 97

propagated 3 times successively, and its presence was confirmed using gram staining. 98

The cells of B. animalis ssp. lactis Bb12 were concentrated by centrifugation of the broth 99

at 14,000 × g for 15 min at 4 oC using Sorval centrifuge (57), and the cell pellet was washed 100

twice with 0.85% of sterilized saline solution. The cell pellet was then resuspended in one fourth 101

of the original volume (5 mL cell pellet added to 15 mL of saline solution). The initial population 102

of bacteria in the suspension was ~2.5 x 1010 CFU/mL. 103

Microencapsulation of probiotic 104

Microencapsulation was carried out using two types of alginate emulsions as per Dianawati and 105

Shah (12). The first formulation contained sodium alginate (at 2.5% w/v of the emulsion) and the 106

second formulation contained sodium alginate and mannitol (each at 2.5% w/v of the emulsion). 107

Canola oil containing 0.5% Tween 80 at 10% was used to develop an emulsion system of both 108

the formulations. Each emulsion was pasteurized by heating at 70 oC for 30 minutes and cooled 109

to 10 oC before an incorporation of B. animalis ssp. lactis Bb12 was carried out (250 mL cells in 110

total 1000 mL of emulsion system). Each mixture containing the bacteria was then dropped into 111

0.1 M CaCl2 solution using a burette to create either the Ca-alginate beads or Ca-alginate-112

mannitol beads with uniform size. Both types of the wet beads were then freeze dried (Freeze 113

drier model FD-300, Airvac Engineering Pty. Ltd., Dandenong, Australia) which was set to 114

achieve -100 Torr of internal pressure before freeze drying at temperature of -88 oC, 44 h of 115

primary freeze drying, and 4 h of secondary freeze drying. Once the freeze drying process was 116

completed, the two type of dehydrated beads namely Ca-alginate containing B. animalis ssp. 117

lactis Bb12 (CAB) and Ca-alginate-mannitol containing B. animalis ssp. lactis Bb12 (CAMB) 118

were obtained. The samples (CAB and CAMB) were stored for 10 weeks in desiccators with aw 119


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adjusted to 0.07, 0.1 and 0.2 using NaOH, LiCl and CH3COOK, respectively, at 25 oC. NaOH 120

(200 g) was added to 45 mL of water (that is equivalent to 111.1 M) to achieve an aw of 0.07; 121

LiCl (200 g) was added to 113 mL of water (that is equivalent to 41.7 M) to achieve an aw of 122

0.11, and CH3COOK (200 g) was added to 65 mL water (that is equivalent to 31.4 M) to achieve 123

aw of 0.23. The samples (CAB and CAMB) kept inside an aluminum foil at room temperature 124

(without aw adjustment) were used as control. 125

Storage at low aw at room temperature of 25 oC 126

An equilibrium between aw of the samples and that of the environment (desiccators) was 127

achieved in two weeks, which was considered as 0 week of storage. The aw was measured using 128

a water activity meter (Decagon CX-2 Serial I/O, Washington DC, USA). Samples were taken at 129

week 10 of storage for measuring changes in cell envelopes and secondary proteins using FTIR 130

spectroscopy. 131

Sample preparation for FTIR spectroscopy 132

Infrared absorption measurements were carried out with an FT-IR spectrometer (Model 133

IRAffinity-1, Shimadzu Corp., Kyoto, Japan) at room temperature (25 oC). Spectra were 134

recorded at a resolution of 4 cm-1 and 20 scans in a wave number range of 4000-500 cm-1. A 135

reference spectrum was measured prior to each experiment to correct the background effects of 136

all the spectra recorded. The instrument was purged with nitrogen gas to reduce the interference 137

of water vapor and CO2 in all the FT-IR measurements. The sample preparation was according to 138

Izutsu and Kojima (27) and Sharma and Kalonia (49). Briefly, a 10 mg sample of CAB or 139

CAMB was mixed with 100 mg of dried KBr powder, and pressed under vacuum using at 10 140

tons of hydraulic pressure (JC hydraulics, KBr Beta Press 6010 & 6102, Buck Sci. Inc. East 141


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Norwalk, Connecticut, USA) using 13 mm pellet die (Specac, PT No. 3000, Orpington, Kent, 142

Britain) was carried out to obtain a transparent pellet of CAB or CAMB. Spectra of fresh B. 143

animalis ssp. lactis Bb12 (harvested after 18 h) was used as a control to observe any change in 144

the frequencies of functional groups of cell envelope (PO2-, (CH3)3N

+ and C-H) and secondary 145

proteins (C-N, N-H) of microencapsulated B. animalis ssp. lactis Bb12. The sample preparation 146

method of the fresh bacteria was carried out according to Santivarangkna et al (47). Briefly, 147

washed cell suspension was spread onto CaF2 windows and was dehydrated at room temperature 148

in vacuum desiccators containing P2O5 for 2 days to reduce any interference of H2O. Spectra 149

were collected from three different batches of samples. Smoothing and normalization of second 150

derivatives of deconvoluted spectra were carried out to develop clearer separation of complex 151

bands using IRsolution software (Shimadzu Corp., Kyoto, Japan). 152

The freeze dried Ca-alginate-mannitol without the bacteria (CAM) was compared with 153

Ca-alginate without bacteria (CA) to study any chemical interaction between Ca-alginate and 154

mannitol as microencapsulant. Na-alginate (SA) powder was used as a control. To examine 155

changes in cell envelope and secondary proteins of microencapsulated B. animalis ssp. lactis 156

Bb12, the spectra of CAMB and CAB was subtracted with those of CAM and CA (22, 37). The 157

subtracted spectra were then compared with those of the freshly harvested B. animalis ssp. lactis 158

Bb12. All FTIR measurements were repeated three times. 159

Determination of Ca-alginate-mannitol, cell envelope and secondary 160

proteins of microencapsulated B. animalis ssp. lactis Bb12 by FTIR 161

spectroscopy 162

163 An interaction between Ca-alginate and mannitol was ascertained by comparing the 164

peaks of CA and CAM at 3000-3700 cm-1 (broaden peak of O-H stretching) and at 1410-1260 165


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(O-H deformation vibration). Sodium alginate (SA) was used as a control (23, 33). An alteration 166

of COO- stretching symmetric vibration and COO- stretching asymmetric vibration of alginate 167

can be detected at ~1420 – 1300 and ~1615-1550, respectively (3, 23, 33, 43). 168

An investigation of cell envelopes of bacteria in CAMB and CAB was carried out in the 169

frequencies in the FTIR spectra of ~1080 cm-1 (P=O of PO2- symmetric stretching) and ~1240 170

cm-1 (P=O of PO2- asymmetric stretching) (16, 17); ~2850 cm-1 and ~2925 cm-1 (CH2 symmetric 171

and asymmetric stretching vibrations, respectively) (47, 60), ~975 cm-1 (CH3)3N+ asymmetric 172

stretching vibration) (44). An observation on secondary proteins was carried out by 173

determination of amide II at 1450-1575 cm-1 (CN stretching, NH bending) (35, 36). 174

ESEM study 175

Morphology of freeze dried microcapsule (CAMB and CAB) was observed after freeze 176

drying using FEI Quanta 200 environmental scanning electron microscope (ESEM). The 177

working distance was 10.2 mm and beam energy was 20.0 kV, spot 5.0, 500 × magnification and 178

pressure 0.98 torr. All bars presented 200.0 µm of size. The microcapsule bead was loaded on a 179

double side carbon tape put on multiple studs before SEM examination. 180

Result 181

Ca-alginate and mannitol interaction in gel bead system 182

FTIR analysis of the CA and CAM was carried out to observe any shift in spectra 183

representing an interaction between alginate and mannitol; thus its mechanism as a protectant 184

could be predicted. The presence of mannitol may influence the frequency shift indicating its 185

interaction with alginate. In this study, wave number alteration of some functional groups of CA 186

and CAM was identified by comparing the shift in –OH stretching and –OH deformation, –COO- 187


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symmetric and asymmetric stretching and –C-O-C- asymmetric stretching of freeze dried CA 188

and CAM as shown in Table 1. Sodium alginate (SA) was used as a control. The alteration of 189

the functional groups of alginate was based on the references (25, 33, 60). A decrease in 190

frequency from 3210 (CA) to 3207 (CAM) and from 1257 (CA) to 1251 (CAM) indicated the 191

presence of –OH stretching and –OH deformation vibration, respectively; meanwhile, SA in the 192

powder form showed higher frequency of –OH stretching and –OH deformation vibration. An 193

alteration to lower frequency indicated an increase in the strength of hydrogen bond (23), 194

possibly due to the presence of mannitol. In addition, no obvious difference between COO- of 195

CA and CAM (both in symmetric and asymmetric vibrations) was detected; meanwhile, SA 196

demonstrated a lower frequency of COO- as compared to that of CA and CAM (Table 1). An 197

alteration to lower frequency indicated an increase in the strength of hydrogen bond (23). It 198

could be due to the presence of hydroxyl groups of mannitol replacing the moisture availability 199

as suggested by Aranda et al. (2) and Santivarangkna et al. (47). 200

In addition, no obvious difference between COO- of CA and CAM (both in symmetric 201

and asymmetric vibrations) was detected; meanwhile, SA demonstrated a lower frequency of 202

COO- as compared to that of CA and CAM (Table 1). This suggests that there was no strong 203

interaction between alginate and mannitol due to the stronger ionic bonding of COO- of alginate 204

with cations. On the other hand, frequencies of C-O-C asymmetric vibration altered from 1160 205

(CA) to 1162 (CAM); while SA showed a slightly higher frequency of C-O-C stretching 206

vibration. 207


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Cell envelopes and secondary proteins of microencapsulated 208

Bifidobacterium animalis ssp. lactis Bb12 after freeze drying and after 209

storage at low aw at room temperature 210

Frequencies of some cell envelope components and amide II of freshly harvested B. 211

animalis ssp. lactis Bb12, CAB and CAMB after freeze drying and after 10 week storage were 212

demonstrated in Table 2, 3 and 4, respectively. P=O and (CH3)3N+ represented the polar site of 213

phospholipid bilayers; while CH2 was the non polar site of phospholipid bilayers. The changes in 214

secondary protein structures were indicated by amide II. 215

A shift of PO2- symmetric stretching vibration of CAB after freeze drying and after 10 216

week storage at various low aw at 25oC is shown in Table 3; meanwhile that of CAMB after the 217

same treatments is revealed in Table 4. There was an interaction between PO2- of cell envelopes 218

and CAMB as shown by an alteration to lower frequency (1043.6) (Table 4) from originally 219

1077.8 of freshly harvested B. animalis ssp. lactis Bb12 (Table 2); while CAB demonstrated a 220

shift to higher frequency (1053.4; Table 3) as compared to that of CAMB. Both frequencies of 221

freeze dried CAB and CAMB were below that of control indicating an interaction of PO2- of 222

phospholipid site of cell lipids with the microcapsule substances via hydrogen bond as suggested 223

by Leslie et al. (35); Oldenhof et al. (42); Aranda et al. (2) and Santivarangkna et al. (47). The 224

similar behaviors were also demonstrated by P=O of PO2- asymmetric stretching vibration of 225

freeze dried CAB and CAMB (Table 3 and Table 4, respectively) as compared to control (1240.4 226

cm-1; Table 2). It indicates that an interaction via hydrogen bond between PO2- of phospholipid 227

bilayers and mannitol was maintained during storage at low aw. On the contrary, there was less 228

interaction between alginate and PO2- of phospholipid bilayers of cell envelopes in CAB kept in 229


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an aluminium foil after 10 week of storage since the frequencies were higher than control (Table 230

3). 231

Choline chain terminal of cell surface could provide more additional information to 232

characterize the microencapsulated bacterial cell envelopes. An alteration of frequency of 233

(CH3)3N+ asymmetric stretching vibration of choline chain terminal of cells within CAB or 234

CAMB after freeze drying and after 10 week of storage at various low aw is shown in Table 3 235

and Table 4, respectively. After 10 week of storage, an increase in frequencies occurred in CAB 236

kept at low aw and in an aluminium foil. This may be due to the effect of -OH of alginate or –OH 237

of residual moisture on choline. Loss in the frequency area of (CH3)3N+ of choline occurred in 238

CAB kept in an aluminium foil. It could be due to an increase in the molecular mobility and 239

chemical reactions along with an increase in aw during storage as suggested by Bell and Labuza 240

(4); therefore an adverse effect on some substances such as choline could not be avoided in 241

freeze dried bacteria that are kept in an aluminium foil. This may be the reason why our previous 242

study (12) demonstrated low survival of bacteria kept in an aluminium foil after 10 week of 243

storage. 244

CH2 symmetric and asymmetric stretching vibration of fatty acids of cell envelopes of B. 245

animalis ssp. lactis Bb12 encapsulated within CAB or CAMB after freeze drying and after 246

storage is also shown in Table 3 and Table 4, respectively. The spectrum of CH2 of fatty acids, 247

an apolar site of phospholipid bilayers of the bacteria was detected at ~2867 and 2926 cm-1. A 248

frequency increase occurred in CAB during storage at various aw and in an aluminium foil; 249

further frequency shift occurred in CAB with aw of 0.07 and CAB kept in an aluminium foil. On 250

the other hand, no increase in frequency was observed in fatty acids of cells kept in CAMB with 251

various aw during storage; instead a slight frequency decrease occurred. In addition, frequencies of 252


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CH2 asymmetric stretching vibration of fatty acids of CAMB increased after storage in an aluminium foil; 253

while those of CAB increased due to storage regardless the presence or an absence of desiccants. 254

Amide II of secondary proteins of B. animalis ssp. lactis Bb12 within CAB or CAMB 255

after freeze drying and that after storage at various aw at room temperature is shown in Table 3 256

and Table 4, respectively. The control showed a native amide II peak at 1541 cm-1. Amide II of 257

CAMB after freeze drying showed lower frequency (1536 cm-1; Table 4) compared to the 258

control; while that of CAB after freeze drying altered to higher frequency (1546 cm-1; Table 3). 259

Further obvious shift of freeze dried CAB after 10 week storage occurred; the peaks were altered 260

to ~1569. On the other hand, the bands of CAMB kept at low aw were relatively unaltered 261

compared to that after freeze drying i.e. at 1534-1538 cm-1; but frequency decreased in CAMB 262

kept in an aluminium foil along with a shoulder formation at 1515. This alteration to ~1510 263

indicates a partially change in secondary proteins from α-helix into β-sheet (52). 264

Microstructure of microcapsules 265

The microstructure of microcapsules containing B. animalis ssp. lactis Bb12 after freeze 266

drying was shown in Figure 1a and Figure 2a, for CAB and CAMB, respectively; while freeze 267

dried CAB and CAMB after 10 weeks of storage at aw of 0.07 was shown in Figure 2a and 2b. 268

The surface of CAB and CAMB microcapsules appeared dense and relatively rough after freeze 269

drying (Figure 1a and 2a) with less wrinkles. After 10 weeks of storage, the wrinkles were more 270

obvious (Figure 1b and 2b) which could be due to residual moisture removal during storage at aw 271

of 0.07. Incorporation of mannitol into alginate gel might soften the bead surface (Figure 1b and 272

2b). The bacteria did not appear on the microcapsule surface indicating that they were entrapped 273

within the matrices. The microstructure of our alginate microcapsules was similar with that of 274


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Muthukumarasamy et al. (41) and Chen et al. (7). Gbassi (19) revealed that probiotic bacteria 275

were randomly distributed in the alginate matrices. 276

Discussion 277

FTIR analysis of the CA and CAM was carried out to observe any shift in spectra 278

representing an interaction between alginate and mannitol; thus its mechanism as a protectant 279

could be predicted. The presence of mannitol may influence the frequency shift indicating its 280

interaction with alginate. Some functional groups such as –OH stretching and –OH deformation, 281

–COO- symmetric and asymmetric stretching and –C-O-C- asymmetric stretching has been used 282

to characterize the Ca-alginate interaction with chitosan or xanthan (33, 43). 283

The formation of matrix of alginate and divalent cations such as Ca2+ has been widely 284

studied (38, 43, 50). Such matrix is known as an “egg-box” formation of alginate-Ca. This 285

matrix formation is mainly due to the interaction between COO- of β-D-mannuronic acid and α-286

L-guluronic acid of alginate and Ca2+ via ionic bond. Our result is in agreement with that of 287

Pongjanyakul and Puttipipatkhachorn (43) who stated that a cross-linking matrix of alginate with 288

calcium ions resulted in an alteration to higher frequencies of COO- of alginate than that bound 289

with sodium ions. Besides ionic bond, a partial covalent bond between calcium and oxygen atom 290

of C-O-C groups of alginate occurs (43); this interaction might cause the difference in C-O-C 291

frequencies of CA and CAM due to the presence of mannitol. In addition, an alteration of COO- 292

stretching peak to lower frequencies was observed owing to xanthan gum incorporation into CA 293

(43). However, our result demonstrated that no obvious difference in frequency alteration of 294

COO- of CA and CAM indicating that –OH of mannitol has less important role in interacting 295

with COO-. Lack of COO- interaction between CA and mannitol could be due to the effect of 296

ionic bond between COO- of alginate and Ca2+ forming a strong cross linking (38). A decrease in 297


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OH vibration and OH deformation frequencies due to mannitol inclusion is in agreement with 298

Hesse and others (23) and Santiavarangkna and others (48) who stated that stronger hydrogen 299

bond was indicated by a shift into lower vibrational frequencies. However, the presence of 300

bacteria influenced the interaction between functional groups of Ca-alginate and that of 301

mannitol. 302

FTIR spectroscopy has been used to determine the chemical components such as lipids, 303

proteins and polysaccharides of microorganisms (53). FTIR permits us to study the molecular 304

structures of colonies or even single cells in situ without any additional reagents or stains (3, 42, 305

48). Second derivative method based on mathematical analysis has been applied to improve the 306

level of separation of molecular spectra, thus specific peaks such as P=O of PO2- of phospholipid 307

bilayers, C-H of fatty acids, (CH3)3N+ of choline and secondary proteins can be more easily 308

recognized (30). 309

PO2- of phospholipid bilayers has been commonly used to recognize any interaction with 310

other substances through hydrogen bond (2, 47). Variability in frequencies after storage at low 311

water activities could be due to the influence of –OH of residual unbound water in CAMB beads 312

besides –OH from mannitol, thus interaction with PO2- can be varied. However, all PO2

- 313

frequencies of cell envelopes of CAMB showed lower frequencies than that of control. 314

Frequency changes of PO2- bands to a lower wave number suggested an increase in hydrogen 315

bond due to the presence of sugars (47). In regard to the lack of interaction between alginate and 316

phospholipid bilayers of CAB, our result is in agreement with that of Oldenhof and others (42). 317

The authors suggested that high molecular weight polysaccharides such as maltodextrin (or 318

alginate) were unable to interact with P=O of PO2- of phospholipid bilayers. Alginate interacted 319

with mannitol through hydrogen bond in the absence of probiotic bacteria (Table 1). However, in 320


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the presence of bacteria, mannitol interacted with PO2- of cell envelopes instead of Ca-alginate as 321

shown by the difference of frequencies between CAB and CAMB (Table 3 and Table 4). 322

An interaction of choline of cell envelopes with OH groups was indicated by an increase 323

in frequencies of N+(CH3)3 (5). Peak at ~970 cm-1 has been identified as asymmetric stretching 324

for (CH3)3N+ of lipids (44, 53). Grdadolnik and Hadzi (20) found a similar trend of the alteration 325

between hydration and sugar incorporation on choline’s trimethylammonium group. It 326

demonstrated that sugars including mannitol could replace water molecules during dehydration 327

and stabilize the polar head region during storage at room temperature at controlled aw. CAMB 328

kept at low aw appears effective in stabilizing CH3)3N+ of phospholipid bilayers; while in the 329

aluminium foil, increase in frequency along with broaden peak was observed likely to be due to 330

strong effect of unbound water. Interaction of surface-exposed choline with water molecules or 331

sugars caused a shift to higher frequencies due to sensitiveness of (CH3)3N+ asymmetric 332

stretching on dipolar interaction (44). Mannitol kept in higher aw (such as in an aluminium foil) 333

might contribute to plasticizing effect and caused conformational changes in polar site of 334

phospholipids due to an increased level of –OH (55). 335

Fatty acids of phospholipid bilayers have also been used to recognize any changes in cell 336

envelope caharacteristics (10). Vibration of CH2 of fatty acids of phospholipid bilayers can be 337

determined from the frequency around 2850 cm-1 and 2930 cm-1 (53); this can be different due to 338

differences between bacterial strains. Freeze drying showed no apparent effect on stability of 339

fatty acids of freeze dried cells within either CAB or CAMB (Table 3 and Table 4, respectively). 340

It indicated that CAB or CAMB was effective in maintaining apolar site of lipids due to a 341

protection effect of CAB or CAMB on the surface site of phospholipid bilayers as shown by PO2- 342

and choline frequency alteration. However, long term storage of CAB at room temperature at 343


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low aw or in an aluminium foil demonstrated an alteration into higher frequencies. The peak 344

alteration into higher frequencies suggests a melting of lipid acyl chains along with gel-liquid 345

crystalline transition (44). It appears that Ca-alginate was not able to preserve fatty acid site of 346

phospholipid bilayers of freeze dried bacterial cell envelopes during storage at room temperature 347

even at low aw. Conversely, storage of CAMB with low aw and in an aluminium foil showed 348

lower frequencies than that of the control. The presence of sugars (including mannitol) which 349

interact with polar site of lipids during dehydration inhibits interior apolar site of lipid changes 350

such as lipid phase transition and fusion (45), whereas storage with low aw conditions maintained 351

the glassy state of sugars (32). Hence lipid stability could be preserved. The alteration to lower 352

frequency (compared to the control of 2867) could be due to the presence of the membrane 353

proteins and glycolipids as a constituent of cell envelopes (47) which became more obvious on 354

water removal. 355

This study used amide II band to examine the changes in secondary proteins instead of 356

amide I, which is commonly examined by FTIR (18, 42). The use of amide I is unreliable as 357

compared to amide II as the presence of C=O stretching vibration of alginate interferes with the 358

amide I bands (36). Amide II bands represent 60% N-H bending and 40% C-N stretching (53). 359

Any changes in amide II bands represent changes in secondary proteins as reported by Carpenter 360

and Crowe (6), Leslie and others (35) and Marcotte and others (36). The amide II band alteration 361

indicated a change in secondary protein structures such as a decrease in native α-helix and an 362

increase in β-sheet (52). Encapsulation of the cells within alginate fortified with mannitol was 363

able to preserve the native conformation of proteins of the cells during freeze drying and during 364

storage at room temperature at low aw. This result was in agreement with that of Leslie and 365

others (35), Garzon-Rodriguez and others (18) and Thomas and others (55). In contrast, CAB 366


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might undergo a failure in protecting secondary proteins of freeze dried bacteria during long term 367

storage at room temperature as indicated by an alteration to higher frequencies (from originally 368

1538 cm-1); this phenomenon appeared independent of aw. High molecular weight carbohydrates 369

have been found to be ineffective in retarding protein unfolding during lyophilization (18). This 370

result showed the importance of mannitol incorporation in preserving secondary protein 371

conformation of probiotic bacteria during storage at room temperature at low aw. This may be 372

the reason why the survival of B. animalis ssp. lactis Bb12 in CAMB was higher than CAB after 373

10-week of storage at aw of 0.1 and 0.2 (12). Our previous study showed that the survival of 374

bacteria of CAMB was 82.6% and 82.0% after 10 week of storage at aw of 0.1 and 0.2, 375

respectively; while those of CAM was 81.1% and 80.2% after storage at the same conditions. 376

Conclusions 377

FTIR study showed an interaction between Ca-alginate and mannitol, mainly between –OH of 378

mannitol and C-O-C groups of alginate via a hydrogen bond. However, mannitol tend to interact 379

with cell components when B. animalis ssp. lactis Bb12 was incorporated; hence, mannitol might 380

act as a protectant instead of an inert bulking substance such as alginate. Mannitol in alginate 381

system was able to interact with headgroups of lipids of cell envelopes of B. animalis ssp. lactis 382

Bb12. CAMB interacted with P=O of PO2- of phospholipid bilayers after freeze drying and after 383

storage at low aw, while CAB was only able to protect this functional groups after freeze drying 384

but no protective effect after 10 week storage at low aw. CAMB also showed an interaction with 385

choline headgroup of lipids and prevented the fatty acids (apolar site of phospholipid bilayers) 386

from gel-liquid crystalline transition. Similarly, CAMB was effective in protecting secondary 387

proteins of the bacteria during freeze drying and during storage at low aw; while CAB failed to 388

protect the cells. In general, Ca-alginate was not effective in protecting cell envelopes and 389


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secondary proteins of the probiotic bacteria during freeze drying and during storage at low aw or 390

kept in an aluminium foil. An incorporation of mannitol was required to improve stability of cell 391

envelopes and secondary proteins of the cells. 392

Acknowledgment 393

The author DD is grateful to Indonesian Department of Higher Education (DIKTI) for providing 394

financial support. 395

396


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Table 1. Assignment of some bands found in CA, CAM and SA spectra (cm-1) 573 Functional groups CA CAM SA

υOH stretching 3210.3+0.3 3207.2+0.2 3237.2+0.2

-OH deformation 1257.2+0.3 1251.0+0.1 1312.2+0.2

υCOO- sym. stretching 1444.2+0.2 1443.5+0.5 1419.3+0.3

υCOO- asym. stretching 1609.2+0.2 1609.2+0.3 1609.4+0.4

υC-O-C asym. stretching 1160.3+0.4 1162.3+0.3 1166.2+0.3 574 575

576


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Table 2. Assignment of some bands of freshly harvested B. animalis ssp. lactis Bb12 (cm-1) 577 Functional groups Fresh B. animalis ssp. lactis Bb12

υP=O sym stretching 1077.8+0.2

υP=O asym stretching 1240.4+0.5

υN+(CH3)3 asym stretching 969.1+0.2

υCH2 sym stretching 2867.0+0.1

υCH2 asym stretching 2925.9+0.2

υamide II 1541.2+0.3 578


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Table 3. Assignment of some bands of CAB after freeze drying and after storage (cm-1) 579

Functional groups CAB after FD FD CAB (aw 0.07)

FD CAB (aw 0.1)

FD CAB (aw 0.2)

FD CAB (in alu. foil)

υP=O sym stretching 1053.4+0.4 1055.5+0.3 1055.9+0.3 1057.7+0.3 1081.6+0.5υP=O asym stretching 1222.8+0.3 1231.8+0.3 1235.9+0.2 1241.5+0.5 1244.0+0.5υN+(CH3)3 asym stretching 980.2+0.3 996.8+0.3 998.8+0.3 1002.8+0.3 undetectableυCH2 sym stretching 2866.8+0.3 2902.3+0.3 2870.6+0.5 2875.9+0.2 2906.6+0.5υCH2 asym stretching 2924.9+0.3 2947.5+0.5 2944.5+0.5 2946.5+0.5 2963.6+0.5

υamide II 1545.8+0.3 1568.5+0.5 1569.8+0.8 1569.8+0.3 1569.3+0.6 580

581


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Table 4. Assignment of some bands of CAMB after freeze drying and after storage (cm-1) 582 Functional groups CAMB after FD CAMB 0.07 CAMB 0.1 CAMB 0.2 CAMB foil υP=O sym stretching 1043.6+0.5 1043.8+0.2 1042.3+0.3 1043.2+0.3 1047.4+0.4υP=O asym stretching 1218.5+0.5 1226.5+0.5 1225.2+0.3 1226.2+0.3 1227.2+0.3υN+(CH3)3 asym stretching 978.2+0.3 979.8+0.3 983.8+0.3 994.7+0.3 995.9+0.2υCH2 sym stretching 2867.4+0.1 2854.3+0.6 2855.5+0.5 2853.2+0.3 2849.0+0.0υCH2 asym stretching 2923.3+0.2 2920.7+0.6 2921.5+0.6 2920.7+0.6 2931.5+0.5

υamide II 1536.4+0.4 1534.5+0.5 1537.8+0.3 1536.5+0.5 1529.8+0.3

and 1515+0.1 583

584


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585 Figure 1a. CAB after FD 586

587 Figure 1b. Freeze dried CAB after 10 weeks of storage at aw of 0.07 588


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589 Figure 2a. CAMB after FD 590 591

592 Figure 2b. Freeze dried CAMB after 10 weeks of storage at aw of 0.07 593


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594


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Documents

Role of Ca-alginate and mannitol in protecting Bifidobacterium