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Effect of drying methods of microencapsulated Lactobacillus acidophilus and Lactococcus lactis ssp. cremoris on secondary protein structure and glass transition temperature as studied by Fourier transform infrared and differential scanning calorimetry

Open ArchivePublished:January 28, 2013DOI:https://doi.org/10.3168/jds.2012-6058

      Abstract

      Protective mechanisms of casein-based microcapsules containing mannitol on Lactobacillus acidophilus and Lactococcus lactis ssp. cremoris, changes in their secondary protein structures, and glass transition of the microcapsules were studied after spray- or freeze-drying and after 10 wk of storage in aluminum foil pouches containing different desiccants (NaOH, LiCl, or silica gel) at 25°C. An in situ Fourier transform infrared analysis was carried out to recognize any changes in fatty acids (FA) of bacterial cell envelopes, interaction between polar site of cell envelopes and microcapsules, and alteration of their secondary protein structures. Differential scanning calorimetry was used to determine glass transition of microcapsules based on glass transition temperature (Tg) values. Hierarchical cluster analysis based on functional groups of cell envelopes and secondary protein structures was also carried out to classify the microencapsulated bacteria due to the effects of spray- or freeze-drying and storage for 10 wk. The results showed that drying process did not affect FA and secondary protein structures of bacteria; however, those structures were affected during storage depending upon the type of desiccant used. Interaction between exterior of bacterial cell envelopes and microencapsulant occurred after spray- or freeze-drying; however, these structures were maintained after storage in foil pouch containing sodium hydroxide. Method of drying and type of desiccants influenced the level of similarities of microencapsulated bacteria. Desiccants and method of drying affected glass transition, yet no Tg ≤25°C was detected. This study demonstrated that the changes in FA and secondary structures of the microencapsulated bacteria still occurred during storage at Tg above room temperature, indicating that the glassy state did not completely prevent chemical activities.

      Key words

      Introduction

      The use of particular drying methods to preserve probiotic bacteria provides some advantages besides its ease of handling, including low cost of transportation and storage at room temperature. Freeze-drying and spray-drying are 2 common drying methods for preservation of bacteria; however, these have many adverse effects on cell envelopes and secondary protein structures (
      • Leslie S.B.
      • Israeli E.
      • Lighthart B.
      • Crowe J.H.
      • Crowe L.M.
      Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying.
      ;

      Mauerer, A. 2006. Secondary structural changes of spray dried proteins with Fourier transform infrared spectroscopy. Doctoral Diss. Department of Pharmaceutics, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany. http://www.pharmtech.uni-erlangen.de/publications/12_Mauerer_06.pdf.

      ). Microencapsulation technology has been developed to overcome these problems. The application of sodium caseinate-glucose to form a glassy Maillard substance, combined with mannitol, is effective in protecting spray-dried probiotic bacteria (
      • Crittenden R.
      • Weerakkody R.
      • Sanguansri L.
      • Augustin M.
      Synbiotic microcapsules that enhance microbial viability during nonrefrigerated storage and gastrointestinal transit.
      ). Mannitol is excellent in protecting probiotic bacteria during storage and exposure to a simulated gastric environment due to its radical scavenging ability and structural stability in low pH (
      • Efiuvwevwere B.J.O.
      • Gorris L.G.M.
      • Smid E.J.
      • Kets E.P.W.
      Mannitol-enhanced survival of Lactococcus lactis subjected to drying.
      ;
      • Telang C.
      • Yu L.
      • Suryanarayanan R.
      Effective inhibition of mannitol crystallization in frozen solutions by sodium chloride.
      ), in spite of its tendency to crystallize (
      • Izutsu K.
      • Kojima S.
      Excipient crystallinity and its protein-structure stabilizing effect during freeze-drying.
      ).
      Mechanisms of dehydrated bacterial protection by sugars can be explained by water replacement theory (
      • Crowe J.H.
      • Crowe L.M.
      • Carpenter J.F.
      • Rudolph A.S.
      • Winstrom C.A.
      • Spargo B.J.
      • Anchordoguy T.J.
      Interactions of sugars with membranes.
      ) or the formation of amorphous state (
      • Santivarangkna C.
      • Aschenbrenner M.
      • Kulozik U.
      • Foerst P.
      Role of glassy state on stabilities of freeze-dried probiotics.
      ). The Fourier transform infrared (FTIR) technique has been used to investigate the role of sugars in retarding conformational changes of bacterial cell envelopes and proteins (
      • Leslie S.B.
      • Israeli E.
      • Lighthart B.
      • Crowe J.H.
      • Crowe L.M.
      Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying.
      ;
      • Oldenhof H.
      • Wolkers W.F.
      • Fonseca F.
      • Passot S.
      • Marin M.
      Effect of sucrose and maltodextrin on the physical properties and survival of air-dried Lactobacillus bulgaricus: An in situ Fourier transform infrared spectroscopy study.
      ;
      • Santivarangkna C.
      • Naumann D.
      • Kulozik U.
      • Foerst P.
      Protective effects of sorbitol during the vacuum-drying of Lactobacillus helveticus: An FT-IR study.
      ). The wave number alteration indicated that the protective mechanism of cell envelopes of bacteria occurs through sugar interaction with phospholipid headgroups via hydrogen bond (
      • Crowe J.H.
      • Crowe L.M.
      • Carpenter J.F.
      • Rudolph A.S.
      • Winstrom C.A.
      • Spargo B.J.
      • Anchordoguy T.J.
      Interactions of sugars with membranes.
      ;
      • Grdadolnik J.
      • Hadzi D.
      FT infrared and Raman investigation of saccharide -phosphatidylcholine interactions using novel structure probes Spectrochim.
      ).
      • Gauger D.R.
      • Binder H.
      • Vogel A.
      • Selle C.
      • Pohle W.
      Comparative FTIR-spectroscopic studies of the hydration of diphytanoylphosphatidylcholine and -ethanolamine.
      stated that certain levels of water activity (aw) at room temperature contributed to conformational disorder of diphytanoylphosphatidylcholine. Protein conformation was also affected by freeze- and spray-drying (
      • Garzon-Rodriguez W.
      • Koval R.L.
      • Chongprasert S.
      • Krishnan S.
      • Randolph T.W.
      • Warne N.W.
      • Carpenter J.F.
      Optimizing storage stability of lyophilized recombinant human interleukin-11 with disaccharide/hydroxyethylstarch mixtures.
      ;
      • Schüle S.
      • Frieß W.
      • Bechtold-Peters K.
      • Garidel P.
      Conformational analysis of protein secondary structure during spray-drying of antibody/mannitol formulations.
      ); drying process and storage at room temperature at low aw might cause the changes in cell envelopes and secondary structure of proteins of bacteria.
      Apart from molecular interaction between cell envelopes and microencapsulants, the physical state of microcapsule matrix is also crucial for bacterial stability. The extremely high viscosity of dehydrated products in the amorphous state is capable of decreasing molecular mobility reducing adverse chemical reactions; however, this solid state is metastable and strongly depends on the glass transition temperature (Tg). Storage at room temperature above Tg might increase the chance of glass transition (
      • Santivarangkna C.
      • Aschenbrenner M.
      • Kulozik U.
      • Foerst P.
      Role of glassy state on stabilities of freeze-dried probiotics.
      ), in which molecular mobility would increase along with the formation of crystalline state. Glass transition temperature is also influenced by aw of storage: an increase in aw results in a decrease in Tg (
      • Higl B.
      • Kurtmann L.
      • Carlsen C.U.
      • Ratjen J.
      • Forst P.
      • Skibsted L.H.
      • Kulozik U.
      • Risbo J.
      Impact of water activity, temperature, and physical state on the storage stability of Lactobacillus paracasei ssp. paracasei freeze-dried in a lactose matrix.
      ;
      • Kurtmann L.
      • Carlsen C.U.
      • Skibted L.H.
      • Risbo J.
      Water activity-temperature state diagrams of freeze dried L. acidophilus (La-5): Influence of physical state on bacterial survival during storage.
      ). The mechanism of bacterial protection by sugars during dehydration has been established, but the effect of long-term storage at room temperature on the changes in phospholipid bilayers and secondary protein structures of bacterial cells has not. The aims of this study were to ascertain the interaction between cell envelopes of bacteria and encapsulant, as well as to determine the changes in the structure of secondary proteins and to establish Tg and moisture content of microcapsules after spray- or freeze-drying and after 10 wk of storage in aluminum foil pouches containing different desiccants at 25°C. One probiotic bacteria (Lactobacillus acidophilus) and one sensitive lactic acid bacteria (Lactococcus cremoris ssp. lactis) were used as models in this study.

      Materials and Methods

       Lb. acidophilus 2401 and Lc. lactis ssp. cremoris R-704 and Their Cultivation

      Pure cultures of Lactobacillus acidophilus 2401 (Lb. acidophilus) and Lactococcus lactis ssp. cremoris R-704 (Lc. cremoris) were obtained from Victoria University stock culture and were confirmed using Gram staining (
      • Ding W.K.
      • Shah N.P.
      An improved method of microencapsulation of probiotic bacteria for their stability in acidic and bile conditions during storage.
      ). Lactobacillus acidophilus was grown in de Man, Rogosa, and Sharpe broth at 37°C for 18 h (
      • Riveros B.
      • Ferrer J.
      • Borquez R.
      Spray-drying of a vaginal probiotic strain of Lactobacillus acidophilus..
      ), whereas Lc. cremoris was grown in M17 supplemented with 0.5% glucose at 30°C for 18 h (
      • Kimoto H.
      • Nomura M.
      • Kobayashi M.
      • Mizumachi K.
      • Okamoto T.
      Survival of lactococci during passage through mouse digestive tract.
      ); both organisms were subcultured 3 times. The cells were concentrated by centrifuging the broth at 14,000 × g for 15 min at 4°C (
      • Vinderola C.G.
      • Reinheimer J.A.
      Lactic acid starter and probiotic bacteria, a comparative “in vitro” study of probiotic characteristics and biological barrier resistance.
      ). The resultant cell pellet was washed twice with 0.85% of sterilized saline solution and then resuspended in the same solution (10 mL of cell pellet was added by 10 mL of saline solution). The initial population of concentrated bacteria was 3.1 × 1010 cfu/mL for Lb. acidophilus and 1.1 × 1010 cfu/mL for Lc. cremoris.

       Preparation of Microcapsules

      Microencapsulation was performed using an oil-in-water emulsion system comprising vegetable oil (10% wt/vol), sodium caseinate (6% wt/vol), fructooligosaccharides from chicory (2% wt/vol), d-glucose (3% wt/vol), and mannitol (3% wt/vol). All of the materials were from Sigma Aldrich Corp. (St. Louis, MO) except vegetable oil, which was obtained from a local supermarket. The materials were mixed and homogenized using a magnetic stirrer, and were heated at 95°C for 30 min to initiate the Maillard reaction. One-fifth of the concentrated bacteria were incorporated to the cold emulsion system (10°C) before spray- or freeze-drying. The emulsion was spray-dried using a Buchi Mini spray drier (model B290, Bern, Switzerland) with Dehumidifier B296 (humidity 86%; temperature −3°C; Buchi). The outlet temperature was 50°C, hence the inlet temperature was set to 99°C with pump 27% (feeding rate = 7.14 mL/min) for the emulsion system containing Lb. acidophilus, and was set to 80°C with pump 20% (feeding rate = 3.03 mL/min) for the emulsion system containing Lc. cremoris. The powder gathered from the collection vessel was then stored in desiccators. For freeze-drying, frozen microcapsules were loaded into a freeze-drier (model FD-300, Airvac Engineering Pty. Ltd., Dandenong, Australia) set to achieve −13,332.2 Pa of internal pressure before freeze-drying at a temperature of −88°C, with 44 h of primary freeze-drying, and 4 h of secondary freeze-drying. Each of the freeze-dried and spray-dried products (Lb. acidophilus and Lc. cremoris) were placed on Petri-disks and kept in desiccators containing a saturated solution of sodium hydroxide (NaOH; aw = 0.07), a saturated solution of lithium chloride (LiCl; aw = 0.11), or silica gel for 2 wk to reach the equilibrium. Once equilibrium was established, the products were transferred to aluminum foil pouches, and NaOH, LiCl, or silica gel was packed inside a semi-permeable membrane and placed inside the pouch. Controls were stored without desiccant, fresh samples were freshly harvested bacteria after being grown in media for 18 h, and prestorage samples were after freeze drying/after spray drying. Storage at 25°C was carried out for 10 wk; after the end of storage period, samples were kept at −80°C until further analysis.

       Sample Preparation for FTIR Spectroscopy

      Solid sample preparation was carried out according to
      • Izutsu K.
      • Kojima S.
      Excipient crystallinity and its protein-structure stabilizing effect during freeze-drying.
      and
      • Sharma V.K.
      • Kalonia D.S.
      Effect of vacuum-drying on protein-mannitol interactions: The physical state of mannitol and protein structure in the dried state.
      . The powdered sample (10 mg) of dehydrated, microencapsulated bacteria was mixed with 100 mg of dried KBr powder. A transparent pellet of the sample KBr mixture was obtained by pressing the mixture under vacuum at 10 tons of hydraulic pressure. The spectra of microcapsules without bacteria were subtracted from those of samples with bacteria (

      Mauerer, A. 2006. Secondary structural changes of spray dried proteins with Fourier transform infrared spectroscopy. Doctoral Diss. Department of Pharmaceutics, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany. http://www.pharmtech.uni-erlangen.de/publications/12_Mauerer_06.pdf.

      ;
      • Han Y.
      • Jin B.-S.
      • Lee S.-B.
      • Sohn Y.
      • Joung J.-W.
      • Lee J.-H.
      Effects of sugar additives on protein stability of recombinant human serum albumin during lyophilization and storage.
      ). Measurement of spectra of functional groups was carried out at room temperature (∼25°C) using an FTIR combined with infrared solution software (Type 8400S, Shimadzu, Kyoto, Japan). All FTIR spectra were recorded using a resolution of 4 cm−1 and 20 scans. Air spectra were recorded before each experiment to correct background effects for all spectra recorded. Spectra were collected from 3 different batches of samples. Smoothing and normalization of the second derivatives of deconvoluted spectra were carried out to develop clearer separation of complex bands (
      • Santivarangkna C.
      • Wenning M.
      • Foerst P.
      • Kulozik U.
      Damage of cell envelope of Lactobacillus helveticus during vacuum-drying.
      ). Spectra of freshly harvested Lb. acidophilus and Lc. cremoris were used as controls to recognize whether cell envelope and secondary structure of proteins of microencapsulated bacteria experienced a change in frequency. Ten microliters of washed bacterial cell suspension was spread onto the surface of a calcium difluoride window and the spectra of cells were determined after being dehydrated in desiccators containing phosphorus pentoxide (
      • Oldenhof H.
      • Wolkers W.F.
      • Fonseca F.
      • Passot S.
      • Marin M.
      Effect of sucrose and maltodextrin on the physical properties and survival of air-dried Lactobacillus bulgaricus: An in situ Fourier transform infrared spectroscopy study.
      ) to reduce interfering spectra of water. All FTIR measurements were repeated 3 times.

       Determination of State of Cell Envelopes and Secondary Proteins of Microencapsulated Lb. acidophilus and Lc. cremoris Using FTIR Spectroscopy

      Wavenumbers (cm−1) of molecular vibrations were detected based on the functional groups of cell envelopes and secondary protein structures of the 2 bacteria. Hydrophobic sites consisted of CH3 (asymmetric and symmetric vibration) of FA in the range of 2950 to 2990 and 2860 to 2890 cm−1, respectively (

      Davis, R., and L. J. Mauer. 2010. Fourier transform infrared (FT-IR) spectroscopy: A rapid tool for detection and analysis of foodborne pathogenic bacteria. Pages 1582–1594 in Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. A. Mendez-Vilas, ed. Formatex Research Center, Badajoz, Spain. http://www.formatex.info/microbiology2/1582-1594.pdf.

      ). Hydrophilic sites consisted of choline group, N+(CH3)3 asymmetric stretching vibration at ∼970 cm−1 (
      • Popova A.V.
      • Hincha D.K.
      Intermolecular interactions in dry and rehydrated pure and mixed bilayers of phosphatidylcholine and digalactosyldiacylglycerol: A Fourier transform infrared spectroscopy study.
      ), and P=O symmetric stretching of phosphodiesters in phospholipids at ∼1080 cm−1 (

      Davis, R., and L. J. Mauer. 2010. Fourier transform infrared (FT-IR) spectroscopy: A rapid tool for detection and analysis of foodborne pathogenic bacteria. Pages 1582–1594 in Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. A. Mendez-Vilas, ed. Formatex Research Center, Badajoz, Spain. http://www.formatex.info/microbiology2/1582-1594.pdf.

      ). Polar/apolar site of C=O stretching of lipid ester was detected at 1715 to 1740 cm−1 (
      • Santivarangkna C.
      • Naumann D.
      • Kulozik U.
      • Foerst P.
      Protective effects of sorbitol during the vacuum-drying of Lactobacillus helveticus: An FT-IR study.
      ). Secondary proteins were detected in the wide range of 1620 to 1700 cm−1 (amide I) reflecting β-sheet, α-helix, β-turn, or unordered structures (
      • Kong J.
      • Yu S.
      FTIR spectroscopic analysis of protein secondary structures.
      ).

       Cluster Analysis

      Hierarchical cluster analysis was carried out according to the modified method of
      • Dziuba B.
      • Babuchowski A.
      • Naecz D.
      • Niklewicz M.
      Identification of lactic acid bacteria using FTIR spectroscopy and cluster analysis.
      . The Ward's algorithm method (
      • Lipkus A.H.
      • Lenk T.J.
      • Chittur K.K.
      • Gendreau R.M.
      Cluster analysis of protein Fourier transform infrared spectra.
      ) was used to analyze the similarities between bacterial spectra (for each Lb. acidophilus and Lc. cremoris) after drying and after 10 wk of storage at room temperature using various desiccants. The fresh bacteria were used as a control.

       Differential Scanning Calorimetry and Residual Moisture Content

      Differential scanning calorimetry (DSC) was performed using a PerkinElmer DSC 7 (PerkinElmer, San Jose, CA) to determine Tg of the samples; samples (8–12 mg) were pressed in standard sealed aluminum DSC pans. Pressed samples were scanned from 5 to 170°C at a heating rate of 5°C/min (
      • Zimeri J.E.
      • Kokini J.L.
      The effect of moisture content on the crystallinity and glass transition temperature of inulin.
      ); measurements were carried out in duplicate. Glass transition temperature was obtained from the temperature of the midpoint of the change in heat capacity scanned at 10°C/min as suggested by
      • Kalichevsky M.T.
      • Blanshard J.M.V.
      A study of the effect of water on the glass transition of 1:1 mixtures of amylopectin, casein and gluten using DSC and DMTA.
      . Residual moisture content of spray- or freeze-dried products was determined gravimetrically at 105°C (
      • Mauer L.J.
      • Smith D.E.
      • Labuza T.P.
      Water vapor permeability, mechanical, and structural properties of edible β-casein films.
      ;
      • Lu J.
      • Wang X.-J.
      • Liu Y.-X.
      • Ching C.-B.
      Thermal and FTIR investigation of freeze-dried protein-excipient mixtures.
      ).

      Results and Discussion

       Cell Envelopes and Secondary Protein Structures of Microencapsulated Bacteria

      The second derivative of spectra of cell envelopes of fresh and microencapsulated Lb. acidophilus and Lc. lactis after spray- or freeze-drying and subsequent storage are shown in Tables 1 and 2 2, respectively. The N+(CH3)3 asymmetric stretching of choline of fresh Lb. acidophilus and Lc. lactis were indicated at 957 and 947 cm−1, respectively. Frequencies of C–H asymmetric and symmetric stretching vibration of FA of cell envelopes of fresh Lb. acidophilus were at ∼2963 and ∼2882 cm−1, respectively; whereas those of fresh Lc. lactis were at ∼2964 and ∼2883 cm−1, respectively. A band of 1768 to 1776 cm−1 indicated C=O located in interface between the polar site of headgroups and the apolar site of tailgroups of phospholipid bilayers (

      Davis, R., and L. J. Mauer. 2010. Fourier transform infrared (FT-IR) spectroscopy: A rapid tool for detection and analysis of foodborne pathogenic bacteria. Pages 1582–1594 in Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. A. Mendez-Vilas, ed. Formatex Research Center, Badajoz, Spain. http://www.formatex.info/microbiology2/1582-1594.pdf.

      ). This functional group is dependent on a hydrogen bond; a decrease in frequencies indicated a stronger C=OH–O bonding (dotted bond = hydrogen bond) interaction (
      • Cacela C.
      • Hincha D.K.
      Low amounts of sucrose are sufficient to depress the phase transition temperature of dry phosphatidylcholine, but not for lyoprotection of liposomes.
      ). Frequencies at 1075 and 1073 cm−1 indicated the vibration of P=O symmetric of fresh Lb. acidophilus and Lc. lactis, respectively.
      Frequency increase in C–H frequencies and asymmetric and symmetric stretching of CH3 of FA of cell envelopes of spray- or freeze-dried Lb. acidophilus were shown in Table 1. A peak alteration of C–H asymmetric of Lb. acidophilus was detected after freeze- and spray-drying from 2963 (fresh cells) to 2972 and 2968, respectively. Storage in a foil pouch containing silica gel appeared less effective than storage in pouches containing NaOH or LiCl, as reflected by the shift to higher wavenumbers, such as 2987 for freeze-dried Lb. acidophilus and 2967 for spray-dried Lb. acidophilus, along with peak broadening. Conversely, no obvious alteration was detected of C–H frequency of microencapsulated Lc. cremoris during storage at low aw (NaOH and LiCl desiccants) compared with that of freshly harvested Lc. cremoris (Table 2). However, spray-dried Lc. cremoris kept in foil pouch without using desiccant (control), as well as freeze-dried Lc. cremoris kept in foil pouch containing silica gel or the control (freeze-dried Lc. cremoris without desiccant), demonstrated an alteration to the higher frequencies. Similar behavior was also observed of peak alteration of C–H symmetric of Lb. acidophilus and Lc. cremoris due to drying and storage at different aw (
      • Gauger D.R.
      • Binder H.
      • Vogel A.
      • Selle C.
      • Pohle W.
      Comparative FTIR-spectroscopic studies of the hydration of diphytanoylphosphatidylcholine and -ethanolamine.
      ).
      Table 1Second derivative of spectra of cell envelopes of microencaspulated Lactobacillus acidophilus (La; means ± SD)
      CH3 asym=CH3 asymmetric stretching vibration of FA; CH3 sym=CH3 symmetric stretching vibration of FA; N+(CH3)3 asym=N+(CH3)3 asymmetric stretching vibration of choline group; P=O sym=P=O symmetric stretching vibration of phosphate group of phospholipids; C=O=C=O stretching vibration of carboxylic ester.
      Functional group
      After FD/after SD=microencapsulated La after freeze drying/spray drying; NaOH=microencapsulated La (under FD/SD) after storage in foil pouch containing NaOH as desiccant; LiCl=microencapsulated La (under FD/SD) after storage in foil pouch containing LiCl as desiccant; silica gel=microencapsulated La (under FD/SD) after storage in foil pouch containing silica gel as desiccant; control=microencapsulated La (under FD/SD) after storage in foil pouch without desiccant; fresh=freshly harvested La after being grown in the medium for 18h.
      Freeze-dried (FD)Spray-dried (SD)
      After FDNaOHLiClSilica gelControlAfter SDNaOHLiClSilica gelControlFresh
      CH3 asym2,972.2 ± 0.32,971.7 ± 0.32,976.5 ± 0.52,986.7 ± 0.32,988.5 ± 0.52,967.5 ± 0.52,966.3 ± 0.32,966.5 ± 0.52,967.2 ± 0.32,978.0 ± 0.52,963.2 ± 0.3
      CH3 sym2,884.0 ± 0.52,886.0 ± 0.52,887.0 ± 0.52,888.8 ± 0.32,890.5 ± 0.52,888.5 ± 0.52,889.1 ± 0.22,898.5 ± 0.52,900.5 ± 0.52,901.8 ± 0.82,882.2 ± 0.3
      N+(CH3)3 asym958.2 ± 0.3957.7 ± 0.8966.5 ± 0.5968.5 ± 0.5970.6 ± 0.4968.7 ± 0.3967.5 ± 0.5967.4 ± 0.4972.5 ± 0.5974.5 ± 0.5957.3 ± 0.2
      P=O sym1,047.1 ± 0.21,044.5 ± 0.51,055.5 ± 0.51,058.1 ± 0.21,058.5 ± 0.51,048.2 ± 0.31,047.0 ± 0.41,057.4 ± 0.41,057.5 ± 0.51,058.7 ± 0.31,075.2 ± 0.3
      C=O1,747.5 ± 0.51,747.3 ± 0.31,748.0 ± 0.51,749.2 ± 0.31,750.5 ± 0.51,716.8 ± 0.81,717.5 ± 0.51,721.5 ± 0.51,722.7 ± 0.61,735.5 ± 0.51,767.8 ± 0.3
      1 CH3 asym = CH3 asymmetric stretching vibration of FA; CH3 sym = CH3 symmetric stretching vibration of FA; N+(CH3)3 asym = N+(CH3)3 asymmetric stretching vibration of choline group; P=O sym = P=O symmetric stretching vibration of phosphate group of phospholipids; C=O = C=O stretching vibration of carboxylic ester.
      2 After FD/after SD = microencapsulated La after freeze drying/spray drying; NaOH = microencapsulated La (under FD/SD) after storage in foil pouch containing NaOH as desiccant; LiCl = microencapsulated La (under FD/SD) after storage in foil pouch containing LiCl as desiccant; silica gel = microencapsulated La (under FD/SD) after storage in foil pouch containing silica gel as desiccant; control = microencapsulated La (under FD/SD) after storage in foil pouch without desiccant; fresh = freshly harvested La after being grown in the medium for 18 h.
      Table 2Second derivative of spectra of cell envelopes of microencaspulated Lactococcus lactis ssp. cremoris (Lc; means ± SD)
      CH3 asym=CH3 asymmetric stretching vibration of FA; CH3 sym=CH3 symmetric stretching vibration of FA; N+(CH3)3 asym=N+(CH3)3 asymmetric stretching vibration of choline group; P=O sym=P=O symmetric stretching vibration of phosphate group of phospholipids; C=O=C=O stretching vibration of carboxylic ester.
      Functional group
      After FD/after SD=microencapsulated Lc after freeze drying/spray drying; NaOH=microencapsulated Lc (under FD/SD) after storage in foil pouch containing NaOH as desiccant; LiCl=microencapsulated Lc (under FD/SD) after storage in foil pouch containing LiCl as desiccant; silica gel=microencapsulated Lc (under FD/SD) after storage in foil pouch containing silica gel as desiccant; control=microencapsulated Lc (under FD/SD) after storage in foil pouch without desiccant; fresh=freshly harvested Lc after being grown in the medium for 18h.
      Freeze-dried (FD)Spray-dried (SD)Fresh
      After FDNaOHLiClSilica gelControlAfter SDNaOHLiClSilica gelControl
      CH3 asym2,965.3 ± 0.22,964.2 ± 0.32,965.5 ± 0.42,985.5 ± 0.52,988.8 ± 0.22,963.0 ± 0.22,965.2 ± 0.22,964.5 ± 0.52,965.2 ± 0.32,973.2 ± 0.22,964.1 ± 0.2
      CH3 sym2,883.2 ± 0.22,881.0 ± 0.22,887.0 ± 0.52,895.9 ± 0.22,904.0 ± 0.62,883.0 ± 0.32,888.0 ± 0.12,900.7 ± 0.42,900.4 ± 0.42,901.3 ± 0.32,883.2 ± 0.3
      N+(CH3)3 asym984.2 ± 0.2982.2 ± 0.3989.0 ± 0.3995.9 ± 0.3998.0 ± 0.3950.3 ± 0.3950.2 ± 0.2952.3 ± 0.3952.4 ± 0.4952.7 ± 0.6947.1 ± 0.1
      P=O sym1,045.1 ± 0.11,055.0 ± 0.41,055.1 ± 0.31,055.9 ± 0.21,057.2 ± 0.21,056.0 ± 0.31,055.2 ± 0.31,056.0 ± 0.21,056.9 ± 0.21,057.3 ± 0.31,073.1 ± 0.1
      C=O1,723.2 ± 0.21,720.5 ± 0.51,744.0 ± 0.31,744.0 ± 0.21,746.2 ± 0.21,729.2 ± 0.31,741.5 ± 0.51,743.3 ± 0.31,743.9 ± 0.31,744.0 ± 0.51,776.2 ± 0.3
      1 CH3 asym = CH3 asymmetric stretching vibration of FA; CH3 sym = CH3 symmetric stretching vibration of FA; N+(CH3)3 asym = N+(CH3)3 asymmetric stretching vibration of choline group; P=O sym = P=O symmetric stretching vibration of phosphate group of phospholipids; C=O = C=O stretching vibration of carboxylic ester.
      2 After FD/after SD = microencapsulated Lc after freeze drying/spray drying; NaOH = microencapsulated Lc (under FD/SD) after storage in foil pouch containing NaOH as desiccant; LiCl = microencapsulated Lc (under FD/SD) after storage in foil pouch containing LiCl as desiccant; silica gel = microencapsulated Lc (under FD/SD) after storage in foil pouch containing silica gel as desiccant; control = microencapsulated Lc (under FD/SD) after storage in foil pouch without desiccant; fresh = freshly harvested Lc after being grown in the medium for 18 h.
      The IR frequency of ∼2955 and of ∼2880 indicates C–H stretching of –CH3 in FA of cell envelopes of bacteria (

      Davis, R., and L. J. Mauer. 2010. Fourier transform infrared (FT-IR) spectroscopy: A rapid tool for detection and analysis of foodborne pathogenic bacteria. Pages 1582–1594 in Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. A. Mendez-Vilas, ed. Formatex Research Center, Badajoz, Spain. http://www.formatex.info/microbiology2/1582-1594.pdf.

      ); however, it depends on the bacterial species and strains. An upshift in C–H frequencies indicated FA conformational changes from lyotropic gel into liquid crystalline phase (
      • Goodrich R.P.
      • Crowe J.H.
      • Crowe L.M.
      • Baldeschwieler J.D.
      Alterations in membrane surfaces induced by attachment of carbohydrates.
      ;
      • Grdadolnik J.
      • Hadzi D.
      FT infrared and Raman investigation of saccharide -phosphatidylcholine interactions using novel structure probes Spectrochim.
      ;
      • Popova A.V.
      • Hincha D.K.
      Intermolecular interactions in dry and rehydrated pure and mixed bilayers of phosphatidylcholine and digalactosyldiacylglycerol: A Fourier transform infrared spectroscopy study.
      ,
      • Popova A.V.
      • Hincha D.
      Effects of cholesterol on dry bilayers: interactions between phosphatidylcholine unsaturation and glycolipid or free sugar.
      ). This occurrence is known as chain melting, which could be induced by the change in water content (
      • Gauger D.R.
      • Selle C.
      • Fritzsche H.
      • Pohle W.
      Chain-length dependence of the hydration properties of saturated phosphatidylcholine as revealed by FTIR spectroscopy.
      ). The intermediate level of hydration, such as relative humidity of 20% (aw = 0.22), causes a steep increase in frequencies reflecting conformational disorder of the acyl chains (
      • Gauger D.R.
      • Binder H.
      • Vogel A.
      • Selle C.
      • Pohle W.
      Comparative FTIR-spectroscopic studies of the hydration of diphytanoylphosphatidylcholine and -ethanolamine.
      ). In our study, the rehydration phenomena could be due to ineffectiveness of silica gel as an adsorbent (aw microcapsules = 0.28), hence, the moisture adsorption from environment by the microcapsules can take place; the presence of water causes adverse chemical activities (
      • Labuza T.P.
      Moisture sorption: Practical aspects of isotherm measurement and use.
      ).
      The P=O frequencies of microencapsulated Lb. acidophilus after freeze- and spray-drying were 1047 and 1048, respectively; whereas those after 10 wk of storage were in the range of 1044 to 1059, depending upon the desiccant type placed in the foil pouch (Table 1a). A slight increase in frequencies of P=O was detected when the freeze- or spray-dried Lb. acidophilus were kept in foil pouch containing silica gel. It showed similar frequency with that kept in foil pouch without desiccant as control. However, all of the P=O symmetric frequencies were lower than that of freshly harvested Lb. acidophilus (1075). Similarly, an interaction of phospholipid bilayers of Lc. cremoris with the polar group of microencapsule materials during storage appears depending on the presence of moisture (Table 1b). The frequencies of P=O symmetric of Lc. cremoris after freeze- and spray-drying were 1045 and 1056, respectively, whereas those of Lc. cremoris after subsequent storage were between 1055 and 1057.
      Decrease in P=O wavenumbers in our findings was in agreement with that of
      • Leslie S.B.
      • Israeli E.
      • Lighthart B.
      • Crowe J.H.
      • Crowe L.M.
      Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying.
      ,
      • Oldenhof H.
      • Wolkers W.F.
      • Fonseca F.
      • Passot S.
      • Marin M.
      Effect of sucrose and maltodextrin on the physical properties and survival of air-dried Lactobacillus bulgaricus: An in situ Fourier transform infrared spectroscopy study.
      , and
      • Santivarangkna C.
      • Naumann D.
      • Kulozik U.
      • Foerst P.
      Protective effects of sorbitol during the vacuum-drying of Lactobacillus helveticus: An FT-IR study.
      . In the dehydrated form, an interaction takes place between molecules of sugars and the polar site of lipids, which decreases the chance of lateral lipid movement (
      • van den Bogaart G.
      • Hermans N.
      • Krasnikov V.
      • Vries A.H.d.
      • Poolman B.
      On the decrease in lateral mobility of phospholipids by sugars.
      ). The role of sugars to replace water during dehydration is important in cell envelope protection mechanism (
      • Goodrich R.P.
      • Crowe J.H.
      • Crowe L.M.
      • Baldeschwieler J.D.
      Alterations in membrane surfaces induced by attachment of carbohydrates.
      ). This theory might explain the relationship between the stability of FA of tailgroups of cell envelopes of Lb. acidophilus and Lc. cremoris kept at low aw (using NaOH, LiCl, or silica gel) and the interaction of microcapsule substances with lipid headgroups. In terms of the protection effect of microencapsulant on phospholipid bilayers, one possible explanation might come from the fortification of mannitol in the formulation. Glucose interacted with proteins (caseins) through formation of Maillard complex substances during microcapsule preparation (
      • Crittenden R.
      • Weerakkody R.
      • Sanguansri L.
      • Augustin M.
      Synbiotic microcapsules that enhance microbial viability during nonrefrigerated storage and gastrointestinal transit.
      ) via interaction between glucose carbonyl groups and primary amino groups of proteins (
      • Blei I.
      • Odian G.
      Organic and Biochemistry – Connecting chemistry to your life.
      ); thus, the chance of glucose to interact directly with P=O of lipid headgroups might be lower than mannitol. Mannitol, which is not able to take a part in Maillard formation, might have more chance to interact with polar surface of phospholipid bilayers. The interaction could be through mannitol's role as a proton donor; hence, a strong hydrogen bond was formed (
      • Grdadolnik J.
      • Hadzi D.
      FT infrared and Raman investigation of saccharide -phosphatidylcholine interactions using novel structure probes Spectrochim.
      ) via sugar hydroxyl–lipid headgroups (
      • Ricker J.V.
      • Tsvetkova N.M.
      • Wolkers W.F.
      • Leidy C.
      • Tablin F.
      • Longo M.
      • Crowe J.H.
      Trehalose maintains phase separation in an air-dried binary lipid mixture.
      ).
      The N+(CH3)3 asymmetric stretching vibration of the choline terminal of spray- and freeze-dried Lb. acidophilus after storage, as well as that of spray-dried and freeze-dried Lc. cremoris, is also demonstrated in Tables 1 and 2, respectively. All of the frequencies were higher than that of freshly harvested bacteria (957 for Lb. acidophilus and 947 for Lc. cremoris). The N+(CH3)3 of spray-dried Lb. acidophilus demonstrated a higher frequencies than that of freeze-dried Lb. acidophilus. The N+(CH3)3 of freeze-dried Lc. cremoris was commonly higher than that of spray-dried Lc. cremoris, which might reflect a stronger interaction with sugars. In addition, storage of spray- or freeze-dried Lc. cremoris in silica gel or without any desiccant showed higher frequencies, which could be due to moisture adsorption from surroundings. The use of silica gel as a desiccant increased wavenumbers indicating an interference of moisture from the environment.
      Our study showed that frequencies of asymmetric N+(CH3)3 stretching vibration of freeze- or spray-dried Lb. acidophilus and Lc. cremoris were higher than that of freshly harvested ones. This might be due to dipolar interaction between choline functional groups and sugars (
      • Popova A.V.
      • Hincha D.K.
      Intermolecular interactions in dry and rehydrated pure and mixed bilayers of phosphatidylcholine and digalactosyldiacylglycerol: A Fourier transform infrared spectroscopy study.
      ). However, storage in silica gel showed higher wavenumbers, which might be related to the ineffectiveness of the desiccant; therefore, the surrounding moisture could be adsorbed and increase the wavenumbers slightly. Both sugars and moisture interaction result in almost similar peak alteration (
      • Cacela C.
      • Hincha D.K.
      Low amounts of sucrose are sufficient to depress the phase transition temperature of dry phosphatidylcholine, but not for lyoprotection of liposomes.
      ). Our results were in agreement with that of
      • Grdadolnik J.
      • Hadzi D.
      FT infrared and Raman investigation of saccharide -phosphatidylcholine interactions using novel structure probes Spectrochim.
      and
      • Popova A.V.
      • Hincha D.K.
      Intermolecular interactions in dry and rehydrated pure and mixed bilayers of phosphatidylcholine and digalactosyldiacylglycerol: A Fourier transform infrared spectroscopy study.
      , who demonstrated a shift to higher wavenumbers due to an interaction between sugars, such as glycerol or glucose, and polar site of phosphatidylcholine. A possible mechanism of frequency alteration of N+(CH3)3 stretching vibration could be explained by the torsional angles theory as proposed by
      • Grdadolnik J.
      • Hadzi D.
      FT infrared and Raman investigation of saccharide -phosphatidylcholine interactions using novel structure probes Spectrochim.
      . The authors suggested that the presence of sugars such as sorbitol or moisture alters rotamer population, resulting in an ap torsion angle increase along with an sc torsion angle decrease, thus indicating an increase in H-bond. This might explain the difference of choline frequencies in our study due to the storage at low aw using various desiccants.
      The C=O double bond of microencapsulated Lb. acidophilus and Lc. cremoris was also demonstrated in Tables 1 and 2, respectively. The frequencies of C=O were lower after freeze- or spray-drying, and after storage in a foil pouch containing NaOH or LiCl, compared to the C=O of the fresh bacteria. Trend of peak alteration of C=O was almost similar to that of P=O, as mentioned above. The wavenumber of C=O stretching vibration of ester carbonyl group as a part of polar/apolar interfacial of bacterial pospholipids varies between 1716 and 1750 cm−1 (
      • Erukhimovitch V.
      • Pavlov V.
      • Talyshinsky M.
      • Souprun Y.
      • Huleihel M.
      FTIR microscopy as a method for identification of bacterial and fungal infections.
      ;
      • Santivarangkna C.
      • Naumann D.
      • Kulozik U.
      • Foerst P.
      Protective effects of sorbitol during the vacuum-drying of Lactobacillus helveticus: An FT-IR study.
      ). Nonhydrogen-bonded or weak and strong hydrogen-bonded C=O were indicated by higher and lower frequencies, respectively (
      • Lewis R.N.A.H.
      • McElhaney R.N.
      The structure and organization of phospholipid bilayers as revealed by infrared spectroscopy.
      ). Decrease in C=O frequencies of freeze- or spray-dried bacteria could be due to water removal along with replacement by sugars (
      • Santivarangkna C.
      • Naumann D.
      • Kulozik U.
      • Foerst P.
      Protective effects of sorbitol during the vacuum-drying of Lactobacillus helveticus: An FT-IR study.
      ).
      Amide I band is mainly related to C=O stretching vibration (70–85%) and C–N group (10–20%). It showed the secondary structure of peptide components such as α-helix, β-sheet and β-turn (

      Gallagher, W. 2011. FTIR analysis of protein structure. http://download.bioon.com.cn/view/upload/201110/10233820_7954.pdf. Retrieved on April 27, 2011.

      ); determination of secondary protein structures was based on
      • Chirgadze Y.N.
      • Brazhnikov E.V.
      • Nevskaya N.A.
      Intramolecular distortion of the alpha-helical structure of polypeptides.
      ,
      • Kong J.
      • Yu S.
      FTIR spectroscopic analysis of protein secondary structures.
      , and
      • Mobili P.
      • Londero A.
      • Maria T.M.R.
      • Eusebio M.E.S.
      • Antoni G.L.D.
      • Fausto R.
      • Gomez-Zavaglia A.
      Characterization of S-layer proteins of Lactobacillus by FTIR spectroscopy and differential scanning calorimetry.
      . Elements of amide I reflecting secondary protein structures of spray-dried Lc. cremoris, freeze-dried Lc. cremoris, spray-dried Lb. acidophilus, and freeze-dried Lb. acidophilus are shown in Table 3. Wave numbers of Lb. acidophilus after freeze- or spray-drying, as well as freeze-dried Lb. acidophilus kept in foil pouch containing NaOH and spray-dried Lb. acidophilus kept in a pouch containing either NaOH or LiCl, indicated the presence of α-helix (from 1649 to 1657 cm−1); whereas storage using silica gel caused the conformational changes from α-helix to β-sheet or β-turn. On the contrary, Lc. cremoris appeared more sensitive to drying processes as indicated by the formation of no-order and β-sheet after freeze-drying, whereas the α-helix structure of Lc. cremoris was maintained after spray-drying. However, storage at room temperature for a long period affected the secondary protein structures of microencapsulated Lc. cremoris, as indicated by frequency changes along with the presence of a new peak (Table 3). For instance, spray-dried Lc. cremoris kept in a foil pouch containing NaOH showed 2 peaks at 1646 and 1684, whereas freeze-dried Lc. cremoris under the same conditions showed peaks at 1650 and 1689 (frequency of fresh Lc. cremoris = 1651).
      Table 3Assignment of components of secondary protein structures of microencapsulated Lactobacillus acidophilus and Lactococcus lactis ssp. cremoris (means ± SD).
      Species and treatment
      After FD/after SD=microencapsulated bacteria after freeze drying/spray drying; FD/SD: NaOH=microencapsulated bacteria (under FD/SD) after storage in foil pouch containing NaOH as desiccant; FD/SD: LiCl=microencapsulated bacteria (under FD/SD) after storage in foil pouch containing LiCl as desiccant; FD/SD: silica gel=microencapsulated bacteria (under FD/SD) after storage in foil pouch containing silica gel as desiccant; FD/SD: control=microencapsulated bacteria (under FD/SD) after storage in foil pouch without desiccant; fresh=freshly harvested bacteria after being grown in the medium for 18h.
      Wavenumber (cm−1)Assignment
      Lb. acidophilus
       After FD1,650.3 ± 0.3α-Helix
       FD: NaOH1,650.2 ± 0.3α-Helix
       FD: LiCl1,624.2 ± 0.2β-Sheet
       FD: silica gel1,629.3 ± 0.3β-Sheet
      1,667.2 ± 0.3β-Turn
       FD: control1,629.9 ± 0.4β-Sheet
      1,668.0 ± 0.2β-Turn
       After SD1,649.2 ± 0.2α-Helix
       SD: NaOH1,655.1 ± 0.1α-Helix
       SD: LiCl1,656.3 ± 0.3α-Helix
       SD: silica gel1,638.0 ± 0.3β-Sheet
      1,672.9 ± 0.1β-Sheet
       SD: control1,633.0 ± 0.2β-Sheet
      1,670.0 ± 0.5β-Turn
       Fresh1,654.2 ± 0.1α-Helix
      Lc. cremoris
       After FD1,649.8 ± 0.5No order
      1,692.0 ± 0.2β-Sheet
       FD: NaOH1,649.9 ± 0.2No order
      1,689.0 ± 0.2β-Sheet
       FD: LiCl1,650.1 ± 0.2α-Helix
      1,690.0 ± 0.2β-Sheet
       FD: silica gel1,642.0 ± 0.2No order
      1,689.5 ± 0.5β-Turn
       FD: control1,640.1 ± 0.2β-Sheet
      1,688.4 ± 0.5β-Turn
       After SD1,656.1 ± 0.2α-Helix
       SD: NaOH1,646.0 ± 0.2No order
      1,684.4 ± 0.4β-Turn
       SD: LiCl1,644.2 ± 0.2No order
      1,671.2 ± 0.2β-Turn
       SD: silica gel1,648.2 ± 0.2No order
      1,688.3 ± 0.3β-Turn
       SD: control1,647.1 ± 0.1No order
      1,683.2 ± 0.2β-Turn
       Fresh1,651.1 ± 0.1α-Helix
      1 After FD/after SD = microencapsulated bacteria after freeze drying/spray drying; FD/SD: NaOH = microencapsulated bacteria (under FD/SD) after storage in foil pouch containing NaOH as desiccant; FD/SD: LiCl = microencapsulated bacteria (under FD/SD) after storage in foil pouch containing LiCl as desiccant; FD/SD: silica gel = microencapsulated bacteria (under FD/SD) after storage in foil pouch containing silica gel as desiccant; FD/SD: control = microencapsulated bacteria (under FD/SD) after storage in foil pouch without desiccant; fresh = freshly harvested bacteria after being grown in the medium for 18 h.
      Table 3 demonstrated that the structure of secondary proteins of microencapsulated bacteria was retained after dehydration. This result was in agreement with that of
      • Oldenhof H.
      • Wolkers W.F.
      • Fonseca F.
      • Passot S.
      • Marin M.
      Effect of sucrose and maltodextrin on the physical properties and survival of air-dried Lactobacillus bulgaricus: An in situ Fourier transform infrared spectroscopy study.
      and
      • Garzon-Rodriguez W.
      • Koval R.L.
      • Chongprasert S.
      • Krishnan S.
      • Randolph T.W.
      • Warne N.W.
      • Carpenter J.F.
      Optimizing storage stability of lyophilized recombinant human interleukin-11 with disaccharide/hydroxyethylstarch mixtures.
      . These authors stated that the use of sugars, such as sucrose, maltodextrin, or disaccharides, combined with starch maintains the native-like secondary protein components after freeze-drying. In addition,
      • Schüle S.
      • Frieß W.
      • Bechtold-Peters K.
      • Garidel P.
      Conformational analysis of protein secondary structure during spray-drying of antibody/mannitol formulations.
      found that mannitol at relatively low concentration protected antibodies during spray-drying with inlet and outlet temperatures of 90°C and 50°C, which was similar to our spray-drying procedure. Similar results were demonstrated by
      • Tzannis S.T.
      • Prestrelski S.J.
      Activity-stability considerations of trypsinogen during spray-drying: Effects of sucrose.
      and
      • Liao Y.-H.
      • Brown M.B.
      • Nazir T.
      • Quader A.
      • Martin G.P.
      Effects of sucrose and trehalose on the preservation of the native structure of spray-dried lysozyme.
      using different sugars as the protein protectant. The protective mechanism during freeze- or spray-drying of protein models in those studies is taken place through water replacement via H-bond (
      • Maury M.
      • Murphy K.
      • Kumar S.
      • Mauerer A.
      • Lee G.
      Spray-drying of proteins: effects of sorbitol and trehalose on aggregation and FT-IR amide I spectrum of an immunoglobulin G.
      ); hence preservation of protein folding occurs (
      • Garzon-Rodriguez W.
      • Koval R.L.
      • Chongprasert S.
      • Krishnan S.
      • Randolph T.W.
      • Warne N.W.
      • Carpenter J.F.
      Optimizing storage stability of lyophilized recombinant human interleukin-11 with disaccharide/hydroxyethylstarch mixtures.
      ). The protective mechanism might be different with ours, as bacterial proteins could be embedded on the surface or within the cell. In our study, an encapsulant containing mannitol and glucose interacted with the polar site of phospholipid bilayers, thus protection effect on proteins from dehydration should be indirect.
      In terms of storage,
      • Garzon-Rodriguez W.
      • Koval R.L.
      • Chongprasert S.
      • Krishnan S.
      • Randolph T.W.
      • Warne N.W.
      • Carpenter J.F.
      Optimizing storage stability of lyophilized recombinant human interleukin-11 with disaccharide/hydroxyethylstarch mixtures.
      found that perturbation of freeze-dried proteins takes place during storage at a temperature of 40°C for 6 mo with no aw adjusted. The change of protein structures was indicated by disappearing of band of α-helix along with the extension of the β-sheet. Their findings might be in agreement with our study regarding the role of low aw in increasing protein stability, however, the difference in storage temperature should be considered. In addition,
      • Maury M.
      • Murphy K.
      • Kumar S.
      • Mauerer A.
      • Lee G.
      Spray-drying of proteins: effects of sorbitol and trehalose on aggregation and FT-IR amide I spectrum of an immunoglobulin G.
      revealed that amide I spectra of spray-dried immunoglobulin G protected by sorbitol and trehalose sealed under dry N2 was not altered after 12 mo at 25°C.
      Hierarchical cluster analysis was used determine the similarities of bacterial spectra and to categorize them into a cluster (
      • Dziuba B.
      • Babuchowski A.
      • Naecz D.
      • Niklewicz M.
      Identification of lactic acid bacteria using FTIR spectroscopy and cluster analysis.
      ). Second-derivative spectra are commonly used for bacterial classification. A second-derivative spectrum helps in separation and resolution of bacterial spectra; thus classification can be done more easily (

      Davis, R., and L. J. Mauer. 2010. Fourier transform infrared (FT-IR) spectroscopy: A rapid tool for detection and analysis of foodborne pathogenic bacteria. Pages 1582–1594 in Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. A. Mendez-Vilas, ed. Formatex Research Center, Badajoz, Spain. http://www.formatex.info/microbiology2/1582-1594.pdf.

      ). Ward's algorithm is a frequent method for cluster analysis algorithms to develop a dendrogram (
      • Lipkus A.H.
      • Lenk T.J.
      • Chittur K.K.
      • Gendreau R.M.
      Cluster analysis of protein Fourier transform infrared spectra.
      ). A dendrogram, or tree diagram, is commonly used to depict the clusters calculated by clustering algorithm (

      Davis, R., and L. J. Mauer. 2010. Fourier transform infrared (FT-IR) spectroscopy: A rapid tool for detection and analysis of foodborne pathogenic bacteria. Pages 1582–1594 in Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. A. Mendez-Vilas, ed. Formatex Research Center, Badajoz, Spain. http://www.formatex.info/microbiology2/1582-1594.pdf.

      ). Fourier transform infrared bands were used to classify 41 strains of 6 lactobacilli isolated from cheese using the hierarchical cluster analysis method (
      • Savic D.
      • Jokovic N.
      • Topisirovic L.
      Multivariate statistical methods for discrimination of lactobacilli based on their FTIR spectra.
      ). More specifically, the use of cluster analysis has been developed to categorize characteristics of the lactobacilli S-layer (
      • Mobili P.
      • Londero A.
      • Maria T.M.R.
      • Eusebio M.E.S.
      • Antoni G.L.D.
      • Fausto R.
      • Gomez-Zavaglia A.
      Characterization of S-layer proteins of Lactobacillus by FTIR spectroscopy and differential scanning calorimetry.
      ). In this study, we classified microencapsulated Lb. acidophilus (Figure 1) and Lc. cremoris (Figure 2) after spray- or freeze-drying and after 10 wk of storage based on the similarities of the cell envelopes and secondary structure of proteins.
      Figure thumbnail gr1
      Figure 1Classification of microencapsulated Lactobacillus acidophilus (La) after drying and after storage at room temperature. After FD/after SD La = microencapsulated La after freeze drying/spray drying; FD/SD La - NaOH = microencapsulated La (under FD/SD) after storage in foil pouch containing NaOH as desiccant; FD/SD La - LiCl = microencapsulated La (under FD/SD) after storage in foil pouch containing LiCl as desiccant; FD/SD La - silica gel = microencapsulated La (under FD/SD) after storage in foil pouch containing silica gel as desiccant; FD/SD La - control = microencapsulated La (under FD/SD) after storage in foil pouch without desiccant; fresh La = freshly harvested La after being grown in the medium for 18 h.
      Figure thumbnail gr2
      Figure 2Classification of microencapsulated Lactococcus lactis ssp. cremoris (Lc) after drying and after storage at room temperature. After FD/after SD Lc = microencapsulated Lc after freeze drying/spray drying; FD/SD Lc - NaOH = microencapsulated Lc (under FD/SD) after storage in foil pouch containing NaOH as desiccant; FD/SD Lc - LiCl = microencapsulated Lc (under FD/SD) after storage in foil pouch containing LiCl as desiccant; FD/SD Lc - silica gel = microencapsulated Lc (under FD/SD) after storage in foil pouch containing silica gel as desiccant; FD/SD Lc - control = microencapsulated Lc (under FD/SD) after storage in foil pouch without desiccant; fresh Lc = freshly harvested Lc after being grown in the medium for 18 h.
      Microencapsulated Lb. acidophilus after freeze-drying and long-term storage formed different clusters than Lb. acidophilus after spray-drying and storage (Figure 1). Lb. acidophilus after freeze-drying had a high similarity with freeze-dried Lb. acidophilus after storage in foil pouch containing NaOH (which was in one cluster with fresh Lb. acidophilus); whereas Lb. acidophilus after storage in a foil pouch containing either LiCl or silica gel had similarities with the control. Conversely, microencapsulated Lb. acidophilus after spray-drying showed similar characteristics of cell envelopes and secondary protein structures with spray-dried Lb. acidophilus kept in a foil pouch containing either NaOH or LiCl. Freeze-dried Lc. cremoris also indicated different characteristics with spray-dried Lc. cremoris, as demonstrated by the formation of different cluster (Figure 2). Lactococcus cremoris after freeze-drying and freeze-dried Lc. cremoris after storage for 10 wk in a foil pouch containing NaOH showed high similarities. Freeze-dried Lc. cremoris after storage in a foil pouch containing silica gel had similar characteristics with that of the control, and was in one cluster with Lc. cremoris after storage in a foil pouch containing LiCl. Interestingly, microencapsulated Lc. cremoris after spray-drying showed different characteristics than spray-dried Lc. cremoris after storage regardless of aw adjustment, as demonstrated by the formation of different sub clusters. In addition, fresh Lc. cremoris was the most isolated, indicating its difference in characteristics as compared to microencapsulated Lc. cremoris after drying and subsequent storage. Even though classification of bacteria based on their cell envelopes and secondary protein structures has been established by
      • Helm D.
      • Labischinski H.
      • Schallehn G.
      • Naumann D.
      Classification and identification of bacteria by Fourier-transform infrared spectroscopy.
      and
      • Dziuba B.
      • Babuchowski A.
      • Naecz D.
      • Niklewicz M.
      Identification of lactic acid bacteria using FTIR spectroscopy and cluster analysis.
      , specific studies related to the similarities of microencapsulated bacteria after dehydration and after subsequent storage have never been carried out.

       Glass Transition Temperature and Residual Moisture Content of Microcapsules

      Glass transition temperature and residual moisture content (RM) of microcapsules (containing Lb. acidophilus and Lc. cremoris) after spray- or freeze-drying are shown in Table 4, whereas those of freeze- or spray-dried microcapsules after storage (10 wk, 25°C, in foil pouches containing different desiccators) are shown in Table 5. The Tg of the microcapsules after spray-drying was lower than that of microcapsules after freeze-drying, whereas the opposite trend occurred for RM. Higher RM of microcapsules after spray-drying than that of microcapsules after freeze-drying was due to relatively low outlet temperature of spray drying (50°C); therefore, reducing the residual water by storage at low aw was essential. Similarly, RM of spray- or freeze-dried microcapsules increased significantly (P = 0.0006), along with significant decrease in Tg (P = 0.0008) due to storage in a foil pouch using different desiccators. Storage in a foil pouch using NaOH or LiCl resulted in relatively higher Tg of microcapsules than Tg using silica gel, with the exception of storage of freeze-dried microcapsules containing Lc. cremoris kept under LiCl. However, all of the different desiccants showed microcapsule Tg >25°C.
      Table 4Glass transition temperature (Tg) and residual moisture content (RM) of Lactobacillus acidophilus and Lactococcus lactis ssp. cremoris after freeze-drying (FD) and spray-drying (SD).
      ItemLb. acidophilusLc. cremoris
      Tg (°C)RM (%)Tg (°C)RM (%)
      After FD50.0
      Means followed by the same letters indicate no statistical difference (P≥0.05).
      3.0
      Means followed by the same letters indicate no statistical difference (P≥0.05).
      42.0
      Means followed by the same letters indicate no statistical difference (P≥0.05).
      2.9
      Means followed by the same letters indicate no statistical difference (P≥0.05).
      After SD41.2
      Means followed by the same letters indicate no statistical difference (P≥0.05).
      4.0
      Means followed by the same letters indicate no statistical difference (P≥0.05).
      40.3
      Means followed by the same letters indicate no statistical difference (P≥0.05).
      3.2
      Means followed by the same letters indicate no statistical difference (P≥0.05).
      SEM2.560.341.580.08
      a,b Means followed by the same letters indicate no statistical difference (P ≥ 0.05).
      Table 5Glass transition temperature (Tg) and residual moisture content (RM) of freeze-dried (FD) or spray-dried (SD) Lactobacillus acidophilus and Lactococcus lactis ssp. cremoris after 10 wk of storage (25°C) in foil pouches containing different desiccators.
      DesiccatorLb. acidophilusLc. cremoris
      Tg (°C)RM (%)Tg (°C)RM (%)
      SDFDSDFDSDFDSDFD
      NaOH47.6
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      53.5
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      2.6
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      2.2
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      50.3
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      47.3
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      1.7
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      2.2
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      LiCl42.8
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      42.7
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      3.3
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      3.4
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      47.4
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      30.0
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      2.7
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      4.5
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      Silica gel41.1
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      35.2
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      4.2
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      4.5
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      44.6
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      28.1
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      3.6
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      5.0
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      Control39.8
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      36.5
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      5.2
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      5.0
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      40.8
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      27.7
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      4.5
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      5.2
      –eMeans followed by the same letters indicate no statistical difference (P≥0.05).
      SEM1.460.272.290.33
      a –eMeans followed by the same letters indicate no statistical difference (P ≥ 0.05).
      It has been widely reported that in a glassy or amorphous state, dehydrated products have liquid characteristics, with random molecule position but high viscosity (≥1012 Pa·s); thus, molecular mobility is limited (
      • Roos Y.H.
      Importance of glass transition and water activity to spray-drying and stability of dairy powders.
      ). This state is unstable and is temperature-dependent. At a certain temperature (known as Tg), the transformation from a solid-like to a liquid-like state initiates along with an increase in molecular mobility; this phenomenon is recognized as a glass transition (
      • Santivarangkna C.
      • Aschenbrenner M.
      • Kulozik U.
      • Foerst P.
      Role of glassy state on stabilities of freeze-dried probiotics.
      ). Therefore, storage at temperature below Tg is considered to be useful in maintaining products in their amorphous state. In addition to Tg, storage at low aw, particularly at its monolayer state, was effective in extending the shelf life of products (
      • Rahman M.S.
      Food stability determination by macro-micro region concept in the state diagram and by defining a critical temperature.
      ). Water activity and Tg of freeze-dried matrix containing lactobacilli has been proven to influence the survival of lactobacilli (
      • Kurtmann L.
      • Carlsen C.U.
      • Skibted L.H.
      • Risbo J.
      Water activity-temperature state diagrams of freeze dried L. acidophilus (La-5): Influence of physical state on bacterial survival during storage.
      ). An increase in aw and moisture content results in decrease in Tg (
      • Kurtmann L.
      • Carlsen C.U.
      • Skibted L.H.
      • Risbo J.
      Water activity-temperature state diagrams of freeze dried L. acidophilus (La-5): Influence of physical state on bacterial survival during storage.
      ,
      • Pehkonen K.S.
      • Roos Y.H.
      • Miao S.
      • Ross R.P.
      • Stanton C.
      State transition ad physicochemical aspects of cryoprotection and stabilization in freeze-drying of L. rhamnosus GG (LGG).
      ,
      • Roos Y.
      Water activity and glass transition temperature: How do they complement and how do they differ?.
      ), and vice versa. The second order transition, from a glassy to a rubbery state, likely occurs due to moisture adsorption during storage at higher aw. Therefore, we hypothesized that freeze- or spray-dried bacteria kept at low aw (using desiccators) would have relatively higher Tg of mixture than storage temperature, hence glass transition would not have taken place during room temperature storage. Our results showed that all Tg were higher than room temperature, which might reflect that no glass transition occurred at 25°C.
      Glass transition temperature determination is critical for this study, as we fortified mannitol into the formulation, whereas mannitol has a low Tg (i.e., 12.6°C;
      • Yu L.
      • Mishra D.S.
      • Rigsbee D.R.
      Determination of the glass properties of D-mannitol using sorbitol as an impurity.
      ). However, due to the presence of casein as a main component (Tg of 120°C at aw 0.11 at storage at 22.5°C;
      • Mauer L.J.
      • Smith D.E.
      • Labuza T.P.
      Water vapor permeability, mechanical, and structural properties of edible β-casein films.
      ), we expected the Tg of the mixture would be higher than the room temperature we used in this study. The combination of mannitol with sodium caseinate appeared useful to increase Tg of the mixture due to the high Tg of sodium caseinate. A similar study showed that incorporation of skim milk into disaccharides increased Tg of freeze-dried Geotrichum candidum (
      • Hamoudi L.
      • Goulet J.
      • Ratti C.
      Effect of protective agents on the viability of Geotrichum candidum during freeze-drying and storage.
      ). In our study, no Tg of pure crystalline mannitol (at 10°C) was detected, indicating that mannitol strongly interacted with other substances (
      • Kalichevsky M.T.
      • Blanshard J.M.V.
      A study of the effect of water on the glass transition of 1:1 mixtures of amylopectin, casein and gluten using DSC and DMTA.
      ;
      • Taylor L.S.
      • Zografi G.
      Sugar polymer hydrogen bond interactions in lyophilized amorphous mixtures.
      ). However, storage above room temperature does not ensure the stability of encapsulated products, as amorphous matrix of microencapsulants is one of several factors influencing the stability of the bacteria (
      • Ananta E.
      • Volkert M.
      • Knorr D.
      Cellular injuries and storage stability of spray-dried Lactobacillus rhamnosus GG.
      ;
      • Higl B.
      • Kurtmann L.
      • Carlsen C.U.
      • Ratjen J.
      • Forst P.
      • Skibsted L.H.
      • Kulozik U.
      • Risbo J.
      Impact of water activity, temperature, and physical state on the storage stability of Lactobacillus paracasei ssp. paracasei freeze-dried in a lactose matrix.
      ). It is still controversial whether glass transition is more important than molecular interaction in preserving dehydrated biomaterials, or vice versa; the relationship between those factors has been proposed by
      • Taylor L.S.
      • Zografi G.
      Sugar polymer hydrogen bond interactions in lyophilized amorphous mixtures.
      . The authors stated that lower glass transition of matrix could be due to less hydrogen bonding involvement in glassy state, and, thus, it affected Tg; our results are in agreement with those of
      • Taylor L.S.
      • Zografi G.
      Sugar polymer hydrogen bond interactions in lyophilized amorphous mixtures.
      ,
      • Garzon-Rodriguez W.
      • Koval R.L.
      • Chongprasert S.
      • Krishnan S.
      • Randolph T.W.
      • Warne N.W.
      • Carpenter J.F.
      Optimizing storage stability of lyophilized recombinant human interleukin-11 with disaccharide/hydroxyethylstarch mixtures.
      , and
      • Maury M.
      • Murphy K.
      • Kumar S.
      • Mauerer A.
      • Lee G.
      Spray-drying of proteins: effects of sorbitol and trehalose on aggregation and FT-IR amide I spectrum of an immunoglobulin G.
      . For instance, storage at low aw using NaOH as a desiccant provided a relatively high Tg (Table 5) as well as lower frequencies of P=O symmetrical (Table 1 and 2), indicating stronger hydrogen bonding interaction between P=O of cell envelopes and sugars (
      • Santivarangkna C.
      • Naumann D.
      • Kulozik U.
      • Foerst P.
      Protective effects of sorbitol during the vacuum-drying of Lactobacillus helveticus: An FT-IR study.
      ). However,
      • Breen E.D.
      • Curley J.G.
      • Overcashier D.E.
      • Hsu C.C.
      • Shire S.J.
      Effect of moisture on the stability of a lyophilized humanized monoclonal antibody formulation.
      stated that Tg is more important than chemical interaction to protect cells; this is in disagreement with our results. In fact, alteration of wavenumbers of FA (Table 1, 2) and secondary proteins (Table 3) still occurred after 10 wk of storage in a foil pouch using different desiccants, even though all Tg values were higher than room temperature of storage. Water activity appeared to have an important role on these phenomena; in this regard our results are similar to that of
      • Garzon-Rodriguez W.
      • Koval R.L.
      • Chongprasert S.
      • Krishnan S.
      • Randolph T.W.
      • Warne N.W.
      • Carpenter J.F.
      Optimizing storage stability of lyophilized recombinant human interleukin-11 with disaccharide/hydroxyethylstarch mixtures.
      . In addition,
      • Maury M.
      • Murphy K.
      • Kumar S.
      • Mauerer A.
      • Lee G.
      Spray-drying of proteins: effects of sorbitol and trehalose on aggregation and FT-IR amide I spectrum of an immunoglobulin G.
      demonstrated that protein stabilization by sorbitol and trehalose occurred through water replacement mechanism instead of amorphous state. Yet, storage in a foil pouch using NaOH is likely preferable to preserve the glassy state of freeze- or spray-dried microcapsules owing to a wide range of actual room temperatures (20–35°C).

      Conclusions

      Our FTIR study showed that all microcapsules interacted with P=O of phospholipid bilayers of the cell envelopes of Lb. acidophilus and Lc. cremoris after spray- or freeze-drying. After 10 wk of storage, the type of desiccant used (indicating the difference in aw) seemed to affect the FA and secondary protein structures of microencapsulated bacteria. Study on glass transition using DSC demonstrated that Tg of encapsulated Lb. acidophilus and Lc. cremoris after freeze-drying was higher than that after spray-drying. The type of desiccant used during 10 wk of storage had significant effect on Tg of dehydrated Lb. acidophilus and Lc. cremoris. This study demonstrated that even though no glass transition was detected at storage at 25°C, changes in cell envelopes and secondary protein structures could still occur.

      Acknowledgements

      Dianawati Dianawati is grateful to Directorate General of High Education of Department of Nation Education, Indonesia for providing financial support.

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