Effect of Drying Methods on Protein and DNA ... - ACS Publications

Article pubs.acs.org/JAFC

Effect of Drying Methods on Protein and DNA Conformation Changes in Lactobacillus rhamnosus GG Cells by Fourier Transform Infrared Spectroscopy Mya M. Hlaing,*,† Bayden R. Wood,‡ Don McNaughton,‡ DanYang Ying,† Geoff Dumsday,† and Mary Ann Augustin† †

CSIRO Agriculture and Food, 671 Sneydes Road, Werribee, Victoria 3030, Australia Centre for Biospectroscopy, School of Chemistry, Monash University, Clayton, Victoria 3800, Australia

‡

S Supporting Information *

ABSTRACT: Microencapsulation protects cells against environmental stress encountered during the production of probiotics, which are used as live microbial food ingredients. Freeze-drying and spray-drying are used in the preparation of powdered microencapsulated probiotics. This study examines the ability of Fourier transform infrared (FTIR) spectroscopy to detect differences in cells exposed to freeze-drying and spray-drying of encapsulated Lactobacillus rhamnosus GG cells. The FTIR analysis clearly demonstrated there were more significant molecular changes in lipid, fatty acid content, protein, and DNA conformation of nonencapsulated compared to encapsulated bacterial cells. The technique was also able to differentiate between spray-dried and freeze-dried cells. The results also revealed the extent of protection from a protein−carbohydrate-based encapsulant matrix on the cells depending on the type drying process. The extent of this protection to the dehydration stress was shown to be less in spray-dried cells than in freeze-dried cells. This suggests that FTIR could be used as a rapid, noninvasive, and real-time measurement technique to detect detrimental drying effects on cells. KEYWORDS: probiotics, microencapsulation, freeze-drying, spray-drying, Fourier transform infrared spectroscopy

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enhancing the viability of cells.11,12 The protection afforded is dependent on the matrix used for encapsulation.13 An understanding of the effects of drying methods on bacterial stress responses and a knowledge of the effects of different drying methods for cell preservation provide useful insights that can be used for the development of probiotic formulations. Traditional culture-based and microscopy techniques are normally applied to assessing the viability of bacterial cells and membrane damage from different drying methods during processing. Techniques to study the mechanisms that lead to adaptive bacterial tolerance responses under stress conditions in the environment also include Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy.14,15 The vibrational spectroscopic techniques provide significant benefits as noninvasive, reagentless, and rapid analytical tools at the single cell level.16−18 FTIR has been used to investigate the spectral changes related to the cell components of bacterial cells during vacuum drying and the role of sugars in hindering conformational changes of bacterial cell membranes and proteins.19,20 FTIR studies have been used to investigate conformational changes in phospholipid bilayers and secondary protein structures of bacterial cells on dehydration. Few vibrational spectroscopic studies have examined the effect of drying methods on spectral changes from the direct measurement of intact microencapsulated cells. We recently showed

INTRODUCTION Probiotics, live microbial food ingredients, have been extensively applied as natural health supplements for humans and animals and as substitutes for chemical supplements or antibiotic growth promotors.1−3 Developing stable probiotics that enable their incorporation into formulations is of interest to the functional food and animal feed industries. For successful application, it is essential for the probiotics to retain viability and efficacy through production to consumption. Therefore, an understanding of microbial criteria such as stress responses and the ability to withstand stress during processing and storage are crucial for industrial probiotics production. Drying methods are normally applied to preserve probiotic bacteria as well as enable ease of handling, low-cost transportation, and long-term storage at room temperature. Freeze-drying is the method most commonly used for the preparation of dried concentrated cultures; however, spray-drying is also of interest as it is a lowcost alternative for dehydration.4,5 Although dehydration of cells is believed to have many adverse effects on cell membranes and secondary protein structures, freeze-drying cells generally has less impact on viability than the harsher processing conditions encountered during spray-drying.6,7 Microencapsulation technology, which is a process to entrap probiotic cells within a matrix, has been developed to protect probiotic cells from detrimental effects during the drying process.8,9 Previous studies have reported the benefits of microencapsulation technology with the use of mannitol being effective in protecting dehydrated bacterial cells10 and the addition of a protective carbohydrate (such as lactose, sorbitol, inulin, xanthan gum, and trehalose) before the drying process © 2017 American Chemical Society

Received: Revised: Accepted: Published: 1724

December 8, 2016 January 26, 2017 January 29, 2017 January 30, 2017 DOI: 10.1021/acs.jafc.6b05508 J. Agric. Food Chem. 2017, 65, 1724−1731

Article

Journal of Agricultural and Food Chemistry

Figure 1. Correction for signal contribution of the matrix using the vector correction routine: (A) Score plot for the first and second principal components (PC1 and PC2) from PCA on the spectra collected from dehydrated microencapsulated LGG cells, Encap-LGG, (before and after matrix correction) together with matrix spectra and (B) average score value plots for the PC1 and PC2. containing a 1:1:1 mixture of whey protein isolate, maltodextrin, and glucose). The final mixture contained 0.4% w/w of bacteria dry mass. The LGG cells embedded in the protein−carbohydrate mixture were then freeze-dried under the same conditions used for nonencapsulated dried cells. A portion of the bacterial sample with the encapsulant matrix was also spray-dried as outlined above. Dried samples were stored at 4 °C (∼24−48 h) and removed for FTIR analysis as required. For preparation of the fresh hydrated samples before the drying process (control hydrated samples), the washed cell suspensions in PBS were kept at 4 °C until FTIR experiments were carried out (∼24 h). Cells were washed and resuspended in sterile Milli-Q water (ultrapurified water, Millipore) before the experiments. This sample preparation and the storage protocols avoid significant deleterious effect on cells’ viability from the one-week storage time after the drying process and eliminate spectral artifacts from the freezing process.23−25 FTIR Measurement. FTIR spectra were recorded using a Bruker Equinox FTIR system equipped with a Golden Gate diamond attenuated total reflectance crystal and a liquid nitrogen cooled mercury−cadmium−telluride detector. The dry powder samples or ∼10 μL of control hydrated samples were placed in contact with the attenuated total reflectance crystal surface of the FTIR system for the measurement. The control hydrated samples were air-dried first on the crystal surface just before the measurement. The FTIR spectra were collected in the wavenumber range of 600−4000 cm−1 with a resolution of 4 cm−1 for 50 scans. The spectra were baseline corrected and analyzed using OPUS software which is integrated in the Bruker Equinox FTIR system. All 50 FTIR spectra (5 different sample preparations × 2 biological replicates × 5 measurements) were collected from control hydrated samples and dried samples with and without microencapsulation. FTIR Data Analysis. Commercially available software (R language, Matlab, and OriginPro) were used for all data processing, including background subtraction,26 vector correction,27 second-order derivative calculation, and principal components analysis (PCA).28,29 The subtraction of FTIR signal contributions of the water peak (in the range of region 3700−3000 cm−1 and/or 1700−1600 cm−1) from spectra of control hydrated samples was accomplished by using an appropriate subtraction factor for the bulk water spectrum to enhance the signal of the adsorbed bacterial related peaks over the water peak and to facilitate the quantitative peak evaluation.30 To subtract FTIR signal contributions of the encapsulant matrix from spectra of bacterial cells entrapped inside the matrix (i.e., dehydrated microencapsulated samples), a nonsubjective vector correction procedure was applied (Supporting Information).27 The combined bacteria and matrix vector was decomposed into a vector parallel and orthogonal to the matrix vector. The desired nonmatrix-related bacterial spectrum was calculated by subtracting the projection of the combined vector

that Raman spectroscopy is useful for examining changes in cell components on freeze-drying of microencapsulated cells.21 Most previous studies on the survival of microencapsulated bacterial cells were based on conventional viability assays such as plate counting and live/dead cell staining methods using rehydrated cells.22 The aims of this study were to determine infrared spectral changes that result from the application of different drying methods on the protein secondary structure (i.e., α-helices and β-sheets) in dormant bacterial cells. Moreover, the dehydration effect on DNA conformation and the degree of these changes in cells with or without microencapsulation were also examined. A lactic acid-producing probiotic bacterium Lactobacillus rhamnosus GG, which is extensively applied in various food industries, was used as a model in this study.

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MATERIALS AND METHODS

Bacterial Growth Conditions and Sample Preparation. The L. rhamnosus GG (LGG) strain ATCC 53103 (Laboratorium Voor Microbiologie, Ledeganckstraat, Belgium) was used throughout this study. De Man Rogosa and Sharpe broth (MRS broth, Thermo Fisher, Australia) was used as a nonselective medium for LGG growth. Bacteria from a −80 °C stock were grown as the primary culture by inoculating one bead of frozen LGG into 10 mL of MRS broth and then incubating the solution at 37 °C under anaerobic conditions. Two milliliters of the overnight primary culture was further inoculated with 200 mL of MRS broth and incubated at 37 °C for 20 h. These bacterial cultures were further incubated until cell growth reached an optical density of 5.0 at A600 nm and the weight of the wet bacterial mass was 0.01 g/mL.13,23 The second culture of LGG cells was harvested by centrifugation at 3500g for 15 min (∼16 °C).23 The harvested cells were then washed with sterile phosphate buffered saline (PBS) using centrifugation under the same conditions to remove any traces of MRS medium. The harvested LGG cells were used directly for freeze-drying and spray-drying processes to prepare nonencapsulated dried bacteria. Briefly, the washed cells were suspended in Milli-Q water (ultrapurified water, Millipore), transferred into 1 mL aliquots, frozen using liquid nitrogen, and dried using a Dynavac freeze drier for 48 h at 20 Pa. A portion of the cell preparation was spray-dried using a laboratory scale Drytec spray dryer (Drytec Engineering, LLC, United States) operated at inlet and outlet temperatures of 160 and 65 °C, respectively. Formulation and preparation of the microencapsulated cells was based on a previously reported procedure.23 Briefly, harvested washed LGG cells were dispersed in an encapsulant matrix (24% w/v 1725

DOI: 10.1021/acs.jafc.6b05508 J. Agric. Food Chem. 2017, 65, 1724−1731

Article


Figure 2. FTIR measurements. Calculated second derivative of FTIR spectra from LGG cells with or without microencapsulation after freeze-drying or spray-drying in the spectral range of (A) 3000−2700 cm−1, (B) 1800−1400 cm−1, and (C) 1300−900 cm−1. Abbreviations: Encap-LGG, microencapsulated LGG; FD, freeze-dried; SD, spray-dried. The spectra of microencapsulated cells were corrected for the matrix contribution.

Table 1. Second Derivative of FTIR Spectra from L. rhamnosus GG Cells (Means ± SD)a freeze-dried peak assignment strasymCH3 strasymCH2 strsymCH3 strsymCH2 δasymCH3 δCH unordered random coils and β-turns α-helix β-sheet β-sheet νasymPO2− νsymPO2− νC−O−O−C of carbohydrates strC−O, strC−C of DNA backbone

fresh hydrated (control cells) 2957.3 2918.7 2889.8 2851.2 1449.2 1492.6

± ± ± ± ± ±

1.9 1.9 2.63 1.5 5.7 0.13

1658.5 ± 1.5 1637.3 ± 1.5 1210.1 1083.8 1047.2 962.3

± ± ± ±

1.9 0.13 0.13 2.0

nonencapsulated 2960.2 2931.3 2873.4 2852.2 1452.1 1514.8

± ± ± ± ± ±

0.5 0.5 0.13 0.13 0.5 1.5

1654.6 ± 0.13 1635.3 ± 0.13 1240.0 1082.8 1035.6 966.2

± ± ± ±

0.5 1.5 0.13 0.13

spray-dried

microencapsulated 2962.1 2917.8 2881.1 2846.4 1456.0 1504.2 1683.6

± ± ± ± ± ± ±

1.9 0.5 0.13 0.13 0.5 0.13 0.13

1635.3 ± 0.13 1621 ± 0.13 1236.2 ± 0.5 1072.2 ± 0.13 1049.1 ± 0.13 979.7 ± 0.13

nonencapsulated 2961.2 2932.2 2873.4 2852.2 1452.1 1515.8

± ± ± ± ± ±

1.9 1.9 0.13 0.13 0.5 0.13

1654.6 ± 0.13 1637.3 ± 0.13 1240.0 1078.0 1022.1 991.2

± ± ± ±

0.5 0.13 0.13 0.13

microencapsulated 2960.2 2921.6 2886.9 2850.3 1454.1 1513.8 1675.8

± ± ± ± ± ± ±

0.5 0.5 0.13 0.13 0.5 0.13 0.13

1645.0 ± 0.13 1627 ± 0.13 1232.3 ± 2.4 1078.0 ± 0.13 1056.8 ± 0.13 985.5 ± 0.13

Abbreviations: asym, asymmetric; δ, deformation; PO2−, phosphate; str, stretching; sym, symmetric; ν, vibration. Assignments are based on studies in the references.33,34,39−44 a

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(bacteria and matrix) on the matrix vector from the combined vector. To minimize spectrum to spectrum variation, the background subtracted spectra26 were first scaled to standard normal variance (zero mean and unit variance) before application of the vector correction method. Validation of the matrix subtraction method was performed to determine the efficiency and reproducibility of the method. The spectra collected from the dehydrated encapsulated LGG cells were vector-corrected. The vector-corrected spectra, the spectra from dehydrated encapsulated cells before correction, and the pure matrix spectra were analyzed using PCA. The intensities of the background-subtracted spectra and matrixcorrected spectra were normalized using total intensity normalization to account for variations in intensity. Second derivatives of the normalized spectra were calculated using nine smoothing points for the Savitsky−Golay process. To perform PCA, the normalized FTIR spectra were then mean-centered to reposition the centroid of the data to the origin. Finally, the mean-centered data were analyzed by calculating the principal components, creating scores and loadings plots of the first and second principal components that relate the scores to specific regions in the original FTIR data. For specific peak intensity analysis, the normalized intensity values of the specific peaks were averaged by adding the maximum intensity and the intensity values of the two neighboring wavenumbers.28

RESULTS AND DISCUSSION The FTIR spectrum obtained directly from dehydrated encapsulated LGG cells contains signal contributions from both bacteria and the encapsulant matrix (Supporting Information, Figure S1). The corresponding matrix’s signal was subtracted from the spectra of bacterial cells entrapped within the matrix using a vector correction procedure, and the matrix-corrected spectra were used for further analysis. The validation results of the matrix subtraction method are shown in Figure 1. The PCA Scores plot showed distinct clustering of the samples with the first principal component (PC1) accounting for 96.5% of the variation in the data set, enabling the differentiation of the matrix-corrected spectra from the original spectra before correction and the matrix spectra (Figure 1A). The average PC1 score value plots from PCA of FTIR spectra clearly demonstrated the differentiation of matrix-corrected spectra from others (Figure 1B). The second derivative of FTIR spectra taken from control hydrated LGG cells samples, nonencapsulated cells, and the matrix-corrected spectra of microencapsulated LGG cells after 1726


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the bacterial cell membrane. Compared to the nonencapsulated cells, the reduced variation observed in the microencapsulated cells indicated that microencapsulation protects bacterial cells from detrimental effects of drying processes. The Amide I and II Regions (1700−1575 cm−1). Amide I and II bands are the two most prominent vibrational bands of the protein backbone.30 The amide I band in the spectral range of 1700−1600 cm−1 is mainly related to CO stretching vibration (70−85%) and C−N group (10−20%). According to previous studies, a major component of amide I in the range of 1662−1645 cm−1 is attributed to α-helices that form part of secondary protein structures.32 The calculated second derivative spectra which cover for the amide I components (such as α-helices, β-sheets, and β-turns) (1700−1600 cm−1) were investigated to identify the changes in protein backbone conformation (Figure 2B and Table 1). The peaks associated with α-helical structures (from 1658 to 1654 cm−1) were prominent in control cells and nonencapsulated dried cells. More than one β-component, which was assigned as a β-sheet (in the spectral region of 1640−1620 cm−1), was observed in the microencapsulated dried cells. Similarly, the peak assignment in the range of 1682−1662 cm−1 (related to β-turns) was apparent only in the microencapsulated cells after freeze- or spray-drying. These differences observed indicated that microencapsulation of LGG cells resulted in protein conformational changes from α-helices to β-sheets or β-turns. The changes could possibly be explained by an interaction between bacterial cell membrane proteins and the encapsulant matrix mixture. The encapsulant mixture used in this study contained whey protein isolate, maltodextrin, and glucose and might interact with polar sites in the phospholipid bilayers of bacterial cell membranes, thereby resulting in changes to secondary protein structures. The Region of Phosphate Groups and Carbohydrates (1280−940 cm−1). Wavenumbers at ∼1210 and ∼1083 cm−1 are assigned to asymmetric and symmetric phosphate stretching bands from the phosphodiester groups of nucleic acids (νasymPO2− and νsymPO2−) in the control hydrated LGG cells (Figure 2C).33,34 Compared to control cells, a wavenumber increase in νasymPO2− was observed in freeze-dried and spraydried LGG cells. A wavenumber decrease was observed for νsymPO2− in all dried cells where the freeze-dried microencapsulated cells showed the greatest decrease among them. These shifts in the PO2− vibrations of nucleic acids indicated structural alterations in the DNA of LGG cells as a consequence of the drying process. It is known that bacteria possess mechanisms to recognize diverse environmental changes and respond to the stress typically involving cellular homeostasis and repair of damaged DNA and proteins to ensure survival. Several reports indicate that DNA structures alter in prokaryotic and eukaryotic cells in response to stress.35,36 The νasymPO2− vibration shift of 30 cm−1 to 1240 cm−1 in dehydrated cells was comparable with the results shown in a previous study of the conformational transition between B- and A-DNA in dormant bacterial cells.37 A peak alteration of C−O stretching coupled with C−O bending of the C−OH of carbohydrates (including glucose, fructose, glycogen, etc.) (νC−O−O−C)38 was apparent in nonencapsulated cells after freeze-drying and spray-drying due to a shift from ∼1047 to 1035 and 1022 cm−1, respectively. Freeze-drying the cells resulted in less structural changes compared to spray-drying, as indicated by the smaller wavelength shift. When the cells were microencapsulated, the

freeze-drying or spray-drying are shown in Figure 2. The second derivative spectra were investigated based on the FTIR signatures associated with the regions of lipid for cell membranes and regions associated with secondary protein structure, carbohydrates, and DNA conformation changes for bacterial cells. The spectral alterations among nonencapsulated and microencapsulated LGG cells after freeze-drying and spraydrying compared with the control were analyzed to investigate the effect of drying methods as well as the effect of microencapsulation (Tables 1). The CH Region (2970−2840 and 1515−1442 cm−1). The spectral range from 2970 to 2838 cm−1, related to the C− H stretching vibrations, was investigated to identify differences in lipid composition between samples (Figures 2A and B). The chemical structure of lipids from bacterial cell membranes as well as intracellular droplets could be observed in this range in control hydrated samples using peak wavenumbers at 2957 cm−1 (asymmetric stretching vibration of CH3 of acyl chains, strasymCH3), 2919 cm−1 (asymmetric stretching vibration of CH2 of acyl chains, strasymCH2), 2890 cm−1 (symmetric stretching vibration of CH3 of acyl chains, strsymCH3) and 2851 cm−1 (symmetric stretching vibration of CH2 of acyl chains, strsymCH2). Wavenumber shifts related to the C−H stretching vibrations were seen in freeze-dried and spray-dried cells compared to control cells (Table 1). A wavenumber increase for strasymCH3 of LGG cells was detected after freeze- and spray-drying with shifts from 2957 cm−1 (control cells) to 2960 and 2961 cm−1, respectively. A similar increase was observed for strasymCH2 in freeze-dried and spray-dried LGG cells. In contrast, a decrease in wavenumber for symmetric C−H stretching vibration (strsymCH3 and strsymCH2) was observed among freeze/spraydried nonencapsulated cells compared to control cells. A smaller wavenumber shift observed in freeze-dried cells compared to that of spray-dried cells revealed that freezedrying seems to cause less structural change in the cells than spray-drying. The absorption at 1449 cm−1 in control hydrated LGG cells is assigned to the asymmetric H−C−H bending of the methyl groups of proteins (δasymCH3). A peak alteration of 1449 cm−1 (fresh cells) to 1452 cm−1 (δasymCH3) was detected in nonencapsulated cells after freeze-drying and spray-drying. Similarly, compared to control cells (1492 cm−1), increased C− H bending vibration and C−H deformation was observed after freeze/spray-drying of nonencapsulated cells. When the cells were protected inside a matrix (i.e., microencapsulated), the frequency shift related to strasymCH2 between dried cells and control cells was less compared to nonencapsulated cells. Freeze-dried microencapsulated cells appeared to be similar to the control cells, while there was a 2−3 cm−1 wavenumber increase in spray-dried microencapsulated cells. Similarly, the frequency increase of δCH was less when cells were microencapsulated after drying, particularly after the freezedrying. Bacterial membranes consist of proteins that are embedded in a lipid matrix known as a phospholipid bilayer. Bacterial survival normally depends on membrane lipid homeostasis and on an ability to adjust lipid composition to aid survival in different environments.31 The peak variations related to CH deformation of acyl chains and asymmetric CH3 bending of the methyl groups in proteins after dehydration suggested fatty acid alteration and protein conformational changes possibly resulted from liquid-crystalline formations in the phospholipid bilayer of 1727


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Figure 3. PCA comparing the FTIR spectra of control hydrated LGG cells versus (i) freeze-dried LGG, (ii) freeze-dried microencapsulated LGG, (iii) spray-dried LGG, and (iv) spray-dried microencapsulated LGG cells. (A) Score plots for the first and second principal components (PC1 and PC2) of PCA and (B) loading value plots for PC1. Abbreviations: asym, asymmetric; δ, deformation; Encap-LGG, microencapsulated LGG; FD, freeze-dried; PO2−, phosphate; SD, spray-dried; str, stretching; sym, symmetric; ν, vibration.

Figure 4. Relative intensity changes of DNA/RNA structure-specific and protein relative peaks of the FTIR spectra taken from nonencapsulated LGG cells and microencapsulated LGG cells after freeze-drying and spray-drying. Relative intensity changes versus control hydrated LGG cells represent the mean changes (±standard deviation) of each sample group which has ten replicates. Asterisks (**) indicate a significant difference (p < 0.05) within respective sample groups relative to the control sample. Abbreviations: asym, asymmetric; δ, deformation; Encap-LGG, microencapsulated LGG; FD, freeze-dried; PO2−, phosphate; SD, spray-dried; str, stretching; sym, symmetric; ν, vibration.

PCA of FTIR Spectra Taken from LGG Cells with or without Microencapsulation. To extract the relevant chemical information from the spectral variation attributed to macromolecular changes during the microencapsulation process, PCA was performed on the matrix-corrected FTIR spectra of microencapsulated LGG cells and the original spectra of nonencapsulated cells after freeze-drying and spray-drying. The results from Scores plots, shown in Figure 3A, illustrate that dehydrating the samples during the drying process significantly affects the FTIR spectra. The results also demonstrated that the spectra taken from control hydrated samples showed much greater variation spectra from dehydrated samples. These

peak shifts compared to control hydrated cells were smaller. In particular, no obvious alteration was detected in νC−O−O−C peaks of microencapsulated LGG cells after freeze-drying when compared to control hydrated LGG cells. The peak at ∼962−964 cm−1 observed in control hydrated LGG cells was due to the carbon−carbon vibration in the DNA backbone (i.e., C−O and C−C stretching (strC−O and strC− C) of phosphodiester and ribose). An increase in wavenumber was observed in the spectra of all freeze-dried and spray-dried cells. Similar to the behavior in the carbohydrate peaks, the peak shift of strC−O and strC−C of deoxyribose was smaller in freeze-dried cells. 1728


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bacteria. Freeze-drying of cells was less detrimental than spraydrying. By analyzing intensity changes in specific peaks related to the DNA backbone, a lesser change in microencapsulated samples indicated that one benefit of microencapsulation is protection against DNA damage during the drying. The results from comparative PCA for spectra taken from freeze- or spray-dried samples and control samples demonstrated that dehydrating the cells causes spectral alterations associated with different biochemical species. Specific peak analyses indicated that there were fewer changes in microencapsulated cells. This indicated that bacterial cells remain relatively undamaged and remain viable inside the encapsulant matrix, thus increasing membrane resistance to temperature stress. These results are comparable with the result reported by Zhu et al.23 that there is a protective effect and an increased survival rate afforded by maltodextrin with either stereoisomer of glucose during the storage of L. rhamnosus GG in the dry state over seven days. Comparing spectral changes between freeze-dried and spray-dried cells indicated larger variations in the biomolecular components of spray-dried cells. Freezedrying of bacterial cells resulted in FTIR spectra with fewer differences compared to the spectra of a control hydrated sample. It is possible that the changes seen in spray-dried cells arise from fluctuations in metabolic activity of the bacterial cells and molecular modification of macromolecules on the cell surface due to the high temperatures used for spray-drying. As rapid and effective investigation of bacterial stress response is of ongoing interest, the study of detailed spectral changes owing to different drying methods provides useful methodological background for FTIR applications in food microbiology. Through an understanding of the detrimental effect on bacterial cells membranes during the drying process, the techniques presented here can also be beneficial for studying the metabolic status of bacteria and their cellular response to other stresses. This study also shows that changes to particular cellular macromolecules can be measured using FTIR. Future studies could use the same technique to characterize changes to other components such as bacterial extracellular polymeric substances in protecting the cells from environmental stress during the freeze-drying process.

greater separations can be explained by the fact that there might be varying degrees of hydration in control hydrated samples. The PC1 was sufficient to differentiate the freeze-dried and spray-dried cells from the fresh hydrated control LGG cells. The separation of freeze-dried and spray-dried cells with or without microencapsulation from control hydrated cells along PC1 demonstrated a common difference in these spectra. Analysis of the PC1 loading plots further confirmed that the separation seen in the Score plots was associated predominantly with the changes in protein and DNA. The positive peaks in the loading value plots of PC1 indicated the corresponding peaks in the FTIR spectra that contributed to the separation of the control hydrated cells from the dried cells (Figure 3B). In contrast, the peaks seen in the negative loading values were related to the separation of the freeze-dried and spray-dried cells from the control cells. The dominant spectral variation in specific molecular species that enabled distinction of control cells from dried cells was seen in the ranges 1700−1575, 1517− 1458, 1257−1176, 1115−1032, and 981−933 cm−1, which are mostly attributable to protein and DNA/RNA. Specific peak analysis was performed for dehydrated and control hydrated samples to determine the subtle changes in FTIR spectra that correspond to changes in protein and DNA conformation (Figure 4). The relative peak intensity changes to the control hydrated samples were further analyzed in microencapsulated and nonencapsulated LGG cells after freezeor spray-drying. The prominent peaks observed in the loadings plot for the control cells and the dried cells with/without microencapsulation (Figure 3B) were selected for the analysis. Upon dehydration of the cells, the analyses showed the changes in intensity of the protein and DNA/RNA related peaks which were selected from the loading plots. More specifically, the intensity changes in the protein-related peaks were associated with asymmetric stretching vibration of CH3 of acyl chains (strasymCH3), changes to the β-sheet structure of amide I, and C−H deformation (δCH). Changes in the carbon−carbon stretching of the DNA backbone (strC−O and νC−C) and the phosphate stretching vibration (νsymPO2− and νasymPO2−) in freshly hydrated control samples and dried dormant cells indicated that changes to DNA conformation had occurred (Figure 4). The decreased intensity value seen in the dehydrated cells for the peak associated with the phosphate stretching vibration illustrated conformation changes from B- to A-DNA. These results were comparable to the findings of Whelan et al.37 The larger intensity changes of the strC-O, νC− C, and νasymPO2− peaks were observed in nonencapsulated cells compared with those in microencapsulated cells. These findings indicated a more intense cellular response of bacteria to dehydration stress in the absence of encapsulation. Furthermore, the freeze-dried cells showed deceased intensity changes compared to the spray-dried cells, indicating that freeze-drying had a lesser detrimental effect on cellular structures compared to that of spray-drying. The PCA results were in good agreement with the observation of the second derivative spectral analysis. These findings suggested that there may be production of more bacterial extracellular polymeric substances (EPS) during freeze-drying, thereby enhancing protection of the cells from environmental stress. In conclusion, this study also demonstrated that FTIR can be used to measure changes to secondary protein structure and DNA conformation after drying. The type of drying process used (freeze or spray) had different effects on the fatty acids and the secondary protein structures of microencapsulated

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05508. Description of vector correction for removal of the matrix signal and additional FTIR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Mya M. Hlaing: 0000-0002-7351-864X Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Fuller, R. Probiotics: The scientific basis; Springer: Netherlands, 1992. (2) Tannock, G. W. Identification of lactobacilli and bifidobacteria. Curr. Issues Mol. Biol. 1999, 1, 53−64.

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