extfieldsofstudy: Medicine Chemistry

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Comparison of Targeted (HPLC) and Nontargeted (GC-MS and NMR) Approaches for the Detection of Undeclared Addition of Protein Hydrolysates in Turkey Breast Muscle

The adulteration of fresh turkey meat by the undeclared addition of protein hydrolysates is of interest for fraudsters due to the increase of the economic gain by substituting meat with low cost ingredients. The aim of this study was to compare the suitability of three different analytical techniques such as GC-MS and 1H-NMR with HPLC-UV/VIS as a targeted method, for the detection of with protein hydrolysates adulterated turkey meat. For this, turkey breast muscles were treated with different plant- (e.g., wheat) and animal-based (e.g., gelatin, casein) protein hydrolysates with different hydrolyzation degrees (15–53%: partial; 100%: total), which were produced by enzymatic and acidic hydrolysis. A water- and a nontreated sample (REF) served as controls. The data analyses revealed that the hydrolysate-treated samples had significantly higher levels of amino acids (e.g., leucine, phenylalanine, lysine) compared with REF observed with all three techniques concordantly. Furthermore, the nontargeted metabolic profiling (GC-MS and NMR) showed that sugars (glucose, maltose) and/or by-products (build and released during acidic hydrolyses, e.g., levulinic acid) could be used for the differentiation between control and hydrolysates (type, degrees). The combination of amino acid profiling and additional compounds gives stronger evidence for the detection and classification of adulteration in turkey breast meat.

Introduction
Meat is an important supplier of high-quality nutrients such as proteins, minerals and vitamins. Since it is sold on the market for a low price, which does not cover the increasing production cost, it is in focus for food fraud. Adulterators look for opportunities to increase the economic gain. Possibilities would be to misrepresent, use illegal supply chains and/or manipulate the food product, e.g., replace/substitute some, or all, premium quality materials with lower-grade, cheaper cuts of meat, meat from other species or nonmeat components (e.g., water, additives) [1][2][3]. The fraud can influence the consumers' satisfaction and confidence (religious, moral, cultural), but worse it can be extremely dangerous for human health, e.g., causing illness, provoke allergies or even causing death (e.g., melamine scandal) [1,4]. Therefore, it is necessary to have reliable analytical methods to detect adulteration.
The water binding capacity of meat is strongly related to the rate of early postmortem metabolism and the ultimate pH value. It is lowest at a pH of 4.9-5.4, but will increase with increasing or decreasing of the pH [5]. The ultimate pH of poultry breast muscle is 5.67-5.69 and therefore, the water uptake capacity is high [6]. A simple exposure to just water can lead to weight gain of the meat and consequently increase the economic gain. For a fair market competition, the detection of such fraudulent practices is required. The traditional method to determine extraneous water in meat is to analyze the water/protein ratio [7]. In an untreated sample is the water/protein ratio of chicken as well as turkey breast muscle <3.40, of chicken legs between 4.05-4.30 and of turkey legs between 3.80-4.05 (Commission regulation No 543/2008) [8]. If water is added, the water/protein ratio will be higher.
For more than a decade an undeclared addition of protein hydrolysates to poultry meat or meat products could be observed. This way, the analytical protein content rises, masking the water addition [3]. Protein hydrolysates consist mainly of amino acids and possibly peptides. They are cheaper, better soluble and harder to detect than protein additions. Besides amino acids, hydrolysates contain additional compounds like carbohydrates, fatty acids and/or side products, which are formed during the hydrolyzation process. A suitable method is the detection of the free amino acid contents using high performance liquid chromatography with ultraviolet-visible detection (HPLC-UV/VIS). This technique is well established in laboratories and is used as an alternative to the official method in Germany ( §64 LFGB: Determination of free amino acids in meat using gas chromatography with flame ionization detection (GC-FID)) [9].
Alternative analytical approaches such as nontargeted metabolomics provide an entire profile (chromatogram, spectrum, fingerprints, etc.) of a suspicious sample [10,11]. These methods have gained more and more in importance in recent years due to strong technical improvements. With these promising and valuable high-throughput tools such as mass spectrometry (MS) based techniques or nuclear magnetic resonance (NMR) spectroscopy, it is possible to identify and quantify small organic molecules with molecular weights of less than 1.5 kDa, including carbohydrates, peptides, nucleotides, lipids and amino acids [12]. In order to analyze such a broad spectrum of metabolites with diverse properties and concentrations (over several magnitudes) in just one single sample, it is important to have techniques, which are robust and sensitive. The major techniques are MS coupled to a chromatographic separation and NMR. The high sensitivity and selectivity make MS a powerful tool to detect molecular masses and fragmentation patterns for chemical structure identification. It is possible to profile myriads of metabolites due to the different combinations of separation, ionization and detection technique. On the other hand, NMR spectroscopy provides characteristic information on the metabolic profile by analyzing small amounts of one sample in a nondestructive, quantitative and short-time period way with convenient sample preparation [13]. However, independent of the used approach (targeted or nontargeted), large reference datasets are required to account for the natural variation in different products due to multiple influencing factors such as feeding or storage conditions (duration and temperature) [3,14].
Chemometrics is a powerful multivariate data analysis tool that reduces a huge amount of generated data by (1) grouping or ordering unknown samples with similar characteristics (qualitative) and (2) ascertaining adulterant analytes in sample (quantitative) or (3) for assessing their quality or authenticity [14]. Chemometrics, and for this purpose used clustering, principal component analyses (PCA) as well as regression analyses, is a routine complement for MS-and NMR-based metabolomics [15].
The objective of this study was to compare the suitability of three different analytical methods such as GC-MS and 1 H-NMR (nontargeted approaches) and HPLC-UV/VIS as a targeted method, for the detection of adulterated turkey breast muscle with protein hydrolysates. HPLC-UV/VIS was selected due to the fact that it requires inexpensive equipment frequently available in every control laboratory. The first part focuses on the identification of specific markers such as amino acids, which are detectable with all three methods. The second part (nontargeted approaches) focuses on the identification of additional markers as well as possible classification of the added hydrolysates.

Sampling and Adulteration of Turkey Breast
Meat. Three female turkeys (BUT Big 6, Meleagris gallopavo) with an average weight of 10.3 kg and in average 112 days old were collected at 4 • C from a slaughterhouse in Germany directly after slaughter. The Musculus pectoralis superficialis was taken after dissection [16]. After sampling, all meat pieces were immediately frozen in liquid nitrogen and stored at −20 • C until further use.
Protein hydrolyzation. The protein powders (1.0 g of casein from bovine milk, gelatin from porcine skin or gluten from wheat) were hydrolyzed at 150 • C in 8 mL of 6 M aqueous HCl solution for 1 h (total hydrolyzation: TH, degree of hydrolyzation: 100%). After that, neutralization with solid 1.0 M NaOH was performed. The final concentration of amino acids was 55.6 g/L (~0.5 M).
The protein hydrolysate powders (gelatin hydrolysate enzymatic, HyPep ® 4601 protein hydrolysate from wheat gluten, protein hydrolysate N-Z-amine ® AS (casein)) were used without further hydrolyzation (partial hydrolyzation: PH). All three bought protein hydrolysates were peptones (enzymatic hydrolysis with pepsin (E.C.3.4.4.1) or acidic hydrolysis). The degree of hydrolyzation was analyzed photometrically as the following describes. From each sample, 250 µL (aqueous AA solution) were added to 250 µL of 4 M acetate buffer (pH 5.5) and mixed. After that, 75 µL of 1 M NaOH and 250 µL ninhydrin solution (174 mg Ninhydrin + 28 mg hydrindantin-dihydrat in 10 mL 2-methoxyethanol) were added. The reaction took place at 95 • C for 20 min and was stopped by cooling the mixture in an ice bath (0 • C) for 20 min. 300 µL of each mixture was diluted with 1000 µL isopropanol solution (50% v/v in water) and measured at room temperature at 570 nm (photometer DU 640, Beckman Coulter GmbH, Krefeld, Germany). The degree of hydrolyzation (DH) was calculated by comparing the free amino groups of the samples with a solution of the same not hydrolyzed protein (0% DH) and total hydrolyzed protein (100% DH). That implies a hydrolyzation degree for gelatin, wheat and casein of 15% ± 3%, 16% ± 2% and 53% ± 2%, respectively. Hydrolysate injection. About 1 mL solution (0.5 M) hydrolyzed protein or commercially available protein hydrolysate or water was injected per g turkey breast meat across and along the muscle fiber. The samples were frozen at −80 • C and lyophilized for GC-MS and 1 H-NMR analyses. All samples were stored at −80 • C until further use.
Sample code. The sample codes were chosen as followed: reference sample without injection is called REF, an additional control sample injected with water is called water. The different protein hydrolysates (gelatin (G), wheat (W) and casein (C)) with different hydrolyzation degree (partial (PH) or total (TH)) are indicated with the following codes: GPH, WPH and CPH for partial, respectively, and GTH, WTH and CTH for total hydrolyzation, respectively. This means, for example, that the sample GPH contained protein hydrolysate gelatin and was partially hydrolyzed.

Sample Preparation for Amino Acid Analysis
The frozen turkey samples (2 g each) were homogenized with an Ultra-Turrax T10 (12,000 rpm, IKA Werke GmbH und CO KG, Staufen, Germany) in 5 mL 0.025 M EDTA/0.100 M Tris buffer (pH 8.0) at −20 • C. As internal standards, l-norleucine and l-thialysine were used with a final concentration of 133 µM and 88 µM, respectively. The proteins and longer peptides were precipitated with 30% v/v of 15% 5-sulfosalicylic acid over 30 min at 4 • C at a resulting pH of about 2.2 (pH less than 2.5 is recommended for cation exchange chromatography). The samples were centrifuged (15 min, 4 • C, 6827× g), filtered (45 µm) and then frozen at −20 • C until further use. The centrifugation and filtration were repeated directly before the analysis.

Amino Acid Content Determination
The samples were analyzed in duplicates with internal (l-norleucine-and l-thialysine-solution, see Section 2.3.1) and external AA standards. The external standards were worked up similar to the meat samples. A regularly calibration of the amino acid analyzer was done with a certified AA standard (amino acid mix solution [17]). For the cation exchange chromatography, a column with 3 µm beads with a separation over a pH-range from 2.9 to 10.4 and ninhydrin post column derivatization was performed. An injection volume of 20 µL (pH 2.2) and a flow rate of 180 µL/min were applied. For the spectrophotometric analysis two photometers with wavelengths of 440 nm and 570 nm were used for detection of the free amino acids. The limit of detection (LOD) and quantification (LOQ) were defined as three and ten times the signal to noise ratio of the external standard solution, respectively. All measured contents of the free amino acids (FAA) were above the LOQ (0.13 mg/100 g-0.33 mg/100 g, depending on the different AA).

Sample Preparation for Metabolomics Study of White Breast Meat
The sample set consisted of study samples, mix samples and blanks. The mix samples were prepared by combining an aliquot from each muscle powder and served for normalization. All sample types were extracted in the same manner. Study samples were extracted in triplicates. In a first step 20 mg of the dried, homogenous meat powder was extracted with 600 µL ice-cold 80% methanol containing 10 internal standards using a bead mill homogenizer (Minilys, Bertin Technologies SAS, Montigny-le-Bretonneux, France) for 2 times 30 s and an ultrasonic bath for 2 min. The raw extract was centrifuged at 15,000× g for 20 min at 4 • C. In a second step the pellet was re-extracted with 600 µL ice-cold methanol:chloroform (2:1 v/v) according to the first extraction step. Both supernatants were combined, mixed and centrifuged at 15,000× g for 20 min at 4 • C. 100 µL of the supernatant were transferred into 2 mL glass vials containing a 200 µL glass insert and evaporated in a vacuum centrifuge (Christ Speedvac RVC 2¨C18 CD plus, Germany). The dried samples were stored under protective argon atmosphere at −80 • C until analysis.

GC-MS Measurements and Data Processing
Prior to measurement samples were derivatized by methoximation and trimethylsilylation. For methoximation samples were shaken in 30 µL of a 20 mg/mL solution of MAH in pyridine at 50 • C for 1 h. Subsequently, 70 µL MSTFA+1%TMCS were added and samples shook at 70 • C for 1 h. GC-MS analysis was performed on a Shimdazu GCMS QP2010 instrument (Shimadzu, Duisburg, Germany) equipped with an OPTIC-4 injector (GL Sciences, Eindhoven, The Netherlands). A 1 µL aliquot of each sample was injected in a 1:7 split ratio. A series of n-alkanes (C7-C30) was used as a retention time standard. Analytes were separated on a 30 m Rxi-5SIL MS column containing a 10 m Integra-Guard column (Restek, 0.25.mm i.d., 0.25 µm film thickness), and with a linear temperature gradient starting from 80 • C to 300 • C with 5 • C/min and a final 5 min hold at 320 • C. Masses between 60 and 600 m/z were scanned.
For data analysis only those features were selected that were not present in blank samples. The annotation of compounds was performed using the NIST 14 library database implemented in GCMSsolution (Shimadzu, Duisburg, Germany). The retention times and selected masses used for relative quantification are listed in Supplementary Table S1. Samples were normalized according to the calculated means of the mix samples for each feature to reduce the impact of device maintenance (instrument tuning, liner exchange and septum exchange during the measurement batch). Briefly, signal intensities of the mix-samples within one measurement period (between device maintenance) were averaged as well as for the whole measurement batch. Using these means a correction factor was determined between the total means and the means for each measurement period between device maintenances. These correction factors were then applied to the corresponding features of study samples and calibration samples.

Amino Acid Quantification
For AA quantification a calibration curve consisting of a reference standard mixture and 8 final concentrations in the range 1.96-250 pmol/µL was applied. These standard mixes were spiked into aliquots from pooled REF samples (50 µL of standard mixture into 50 µL of REF mixture) to reduce for the impact of the sample matrix. The calibration samples were prepared in duplicates. Calibration samples were evaporated in a vacuum centrifuge and prepared for GC-MS measurement according to Section 2.4.2. For quantification, the calibration samples were at first normalized and then the mean values (ion counts) of selected quantitative ions from calibration-reference samples were subtracted from calibration samples containing the standard mixtures. Calibration coefficients were R 2 > 0.99 with acceptance of alanine which had an R 2 = 0.97). The retention times and selected masses used for quantification are listed in Supplementary Table S2.

Sample Preparation for Metabolomics Study of White Breast Meat
The same dried, homogenous breast muscle samples as used for GC-MS were prepared for NMR metabolomics as described previously by Wagner et al. [18] with slight modifications. In brief, 20 mg of lyophilized, grinded, homogeneous muscle powder was extracted using first ice-cold methanol, then ice-cold chloroform and finally ice-cold water (400 µL of each solvent). The samples were vortexed for 1 min and stored on ice for 10 min between each step, and then stored at 4 • C overnight for separation and finally after centrifugation (2000× g, 4 • C, 30 min) the aqueous phase was collected in a new tube. The collected samples (750 µL) were dried using a vacuum centrifuge. The dried samples were redissolved with 550 µL D 2 O, 25 µL MilliQ water and 25 µL D 2 O containing 0.05 wt% TSP as internal standard for quantification and chemical shift reference.

1 H NMR Spectroscopy, Data Processing and Identification of the Signals
All samples were analyzed with a Bruker 400 MHz spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). For the aqueous white breast muscle, a noesygppr1d pulse program at 25 • C with 64 scans, a spectral width of 8224 Hz collected into 65,536 data point and acquisition time of 3.98 s and an interscan relaxation delay of 4 s was used. 1 H-1 H COSY, 1 H-1 H TOCSY and 1 H-13 C HSQC were obtained on one representative muscle sample for metabolite identification purposes.
All data were processed using Bruker Topspin 3.6.0 software (Bruker), Fourier-transformed after multiplication by line broadening of 0.30 Hz and subsequently referenced to standard peak TSP at 0.00 ppm. After spectral phase and baseline were corrected, each NMR spectrum was integrated using Matlab R2017b (Mathworks, Natick, MA, USA) into 0.01 ppm integral regions (buckets) between 8.60 ppm and 0.80 ppm (area between 4.75 ppm and 4.80 ppm corresponding to the water signal was excluded). Each muscle spectrum region was scaled to the intensity of internal standard (TSP) for quantitative measurements. Afterwards, the signals were identified using ChenomX NMR Suite 8.4 library (ChenomX Inc., Edmonton, AB, Canada), the Human Metabolome Database (www.hmdb.ca) and previous literature [18][19][20] and confirmed with 2D-NMR in case of multiplicity. For quantification (profiling approach), 86 metabolites were identified by overlapping with standard spectra (Supplementary Table S3) and their concentrations (µmol/mg) were calculated using ChenomX NMR Suite 8.4 library after accounting for overlapping signals. The absolute concentrations were presented as mg/100 g wet weight (Supplementary Table S4).

Statistical Analysis
Multivariate data analyses were performed for the GC-MS data, the NMR spectral data (buckets) and the absolute concentrations of the metabolites (profiling approach) using the Simca-P+ software (version 13.0; Umetrics, Umeå, Sweden). All variables were centered and "pareto-scaled" (Par) (GC-MS data and NMR spectral data) or "unit variance" (UV)-scaled (NMR data, absolute concentrations). Principal component analysis (PCA) was used to screen the data and search for outliers. Outliers were determined using PCA-Hotelling T 2 Ellipse (95% confidential interval (CI)).
All statistical calculations such as one-way analysis of variance (ANOVA) and Dunnett's test were done in JMP (13.1.0, SAS Institute Inc., Cary, NC, USA).
The amino acid contents (alanine, leucine, methionine, phenylalanine, proline, serine, tyrosine, histidine, lysine and glutamate) determined by HPLC, GC-MS and NMR are presented as mg/100 g wet weight, henceforth referred to as mg/100 g. All data presented are mean ± standard deviation and differences were considered significant when p < 0.01.

Results and Discussion
The aim of this study was to compare different analytical techniques in respect to their performance for the detection of undeclared protein hydrolysates in fresh turkey breast. For this purpose, a traditional HPLC-UV/VIS approach focusing on the detection of free proteinogenic amino acids was compared with two nontargeted metabolic profiling techniques, GC-MS and 1 H-NMR. Additionally, both nontargeted approaches were compared for their suitability in the detection of the adulterated turkey breast muscle with protein hydrolysates.

Capability of Amino Acid Profiling for the Detection of Added Protein Hydrolysates
The contents of the ten free amino acids (FAA) alanine, leucine, methionine, phenylalanine, proline, serine, tyrosine, histidine, lysine and glutamate out of the 20 proteogenic AA were analyzed by all methods. Those ten AA were selected as exemplary free amino acids with different properties, e.g., aliphatic (alanine, leucine, proline), aromatic (phenylalanine, tyrosine), acidic (glutamate), basic (lysine, histidine), hydroxylic (serine) and sulfur-containing (methionine). The results of five groups (REF and addition of water, gelatin-, wheat-and casein-hydrolysates) were compared depending on the hydrolyzation degree (PH or TH).
The contents of FAA for the addition of partial and total protein hydrolysates are shown in Figures 1 and 2, respectively. All these data are also summarized in the Supplementary Table S5. The reference samples (REF) were not modified and variations were therefore only occurring through the analytical error and natural variations of the FAA contents. The natural FAA contents depend on several conditions, e.g., the gender of the birds [21] or special feed additives [22].
It is obvious that the amount of FAA contents determined by HPLC-UV/VIS (in mg/100 g) and GC-MS as well as 1 H-NMR (in mg/100 g) are quite different. The FAA contents determined by GC-MS and 1 H-NMR are on average 3.8 times (range: 0.6-fold to 7.0-fold) and 3.3 times (range: 0.1-fold to 10.2-fold) higher, respectively, compared with the contents determined by HPLC-UV/VIS. The differences can be arising from the different sample preparations. One possibility could be that the homogenization method (HPLC-UV/VIS) was not able to dissolve all FAA. The homogenization method for HPLC did not contain any specific extraction step with solvents, whereas for GC-MS and 1 H-NMR the samples were extracted using mixtures of water, methanol and chloroform. Moreover, it is possible that the extraction method (GC-MS and 1 H-NMR) led to protein hydrolysis. However, the different quantification procedures could also have caused these differences. Comparative experiments (e.g., with protease inhibitors) clearly demonstrated that no protein hydrolysis occurred by using the homogenization method (manuscript in preparation). This method was also used to determinate 18 of the 20 proteinogenic FAA contents of chicken breast meat (manuscript in preparation) and the contents were in agreement with Rikimaru and Takahashi [23].
The addition of water to the turkey breast muscle resulted in tendentially lower contents of FAA, but statistical significance was not reached by using the Dunnett's test (Figures 1 and 2 and Supplementary Table S5). The reduced mean values in water treated samples could be explained as dilution or even wash-out effect. When the amount of water injected to the sample exceeds its water binding capacity, some endogenous compounds (e.g., FAA) might be washed out.

Comparison of Partial Hydrolyzed Wheat-, Gelatin-and Casein-Hydrolysates
Partial enzymatic hydrolysates from gelatin (GPH, hydrolyzation degree 15% ± 3%), wheat (WPH, hydrolyzation degree: 16% ± 2%) and casein (CPH, hydrolyzation degree 53% ± 2%) were added to the meat samples, respectively ( Figure 1). WPH and GPH were only slightly hydrolyzed and therefore most of the protein was converted to peptides. Therefore, the amount of FAA in these two hydrolysates was lower compared to CPH, which was hydrolyzed to a higher hydrolyzation degree.
Hence, nearly no significant differences were found for the FAA contents of GPH and WPH related to the REF. Only the content of free lysine ( 1 H-NMR, from 5.26 mg/100 g ± 0.45 mg/100 g (REF) to 26.9 mg/100 g ± 3.37 mg/100 g) for GPH, as well as the free methionine (HPLC-UV/VIS) and free leucine contents ( 1 H-NMR) for WPH showed significant differences (Supplementary Table S5).
Contrary to this, the CPH showed clearly significant different FAA contents compared to the REF. As determined with HPLC-UV/VIS-method, the FAA contents of leucine, methionine, phenylalanine and histidine were highly significant different (p < 0.001) and for serine significant different (p < 0.01). For example, the FFA content increased for leucine from 1.97 mg/100 g ± 0.33 mg/100 g (REF) to 19.9 mg/100 g ± 2.39 mg/100 g (CPH). The FAA contents analyzed with GC-MS showed significant differences for five of the ten listed FAA. Leucine was also highly increased (6.97 mg/100 g ± 2.78 mg/100 g for REF to 111 mg/100 g ± 26.4 mg/100 g (CPH), p < 0.001), as well as methionine, phenylalanine and lysine. The histidine content was increased significantly (p < 0.01). The analysis with 1 H-NMR showed seven increased FAA contents: leucine, methionine, phenylalanine, proline and lysine increased highly (p < 0.001), whereas alanine and serine increased significantly (p < 0.01). For example, leucine increased from 4.26 mg/100 g ± 1.25 mg/100 g (REF) to 78.6 mg/100 g ± 19.6 mg/100 g (CPH).   Partial enzymatic hydrolysates from gelatin (GPH, hydrolyzation degree 15% ± 3%), wheat (WPH, hydrolyzation degree: 16% ± 2%) and casein (CPH, hydrolyzation degree 53% ± 2%) were added to the meat samples, respectively (Figure 1). WPH and GPH were only slightly hydrolyzed and therefore most of the protein was converted to peptides. Therefore, the amount of FAA in these two hydrolysates was lower compared to CPH, which was hydrolyzed to a higher hydrolyzation degree. For a clear proof, several FAA contents should differ significantly from the reference sample. In this way other reasons for different FAA contents (e.g., feed supplementation with AA) can be excluded with higher probability. It can be concluded in this study, that in case of partial hydrolysate treatment the detection of fraud could not be ensured by using only these ten FAA contents.

Comparison of Total Hydrolyzed Wheat-, Gelatin-and Casein-Hydrolysates
All the total hydrolyzed proteins (GTH, WTH, CTH) showed a hydrolyzation degree of 100% and were therefore only composed of AA. As expected, the FAA contents of all hydrolysate-treated samples were increased dramatically compared to the REF (Figure 2). The analysis with HPLC-UV/VIS revealed two significant and seven highly significant increases of FAA contents for GTH, eight highly significant increased FAA contents for WTH and nine rises of the amounts of FAA for CTH (p < 0.001). For example, the content of proline changed from 2.56 mg/100 g ± 0.42 mg/100 g (REF) to 52.5 mg/100 g ± 20.4 mg/100 g (GTH), 44.9 mg/100 g ± 16.8 mg/100 g (WTH) and 39.2 mg/100 g ± 13.8 mg/100 g (CTH), respectively. With GC-MS, only highly significant changes were found: for GTH seven, for WTH nine and for CTH all ten FAA contents were increased. As for HPLC-UV/VIS, the proline content was raised obviously: from 10.0 mg/100 g ± 5.81 mg/100 g to 134 mg/100 g ± 71.4 mg/100 g (GTH), 161 mg/100 g ± 31.8 mg/100 g (WTH) and 160 mg/100 g ± 30.5 mg/100 g (CTH), respectively. The same results were also found for the 1 H-NMR analysis: one significant and eight highly significant increases for GTH (proline: from 5.06 mg/100 g ± 0.78 mg/100 g (REF) to 77.5 mg/100 g ± 16.3 mg/100 g), and nine highly significant differences for WTH (proline: 72.8 mg/100 g ± 12.1 mg/100 g) and CTH (proline: 53.8 mg/100 g ± 16.1 mg/100 g). Therefore, only tyrosine (GTH), alanine and lysine (WTH) as well as alanine (CTH) showed no significant differences determined by HPLC-UV/VIS. For the GC-MS-method, only methionine, tyrosine, histidine (GTH) and lysine (WTH) were not significantly different. The analysis by 1 H-NMR revealed also nearly exclusive significant differences with only few exceptions (tyrosine, histidine for GTH, histidine for WTH and CTH).
Depending on the hydrolysate type, different AA were more affected. The addition of GTH resulted in higher levels of alanine, whereas WTH showed higher levels of glutamate and CTH higher levels of leucine, methionine, tyrosine and lysine (Figure 2 and Supplementary Table S5). The latter AA might be used as an indicator for animal-based protein origins whereas glutamate could indicate plant-based protein origins.
It was shown in this study that in case of total hydrolysate treatment a general detection of fraud is possible.

General Aspects Regarding the Detection of Free Amino Acids in Treated Breast Muscles
All three methods used (HPLC-UV/VIS, GC-MS, 1 H-NMR) showed comparable results. Although the FAA contents of the first method were about three-to fourfold lower compared to the other two methods, the validity was given. This is due to the fact that all samples were compared to the corresponding REF, determined with the same method. Hence, the method of sample preparation plays an important role for absolute quantities, whereas regarding the differentiation between REF and hydrolyzed-treated samples (rations are kept independently of the sample preparation) the method has no impact.
It can be concluded that the differentiation of hydrolysate addition depends on the degree of hydrolyzation. If breast muscles were treated with low degree hydrolysates, the additional injected FAA might not induce a significant increase over the range of natural variation. It was shown that a high hydrolyzation degree significantly increased the free AA content of several AA independently of which analytical technique (HPLC-UV/VIS, GC-MS or 1 H-NMR) was used.
Specific FAA profiles might be used for a tentative classification of the origin of the hydrolysate type (e.g., plant-based vs. animal-based protein origins). Nevertheless, a clear classification and identification of the protein used for hydrolyzation was not possible. Thus, it is of interest whether further information about additional compounds might be helpful for the detection and classification of the hydrolysates. For this, the following hypotheses for section two were postulated: (1) Original protein sources are not clean and contain additional compounds, which can be introduced into the breast meat. (2) Acidic hydrolysis leads to formation of byproducts and these compounds are also possible to be found in the breast meat. (3) Additional metabolites can be washed out from the breast meat and (4) therefore, information from metabolite profiling might be of interest and was included in the analysis.

Metabolomics Approaches to Obtain Additional Information Regarding Hydrolysate-Treated Samples Independently of the Hydrolyzation Degree
The detection of hydrolysate treatment in turkey breast muscle by amino acid profiling largely depends on the hydrolyzation degree. Our results clearly indicated that an addition of total hydrolyzation increases the free amino acid content tremendously (Figure 2) so that a detection with all three presented methods was possible. However, the lower the hydrolyzation degree the more uncertain is the validity of amino acid profiles between the natural variation and the differentiation due to hydrolysate treatment. Therefore, we applied two nontargeted metabolite profiling approaches (GC-MS and 1 H-NMR) to test for their suitability in the detection of hydrolysate treatment in turkey breast muscle. Both approaches allow to detect additionally several metabolites besides the proteogenic amino acids like carbohydrates, organic acids, lipids, et cetera. For both techniques PCA was used to check for the variation of metabolite profiles between controls and treatments and between the different types of hydrolysates ( Figure 3).  The total hydrolysate treated samples clearly separated from the controls in PC1 observed with both techniques. Interestingly, with GC-MS analysis the wheat and casein origins showed higher similarities to each other compared to gelatin (GTH). Whereas for NMR analyses, higher similarities were observed between GTH and WTH. Obviously, independently of the analytical technique, there is a clear separation between gelatin (GTH) and casein (CTH). From the loading plot (Figure 3c,d), it can be deduced that proteinogenic amino acids particularly contribute to the differentiation of the total hydrolysate treated samples and the controls (in PC1), which is in accordance to the results presented in 3.1. Nevertheless, besides the amino acids other compounds could be identified which play an additional role for the variation in PC1 such as hydroxyproline, levulinic acid, ornithine or glycerol (the complete feature tables are presented in Supplementary Tables S3 and S4). Compounds, which were additionally detected by 1 H-NMR were pyruvate and acetate. Further compounds detected by GC-MS were 5-hydroxylysine, 3-MCPD or aminomalonic acid among several nonidentified molecular features. The total hydrolysate treated samples clearly separated from the controls in PC1 observed with both techniques. Interestingly, with GC-MS analysis the wheat and casein origins showed higher similarities to each other compared to gelatin (GTH). Whereas for NMR analyses, higher similarities were observed between GTH and WTH. Obviously, independently of the analytical technique, there is a clear separation between gelatin (GTH) and casein (CTH). From the loading plot (Figure 3c,d), it can be deduced that proteinogenic amino acids particularly contribute to the differentiation of the total hydrolysate treated samples and the controls (in PC1), which is in accordance to the results presented in 3.1. Nevertheless, besides the amino acids other compounds could be identified which play an additional role for the variation in PC1 such as hydroxyproline, levulinic acid, ornithine or glycerol (the complete feature tables are presented in Supplementary Tables S3 and S4). Compounds, which were additionally detected by 1 H-NMR were pyruvate and acetate. Further compounds detected by GC-MS were 5-hydroxylysine, 3-MCPD or aminomalonic acid among several nonidentified molecular features. These additional compounds represent characteristics of the protein origin or are byproducts formed ), Water ( The total hydrolysate treated samples clearly separated from the controls in PC1 observed with both techniques. Interestingly, with GC-MS analysis the wheat and casein origins showed higher similarities to each other compared to gelatin (GTH). Whereas for NMR analyses, higher similarities were observed between GTH and WTH. Obviously, independently of the analytical technique, there is a clear separation between gelatin (GTH) and casein (CTH). From the loading plot (Figure 3c,d), it can be deduced that proteinogenic amino acids particularly contribute to the differentiation of the total hydrolysate treated samples and the controls (in PC1), which is in accordance to the results presented in 3.1. Nevertheless, besides the amino acids other compounds could be identified which play an additional role for the variation in PC1 such as hydroxyproline, levulinic acid, ornithine or glycerol (the complete feature tables are presented in Supplementary Tables S3 and S4). Compounds, which were additionally detected by 1 H-NMR were pyruvate and acetate. Further compounds detected by GC-MS were 5-hydroxylysine, 3-MCPD or aminomalonic acid among several nonidentified molecular features. These additional compounds represent characteristics of the protein origin or are byproducts formed The total hydrolysate treated samples clearly separated from the controls in PC1 observed with both techniques. Interestingly, with GC-MS analysis the wheat and casein origins showed higher similarities to each other compared to gelatin (GTH). Whereas for NMR analyses, higher similarities were observed between GTH and WTH. Obviously, independently of the analytical technique, there is a clear separation between gelatin (GTH) and casein (CTH). From the loading plot (Figure 3c,d), it can be deduced that proteinogenic amino acids particularly contribute to the differentiation of the total hydrolysate treated samples and the controls (in PC1), which is in accordance to the results presented in 3.1. Nevertheless, besides the amino acids other compounds could be identified which play an additional role for the variation in PC1 such as hydroxyproline, levulinic acid, ornithine or glycerol (the complete feature tables are presented in Supplementary Tables S3 and S4). Compounds, which were additionally detected by 1 H-NMR were pyruvate and acetate. Further compounds detected by GC-MS were 5-hydroxylysine, 3-MCPD or aminomalonic acid among several nonidentified molecular features. These additional compounds represent characteristics of the protein origin or are byproducts formed The total hydrolysate treated samples clearly separated from the controls in PC1 observed with both techniques. Interestingly, with GC-MS analysis the wheat and casein origins showed higher similarities to each other compared to gelatin (GTH). Whereas for NMR analyses, higher similarities were observed between GTH and WTH. Obviously, independently of the analytical technique, there is a clear separation between gelatin (GTH) and casein (CTH). From the loading plot (Figure 3c,d), it can be deduced that proteinogenic amino acids particularly contribute to the differentiation of the total hydrolysate treated samples and the controls (in PC1), which is in accordance to the results presented in 3.1. Nevertheless, besides the amino acids other compounds could be identified which play an additional role for the variation in PC1 such as hydroxyproline, levulinic acid, ornithine or glycerol (the complete feature tables are presented in Supplementary Tables S3 and S4). Compounds, which were additionally detected by 1 H-NMR were pyruvate and acetate. Further compounds detected by GC-MS were 5-hydroxylysine, 3-MCPD or aminomalonic acid among several nonidentified molecular features. These additional compounds represent characteristics of the protein origin or are byproducts formed The total hydrolysate treated samples clearly separated from the controls in PC1 observed with both techniques. Interestingly, with GC-MS analysis the wheat and casein origins showed higher similarities to each other compared to gelatin (GTH). Whereas for NMR analyses, higher similarities were observed between GTH and WTH. Obviously, independently of the analytical technique, there is a clear separation between gelatin (GTH) and casein (CTH). From the loading plot (Figure 3c,d), it can be deduced that proteinogenic amino acids particularly contribute to the differentiation of the total hydrolysate treated samples and the controls (in PC1), which is in accordance to the results presented in 3.1. Nevertheless, besides the amino acids other compounds could be identified which play an additional role for the variation in PC1 such as hydroxyproline, levulinic acid, ornithine or glycerol (the complete feature tables are presented in Supplementary Tables S3 and S4). Compounds, which were additionally detected by 1 H-NMR were pyruvate and acetate. Further compounds detected by GC-MS were 5-hydroxylysine, 3-MCPD or aminomalonic acid among several nonidentified molecular features. These additional compounds represent characteristics of the protein origin or are byproducts formed ), GTH ( The total hydrolysate treated samples clearly separated from the controls in PC1 observed with both techniques. Interestingly, with GC-MS analysis the wheat and casein origins showed higher similarities to each other compared to gelatin (GTH). Whereas for NMR analyses, higher similarities were observed between GTH and WTH. Obviously, independently of the analytical technique, there is a clear separation between gelatin (GTH) and casein (CTH). From the loading plot (Figure 3c,d), it can be deduced that proteinogenic amino acids particularly contribute to the differentiation of the total hydrolysate treated samples and the controls (in PC1), which is in accordance to the results presented in 3.1. Nevertheless, besides the amino acids other compounds could be identified which play an additional role for the variation in PC1 such as hydroxyproline, levulinic acid, ornithine or glycerol (the complete feature tables are presented in Supplementary Tables S3 and S4). Compounds, which were additionally detected by 1 H-NMR were pyruvate and acetate. Further compounds detected by GC-MS were 5-hydroxylysine, 3-MCPD or aminomalonic acid among several nonidentified molecular features. These additional compounds represent characteristics of the protein origin or are byproducts formed The total hydrolysate treated samples clearly separated from the controls in PC1 observed with both techniques. Interestingly, with GC-MS analysis the wheat and casein origins showed higher similarities to each other compared to gelatin (GTH). Whereas for NMR analyses, higher similarities were observed between GTH and WTH. Obviously, independently of the analytical technique, there is a clear separation between gelatin (GTH) and casein (CTH). From the loading plot (Figure 3c,d), it can be deduced that proteinogenic amino acids particularly contribute to the differentiation of the total hydrolysate treated samples and the controls (in PC1), which is in accordance to the results presented in 3.1. Nevertheless, besides the amino acids other compounds could be identified which play an additional role for the variation in PC1 such as hydroxyproline, levulinic acid, ornithine or glycerol (the complete feature tables are presented in Supplementary Tables S3 and S4). Compounds, which were additionally detected by 1 H-NMR were pyruvate and acetate. Further compounds detected by GC-MS were 5-hydroxylysine, 3-MCPD or aminomalonic acid among several nonidentified molecular features. These additional compounds represent characteristics of the protein origin or are byproducts formed ) and CTH ( The total hydrolysate treated samples clearly separated from the controls in PC1 observed with both techniques. Interestingly, with GC-MS analysis the wheat and casein origins showed higher similarities to each other compared to gelatin (GTH). Whereas for NMR analyses, higher similarities were observed between GTH and WTH. Obviously, independently of the analytical technique, there is a clear separation between gelatin (GTH) and casein (CTH). From the loading plot (Figure 3c,d), it can be deduced that proteinogenic amino acids particularly contribute to the differentiation of the total hydrolysate treated samples and the controls (in PC1), which is in accordance to the results presented in 3.1. Nevertheless, besides the amino acids other compounds could be identified which play an additional role for the variation in PC1 such as hydroxyproline, levulinic acid, ornithine or glycerol (the complete feature tables are presented in Supplementary Tables S3 and S4). Compounds, which were additionally detected by 1 H-NMR were pyruvate and acetate. Further compounds detected by GC-MS were 5-hydroxylysine, 3-MCPD or aminomalonic acid among several nonidentified molecular features. These additional compounds represent characteristics of the protein origin or are byproducts formed With GC-MS a total of 129 features were considered for PCA. The first (horizontal) and second (vertical) components explained 38.0% and 15.7% of the variation, respectively, with R 2 X = 98.4%, Q 2 = 72.8% (Figure 3a). A clear separation between the controls (REF and water-treated control) and five of the hydrolysate-treated sample groups was observed. GPH did not segregate from the controls. WPH varied only in PC2 direction whereas CPH, with 53% hydrolyzation degree, stronger differentiated in PC1 direction. The total hydrolysate treated sample groups showed most variation in PC1 direction.
In addition, the 1 H-NMR spectra obtained were compared by PCA (PC1 vs. PC2) (Figure 3b). The first component (horizontal) which is explained by 40.8% of spectral variation clearly separates controls (REF and water-treated control, left) with total hydrolyzed-treated samples (right) and partial hydrolyzed-treated samples (middle). The second component explained 21.9% of variation and separates the controls and total hydrolyzed-treated samples (top) from partial hydrolyzed (bottom) samples. The model parameters were the following: R 2 X = 98.5%, Q 2 = 93.4%, 16 components. In order to identify metabolic changes, the absolute concentrations of 86 metabolites were quantified through a profiling approach from 1 H-NMR spectra.
The total hydrolysate treated samples clearly separated from the controls in PC1 observed with both techniques. Interestingly, with GC-MS analysis the wheat and casein origins showed higher similarities to each other compared to gelatin (GTH). Whereas for NMR analyses, higher similarities were observed between GTH and WTH. Obviously, independently of the analytical technique, there is a clear separation between gelatin (GTH) and casein (CTH). From the loading plot (Figure 3c,d), it can be deduced that proteinogenic amino acids particularly contribute to the differentiation of the total hydrolysate treated samples and the controls (in PC1), which is in accordance to the results presented in Section 3.1. Nevertheless, besides the amino acids other compounds could be identified which play an additional role for the variation in PC1 such as hydroxyproline, levulinic acid, ornithine or glycerol (the complete feature tables are presented in Supplementary Tables S3 and S4). Compounds, which were additionally detected by 1 H-NMR were pyruvate and acetate. Further compounds detected by GC-MS were 5-hydroxylysine, 3-MCPD or aminomalonic acid among several nonidentified molecular features. These additional compounds represent characteristics of the protein origin or are byproducts formed during the acidic hydrolyzation process. In addition to the amino acid profiles these byproducts might contribute to a better classification of the protein sources.
The wheat protein source contained higher amounts of sugars, which was also observed for the WPH treated breast samples (see below). During the acidic hydrolysis of the protein source, the sugars contained therein such as maltose, saccharose, glucose or fructose are converted to levulinic acid in presence of hydrochloride and under high temperature [24,25]. Thus, the high levels of levulinic acid detected in our analyses could be used to differentiate plant-based hydrolysates and as a marker for acidic hydrolyzation treatment. Nevertheless, other plant-based hydrolysates need to be tested for their carbohydrate content in contrast to animal-based protein sources.
The total protein hydrolysate from gelatin contained higher amounts of AA derivates such as hydroxyproline and hydroxylysine. Gelatin is the denatured form of collagen, which is one of the most abundant proteins in meat, ranging between 2 and 4 mg/g in chicken breast meat [26]. Most abundant amino acids of collagen are glycine, proline, glutamate and hydroxyproline [27]. Hydroxyproline is specific to collagen and its concentration in collagen is rather constant with 12% [28]. Therefore, hydroxyproline is used to estimate the connective tissue content [29,30]. Regarding the treatment of turkey breast meat with protein hydrolysates, the hydroxyproline content can serve as a marker for animal-based protein sources such as gelatin. Hydroxylysine is another modified amino acid, which is unique to collagens. Similar to hydroxyproline, this amino acid becomes posttranslational hydroxylated and subsequently glycosylated forming the α-helical structure of collagens [31]. Therefore, 5-hydroxylysine might serve as an additional indicator of gelatin hydrolysate treatment. Aminomalonic acid that was most abundant in GTH followed by CTH treated samples represents an amino acid derivative, whose origin is suspected to be related to protein oxidation processes [32] and to play a role in the serine-glycine interconversion [33]. According to our results, the acidic hydrolysis process might increase the formation of aminomalonic acid in dependence of the protein source, namely the glycine-rich gelatin.
For casein, the second tested animal derived hydrolysate, it was not that a particular molecule was strongly increased, but the combination of several molecular features could hint towards this treatment. In addition to the amino acid profile, the casein treated samples had higher levels of 3-MCPD and a number of not-identified molecular features (Supplementary Table S1).
Interestingly, the acidic treatment of the different protein sources led to the formation of 3-MCPD. This compound can be found in numerous foodstuffs and is described to be present in acidic hydrolysates of proteins [34]. Depending on the remaining lipids in the original protein sources, different amounts of 3-MCPD and 3-MCPD fatty acid esters might be formed and injected into the breast meat. Thus, 3-MCPD represents an additional marker for acidic hydrolysis, similar to levulinic acid.
From the score plots of the partial hydrolysate treated breast muscles, we observed a clear separation of all three sample groups using 1 H-NMR technique, whereas by GC-MS analysis the GPH group largely overlapped with the controls. The WPH treated samples were in a medium distance to the controls, and the highest variation to the controls was observed for CPH treated samples. Those observations are in accordance with the different degrees of hydrolyzation in the partial hydrolysates with GPH having a hydrolyzation degree of 15% and CPH having a hydrolyzation degree of 53%. Even though WPH has a hydrolyzation degree of only 16%, the better separation compared to GPH might be explained by the plant-based origin and the present additional metabolites.
A closer look at the loading plots from the PCA models for control samples and partial hydrolysate treated samples indicated that proteinogenic amino acids play a minor role for the variation between controls (and GPH) and WPH, both having a low hydrolyzation degree (15% and 16%, respectively). The variation of CPH from controls, which was to 53% hydrolyzed, was already dominated by proteinogenic amino acids. Especially the plant-based hydrolysate contained additional sugars such as maltose ( Figure 4) and hexoses like glucose, detected with both technical approaches. In addition, higher levels of glycerol were detected in WPH. As mentioned above, future studies will have to elucidate to which extent different sugars are present in plant-based protein extracts used for hydrolysis. With the animal-based protein hydrolysates, GPH and CPH, the contents of ornithine ( Figure 4) were increased as detected with 1 H-NMR and GC-MS. A low level of levulinic acid and 3-MCPD was observed in CPH treated breast muscle, which might be related to the CPH production process (CPH was commercially obtained). Using 1 H-NMR technique, higher amounts of acetate (GPH, WPH, CPH), butyrate (CPH), carnitine (WPH, CPH), citrate (WPH, CPH), glutathione (WPH, CPH), pantothenate (CPH) and putrescine (GPH, WPH, CPH), myo-inositol (GPH, WPH) were additionally detected ( Figure 4 and Supplementary Table S4), whereas with GC-MS analysis we obtained increased levels of oxoproline (CPH), urea (CPH), hydroxylysine (GPH) and malic acid (GPH, WPH, CPH) among a few nonidentified compounds (Figure 4 and Supplementary Table S1). It can be concluded that the lower the hydrolyzation degree the more important are the additional compounds from the protein origins for the differentiation of nontreated samples and hydrolysate treated samples.  In accordance with the reduced AA content in water treated samples, we observed for several endogenous metabolites of turkey breast muscle a similar reduction when samples were injected with the different kinds of hydrolysates. This effect was particularly obvious for highly water-soluble compounds such as creatinine and lactate, which were detected by GC-MS and 1 H-NMR. Additionally, reduced levels (not significant) of myo-inositol and the peptides glutathione and anserine were detected In accordance with the reduced AA content in water treated samples, we observed for several endogenous metabolites of turkey breast muscle a similar reduction when samples were injected with the different kinds of hydrolysates. This effect was particularly obvious for highly water-soluble compounds such as creatinine and lactate, which were detected by GC-MS and 1 H-NMR. Additionally, reduced levels (not significant) of myo-inositol and the peptides glutathione and anserine were detected by 1 H-NMR. With GC-MS profiling we detected reduced levels of 4-hydroxybutanoic acid, myo-inositol, inosine and uracil among several nonidentified molecular features (Supplementary Table S1). In our approach, sample preparation was performed using~2 g fresh turkey breast meat to minimize the effect of natural variation when comparing different hydrolysate types. Whether the observed wash-out effect can also be detected by using whole breast muscles has to be validated by further studies. It can be suspected that the natural variation has a greater impact than the detected small levels of a wash-out.

Conclusions
This study aimed at a comparison between different analytical methods and their possibility to detect adulteration of turkey breast meat with different hydrolysates. It showed that FAA profiling allows for the detection of protein hydrolysate treatments only above a certain threshold which is mainly related to the degree of hydrolyzation. The samples naturally strongly differ in their free amino acid contents as a result of feeding, genotype and meat age. Therefore, the FAA analyses under these conditions (e.g., determination of only ten FAA contents) were not suitable for the detection of food fraud in the case of partial hydrolysates. To overcome this limitation, the contents of more than ten FAA of the 20 proteinogenic AA should be analyzed. Further on, a much higher quantity of samples ought to be measured. The evaluation of these datasets enables the reduction of the variations and therefore more significant differences. The additions of hydrolysates with high amounts of AA to breast meat were easily proofed within this study.
The different profiling techniques revealed that protein sources contain different metabolites, which can be used as biomarkers for the detection of partial hydrolysates.
Furthermore, byproducts formed during acidic hydrolysis provide additional evidence for the treatment of breast meat with protein hydrolysates. Therefore, a combination of FAA and metabolite (by-products) profiling makes it possible to identify and classify the addition of nondeclared hydrolysates to turkey breast meat. In addition, an advantage of this comprehensive analysis is that it might be possible to proof the addition of animal-based proteins or animal-based hydrolysates to vegetarian or vegan products. According to the advantages of NMR in terms of sample throughput and direct quantification of the identified compounds, 1 H-NMR is used in further/detailed studies analyzing food fraud of turkey breast meat.
Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/9/8/1084/s1, Table S1: Molecular features used for GC-MS profiling. Annotation was performed with the NIST 14 library implemented in the GCMSsolution software (Shimadzu, Duisburg, Germany). The signal intensities of the quantifier ions were used for the relative quantitation and comparison between sample groups. According to the presented FAA contents the Dunnett's test was used for a comparison between control (REF) and the different treatments.; Table S2: Quantification of amino acids via GC-MS was performed using the following parameters.; Table S3: Assignment of 1 H-NMR signals which could be identified via the software ChenomX NMR Suite 8.4 library. 86 metabolites were identified and exemplary one NMR signal (ppm) was chosen which was the obvious signal for identification.; Table S4: Significantly different absolute concentrations of metabolites in turkey breast meat treated with different hydrolysates and analyzed via 1 H-NMR (mg/100 g).; Table S5: Significantly different absolute concentrations (mg/100 g) of amino acids in turkey breast meat treated with different hydrolysates and analyzed via HPLC-UV/VIS, GC-MS and 1 H-NMR.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Storage stability of freeze-dried Lactobacillus plantarum S20 starter culture as affected by various formulations of drying medium, and its fermentation characteristics in mung bean (Vigna radiata L.) slurry

Development of an active starter culture is essential in the production of safe and highquality fermented foods. Freeze-drying is an effective microbial cell immobilization technology intended to produce active and stable starter cultures for long-term storage. To determine the influence of various drying medium formulations with mung bean powder and disaccharides on the viability of L. plantarum S20 during freeze-drying and subsequent storage at 6±2°C and 30±1°C, three treatments of drying medium consisting of mung bean powder without disaccharides (MBP), MBP with 5% (w/v) trehalose (MBP+T), and MBP with 5% (w/v) sucrose (MBP+T) were evaluated. Results showed that L. plantarum S20 viability after freeze-drying ranged from 9.56 to 10.03 log (CFU/g) with viability loss ranging from 0.20 to 0.51 log (CFU/g), with MBP being the least effective (p<0.05) in minimizing viability loss. Supplementing MBP with either 5% sucrose or 5% trehalose improved (p<0.05) survival of freeze-dried L. plantarum S20; and both disaccharides exhibit similar (p>0.05) cryoprotective effects on L. plantarum S20 during freeze-drying and subsequent storage. Storage at 6±2°C for 180 days resulted in significant decreases of 2.15, 2.17, and 3.27 log (CFU/g) in L. plantarum S20 population in MBP+T, MBP+S, and MBP, respectively. A more drastic decline [>6.0 Log (CFU/g)] in L. plantarum S20 population was observed in freeze-dried culture stored at 30±1°C for 60 days. Fermentation characteristics of freeze-dried L. plantarum S20 in mung bean slurry were also determined. Freeze-dried L. plantarum S20 in MBP with 5% (w/v) sucrose was able to acidify mung bean slurry from pH 6.8 to pH 3.8 after 8 hrs with maximum LAB population of 9.2 log (CFU/mL). Acidification of mung bean slurry by fresh culture (non-freeze-dried) on the other hand was relatively slower where pH dropped from 6.8 to pH 3.72 after 24 hrs. Current work suggested that lyophilized L. plantarum S20 could be used as a starter culture for mung bean-based fermentations but further research on this area is needed.

Introduction
Fermentation is an old but useful form of food preservation, and it is valuable in developing functional foods. It is commonly used in food processing to enhance the safety, nutritional content, flavor, and texture of food. The most popular fermented foods are those achieved by lactic acid bacteria (LAB) (Rezac et al., 2018), which is a diverse group of Gram-positive bacteria capable of converting carbohydrates into lactic acid as the major end product (Paul et al., 2017). A variety of LAB species, particularly those that belong in the genus Lactobacillus, can be found in a variety of fresh produce, fermented foods, and mammalian digestive tracts. The species Lactobacillus plantarum is the most studied Lactobacillus due to its ability to thrive in a wide range of environmental niches. Different strains of L. plantarum have emerged as functional LAB exhibiting a long history of safe use. It is considered as "generally recognized as safe" (GRAS) and has qualified presumption of safety (Ray and Joshi, 2014;Ricci et al., 2017). The versatility of L. plantarum makes it a good functional organism to be used in a wide range of applications in the food industry, especially as a starter culture.
The use of starter cultures plays an essential role in controlled fermentations to ensure the safety and quality of commercially produced fermented foods. A starter culture is composed of either a single species or a mixture of microorganisms that are added directly into a food matrix to start the fermentation process, and to obtain a desired and consistent quality of the finished product (Durso and Hutkins, 2003). The production of starter cultures aims to ensure long-term delivery of viable and stable cultures, which can be achieved through the application of preservation technologies (Carvalho, 2003a). Freeze-drying is a cell immobilization technology that can be used to preserve active and stable starter cultures. It has been established in the food industry as the best method of preserving bacterial cultures in a powdered/crystalline form for long -term storage. However, freeze-drying can result in cell injury that could lead to cell death. Thus, selecting appropriate drying medium as a protective matrix can produce a stable final formulation of freeze-dried cell culture with minimal viability loss. Several studies have investigated the use of various protective agents during feeze-drying consisting of additives (Carvalho et al., 2002), disaccharides, and skim milk (Carvalho et al., 2003b;Jalali et al., 2012;Mendoza et al., 2013;Gisela et al., 2014;Jofré et al., 2015) in order to minimize viability loss of LAB during cryopreservation. In addition, drying media also serve as carriers of freezedried starter cultures for fermentation applications. The use of skim milk supplemented with disaccharides as drying medium for dairy starter cultures has been successful in minimizing bacterial cell death during freeze-drying and subsequent storage (Carvalho et al., 2004). Food-grade plant-based materials were also utilized as components of freeze-drying media for probiotic bacteria and non-dairy starter cultures and have shown to reduce microbial viability loss. A number of these plant-based materials include plant protein powder (Savedboworn et al., 2017), fruit powders (Trachoo et al., 2008;Nualkaekul et al., 2012), soybean flour (Trachoo et al., 2008), plant extracts and fibers (Hongpattarakere et al., 2012). Starchy materials like flours of rice, cassava, yam, millet, maize, and sorghum have been utilized as cryoprotective drying media for non-dairy starter cultures for gari and beer fermentations (Okafor et al., 1999;N'Guessan et al., 2016). Mahidsanan et al. (2017) also reported that a novel soybean flour as an inexpensive cryoprotectant option showed enhanced protection on Bacillus subtilis SB-MYP-1 starter culture for Thu-nao (fermented soybean) processing.
Mung bean (Vigna radiata L.) or munggo, locally known in the Philippines, is a pulse crop with high protein content (21-31%) and considered as a poor man's meat (Anwar et al., 2007). Mung bean is also known for its high carbohydrates content (64.2%) with small portions of dietary fiber (14.6%), ash (3.9%) and some traces of vitamins and minerals (BAFS, 2012). In food processing applications, mung bean has established its popularity due to its good quality starch, which comprises a majority of total carbohydrates. Starch in mung bean comprises more than 50% of the total seed weight (Li et al., 2011). It has the excellent gelling capability, pasting property, and cohesiveness, becoming it a better choice for film-forming biopolymers and starch noodles (Kim et al., 2007;Li et al., 2015;Rompothi et al., 2017). Fermentation of mung bean with L. plantarum strains was associated with the release of angiotensin I-converting enzyme (ACE) inhibitory peptides, which are drugs used to lower blood pressure. Fermentation of pea with L. plantarum 299 has produced bioactive metabolites that facilitated the release of angiotensin I-converting enzyme (ACE) inhibitory peptides during in vitro gastrointestinal process (Torino et al., 2013). L. plantarum B1-6-fermented mung bean milk was shown to produce significantly higher ACE inhibitory activity (67.5%) at the end of fermentation (Wu et al., 2015). In addition, Wu et al. (2015) also demonstrated that mung bean milk could be a carrier matrix of probiotics. Thus, it is interesting to explore the potential of utilizing of mung bean as starter culture drying medium and fermentation substrate for L. plantarum.
Freeze-drying at commercial scale is an expensive dehydration technology. However, it is still the most used method at commercial scale for the production of starter cultures. Thus, utilizing freeze-drying media formulated with low-cost ingredients like mung bean should be able to reduce a portion of the cost. To date, no studies have been conducted in utilizing mung bean as a drying medium component that will also serve as a carrier matrix for L. plantarum starter culture. This study aimed to evaluate the influence of various protective agents formulated with mung bean powder and disaccharides on the freeze-and post-freeze-drying survival of L. plantarum S20 starter, which can be used in mung bean-based fermentation. Results of the study will provide baseline data necessary to produce starter culture by utilizing cheap food-grade materials like mung bean as a component of drying medium for the production of novel functional non-dairy fermented foods.  (Nguyen et al., 2010). It was isolated using streak plating technique and identified by its 16s rDNA sequence. L. plantarum S20 was maintained in 7.0 mL deMann Rogosa Sharpe (MRS) broth (HiMedia, Mumbai, India) at 4±1°C and sub-cultured every 2 weeks.

Preparation of mung bean powder
Whole green mung bean seeds (NSIC MG-14) were purchased from the Bureau of Plant Industries-National Crop Research and Development Center of Los Baños, Laguna. The seeds are characterized to be dull-green and can be planted and harvested during the wet and dry season. Seeds were dried at 70°C for 2 hrs (moisture content= 9.33±0.40%) and were then pulverized with the fabricated stainless steel pin mill machine (50 Kg capacity; Alameda, Tondo, Manila, Philippines) and screened through 80-mesh sieve. Mung bean powder was then packed and vacuum-sealed in Polyethylene bags and stored at room temperature until use.

Preparation of drying medium
A total of three treatments of drying medium formulated with mung bean base mixture and two types of disaccharides (sucrose and trehalose) were prepared. The treatments include mung bean with 5% (w/v) trehalose (MBP+T), mung bean with 5% (w/v) sucrose (MBP+S), and mung bean powder only (MBP). A mixture of mung bean powder and sterile 0.85% (w/v) NaCl solution was used as base mixture ingredients of the freeze-drying medium. The proportion used in preparing the base mixture of drying medium was based on the optimized cryoprotectant formulation using skim milk described by Gisela et al. (2014) with some modifications. About 25 g of mung bean powder was diluted with 0.85% (w/v) NaCl solution to 100 mL. The mixture was then transferred to a sterile media bottle and pasteurized at 71±1°C for 66±1 mins in order to inactivate pathogenic bacteria and reduce population spoilage microorganisms. The hot mixture was then assigned to one of the three treatments: MBP, MBP+T, and MBP+S. Hot mixture assigned to MBP with disaccharides was prepared by supplementing it with either trehalose (Hayashibara, Japan) or sucrose (Ajax-FineChem Univar, Australia) at 5% (w/v) inclusion rate. All treatments were then allowed to cool down at room temperature.

Preparation and freeze-drying of starter culture
The preparation of starter culture was based on the procedures described by Yao et al. (2009) with major modifications. About 1 mL of overnight (16-18 hrs at 37°C) MRS-cultured L. plantarum S20 was propagated in fresh 100 mL MRS broth at 37°C for 18 hrs. The L. plantarum S20 cells were then harvested by centrifugation at 4300 x g at 4°C for 15 mins and washed with sterile 0.85% NaCl solution twice. Washed cells were then re-suspended in 5 mL of sterile 0.85% NaCl solution. The resulting cell suspension was then aseptically transferred onto a media bottle containing one of the previously prepared freeze-drying media. All mixtures were then aseptically transferred onto sterile petri dishes and were immediately frozen for 24 hrs at -20°C. All treatments were then lyophilized at a vacuum pressure of 0.20 Torr (ULVAC DF-01H, ULVAC Corp.), and secondary drying temperature set at 40°C for 72 hrs. Preparing a freeze-drying medium devoid of any of the cryoprotectants and L. plantarum S20 was used as a negative control.

Packaging and storage of freeze-dried starter culture
Under a sterile laminar flow cabinet, freeze-dried cultures were pulverized aseptically using a sterile spatula and transferred onto pre-sterilized amber glass vials. Sample vials were immediately sealed with sterile rubber stopper and stored either at 6±2°C for 180 days in the chiller or at 30±°C for 60 days in the incubator.

Evaluation of fermentation performance of the starter culture on mung bean slurry 2.2.1 The mung bean slurry preparation
Whole green mung bean seeds (NSIC MG-14) were soaked in water for 9-10 hrs. Soaked beans were manually de-hulled and a 200 g portion was transferred onto a clean stainless steel stockpot. About 1.5 L of sterile distilled water was added into the beans and the mixture was heated to boiling at approx. 100°C. Immediately after boiling, the heat was adjusted to the medium low setting and the mixture was heated to approx. 100°C for another 20 mins or until beans become soft. The mung bean mixture was then transferred into a blender and an additional 500 mL of sterile distilled water was used to wash the remaining mixture. The mixture was then homogenized for 2 mins and transferred into a clean stainless stockpot. The mixture was then added with 500 mL sterile distilled water and was brought to boil. About 250 mL portion of mung bean slurry was immediately transferred into a FULL PAPER sterile screw-capped media bottle glass container and was supplemented with sucrose at 5% (w/v) inclusion rate. The mixture was then transferred to the boiling water bath and was allowed to be pasteurized at 71±1°C for 66 mins and was allowed to cool at room temperature.

Fermentation of mung bean slurring using freeze-dried Lactobacillus plantarum S20 culture
Three fermentation treatments containing 250 mL mung bean slurry were prepared for fermentation experiment. The first treatment of mung bean slurry was supplemented with fresh L. plantarum S20 culture. This was done by harvesting L. plantarum S20 cells from 10 mL overnight (37°C for 18 hrs) culture of L. plantarum S20 in MRS broth, using the procedure described in the preparation of starter culture for freeze-drying (Section 2.1.4). Harvested cells from 10 mL of overnight culture (approx. 10 10 CFU/mL) in MRS broth were re-suspended in 9 mL 0.85% NaCl. About 5 mL of the cell suspension was added to previously prepared mung bean slurry to contain initial LAB population of 7 log (CFU/mL). The second fermentation treatment of mung bean slurry was supplemented with freeze-dried L. plantarum S20 culture. This was done by rehydrating 1 g of 2-day chilled freeze-dried L. plantarum S20 culture (approx. 10 10 CFU/g) in 9 mL 0.85% NaCl solution for 15 min. About 5 mL of the rehydrated cells were transferred onto 250 mL pasteurized mung bean slurry to contain initial LAB population of 7 log (CFU/mL). Lastly, the third fermentation treatment of mung bean slurry was not supplemented with L. plantarum S20 culture and served as the control of the experiment.

Sample collection from the fermentation mixture
During fermentation, sample aliquots for LAB viability test, pH, and total soluble solids (TSS) measurements were aseptically drawn from the fermenting mung bean slurry at 0, 4, 8, 16, 24, and 32 h of fermentation at 37°C.

Analytical procedures and LAB viability test 2.3.1 Freeze-drying and storage viability of L.actobacillus plantarum S20
The ability of L. plantarum S20 to survive freezedrying and storage conditions in mung bean powder with either trehalose or sucrose was determined using standard spread plating technique. To determine the influence of cryoprotectants on L. plantarum S20 viability in mung bean powder during freeze-drying, about 1 g of freeze-dried culture was aseptically drawn from each freeze-drying medium treatment before and after the freeze-drying process. To assess the stability of freeze-dried L. plantarum S20 during storage, the same amount of freeze-dried culture was aseptically drawn at 0, 60, 120, and 180 days of 6±2°C storage and at 0, 20, 40, and 60 days of 30±1°C storage. Each sample obtained was serially diluted in 0.1% peptone (HiMedia Sa, Mumbai, India). About 0.1 mL sample aliquot from the diluted sample was spread plated onto MRS agar and overlaid with 5.0 mL molten MRS agar. Plates were then incubated at 37°C for 48 hrs. Viable cell count was reported as Log (CFU/g).

Enumeration of LAB population during fermentation
Population of lactic acid bacteria during fermentation of mung bean slurry with L. plantarum S20 was enumerated using a standard pour plating technique on MRS agar. About 1 mL sample aliquot drawn from fermenting mung bean slurry (section 2.2.3) was serially diluted in 0.1% peptone. About 1 mL of the diluted sample was pour plated with molten MRS agar. Plates were then incubated at 37°C for 48 hrs. Viable LAB counts were reported as log CFU/mL.

Water activity of freeze-dried starter during storage
About 2 g of sample was obtained from freeze-dried L. plantarum S20 before and after 180 and 60 d storage at 6±2°C and 30±1°C, respectively. Water activities of freeze-dried samples were then measured using a water activity meter (LabSwift-a w meter; Novasina, Hamilton, NJ, USA) at room temperature.

Total soluble solids and pH during mung bean slurry fermentation
About 20 mL of sample was used for TSS and pH measurement. The total soluble solids content of samples was measured using a handheld refractometer (Atago, Japan) and the results were expressed as °Brix. The pH of samples was measured using a hand-held pH meter fitted with spear-shaped probe (PH60S; Apera Instruments) calibrated with pH 7.00 and pH 4.00 buffer solution. All readings for each parameter were done in duplicate for each sample.

Statistical analysis
Analysis of variance (ANOVA) with repeated measures was used to determine treatments significantly differ from each other, using 5% level of significance. A pairwise comparison using Tukey's HSD (honestly significant difference) was used to compare means using 5% level of significance. The analysis was carried out using SPSS Statistics (Version 25; IBM, USA). Both freeze-drying and storage stability experiments were FULL PAPER eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources independently replicated three times with two independent samples used in each replication. Fermentation experiment, on the other hand, was replicated four times with one independent sample used in each replication.

Results and discussion
3.1 Effect of protective agents on Lactobacillus plantarum S20 during freeze-drying The population L. plantarum S20 in mung bean powder with or without cryoprotectants after freezedrying is shown in Figure 1. It is shown that viability after freeze-drying ranged from 9.56 to 10.03 log (CFU/ g) with viability loss ranging from 0.20 to 0.51 log (CFU/g). Addition of either sucrose or trehalose at 5% (wt/v) exhibit improved protection on L. plantarum S20 during freeze-drying where higher (p<0.05) level of surviving L. plantarum population remained after freezedrying was observed.
Viability loss observed in the current study was expected due to cell injury caused by the freezing and dehydration process. Several studies reported that cellular damage during freeze-drying was largely associated with cell membrane damage due to ice crystal formation that leads to high osmolarity of the cell (Telang et al., 2003), macromolecule denaturation (Thammavongs et al., 1996), and the removal of water (Allison et al., 1999;De Paz et al., 2002). Viability loss in L. plantarum S20 population observed in this study was almost similar to the values reported by Hongpattarakere et al. (2012). They reported that freezedrying of L. plantarum suspended in MRS growth medium added with and without mung bean fiber has resulted in 0.25 and 0.51 log (CFU/g) viability loss. In the present work, all treatments were exposed at -20°C for 24 hrs freezing temperature prior to freeze-drying. This freezing temperature may have resulted in bigger ice crystals that resulted in cell injury. Hongpattarakere et al. (2012) and Perry (1998) noted that slow freezing occurring at temperatures above −30°C may result in poor survival due to loss of water from the cell while increasing the effects of solute concentration. Exposure of L. plantarum culture to extremely low freezing temperature of -196°C have shown to have the significantly lower freeze-drying viability loss of 0.18 log (CFU/g) as compared to -80 and -20°C with cell reduction of 0.57 and 0.38 log (CFU/g), respectively (Hongpattarakere et al., 2012).
Current results showed that using mung bean powder only as drying medium exhibit significantly lower viability compared to those formulated with either sucrose or trehalose. The surviving L. plantarum S20 in mung bean drying medium after freeze-drying was 9.56 log (CFU/g). This survival level is equivalent to approx. 31% of the initial population level before freeze-drying. This survival rate was slightly better compared to the survival rate (23%) of L. plantarum TISTR 2075 when 15% plant protein was used as a protective and drying medium (Savedboworn et al., 2017). In the current study, the use of ground mung bean may have served as inert bulking agent forming viscous layer that provided some protection on L. plantarum S20 cells during the freezedrying process. Carvalho et al. (2004) noted that some high molecular weight substances present in the drying medium like polysaccharides and proteins have no capability to penetrate the cell wall or interact with the cell wall or membrane. Their protective effect lies on their ability to be adsorbed on the surfaces of microorganisms forming viscous layer, which could cause partial removal of water from the cell to the surroundings. This would inhibit ice crystal growth while keeping the structure of amorphous ice in the close proximity of the cell (Hubálek, 1996). Consequently, this will decrease the amount of ice crystals inside the cell, hence could prevent cell death.
The addition of either trehalose or sucrose at 5% (w/ v) in the current study appeared to have increased the resistance of L. plantarum S20 to freeze-drying. Previous studies showed that these two disaccharides were FULL PAPER Figure 1. Population of L. plantarum S20 in various freezedrying media before and after lyophilization. Data presented as mean Log (CFU/g) from 6 independent samples. Drying medium treatments: MBP+T= mung bean powder with 5% trehalose; MBP+S= mung bean powder with 5% sucrose; MBC= mung bean powder only. ab Values within each treatment not sharing the same letters are different from each other (p<0.05) analyzed using Tukey's HSD post hoc test; XYZ Values across treatments not sharing the same letters are different from each other (p<0.05) analyzed using Tukey's HSD post hoc test; Error bars represent standard error of the mean.
eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources effective as protective agents for cryopreservation of lactic acid bacteria (Chavarri et al., 1988;Carvalho et al., 2002;Carvalho et al., 2003b;Gisela et al., 2014). Cryoprotectants in freeze-drying serve as antifreeze agents, which minimize cell death by lowering the melting point of freeze-drying media (Polge et al., 1949). This would eventually freeze the medium surrounding the cell faster than the intracellular components, hence, preventing cell death. Carvalho et al. (2004) noted that trehalose and sucrose have the ability to decrease the transition temperature of dry membranes by replacing water between the lipid head groups. This phenomenon prevents phase transition, and its accompanying leakage upon rehydration (Leslie et al., 1995;Castro et al., 1997). An earlier study on cryopreservation of biomaterials suggested that trehalose was more effective cryoprotectant than other sugars (Crowe et al., 1996). On the contrary, current work did not observe a significant difference between the cryoprotective ability of sucrose and trehalose. Current observation is supported by a previous study conducted by Strasser et al. (2009) where trehalose and sucrose demonstrated similar cryoprotective effects on L. plantarum strain IFA No. 278 during freeze-drying. In addition, Strasser et al. (2009) also reported that these two cryoprotectants were the most effective among the cryoprotectants tested in enhancing the survival of L. plantarum strain IFA No. 278 during lyophilization.

Stability of lyophilized Lactobacillus plantarum S20 during storage
Post-freeze-drying survival of L. plantarum S20 during chilled and ambient temperature storage is shown in Figure 2. It is shown that storage of freeze-dried cultures for either 180 days at 6±2°C or 60 days at 30±1°C resulted in a significant decrease on the viability of lyophilized L. plantarum S20 cells. At the end of 180 d chilled storage period, viable L. plantarum population in MBP+T, MBP+S, and MBP decreased (p<0.05) by 2.15, 2.17, and 3.27 log (CFU/g), respectively (Figure 2A). Meanwhile, L. plantarum S20 did survive poorly during storage at a much higher temperature ( Figure 2B). Ambient storage for 60 d resulted in >4 log CFU/g decrease in viability of L. plantarum S20. Results also show that for both storage temperatures, the addition of either 5% sucrose or 5% trehalose did improve survival of L. plantarum S20 during storage with lower population reduction as compared to drying medium containing ground mung bean only. No difference (p>0.05) in viability was observed in L. plantarum S20 population in MBP+T and MBP+S during ambient and chilled storage indicating that both cryoprotectants have similar protective effects on L. plantarum S20 survival at post-freeze-drying conditions. In addition, MBP exhibit lowest (p<0.05) survival after 180 d (cell reduction 3.78 log CFU/g) and 60 days (5.48 log CFU/g) storage at chilled and ambient storage, respectively. It appears that storage of freeze-dried cultures with mung bean powder and cryoprotectants at 6±2°C for 60 and 120 d manage to preserve L. plantarum S20 survival >8.0 Log (CFU/g) with only <0.70 and <1.70 log (CFU/g) reduction in cell population, respectively.
The drastic decline in L. plantarum S20 population during chilled and ambient storage could be attributed to relatively high water activities of the freeze-dried samples. Table 1 shows the water activity values of freeze-dried L. plantarum S20 in ground mung bean using different cryoprotectants before and after 180 and 60 d of storage at 6±2°C and 30±1°C, respectively. It is also shown that water activities slightly increase (p<0.05) after storage at 6±2°C and 30±1°C. Generally, dehydrated microorganisms survive better at much lower water activity. Stability of dried LAB upon storage is FULL PAPER Figure 2. Population of lyophilized L. plantarum S20 culture in various freeze-drying media during storage at 6±2°C for 180 days (A) and at 30±1°C for 30 (B). Data presented as mean Log (CFU/g) from 6 independent samples. Drying medium treatments: Open diamond = MBP+T (mung bean powder with 5% trehalose); Open circle= MBP+S (mung bean powder with 5% sucrose); Closed triangle with dash line = MBP (mung bean powder only). eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources shown to be optimal at 0.1 to 0.2 aw (Champagne et al., 1996). In the current study, the slightly higher water activity of the dried cultures may have largely affected the viability of the L. plantarum cells during storage. Previous studies indicated that lipid oxidation of the membrane fatty acid was predominantly responsible for cell death during storage (Teixeira et al., 1996;Kurtmann et al., 2009). Thus, decreasing water activity and oxygen in the packaging have shown to stabilize freeze-dried cultures (Kurtmann et al., 2009). In addition, the temperature can also influence the rate and magnitude of lipid oxidation. Current work observed higher mortality in L. plantarum population during storage at ambient temperature. A number of studies have shown that storage of freeze-dried cultures at higher temperatures resulted in a higher loss in viability a shorter shelf life. For instance, storage of vacuumpackaged freeze-dried P. aglomerans stored at 4°C for 90 d showed no more than 0.5 log viability loss, while those that were stored at 25°C resulted in 3 log decrease in viability after 28 d (Costa et al., 2002). Moreover, previous work of Champagne et al. (1996) showed that viability loss of a freeze-dried culture containing Bifidobacrium longum at 20°C were 100x greater than those that were stored at 4°C. Similar observation was reported by Dhewa et al. (2014) where a considerable reduction in viability of L. plantarum was observed in freeze-dried symbiotic formulation stored at 25°C for 90 d while those that were stored at 4°C exhibit higher L. plantarum survival. Selmer-Olsen et al. (1999) recommended 0-5°C storage temperature for freeze-dried cultures because low temperatures maintain lower metabolic rate, thus increases storage stability.

Fermentation characteristics of freeze-dried Lactobacillus plantarum S20
Changes in lactic acid bacteria (LAB) population, pH, and total soluble sugar (TSS) of mung bean slurry during 32 hrs of aerobic fermentation at 37°C are presented in Figure 3. Results show that LAB population changes in mung bean slurry with fresh and freeze-dried starter culture showed a similar trend ( Figure 3A). It appears that LAB in both treatments entered exponential growth phase any time between 0 and 4 th hr of fermentation due to the added L. plantarum S20 inoculum. Exponential growth phase for mung bean slurry with freeze-dried L. plantarum S20 extended up to 8 hrs of fermentation where about 2.0 log cycle increases in LAB population was observed, from an initial LAB population of approx. 7.0 log (CFU/mL) to 9.30 log (CFU/mL). It is evident that the maximum LAB population in mung bean with freeze-dried culture was reached faster compared to mung bean slurry with fresh culture. The LAB population in mung bean slurry with freeze-dried culture peaked after 8 hrs with cell population of 9.30 log (CFU/mL), while LAB in mung bean with fresh culture of L. plantarum S20 peaked after 16 and 18 hrs with cell population 9.07 and 9.12 log (CFU/g). The significant increases in LAB population in both treatments caused a significant drop in mung bean slurry pH and TSS. Highest pH drop (from pH 6.78 to pH 3.82) was observed after 8 hrs of fermentation in mung bean slurry inoculated with freeze-dried starter, while pH drop in treatment with fresh starter culture was slower from pH 6.81 to pH 3.72, which extended up to 16 and 24 hrs. It is observed that TSS in mung bean slurry with either fresh or freeze-dried L. plantarum S20 starter decreased (p<0.05) exponentially over time from 0 to 16 hrs and leveled off until the end of fermentation ( Figure 3C).
Higher (p<0.05) drop in TSS was observed in mung bean slurry with a freeze-dried starter. The LAB growth curve trend corresponding to pH and TSS drop in mung bean slurry with L. plantarum S20 indicates sugar utilization of L. plantarum inoculum added in the mung bean slurry. This trend was not observed in the control where natural fermentation was used. The LAB population present in the natural microflora of mung bean was only observed after 4 hrs of fermentation and exponential growth phase was extended up to 16 hrs where the maximum population of 7.15 log (CFU/mL) was reached. This population level is significantly lower than the maximum population achieved by mung bean slurry with L. plantarum S20. This trend also FULL PAPER corresponds to higher minimum pH achieved by mung bean slurry without starter culture as compared to mung bean inoculated with fresh or freeze-dried L. plantarum S20 starter. Interestingly, unlike the other treatments, TSS gradually increased in mung bean slurry without a starter culture. This can be attributed to biological activities by some LAB species naturally present in mung bean that survived pasteurization. For instance, heat-resistant, spore-forming, and lactic acid-producing Bacillus spp. (e.g., Bacillus subtilis) was reported to be the most predominant fermenting species in soybean and mung bean (Shrestha et al., 2013;John and Olusegun, 2016). Bacillus species can produce a variety of enzymes capable of hydrolyzing macromolecules like starch into soluble sugars like oligosaccharides and simple sugars (Neway, 1989;Shrestha et al., 2013), and this may have contributed to the increasing trend in TSS observed in the current study. This additional step could have slowed down the conversion of soluble sugars (sucrose, glucose, etc.) into lactic acid, hence resulted in a much slower pH drop.
Previous work by Wu et al. (2015) on L. plantarum fermentation of mung bean milk showed slightly different results due to different mung bean preparation, sucrose concentration, and L. plantarum strain used. They reported that L. plantarum B1-6 fermentation mung bean beverage (without okara) supplemented with 5.55% sucrose and 5.19% inoculum concentration reached optimum acidity and highest LAB population of 8.96 log (CFU/mL) after 4.86 h of fermentation at 35.4°C.
Previous studies have demonstrated that mung bean could be used as a fermentation substrate for L. plantarum. L. plantarum has been found to dominate early stages of a 4-stage of fermentation of mung bean as a base material for lu-doh-huang (Taiwanese herbal medicine) (Chao et al., 2013). In addition, a number of studies have documented the use of L. plantarum as a starter culture for mung bean-based fermentations (Wu et al., 2015;Landete et al., 2015;Gan et al., 2017;Świeca et al., 2018). Current work showed that the use of freezedried L. plantarum S20 starter was able to rapidly decrease pH of fermenting mung bean below pH 4.0 corresponding to maximum population of 9.3 log after 8 hrs. This indicates that freeze-dried L. plantarum S20 has successfully started the fermentation process. Previous studies documented the ability of rapid acidification of freeze-dried L. plantarum starter for cassava fermentation has been previously documented (Yao et al., 2009). An important characteristic of potential starter strains is their ability to acidify their environment rapidly, which could inhibit the growth of contaminating spoilage and pathogenic microorganisms (Smulders et al., 1986). Leroy and De Vuyst (2004) reported that the direct addition of selected starter culture to raw materials FULL PAPER Figure 3. Changes in lactic acid bacteria population (A), pH (B), and total soluble sugars (TSS) (C) during 32 hrs mung bean slurry fermentation at 37°C with lyophilized culture, fresh, and without culture. Data presented as means from 4 independent samples. Error bars represent standard error of the mean (n=4). eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources resulted in dominance by the best-adapted strains. Furthermore, it is worth noting that freeze-dried L. plantarum S20 starter culture developed in the current study showed faster substrate acidification and LAB growth compared to MRS-grown living cells. This finding was inconsistent to previous reports, which showed that freeze-drying could impair metabolic activities of microorganisms upon rehydration (Meng et al., 2008;Mahidsanan et al., 2017) and could decrease growth and acidification activity (Fonseca et al., 2000;Santivarangkna et al., 2007). In the current study, sublethal stress due to freezing and freeze-drying process may have a role in rapid acidification and growth observed when freeze-dried L. plantarum S20 culture was used to ferment mung bean slurry. Previous work on the pre-inoculation treatment of Lactobacillus sakei starter culture with sublethal stress (cold shock, heat shock, salt shock) resulted in increased growth and acid production during sausage fermentation (Hüfner and Hertel, 2008). However, the extent of damage to microbial cells exerted by sublethal stresses applied to L. sakei is not as damaging compared to freeze-drying.
Since current work has limited scope, current findings on this area warrant further study.

Conclusion
Results of the current study were able to demonstrate that mung bean powder formulated with either trehalose or sucrose could be used as a freeze-drying medium for L. plantarum S20. Results indicate that drying medium formulation and storage temperature greatly affected L. plantarum S20 viability during freeze-drying and subsequent storage. Supplementing of mung bean powder with any of the two disaccharides at 5% (w/v) has improved tolerance of L. plantarum S20 to freezedrying. In addition, no difference was observed between sucrose and trehalose in terms of their cryoprotective ability on L. plantarum S20 during freeze-drying and subsequent storage. Low temperature storage at 6±2°C performed better than ambient storage (30±1°C) in minimizing viability loss of L. plantarum S20. However, drying medium formulations used in the study was not able to maintain the stability of L. plantarum S20 throughout storage. A significant decline of 2.0 to >4.0 log (CFU/g) in L. plantarum S20 population was observed in all drying medium treatments during cold and ambient storage. In future research, it will be useful to optimize the formulation of freeze-drying medium preparations, packaging conditions, and freeze-drying parameters to achieve higher recovery and stability for L. plantarum S20. In addition to mung powder and disaccharides, the incorporation of additives must be considered to further improve the shelf stability of freeze -dried culture. When freeze-dried L. plantarum S20 was used as a starter culture in mung bean slurry, it was able to acidify the slurry from pH 6.8 to pH 3.8 after 8 hrs with maximum LAB population of 9.2 log (CFU/mL). Acidification of mung bean slurry by fresh culture (nonfreeze-dried) on the other hand appeared to be slower than the freeze-dried culture. This observation contradicts previous findings. While freeze-dried L. plantarum S20 culture developed in the current study could be used as a starter culture for mung bean-based fermentations, its ability to perform better than nonfreeze-dried culture by rapidly growing and acidifying mung bean slurry warrant further research.

Conflict of Interest
Authors declare no conflict of interest.