Genomic relationship and physiochemical properties among raw materials used for Thai black garlic processing

Abstract Raw materials used for black garlic (BG) processing were collected from the major garlic production areas in Northern Thailand. Five of those were identified as of Thai origin (accession G1–G5), and accession G6 was of the Chinese variety. They were initially analyzed for varietal differences using morphological characteristics and genetic variation. Fresh materials from each accession were dried to the same moisture content (55%–60%) and BG processed at 75°C, 90% relative humidity (RH) for 15 days. Thereafter, physiochemical and chemical profiles were analyzed and compared. The dendrogram from random amplified polymorphic DNA fingerprints grouped G2, G3, G4, and G5 as closely related while G1 and G6 were out‐groups. Prior to BG processing, the pH of fresh garlic was approximately 6.3 and decreased to 3.7, thereafter. The contents of chemical properties were independent with genotypes. BG processing improved phenolic, flavonoid, and antioxidant but the content of thiosulfinate was minimized in all BG samples. Overall, result indicated that garlics grown in Northern Thailand were genotypically variable. BG processing altered physical and chemical appearance, and these changes were independent with the genotypes.


| INTRODUC TI ON
Garlic is known as culinary herb that had been traditionally used in the recipe of food in many cultures around the world (Bae, Cho, Won, Lee, & Park, 2014;Butt, Sultan, Butt, & Iqbal, 2009; Dufoo-Hurtado, Huerta-Ocampo, Barrera-Pacheco, Barba de la Rosa, & Mercado-Silva, 2015). For more than thousand years, garlic was recorded in the historical papyrus as medicine to cure many diseases and aliments (Block, 1985). Having been regarded as essential in all kitchens, garlic is today claimed as one of the modern "super foods" with long listed bioactive functionalities (Capasso, 2013;Choi, Cha, & Lee, 2014;Morihara et al., 2007;Nicastro, Ross, & Milner, 2015;Rahman, 2003;Rahman & Lowe, 2006;Sierpina et al., 2013;Zakarova et al., 2014). Approximately 7,000 tons of fresh garlic are produced annually in Thailand and the majority of the production was located mainly in the north including Mae hong son, Lamphun, Lampang, and Chiang rai. In recent years, due to the influx of foreign produce mainly from China, Thai garlic has faced challenges of over-supply for the domestic consumption. Therefore, attempts have been made toward value-adding Thai garlic through processing for much preferably healthy products (Bae et al., 2014;Li, Pei, & Qiao, 2014).
Black garlic (BG) processing involves heating the raw material at high temperature and saturated humidity for 14-40 days without additive until the color of the flesh turns to black by either Maillard reaction or caramelization (Bae, Cho, Won, Lee, & Park, 2012;Choi et al., 2014;Hee Kim, Hyun Nam, Rico, & Young Kang, 2012;Lu, Li, Qiao, Qiu, & Liu, 2017). There by, its texture becomes more elastic with sweet-sour taste and less offensive odor (Bae et al., 2014;Choi et al., 2014;Sasaki, 2015;Shin et al., 2008). BG improves amount of reducing sugars, amino acids, and antioxidative compounds which are being recognized as a functional food product in the global market (Kimura et al., 2017;Queiroz, Ishimoto, Bastos, Sampaio, & Torres, 2009). This process also reduces the pungent odor as sulfur volatiles (i.e., allicin) and is converted into water soluble antioxidants which is preferable by consumers who dislike strong garlic smell (Kim, Kang, & Gweon, 2013). The antioxidative compounds in BG in particular possess long lists of functionalities including antibacterial, antibiotic, antifungal, antiviral, anticancer, and antioxidant properties (Choi et al., 2014). Also many studies have shown that aged garlic prevents carcinogenesis, cardiovascular, and age-related diseases (Rahman, 2003). This process has helped Thai food producers to restore the market position of Thai garlic raw material. Nonetheless, according to our survey, the quality of the BG in Thai markets vastly diverged from one producer to the others. The major problem is that no standardization criteria for raw material quality control have been set up. Previous studies indicated that differences in genotyping had an effect on chemical compositions in both fresh garlic and finished products (Naheed, Cheng, Wu, Wen, & Ding, 2017;Sommano, Saratan, Suksathan, & Pusadee, 2016). Even though other factors could also have forthright effect on BG qualities, for example, temperature, relative humidity during the thermal processing, and pretreatment process (Kimura et al., 2017;Li et al., 2014), the data on varietal differences of raw materials used for BG processing, especially, among the northern Thai food producers are very slim. The research aim of this study was, therefore, to investigate differences in genotyping among the raw materials used for BG processing along with their physiochemical compositions. The key finding from this research will be useful toward raw material selection to ensure the constant quality during commercial processing of Thai BG.

| Plant material
Unless otherwise stated, all Thai garlic samples listed in Table 1 were collected at the same commercial harvesting stage (between 90-120 days after cultivated) in March 2019 in which leaves appeared dry and the bulb was pinkish-white, clean without disease and pest damage. They were transported fresh to the laboratory immediately for the experiments. Bulb morphology (viz. size, weight, number of cloves, and their weights) of garlic samples from different cultivation sites in the north of Thailand was recorded (Sommano et al., 2016).

| DNA extraction
Cloves of each sample were separated and propagated as seedling in a wet sponge for 20 days. Young leaf tissue from the seedling was then used for DNA extraction. Total genomic DNA was extracted using the cetyl-trimethylammoniumbromide (CTAB) method with some modification (Doyle & Doyle, 1986

G6
Chinese garlic Thai local market centrifuged at 6,800 g for 10 min. The supernatant was transferred to a fresh vial, and isopropanol was added to achieve DNA separation. The DNA fraction was air-dried at room temperature, and then, this pellet was resuspended in 100 μl TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA and pH 8.0). The DNA samples were stored at −20°C prior to random amplified polymorphic DNA (RAPD) analysis.
The DNA solution was diluted with deionized water for further polymerase chain reaction (PCR) and kept in −20°C until used.
Consequently, the sample was separated in a 1.5% agarose gel in 1× TBE buffer. The PCR products were stained with the green stain and run at 100 V for 65 min. The gels were then visualized using the LED transilluminator, accordingly.
Black garlic was processed in a rice cooker (KS11E; Federal Electric Corporation) using warm mode (at 75°C, 80% RH) for 15 days according to method of Sasaki (2015) with some modification; then, the BGs were removed. The BG samples were lyophilized then ground to powder and stored at −20°C for further phytochemical analyses.

| Moisture content
The moisture content of the garlic samples was determined by measuring the weight loss percentage after 12 hr at 105°C in a hot-air oven according to the Association of Official Analytical Chemists (Cunniff, 1996).

| pH and color intensity
Flesh of samples (10 g) was blended with 100 ml of distilled water.
Then, the pH was measured using a pH meter (Five easy F20; Mettler Toledo) (Choi et al., 2014), and browning intensity was measured at an absorbance of 420 nm using a spectrophotometer (SPECTROstar Nano; BMG LABTECH; Kang, 2016).

| Scanning electron microscopy of cell structure
The garlic cloves were cross-section into 3 mm-thick then immediately immersed in cold formalin-acetic acid-alcohol (FAA solution; formaldehyde: acetic acid: ethanol; 1:1:10) for 72 hr. The fixed samples were then dehydrated in a series of aqueous ethanol solutions with successively increasing ethanol concentrations from 30% to 100%, v/v and dried in freeze dryer. Dry samples were coated with platinum using a vacuum sputter coater to increase the conductivity of the samples (Wang & Rhim, 2019). The images were taken using Scanning electron microscope (SEM; TM3000, Hitachi, Ltd).

| Thiosulfinate content
Garlic powder sample (0.5 g) was added to 25 ml of distilled water and shaken for 10 min.  Parvin et al. (2008) filter paper, and the supernatant was separated to a new Erlenmeyer flask. Then, thiosulfinate from the mixture was extracted by using 10 ml of hexane, swirled the mixture gently, and separated the hexane layer. The water layer was re-extracted with 5 ml of hexane.
The first and second extracts were combined, and the absorbance of the hexane solution was measured at 254 nm (Kang, 2016). The thiosulfinate content of the hexane solution was calculated using the following equation: where A is the absorbance; b is the path length (cm); C is the solution concentration (μM/g); ε is the molar absorptivity of thiosulfinate solution at 254 nm (0.014 g/μM cm).

| Methanol extraction
The sample powder (0.1 g) was added into 5 ml of 80% methanol in a test tube then heated at 70°C for 30 min. The supernatant was collected after centrifugation at 6,800 g for 12 min. The extraction step was repeated five times, and all supernatants were combined.
Finally, the resulted extract was adjusted to the total volume of 25 ml with 80% methanol and used as methanol extracts for the subsequent chemical analyses.

| Total phenolic content
The total phenolic content was determined using gallic acid as a standard according to the method described by the International Organisation for Standardisation (ISO, 2005) with minor modification. Briefly, methanol extract (30 µl) was mixed with 150 µl of Folin-Ciocalteu reagent then 120 µl of 7.5% w/v NaCO 3 solution was added, mixed and left in darkness at room temperature for 60 min.
The absorbance at 765 nm was measured using the spectrophotometer and total phenolic content was expressed as milligram of gallic acid equivalents per gram of dried garlic sample.

| Total flavonoid content
The total flavonoid content was analyzed using catechin as a standard according to the method described by Kim et al. (2013) with some modification. The methanol extract (25 µl) was mixed with 125 µl of distilled water; then, 7.5 µl of 5% NaNO 2 solution was added. The mixture was allowed to react at room temperature for 5 min before adding 15 µl of 10% AlCl 3 ·6H 2 O solution. After 6 min of the incubation, 1 M of NaOH solution (50 µl) and distilled water (27.5 µl) were added. The absorbance was measured at 510 nm using the spectrophotometer. The total flavonoid content was expressed as milligram catechin equivalents per gram of dried garlic sample.
After incubated at room temperature in darkness for 30 min, the absorbance was measured at 550 nm using the spectrophotometer.
where A control is the absorbance of DPPH and A sample is the absorbance of sample. (1)

| Statistical analysis
All experiment data were expressed as mean (n = 9) ± SE. One-way

| Morphology
The morphological data of six garlic samples used in this experiment are shown in Table 3. The sizes of bulb and clove of foreign sample (G6) was the biggest as compared with the others. All  samples of Lampang and Mae hong son. The second cluster contained samples of Chiang Mai, Lamphun, and Chiang Rai which were higher in number of clove and bulb size while Chinese garlic was separated into different cluster due to bulb and clove weights. Chen et al. (2013), however, urged that morphological traits alone were unable to fully describe garlic variety, and thus, differences in genotyping should be analyzed.

| Genetic variation of garlic samples
The RAPD-PCR fingerprints were generated from DNA extract of all garlic samples using 12 randomly 10-oligonucleotide primers as shown in Table 2. Only nine primers, however, provided band patterns. Results indicated that there were 69 fragments based on the RAPD (Figure 3a) which later on were used to create a dendrogram as shown in Figure 3b.

| Physicochemical properties of BG
After BG processing, the moisture content of BG, however, was independent with the initial moisture contents but variety differences had greater impact on the finished product moisture content, average 30% in Thai garlic samples and 53% in foreign garlic (Table 4).
This was in-line with the work of Naheed et al. (2017) who advised that the varietal variation could affect weight loss during processing.
Geometrical shape of the bulb also influences moisture loss activity which much more labile in the outermost layers reached there after the most difficult part of the inner core (Bloem, Haneklaus, & Schnug, 2011). From our result, the Chinese cultivar gave the lowest water loss during the processing as the bulb was 1.3-1.8 times bigger than other Thai cultivars. SEM images illustrated that the greater moisture content of the product could lead to higher degree of damaged to the cell structure ( Figure 4).
The pH of all fresh garlics was detected just below seven and decreased thereafter BG processing (~3.8) as shown in Table 4.
The pH was also varietal independent as all sample types pos-  which was naturally produced during heat processing (Yilmaz & Toledo, 2005). This generated the dark brown pigments as a result of the reaction (Choi et al., 2014). In BG, fructan is mainly responsible for this reaction. Rate of the reaction depends on many factors, for example, temperature, time, pH, and water activity. Cuzzoni, Stoppini, and Gazzani GandMazza (1988) studied the effect of water activity on heated ribose-lysine and glucose-lysine, and the result showed that at high water activity, the rate of Maillard reaction was low. Bae et al. (2014) suggested that decrease in pH is in association with browning mechanism during heat process. The UV absorbance at 420 nm is regularly used to measure the brown color formation of biochemical reaction (Yuan, Sun, Chen, & Wang, 2018). In the experiment, brown color intensity increased the greatest in G3 but less in other Thai samples. The browning intensity was the lowest in the foreign garlic (G6), which apparently was well corresponded with the higher moisture content of the BG (53%). Browning intensity could therefore be as consequences of low pH and higher moisture content. Accumulation brown pigments was formed as a result of Maillard reaction between the products of cell structure as shown in Figure 4a,c (red arrows pointed).
From the PCA analysis which deposits 42.83% in PC1 and 34.95% in PC2, the data were categorized into three clusters
Thiosulfinate presents the unique garlic flavor, and the content usually decreases during heat processing due to the alteration of thiosulfinate to S-allyl cysteine, S-allyl mercapto-cysteine, and arginine. Kang (2016), Choi et al., (2014)  Total phenolic content in the BGs was significantly higher than that of fresh garlics and the content increased up to 13-fold after BG processing as shown in Table 4. BG3, BG4 and BG5 showed the highest total phenolic content approximately 6.42 mg/g of all processed samples. Kang (2016) and Robards, Prenzler, Tucker, Swatsitang, and Glover (1999) explained that total phenolic content increased due to the release of the bound of polyphenol compound to phenolic acid after high temperature processing. Cheng, Dai, Zhou, Yang, and Liu (2007) also reported that the phenolic compounds of bound form can be separated from cell wall by heat processing. As a result, the content of oxidative phenolic acid is usually higher with this process. This result was compatible with the study of Scalzo, Iannoccari, Summa, Morelli, and Rapisarda (2004), who reported that high temperature processing increased phenolic substances of fruit and vegetable crops.
Flavonoids belong to the phenolic group, and the flavones are generally found to a greater extent in fresh garlic. The total flavonoid content was similar among the fresh garlic samples (~0.28 mg/g). BG processing increased the content of the total flavonoid and the level was much higher in the BG4 (~1.3 mg/g). Flavonoid was higher in BG wherewith the heat treatment discharged the bound polyphenolic and flavonoid compounds (Choi et al., 2014). Flavonoid content alteration depends on the processing condition and their sensitivity to heat treatment (Ioannou, Hafsa, Hamdi, Charbonnel, & Ghoul, 2012).
During the BG processing, the flavanols and flavanones are the main metabolites as they illustrate thermostable characteristic (Kim et al., 2013).
The DPPH radical-scavenging activities increased up to 86.21% after heat processing. The study of Stratil, Klejdus, and Kubáň (2006) demonstrated that there was a greatly significant relationship between total phenolic content and antioxidant activity. Accordingly, the trend of DPPH scavenging activity in this study was assimilative to total phenolic and flavonoid content.
Moreover, all those properties were correlated with processing temperature (Bae et al., 2014). Many studies demonstrated that antioxidants played an important role in preventing diseases F I G U R E 4 (a) Scanning electron microscope images of black garlic from Lampang origin (b) fresh raw material of Lampang origin (G3) and (c) black garlic from raw material of the Chinese origin. Arrows indicate blackening pigments accumulated within cell structure after processed caused by free radicals as well as the memory and nervous systems.
We used PCA to explain relationship of the samples based on their chemical properties. For fresh garlic samples, the PCA warranted total of 80.30% PC1 and PC2 as shown in Figure 6a. From the biplot analysis, it only showed the distinct separation of Lampang and Chiang Mai, while other samples were grouped together. However, after aged processing we were only able to see the separation of Mae hong son garlic in Figure 6b. Again, the results established that genotype had no influence on chemical property changes after BG processing.

| CON CLUS ION
In an attempt to explore the genotype differences of raw materials used in BG processing in association with the phytochemical properties before and after processing, this study demonstrated that there were morphological differences and genetic variations between garlic samples cultivated in different areas of the northern Thailand. In addition, the physicochemical and chemical properties of both fresh and BGs were dependant with genotype. Future direction should look into the other factors that may influence BG qualities such as postharvest handling condition of raw material prior to processing.

| E THI C AL G U IDELINE S
Ethics approval was not required for this research.

ACK N OWLED G EM ENTS
This research was partially supported by Chiang Mai University.
The student fund in the forms of scholarship and Teaching Assistant and Research Assistant (TA/RA) was granted to PS from Graduated school, Chiang Mai University.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest with respect to the research.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.