The Occurrence of Propyl Lactate in Chinese Baijius (Chinese Liquors) Detected by Direct Injection Coupled with Gas Chromatography-Mass Spectrometry

As one of the oldest distillates in the world, flavor compounds of Chinese Baijiu (Chinese liquor) were extremely complex. Propyl lactate was firstly detected by direct injection and gas chromatography-mass spectrometry (GC-MS) in 72 Chinese Baijius. The objectives were to detect the contents of propyl lactate and evaluate its contribution to the aroma of Chinese Baijiu based on odor activity values (OAVs). The levels of propyl lactate in these distillates were determined by internal standard method and selective ion monitoring (SIM), which ranged from 0.050 to 1.900 mg∙L−1 under investigation. Its detection threshold was determined by Three-Alternative Forced-Choice (3-AFC) and curve fitting (CF), which was 0.740 mg∙L−1 in 38% ethanol solution. The contribution of propyl lactate on the aroma of these distillate drinks was evaluated by their odor activity values (OAVs), which varied from 0.066 to 4.440. The OAVs of propyl lactate were found to exceed 1 in 13 Chinese Baijius, including 50° Jingzhi Guniang 5 years (4.440), 52° Jingzhi Guniang 10 years (3.024), Jingyanggang (2.568), Xianghe Ronghe Shaofang (2.313), and 1956 Laolang (1.431), which indicated that propyl lactate was one of odor-active components in these Chinese Baijius.


Introduction
Chinese Baijiu (Chinese liquor) is one of the oldest distillates in the world, and it is the most popular spirits in China with the annual production of about 4 million metric tons. The general process for the production of Chinese Baijiu is as follows. At first, the grain raw materials, such as wheat, sorghum, corn, rice or glutinous rice, are cooked with steam. And then some saccharification and fermentation agents ("DaQu" or "XiaoQu") are added into the cooked grain matrix. Finally, the liquors are distilled out with steam from the fermentation products after several months or years of fermentation [1]. The fresh distillates need to be aged for a long time in order to balance the flavors. The final commercialized products are blended with aged distillate drinks, fresh distillate drinks and water based on certain ratios according to different formulations of spirit drinks [2].
Flavor compounds of Chinese Baijiu are extremely complex due to different raw materials, various microorganisms and diverse procedures in different production regions, and a great number of compounds have been studied extensively [3][4][5][6], such as esters, alcohols, ketones, acids, and so on. Lactates have been reported to occur in Chinese liquors widely, such as methyl lactate [4], ethyl lactate [7], butyl lactate [8], hexyl lactate [9], isopropyl lactate [10], isobutyl lactate [4] and isoamyl lactate [4]. Lactates are recognized to be important flavor compounds in Chinese Baijiu [11]. Ethyl lactate and butyl lactate were detected in "Gujing" Baijiu during investigation carried out in our lab. In the meantime, lactic acid and propanol were also found in this distillate, which had been reported in other Chinese Baijius [12,13]. It is a little strange that propyl lactate has not been reported in Chinese Baijiu up to now. The same are other alcoholic beverages, except distilled Calvados [14] and Chinese rice wine [15]. Meanwhile, the odor properties of propyl lactate and its contribution to aroma of these spirits are also not known. We checked the newest edition (2011 edition) of NIST (National Institute of Standards and Technology) library and found that the mass spectrogram of propyl lactate was not included in this library. The occurrence of propyl lactate in Chinese Baijiu might be overlooked since the identification of volatile components was usually performed by searching NIST library. The objective of this work were (i) the detection of the occurrence of propyl lactate in 72 Chinese Baijius and (ii) the evaluation of its contribution to the aroma of Chinese Baijius based on odor activity values (OAVs).

Identification of Propyl Lactate in Distillate Samples
The mass spectra of an unknown compound shown at 17.4 min in sample 33 and propyl lactate standard were presented as Figure 1, and the differential spectrum of them was located at the bottom, which indicated the unknown compound shown at 17.4 min being matched with propyl lactate. The two magnified TICs of the unknown compound in sample 33 (a TIC) and propyl lactate standard (b TIC) at 17.4 min were shown in Figure 2. These two peaks were completely overlapped.

Detection Threshold of Propyl Lactate and OAV Analysis
The determination of detection threshold of propyl lactate was conducted by the statistical analyses and the Curve Fitting, and the result was shown in Figure 3. The calibration curve equation was y = 0.1623x + 0.6877, and the correlation coefficient R 2 = 0.9663. The threshold was the corresponding X value (0.740 mg·L −1 ) when Y = 66.67% [17].

Discussion
The unknown compound shown at 17.4 min in sample 33 was identified to be propyl lactate based on the comparison of mass spectra and retention time with the standard. Propyl lactate was also discovered in all other samples under investigation by the same method.
Propyl lactate was possibly formed through the esterification of lactic acid with propanol, both of which have been reported during the fermentation. There were a great number of lactobacilli during the fermentation process of Chinese spirit drinks, which could convert sugars to lactic acid [18,19]. Higher alcohols could be formed during the fermentation under aerobic condition from sugar or under anaerobic conditions from amino acids [20] since the raw materials, sorghum, rice, sticky rice, wheat and corn, were rich sources of amino acids. Propanol can be produced from threonine by yeast via the Ehrlich metabolic pathway [21]. Small amounts of propanol could also be formed by yeast through reduction of propanal. The esterification of lactic acid with propanol could be taken place directly or catalyzed by esterases during the fermentation and aging process of Chinese liquor production [20]. The esterases might be from yeasts, molds, or bacteria, which existed in "Daqu" or "Xiaoqu" [21]. Fan [22] also reported that the "Daqu" had high esterase activities. The main formation process of propyl lactate was as Figure 4. The odor activity values (OAV) equaled to the ratio of the concentration of propyl lactate and its detection threshold value. If a compound has an OAV > 1.0, then it would contribute to the flavor of a product [23]. The OAVs of propyl lactate in 82 Chinese Baijius were listed in Table 1. There were 13 distillate samples with the OAVs of propyl lactate higher than 1, including 50° Jingzhi Guniang 5 years (4.440), 52° Jingzhi Guniang 10 years (3.024), 38° Jingyanggang (2.568), 53° Xianghe Ronghe Shaofang (2.313), and so on, whereas other 69 liquor samples with the OAVs lower than 1. Propyl lactate had a grape-like fruity, milk and ester odor [17]. The results indicated that propyl lactate was one of odor-active components of these 13 Chinese Baijius. Whether propyl lactate was a key odor compound or not based on more experiments and proofs, which we would be to study next.

Chemicals
Chemicals and standards were GC grade with a high purity (>99.0%).The water was boiling for at least 0.5 h and redistilled twice before use. Methyl lactate (PubChem CID: 11040), used as internal standard (IS), and propyl lactate (PubChem CID: 92821) were obtained from Tokyo Chemical Industry CO., Ltd. (Shanghai, China). Absolute ethanol (PubChem CID: 702) was obtained from Merck (Darmstadt, Germany).

Spirit Drink Samples
A total of 72 spirit drinks, shown as Table 1, were obtained from different factories in China, or supermarkets, such as Wal-Mart and Carrefour in Beijing, China.

Qualitative and Quantitative Analysis by GC-MS
1.0 µL of Chinese spirit drinks was injected and analyzed by GC-MS with the full scan mode, and the occurrence of propyl lactate was confirmed by comparing its retention time and mass spectrum with the standards. The concentrations of propyl lactate in these distillates were determined by the internal standard method with the selected ions monitoring mode of GC-MS. At first, a series of the standard solutions, such as 5.000 mg·L −1 , 2.500 mg·L −1 , 1.250 mg·L −1 , 0.625 mg·L −1 , 0.313 mg·L −1 , 0.156 mg·L −1 , 0.078 mg·L −1 , 0.039 mg·L −1 and 0.020 mg·L −1 , were prepared with absolute ethanol and analyzed by GC-MS. Then 1.0 mL of each distillate drink sample with 10.0 µL of methyl lactate solution (100.000 mg·L −1 ) were placed in 72 tightly closed sample vials, numbered 1 to 72, for GC-MS analysis. Finally, the concentrations of propyl lactate were calculated by the software of GC-MS. The conditions of GC-MS (6890A-5975C, Agilent technologies Co., Ltd., Beijing, China) were as follows.
MS conditions: electron ionization source, 70 eV; ion source and quadruple temperatures, 230 °C and 150 °C, respectively; The monitored ions and other parameters of selected-ion-monitoring (SIM) mode were listed in Table 2.

Determination of the Detection Threshold of Propyl Lactate
3-AFC test was recommended as the national standard method for determination of Chinese Baijiu flavors thresholds by GB/T 22366-2008 [16], which is a general guidance for measuring odor, flavor and taste detection threshold, because of its higher efficiency and accuracy. Meanwhile, CF method was adopted as threshold calculation method, which was recommended by ASTM E1432-2004 Standard Practice. So 3-AFC and CF were selected to determine the detection threshold of propyl lactate in Chinese spirit drinks.
There were eighteen samples for sensory evaluation, which were equally divided into six groups. All these samples were placed in 10 mL tulip-like glass wine cups. Two samples of each group were the control samples and the remaining one had different concentration of propyl lactate in it. Each sample was marked in a random four-digit number. A group of 30 untrained and normal olfaction assessors were invited to determine propyl lactate detection threshold. All samples were assessed at room temperature. For each test, the assessors needed to pick out the one which was "very different from the references", and wrote down the number. Each test was replicated 3 times, so that 90 responses were obtained for each testing concentration. Then the Curve Fitting (CF) was conducted by the statistical analyses, and the curve was drawn. When Y = 66.7%, the corresponding X value was the detection threshold value of propyl lactate.