Shsh Qi, Ping Zhn*, Honglei Tin*, Peng Wng Xueping M, Kixun Li

a School of Food Science and Technology, Shihezi University, Shihezi, Xinjiang 832000, China

b College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, Shaanxi 710119, China

Keywords:

Mutton broth

Thyme

Volatile compounds

Free fatty acids

GC-MS

PLSR

A B S T R A C T

To investigate the effects of thyme (Thymus vulgaris L.) addition on the flavor modification of mutton broth during boiling, three mutton-broth samples with various thyme contents 0.06% (S1), 0.30% (S2), and 1.50% (S3) were analyzed and compared, without thyme as control (0.00% , S0). The meaty, fatty, pastoral flavor and spicy were chosen as sensory attributes to evaluate the flavor of the mutton-broth samples.Sensory results were significantly different (P ＜ 0.001), with S2 having the optimum overall acceptability.A total of 99 volatile compounds were identified by gas chromatography-mass spectrometry, among which 19 compounds were considered as the odor-active compounds according to their odor activity values.Significant changes (P ＜ 0.05) appeared in most volatiles in S0 with thyme addition, especially aldehydes.Free fatty acids (FFAs) were also identified, and all of them significantly increased with increased thyme(P ＜ 0.05). Correlation analysis of odor-active compounds, FFAs, and sensory attributes through partial least squares regression indicated the important volatiles and FFAs remarkably contributed to the mutton broth samples, and further confirmed that the 0.30% of thyme may be a desirable addition amount for the sensory characteristics of mutton broth.

1. Introduction

Broth, with various forms and different flavors, plays an important role in Chinese food culture. Previous studies have shown that broth can improve gastric motility, increase consumer appetite,and reduce the probability of obesity [1,2]. Broth can easily be digested and absorbed and has beneficial effects for human health and healing considering its various water-soluble nutrients (amino acids,nucleotides, peptides and minerals, etc.) [3,4]. Nutrition of broth is important, whereas its flavor is also worth concerning because the flavor is an important indicator for evaluating consumers’ preference and choice of broth [5]; a complex set of thermally induced reactions,such as Maillard reaction, lipid oxidation, and their interaction, result in the generation of abundant volatile compounds and the formation of various flavors [6].

Regarding mutton is much tenderer than other meats, rich in polyunsaturated fatty acids (PUFAs) [7], regardless of religious taboos, it is widely used in making broth. However, a speciesspecific odor (unpleasant odor, described as a pastoral flavor here) of mutton can be released during heating, which might contribute to the decreasing acceptability among consumers [8]. Cooking meat with suitable spices is one of the key methods to improve the meat and broth flavor, as proven by several papers. Li et al. [9]used ginger and garlic for grass carp soup, Soto-Simental et al. [10]added maguey leaves while cooking lamb, Sun et al. [11]found that addition of star anise could affect the flavor of stewed chicken, and Mielnik et al. [12]used aqueous extracts of rosemary, sage, and thyme to marinade turkey thigh, which significantly reduced the warmed-over flavor.

Thyme (Thymus vulgarisL.), which belongs to the Lamiaceae family, has been wildly used as a culinary spice to add flavor to food and has some pharmacological properties [13,14]. Besides,thyme has a significant antioxidant capacity and antibacterial ability because of its high content of phenolic substances, such as thymol and carvacrol [15,16]. A traditional dish in Shaanxi Province of China named thyme-mutton broth is obtained by boiling mutton in a pan with the addition of thyme. It is famous for its delicious taste and as a unique diet for thousands of years. Nevertheless, the effect of thyme on mutton flavor has always lacked a scientific basis, resulting in considerable restrictions on the promotion and industrial production of thyme-mutton broth.

This study aimed to evaluate the effects of mutton in direct contact with thyme on the volatile compounds of the mutton broth during boiling, and investigate the reasons for flavor differences among mutton broth samples with and without thyme.

2. Materials and methods

2.1 Materials and reagents

The raw mutton materials were chosen from the hind legs and tails of three 12 months-old Sunit male sheep, which were purchased from the Zhu Que Market (Xi’an, China). The sheep were slaughtered in an abattoir following standard protocols. After aging at 4 °C for 24 h, the same part gathered from the 3 sheep per breed was combined as one sample. The hind legs and tails of carcasses were vacuum-packed and kept at –18 °C. Thyme (dry leaves) was provided by an online shop named Niao ge (Yulin, China). Re fined mutton suet and ram sheep tail were provided by Tianyuan Grease Co., Ltd. (Tianjin, China).

Methanol and 1,2-dichlorobenzene (99% purity) were of chromatography grade provided by TCI Development Co., Ltd.(Shanghai, China). Chloroform,n-hexane, andn-alkane standards(C7–C40, ＞ 97.1% purity) were of chromatography grade, with boron trifluoride (analytical reagent) were all purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Undecanoic acid(chromatography grade) was purchased from Tokyo Chemical Industry Co., Ltd.

2.2 Sample preparation

Skin, visible fat, and connective tissue were removed from the hind legs, and skin was removed from the tails after the frozen sample thawed at 4 °C for 24 h, then lean and tail fat were washed with water several times until clean and then cut into cubes of uniform size of approximately (2 ± 0.5) cm.

One hundred grams of raw meat (fat/lean 3:7), 150 mL of water,1 g of NaCl and thyme with different concentrations (based on raw meat weight) were separately put into a pressure bottle, capped tightly and heated in a thermostatic oil bath with magnetic stirring(300 r/min) at 120 °C from room temperature ((22 ± 0.5) °C) to broth boiling temperature; such condition was maintained for 2 h.Afterward, the bottles were immediately cooled in ice water to stop the reaction. After filtering with gauze, approximately 170 mL of broth was collected and stored at –18 °C until used. All samples were prepared in triplicate. The samples were named S0 (control group,without thyme), S1-S3 (addition group, S1: 0.06% thyme, S2: 0.30% thyme, S3: 1.50% thyme). Some samples were freeze-dried and frozen at -18 °C for fatty acid analysis.

2.3 Sensory evaluation

Sensory analysis was conducted by a trained panel consisting of 8 panelists at the age of 20-38 (4 males and 4 females) who were in good health, had no smoking habits, and did not suffer from rhinitis.They had sensory evaluation experience and were generically trained following the procedures of the ISO standard (ISO 8589: 2007). In addition, the panelists were trained specially for 1 week to ensure the results of this experiment more accurate referring to the method of Pu et al. [17]with some minor modification. They were required to descriptively analyze the following 4 odor attributes of the sample and required to be able to accurately identify the intensity of each odor attribute. Each of the above training was conducted 3 times, lasted for 20 min, to ensure that each panelist could competent for the sense evaluation work. The sensory attributes here included meaty, fatty,pastoral flavor, spicy and overall acceptability. Each attribute was scored on 5 cm non-structured lines with anchor points at each end(0 = absent, 5= very strong). The experiment was conducted in a sensory laboratory. Approximately 50 mL of broth packed in a transparent glass cup was served to each member. The cups were kept heated in a 50 °C water bath to simulate the environment in which people usually feel the flavor of the broth. Each sensory term was defined according to Song et al. [18]with some minor modification:refined mutton suet for “fatty”; defatted lean mutton boiled in water for 2 h for “meaty”; ram sheep tail boiled in water for 2 h for “pastoral flavor”; thyme boiled in water for 2 h for “spicy”; and the mutton broth product obtained from the local famous restaurant was labeled “overall acceptability”. The evaluations were performed in triplicate.

2.4 Analysis of volatile compounds

Volatile compounds were extracted by solid-phase microextraction(SPME) and analyzed by gas chromatography-mass spectrometry(GC-MS). 5.0 g of each sample was placed in a 15 mL headspace glass vial sealed with a polytetra fluoroethylene (PTFE)-faced silicone septum. Meanwhile, 2 μL of 1,2-dichlorobenzene (0.130 7 μg/μL in methanol) was an internal standard for each sample for quantitative analysis before the trap. Vials were placed in a block heater at 50 °C for both equilibration (15 min) and extraction (25 min). A 2 cm × 50/30 μm Stable-Flexdivinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS)-coated fiber (Supelco, Bellefonte, PA, USA) was used for the extraction. The fiber was then inserted into the GC injection port(Agilent 7890B) for desorption (250 °C/7 min in splitless mode).

Chromatographic separation was carried out in a DB-WAX column(30 m × 0.25 mm × 0.25 μm), and the temperature program was as follows: the column temperature program started at 45 °C and held for 6 min, increased to 53 °C at 2 °C/min for 1 min, then increased to 60 °C at 2 °C/min and maintained for 2 min, later increased to 100 °C at 3 °C/min holding for 3 min, subsequently, increased to 115 °C at 3 °C/min holding for 2 min, finally, increased to 170 °C at 2 °C/min and maintained for 2 min. Detection was performed by MS (Agilent 5977B).The electron impact ionization of the MS detector was 70 eV, with the quadrupole in full-scan mode (total ion currents from 45 to 350m/zat 1 scan/s). The interface, source, and quadrupole temperatures were 250, 230, and 150 °C, respectively.

Compound identification was carried out by spectral comparison using the NIST/EPA/NIH Mass Spectral Library (NIST14 or by calculation of retention indexes (RI) relative to a series ofn-alkanes(C7–C40)). The compound was calculated according to the formula described in the literature [19]after the conversion unit, and the result meant the amount of the compound containing in the sample (relative to the internal standard).

2.5 Analysis of fatty acids

Each sample was weighed accurately, using 1.0 mL (4.359 mg/mL)of undecanoic acid as an internal standard. 20, 10, and 10 mL of a mixed solvent (chloroform/methanol: 2/1) were added respectively,extracted 3 times, filtered, and combined to dry. 2 mL of sodium hydroxide methanol (0.50 mol/L) solution was added to the above samples, and then bathed in the water at 60 °C for 30 min. Following cooling, 2 mL of boron tri fluoride methanol (25% ) solution was added and then bathed in water at 60 °C for 20 min. Following the second cooling,n-hexane (2 mL) and saturated sodium chloride solution(2 mL) were added. At last, the mixture was shaken, extracted, stood still to stratification. The upper layer was harvested for GC (Shimadzu,GC-2030) analysis.

A DB-WAX column (30 m × 0.32 mm × 0.25 μm) was used. The column temperature program started at 100 °C and held for 3 min,increased to 180 °C at a rate of 10 °C/min and maintained for 1 min,at last, increased to 240 °C at a rate of 3 °C/min, holding for 9 min.Other chromatographic conditions were referred to Zhang et al. [20],slightly modified. The fatty acid methyl esters were identified by the NIST 15 mass spectra database and quantitated using undecanoic acid as an internal standard.

2.6 Odor activity values (OAVs)

The OAVs were used to evaluate the contribution of each compound to the flavor of the mutton broth and was calculated as the ratio of the concentration of each compound to its odor threshold in water.

2.7 Data processing

Differences between means were handled by one-way ANOVA with Duncan’s multiple comparison tests with SPSS 21.0 for windows(SPSS Inc., USA). AP-level less than 0.05 was considered a significant difference. The correlations between free fatty acids, odor-active compounds, and sensory data were analyzed by partial least squares regression (PLSR) using Unscrambler version 9.7 (CAMO ASA,Oslo, Norway).

3. Results and discussion

3.1 Sensory evaluation

The sensory evaluation of samples is shown in Fig. 1. There was a significant difference among the 4 samples (P＜ 0.001). The contours of S0 were similar to those of S1, and no significant difference(P＞ 0.05) existed between their indicators, except for spicy attribute. The reason might be explained by the observation that the level of thyme here was excessively low to affect the flavor noticeably. These two samples scored higher on pastoral flavor,meaty and fatty attributes while scored lower on the attribute of overall acceptability. Similar to the situation encountered in daily cooking, mutton without adding spice produced a strong unpleasant odor (known as pastoral flavor), and that might be partly related to the high content of aldehydes, especially unsaturated aldehydes, as reported in the literature that unsaturated aldehydes were likely to have a certain effect on the species-characteristic flavor [21]. On the contrary, S3 with 1.50% thyme exhibited the lowest pastoral flavor, meaty and fatty notes, but showed the highest spicy note, thus resulting in the worst overall acceptability. This might be attributed to the high addition sample S3 generating strong spicy smell, and thus dramatically covering up the pastoral flavor, meaty and fatty attributes. Another reason might be the fact that the high amounts of antioxidant compounds in thyme hindered the production of the volatiles from lipid oxidation, among which were described as the main contributors to species-specific mutton flavor.

Fig. 1 Results of sensory evaluation of four different mutton broth. *** means significance at P ＜ 0.001, which can more intuitively show the difference in aroma of all the samples in the sensory attributes.

Notably, S2 showed the highest score in overall acceptability, as its moderate scores in meaty, fatty and spicy notes, and very weak in pastoral flavor. This finding may be attributed to the appropriate addition level of thyme to modify the balance of odorous compounds.On one hand, the risk of high content of aldehydes to fatty and pastoral flavor could be reduced by properly regulating the lipid oxidation reaction; on the other hand, the introduction of various flavor substances in thyme (e.g., terpenoids) could also contribute to modification and enhancement the meat flavor via direct or indirectly participating in further various reactions [7].

3.2 Free fatty acids (FFAs)

As the important precursors of meat flavor, FFAs were associated with the characteristic flavor of meat [6]. The content and composition of FFAs in mutton broth from the 3 different addition groups (S1-S3) and control group (S0) are listed in Table 1. A total of 16 FFAs were detected in all samples, including 9 saturated fatty acids (SFAs),4 monounsaturated fatty acids (MUFAs), and 3 PUFAs. The most abundant PUFA, MUFA, and SFA of each sample were linoleic acid (C18:2), oleic acid (C18:1) and palmitic acid (C16:0), respectively.ANOVA of the FFAs results revealed that significant differences(P＜ 0.01) existed among different samples due to the addition of thyme. Total FFAs varied from 85.529 mg/g in control (S0, without thyme) to 392.326 mg/g in S3 with 1.50% thyme. As shown in Table 1,the contents of SFA in S1-S3 were 1.2, 3.8, and 4.4 times of their levels in S0, respectively; with the contents of MUFA in S1-S3 were 1.3, 4.2 and 4.7 times of that in S0, respectively. Similarly, the PUFA contents also showed an upward trend with the increasing thyme addition,reaching approximately 1.5, 3.8, and 4.3 fold higher than that of control group S0, respectively. Thyme is rich in phenolic compounds, which have remarkable antioxidant and antibacterial activities [15], and good effects on inhibiting oil oxidation under heating conditions [22].Therefore, the increase in FFAs was primarily due to the natural antioxidants in thyme that inhibited fat oxidation during boiling.

Table 1FFAs in freeze-dried broth with different treatments (n = 3).

Comparing the control group S0, all FFAs exhibited group insignificant (P＞ 0.05), except for arachidonic (C20:4), when thyme was added at a lower level as in the sample S1. Arachidonic had the highest degree of unsaturation, and the fastest thermal oxidation rate;thus this fatty acid was prone to specific influence by the antioxidant components. However, FFAs in S2 (added at a moderate level) and S3(added at a high level) showed a significant increase compared with that in S0 (P＜ 0.05). This result indicated that the reactions of FFAs oxidation and degradation might be inhibited with the continuous increase in the content of certain antioxidants of thyme. The content of FFAs, except for pentadecanoic acid (C15:0) and eicosenoic acid (C20:1),in S3 did not increase significantly compared with that in S2 (P＞ 0.05),which might be due to that the thyme concentration was already close to saturation in the sample S2.

The above-mentioned results supported the speculation that the differences for fatty and pastoral flavor among the samples were arising from different degrees of lipid oxidation inhibition caused by thyme addition during boiling.

3.3 Volatile compounds by GC-MS

A total of 99 volatile compounds, mainly including aldehydes,ketones, alcohols, esters, phenol, aromatic compounds, furans, alkenes,nitrogen- and sulfur-containing compounds, and alkanes, were identified, as shown in Table 2. Alcohols were in the largest amount,followed by alkenes and aldehydes. Table 3 lists the chemical classes,quantities, proportions and the possible formation ways of volatile compounds in different samples. As shown, 55 compounds were found in S0, in which the largest number belonged to aldehydes. Compared with the control group S0 (55), the number of volatiles in addition groups(S1-S3) increased to numbers of 60, 58 and 70, respectively. Among these 3 addition groups, the numbers of aldehydes in S1 had a slight change compared to S0, remained in the highest quantities; alkenes increased to 3 times than those in S0. As the amount of thyme continued to increase,alcohols became the most abundant compounds in S2, followed by aldehydes. However, alcohols and alkenes were in the highest numbers in S3, while the number of aldehydes dropped to the third-highest. Detailed information on the major families of volatiles is as follows:

Table 2Volatile compounds identified in mutton broth with different concentration of thyme by GC-MS.

Table 2 (Continued)

Table 2 (Continued)

Table 3Quantities and ratios of volatile composition of different samples.

Aldehydes were mainly derived from lipid oxidation, and could also be formed by Streck degradation [23,24]; these volatiles were generally described as green, fruity, and dainty at low concentration, but exhibited rancid, fatty, pastoral flavor or other unpleasant flavors at high contents [25].As previously reported in the study, aldehydes in cooked mutton or lamb were in a high total amount and a large number [10,26,27].This was consistent with our results, aldehydes were the most volatiles (226.783 μg/kg) in control group S0 (Table 2), including 8 saturated aliphatic aldehydes, 9 unsaturated aliphatic aldehydes, and 1 aromatic aldehyde. Heptanal had the highest level in the saturated aliphatic aldehydes (62.440 μg/kg), followed by hexanal, nonanal and octanal, which were all in a considerable level (＞ 30 μg/kg), whereas unsaturated aldehydes were in the most abundant in S0, in which,(E)-2-heptenal, (E)-2-octenal, (E)-2-nonenal and (Z)-2-decenal were also in high levels (＞ 5 μg/kg). Octanal, nonanal, and (Z)-2-decenal were derived from the oxidation of oleic acid, whereas hexanal,heptanal, (E)-2-heptenal, (E)-2-octenal and (E)-2-nonenal were generated from the oxidation of linoleic acid [28,29]. Benzaldehyde could be derived from the degradation ofα-linolenic acid [30]. The same as previous research [31,32], hexanal in the control group S0 was the predominant aldehyde. Hexanal was a more effective indicator of meat lipid oxidation than any other volatile compounds [33]. As shown in Table 2, hexanal (N2) decreased significantly in the addition groups (P＜ 0.05), from 42.143 μg/kg to 0.521 μg/kg, which indicated that thyme inhibited the oxidation of linoleic acid. It was consistent with the FFAs results (Table 1). The down-regulation of aldehyde levels in the addition group also confirmed this speculation. A similar result appeared in the research of Nieto et al. [34], who found that the amount of hexanal could be significantly reduced in cooked sheep meat by adding thyme leaves to the animal diet. Similar results were found in other aldehydes, especially heptanal (N3), octanal (N5), nonanal(N8), (E)-2-octenal (N9) and (E)-2-nonenal (N13), as clearly shown in the gas chromatogram (Fig. 2). Furthermore, the high content of these aldehydes was most likely the cause of the pastoral flavor and the lowest overall acceptability of the control group.

Fig. 2 Gas chromatogram of 4 different mutton broth. S0-S3 shows photos of the samples; codes represent compounds shown in Table 2. Codes marked in red represent compounds that are significantly reduced after the addition of thyme; blue rectangles represent compounds that are directly introduced or significantly increased due to thyme addition.

Another major product of lipid oxidation and degradation was ketones. Only 4 ketones (0.782 μg/kg) were found in sample S0,and the contents were all at a low level. Compared with the control group S0, 3-ethylcyclopentanone and 2-tridecanone were significantly decreased in the addition group (P＜ 0.05). However, 2-decanone had no significant differences (P＞ 0.05) and 2-undecanone presented irregular changes, and the reason for this was unclear.

Alcohols might be produced in the process of secondary decomposition of hydroperoxide of fatty acids [35]. Twelve alcohols(29.976 μg/kg), including isoborneol and 11 aliphatic alcohols,were detected in S0. Among them, 4 alcohols with higher contents(＞ 5 μg/kg) were 1-hexanol, 1-octen-3-ol, 1-heptanol, and 1-octanol,respectively. 1-Octen-3-ol, 2-nonen-1-ol, and isoborneol showed significantly higher levels in the addition groups than that in the control group (P＜ 0.05). These 3 alcohols had inherently high levels in thyme(data not shown) might be the reasons for such a phenomenon. The 9 other alcohols showed opposite changes, that might be some lipid oxidation processes were inhibited by thyme. Three liner alcohols(1-hexanol (N34), 1-heptanol (N38) and1-octanol (N44)) in S0 had obviously decreased as in the addition groups (Fig. 2, red line), but they were considered to have little contribution to meat products as their high threshold.

It should be noted that the gas chromatographic peak areas of 4 alcohols had obviously increased in the chromatogram after adding thyme (Fig. 2); they were eucalyptol (N31), linalool (N42),isoborneol (N52), and 1-octen-3-ol (N37), respectively. 1-Octen-3-ol was widely present in various types of meat and meat products,playing an important role in the formation of their overall aroma, and was generally considered a kind of meat flavor modifier [7,36,37].Terpenols, represented by N31, N42, and N52, were naturally occurring aromatic compound in thyme; they could provide a variety of aroma when at a suitable concentration.

Four furans (27.436 μg/kg), and 3 nitrogen- and sulfur-containing compounds (1.276 μg/kg) were found in S0. Obviously, the levels of these compounds in the addition group had significantly changed(P＜ 0.05). All of the furans, including 2-ethylfuran, 2-butylfuran,2-pentylfuran, and 2-hexylfuran, underwent a significant decrease(P＜ 0.05). Likewise, methoxy-phenyl-oxime and benzothiazole showed the same trend. The level of 3-thiophenecarboxaldehyde increased only in S3. Furans and sulfur-containing compounds were considered important compounds in meat products with a relatively low threshold [38], and they were primarily formed by the Maillard reaction [39,40]. As a noncarboxylic compound derived from the oxidization of linoleic acid and othern-6 fatty acids [41], 2-pentylfuran(N66) showed the highest value (24.181 μg/kg) compared with other heterocyclic compounds in S0, and its peak area underwent a significant reduction in the chromatogram after adding thyme (Fig. 2).

Only 3 benzene compounds (21.678 μg/kg) were found in S0,includingp-cymene, toluene, and naphthalene. Among them,p-cymene showed the highest level (20.782 μg/kg), which was significantly increased in the addition group (P＜ 0.05).p-Cymene presented in S0 might be derived from the thermal decomposition of hydrocarbons, fats,and proteins, and that was in accordance with the source of aromatic hydrocarbons [42]. However, its level had greatly increased in the addition group (Fig. 2, N60) because it was presented in a large amount in thyme (data not shown).p-Cymene had a characteristic “solvent,gasoline, and citrus” odor (Table 4), and it might play an important role in the overall flavor of mutton broth. No significant difference (P＞ 0.05)was determined in the level of toluene, whereas the level of naphthalene was significantly decreased in the addition groups (P＜ 0.05).

Table 4Odor-active compounds (OAVs ＞ 1) in broth with different treatments.

The content (9.408 μg/kg) and species (only 3) of alkenes were at a low level in S0 compared with those in S2; the highest content wasγ-terpinene (1.218 μg/kg), followed by 3-ethyl-2-methyl-1, 3-hexadiene,and (Z)-5-tridecene.γ-Terpinene in S0 might be derived from the mutton diet, and its content was obviously increased by the increase of thyme (P＜ 0.05). The content ofγ-terpinene in thyme was abundant(data not shown). Besides, two other alkenes were significantly decreased (P＜ 0.05) in the addition group, which could also be caused by the inhibition of lipid oxidation. Noticeably, 6 terpenes (α-pinene(N69), camphene (N70),β-myrcene (N75), terpinolene (N76),D-limonene (N77), and sabinen (N78)) with a high content appeared in the addition group (Fig. 2), and they all originated from thyme. These terpenes were one of the main sources of spice attributes in sensory evaluation of mutton broth.

In addition, 1 ester (0.015 μg/kg) namedγ-dodecalactone, 4 alkanes(13.577 μg/kg), and 3 unknown compounds were detected in the control group S0. Alkanes were generally considered to have a minimal contribution to flavor because of their high threshold, and the content ofγ-dodecalactone was too low to effectively affect the flavor.

3.4 Odor-active compounds of mutton broth

Concentration was considered in assessing the contribution of individual compounds to the overall flavor, and the threshold was an important factor. The odor-active compounds in this study were defined as the compounds with OAVs ＞ 1. A total of 19 odor-active compounds are listed in Table 4, including 10 aldehydes, 4 alcohols,3 terpenes, 1 furan, and 1 benzene compound. Obviously, all of them were affected by thyme addition. The OAVs of the 10 aldehydes and 1 furan showed a decreasing trend; however, this condition did not mean that all pleasant aroma substances were reduced. For example,hexanal showed a grassy aroma at a low concentration which had a positive effect on meat products, while it displayed a rancid taste at a high concentration, and which exhibited a negative effect on the meat flavor [28]. Therefore, maintaining a proper level of aldehydes would produce a pleasant odor.

By contrast, the OAVs of terpenes (except forα-pinene), alcohols,andp-cymene showed a regular increase with an increase in thyme concentration. Eucalyptol (green, woody, sweet), linalool (flower,lavender),α-pinene (pine, turpentine), andβ-myrcene (pepper, spicy,plastic) were derived from thyme, which provided various aroma compounds to the mutton broth. The OAVs of these compounds could reach 32.5, 1.7, 21.3 and 12.3, respectively, when the concentration of thyme reached S2. Two other compounds with relatively high OAVs (＞ 10)were 1-octen-3-ol andp-cymene. The OAV of 1-octen-3-ol showed an increase, which might compensate for the loss of meat aroma caused by the reduction of furan and certain aldehydes to some extent. The reason for such condition was according to one research that 1-octen-3-ol(mushroom, smoke) might be a major contributor to the overall meat aroma [43,44].p-Cymene is in high content and low threshold (5.01) in thyme and many other spices, which makes it an important part of the unique flavor of spice- meat.

3.5 FFAs, odor-active compounds and sensory profiles by PLSR

PLSR was conducted to show the relationship among FFAs,odor-active compounds and sensory attributes in mutton broth intuitively after adding thyme. TheX-matrix was designed as 16 FFAs(Table 1) and 19 odor-active compounds (Table 4), whereas theY-matrix was designed as 5 sensory notes. Fig. 3 shows that the calibrated explained variance for this model was PC1 = 84% and PC2 = 5% . 4 samples were well distinguished in the plot, and they were located in the 4 quadrants, respectively, which was consistent with the results of sensory evaluation. Fatty, meaty and pastoral flavor attributes were on the left side of the plot,while spicy, overall acceptability attributes and FFAs were on the opposite side. All the sensory attributes, FFAs and odor-active compounds, except for decanal (N11), were located between the inner and the outer ellipses, respectively, which represented that they could be well explained by this PLSR model (the correlation coefficient was 0.964 520).

Fig. 3 Correlation loadings plot for FFAs, odor-active compounds (X-matrix) and sensory scores (Y-matrix) of mutton broth.

Three sensory attributes (meaty, fatty, and pastoral flavor) located in the negative direction of PC1, near S0 and S1 on the same side,indicating that they were relatively highly correlated with these two samples. It was well explained by the data of FFAs (Table 1) and volatile compounds (Table 2); most of the compounds in S0 and S1 were insignificantly different (P＞ 0.05). 7 aldehydes (hexanal (N2),octanal (N5), (E)-2-octenal (N9), decanal (N11), (E)-2-nonenal(N13), (E,E)-2,4-nonadienal (N16), (E,E)-2,4-decadienal (N18))and 2-pentylfuran (N66) showed a significant correlation with the pastoral flavor. In addition, these 8 compounds together with heptanal (N3), nonanal (N8) and (E)-2-undecenal (N17) were related to meaty and fatty attributes. This also agrees with the results of sensory results.

In the positive direction of PC1, it seems that spicy became the characteristic flavor of S3. Meanwhile, eucalyptol (N31), 1-octen-3-ol (N37), linalool (N42), isoborneol (N52),p-cymene (N60),α-pinene (N69),β-myrcene (N75) andγ-terpinene (N79) of odoractive compounds were significantly correlated with spicy attribute.Their contents were significantly increased with an increase in thyme concentration, as shown in Table 2 (P＜ 0.05). Interestingly, the 16 FFAs were concentrated in the quadrants to which S2 and S3 belonged.It was mainly because the oxidative degradation of fatty acids was more sufficiently suppressed as the level of thyme increased to a certain extent, which was consistent with the results in Table 1. Eleven FFAs,namely C10:0(A1), C14:0(A3), C15:0(A4), C16:0(A5), C16:1(A6), C17:0(A7),C18:1(A10), C18:2(A11), C18:3(A12), C20:1(A14) and C20:4(A15), were significantly correlated with sensory variables (P＜ 0.05).

4. Conclusions

The sensory attributes of mutton broth were affected significantly by the addition of thyme during boiling, and the overall acceptability was greatly improved. One of the reasons was that the oxidation and degradation of mutton suet were inhibited by thyme, which induced significant changes in the content of volatile compounds; the other was that abundant terpenoids were introduced to make the flavor of mutton broth more pleasant. 19 odor-active compounds of mutton broth were identified by OAVs. Correlation analysis of odor-active compounds,FFAs, and sensory attributes through PLSR indicated the important volatiles and FFAs remarkably contributed to the mutton broth samples,and further confirmed that the 0.30% of thyme may be a desirable addition amount for the sensory characteristics of mutton broth. The information provided an important reference for the flavor control of spiced mutton and broth products in the future.

Acknowledgments

This work was supported by the National key research and development program (grant number 2016YFD0400705).

Declaration of Competing Interest

None.