# Effect of Chilled Ageing Conditioning at 4°C in Lamb Longissimus Dorsi Muscles on Water-Soluble Flavour Precursors as Revealed by a Metabolomic Approach

Authors: Liqin You; Ruiming Luo
Journal: Journal of Food Quality
 (2019)

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## Abstract

The objective of this study was to investigate, by a metabolomic approach, the effects of chilled ageing conditioning at 4°C in lamb longissimus dorsi (LD) muscles on water-soluble flavour precursors. The results showed that the content of nucleotide degradation products significantly increased (P<0.05) due to the adjusted biosynthesis of alkaloids derived from histidine and purine from day 0 to day 4. Additionally, the content of glycolytic compounds significantly increased (P<0.05) due to enhanced glycolysis, and the content of organic acid increased (P<0.05) because of the altered tricarboxylic acid cycle (TCA) from day 0 to day 4. In addition, the content of total free amino acids significantly increased (P<0.05), owing to the altered biosynthesis of amino acids from day 4 to day 8. These results are significant proof that there were quantitative changes observed in lamb flavour precursors during chilled ageing.


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## Body

## 1. Introduction

Lambs are important meat-producing animals worldwide [1]. Flavour is an important quality evaluation parameter for cooked meat and is developed through a series of complex reactions among flavour precursors in the raw meat [2]. Water-soluble components and lipids are two major categories of flavour precursors [3, 4]. Ageing (postmortem) affects the contents of flavour precursors mainly through the action of cell metabolism and endogenous enzymes [5]. Meat shows a significant alteration in various chemical components like free ribose, hypoxanthine, and phosphate from the breakdown of ribonucleotides, free amino acids, and peptides through proteolysis, low molecular weight, and sugar-related metabolites by depletion of glycogen [6]. These compounds may be used as flavour precursors or intermediates to form meaty aroma during cooking [3].Metabonomics is defined as the study of the endogenous metabolites present in an organism and the changes that occur in these compounds [7]. Metabolomics is a common analytical method used in the field of food science, including nuclear magnetic resonance spectroscopy (NMR) [8], gas chromatography-mass spectrometry (GC-MS) [9], and liquid chromatography-mass spectrometry (LC-MS) [10]. Of the various profiling techniques in metabolomics, GC-MS platform offers higher resolution and sensitivity detection and convenient mass spectral data [11]. Two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC × GC-TOF/MS) has the advantages of increased peak capacity, sensitivity, and selectivity for the qualitative and quantitative analysis of complex matrices [9]. In meat science, metabolomics has been used to investigate the confinement odour of lamb and assess the spoilage of foods [12]. The method was also used to detect mechanically recovered meat in food products [13], evaluate beef conservation and metabolite profiles during ageing [14–16], and determine the processing conditions of dry-cured hams [17].Although many studies have reported changes in flavour precursors during the postmortem aging, most reports determine flavour precursors mainly by the traditional chromatography method to evaluate the influence of aging on flavour precursors during the postmortem ageing [18–21]. However, the past methods could not comprehensively assess the existing metabolites and their related metabolic pathways and their variations in the biological system of organisms. Therefore, this study was aimed to determine the effects of postmortem chilled ageing up to 8 days at 4°C on flavour-related metabolites of lamb LD by a metabolomic approach and investigate the potential impact of the key metabolic pathways on water-soluble flavour precursors.


## 2. Materials and Methods

### 2.1. Materials and Samples Collection

A total of 8 LD muscles (c.1.5 kg each) obtained from approximately 6-month-old Tan sheep were bought from a commercial meat company (Yanchi, Yinchuan, China). The samples were vacuum-packed and transferred in portable coolers (4°C) to the laboratory. The samples were stored at 4°C and a relative humidity of 80% in polystyrene trays sealed with polyethylene films for 8 days. Sampling consisted of cutting a 20 cm × 20 cm slice from the whole LD using an alcohol-sterilized knife on day 0, 4, and 8. Samples (n = 8, each for 200 mg) taken from different time points were immediately frozen at −80°C for metabolic analysis.


### 2.2. Sample Derivation

The experimental protocols for sample derivation followed our previously published work with minor modifications [22]. Samples (30 mg) were placed into 2-mL EP tubes, treated with methanol-chloroform (Meryer Technologies Co., Ltd., Shanghai, China; 0.4 mL, 3 : 1, v/v), and L-2-chlorophenylalanine (20 μL, Hengbai Biotech Co., Ltd., Shanghai, China; 1 mg/mL stock in d H2O) was added as an internal standard. The suspension was homogenized using a ball mill (3 min, 65 Hz) and centrifuged 14,000 × g for 15 min at 4°C. QC sample was prepared by mixing aliquots of all the samples to be a pooled sample. The supernatant (300 μL) was transferred to GC-MS glass vials (2 mL), vacuum dried at 37°C for about 3.5 h, and methoxylamine hydrochloride (80 μL) was added. Methoxylamine hydrochloride (Meryer Technologies Co., Ltd., Shanghai, China) was initially dissolved in pyridine for a final concentration of 20 mg/mL. Samples were incubated at 80°C for 20 min following mixing and sealing. After incubation, the vials were opened and BSTFA (80 μL; Regis Technologies, Inc., Morton Grove, USA) and hexane (20 μL; Hengbai Biotech Co., Ltd., Shanghai, China) were added into each sample and vortexed vigorously for 2 min. Vials were sealed again, incubated at 70°C for 1 h, and then cooled to room temperature for subsequent GC-MS analysis.


### 2.3. GC × GC-TOF/MS Metabolite Analysis

Samples were analyzed by GC × GC-TOF/MS based on the reports of Peng et al. [23] and Jin et al. [9], with minor modifications. The GC × GC-TOF/MS system used was a gas chromatograph 6890N Agilent (Agilent Technologies) with a LECO Pegasus 4D time-of-flight mass spectrometric (TOF MS) system (St. Joseph, MI, USA). The first-dimensional chromatographic separation column was a DB-5MS (30 m × 0.25 mm i.d., 0.25 µm df, J&W Scientific, Folsom, CA). The second-dimensional column was DB-17HT (2 m × 0.1 mm i.d., 0.15 μm df, J&W Scientific, Folsom, CA). Highly purified helium (99.999%) was selected as the carrier gas, with a flow rate of 1.0 mL/min. All samples (1 µL) were introduced into the GC × GC inlet system by an auto sampler using splitless mode injection at 260°C. The initial oven temperature was 80°C, held at 80°C for 2 min, ramped to 180°C at a rate of 10°C/min to 240°C at a rate of 5°C/min and to 280°C at a rate of 20°C/min, and finally held at 280°C for 9 min. The TOF/MS was operated in the electronic impact ionization mode at 70 eV at an acquisition rate of 100 spectra/s and a mass range of m/z 30–600. The ion source temperature was 220°C, and the interface temperature was 280°C, respectively. The quality control samples (QCs) were injected at regular intervals (every 10 samples) throughout the analytical run to provide a set of data from which repeatability could be assessed.


### 2.4. Statistical Analyses

Data on GCxGC-TOF/MS were transferred into the SIMCA software package (14.0, Umetrics, Umeå, Sweden). Principle component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) were performed to visualize the metabolic differences among three groups. The Hotelling’s T2 region is shown as an ellipse in score plots of the models, which defines the 95% confidence interval of the modelled variation. Variable importance in the projection (VIP) ranks the overall contribution of each variable to the OPLS-DA model, and the variables with “VIP > 1.00” and “P<0.05” were considered relevant for group discrimination.Differential metabolites from GC × GC-TOF/MS were further validated by searching the online databases including the Kyoto Encyclopedia of Genes and Genomes (KEGG) and theOvis aries metabolome database. Each differential metabolite was then cross-matched with the pathways in the KEGG, and the key altered pathways were identified and finally integrated according to the potential functional analysis.The effect of ageing on flavour precursors was analysed by ANOVA using SPSS 19.0 statistical software (SPSS Inc., Chicago, IL, USA.).


## 2.1. Materials and Samples Collection

A total of 8 LD muscles (c.1.5 kg each) obtained from approximately 6-month-old Tan sheep were bought from a commercial meat company (Yanchi, Yinchuan, China). The samples were vacuum-packed and transferred in portable coolers (4°C) to the laboratory. The samples were stored at 4°C and a relative humidity of 80% in polystyrene trays sealed with polyethylene films for 8 days. Sampling consisted of cutting a 20 cm × 20 cm slice from the whole LD using an alcohol-sterilized knife on day 0, 4, and 8. Samples (n = 8, each for 200 mg) taken from different time points were immediately frozen at −80°C for metabolic analysis.


## 2.2. Sample Derivation

The experimental protocols for sample derivation followed our previously published work with minor modifications [22]. Samples (30 mg) were placed into 2-mL EP tubes, treated with methanol-chloroform (Meryer Technologies Co., Ltd., Shanghai, China; 0.4 mL, 3 : 1, v/v), and L-2-chlorophenylalanine (20 μL, Hengbai Biotech Co., Ltd., Shanghai, China; 1 mg/mL stock in d H2O) was added as an internal standard. The suspension was homogenized using a ball mill (3 min, 65 Hz) and centrifuged 14,000 × g for 15 min at 4°C. QC sample was prepared by mixing aliquots of all the samples to be a pooled sample. The supernatant (300 μL) was transferred to GC-MS glass vials (2 mL), vacuum dried at 37°C for about 3.5 h, and methoxylamine hydrochloride (80 μL) was added. Methoxylamine hydrochloride (Meryer Technologies Co., Ltd., Shanghai, China) was initially dissolved in pyridine for a final concentration of 20 mg/mL. Samples were incubated at 80°C for 20 min following mixing and sealing. After incubation, the vials were opened and BSTFA (80 μL; Regis Technologies, Inc., Morton Grove, USA) and hexane (20 μL; Hengbai Biotech Co., Ltd., Shanghai, China) were added into each sample and vortexed vigorously for 2 min. Vials were sealed again, incubated at 70°C for 1 h, and then cooled to room temperature for subsequent GC-MS analysis.


## 2.3. GC × GC-TOF/MS Metabolite Analysis

Samples were analyzed by GC × GC-TOF/MS based on the reports of Peng et al. [23] and Jin et al. [9], with minor modifications. The GC × GC-TOF/MS system used was a gas chromatograph 6890N Agilent (Agilent Technologies) with a LECO Pegasus 4D time-of-flight mass spectrometric (TOF MS) system (St. Joseph, MI, USA). The first-dimensional chromatographic separation column was a DB-5MS (30 m × 0.25 mm i.d., 0.25 µm df, J&W Scientific, Folsom, CA). The second-dimensional column was DB-17HT (2 m × 0.1 mm i.d., 0.15 μm df, J&W Scientific, Folsom, CA). Highly purified helium (99.999%) was selected as the carrier gas, with a flow rate of 1.0 mL/min. All samples (1 µL) were introduced into the GC × GC inlet system by an auto sampler using splitless mode injection at 260°C. The initial oven temperature was 80°C, held at 80°C for 2 min, ramped to 180°C at a rate of 10°C/min to 240°C at a rate of 5°C/min and to 280°C at a rate of 20°C/min, and finally held at 280°C for 9 min. The TOF/MS was operated in the electronic impact ionization mode at 70 eV at an acquisition rate of 100 spectra/s and a mass range of m/z 30–600. The ion source temperature was 220°C, and the interface temperature was 280°C, respectively. The quality control samples (QCs) were injected at regular intervals (every 10 samples) throughout the analytical run to provide a set of data from which repeatability could be assessed.


## 2.4. Statistical Analyses

Data on GCxGC-TOF/MS were transferred into the SIMCA software package (14.0, Umetrics, Umeå, Sweden). Principle component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) were performed to visualize the metabolic differences among three groups. The Hotelling’s T2 region is shown as an ellipse in score plots of the models, which defines the 95% confidence interval of the modelled variation. Variable importance in the projection (VIP) ranks the overall contribution of each variable to the OPLS-DA model, and the variables with “VIP > 1.00” and “P<0.05” were considered relevant for group discrimination.Differential metabolites from GC × GC-TOF/MS were further validated by searching the online databases including the Kyoto Encyclopedia of Genes and Genomes (KEGG) and theOvis aries metabolome database. Each differential metabolite was then cross-matched with the pathways in the KEGG, and the key altered pathways were identified and finally integrated according to the potential functional analysis.The effect of ageing on flavour precursors was analysed by ANOVA using SPSS 19.0 statistical software (SPSS Inc., Chicago, IL, USA.).


## 3. Results and Discussion

### 3.1. Multivariate Statistical Analysis

The Chroma TOF4.3X software (LECO) and LECO-Fiehn Rtx5 database were used to analyze the data. For all group samples, the missing value of the original data was simulated by filling half of the minimum value and noise removal was used by an interquartile range to filter data. The data were normalized to the total peak area of each sample and multiplied by 10000, and the peaks from the same metabolite were combined. The datasets consisted of 102 variables obtained from GC × GC-TOF/MS analyses after applying the above-described quality assurance criteria.These datasets were used to build PCA and OPLS-DA in order to visualize group trends during meat chilled ageing. The PCA score plot (Figures1(a) and 1(d)) and OPLS-DA (Figures 1(c) and 1(f)) showed a clear separation of all sampling periods. The results indicated that the ageing time had a significant impact on the changes of metabolites. The fit parameter (R2) and prediction parameter (Q2) values shown in Figures 1(b) and 1(e) were calculated based on 7-fold cross validation and 200-response permutation testing that represents the quality of the obtained OPLS-DA model. The corresponding R2 values obtained between day 0 and day 4 and day 4 and day 8 were 0.983 and 0.990; Q2 values were −0.158 and −0.120, respectively. The permutation tests among the 3 groups were all in a good range, indicating a satisfactory effectiveness of the model.Figure 1
PCA, corresponding validation plots of OPLS-DA, and OPLS-DA score plots derived from the GC × GC-TOF/MS between day 0 and day 4 and day 4 and day 8 during chilled ageing. Day 0: triangles; day 4: circles; day 8: squares.
(a)
(b)
(c)
(d)
(e)
(f)All the samples in the score plots of PCA and OPLS-DA were inside the 95% Hotelling T2 ellipse. Clear separation and discrimination indicated that the OPLS-DA model can be used to identify the difference between groups.


### 3.2. Differential Characteristic Metabolites

The reproducible ionization method used for GC × GC-TOF/MS qualitative identification of compounds is less complicated due to the availability of universal mass spectral libraries. Based on the NIST 11 database and Fiehn metabolomics library, the majority of the peaks were endogenous metabolites and some of these peaks may be attributed to the derivatives of byproducts. After screening with “VIP > 1.00,” “P<0.05,” and “similarity > 700,” significantly different metabolites were identified (Table 1).Table 1
Identification of significantly different metabolites in lamb samples using GC × GC-TOF/.
Metabolite name
RT
Mass
Similarity
VIP
P value
FC
0 d versus 4 d
Nicotinoyl-glycine
7.28
207
912
1.26
0.05
1.34
Pyruvic acid
7.46
174
885
1.56
0.04
0.64
Hydroxylamine
8.29
146
870
1.74
<0.01
1.21
3-Hydroxybutyric acid
8.64
117
925
1.45
0.02
0.70
Glycerol
9.91
205
933
1.36
0.04
1.26
N-Epsilon-acetyl-L-lysine
10.13
200
912
1.75
<0.01
0.75
Succinic acid
10.49
247
899
1.80
<0.01
1.49
D-Glyceric acid
10.62
189
917
2.26
<0.01
2.36
Methionine
13.32
176
797
1.30
0.04
1.49
Glucose-1-phosphate
16.95
217
900
1.26
0.05
1.20
Tyrosine
17.21
226
904
1.33
0.04
0.73
Citric acid
17.79
273
910
1.15
0.05
1.51
Hypoxanthine
18.05
265
797
1.07
<0.01
1.93
N,N-Dimethyl-P-phenylenediamine
22.91
265
912
1.35
0.01
15.20
Fructose-6-phosphate
24.03
459
914
1.86
<0.01
1.70
Inosine
26.03
230
913
1.40
0.04
1.36
4 d versus 8 d
Lactic acid
7.40
117
825
1.87
<0.01
0.94
Maleamate
7.89
151
797
1.56
<0.01
0.87
Methylmalonic acid
8.37
218
721
1.86
<0.01
0.86
Valine
9.31
144
918
1.42
0.02
1.52
N-Epsilon-acetyl-L-lysine
10.13
200
912
1.56
0.01
0.78
Isoleucine
10.25
158
908
1.41
0.03
1.56
D-Glyceric acid
10.62
189
917
1.28
0.04
1.42
Threonine
11.36
219
904
1.38
0.03
1.33
N-Alpha-acetyl-L-ornithine
12.49
154
912
1.42
0.03
0.89
Aminomalonic acid
12.54
218
778
1.40
0.03
1.34
Aspartate
13.21
232
701
1.62
0.02
1.90
Methionine
13.32
176
797
1.95
<0.01
2.66
Phenylalanine
14.97
218
702
1.53
0.02
2.03
Tyrosine
17.21
226
904
2.03
<0.01
1.82
O-Phosphorylethanolamine
17.23
73
766
1.59
0.01
1.28
Hypoxanthine
18.05
265
797
1.44
0.03
1.14
N-Acetyl-beta-D-mannosamine
21.98
202
912
1.99
<0.01
4.66
Inosine
26.03
230
913
2.14
<0.01
1.51
RT = retention time; FC = fold change. The values of FC are mean value of peak area obtained from day 4 group/mean value of peak area obtained from day 0 group, day 8 group/day 4 group, respectively.Nucleotide degradation compounds produced by ATP breakdown are associated with flavour formation in pork [19]. In this study, the contents of hypoxanthine significantly increased from day 0 to day 4 (P<0.05), whereas the inosine content in lamb LD significantly increased during the whole period of ageing as shown in Table 2 (P<0.05). These findings indicate that sufficient IMP was further degraded to inosine, hypoxanthine, and ribose until day 8 of ageing. This is consistent with the effect of flavour precursors in Bison bison longissimus dorsi muscle during chilled storage at 4°C [21]. Kang et al. [24] showed that ageing highly affected the nucleotide-related compounds among water-soluble flavour precursors in loin and top round from Hanwoo cattle. Oltra et al. [25] reported that free ribose and phosphoribose were formed by nucleotides and nucleosides which were involved in the Maillard reaction. Therefore, the total content of nucleotide degradation compounds in postmortem muscle may potentially affect flavour composition during cooking of lamb.Table 2
Effects of chilled ageing conditioning on water-soluble flavour precursors.
Metabolite name
Storage days
0 d
4 d
8 d
Nucleotide degradation products
Inosine
38.99 ± 13.66a
53.17 ± 10.97b
80.03 ± 5.57c
Hypoxanthine
5.41 ± 4.22a
10.43 ± 1.04b
11.91 ± 1.30b
Glycolytic compounds
Glucose-1-phosphate
14.32 ± 2.03a
17.16 ± 3.08a
16.95 ± 2.82a
Fructose-6-phosphate
4.34 ± 2.17a
7.38 ± 0.83b
6.75 ± 2.07b
Organic acids
Succinic acid
1.90 ± 0.63a
2.83 ± 0.32b
3.53 ± 0.87c
3-Hydroxybutyric acid
10.94 ± 2.94a
7.61 ± 1.83b
6.51 ± 1.11b
Pyruvic acid
14.82 ± 3.21a
9.42 ± 5.89b
6.42 ± 1.92b
Citric acid
9.49 ± 3.69a
14.35 ± 5.12a
13.71 ± 4.88a
Lactic acid
3159.49 ± 94.16a
3121.1 ± 88.72a
2918.62 ± 115.28b
Free amino acids
N-Epsilon-acetyl-L-lysine
1.69 ± 0.25a
1.27 ± 0.24b
0.99 ± 0.12c
Methionine
0.87 ± 0.20a
1.30 ± 0.51a
3.46 ± 1.69b
Nicotinoyl-glycine
27.69 ± 1.77a
37.11 ± 12.17b
46.27 ± 3.33c
Tyrosine
1.32 ± 0.40a
0.96 ± 0.23a
1.74 ± 0.41b
Aspartate
0.78 ± 0.36a
2.54 ± 1.44b
4.83 ± 2.04c
Phenylalanine
2.83 ± 0.61a
3.50 ± 1.50a
7.12 ± 3.66b
Valine
18.28 ± 2.78a
21.64 ± 6.27a
32.96 ± 11.06b
N-Alpha-acetyl-L-ornithine
51.36 ± 4.07a
52.09 ± 5.31a
46.45 ± 3.56b
Isoleucine
10.87 ± 1.82a
13.85 ± 4.52a
21.66 ± 8.06b
Threonine
5.60 ± 1.43a
5.71 ± 1.42a
7.61 ± 1.78b
The values followed by different superscript letters were significantly different (P<0.05).The content of fructose-6-phosphate significantly increased (P<0.05) from day 0 to day 4 (Table 2). Koutsidis et al. [6] reported that glucose, fructose, and mannose were the main carbohydrates in raw lamb. Kosowska et al. [26] pointed out that the synthesis of glucose and glucose-6-phosphate in meat was due to glycogenolysis and glycolysis. The increase in glycolytic compounds implies that glycogenolysis and glycolysis were enhanced from day 0 to day 4 in the present study.The contents of 3-hydroxybutiric acid and pyruvic acid significantly decreased from day 0 to day 4 (P<0.05). The lactic acid content decreased during aging and significantly decreased from day 4 to day 8 (P<0.05), while the succinic acid significantly increased from day 0 to day 8 (P<0.05). No significantly differences in citric acid were found. It showed that the contents of organic acid were accumulated at day 4. Hou et al. [18] showed that a special sour and umami taste occurred when succinic acid was combined with lactic and citric acids. Thus, the changes in succinic acid content may affect the strength of the sour and umami taste of lamb as ageing time increases. Kim et al. [16] analysed these water-soluble metabolites, including sugars, sugar acids, and citric acid cycle intermediates, and found that they contributed to the flavour of cooked meat. Therefore, the altered concentration of glycolytic compounds and organic acids in raw meat may affect the sour and umami taste of the cooked meat.Free amino acids (FAAs) play important roles in the formation of volatile aromatics and taste characteristics [20]. As shown in Table 2, the most of free amino acid contents significantly increased from day 4 to day 8 (P<0.05), such as tyrosine, methionine, nicotinoyl-glycine, aspartate, phenylalanine, valine, isoleucine, and threonine. These findings indicate that postmortem ageing promoted the release of FAAs. Madruga et al. [27] studied the water-soluble amino acid precursors of mutton and found primarily glutamine, alanine, glycine, glutamic acid, leucine, and lysine. Kim et al. [16] performed a metabolic analysis of beef during the ageing process. The amino acids related to the taste and flavour of the meat included alanine, leucine, glutamine, glutamic acid, proline, methionine, tyrosine, isoleucine, and phenylalanine. This could be caused by the release of FAAs by the actions of endogenous enzymes [28]. Thus, the total content of FAAs was increased with prolonged aging so that taste characteristics would be abundant in the cooked meat.


### 3.3. Key Metabolic Pathways

The annotated metabolites in the datasets obtained from GC × GC-TOF/MS were combined into a new data set (34 significantly different metabolites in total, in which there were 16 and 18 metabolites from day 0 to day 4 and day 4 to day 8, respectively). According to the KEGG database, the TCA cycle and biosynthesis of alkaloids derived from histidine and purine were the key metabolic pathways in lamb refrigerated at 4°C from day 0 to day 4, whereas biosynthesis of amino acids was important from day 4 to day 8.Based on the KEGG maps, three integrated metabolic pathways are associated with each other through several metabolites (Figure2). The cessation of blood circulation ends the O2 supply to the muscle, and anaerobic conditions start to develop. The energy-rich phosphates, such as creatine phosphate, ATP, and ADP, are degraded. The accumulation of IMP results from AMP deaminase in stored meat [21]. The enhanced glycolysis in the meat leads to reduced nucleotides and accumulated lactic acid. The flavour of the meat is of low quality during this early period of storage [29]. The altered purine metabolism in the meat by day 4 mainly resulted in IMP that was degraded into inosine and hypoxanthine; therefore, the fresh taste of the meat increased by accumulated inosine and hypoxanthine. The adjusted biosynthesis of amino acids in the meat by day 8 mainly resulted in the increased flavour precursors of the free amino acids, such as tyrosine, phenylalanine, valine, methionine, and threonine so that the meat was tenderized and the flavour was enhanced [5].Figure 2
Kyoto Encyclopedia of Genes and Genomes pathways are manually linked together. The map illustrates significantly different metabolites in meat samples between two compared groups and three key metabolic pathways including the TCA cycle, biosynthesis of alkaloids derived from histidine and purine, and biosynthesis of amino acids.The integrated metabolic pathways contain interactive networks, as well as related metabolites, and provide information during chilled ageing. This information extends beyond metabolic relevance and has effects as demonstrated in the pathways and network analyses applied in the metabolomics analyses [30]. The detailed construction of the altered TCA cycle, biosynthesis of alkaloids derived from histidine and purine, and biosynthesis of amino acids with the related metabolites can be used to understand the effects of different storage time points on the altered flavour precursors and provide insights for future mechanistic exploration.


## 3.1. Multivariate Statistical Analysis

The Chroma TOF4.3X software (LECO) and LECO-Fiehn Rtx5 database were used to analyze the data. For all group samples, the missing value of the original data was simulated by filling half of the minimum value and noise removal was used by an interquartile range to filter data. The data were normalized to the total peak area of each sample and multiplied by 10000, and the peaks from the same metabolite were combined. The datasets consisted of 102 variables obtained from GC × GC-TOF/MS analyses after applying the above-described quality assurance criteria.These datasets were used to build PCA and OPLS-DA in order to visualize group trends during meat chilled ageing. The PCA score plot (Figures1(a) and 1(d)) and OPLS-DA (Figures 1(c) and 1(f)) showed a clear separation of all sampling periods. The results indicated that the ageing time had a significant impact on the changes of metabolites. The fit parameter (R2) and prediction parameter (Q2) values shown in Figures 1(b) and 1(e) were calculated based on 7-fold cross validation and 200-response permutation testing that represents the quality of the obtained OPLS-DA model. The corresponding R2 values obtained between day 0 and day 4 and day 4 and day 8 were 0.983 and 0.990; Q2 values were −0.158 and −0.120, respectively. The permutation tests among the 3 groups were all in a good range, indicating a satisfactory effectiveness of the model.Figure 1
PCA, corresponding validation plots of OPLS-DA, and OPLS-DA score plots derived from the GC × GC-TOF/MS between day 0 and day 4 and day 4 and day 8 during chilled ageing. Day 0: triangles; day 4: circles; day 8: squares.
(a)
(b)
(c)
(d)
(e)
(f)All the samples in the score plots of PCA and OPLS-DA were inside the 95% Hotelling T2 ellipse. Clear separation and discrimination indicated that the OPLS-DA model can be used to identify the difference between groups.


## 3.2. Differential Characteristic Metabolites

The reproducible ionization method used for GC × GC-TOF/MS qualitative identification of compounds is less complicated due to the availability of universal mass spectral libraries. Based on the NIST 11 database and Fiehn metabolomics library, the majority of the peaks were endogenous metabolites and some of these peaks may be attributed to the derivatives of byproducts. After screening with “VIP > 1.00,” “P<0.05,” and “similarity > 700,” significantly different metabolites were identified (Table 1).Table 1
Identification of significantly different metabolites in lamb samples using GC × GC-TOF/.
Metabolite name
RT
Mass
Similarity
VIP
P value
FC
0 d versus 4 d
Nicotinoyl-glycine
7.28
207
912
1.26
0.05
1.34
Pyruvic acid
7.46
174
885
1.56
0.04
0.64
Hydroxylamine
8.29
146
870
1.74
<0.01
1.21
3-Hydroxybutyric acid
8.64
117
925
1.45
0.02
0.70
Glycerol
9.91
205
933
1.36
0.04
1.26
N-Epsilon-acetyl-L-lysine
10.13
200
912
1.75
<0.01
0.75
Succinic acid
10.49
247
899
1.80
<0.01
1.49
D-Glyceric acid
10.62
189
917
2.26
<0.01
2.36
Methionine
13.32
176
797
1.30
0.04
1.49
Glucose-1-phosphate
16.95
217
900
1.26
0.05
1.20
Tyrosine
17.21
226
904
1.33
0.04
0.73
Citric acid
17.79
273
910
1.15
0.05
1.51
Hypoxanthine
18.05
265
797
1.07
<0.01
1.93
N,N-Dimethyl-P-phenylenediamine
22.91
265
912
1.35
0.01
15.20
Fructose-6-phosphate
24.03
459
914
1.86
<0.01
1.70
Inosine
26.03
230
913
1.40
0.04
1.36
4 d versus 8 d
Lactic acid
7.40
117
825
1.87
<0.01
0.94
Maleamate
7.89
151
797
1.56
<0.01
0.87
Methylmalonic acid
8.37
218
721
1.86
<0.01
0.86
Valine
9.31
144
918
1.42
0.02
1.52
N-Epsilon-acetyl-L-lysine
10.13
200
912
1.56
0.01
0.78
Isoleucine
10.25
158
908
1.41
0.03
1.56
D-Glyceric acid
10.62
189
917
1.28
0.04
1.42
Threonine
11.36
219
904
1.38
0.03
1.33
N-Alpha-acetyl-L-ornithine
12.49
154
912
1.42
0.03
0.89
Aminomalonic acid
12.54
218
778
1.40
0.03
1.34
Aspartate
13.21
232
701
1.62
0.02
1.90
Methionine
13.32
176
797
1.95
<0.01
2.66
Phenylalanine
14.97
218
702
1.53
0.02
2.03
Tyrosine
17.21
226
904
2.03
<0.01
1.82
O-Phosphorylethanolamine
17.23
73
766
1.59
0.01
1.28
Hypoxanthine
18.05
265
797
1.44
0.03
1.14
N-Acetyl-beta-D-mannosamine
21.98
202
912
1.99
<0.01
4.66
Inosine
26.03
230
913
2.14
<0.01
1.51
RT = retention time; FC = fold change. The values of FC are mean value of peak area obtained from day 4 group/mean value of peak area obtained from day 0 group, day 8 group/day 4 group, respectively.Nucleotide degradation compounds produced by ATP breakdown are associated with flavour formation in pork [19]. In this study, the contents of hypoxanthine significantly increased from day 0 to day 4 (P<0.05), whereas the inosine content in lamb LD significantly increased during the whole period of ageing as shown in Table 2 (P<0.05). These findings indicate that sufficient IMP was further degraded to inosine, hypoxanthine, and ribose until day 8 of ageing. This is consistent with the effect of flavour precursors in Bison bison longissimus dorsi muscle during chilled storage at 4°C [21]. Kang et al. [24] showed that ageing highly affected the nucleotide-related compounds among water-soluble flavour precursors in loin and top round from Hanwoo cattle. Oltra et al. [25] reported that free ribose and phosphoribose were formed by nucleotides and nucleosides which were involved in the Maillard reaction. Therefore, the total content of nucleotide degradation compounds in postmortem muscle may potentially affect flavour composition during cooking of lamb.Table 2
Effects of chilled ageing conditioning on water-soluble flavour precursors.
Metabolite name
Storage days
0 d
4 d
8 d
Nucleotide degradation products
Inosine
38.99 ± 13.66a
53.17 ± 10.97b
80.03 ± 5.57c
Hypoxanthine
5.41 ± 4.22a
10.43 ± 1.04b
11.91 ± 1.30b
Glycolytic compounds
Glucose-1-phosphate
14.32 ± 2.03a
17.16 ± 3.08a
16.95 ± 2.82a
Fructose-6-phosphate
4.34 ± 2.17a
7.38 ± 0.83b
6.75 ± 2.07b
Organic acids
Succinic acid
1.90 ± 0.63a
2.83 ± 0.32b
3.53 ± 0.87c
3-Hydroxybutyric acid
10.94 ± 2.94a
7.61 ± 1.83b
6.51 ± 1.11b
Pyruvic acid
14.82 ± 3.21a
9.42 ± 5.89b
6.42 ± 1.92b
Citric acid
9.49 ± 3.69a
14.35 ± 5.12a
13.71 ± 4.88a
Lactic acid
3159.49 ± 94.16a
3121.1 ± 88.72a
2918.62 ± 115.28b
Free amino acids
N-Epsilon-acetyl-L-lysine
1.69 ± 0.25a
1.27 ± 0.24b
0.99 ± 0.12c
Methionine
0.87 ± 0.20a
1.30 ± 0.51a
3.46 ± 1.69b
Nicotinoyl-glycine
27.69 ± 1.77a
37.11 ± 12.17b
46.27 ± 3.33c
Tyrosine
1.32 ± 0.40a
0.96 ± 0.23a
1.74 ± 0.41b
Aspartate
0.78 ± 0.36a
2.54 ± 1.44b
4.83 ± 2.04c
Phenylalanine
2.83 ± 0.61a
3.50 ± 1.50a
7.12 ± 3.66b
Valine
18.28 ± 2.78a
21.64 ± 6.27a
32.96 ± 11.06b
N-Alpha-acetyl-L-ornithine
51.36 ± 4.07a
52.09 ± 5.31a
46.45 ± 3.56b
Isoleucine
10.87 ± 1.82a
13.85 ± 4.52a
21.66 ± 8.06b
Threonine
5.60 ± 1.43a
5.71 ± 1.42a
7.61 ± 1.78b
The values followed by different superscript letters were significantly different (P<0.05).The content of fructose-6-phosphate significantly increased (P<0.05) from day 0 to day 4 (Table 2). Koutsidis et al. [6] reported that glucose, fructose, and mannose were the main carbohydrates in raw lamb. Kosowska et al. [26] pointed out that the synthesis of glucose and glucose-6-phosphate in meat was due to glycogenolysis and glycolysis. The increase in glycolytic compounds implies that glycogenolysis and glycolysis were enhanced from day 0 to day 4 in the present study.The contents of 3-hydroxybutiric acid and pyruvic acid significantly decreased from day 0 to day 4 (P<0.05). The lactic acid content decreased during aging and significantly decreased from day 4 to day 8 (P<0.05), while the succinic acid significantly increased from day 0 to day 8 (P<0.05). No significantly differences in citric acid were found. It showed that the contents of organic acid were accumulated at day 4. Hou et al. [18] showed that a special sour and umami taste occurred when succinic acid was combined with lactic and citric acids. Thus, the changes in succinic acid content may affect the strength of the sour and umami taste of lamb as ageing time increases. Kim et al. [16] analysed these water-soluble metabolites, including sugars, sugar acids, and citric acid cycle intermediates, and found that they contributed to the flavour of cooked meat. Therefore, the altered concentration of glycolytic compounds and organic acids in raw meat may affect the sour and umami taste of the cooked meat.Free amino acids (FAAs) play important roles in the formation of volatile aromatics and taste characteristics [20]. As shown in Table 2, the most of free amino acid contents significantly increased from day 4 to day 8 (P<0.05), such as tyrosine, methionine, nicotinoyl-glycine, aspartate, phenylalanine, valine, isoleucine, and threonine. These findings indicate that postmortem ageing promoted the release of FAAs. Madruga et al. [27] studied the water-soluble amino acid precursors of mutton and found primarily glutamine, alanine, glycine, glutamic acid, leucine, and lysine. Kim et al. [16] performed a metabolic analysis of beef during the ageing process. The amino acids related to the taste and flavour of the meat included alanine, leucine, glutamine, glutamic acid, proline, methionine, tyrosine, isoleucine, and phenylalanine. This could be caused by the release of FAAs by the actions of endogenous enzymes [28]. Thus, the total content of FAAs was increased with prolonged aging so that taste characteristics would be abundant in the cooked meat.


## 3.3. Key Metabolic Pathways

The annotated metabolites in the datasets obtained from GC × GC-TOF/MS were combined into a new data set (34 significantly different metabolites in total, in which there were 16 and 18 metabolites from day 0 to day 4 and day 4 to day 8, respectively). According to the KEGG database, the TCA cycle and biosynthesis of alkaloids derived from histidine and purine were the key metabolic pathways in lamb refrigerated at 4°C from day 0 to day 4, whereas biosynthesis of amino acids was important from day 4 to day 8.Based on the KEGG maps, three integrated metabolic pathways are associated with each other through several metabolites (Figure2). The cessation of blood circulation ends the O2 supply to the muscle, and anaerobic conditions start to develop. The energy-rich phosphates, such as creatine phosphate, ATP, and ADP, are degraded. The accumulation of IMP results from AMP deaminase in stored meat [21]. The enhanced glycolysis in the meat leads to reduced nucleotides and accumulated lactic acid. The flavour of the meat is of low quality during this early period of storage [29]. The altered purine metabolism in the meat by day 4 mainly resulted in IMP that was degraded into inosine and hypoxanthine; therefore, the fresh taste of the meat increased by accumulated inosine and hypoxanthine. The adjusted biosynthesis of amino acids in the meat by day 8 mainly resulted in the increased flavour precursors of the free amino acids, such as tyrosine, phenylalanine, valine, methionine, and threonine so that the meat was tenderized and the flavour was enhanced [5].Figure 2
Kyoto Encyclopedia of Genes and Genomes pathways are manually linked together. The map illustrates significantly different metabolites in meat samples between two compared groups and three key metabolic pathways including the TCA cycle, biosynthesis of alkaloids derived from histidine and purine, and biosynthesis of amino acids.The integrated metabolic pathways contain interactive networks, as well as related metabolites, and provide information during chilled ageing. This information extends beyond metabolic relevance and has effects as demonstrated in the pathways and network analyses applied in the metabolomics analyses [30]. The detailed construction of the altered TCA cycle, biosynthesis of alkaloids derived from histidine and purine, and biosynthesis of amino acids with the related metabolites can be used to understand the effects of different storage time points on the altered flavour precursors and provide insights for future mechanistic exploration.


## 4. Conclusions

Chromatography methods were used to investigate the metabolite profiles in lamb longissimus dorsi muscles stored at 4°C for 0, 4, and 8 days to investigate the potential impact of chilled ageing conditioning on water-soluble flavour precursors. Flavour-related metabolites, such as nucleotide degradation products, glycolytic compounds, organic acids, and free amino acids, were evaluated, and three key metabolic pathways were identified during aging. The results showed that flavour precursors were more abundant after completion of the TCA cycle, through the biosynthesis of alkaloids derived from histidine and purine, and through the biosynthesis of amino acids that occurred during the prolonged ageing of chilled lamb. Therefore, the results of this study may provide a strong evidence for further investigations of the effect and mechanism of ageing on the flavour of meat products.



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*Source: 4529830-2019-09-02.xml*