Exploring the formation mechanism of off-flavor of irradiated yak meat based on metabolomics

Highlights

• •The dosage of the irradiation was more than 5 kGy could result in yak meat quality degradation.
• •Allyl methyl sulfide, benzaldehyde, 4-methylthiazole etc. were critical sources of off-flavor.
• •The content of cysteine and methionine showed significant differences with irradiation dose.
• •The oxidative of sulfur-containing amino acids and unsaturated fatty acids was the principal cause of off-flavors.

Abstract

Irradiation's effects on quality, volatile compounds, and differential metabolites of yak meat were studied. Irradiation dose at 3 kGy had no effect on yak meat quality, however irradiation dose at 5 kGy resulted in yak meat quality deterioration as well as considerable irradiated off-flavors. And the level of the off-odor was strongly associated with the irradiation dose, and allyl methyl sulfide, octanal, nonanal, benzaldehyde, and 4-methylthiazole were all significant producers of off-odor. Meanwhile, with the increased of radiation dose, the amounts of cysteine, methionine, proline, linoleic acid, stearic acid changed obviously. The main generation pathway of irradiated off-flavors in yak meat were thought to be cysteine and methionine metabolism, and linoleic acid metabolism. The oxidative decomposition of sulfur-containing amino acids and unsaturated fatty acids may cause the off-flavor of irradiation yak meat. This research established a theoretical foundation for future control systems to prevent flavor quality alterations during irradiation preservation.

Introduction

Compared with other beef, yak meat has higher protein content and lower fat content. Moreover, yak meat is rich in amino acids and its structural ratio is like to the human body, which makes it an excellent meat product for people. However, yaks live all year round in the Tibetan plateau region of China, where there is lack of access to transportation (Huang et al., 2022). Therefore, yak meat is susceptible to contamination by Salmonella, E. coli, Clostridium botulinum and other pathogenic bacteria during post-slaughter processing, transportation, and storage, resulting in quality decline of the yak meat. Thus, it is a hot issue of current research to inhibit the propagation of microorganisms and prolong the shelf life of yak meat.

Irradiation, as a new non-thermal processing technology, has a wide range of applications in the field of food preservation, especially extending the shelf life of fresh meat and meat products. Irradiation is also considered the safest and most effective method to improve the shelf life of meat products. Irradiation technology, as an effective method can be used to control microorganisms and extend the shelf life of meat products, has been approved in many countries (Dyckman, 2000). Hassanzadeh et al. (2017) irradiated (2.5 kGy) chicken meat and stored at 4 ℃. The results showed that shelf life of irradiated group was significantly increased than comparison group and its sensory characteristics were also better than the control group.

After irradiation, a large number of free radicals emerged in the meat, which can inactivate microorganisms by damaging the chromosomal DNA of cells (David & Steele, 2001). However, these free radicals can also attack muscle proteins, inducing the breakage of covalent bonds in protein polypeptide chains, which could be the reason for the poor organoleptic quality of meat. Komolprasert and Morehouse (2004) have shown that many chemical and biological changes in irradiated meat are associated with the reaction of free radicals, especially the oxidation of lipids and proteins. Jo and Ahn (2000) identified that the lipolysis and protein oxidation triggered by irradiation has a key role in the formation of irradiated off-flavors.

After irradiation, significant irradiation odors emerged in meat and meat products, even some off-flavor like to rotten eggs and sulfur flavors, especially in high-protein foods. Which seriously limited the application of irradiation technology in meat products (Kim et al., 2004). In recent years, research on irradiation odor mainly focused on the qualitative analysis of odor (Nam & Ahn, 2003), and few studies reported the source of irradiation odor and its formation mechanism. Metabolomics, as a method that can analyze multiple metabolites and their metabolic pathways in complex biological systems, has been widely used in the field of food testing (Setyabrata et al., 2021).

Therefore, the effects of irradiation on the quality of yak meat were analyzed by physical and chemical indicators such as the volatile basic nitrogen (TVB-N) and thiobarbituric acid reactive substances (TBARS) values after processing of different irradiation dose. In this study, the gas chromatography-mass spectrometer (GC–MS) technique was used to characterize the volatile odor substances produced by yak meat during irradiation and metabolomics technique to elucidate the source and formation mechanism of irradiation odor. This will be an important guidance for the development of new methods to inhibit the off-odor substance during irradiation in the future. And it was very important for us to overcome the technical bottleneck of commercial application by irradiation.

Materials and methods

Materials and chemicals

Maiwa yaks from the natural pasture of Chuanbei Ge, Ruoerge County, Aba Prefecture, Sichuan Province, China. The hind leg meat was cut into 500 ± 10 g pieces, vacuum packed and quick frozen in −40 ℃ cold storage, and then transported to our laboratory for frozen storage (−20 ℃) on the same day. The samples were thawed at 4 ℃ for 12 h before use. The yak meat was divided into meat pieces of uniform quality (5 ± 0.1 g) after thawing, cleaning, and removing the excess fat and connective tissue. Then, the meat was cleaned with running water, and the water on the surface was dried with paper towels, and then put into a refrigerator at 4 ℃ for later use.

Irradiation processing

The pre-treated yak meat samples were randomly divided into 4 portions. Paper towels were used to absorb moisture from the surface of the meat pieces, and all samples were vacuum packed in airtight nylon/polyethylene sterile bags. They were placed in a foam box with several ice bags, labeled with different doses of irradiation, and sent to the Rice Research Institute of Fujian Academy of Agricultural Sciences (Fuzhou, Fujian Province, China) for irradiation treatment (⁶⁰Co irradiation source). The samples were irradiated at 0, 3, 5 and 7 kGy then immediately returned to the laboratory and chilled in a −20 °C refrigerator. Three replicates were set up for all experimental and control groups.

TVB-n

The volatile basic nitrogen (TVB-N) was measured as previously reported with some modifications (Dong et al., 2020). The meat samples were cut up, dissolved with water and filtered. The Nessler was added into the filtrate. The absorbance of the solution was measured at 420 nm using water as control.

TBARs

TBARS was according to the method of Luo et al. (2021). Meat sample (0.3 g) was mixed with 3 mL TBA and 17 mL TCA-HCI in boiling water bath for 30 min, then 5 mL chloroform was added. And the absorbance value was measured at 532 nm after centrifugation (3000g, 10 min) (H1650R, Xiangyi Co., Ltd., Hunan, China). The amounts of TBARS were expressed as milligrams of malondialdehyde per kilogram of meat.

Identification and analysis of volatile compounds

The grinded sample (2 g) was packed in a 20 mL headspace vial, purified with nitrogen for 1 min, sealed with a cap, and placed in a refrigerator at 4 ℃ to be measured. The volatile flavors were extracted with 50/30 μm DVB/CAR on PDMS (Supelco, Bellefonte, PA, USA). The analysis was performed using a CTC trinity autosampler with the heating chamber temperature of 50 ℃, shaking time of 15 min, extraction time of 30 min, shaking speed of 250 rpm, resolution time of 5 min and circulation time of 50 min.

Volatile flavor was analyzed and identified on Agilent GC–MS 7890B 5977B gas chromatograph-mass spectrometer equipped (Agilent, PaloAlto, USA) with DB-wax chromatographic column (30 m × 0.25 mm × 0.25 µm). The GC operation condition was as follows: Inlet temperature of 260 ℃; Helium (purity: 99.999 %); carrier gas flow rate of 1 mL/min; column temperature of 40 ℃ for 5 min, followed by 5 ℃/min to 220 ℃, and then programmed to 250 ℃ at 20 ℃/min, and held for 2.5 min; interface temperature of 260 ℃, ion source temperature of 230 ℃. Mass range: charge to mass ratio (m/z) = 20 ∼ 400.

The data obtained for the qualitative analysis of the compounds were searched and matched in NIST2014 spectral library, and substances with a match higher than 80 % were selected. The total ion flux chromatogram was quantified using peak area normalization to yield the relative content of each component.

Metabolite analysis and identification

The extraction of metabolites was carried out according to the method of Want et al. (Want et al., 2013). Irradiated yak meat samples (100 ± 2 mg) were weighed into a 2 mL centrifuge tubes, about 1000 µL of tissue extract was added to the samples. Then placed in a tissue grinder (TISSUELYSER-II, Jingxin Pharmaceutical Machinery Co., Ltd., Shanghai, China) and ground at 50 Hz for 60 s. The process was repeated 2 times. The mixture was sonicated for 30 min and stored at 0 ℃ for 30 min; then the mixture was centrifuged at 12,000 g for 10 min at 4 ℃. Then 200 μL of 2-chloro-l-phenylalanine solution (4 ppm) (4 ℃) was added to re-solubilize the sample. The supernatant bottle was collected and used for LC-MS analysis.

LC-MS analysis was performed using an ACQUITY UPLC® HSS T3 column (2.1 × 150 mm, 1.8 µm) (Waters, Milford, MA, USA). Flow rate of 0.25 mL/min, column temperature of 40 ℃, injection volume of 2 μL. The mobile phase was positive ion 0.1 % formic acid acetonitrile (c)-0.1 % formic acid water (d); Anion acetonitrile (a)-5 mM ammonium formate water (b) (Wang et al., 2021, Liu et al., 2022).

MS Conditions: The positive and negative ion modes were applied with the capillary voltages of 3.5 kV and 2.5 kV, respectively, using an electrospray ion source (ESI). The optimal ionization source parameters were as follows: sheath gas flow rate 30 arb; auxiliary gas flow rate 10 arb; capillary temperature 325 ℃; auxiliary gas heater temperature 325 ℃. A full MS scan was applied (m/z 81 to 1,000) with a resolution of 70,000. HCD was used for secondary cracking with a collision voltage of 30 eV, and the unnecessary MS/MS information was removed by dynamic elimination.

Multivariate statistical analysis and data processing

To further discover the effect of irradiation intensity on flavor substances of yak meat. Principal component analysis (PCA) was used to analyze the differences in volatile flavor components of irradiated samples by SIMCA 14.1 (Umetrics Inc., Stockholm, Sweden). Orthogonal-partial least squares discriminant analysis (OPLS-DA) was used to downscale and categorize the metabolic data to obtain more reliable and intuitive results. All metabolites were obtained from the following databases: Metlin (https://metlin.scripps.edu/), MoNA (https://mona.fiehnlab.ucdavis.edu//) and Panomic’s own standard database (Suzhou BioNovoGene Biomedical Tech Co., Ltd., Suzhou, China). All metabolic pathways were obtained from the KEGG database (https://www.genome.jp).

The measurements data were presented as the mean values of at least triplicate experiments, and the data were analyzed and plotted by GraphPad Prism 7.05 software (San Diego, CA, USA). One-way ANOVA was used to estimate the difference between the means (P < 0.05).

Results and discussion

TVB-N and TBARS

TVB-N value was the content of alkaline volatile nitrogenous compounds in yak beef, which was mainly caused by alkaline nitrogenous substances such as ammonia and amines produced by protein degradation (Zhang, Xu, & Zhi, 2012). Meanwhile, TVB-N value was the sole physical and chemical index that can be used to evaluate the freshness of meat in Chinese standards (Dong et al., 2020). The TVB-N values of yak meat were higher than the control group after irradiation treatment, and the TVB-N values increased gradually with the irradiation dose (Fig. 1a). It can be attributed to the free radicals that are generated by irradiation accelerate the oxidation of proteins, which causes protein breakdown to produce alkaline nitrogenous substances such as ammonia and amines.

Fig. 1.

Effects of different irradiation doses on TVB-N and TBARS of yak meat.

TBARS value can evaluate the degree of lipid oxidation accurately. (Wang et al., 2021) There were significant promotion effects of irradiation on lipid oxidation in yak meat, especially the TBARS value of yak meat samples increased significantly when the irradiation dose raised from 3 kGy to 7 kGy (P < 0.05). It is due to that irradiation caused the decomposition of small molecules in yak meat and produced active substances such as free radicals, which triggered a chain reaction of free radicals and accelerated the oxidation of fat in yak meat.

Something interesting is that the TBARS and TVB-N values of yak meat samples were not markedly different in the control group compared to the irradiation dose 3 kGy group (P > 0.05). Remarkable changes in TBARS and TVB-N values were only observed after the irradiation dose exceeded 5 kGy. It can be mainly attributed to the fact that low doses of irradiation did not induce the changes in yak meat quality strikingly, and the quality of yak meat was only significantly affected after the irradiation dose over 5 kGy. This was also similar to the WHO recommendation of allowing a maximum of 3.5 kGy of irradiation intensity for fresh meat (Brewer, 2009, Sebranek, 2022).

GC–MS analysis

Qualitative analysis of irradiated flavor substances of yak meat

“Flavor” is the result of the combination between basic flavors produced by water-soluble compounds in foods and their own volatile substances. In addition to killing the microorganisms in yak meat, irradiation also caused a large number of reactive free radicals to be generated in the meat. The free radicals act on the protein and fat components of the meat, resulting in flavor changes of yak meat (Brewer, 2009). The yak meat would generate heavy odor especially after high dose irradiation, which not only destroyed the sensory quality of the meat, but also caused the loss of nutritional value.

In this study, the volatile flavor compounds in yak meat after irradiation at different doses were detected by SPME/GC–MS technique (Table 1). A total of 63 volatiles, including acids(4), alcohols(11), aldehydes(8), esters(6), ketones(6), ethers(2), alkanes(11), olefins(4), benzenes(3), amides(2), furans(2), and 4 other compounds were identified.

Table 1.

Effects of different irradiation doses on volatile flavor substances of yak meat.

Name	Molecular formula	Molecular weight	Retention Time	0 kGy	3 kGy	5 kGy	7 kGy
Pentane	C5H12	72.15	1.6960	100.69 ± 4.01	184.9 ± 15.27	255.62 ± 18.41	356.76 ± 29.83
1-Heptene	C7H14	98.18	2.1308	661.08 ± 33.6	894.39 ± 44.75	1456.43 ± 49.52	1416.86 ± 81.84
2,4-Dimethylheptane	C9H20	128.26	2.4408	175.9 ± 11.11	271.41 ± 13.52	305.82 ± 6.47	432.47 ± 35.69
Acetone	C3H6O	58.08	2.5260	202.13 ± 12.37	36.05 ± 1.33	46.59 ± 3.1	85.03 ± 3.12
2-Butanone	C4H8O	72.10	3.4013	161.95 ± 9.76	340.09 ± 42.02	478.86 ± 28.3	417.9 ± 5.87
3-methyl-Butanal	C5H10O	86.13	3.6603	52.58 ± 3.53	92.39 ± 4.73	132.42 ± 3.69	124.97 ± 4.73
Trichloromethane	CHCl3	119.38	6.1461	100.65 ± 6.57	176.18 ± 2.87	196.03 ± 8.23	164.24 ± 6.99
Toluene	C7H8	92.14	6.6273	100.86 ± 3.76	390.06 ± 7.66	410.3 ± 1.92	427.75 ± 6.77
Hexanal	C6H12O	100.16	8.0015	61.06 ± 1.82	78.44 ± 1.28	300.73 ± 18.03	468.14 ± 10.95
1-Butanol	C4H10O	74.12	10.7203	5.5 ± 0.39	10.51 ± 1.19	10.52 ± 0.25	14.59 ± 0.59
1-Penten-3-ol	C5H10O	86.13	11.0420	101.13 ± 3.29	256.59 ± 7.79	363.49 ± 9.56	504.99 ± 7.93
Heptanal	C7H14O	114.19	11.2460	104.5 ± 5.85	194.14 ± 8.74	300.1 ± 6.33	480.68 ± 9.96
Hexanoic acid ethyl ester	C8H16O2	144.21	12.7760	3.35 ± 0.27	15.32 ± 1.68	26.94 ± 2.16	17.74 ± 3.77
1-Pentanol	C5H12O	88.15	13.7603	4.59 ± 0.8	24.68 ± 0.6	86.89 ± 2.37	126.45 ± 9.22
Nonanal	C9H28O	142.24	17.5415	30.01 ± 1.66	37.93 ± 1.45	73.5 ± 2.46	115.7 ± 7.34
1-Octen-3-ol	C8H16O	128.21	19.2520	19.08 ± 0.62	30.34 ± 0.80	92.08 ± 4.9	155.86 ± 5.68
Heptacosane	C7H16	100.2	20.3960	5.52 ± 0.65	13.08 ± 1.33	24.83 ± 2.57	50.10 ± 3.34
Benzaldehyde	C7H6O	106.12	20.8968	26.42 ± 2.48	35.05 ± 1.00	42.51 ± 0.70	65.06 ± 2.01
Formic acid octyl ester	C7H12O3	144.17	21.9987	12.49 ± 0.99	17.79 ± 1.20	31.98 ± 1.61	53.13 ± 0.99
2-Octen-1-ol	C8H16O	128.21	23.3060	2.49 ± 0.14	6.33 ± 0.15	12.5 ± 0.85	8.56 ± 0.71
Butanoic acid	C4H8O2	88.11	24.0240	3.53 ± 0.07	9.68 ± 0.19	17.98 ± 1.19	35.74 ± 0.93
Hexanoic acid	C6H12O2	116.16	28.9223	12.07 ± 0.96	15.84 ± 0.79	28.1 ± 1.74	63.82 ± 1.78
Tributyl phosphate	C12H27PO4	266.32	30.0251	67.58 ± 0.89	73.94 ± 3.84	85.93 ± 3.14	83.7 ± 0.79
3,4-Dihydroxybenzyl alcohol	C7H8O3	140.12	30.6600	6.79 ± 0.39	17.01 ± 1.91	34.46 ± 1.79	22.13 ± 1.43
Propanoic acid	C3H6O2	74.00	21.7528	21.21 ± 1.21	13.33 ± 0.56	25.94 ± 1.73	9.01 ± 0.46
N,N-dimethyl-Methylamine	C3H7NO	73.09	1.7440	nd	75.07 ± 12.02	178.48 ± 5	181.02 ± 5.99
Octane	C8H18	114.23	2.4197	nd	265.85 ± 25.77	282.59 ± 12.94	389.00 ± 70.89
Allyl ethyl ether	C6H10O	98.15	3.5893	nd	51.12 ± 1.13	58.70 ± 0.85	63.41 ± 2.19
Allyl methyl Sulfide	C4H8S	88.17	4.3883	nd	5.13 ± 0.14	10.41 ± 0.74	15.96 ± 1.45
1-methoxy-2-Propanol	C4H10O2	90.12	10.1670	nd	2.63 ± 0.30	8.10 ± 0.14	13.73 ± 1.01
2-pentyl-Furan	C9H14O	138.08	12.5987	nd	8.25 ± 0.88	14.82 ± 1.61	19.18 ± 2.06
2,4-Dithiapentane	C5H8S2	132.23	14.2951	nd	6.63 ± 0.66	11.71 ± 0.85	9.16 ± 0.15
Octanal	C8H16O	128.21	14.4862	nd	14.93 ± 0.51	24.67 ± 1.11	36.63 ± 1.79
3-Methylthio-2-butanone	C7H14N2O2S	190.26	15.2583	nd	5.23 ± 0.46	8.73 ± 0.75	12.04 ± 0.69
n-Caproic acid vinyl ester	C8H14O2	142.2	15.6075	nd	9.21 ± 0.44	53.85 ± 4.48	109.91 ± 3.31
1-Hexanol	C6H14O	102.18	16.6788	nd	12.25 ± 1.02	28.41 ± 1.48	130.44 ± 7.82
Succinimide	C4H5NO2	99.09	39.9888	nd	0.64 ± 0.05	0.84 ± 0.04	1.99 ± 0.22
Hexadecanal	C16H32O	240.42	34.2160	nd	5.99 ± 0.43	12.68 ± 0.78	27.57 ± 1.11
1-Octene	C8H16	112.21	2.7511	nd	nd	34.19 ± 4.2	44.76 ± 6.7
2-Octene	C8H16	112.21	3.0368	nd	nd	56.79 ± 5.45	121.77 ± 4.48
2-ethyl-Furan	C6H8O	96.14	4.3570	nd	nd	5.11 ± 0.56	8.46 ± 0.13
1,2-dimethyl-Cyclooctane	C10H20	140.26	6.8695	nd	nd	68.46 ± 1.56	148.97 ± 6.31
Undecane	C11H24	156.31	8.4130	nd	nd	12.14 ± 0.54	18.05 ± 0.64
Pyridine	C5H5N	79.1	11.5110	nd	nd	10.9 ± 1.26	28.69 ± 1.92
2-Octanone	C8H16O	128.212	14.4423	nd	nd	5.47 ± 0.18	8.69 ± 0.43
4-Nonanone	C9H18O	142.24	15.5885	nd	nd	54.74 ± 5.39	82.82 ± 3.24
Formic acid heptyl ester	C8H16O2	144.21	19.4165	nd	nd	26.45 ± 1.82	47.20 ± 5.09
Pentadecane	C15H32	212.41	20.3760	nd	nd	9.73 ± 0.19	15.47 ± 0.66
4-Methylthiazole	C6H6ClNO	143.57	23.7815	nd	nd	3.95 ± 0.07	25.35 ± 1.88
N,N-dibutyl-Formamide	C9H19NO	157.25	26.9350	nd	nd	162.44 ± 12.13	306.02 ± 22.39
2-phenoxy-ethanol	C8H10O2	138.16	34.3280	nd	nd	2.44 ± 0.22	6.39 ± 0.50
p-Xylene	C8H10	106.17	9.5550	nd	nd	nd	6.49 ± 0.73
Benzylalcohol	C7H8O	108.13	29.0977	nd	nd	nd	16.55 ± 0.73
Tridecane	C13H28	184.41	14.7580	nd	nd	64.22 ± 0.6	58.11 ± 2.06
Styrene	C8H8	104.15	13.4103	nd	25.24 ± 0.27	17.48 ± 2.08	10.14 ± 0.64
Ethanol	C2H6O	46.07	4.0829	3.59 ± 0.26	390.1 ± 14.65	347.94 ± 8.87	337.11 ± 7.19
Acetaldehyde	C2H4O	44.05	1.9780	309.23 ± 4.31	265.29 ± 8.37	187.76 ± 6.56	101.91 ± 2.87
Ethyl Acetate	C4H8O2	88.11	3.2362	212.35 ± 19.04	94.80 ± 10.19	88.38 ± 0.91	69.23 ± 3.43
2,3-Butanedione	C4H6O2	86.09	4.9230	76.22 ± 1.22	67.67 ± 1.08	44.86 ± 1.21	12.13 ± 1.85
Dodecane	C12H26	170.34	11.4063	51.43 ± 0.62	38.35 ± 0.22	15.46 ± 2.22	8.61 ± 0.29
Butylated Hydroxytoluene	C15H24O	220.35	29.8294	90.33 ± 1.18	58.08 ± 2.16	26.38 ± 1.90	14.76 ± 0.76
Carbamodithioic acid	CH2S2	78.15	31.8585	150.5 ± 0.81	104.45 ± 5.87	53.00 ± 2.62	49.08 ± 1.00
Oxime-methoxy-phenyl	C8H9NO2	151.16	26.7817	282.36 ± 9.25	126.32 ± 7.54	67.02 ± 2.62	nd

Compared with unirradiated yak meat (0 kGy), a series of new volatiles were produced in the irradiated yak meat (Table 1). A total of 33 compounds were detected in the yak meat samples of 0 kGy group, 47 compounds in the 3 kGy group, 61 compounds in the 5 kGy group and 62 compounds in the 7 kGy group. The content of most volatiles gradually increased with the irradiation dose. Which might be caused by the ions and free radicals generated by irradiation promoted the oxidation of lipids and proteins, thus facilitating the formation of volatile flavor substances.

Principal component analysis of volatile flavor substances

PCA of the GC–MS results was established for the purpose of further analyzing the effect of different irradiation doses on the composition of volatiles of yak meat, and the results were shown in Fig. 2a. The cumulative variance contribution of the principal components PC1 and PC2 was 75.6 %. The results indicated that PC1 and PC2 could respond well to the information of the raw data and can be used for the downscaling analysis of volatiles of yak meat irradiated with different doses. As can be seen from Fig. 2a, four groups of samples can be clearly distinguished in the PCA plots, and there were some differences between each different irradiation treatment groups. It was shown that the volatiles in yak meat were significantly changed through different doses of irradiating.

Fig. 2.

PCA plot (a), OPLS-DA plot (b) and cluster heat map analysis of volatile flavor substances (c) of yak meat samples at different irradiation doses.

Differences in volatile flavor substances of yak meat after irradiation

In order to visualize the differences between volatiles of diverse irradiation doses, the cluster heat map analysis was performed for 63 metabolites, and the consequences were presented in Fig. 2c.

Compared with the control group (0 kGy), yak meat treated with 3 kGy irradiation produced 13 new volatiles (Fig. 2c), including octane, octanal, allyl ethyl ether, allyl methyl sulfide, 2-pentyl furan, 2,4-dithiopentane, N, N-dimethylformamide, 3-mercapto-2-butanone, etc. Among them, glucose was degraded by enzymes to produce pyruvate. Pyruvate was synthesized as α-acetyl lactate by the action of enzymes. α-acetyl lactate produced 3-hydroxy-2-butanone by oxidative decarboxylation, and then 3-Mercapto-2-butanone was formed by thio-reaction. Some of the alkoxy radicals in 3-hydroxy-2-butanone were broken, and combined with the propyl radicals, and then the furans were produced after removing the hydroxyl radicals. Consequently, the content of 2-pentyl furan gradually raised.

In our study, it was found that sulphur-containing volatiles such as allyl methyl sulphide were generated by the induction of irradiation, and their content was significantly increased with irradiation dose. Previous reports indicated that allyl methyl sulfide was derived from oxidative degradation of proteins and interactions with reducing sugars (Zhu, Lee, Mendonca, & Ahn, 2004). The reaction of sulfur-containing amino acids with free radicals produced by irradiation appeared to be the source of odor-causing sulfides and other volatile sulfur-containing compounds. Sulfur-containing compounds and their range of carbonyl compounds had low odor thresholds, and these substances also contributed significantly to irradiation odors (Houser, Sebranek, Maisonet, Cordray, & Lee, 2010). Moreover, phospholipids were a source of several sulfides, and 4-methylthiazole was generated by their reaction with cysteine or ribose (Rowe, 2002). 3-Methylbutyraldehyde was also a product of phospholipid oxidation, and its content increased in a dependent manner with irradiation dose. Cha et al. (2000) revealed that irradiation doses have shown a remarkable effect on fatty acids derived from phospholipids in chicken meat.

It was observed that 1-octene and 2-octene were produced in yak meat after irradiation at 5 kGy. At the same time, the content of 1-heptene increased with irradiation dose, which was also similar to the research results of Jo and Ahn (2000) in beef. The content of alkanes such as undecane, tridecane and pentadecane also showed a positive correlation with the irradiation dose, which seemed to be the results of decomposition from unsaturated fatty acids and amino acids (M. Du, Hur, Nam, Ismail, & Ahn, 2001).

The irradiation also induced yak beef to produce a series of low molecular weight aldehydes, including two long carbon chain aldehydes (hexanal, heptanal, octanal and nonanal) and a Strecker aldehyde (benzaldehyde), which strikingly increased with irradiation dose. Therefore, we deduced that irradiation triggered oxidative protein degradation and produced a series of irradiation products with secondary oxidation products.

In the previously published reports, hexanal content was considered as a useful marker for TBARS and lipid oxidation (Brewer, 2009). In our study, the hexanal content increased prominently with irradiation dose, but the difference between the control (0 kGy) and 3 kGy irradiation groups was not significant, which was also like to the results of the TBARS value. Hexanal and heptanal, on the other hand, had a putrid odor, nonanal had a buttery odor, and benzaldehyde also had an unpleasant odor. And low odor threshold allowed all the molecules to contribute to the irradiation odor.

Irradiation also culminated in the synthesis of a number of ketones, including 2-octanone, 2-butanone, 2,3-butanedione, and so on, which could be the products of carboxylic acid breakdown (Renz, 2005). Aside from ketones, some branched alcohols like 1-penten-3-ol and 1-octen-3-ol were found in linoleic acid oxidation products, which had a mushroom/metal odor (Yang, Shewfelt, Lee, & Kays, 2008). Changes in 1-octen-3-ol content were traditionally used to reflect the degree of rancidity of meat products (Q. Zhang, Ding, Gu, Zhu, Zhou, & Ding, 2020). In our study, 1-octen-3-ol was found in the irradiated group samples, especially in higher concentrations in the 7 kGy irradiated group. Therefore, the pathway of irradiation-induced bulk production of alcohols includes two aspects: (1) fatty acids underwent direct carbon chain scavenging followed by oxidation with hydroxyl radicals to produce alcohols (Brewer, 2009); (2) reduction of aldehydes to alcohols by free hydrogen atoms.

After irradiaton, oxidizing active ligands like –OH would be created based on water in yak meat system and as well as the reducing active ligands like water electron eaq- and free hydrogen atom -H (M. S. Brewer, 2009, Thakur and Singh, 1994). As a result, we speculated that irradiation-induced formation of alcohols like 1-octen-3-ol might be more inclined to the reduction of the aldehydes' hydrogen atom. In reality, lipid oxidation and the further oxydative degradation of amino acids should account for the great majority of the metabolism pathway of the branched alkanes in irradiation group. Lipid oxidation and oxydative degradation of proteins (amino acids) induced by irradiation and the new volatiles created by their interactions would play an important role in irradiation off-odor.

Metabolomics analysis

Qualitative results of untargeted metabolomics of irradiated yak meat

Multivariate data analysis was performed using chemometric methods such as OPLS-DA to better obtain differential metabolites between yak meat samples with different irradiation intensities, analyzed the effect of irradiation on yak meat metabolites, and identified biological metabolites that caused differences between control and different irradiation intensity groups. As shown in Fig. 2b, principal components 1 and 2 in OPLS-DA explained 36.5 percent and 22.5 percent of the overall variance, respectively, suggesting that yak meat with different irradiation intensities had a substantial difference (R²X = 0.662, Q² = 0.967). The data all formed a better distribution than the statistics obtained based on the original data in the OPLS-DA, showing that the model had a high degree of prediction and discrimination and can be used for the next step of metabolite screening (Wang, Fang, He, Dai, & Fang, 2017).

VIP values were commonly employed to describe the relative importance of the X and Y data sets. Thus, the OPLS-DA model's projected importance of variables (VIP) was utilized to determine the differential metabolites of samples with varying irradiation intensities. To sift the difference metabolites in this investigation, VIP > 1 and P < 0.05 were carried as criteria. Finally, a total of 123 significantly distinct metabolites were examined, with 69 being positive ions mode and 54 being negative ions (Table 2). As shown in Fig. 3a, there were 31 organic acids, 28 amino acids, 3 peptides, 2 amides, 3 ketones, 3 aldehydes, 11 sugars, 3 lipids, 4 esters, 10 cofactors, 4 pyrimidines, 6 purines, and 16 other chemicals.

Table 2.

Effects of different irradiation doses on the differential metabolites of yak meat.

No.	Compounds	Specific charge	Retention time(s)	Molecular formula	Irradiation intensity				p value	VIP	Mode
					0 kGy	3 kGy	5 kGy	7 kGy
1	Betaine aldehyde	102.09	89.2	C5H12NO	475.07 ± 58.77	646.22 ± 33.32	675.79 ± 43.01	690.89 ± 51.48	6.27E−05	1.4082	pos
2	m-Cresol	109.10	552.0	C7H8O	82.3 ± 8.86	98.09 ± 7.44	179.54 ± 16.93	236.37 ± 5.93	1.90E−02	1.1866	pos
3	Creatinine	114.07	101.3	C4H7N3O	8751.3 ± 186.82	10671.24 ± 204.07	16208.1 ± 208.66	27200.88 ± 221.3	2.87E−04	1.5816	pos
4	Aminomalonic acid	119.02	105.5	C3H5NO4	271.88 ± 14.5	331.75 ± 13.69	360.01 ± 14.24	417.69 ± 32.3	4.09E−06	1.2459	pos
5	4-Hydroxybenzaldehyde	123.04	142.0	C7H6O2	1603.08 ± 61.28	1954.49 ± 52.24	2073.66 ± 103.83	2208.19 ± 81.61	2.04E−06	1.2704	pos
6	trans-Cinnamate	131.05	338.4	C9H8O2	1193.81 ± 50.94	1364.43 ± 65.05	1593.25 ± 82.43	1606.64 ± 73.34	7.61E−06	1.5253	pos
7	5,6-Dihydro-5-fluorouracil	133.03	143.5	C4H5FN2O2	1131.67 ± 83.68	1265.67 ± 68.03	1467.73 ± 57.2	1608.59 ± 44.74	2.09E−04	1.5741	pos
8	Phthalic acid	149.02	567.4	C8H6O4	49.81 ± 2.65	69.47 ± 3.8	75.34 ± 2.02	86.42 ± 3.51	1.43E−07	1.0137	pos
9	l-Methionine S-oxide	166.05	89.9	C5H11NO3S	3988.85 ± 51.13	4854.42 ± 47.73	5903.06 ± 63.04	7083.56 ± 73.09	1.55E−06	1.6517	pos
10	Xanthine	153.04	318.7	C5H4N4O2	1137.52 ± 63.31	1185.88 ± 40.36	1525.23 ± 52.12	1849.87 ± 60.53	9.68E−03	1.5235	pos
11	2′,4′-Dihydroxyacetophenone	153.10	510.9	C8H8O3	18.23 ± 3.58	49.88 ± 9.23	74.98 ± 9.23	91.5 ± 8.17	2.44E−08	1.4110	pos
12	l-Histidine	156.08	85.5	C6H9N3O2	25132.02 ± 200.99	29446.2 ± 255.63	48676.18 ± 232.63	61181.02 ± 320.35	1.75E−02	1.1653	pos
13	Suberic acid	157.08	118.2	C8H14O4	24.74 ± 9.9	128.55 ± 38.34	210.74 ± 10.59	266.43 ± 11.91	1.19E−05	1.4417	pos
14	Methacholine	160.13	133.2	C8H18NO2	406.19 ± 64.22	785.53 ± 53.24	1479.24 ± 99.15	3571.08 ± 109.88	3.34E−03	1.2804	pos
15	N5-Methyl-l-glutamine	161.09	99.0	C6H12N2O3	587.65 ± 2.39	797.6 ± 52.78	817.68 ± 64.25	864.72 ± 84.39	2.99E−06	1.4533	pos
16	l-Carnitine	162.11	143.3	C7H15NO3	2223.87 ± 183.28	4537.63 ± 184.45	6175.77 ± 185.5	7130.82 ± 126.88	6.62E−03	1.3601	pos
17	4-Hydroxycoumarin	163.04	567.4	C9H6O3	37.41 ± 3.78	42.4 ± 2.65	53.52 ± 3.4	67.04 ± 3.36	2.00E−07	1.0465	pos
18	3,4-Dihydroxyphenylpropanoate	165.05	142.0	C9H10O4	13535.87 ± 100.81	16530.31 ± 64.21	18056.69 ± 52.54	18982.7 ± 51.08	9.99E−07	1.3463	pos
19	d-Phenyllactic acid	166.06	142.0	C9H10O3	1336.97 ± 4.73	1684.01 ± 6.73	1712.49 ± 2.8	1783.31 ± 1.06	9.35E−08	1.2408	pos
20	l-Phenylalanine	166.08	338.4	C9H11NO2	126086.55 ± 200.9	145720.87 ± 106.27	167924.7 ± 213.06	168170.5 ± 213.17	3.51E−05	1.5025	pos
21	d-synephrine	168.09	338.4	C9H13NO2	288.88 ± 13.44	327.13 ± 13.19	488.22 ± 13.2	520.29 ± 21.26	6.99E−06	1.5082	pos
22	Norepinephrine	169.10	350.9	C8H11NO3	315.88 ± 18.84	383.79 ± 21.43	648.82 ± 47.57	798.67 ± 26.57	2.50E−04	1.2955	pos
23	l-Arginine	174.11	103.8	C6H14N4O2	1116.34 ± 32.69	1296.02 ± 52.19	1373.54 ± 51.07	1390.08 ± 60.61	2.04E−06	1.1523	pos
24	Metanephrine	180.10	307.3	C10H15NO3	177.02 ± 33.82	204.79 ± 18.37	264.98 ± 25.37	395.39 ± 12.3	3.12E−03	1.4007	pos
25	(S)-beta-Tyrosine	182.08	142.0	C9H11NO3	44305.93 ± 201.03	54724.48 ± 204.00	59269.07 ± 200.84	62416.86 ± 401.17	2.33E−07	1.3844	pos
26	l-Tyrosine	182.08	198.9	C9H11NO3	12194.66 ± 104.18	15674.67 ± 204.57	18584.44 ± 404.04	20895.01 ± 305.6	5.20E−06	1.6066	pos
27	Asymmetric dimethylarginine	203.15	95.6	C8H18N4O2	1108.51 ± 55.24	1129.11 ± 63.73	1549.46 ± 105.41	1702.07 ± 101.04	2.16E−06	1.5893	pos
28	5-Hydroxyindoleacetic acid	192.07	334.7	C10H9NO3	339.57 ± 0.59	441.49 ± 13.77	480.1 ± 13.27	497.08 ± 12.37	6.90E−07	1.4188	pos
29	3-Carbamoyl-2-phenylpropionaldehyde	193.07	183.0	C10H11NO3	113.88 ± 19.69	140.55 ± 15.34	202.08 ± 12.67	278.3 ± 20.38	2.17E−04	1.4444	pos
30	Spermine	203.22	66.6	C10H26N4	1346.15 ± 24.05	1896.41 ± 13.39	2505.51 ± 54.27	3028.83 ± 44.07	3.89E−06	1.6080	pos
31	Indolelactic acid	206.08	549.4	C11H11NO3	65.76 ± 1.58	71.97 ± 1.91	77.58 ± 4.15	83.01 ± 2.39	1.40E−05	1.2113	pos
32	l-Kynurenine	209.09	334.7	C10H12N2O3	969.94 ± 10.91	1193.33 ± 12.5	1258.8 ± 12.71	1275.68 ± 12.44	1.26E−06	1.4014	pos
33	3-Methyl-l-tyrosine	195.10	823.5	C10H13NO3	506.86 ± 15.28	781.07 ± 10.17	1222.9 ± 17.92	1743.83 ± 52.48	2.68E−05	1.3716	pos
34	Allocystathionine	222.08	434.3	C7H14N2O4S	50.89 ± 3.65	73.7 ± 5.44	81.28 ± 2.31	83.14 ± 2.03	5.70E−07	1.4400	pos
35	Butyryl-l-carnitine	232.15	374.9	C11H21NO4	216468.65 ± 204.94	295737.83 ± 305.35	303603.79 ± 404.29	321498.4 ± 303.29	6.02E−05	1.0158	pos
36	(5-l-Glutamyl)-l-glutamate	259.09	161.8	C10H16N2O7	185.91 ± 9.35	204.88 ± 17.01	215.63 ± 14.58	280.49 ± 15.19	3.46E−04	1.1801	pos
37	N-Acetylaspartylglutamic acid	305.10	154.5	C11H16N2O8	307.39 ± 13.78	416.25 ± 23.42	419.08 ± 33.63	420.68 ± 33.7	1.14E−05	1.2727	pos
38	N-Acetyl-a-neuraminic acid	310.11	98.9	C11H19NO9	194.11 ± 3.87	253.56 ± 16.83	282.66 ± 15.99	298.59 ± 15.57	4.52E−03	1.2519	pos
39	S-(Hydroxymethyl)glutathione	320.09	178.8	C11H19N3O7S	66.01 ± 3.18	170.93 ± 8.06	378.84 ± 12.3	339.46 ± 36.15	5.48E−08	1.5316	pos
40	Erucic acid	338.34	761.6	C22H42O2	78.34 ± 15.76	106.29 ± 5.69	401.32 ± 23.28	666.01 ± 21.96	1.00E−03	1.5361	pos
41	N-a-Acetylcitrulline	218.11	88.6	C8H15N3O4	53.29 ± 1.15	62.36 ± 6.83	99.73 ± 7.82	149.39 ± 13.1	2.31E−05	1.5175	pos
42	Retinoyl b-glucuronide	476.28	800.1	C26H36O8	71.07 ± 16.24	116.9 ± 38.04	106.99 ± 11.62	172.8 ± 20.78	3.01E−02	1.2450	pos
43	l-Valine	116.07	109.8	C5H11NO2	1105.82 ± 27.25	1353.5 ± 108.37	1446.04 ± 105.19	1388.6 ± 105.49	3.23E−02	1.1235	neg
44	Succinic acid	117.02	69.7	C4H6O4	59.88 ± 10.3	102.41 ± 12.25	106.96 ± 11.24	84.87 ± 14.95	1.20E−04	1.0183	neg
45	Methylmalonic acid	116.93	816.7	C4H6O4	6687.16 ± 52.81	6995.02 ± 106.44	8052.16 ± 149.67	9528.1 ± 190.69	9.91E−08	1.5760	neg
46	Erythritol	121.03	500.6	C4H10O4	80.17 ± 3.66	89.65 ± 9.12	93.14 ± 10.75	107.25 ± 22.07	3.07E−05	1.1244	neg
47	1,2,3-Trihydroxybenzene	126.03	433.7	C6H6O3	153.86 ± 3.86	190.43 ± 6.48	534.23 ± 32.56	957.89 ± 59.5	2.31E−04	1.3890	neg
48	Maltol	126.03	694.0	C6H6O3	30.87 ± 3.87	66.08 ± 3.23	114.58 ± 13.52	169.1± 18.79	4.04E−03	1.3471	neg
49	l-Malic acid	133.01	113.1	C4H6O5	421.86 ± 12.54	850.78 ± 58.12	1118.34 ± 109.06	2352.13 ± 101.66	2.48E−03	1.4834	neg
50	Salicylic acid	137.02	371.2	C7H6O3	27.97 ± 13.48	31.92 ± 8.41	103.12 ± 16.43	242.54 ± 18.29	3.23E−02	1.3669	neg
51	4-Guanidinobutanoic acid	145.09	399.1	C5H11N3O2	10.89 ± 3.74	17.45 ± 4.75	24.57 ± 5.23	37.17 ± 8.78	2.01E−07	1.6381	neg
52	l-Arabinose	149.99	709.1	C5H10O5	36.17 ± 7.8	74.59 ± 7.32	119.99 ± 13.53	163.05 ± 16.77	2.12E−02	1.2693	neg
53	Pelargonic acid	157.12	765.1	C9H18O2	66.28 ± 9.21	87.68 ± 5.54	169.6 ± 20.52	363.29 ± 16.18	7.06E−03	1.5201	neg
54	Nicotine	160.94	808.5	C10H14N2	157.93 ± 15.6	202.69 ± 15.01	476.89 ± 23.65	682.91 ± 51.33	2.18E−03	1.3117	neg
55	Capric acid	171.14	810.7	C10H20O2	133.22 ± 33.2	174.48 ± 26.59	258.06 ± 23.42	418.45 ± 22.02	3.65E−03	1.5854	neg
56	Argininosuccinic acid	291.13	87.8	C10H18N4O6	41.74 ± 5.51	56.87 ± 7.81	148.96 ± 17.01	246.1 ± 15.95	1.02E−03	1.5205	neg
57	4-Quinolinecarboxylic acid	173.12	542.5	C10H7NO2	37.96 ± 2.2	49.39 ± 15.98	68.28 ± 13.26	114.48 ± 23.64	7.22E−03	1.3657	neg
58	Gluconolactone	177.04	74.7	C6H10O6	49.62 ± 7.21	188.73 ± 19.4	530.49 ± 53.53	1030.83 ± 107.51	2.47E−04	1.5617	neg
59	Phenylacetylglycine	192.07	373.8	C10H11NO3	370.1 ± 53.35	563.27 ± 55.1	1571.46 ± 109.84	3034.04 ± 105.53	4.75E−07	1.4999	neg
60	Methyl beta-d-galactoside	194.08	806.6	C7H14O6	30.91 ± 5.65	53.15 ± 2.19	106.1 ± 6.81	303.02 ± 8.24	8.34E−03	1.3726	neg
61	5-Hydroxy-l-tryptophan	219.08	332.5	C11H12N2O3	19.26 ± 9.64	37.03 ± 1.32	48.65 ± 0.94	56.2 ± 2.41	3.28E−05	1.5613	neg
62	Thymidine	223.03	758.5	C10H14N2O5	267.66 ± 19.05	348.41 ± 58.18	3381.65 ± 108.86	6330.24 ± 147.79	1.71E−03	1.4786	neg
63	Methyl jasmonate	223.13	761.4	C13H20O3	25.17 ± 4.39	34.5 ± 9.33	103.65 ± 9.14	150.61 ± 15.82	1.77E−02	1.3362	neg
64	Palmitic acid	255.23	816.6	C16H32O2	279.37 ± 13.59	407.88 ± 49.69	572.41 ± 39.88	1003 ± 83.23	1.11E−03	1.4807	neg
65	Dehydroepiandrosterone	269.21	808.9	C19H28O2	155.04 ± 19.97	278.00 ± 12.52	355.27 ± 11.16	575.26 ± 18.47	3.42E−03	1.4701	neg
66	Gingerol	293.18	740.2	C17H26O4	69.31 ± 7.58	49.19 ± 11.22	175.69 ± 13.03	227.04 ± 12.01	4.03E−02	1.3036	neg
67	9,10-Epoxyoctadecenoic acid	295.23	839.5	C18H32O3	6290.34 ± 83.87	8644.6 ± 103.22	10841.04 ± 105.62	26486.22 ± 207.83	2.72E−03	1.3416	neg
68	11-Dehydrocorticosterone	325.18	802.4	C21H28O4	2099.44 ± 84.57	4056.77 ± 88.46	6957.36 ± 106.69	8350.58 ± 107.72	1.91E−04	1.3145	neg
69	Fructose 1,6-bisphosphate	339.20	819.2	C6H14O12P2	2543.34 ± 89.79	4390.86 ± 103.34	8574.31 ± 109.71	10865.02 ± 156.9	1.24E−05	1.4503	neg
70	dGMP	346.05	121.3	C10H14N5O7P	2236.85 ± 96.32	2600.31 ± 101.32	12384.16 ± 103.22	25859.36 ± 155.4	2.49E−02	1.3417	neg
71	AMP	346.05	147.5	C10H14N5O7P	292.41 ± 23.49	498.17 ± 22.04	1166.35 ± 90.18	3991.54 ± 130.15	2.00E−03	1.3341	neg
72	NADH	664.12	126.5	C21H29N7O14P2	1216.67 ± 94.69	3376.00 ± 119.41	5296.93 ± 120.31	10169.39 ± 127.69	2.28E−05	1.5508	neg
73	Benzaldehyde	107.05	626.8	C7H6O	48.97 ± 1.41	42.5 ± 2.73	35.75 ± 4.62	24.61 ± 5.57	1.11E−02	1.2591	pos
74	PyrrolE−2-carboxylic acid	111.52	703.4	C5H5NO2	218.22 ± 18.45	134.85 ± 34.16	60.46 ± 14.98	38.45 ± 12.69	8.36E−04	1.2982	pos
75	Betaine	118.12	92.1	C5H11NO2	877.62 ± 58.95	493.54 ± 43.97	326.1 ± 12.65	209.09 ± 8.76	9.83E−12	1.4633	pos
76	1-Methylhistamine	126.10	73.0	C6H11N3	1270.75 ± 58.74	1236.59 ± 61.71	1052.42 ± 42.49	904.63 ± 52.99	5.09E−04	1.3645	pos
77	Taurine	126.02	86.9	C2H7NO3S	2139.86 ± 122.95	1463.1 ± 114.93	1385.06 ± 113.37	1323.39 ± 112.51	6.92E−07	1.4366	pos
78	cis-4-Hydroxy-d-proline	132.07	87.5	C5H9NO3	1788.07 ± 115.4	1767.03 ± 121.91	1630.91 ± 122.82	1610.26 ± 120.15	1.19E−02	1.3630	pos
79	4-Hydroxycinnamic acid	146.98	837.2	C9H8O3	4713.27 ± 185.22	1504.24 ± 191.12	924.28 ± 129.92	712.4 ± 128.95	2.68E−03	1.4742	pos
80	l-Glutamine	147.08	86.9	C5H10N2O3	29181.26 ± 181.85	24847.71 ± 112.38	21213.73 ± 110.6	20511.1 ± 112.73	9.62E−08	1.6263	pos
81	2,3-Butanediol	154.99	58.8	C4H10O2S2	661.6 ± 12.06	501.88 ± 17.9	289.18 ± 18.77	235.52 ± 11.57	1.13E−02	1.3134	pos
82	Gentisic acid	154.99	632.7	C7H6O4	2708.02 ± 38.22	1974.78 ± 25.17	782.92 ± 20.98	303.98 ± 32.77	2.61E−03	1.2333	pos
83	Scopoline	156.10	107.6	C8H13NO2	437.4 ± 17.41	386.3 ± 11.81	384.54 ± 10.81	343.87 ± 11.12	7.26E−04	1.0176	pos
84	l-Methionine	150.06	143.6	C5H11NO2S	1097.63 ± 14.99	1067.85 ± 28.81	704.55 ± 17.38	671.49 ± 22.97	5.00E−06	1.5502	pos
85	3-(2-Hydroxyphenyl)propanoic acid	167.07	823.5	C9H10O3	7457.69 ± 73.39	7186.08 ± 82.27	7173.12 ± 61.59	6382.78 ± 52.51	6.81E−04	1.3351	pos
86	beta-Alanyl-l-lysine	217.15	320.4	C9H19N3O3	15163.97 ± 106.13	14783.67 ± 104.97	14716.18 ± 101.9	13333.26 ± 91.04	2.07E−02	1.1424	pos
87	S-Glutathionyl-l-cysteine	427.09	98.4	C13H22N4O8S2	483.27 ± 10.48	423.78 ± 14.61	375.7 ± 7.63	345.69 ± 12.36	1.51E−02	1.1392	pos
88	S-Adenosylhomocysteine	385.13	182.8	C14H20N6O5S	1027.17 ± 38.68	936.33 ± 42.51	742.98 ± 34.33	718.05 ± 45.16	1.30E−02	1.0154	pos
89	Cytidine	244.09	119.8	C9H13N3O5	3196.41 ± 51.47	2604.34 ± 45.49	2484.6 ± 31.69	1988.39 ± 41.73	6.30E−07	1.1380	pos
90	Gamma-Linolenic acid	278.19	448.8	C18H30O2	281.61 ± 14.58	251.43 ± 14.66	197.1 ± 16.98	188.48 ± 12.53	6.54E−04	1.0236	pos
91	Sulfamethazine	279.09	749.7	C12H14N4O2S	88.4 ± 9.46	67.55 ± 8.25	63.45 ± 9.33	57.53 ± 6.43	1.09E−04	1.2748	pos
92	Stearic acid	284.19	430.8	C18H36O2	182.38 ± 11.67	170.76 ± 12.68	150.04 ± 9.45	107.68 ± 8.00	8.08E−04	1.4219	pos
93	N-Acetylleucine	172.10	348.9	C8H15NO3	324.93 ± 13.25	312.6 ± 13.27	283.23 ± 10.32	175.91 ± 10.35	2.50E−04	1.3797	pos
94	Dihydrocapsaicin	308.22	613.5	C18H29NO3	46.72 ± 2.91	32.77 ± 4.81	31.62 ± 4.65	31.00 ± 15.76	8.32E−04	1.3710	pos
95	Sucrose	343.29	736.3	C12H22O11	150.06 ± 12.86	107.08 ± 12.36	98.6 ± 9.47	82.8 ± 8.85	4.23E−04	1.1670	pos
96	IMP	349.06	125.0	C10H13N4O8P	50550.62 ± 106.67	40096.32 ± 156.45	37993.82 ± 186.53	8358.3 ± 185.07	1.95E−06	1.1847	pos
97	Nobiletin	403.14	772.1	C21H22O8	117.02 ± 3.03	79.59 ± 5.05	20.32 ± 5.48	15.77 ± 2.86	8.67E−04	1.3967	pos
98	Lanosterin	427.27	467.1	C30H50O	82.20 ± 6.59	63.93 ± 2.99	47.38 ± 5.46	28.86 ± 3.21	4.90E−05	1.4470	pos
99	Glycyl-leucine	189.12	363.3	C8H16N2O3	234.98 ± 10.00	213.31 ± 5.17	179.77 ± 5.13	170.3 ± 1.14	1.39E−03	1.4447	pos
100	2-Ketobutyric acid	101.02	232.3	C4H6O3	61.54 ± 6.46	56.83 ± 4.52	44.68 ± 4.85	37.92 ± 3.54	1.77E−05	1.4881	neg
101	Cytosine	111.05	398.4	C4H5N3O	31.94 ± 4.48	22.16 ± 4.26	15.49 ± 4.67	13.58 ± 3.18	1.01E−07	1.6173	neg
102	(S)-3-Methyl-2-oxopentanoic acid	129.06	333.5	C6H10O3	151.44 ± 4.54	77.12 ± 7.53	65.8 ± 8.13	32.37 ± 2.07	1.16E−02	1.3448	neg
103	l-Aspartic acid	132.03	81.3	C4H7NO4	420.67 ± 6.68	409.09 ± 2.73	401.09 ± 9.15	194.08 ± 10.41	5.58E−05	1.2670	neg
104	Ribitol	133.05	322.0	C5H12O5	148.82 ± 45.81	87.39 ± 13.93	72.54 ± 7.94	28.65 ± 3.61	1.93E−04	1.5606	neg
105	Hypoxanthine	135.03	126.5	C5H4N4O	27276.09 ± 105.03	23555.63 ± 103.17	19204.07 ± 108.68	13318.21 ± 158.44	5.01E−06	1.3288	neg
106	d-Ribose	149.05	82.7	C5H10O5	385.41 ± 36.86	198.86 ± 44.62	137.68 ± 20.67	83.62 ± 11.69	2.37E−03	1.3556	neg
107	3-Hydroxyanthranilic acid	152.04	682.4	C7H7NO3	110.22 ± 11.6	49.71 ± 6.16	21.63 ± 2.51	18.35 ± 1.93	8.28E−03	1.2677	neg
108	Oxoadipic acid	158.98	118.4	C6H8O5	505.78 ± 19.13	455.19 ± 16.69	322.63 ± 15.71	269.31 ± 16.94	1.11E−04	1.5625	neg
109	3-Hydroxyvalproic acid	159.10	470.3	C8H16O3	105.44 ± 12.29	80.94 ± 11.35	40.61 ± 9.52	25.04 ± 9.08	9.80E−03	1.3255	neg
110	d-Fructose	161.04	636.8	C6H12O6	219.78 ± 24.23	163.95 ± 19.00	102.6 ± 12.79	46.12 ± 16.61	1.90E−02	1.1448	neg
111	Shikimic acid	172.99	391.3	C7H10O5	21.1 ± 6.97	17.14 ± 5.9	14.36 ± 6.29	12.39 ± 5.95	3.61E−04	1.5734	neg
112	Alpha-d-Glucose	179.06	579.4	C6H12O6	193.12 ± 13.8	133.06 ± 9.19	105.04 ± 10.63	102.14 ± 9.31	3.18E−02	1.0578	neg
113	2,4-Dinitrophenol	183.00	469.8	C6H4N2O5	149.54 ± 14.02	117.66 ± 5.37	74.44 ± 7.56	55.00 ± 17.38	7.33E−05	1.5850	neg
114	Citric acid	191.02	82.2	C6H8O7	346.53 ± 3.37	253.82 ± 7.53	109.33 ± 5.96	88.69 ± 6.63	1.01E−04	1.5004	neg
115	Gluconic acid	196.05	82.4	C6H12O7	320.7 ± 6.33	153.46 ± 2.29	89.95 ± 3.03	51.17 ± 4.04	6.48E−05	1.2450	neg
116	N-Acetyl-l-phenylalanine	206.08	384.2	C11H13NO3	45.54 ± 9.62	31.03 ± 2.99	20.02 ± 3.03	19.51 ± 5.79	1.35E−03	1.3106	neg
117	Galactaric acid	209.03	73.3	C6H10O8	208.51 ± 6.35	108.95 ± 5.33	86.37 ± 8.44	15.37 ± 9.76	3.59E−03	1.4358	neg
118	Sedoheptulose 7-phosphate	289.03	77.8	C7H15O10P	7659.94 ± 170.89	7150.65 ± 106.1	2872.96 ± 128.61	1205.94 ± 120.46	2.24E−03	1.2115	neg
119	5′-S-Methyl-5′-thioinosine	297.07	399.3	C11H14N4O4S	80.66 ± 11.75	40.32 ± 3.09	35.61 ± 1.93	22.19 ± 2.73	3.96E−02	1.1322	neg
120	CMP	322.04	81.9	C9H14N3O8P	177.17 ± 17.04	124.58 ± 6.33	84.74 ± 3.52	65.34 ± 1.45	8.70E−03	1.1090	neg
121	12-Keto-leukotriene B4	333.21	780.1	C20H30O4	200.36 ± 12.65	178.55 ± 11.7	69.23 ± 8.52	49.06 ± 2.1	4.18E−04	1.5087	neg
122	Glycocholic acid	464.30	606.4	C26H43NO6	277.7 ± 12.94	248.68 ± 6.88	152.76 ± 12.37	118.66 ± 7.64	3.99E−02	1.2726	neg
123	Uridine diphosphate glucose	565.04	81.2	C15H24N2O17P2	163.3 ± 17.18	143.24 ± 5.2	105.25 ± 5.82	40.94 ± 1.18	1.93E−02	1.0374	neg

Fig. 3.

Analysis of metabolite species distribution (a), differential heat map (b) and metabolic pathways (c) of yak meat at different irradiation doses.

Changes in differential metabolites with different irradiation intensities

A cluster heat map analysis was utilized to visualize the differences directly in metabolites of yak beef at different irradiation intensities. The hue of the heat map represented the metabolites' relative content. Different color gradients were used to depict the differences in the data, and the results were displayed in Fig. 3b.

In general, the flavor of yak meat was determined by non-volatile flavoring chemicals (non-volatile flavoring substances). However, in general, these non-volatiles were precursors to volatile flavor compounds. Unsaturated fatty acids, free amino acids, inosinic acids, inorganic salts, ribose, peptides, and organic acids were the most common taste-presenting compounds.

Yak meat was high in unsaturated fatty acids, which could interact with free radicals to form a considerable number of peroxides under irradiation conditions. The peroxides would continue to decomposition and polymerization to generate aldehydes, alkenes, alcohols, and other taste compounds due to its unstable attribution. Which indicated that unsaturated fatty acids had a significant role in the creation of yak meat's irradiated flavor. After irradiation, the amounts of linoleic acid, γ-linolenic acid, and stearic acid in yak meat were strikingly altered. After 7 kGy irradiation, the intensity of γ-linolenic acid decreased by 33.07 percent, and the intensity of stearic acid decreased by 40.96 percent. Compared to unirradiated samples, whereas the content of 9,10-epoxy linoleic acid increased by 321.06 percent year-on-year, indicating that the yak meat had spoiled after 7 kGy irradiation. After 3 kGy irradiation, linoleic acid, linolenic acid, and stearic acid levels were all identical to the unirradiated group, indicating that the 3 kGy irradiated yak meat samples were like to the unirradiated samples.

Amino acids were mainly used to produce volatile flavor substances through the Maillard reaction and Strecker degradation, which had an effect on the flavor of yak meat. As a result, the alterations in all 28 differential amino acids observed during irradiation were quite significant. When compared to the unirradiated group, the levels of cis-4-hydroxy-d-proline and N-acetylleucine reduced significantly (P < 0.05), and the leucine levels underwent a 45.86 percent reduction after 7 kGy irradiation. We supposed that the reason could be attributed to the irradiation-induced free radicals such as amino acid residues (e.g., proline residues) oxidize myogenic fibrous proteins in yak meat, causing protein aggregation and other problems (Estévez, 2011).

Furthermore, the concentration of both sulfur-containing amino acids (cysteine and methionine) was discovered to decline with the irradiation dose increasing. This could be because the sulfur atoms of both cysteine and methionine were particularly vulnerable to occur single or two electron oxidation. Meanwhile, the residues of methionine were susceptible to be oxidized by free radicals to form compounds like sialic acid (Ahn et al., 2016b). What’s more, it has been shown that cysteine and methionine are the most vulnerable amino acids to irradiation radical action, resulting in the production of the sulfur-containing volatiles, which were also considered as contributors to irradiation scents (Estévez et al., 2011, Uk Ahn et al., 2016). However, we discovered that the levels of cysteine and methionine did not change considerably after the 3 kGy irradiation treatment. Therefore, we speculated that the 3 kGy irradiation therapy likely maintained the levels of simple amino acids for a short period by decomposing complex amino acids.

Nucleotides, as an essential taste-presenting ingredient, can affect the formation of scent by interacting with reducing sugar, as well as forming the umami of yak meat by synergizing with glutamic acid and other umami amino acids (Koutsidis, Elmore, Oruna-Concha, Campo, Wood, & Mottram, 2008). The concentration of IMP, GMP, CMP, and other nucleotides fell dramatically as the irradiation dose was raised, whereas the content of AMP grew significantly (Table 2). It could be owing to the phosphorylation reactions that degrade IMP and ribonucleotides to produce compounds like hypoxanthine (Dashdorj, Amna, & Hwang, 2015), and the boost in AMP concentration could be due to the facilitation of nucleotide degradation during irradiation (H. Du et al., 2020).

Reducing sugars were also a key component of meat flavor precursor substance. In comparison to the control group, the content of reducing sugars in irradiation yak meat changed drastically. The concentration of reducing sugars such as glucose, fructose, and galactose and so forth were greatly reduced, which primarily produced a huge amount of volatile taste compounds via the Maillard reaction, whereas sucrose could generate the analogous reducing sugars via the hydrolysis process.

Differential metabolic pathway analysis

To better visualize the internal linkages of metabolites, a metabolic pathway analysis was done on 126 differential metabolites by using KEGG. A total of 20 major metabolic pathways were sifted, and the Pathway Impact of all these remarkable enrichment pathways was greater than 0.05 (Fig. 3c). The metabolism of alanine, aspartic acid, and glutamic acid had the most impact on the flavor of yak meat among them. As representations of umami amino acids, aspartic acid and glutamic acid had a dramatic impact on the flavor of yak meat. However, during the irradiation of yak meat, the free radicals generated with the increase of irradiation intensity caused the oxidative decomposition of these umami amino acids, thus affecting the flavor of yak meat. Protein digestion and absorption was regarded as one of the metabolic pathways that could strikingly influence the irradiation flavor of yak meat markedly. As a result, we speculated that irradiation-induced oxidation of protein side chains or protein backbone breaking was likely to be a key route causing the off-flavor of irradiated yak meat.

Furthermore, cysteine and methionine, as typical sulfur-containing amino acids, were particularly vulnerable to irradiation radical assault, resulting in sulfur-containing volatiles, and its metabolism could affect the irradiation flavor of yak meat dramatically. Sulfur-containing volatiles also showed very low olfactory thresholds, suggesting that they might play a substantial role in irradiated off-flavors. The flavor characteristics produced by the irradiation degration of methionine and cysteine were most similar to the irradiated off-flavors, and the sensitivity of the irradiated degradation of cysteine was more obvious than methionine (Uk Ahn et al., 2016, Ahn et al., 2016a). According to our hypothesis, irradiation triggered an oxidative protein degradation process, causing the formation of a sequence products of irradiation and secondary oxidation. Hence, cysteine and methionine metabolism might play a critical role in the development of irradiation off-flavors.

Correlation analysis of irradiated flavor and differential metabolites

Yak meat can be irradiated to lengthen its shelf life and improve its quality. Irradiation, on the other hand, can also affect the flavor of yak meat and result in an unpleasant “irradiated taste,” which is one of the main reasons why irradiation technology in meat processing should be limited. The decomposition of sulfur-containing amino acids in yak meat to form chemicals with disagreeable odor, and the oxidative degradation of fatty acids to produce odorous compounds, are the two main causes of odor following irradiation.

Irradiated free radicals were particularly sensitive to assault by sulfur-containing amino acids such as methionine and cysteine, resulting in the oxidation of several sulfur-containing volatile compounds. As shown in Fig. 4, cysteine could combine with Strecker's aldehydes (products of the Maillard reaction) to generate sulfides, and react with phospholipids to form the sulfide: 4-methylthiazole. Transmethylation of methionine could create residues that could be oxidized by free radicals to become sulphurous, and then formed sulfur-containing volatiles, for example: allyl methyl sulfide (Feng, Lee, Nam, Jo, Ko, & Ahn, 2016). Sulfur-containing volatiles had very low thresholds, so they could contribute remarkably to irradiation scents. Consequently, yak meat irradiation off-flavors were believed to be caused by the oxidation of cysteine and methionine residues.

Fig. 4.

Association analysis of volatile flavor substances and metabolites in yak meat at different irradiation doses.

Fat oxidation in irradiated meat products was thought to be linked to the generation of irradiated off-flavors by the majority of researchers (Schlüter et al., 2013). Yak meat was rich in unsaturated fatty acids and lacked electrons out of both carbonyl and carbon–carbon double bonds. Hence, the irradiation could generate many lipid radicals, which could trigger free radical chain reactions and accelerate oxidation and hydrolysis of lipids, resulting in the reduction of fatty acids and the generation of a series of lipid oxidation products and contributing to the irradiated off-flavors. At the same time, irradiation could also enhance the concentration of aldehydes of low molecular weight, such as heptanal, octanal, nonanal, and benzaldehyde. Sour, unpleasant, and putrid scents were produced by volatile products such as heptanal, octanal, and nonanal generated by oleic acid oxidation and compounds such as pentane, hexanal, and octanal created by linoleic acid autoxidation. As a result, irradiation-induced fat oxidation was linked to the formation of the irradiation odor.

Conclusion

The TVB-N and TBARS values of yak meat were unaffected by 3 kGy irradiation, while the dosage of the irradiation was more than 5 kGy could result in yak meat quality degradation. Allyl methyl sulfide, octanal, nonanal, benzaldehyde, and 4-methylthiazole were critical sources of off-flavor that spring from irradiated yak beef, and their levels were strongly linked with the irradiation dose (P < 0.05). The LC-MS results indicated that the difference metabolites in yak meat were primarily derived from the metabolism of alanine, aspartic acid, and glutamic acid, with cysteine and methionine metabolism also contributing significantly. The concentrations of cysteine and methionine differed considerably with irradiation dose (P < 0.05), but the contents of proline and leucine fell dramatically as the irradiation dose augmented. In summary, the oxidative decomposition of sulfur-containing amino acids and unsaturated fatty acids was the principal cause of off-flavors in irradiated yak meat. The present research clarified the causes and mechanisms of off-flavors in yak meat during irradiation preservation, as well as providing a theoretical foundation for developing a regulation system to prevent flavor quality alterations during irradiation preservation in the future.

CRediT authorship contribution statement

Qia Wang: Investigation, Formal analysis, Writing – original draft, Writing – review & editing. Kai Dong: Investigation, Formal analysis, Writing – original draft, Writing – review & editing. Yongyan Wu: Investigation, Methodology, Formal analysis. Fengping An: Data curation, Software. Zhang Luo: Funding acquisition, Supervision. Qun Huang: Validation, Supervision. Shaofeng Wei: Funding acquisition, Conceptualization.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.