
==== Front
Ultrason Sonochem
Ultrason Sonochem
Ultrasonics Sonochemistry
1350-4177
1873-2828
Elsevier

S1350-4177(24)00308-0
10.1016/j.ultsonch.2024.107060
107060
Original Research Article
The improvement mechanism of volatile for cooked Tibetan pork assisted with ultrasound at low-temperature: Based on the differences in oxidation of lipid and protein
Cheng Lujie ab1
Li Xin a1
Li Xiefei a
Wu Yingmei a
An Fengping b
Luo Zhang c
Geng Fang d
Huang Qun huangqunlaoshi@126.com
abc⁎
Liu Zhendong liu304418091@126.com
c⁎
Tian Yuting etingtian@hotmail.com
b⁎
a School of Public Health, Guizhou Province Engineering Research Center of Health Food Innovative Manufacturing, Guizhou Medical University, Guiyang 550025, China
b College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
c College of Food Science, Tibet Agriculture and Animal Husbandry University, Linzhi, Tibet Autonomous Region 860000, China
d School of Food and Biological Engineering, Chengdu University, Chengdu 610106, China
⁎ Corresponding authors at: Guizhou Medical University, Gui ’an New District, Guizhou Province 550025, China. huangqunlaoshi@126.comliu304418091@126.cometingtian@hotmail.com
1 Authors contributed equally to this work.

05 9 2024
11 2024
05 9 2024
110 10706012 7 2024
1 9 2024
4 9 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Low-temperature cooking causes flavor weakness while improving the texture and digestive properties of meat. To enhance the flavor of low-temperature cooked Tibetan pork, samples were cooked at low-temperature with or without ultrasound-assisted (UBTP, BTP) for different times (30 min, 90 min) and then analyzed using GC–MS and LC-MS. The results showed that ultrasound-assisted cooking caused a significant increase in lipid oxidation by 9.10% in the early stage of the treatment. Additionally, at the later stage of ultrasound-assisted processing, proteins were oxidized and degraded, which resulted in a remarkable rise in the protein carbonyl content by 6.84%. With prolonged effects of ultrasound and low-temperature cooking, the formation of phenylacetaldehyde in UBTP-90 sample originated from the degradation of phenylalanine through multivariate statistics and correlation analysis. Meanwhile, trans, cis-2,6-nonadienal and 1-octen-3-one originated from the degradation of linolenic acid and arachidonic acid. This study clarified the mechanism of ultrasound-assisted treatment improving the flavor of low-temperature-cooked Tibetan pork based on the perspective of lipids and proteins oxidation, providing theoretical supports for flavor enhancement in Tibetan pork-related products.

Keywords

Ultrasound-assisted cooking
Low-temperature cooking
Oxidation differences
Flavor
Metabolomics
==== Body
pmc1 Introduction

Low-temperature cooking plays a positive role in improving the texture and reducing the loss of nutrients in meat products, as well as reducing the generation of harmful substances, etc. [1]. Recently, the demand of consumers for healthy food has become more and more intense with the gradual development of the economy. As a result, researchers focus their attention on low-temperature cooking methods. However, the degradation of lipids and proteins is limited due to the reduction of temperature, which makes it resistant to forming a desirable flavor. Therefore, it is necessary to search for an appropriate method to enhance the flavor of meat products cooked at low temperatures.

Low-temperature cooking refers to a stewing temperature of 53–81 °C [2]. It was previously found that low-temperature cooking can enhance the texture of pork by raising the water-holding ability of the muscle tissue to be tenderized and juicy [3]. Besides, low-temperature cooking has also been reported to improve the digestive ability of proteins and boost the bioaccessibility of food products by decreasing protein aggregation as compared to traditional cooking methods [4]. However, since it is difficult to develop a desirable characteristic flavor, the researchers enhanced the aroma of the meat products by prolonging the treatment time. Zhang et al. [2] found that the appropriate cooking temperature and duration (70 °C, 6 h) can improve the flavor of the meat products, resulting in duck products with a higher fatty and umami taste. The increased cost of time has caused researchers to turn their attention to assisted cooking techniques.

Ultrasound is widely applied in meat products for its advantages of strong penetration and fast transmission speed. Silva et al. [5] found that the time required to reach a specific temperature with ultrasound-assisted cooking was 50% less than that with traditional cooking methods. Regarding the storage of food, ultrasound treatment may accelerate the freezing of food and generate tiny and uniform ice crystals [6], which reduces the thawing loss rate of frozen food. Moreover, it has been reported that the right ultrasound power (400 W) can effectively shorten the thawing time of white yak meat, prevent the loss of nutrients during thawing, and retain the integrity of the microstructure [7]. Besides, ultrasound has also been utilized for the marination of food products. Tong et al. [8] found that ultrasound mechanically broke down the structure of muscle fibers and facilitated the uniform distribution of water, which promoted the curing rate and tenderness of chicken as well as improved the flavor of chicken. Apart from improving the textural properties, ultrasound has also been successfully utilized to enhance the meat flavor. It can induce the production of substantial amounts of free radicals, causing elevated levels of lipid and protein oxidation in meat products during the process [9]. Generally, the oxidation of lipids and proteins in meat results in significant alterations to the flavor of food [10]. Qi et al. [11] demonstrated that ultrasound-assisted cooking positively affected the stability of chicken broth and the formation of flavor by microscopic means. They also found that fat oxidation was not the only factor determining the formation of volatile flavor compounds in chicken broth. Wang et al. [10] compared the differences in flavor of salted egg yolks with or without ultrasonic treatment. According to research, cooking with ultrasonic assistance dramatically raised the thiobarbituric acid reactive substances (TBARS) value and positively affects the production of volatile flavor compounds. In addition to modifying the volatile aromas, ultrasound optimized the taste of food products. Zhang et al. [12] showed that ultrasound-assisted frying not only boosted the aroma of meatballs, but also promoted the formation of nucleotides and improved the overall flavor of fried meatballs.

Based on the references reported, it can be reasonably hypothesized that the oxidative levels of lipids and proteins in low-temperature cooked Tibetan pork may be raised by ultrasound-assisted treatment, which would improve the flavor of low-temperature cooked Tibetan pork. Tibetan pork features a high content of unsaturated fatty acids, which are more susceptible to oxidation to generate flavor substances. However, as far as we know, there are few literature reports on the effect of ultrasound-assisted cooking at low temperature on the flavor of Tibetan pork. Therefore, this study aimed to assess the improvement of flavor in Tibetan pork by ultrasonic treatment. Also, oxidation indexes were combined with GC–MS and LC-MS techniques to investigate the flavor variations of cooked Tibetan pork with or without ultrasound-assisted treatment.

2 Materials and methods

2.1 Samples preparation and grouping

Tibetan pigs (12‐month‐old, male, castrated) were provided by the Abazhou Bowen Husbandry Technology Co., Ltd., Sichuan, China. Three independent hind legs from three Tibetan pigs were collected and transported back to the laboratory by cold chain and stored at −80 °C. After preliminarily thawing at 4 °C for 10 h, each sample was cut into 1.5 cm thick slices, then placed in a vacuum cooking bag (9.5 × 8.0 × 1.5 cm) after complete thawing. RAW group: the Tibetan pork without treatment. BTP-30 group: the cooking bag containing slices of Tibetan pork was placed in a water bath (DK-S24, Jinghong Experimental Equipment Co., Ltd., Shanghai, China) at 80 °C for 30 min. BTP-90 group: the cooking bag containing slices of Tibetan pork was placed in a water bath at 80 °C for 90 min. UBTP-30: the cooking bag containing slices of Tibetan pork was placed in an ultrasonic cleaner (KQ-250 V, Kunshan Ultrasonic Instrument Co., Ltd., Kunshan, China) and processed at 80 °C for 30 min at an ultrasonic power of 250 W and a frequency of 40 kHz. UBTP-90: the cooking bag containing samples was placed in an ultrasonic cleaner and processed at 80 °C for 90 min at an ultrasonic power of 250 W and a frequency of 40 kHz. All the samples were cooled to room temperature, minced with a meat grinder and stored in a freezer at −80 °C.

2.2 Oxidation indices

2.2.1 Carbonyl content

The carbonyl content was determined by the DNPH method [13]. Myofibrillar protein (MP) was diluted to 5 mg/mL by phosphate buffer (0.04 mol/L, pH 7.0). The solution of 0.5 mL of MP was placed in a centrifuge tube, adding 0.2% of DNPH (2 mL) and 2 mol/L of HCI (2 mL), respectively, and reacted at room temperature for 1 h. Then, 20% trichloroacetic acid (2 mL) was added, and the mixture was centrifuged at 4 °C for 10 min at 9,000×g (H1605R, Xiang Yi Co., Ltd., Hunan, China), and the supernatant was abandoned. The precipitate was washed three times with a mixture of 2 mL of anhydrous ethanol and ethyl acetate (v:v = 1:1). Finally, the guanidine hydrochloride (6 mol/L, 3 mL) was added to the precipitate and incubated in a water bath at 37 °C for 30 min. The supernatant was centrifuged (9,000×g, 4 °C, 10 min) and measured at 370 nm for absorbance (Bio Tek Synergy H1, Bio Tek Instruments, Inc., Vermont, USA).

2.2.2 TBARS

TBARS was analyzed by the method of Luo et al. [14]. The reaction of samples (0.3 g) with 3 mL of TBA solution and 17 mL of TCA-HCL solution was carried out in a boiling water bath for 30 min. After it was cooled down to room temperature, chloroform was added and centrifuged in a TGL-16 centrifuge (Shu Ke Co., Ltd., Sichuan, China) for 10 min (3,000×g). The absorbance of the liquid was measured in a Bio Tek Synergy H1 (Bio Tek Instruments, Inc., Vermont, USA) at 532 nm.

2.3 Identification of volatile compounds

The volatile flavor compounds (VOCs) of Tibetan pork were identified by referring to Wang et al. [15]. The minced meat (3.00 g) and 10 μL of internal standard (1,2-dichlorobenzene, 5 mg/L) were equilibrated with a headspace bottle in a water bath at 60 °C for 5 min, followed by the insertion of a needle (50/30 μm DVB/CAR/PDMS) for adsorption for 40 min. Then the needle was performed on an injection port of a gas chromatography-mass spectrometer (GC–MS) (SHIMADZU GC–MS-QP2010Ultra, Shimadzu, Kyoto, Japan) with a DB-5MS capillary column (30.0 m × 0.25 mm × 0.25 μm) and resolving the extract at 230 °C for 5 min. The initial column temperature was 40 °C and kept for 5 min, followed by increasing to 50 °C at a rate of 10 °C/min and holding for 2 min, then increasing to 120 °C at a rate of 4 °C/min and staying for 2 min, and finally rising to 230 °C at a rate of 12 °C/min and maintaining for 5 min, with a total program time of 41.67 min. The ion source temperature was 230 °C, the interface temperature was 210 °C, and the solvent delay time was 3 min.

2.4 Identification of flavor precursors

The sample of Tibetan pork (100 ± 2 mg) was placed in a 2 mL centrifuge tube, and 1 mL of tissue extract (75% methanol: chloroform = 9:1, 25% H2O) was added and ground (50 Hz, 60 s), which was repeated two times. The supernatant was taken after centrifugation (4 °C, 12,000 r/min, 10 min) and dried. Two hundred μL of 2-chloro-L-phenylalanine solution (4 ppm, 4 °C) was reconstituted and filtered for LC-MS detection [16].

Two μL extracts were used to identify the flavor precursors compounds of Tibetan pork in an ACQUITY UPLC® HSS T3 (2.1 × 150 mm, 1.8 µm) (Waters, Milford, MA, USA) column at a flow rate of 0.25 mL/min and a column temperature of 40 °C. The mobile phases were 0.1% formic acid acetonitrile (C) and 0.1% formic acid water (D) in positive ionization mode, as well as acetonitrile (A) and 5 mM ammonium formate water (B) in negative ionization mode. The positive and negative ion spray voltages were 3.50 kV and 2.50 kV, respectively, and the capillary temperature was 325 °C. The full scan was performed at a resolution of 70,000 with a scanning range of 81 ∼ 1,000, and HCD was utilized for secondary cleavage with a collision voltage of 30 eV.

2.5 Data processing

Differences between means were estimated by one-way ANOVA (P < 0.05) using SPSS 27.0 (SPSS Inc., Chicago, IL, USA), and results were expressed as mean ± standard deviation for all data except E-nose and E-tongue data. Origin 2023 (OriginLab Co., Northampton, MA, USA) and GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA) were used to analyze the variations in protein and fat oxidation. Clustered heat map analyses were performed using OmicStudio tools (https://www.omicstudio.cn/tool) to visualize the data of volatile flavor compounds and flavor precursors compounds. PLS and OPLS analyses were carried out using Metaboanalyst 5.0 (https://www.metaboanalyst.ca/) to clarify the variations in volatile flavor compounds (GC–MS) and flavor precursors (LC-MS) of BTP and UBTP samples. Pearson correlation heatmap analysis of volatile flavor compounds and precursor substances was performed using Origin 2023 (OriginLab Co., Northampton, MA, USA).

3 Results and discussion

3.1 Differential oxidation of lipids and proteins in BTP and UBTP

Lipid and protein oxidation contribute significantly to the flavor of cooked Tibetan pork. It can be observed from Fig. 1a that the TBARS value of low-temperature cooked Tibetan pork increased markedly with the longer treatment time, and the level of secondary oxidation of lipids in cooked Tibetan pork rose dramatically when combined with the action of ultrasound. This phenomenon may be explained by the ultrasonic cavitation effect, which boosted the temperature and pressure in the cavitation area and produced more reactive oxygen radicals, accelerating the oxidation of lipids [12]. Furthermore, Fig. 1b illustrated that there was no significant variation in the carbonyl content of UBTP and BTP at 30 min. The effects of ultrasound were apparent over time, which promoted the oxidation of sensitive amino acids such as arginine and proline in Tibetan pork, causing a dramatic increase in the carbonyl content of UBTP-90.Fig. 1 Effect of low-temperature cooking with or without ultrasound on oxidation difference of Tibetan pork. TBARS (a and c) and carbonyl content (b and d). (* represents significant (P < 0.05) oxidation values of BTP and UBTP samples at the same treatment time, and % represents multiplicative increase in the oxidation values of samples UBTP over BTP at the same treatment time.)

Interestingly, during the initial phase of thermal treatment, ultrasound-assisted cooking increased the level of lipid oxidation by 9.10%, while hardly improving protein oxidation. As time went on, there was a noticeable rise in protein oxidation of 6.48% after 90 min of ultrasound-assisted cooking, whereas the lipid oxidation level increased just 2.61% (Fig. 1c and d). The reasons for this phenomenon may have two aspects. On the one hand, it can be attributed to the fact that malondialdehyde interacted with the side chains of amino acids to form carbonyl groups in the later stage of heat treatment, which elevated the carbonyl content and dropped the TBARS value [17]. On the other hand, lipid oxidation occurred more readily compared to protein oxidation and was able to promote protein oxidation partly [18]. Therefore, lipid oxidation was remarkably enhanced during the early stage of ultrasonic-assisted cooking at low temperature, and with the reaction, lipid free radicals were generated from lipid oxidation, which facilitated protein oxidation and notably raised the carbonyl content of the UBTP-90 samples.

3.2 The variation of volatile compounds in BTP and UBTP

3.2.1 The generation of VOCs in UBTP induced by ultrasound: a comparison of samples treated for 30 min and 90 min

A total of 73 volatile flavor compounds, including 25 aldehydes, 13 alcohols, 6 ketones, 10 esters, 16 hydrocarbons, and 3 others, were identified in the samples of low-temperature cooked with or without ultrasound (Fig. 2a, Table 1). The PLS-DA analysis was utilized to observe the differences in the samples from each treatment (Fig. 2b). The results showed that the raw meat was located on the right side of the model, while the cooked Tibetan pork samples were distributed on the left side of the raw meat and gradually shifted to the right as oxidation increased on PC1. It might be attributed to the difference in the content of hexanal, 1-octen-3-ol, 2,3-octanedione, and nonanal among them. It can be inferred that the differences in content of volatile compound may be caused by ultrasound or treatment times. Besides, the low-temperature cooked Tibetan pork samples gradually moved upward with the assistance of ultrasound on the PC2. It is obvious that hexanal, pentanal, octanal, and 1-pentanol possessed high VIP values and presented significant differences in quantity (Fig. 2d), which might be responsible for the flavor variations in the ultrasonically treated group. Moreover, Zhou et al. [19] also discovered that ultrasonic treatment was positively regulating the generation of volatile flavor components in ham while using ultrasound-assisted cooking.Fig. 2 Differences in volatile flavor compounds in RAW, BTP, and UBTP samples. Heat map of volatile flavor compounds in five Tibetan pork samples (a), PLS score plot of five samples of Tibetan pork (b), VIP value of the PC1 (c), VIP value of the PC2 (d).

Table 1 Changes of volatile flavor compounds in low-temperature Tibetan pork samples with or without ultrasound-assisted.

Number	Name	Molecular formula	RT	Concentrations (μg/kg)	
RAW	BTP-30	BTP-90	UBTP-30	UBTP-90	
A1	Pentanal	C5H10O	3.02	N.D.	N.D.	N.D.	15.81 ± 0.49a	15.90 ± 0.61a	
A2	Hexanal	C6H12O	5.40	2.28 ± 0.14e	722.15 ± 48.76b	554.32 ± 24.05c	850.37 ± 30.92a	397.84 ± 9.75d	
A3	Heptanal	C7H14O	9.37	N.D.	43.00 ± 4.37b	34.03 ± 1.17c	57.27 ± 2.60a	25.58 ± 0.19d	
A4	2-Heptenal	C7H12O	11.87	N.D.	38.59 ± 1.60b	31.91 ± 1.29c	42.41 ± 3.42a	31.03 ± 0.83c	
A5	Octanal	C8H16O	14.02	1.51 ± 0.02e	32.75 ± 2.33b	27.30 ± 1.21c	48.14 ± 2.32a	21.28 ± 0.43d	
A6	Phenylacetaldehyde	C8H8O	15.64	N.D.	N.D.	N.D.	N.D.	1.55 ± 0.11	
A7	Nonanal	C9H18O	18.30	12.73 ± 0.41e	105.15 ± 6.54b	76.50 ± 3.60c	138.80 ± 12.87a	54.05 ± 2.62d	
A8	Decanal	C10H20O	22.25	1.56 ± 0.07d	3.41 ± 0.13b	2.56 ± 0.02c	4.16 ± 0.66a	2.60 ± 0.10c	
A9	Tridecanal	C13H26O	29.49	N.D.	5.61 ± 0.49ab	5.08 ± 0.23b	6.20 ± 0.59a	3.91 ± 0.04c	
A10	Tetradecanal	C14H28O	34.13	1.74 ± 0.09d	3.64 ± 0.20b	N.D.	5.45 ± 0.34a	2.82 ± 0.10c	
A11	Pentadecanal	C15H30O	32.96	N.D.	N.D.	N.D.	0.50 ± 0.04a	0.40 ± 0.03b	
A12	Octadecanal	C18H36O	35.22	0.92 ± 0.10b	1.11 ± 0.05b	2.99 ± 0.33a	N.D.	N.D.	
A13	(E, E)-2,4-Heptadienal	C7H10O	14.29	N.D.	5.55 ± 1.39a	3.43 ± 0.38b	5.09 ± 0.15a	2.80 ± 0.15b	
A14	(2E)-2-Nonenal	C9H16O	15.85	N.D.	47.44 ± 4.12b	41.48 ± 1.58c	60.09 ± 4.58a	31.88 ± 1.15d	
A15	(2E)-2-Octenal	C8H14O	16.35	N.D.	2.85 ± 0.11a	2.95 ± 0.12a	2.97 ± 0.11a	1.94 ± 0.18b	
A16	(E)-4-Nonenal	C9H16O	17.99	N.D.	N.D.	N.D.	N.D.	1.28 ± 0.03	
A17	trans-2, cis-6-Nonadienal	C9H14O	20.15	N.D.	N.D.	N.D.	N.D.	0.35 ± 0.05	
A18	cis-4-Decenal	C10H18O	21.78	N.D.	6.80 ± 0.46a	5.41 ± 0.46b	7.31 ± 0.92a	N.D.	
A19	(4E)-4-Undecenal	C11H20O	21.78	N.D.	N.D.	N.D.	N.D.	3.44 ± 0.33	
A20	2,5-Dimethylbenzaldehyde	C9H10O	22.45	4.68 ± 1.11a	1.69 ± 0.55b	2.35 ± 0.61b	2.57 ± 0.53b	1.43 ± 0.29b	
A21	(2E,4E)-2,4-Nonadienal	C9H14O	22.56	N.D.	8.63 ± 0.68b	7.14 ± 0.63c	12.53 ± 1.05a	4.38 ± 0.08d	
A22	(2E)-2-Decenal	C10H18O	24.28	N.D.	8.90 ± 1.09b	8.28 ± 0.41b	16.95 ± 0.96a	4.89 ± 0.18c	
A23	(2E,4E)-2,4-Decadienal	C10H16O	25.42	1.53 ± 0.10d	10.00 ± 0.27b	9.97 ± 0.40b	13.81 ± 1.26a	7.75 ± 0.55c	
A24	2-Undecenal	C11H20O	28.15	N.D.	7.90 ± 0.56b	5.73 ± 0.41c	10.94 ± 0.63a	3.54 ± 0.44d	
A25	5,9,13-Trimethyl-4,8,12-tetradecatrienal	C17H28O	35.36	N.D.	N.D.	N.D.	N.D.	1.37 ± 0.03	
B1	Pentanol	C5H12O	4.37	N.D.	37.24 ± 3.37a	13.12 ± 0.59b	39.75 ± 4.19a	4.81 ± 0.53c	
B2	1-Heptanol	C7H16O	12.57	N.D.	10.17 ± 0.40b	7.22 ± 0.35c	18.20 ± 1.08a	4.90 ± 0.16d	
B3	1-Octanol	C8H18O	16.93	1.80 ± 0.20e	16.97 ± 0.45b	10.84 ± 0.58c	27.54 ± 0.92a	6.15 ± 0.42d	
B4	1-Pentadecanol	C15H32O	30.35	0.64 ± 0.06a	N.D.	N.D.	N.D.	0.40 ± 0.05b	
B5	1-Dodecanol	C12H26O	30.86	N.D.	N.D.	N.D.	0.93 ± 0.08a	0.41 ± 0.03b	
B6	1-Octen-3-ol	C8H16O	13.01	1.76 ± 0.09e	97.29 ± 1.50b	76.00 ± 4.20c	105.91 ± 2.93a	52.58 ± 0.61d	
B7	2,4-Dimethyl-Cyclohexanol	C8H16O	15.38	N.D.	N.D.	N.D.	N.D.	1.41 ± 0.12	
B8	trans-2-Octen-1-Ol	C8H16O	16.75	N.D.	25.92 ± 1.17b	18.28 ± 0.53c	29.44 ± 1.56a	9.82 ± 0.52d	
B9	2,3-Octanediol	C8H18O2	17.80	N.D.	10.63 ± 0.57a	6.39 ± 0.44c	8.03 ± 0.83b	3.33 ± 0.34d	
B10	2-Butyl-1-Octanol	C12H26O	23.52	N.D.	N.D.	N.D.	N.D.	0.57 ± 0.03	
B11	2-Ethylhexanol	C8H18O	24.43	N.D.	2.54 ± 0.12b	N.D.	3.01 ± 0.07a	N.D.	
B12	2-Hexyl-1-decanol	C16H34O	29.62	N.D.	N.D.	N.D.	N.D.	0.62 ± 0.03	
B13	(E)-2-Hepten-4-ol, 6-methyl-,	C8H16O	17.89	N.D.	6.30 ± 0.22b	6.56 ± 0.24b	10.44 ± 0.70a	4.54 ± 0.24c	
C1	1-Octen-3-one	C8H14O	12.81	N.D.	N.D.	N.D.	N.D.	2.07 ± 0.05	
C2	2,3-Octandione	C8H14O2	13.27	N.D.	283.78 ± 13.75a	203.79 ± 10.41c	224.08 ± 4.34b	155.64 ± 6.27d	
C3	3-Octen-2-one	C8H14O	15.48	N.D.	6.57 ± 0.20a	4.91 ± 0.27c	6.01 ± 0.44b	3.19 ± 0.19d	
C4	2-Tridecanone	C13H26O	31.22	N.D.	N.D.	N.D.	N.D.	0.83 ± 0.05	
C5	2-Pentadecanone	C15H30O	33.97	N.D.	N.D.	N.D.	N.D.	2.18 ± 0.14	
C6	2-Heptadecanone	C17H34O	35.99	N.D.	N.D.	N.D.	N.D.	0.22 ± 0.07	
D1	Undecane	C11H24	18.13	4.50 ± 0.12b	4.48 ± 0.18b	4.41 ± 0.35b	6.13 ± 0.35a	3.22 ± 0.31c	
D2	Dodecane	C12H26	22.04	7.33 ± 0.51a	5.80 ± 0.27b	6.00 ± 0.34b	6.40 ± 0.44b	4.83 ± 0.38c	
D3	Tridecane	C13H28	25.67	1.79 ± 0.10d	2.89 ± 0.10b	2.60 ± 0.08c	3.30 ± 0.15a	2.71 ± 0.03c	
D4	Pentadecane	C15H32	29.27	1.82 ± 0.03b	1.58 ± 0.08c	2.22 ± 0.08a	1.78 ± 0.12b	1.29 ± 0.01d	
D5	Hexadecane	C16H34	31.33	6.13 ± 0.59ab	6.88 ± 0.48a	5.50 ± 0.25b	6.97 ± 0.68a	4.56 ± 0.26c	
D6	4-Methyldodecane	C13H28	22.84	0.25 ± 0.04d	1.16 ± 0.01a	1.18 ± 0.06a	0.82 ± 0.06b	0.60 ± 0.03c	
D7	4,6-Dimethyl-dodecane	C14H30	24.74	4.55 ± 0.19a	1.27 ± 0.06c	1.09 ± 0.06d	1.61 ± 0.07b	0.85 ± 0.05e	
D8	3,8-Dimethyl-decane	C12H26	28.40	2.46 ± 0.06	2.51 ± 0.12a	2.50 ± 0.13a	N.D.	N.D.	
D9	3-Methyl-pentadecan	C16H34	32.40	1.43 ± 0.01a	1.09 ± 0.09b	0.99 ± 0.04b	1.05 ± 0.08b	0.72 ± 0.05c	
D10	Phytane	C20H42	32.40	0.43 ± 0.05b	N.D.	N.D.	0.78 ± 0.03a	0.40 ± 0.04b	
D11	3-Ethyl-3-Methylheptane	C10H22	16.22	0.35 ± 0.01e	1.69 ± 0.07c	2.04 ± 0.06b	2.74 ± 0.09a	1.39 ± 0.12d	
D12	2,6,10,14-Tetramethyl-Pentadecane,	C19H40	33.38	0.22 ± 0.01d	N.D.	0.56 ± 0.04c	1.07 ± 0.07a	0.91 ± 0.04b	
D13	1-Tetradecene	C14H28	29.14	N.D.	1.52 ± 0.06b	1.32 ± 0.10c	1.68 ± 0.03a	1.08 ± 0.12d	
D14	α-Cedrene	C15H24	29.56	6.30 ± 0.24a	4.34 ± 0.20b	4.29 ± 0.43b	6.22 ± 0.04a	2.63 ± 0.13c	
D15	β-Cedrene	C15H24	29.75	3.61 ± 0.39a	1.47 ± 0.07d	1.97 ± 0.05c	2.42 ± 0.06b	1.19 ± 0.06d	
D16	1,2,3,5-Tetramethylbenzene	C10H14	18.72	N.D.	0.77 ± 0.08c	0.93 ± 0.02b	1.16 ± 0.02a	0.63 ± 0.05d	
E1	Vinyl hexanoate	C8H14O2	17.37	N.D.	9.38 ± 1.22a	7.30 ± 0.68b	6.90 ± 0.56b	5.44 ± 0.43c	
E2	Methyl octylate	C9H18O2	19.08	8.54 ± 0.55	N.D.	N.D.	N.D.	N.D.	
E3	Dibutyl oxalate	C10H18O4	20.73	50.80 ± 2.90b	41.97 ± 3.19c	41.46 ± 8.80c	63.85 ± 3.72a	28.15 ± 0.53d	
E4	Allyl heptylate	C10H18O2	21.27	N.D.	N.D.	N.D.	N.D.	0.97 ± 0.05	
E5	Methyl nonanoate	C10H20O2	22.91	4.30 ± 0.39	N.D.	N.D.	N.D.	N.D.	
E6	Methyl Caprate	C11H22O2	26.53	3.63 ± 0.12	N.D.	N.D.	N.D.	N.D.	
E7	6-Pentyloxan-2-one	C10H18O2	31.12	N.D.	N.D.	N.D.	N.D.	1.29 ± 0.06	
E8	6-Heptyltetrahydropyran-2-one	C12H22O2	34.07	N.D.	N.D.	N.D.	N.D.	0.49 ± 0.06	
E9	Methyl hexoate	C7H14O2	10.41	3.44 ± 0.34	N.D.	N.D.	N.D.	N.D.	
E10	Diisobutyl phthalate	C16H22O4	35.61	0.75 ± 0.08b	N.D.	1.36 ± 0.04a	N.D.	0.56 ± 0.08c	
F1	Diallyl disulfide	C6H10S2	17.13	18.13 ± 0.35a	6.80 ± 0.71c	5.21 ± 0.55d	8.71 ± 0.37b	7.30 ± 1.01c	
F2	N,N-Dibutylformamide	C9H19NO	25.55	N.D.	N.D.	0.85 ± 0.06b	1.52 ± 0.12a	0.69 ± 0.04c	
F3	2,4-Ditert-butylphenol	C14H22O	31.40	1.22 ± 0.06a	0.74 ± 0.04c	0.93 ± 0.03b	0.96 ± 0.06b	0.82 ± 0.09c	
a–d Different letters in peer data indicate significant differences between groups (P < 0.05).

N.D. represents not detected.

It is obviously noticed from Fig. 3a that the samples cooked for 30 min with and without ultrasound presented a left–right distribution in PC1. Meanwhile, the amounts of substances in cooked Tibetan pork for 30 min that included pentanal, 1-octanol, 3,8-dimethyldecane, and octadecanal altered greatly. Among them, compared to the BTP-30, the contents of compounds in UBTP-30 such as pentanal, pentadecanal, trans-2-decenal, 1-dodecanol, and 1-heptanol rose dramatically, whereas the contents of compounds such as 2,3-octanedione and octadecanal dropped significantly. Furthermore, the impact of ultrasound on volatile flavor compounds of cooked Tibetan pork was further confirmed by samples cooked for 90 min (Fig. 3c and d). The samples cooked for 90 min with and without ultrasound exhibited the same left–right distribution in the OPLS model as those cooked for 30 min. Ultrasound-assisted cooking expanded the abundance of pentanal, (E)-4-nonenal, tetradecanal, and 1-octen-3-one, while reduced the levels of trans-2-octen-1-ol, (Z)-4-decenal, and 1-pentanol.Fig. 3 OPLS score plot and VIP values of volatile flavor compounds in Tibetan pork samples cooked for 30 min with and without ultrasound-assisted (a and b), OPLS score plot and VIP values of volatile flavor compounds in Tibetan pork samples cooked for 90 min with and without ultrasound-assisted (c and d) and Venn diagram of up-regulated volatile flavor compounds in UBTP samples (e).

Except for the ultrasound-assisted treatment, the synergism of ultrasound and low-temperature cooking at different times also affected the flavor of Tibetan pork. In order to exclude the impact of time, a comparison was made among compounds whose abundance rose in the ultrasound group. It can be found that the levels of five compounds were remarkably elevated after ultrasonication at different times, namely, pentanal, pentadecanal, tetradecanal, 1-dodecanol, and phytane. It is certain that the formation of the above compounds was induced by ultrasound during the cooking treatment.

3.2.2 The production of VOCs in UBTP induced by synergism of ultrasound and low-temperature cooking

The distribution of cooked Tibetan pork in PLS showed that the samples gradually displaced to the right on PC1 as the lipid oxidation level increased (Fig. 2b). Moreover, BTP-30 and BTP-90 were closer to one another, and Table 1 revealed that the kinds of volatile flavor compounds were not clearly distinguished between them. Although there was a significant difference in the degree of oxidation between them (Fig. 1a and b), increasing the low-temperature cooking time without ultrasound would only induce changes in the content of volatile flavor compounds and have no significant effect on the formation of new flavor compounds. Interestingly, there was no remarkable variation (P < 0.05) in the level of lipid oxidation of samples UBTP-30 and UBTP-90, yet UBTP-90 was individually distributed on the positive side of PC1 (Fig. 2b). Meanwhile, it was found that sample of UBTP-90 exhibited a substantially larger carbonyl content than UBTP-30. Therefore, it can be reasonably hypothesized that free radicals generated from lipid oxidation during the late stage of ultrasound-assisted cooking encouraged the oxidation of proteins and produced volatile flavor compounds [20].

As can be seen from Fig. 3b, 26 compounds were noticeably up-regulated and down-regulated in the samples of Tibetan pork cooked with and without ultrasound for 30 min, respectively. In particular, 21 compounds including octanol, heptanol, 1-octen-3-ol, and 2-undecenal were produced in a short time by the synergism of ultrasound and low-temperature cooking (Fig. 3e), and these compounds mainly originated from the degradation of fatty acids. The phenomenon confirmed the results of the TBARS values and carbonyl content in the early experiments, which indicated that flavor generation during the initial stage of ultrasound-assisted cooking was mostly dependent on lipid oxidation. Similarly, by comparing the samples cooked for 90 min with and without ultrasound-assisted (Fig. 3d), it was discovered that 19 compounds such as 1-octen-3-one, (E)-4-nonenal, tetradecanal, 2-pentadecanone, phenylacetaldehyde, 2,4-dimethylcyclohexanol, allyl heptanoate, 2-tridecanone, and 2-hexyl-1-decanol, etc., were markedly down-regulated and up-regulated in the BTP and the UBTP, respectively. There, a total of 14 compounds like phenylacetaldehyde, 1-octen-3-one, and tridecanal were derived from the synergism of ultrasound and low-temperature cooking for a long time. Besides, phenylacetaldehyde and diallyl disulfide, which were proven to stem from protein oxidation, contributed greatly to the Tibetan pork cooked for 90 min [21]. However, they were not detected in the samples of BTP-30 and UBTP-30. This phenomenon suggested that protein oxidation played an important role in flavor formation during the later stage of ultrasound-assisted cooking.

Pentanal has been identified as a volatile flavor compound derived from ultrasound-induced, which is described as a nutty flavor [22] and is thought to generate from the oxidation of unsaturated fatty acids [23]. Additionally, octanal, heptanal, and other linear aldehydes are also derived from the oxidation of fatty acids [24], which were detected in all samples. It is evident from Fig. 2a that majority of the aldehydes diminished with a higher ultrasound time. It could be attributed to the interaction of aldehydes with amino acids under ultrasound [25]. This process resulted in a decrease in aldehydes’ content and an increase in protein oxidation in cooked Tibetan pork during the later stage of ultrasound-assisted cooking. Notably, phenylacetaldehyde has been identified only in the UBTP-90 sample. Phenylalanine was degraded to phenylacetaldehyde under the synergism of ultrasound and low-temperature cooking for a long time [26], while a short synergism did not produce this result. Also, diallyl disulfide was identified as a high contributor in samples cooked for 90 min, and the content increased significantly after sonication. It indicates that ultrasound treatment may aid its production, which is consistent with the data reported by Zhu et al. [27].

Additionally, ketones are also significant volatile flavor compounds in cooked Tibetan pork. 1-Octen-3-one with mushroom odor, generally coming from the oxidation of linoleic acid and arachidonic acid, was only identified in UBTP-90. Which suggested that fat oxidation was still an important contributor to the flavor of Tibetan pork in the later stage of the cooking treatment. As mentioned by Wang et al. [28], lipid oxidation is the primary source of flavor components in pork. However, the role of protein oxidative degradation in ultrasound-assisted cooking at low-temperature cannot be ignored. In fact, the production of phenylacetaldehyde mentioned above provided convincing proof that proteins degrade into flavor compounds in the later stage of ultrasound-assisted cooking [29].

Furthermore, trans-2-octen-1-ol, which was examined in all of the cooked Tibetan pork samples, was commonly characterized as an unpleasant plastic odor that negatively impacted the flavor of Tibetan pork [30]. Fortunately, the content of trans-2-octen-1-ol was significantly weakened after a prolonged ultrasound-assisted treatment, which reducing the off-flavors of cooked Tibetan pork.

In summary, lipid oxidation was essential to the flavor of Tibetan pork cooked with ultrasound at low temperature. At the early stage of ultrasound-assisted cooking (UBTP-30), the flavor of cooked Tibetan pork was mainly contributed by lipid oxidation. At the later stage of ultrasound-assisted cooking (UBTP-90), proteins were attacked by lipid oxidation products (aldehydes and lipid free radicals), forming novel aroma compounds and enriching the odor of Tibetan pork.

3.3 The variation of fatty acids and amino acids in BTP and UBTP

3.3.1 The degradation of fatty acids and amino acids in UBTP induced by ultrasound: a comparison of samples treated for 30 min and 90 min

Metabolomics was utilized to identify the variations of fatty acids and amino acids in Tibetan pork cooked with or without ultrasound at low temperature (Table 2). The variable importance in projection (VIP) was used to determine their differential precursors in the cooked Tibetan pork (VIP > 1, P < 0.05). A total of 30 flavor precursors were identified and the PLS-DA (Fig. 4b) was utilized to visualize their overall changes with different treatments. It was found that raw and cooked Tibetan pork were located in the upper-left and below. Moreover, the Tibetan pork samples kept moving to the right on the PC1 with time and ultrasound added. Besides, the samples of UBTP-90 were placed on the positive side of PC1, and the other cooked Tibetan pork samples were located on the negative side of PC2. The allocation of cooked Tibetan pork samples in the PLS model may be explained by the fact that ultrasound-assisted cooking modified the levels of methionine, lysine, glycerol-3-phosphate, and other small-molecule precursors in Tibetan pork (Fig. 4c).Table 2 Changes of flavor precursor substances (fatty acids and amino acids) in low-temperature Tibetan pork samples with or without ultrasound-assisted.

Number	Name	Formula	Intensity (a.u. ×106)	p value	VIP	Mode	
RAW	BTP-30	UBTP-30	BTP-90	UBTP-90	
1	L-Valine	C5H11NO2	29.85 ± 1.71d	42.64 ± 2.41c	48.89 ± 2.82ab	45.82 ± 3.40b	52.32 ± 1.62ac	0.006613179	1.317519	pos	
2	L-Isoleucine	C6H13NO2	70.45 ± 2.85d	86.44 ± 0.82c	104.60 ± 7.05b	88.29 ± 1.24c	113.27 ± 2.65a	0.006598951	1.294969	pos	
3	L-Leucine	C6H13NO2	2669.39 ± 98.42a	1883.36 ± 115.75c	2198.61 ± 278.98b	1723.41 ± 51.79c	2836.32 ± 136.44a	0.004118466	1.62552	pos	
4	L-Lysine	C6H14N2O2	160.54 ± 0.59d	184.48 ± 7.62c	356.37 ± 7.83a	246.67 ± 14.89b	347.63 ± 4.28a	0.006177765	1.141644	pos	
5	L-Glutamic acid	C5H9NO4	165.57 ± 4.92a	49.94 ± 2.37d	55.24 ± 2.55c	60.76 ± 1.62b	37.02 ± 0.40e	5.76E-11	1.509698	pos	
6	L-Histidine	C6H9N3O2	52.19 ± 0.23a	50.59 ± 0.09c	51.00 ± 0.47bc	50.68 ± 0.70c	51.48 ± 0.11ab	0.001128468	1.374227	pos	
7	L-Phenylalanine	C9H11NO2	8.67 ± 0.21c	10.05 ± 0.19b	10.10 ± 0.45b	12.07 ± 0.69a	10.12 ± 0.50b	0.004041906	1.673606	pos	
8	L-Tryptophan	C11H12N2O3	14.46 ± 1.39a	13.26 ± 0.83ab	12.97 ± 0.36b	13.74 ± 0.34ab	12.75 ± 0.19b	0.002373562	1.085963	pos	
9	L-Proline	C5H9NO2	31.64 ± 0.79a	10.87 ± 0.17e	15.92 ± 0.19b	13.88 ± 0.72c	12.61 ± 0.93d	0.008871088	1.002756	pos	
10	L-Methionine	C5H11NO2S	2740.80 ± 67.78a	1567.00 ± 36.82d	1523.06 ± 41.67d	1682.29 ± 9.84c	1759.21 ± 13.58b	9.64E-14	1.725066	pos	
11	L-Aspartic acid	C4H7NO4	2.59 ± 0.14a	2.35 ± 0.13a	1.97 ± 0.16b	1.75 ± 0.11b	1.73 ± 0.10b	0.034099612	1.375671	neg	
12	L-Arginine	C6H14N4O2	52.01 ± 1.34a	35.94 ± 0.50d	39.37 ± 0.59c	46.18 ± 1.62b	40.73 ± 0.50c	0.007291932	1.0359	neg	
13	L-Kynurenine	C10H12N2O3	1.87 ± 0.05e	7.20 ± 0.13d	7.70 ± 0.19c	8.68 ± 0.20b	8.99 ± 0.15a	0.001084943	1.465898	pos	
14	Dodecanoic acid	C12H24O2	6.95 ± 0.22c	8.51 ± 0.27b	6.61 ± 0.11d	4.91 ± 0.11e	9.14 ± 0.09a	0.000208153	1.264042	pos	
15	gamma-Linolenic acid	C18H30O2	2.27 ± 0.07a	2.26 ± 0.17a	1.69 ± 0.04c	1.98 ± 0.05b	1.38 ± 0.09d	0.003840083	1.057491	pos	
16	alpha-linolenic acid	C18H30O2	43.19 ± 1.71e	77.58 ± 0.85c	93.04 ± 2.60a	60.59 ± 0.80d	86.16 ± 1.84b	0.003307058	1.448303	pos	
17	Linoleic acid	C18H32O2	9.29 ± 0.19d	10.54 ± 0.37b	11.91 ± 0.37a	9.85 ± 0.11c	8.31 ± 0.25e	0.001859738	1.53723	pos	
18	Stearic acid	C18H36O2	2.34 ± 0.06e	7.34 ± 0.19c	9.11 ± 0.02a	4.15 ± 0.12d	8.79 ± 0.12b	0.004565646	2.061486	pos	
19	Oleic acid	C18H34O2	29.73 ± 1.52a	16.57 ± 2.57b	16.94 ± 0.74b	9.18 ± 0.06c	18.63 ± 0.54b	0.003768208	1.431868	pos	
20	Arachidonic acid	C20H32O2	4.25 ± 0.09e	14.79 ± 0.23b	18.56 ± 1.16a	12.38 ± 0.94c	10.78 ± 0.27d	3.33236E-05	1.433282	pos	
21	Palmitoleic acid	C16H30O2	16.16 ± 0.25a	11.96 ± 1.13c	13.73 ± 0.83b	14.95 ± 0.71ab	16.06 ± 0.11a	0.00393845	1.358885	pos	
22	Arachidic acid	C20H40O2	284.75 ± 5.54b	240.09 ± 9.05d	364.94 ± 10.52a	366.78 ± 17.56a	260.41 ± 8.19c	0.002950217	1.718808	pos	
23	Erucic acid	C22H42O2	20.46 ± 0.54d	23.07 ± 0.89c	27.28 ± 0.87a	21.99 ± 0.69c	24.81 ± 0.44b	0.001918566	1.50397	pos	
24	Stearidonic acid	C18H28O2	23.42 ± 1.22e	44.39 ± 1.03d	50.30 ± 1.92c	62.13 ± 0.73b	64.42 ± 0.95a	0.000655998	1.361655	pos	
25	Eicosadienoic acid	C20H36O2	21.65 ± 1.64a	1.15 ± 0.14d	6.86 ± 0.18b	4.10 ± 0.23c	7.67 ± 0.26b	1.9745E-11	1.970256	pos	
26	Glycerol 3-phosphate	C3H9O6P	18.20 ± 0.76e	552.94 ± 6.19a	359.41 ± 7.73b	254.95 ± 4.85c	232.33 ± 10.84d	4.20929E-08	1.17145	neg	
27	Palmitic acid	C16H32O2	25.79 ± 0.45a	21.68 ± 1.53c	22.67 ± 0.96bc	22.12 ± 1.03c	24.43 ± 0.53ab	0.003227813	1.129958	neg	
28	Bovinic acid	C18H32O2	136.33 ± 13.79c	115.56 ± 11.74d	155.54 ± 11.81b	102.52 ± 0.06d	187.26 ± 7.72a	5.13619E-05	2.477497	neg	
29	Adrenic acid	C22H36O2	2132.63 ± 49.41d	2333.21 ± 62.81c	2677.35 ± 154.95b	2668.56 ± 138.15b	3955.59 ± 68.56a	0.005165042	1.848749	neg	
30	cis-4-Hydroxy-L-proline	C5H9NO3	165.31 ± 13.16c	224.43 ± 12.18a	197.72 ± 7.29b	134.23 ± 7.64d	122.76 ± 14.87d	0.003061984	1.540102	pos	
a–d Different letters in peer data indicate significant differences between groups (P < 0.05).

Fig. 4 Differences in flavor precursor substances (fatty acids and amino acids) in RAW, BTP, and UBTP samples. Heat map of flavor precursor substances in five Tibetan pork samples (a), PLS score plot of five samples of Tibetan pork (b), VIP value of the PC1 (c), VIP value of the PC2 (d).

To further clarify the role of ultrasound in cooked Tibetan pork, the samples of Tibetan pork at 30 min and 90 min were analyzed by OPLS, respectively (Fig. 5a–d). The results demonstrated that in samples treated for 30 min, ultrasound-assisted cooking resulted in a significant reduction in the content of seven compounds, including glycerol-3-phosphate, methionine, lauric acid, and γ-linolenic acid (Fig. 5b). This reduction may be due to the action of ultrasound alone or to the synergistic effect of ultrasound and low-temperature cooking in a short time. In the samples treated for 90 min, ultrasonication produced a remarkable decline in the level of 12 precursor substances (Fig. 5d). This similarly originated with the action of ultrasound alone or the synergistic effect of ultrasound and low temperature cooking over a long time. Further, a comparison of the above 7 compounds (compounds with lower levels in UBTP-30 samples than BTP-30) and 12 compounds (compounds with lower levels in UBTP-90 samples than BTP-90) revealed that 5 compounds (glycerol-3-phosphate, γ-linolenic acid, aspartic acid, hydroxyproline, and tryptophan) overlapped. It indicated that the degradation of the five flavor precursors came from the action of ultrasound.Fig. 5 OPLS score plot and VIP values of flavor precursor substances in Tibetan pork samples cooked for 30 min with and without ultrasound-assisted (a and b), OPLS score plot and VIP values of flavor precursor substances in Tibetan pork samples cooked for 90 min with and without ultrasound-assisted (c and d) and Venn diagram of down-regulated flavor precursor substances in UBTP samples (e).

3.3.2 The degradation of fatty acids and amino acids in UBTP induced by synergism of ultrasound and low-temperature cooking

In order to investigate the effect of ultrasound on the flavor formation of low-temperature cooked Tibetan pork at different times, the precursors of BTP and UBTP samples were analyzed by OPLS (Fig. 5a–d). As shown in Fig. 5a, BTP and UBTP were present in a side-to-side distribution in Tibetan pork samples processed for 30 min. The short duration of ultrasound accelerated the degradation of seven compounds, including glycerol 3-phosphate, lauric acid, methionine, γ-linolenic acid, etc. Among them, the breakdown of lauric acid and methionine was attributed to the synergism of ultrasound and low-temperature cooking within a short time (Fig. 5e). Glycerol 3-phosphate, the primary component of glycerol phosphate esters, was identified as the precursor substance with a significant contribution to the samples cooked for 30 min. The amount of 3-phosphoglycerol remarkably declined after ultrasound-assisted cooking, which suggested that lipids were greatly degraded to fatty acids during the preliminary stage of treatment, and fatty acids were also oxidized to volatile flavor compounds. Notably, the majority of the fatty acids were found to be considerably more abundant in the UBTP-30 sample in contrast to the BTP-30 sample, proving the breakdown of 3-phosphoglycerol caused by ultrasound. Meanwhile, most amino acids show an upward trend. This phenomenon suggested that in the preliminary stage of ultrasound-assisted cooking, proteins were extensively broken down into free amino acids. Furthermore, ten compounds, including arachidonic acid, eicosadienoic acid, lauric acid, methionine, lysine, etc., were identified as flavor precursors with large contributions in the cooked samples for 30 min (Fig. 5b). Eicosadienoic acid and octadecadienoic acid are polyunsaturated fatty acids that serve as precursors for the production of volatile flavor compounds during processing, including alcohols, ketones, and aldehydes [31]. Lysine is extremely susceptible to oxidation during heat treatment because of its sensitivity [18]. Methionine is a major precursor substance for volatile sulfides. The concentration of free amino acids such as methionine, proline, and leucine declined dramatically after treated for 30 min, while their levels were higher in UBTP-30 samples than in BTP-30 samples. The phenomenon could be attributed to amino acids in Tibetan pork being lost with the juices during the early period of processing. With the addition of ultrasound, proteins were broken down into free amino acids abundantly, which caused an increase in the contents of the UBTP-30 samples. Nevertheless, there was only little or even no degradation of free amino acids to volatile flavor compounds during the early stage of ultrasound-assisted cooking. Therefore, lipid oxidation played a vital role in the flavor development of Tibetan pork during the initial phase of ultrasound-assisted cooking.

It is noteworthy that the levels of free fatty acids and amino acids varied greatly in the samples cooked for 90 min with or without ultrasound-assisted, which led to an increase of their VIP values in the OPLS model. It can be seen from Fig. 5d that prolonged ultrasound treatment encouraged the degradation of 12 compounds such as glutamic acid, arachidic acid, γ-linolenic acid, linoleic acid, and so on. Among them, seven important precursors of volatile compounds, including glutamic acid, arachidonic acid, linoleic acid, arachidonic acid, and arginine, originated from the synergism of ultrasound and low-temperature cooking for a long time (Fig. 5e). Moreover, the levels of fatty acids such as arachidonic acid, linoleic acid, γ-linolenic acid, and arachidonic acid, were significantly lower in the UBTP-90 samples than in the samples of BTP-90 and UBTP-30. It can be inferred that prolonged ultrasonication accelerated the degradation of those fatty acids, which might be the reason that compounds such as trans, cis-2,6-nonadienal, 1-octen-3-one, etc. were only observed in the samples of UBTP-90 [32], [33]. The findings suggested that lipid oxidation remained an important pathway for flavor formation in Tibetan pork at the later stage of ultrasound-assisted cooking. Interestingly, a total of five amino acids were degraded in the samples of UBTP-90, especially phenylalanine, proline, and arginine (Fig. 4a). Amino acids are precursors of branched short-chain aldehydes and sulfides [34], yet the production of branched aldehydes and sulfides was not detected in significant amounts in this study, possibly caused by the limitations of the flavor extraction method and instrumentation during the determination [35], [36]. Furthermore, the concentration of phenylalanine remarkably reduced with the addition of ultrasound in the samples treated for 90 min (P < 0.05). Also, it was identified as a high VIP value amino acid in the OPLS analysis, which became powerful evidence for the degradation of phenylalanine. The results further confirmed the previous speculation about the role of protein oxidation in flavor during ultrasound-assisted cooking for 90 min. Therefore, lipid and protein oxidation played essential roles in the flavor of cooked Tibetan pork at the later stage of ultrasound-assisted cooking.

3.4 Correlation between fatty acids and amino acids with volatile flavor compounds induced by ultrasonic-assisted cooking at low temperature in various times

Pearson's correlation analysis among the up-regulated volatile compounds with fatty acids and amino acids was performed to explore the potential relationship between flavor precursors and volatiles in Tibetan pork cooked with ultrasound at low temperature. It is evident that the majority of the volatile flavor compounds showed positive correlations with fatty acids during the initial period of ultrasound-assisted cooking at low temperature (Fig. 6a). This occurrence implied that there was a noticeable degradation of lipids concurrent with the generation of volatiles during the pre-heat treatment stage. Additionally, it was obvious that there was less correlation between amino acids and volatile flavor compounds, especially phenylalanine and tryptophan. This phenomenon validated the hypothesis that proteins contributed very little to the flavor of Tibetan pork at the beginning of cooking. Besides, oleic acid and palmitic acid did not present obvious correlations with volatile flavor compounds in the pre-thermal treatment period. It indicated that gentle treatment conditions could not promote the conversion of oleic acid, palmitic acid, and some amino acids to volatile flavor compounds in a short time. Pentanal presented a strong correlation with most of the unsaturated fatty acids, meaning that it might be derived from the degradation of unsaturated fatty acids such as oleic, linolenic, and arachidonic acids. Differently, volatile flavor compounds exhibited a stronger correlation with flavor precursors compounds at the later stage of ultrasound-assisted cooking (Fig. 6b). Moreover, there was a growing negative correlation between volatile and flavor precursors. It indicated that with the expansion of ultrasound-assisted cooking time, several fatty acids and amino acids were decomposed largely and generated to volatiles, which resulted in a negative correlation among them. Notably, phenylalanine showed a remarkable correlation with volatile flavor compounds. Phenylalanine was degraded by Skrecker to produce phenylacetaldehyde with a fruity odor [19], and a stronger negative correlation between them was found in the study. There was no prominent correlation between diallyl disulfide and sulfur-containing amino acids such as methionine, which might be explained by its pyrolysis during processing. Furthermore, 1-octen-3-one was exclusively detected in UBTP-90, which presented a close correlation with linoleic acid and arachidonic acid. Similarly, trans, cis-2,6-nonadienal displayed a strong correlation with α-linolenic acid and γ-linolenic acid and was only identified in UBTP-90.Fig. 6 Heat map of the correlation between up-regulated volatile flavor compounds and fatty acids and amino acids. Pearson correlation analysis between samples cooked at low temperature for 30 min with or without ultrasound-assisted (a), Pearson correlation analysis between samples cooked at low temperature for 90 min with or without ultrasound-assisted (b).

3.5 Prediction of flavor formation pathways in Tibetan pork induced by ultrasound-assisted cooking at low temperature

The flavor formation pathways of low-temperature cooked Tibetan pork with or without ultrasound assistance are shown in Fig. 7. The flavor development of ultrasound-assisted cooked Tibetan pork at low temperature was mainly impacted by the oxidation of lipids and proteins. Lipid oxidation was the primary pathway for the formation of volatile flavor compounds in cooked Tibetan pork. Thermal action could break the fat in Tibetan pork into fatty acids during the cooking procedure. Unsaturated fatty acids are readily oxidized to aldehydes, alcohols, ketones, and other volatile flavor compounds based on the presence of carbon–carbon double bonds. Besides, ultrasound induced the cracking of water molecules to produce hydroxyl radicals (·OH), which promoted interactions among molecules and influenced the flavor attributes of cooked Tibetan pork [37].Fig. 7 Prediction of flavor formation pathway in Tibetan pork at the early and late stages of ultrasonic-assisted cooking at low temperature.

In the process of ultrasound-assisted cooking at low temperature, a large number of lipids and proteins were hydrolyzed under the effects of thermal and ultrasound, resulting in the formation of flavor precursors such as free fatty acids and free amino acids. Subsequently, the precursor materials were broken down into volatile flavor compounds with characteristic aromas through pyrolysis and ultrasonic generation of free radicals. Lipid oxidation was the primary process for the formation of volatile flavor compounds in cooked Tibetan pork at low temperature for 30 min with or without ultrasound assistance. Moreover, water molecules were split into hydroxyl radicals by ultrasound [38], which enhanced the oxidation of fatty acids and resulted in more volatile flavor compounds. Straight-chain aldehydes such as pentanal, hexanal, heptanal, octanal, and nonanal originate from the oxidation of n-6 (linoleic acid, arachidonic acid) and n-9 (oleic acid, erucic acid) unsaturated fatty acids, which are typically described as fruity or grassy aromas [39], [40]. Trans aldehydes stem from the oxidation of linoleic and oleic acids and provide fatty and meaty aroma, such as (2E)-2-nonenal, (2E)-2-octenal, (2E,4E)-2,4-nonadienal, (2E)-2-decenal, (2E,4E)-2,4-decadienal, and others [11].

In the samples of UBTP-90, aldehydes and lipid free radicals generated by lipid oxidation caused the conversion of amino acid into volatile flavor compounds owing to prolonged heating and ultrasonication [41], thus leading to a loss of some aldehydes. In the later stage of ultrasound-assisted cooking, extended ultrasonication created new aroma-active compounds, including phenylacetaldehyde, trans, cis-2,6-nonadienal, and 1-octen-3-one. Phenylacetaldehyde originated from the degradation of phenylalanine gives a fruity flavor. Trans, cis-2,6-nonadienal is the main flavor substance in cuke and has a natural cucumber flavor derived from the degradation of linolenic acid [42]. 1-Octen-3-one is obtained from the oxidation of arachidonic acid. These three compounds were identified in UBTP-90 samples, which indicated that the generation of phenylacetaldehyde, trans, cis-2,6-nonadienal, and 1-octen-3-one required higher-intensity cooking and that the sustained operation of ultrasound and free radicals aided the production of aroma compounds in Tibetan pork. Furthermore, trans-2-octen-1-ol is thought to derive from the oxidation of linoleic acid [43]. Its variation during different periods of ultrasound-assisted cooking revealed that trans-2-octen-1-ol is more temperature-sensitive and can be created during low-intensity cooking as well as degraded and lost by high-intensity cooking.

4 Conclusion

Ultrasound-assisted cooking technique significantly changed the flavor of Tibetan pork cooked at low-temperature. The TBARS value of cooked Tibetan pork was remarkably raised by 9.10% in the early stage of ultrasound-assisted treatment, lipid oxidation was the main source of flavor of cooked Tibetan pork. At the later stage of ultrasound-assisted cooking, the carbonyl content increased dramatically by 6.48%, the oxidation of lipids and proteins became the major source of flavor in cooked Tibetan pork. Concretely, fatty acids such as linoleic acid and oleic acid were degraded a lot in the early stage of ultrasonic-assisted ripening, generating volatiles such as hexanal, nonanal, and (2E)-2-decenal. However, proteins were hardly broken down into volatile flavor compounds. During the later stage of processing, the synergistic effect of ultrasound and prolonged low-temperature cooking promoted the conversion of phenylalanine, linolenic acid, and arachidonic acid to specific volatiles, with the addition of new aroma compounds such as phenylacetaldehyde, trans–cis-2,6-nonadienal, and 1-octen-3-one. Meanwhile, the content of the irritating volatile trans-2-octen-1-ol was greatly decreased, which improved the flavor quality of cooked Tibetan pork. In summary, ultrasound-assisted cooking modified the flavor of low-temperature cooked Tibetan pork by enhancing oxidative differences. Among them, extended ultrasound-assisted cooking at low temperature could boost the conversion of amino acids to flavor compounds and optimize the content of volatile flavor compounds, which had a remarkable ability to improve the overall flavor of Tibetan pork.

CRediT authorship contribution statement

Lujie Cheng: Writing – original draft, Visualization, Investigation, Formal analysis. Xin Li: Writing – review & editing, Methodology, Formal analysis. Xiefei Li: Writing – review & editing, Validation. Yingmei Wu: Investigation, Formal analysis, Data curation. Fengping An: Writing – review & editing. Zhang Luo: Writing – review & editing, Conceptualization. Fang Geng: Writing – review & editing, Validation. Qun Huang: Supervision, Project administration. Zhendong Liu: Supervision, Funding acquisition. Yuting Tian: 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.

Acknowledgements

This study was financially supported through grants from the 10.13039/501100012166 National Key Research and Development Program of China (2022YFD1600905 ), the 10.13039/501100001809 National Natural Science Foundation of China (32460604 ), and the Joint Project of 10.13039/501100007548 Northwest A & F University -Xizang Agriculture and Animal Husbandry College (XNLH2022-03 ).
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References

1 Liu J. Li X. Jing R. Huang X. Geng F. Luo Z. Shang P. Liu Z. Huang Q. Effect of prolonged cooking at low temperatures on the eating quality of Tibetan pork: meat quality, water distribution, and microstructure Food Qual. Saf. 2024 fyae025 10.1093/fqsafe/fyae025
2 Zhang M. Chen M. Fang F. Fu C. Xing S. Qian C. Liu J. Kan J. Jin C. Effect of sous vide cooking treatment on the quality, structural properties and flavor profile of duck meat Int. J. Gastron. Food Sci. 29 2022 100565 10.1016/j.ijgfs.2022.100565
3 Becker A. Boulaaba A. Pingen S. Krischek C. Klein G. Low temperature cooking of pork meat—physicochemical and sensory aspects Meat Sci. 118 2016 82 88 10.1016/j.meatsci.2016.03.026 27060409
4 Liu F. Dong X. Shen S. Shi Y. Ou Y. Cai W. Chen Y. Zhu B. Changes in the digestion properties and protein conformation of sturgeon myofibrillar protein treated by low temperature vacuum heating during in vitro digestion Food Funct. 12 2021 6981 6991 10.1039/d0fo03247f 34137398
5 da Silva J.S. Voss M. de Menezes C.R. Barin J.S. Wagner R. Campagnol P.C.B. Cichoski A.J. Is it possible to reduce the cooking time of mortadellas using ultrasound without affecting their oxidative and microbiological quality? Meat Sci. 159 2020 107947 10.1016/j.meatsci.2019.107947
6 Zhang M. Haili N. Chen Q. Xia X. Kong B. Influence of ultrasound-assisted immersion freezing on the freezing rate and quality of porcine longissimus muscles Meat Sci. 136 2018 1 8 10.1016/j.meatsci.2017.10.005 29055228
7 Guo Z. Ge X. Yang L. Ma G. Ma J. Yu Q.-L. Han L. Ultrasound-assisted thawing of frozen white yak meat: effects on thawing rate, meat quality, nutrients, and microstructure Ultrason. Sonochem. 70 2021 105345 10.1016/j.ultsonch.2020.105345
8 Tong H. Cao C. Du Y. Liu Y. Huang W. Ultrasonic-assisted phosphate curing: a novel approach to improve curing rate and chicken meat quality Int. J. Food Sci. Technol. 57 2022 2906 2917
9 Kang D.-C. Zou Y.-H. Cheng Y.-P. Xing L.-J. Zhou G.-H. Zhang W.-G. Effects of power ultrasound on oxidation and structure of beef proteins during curing processing Ultrason. Sonochem. 33 2016 47 53 10.1016/j.ultsonch.2016.04.024 27245955
10 Wang X. Huang Y. Zhou B. Xu W. Xiang X. Huang Q. Li S. Improvement of quality and flavor of salted egg yolks by ultrasonic assisted cooking Ultrason. Sonochem. 75 2021 105579 10.1016/j.ultsonch.2021.105579
11 Qi J. Jia C.-K. Zhang W.-W. Yan H.-M. Cai Q.-Y. Yao X.-N. Xu K. Xu Y. Xu W.-P. Xiong G.-Y. Li M.-Q. Ultrasonic-assisted stewing enhances the aroma intensity of chicken broth: a perspective of the aroma-binding behavior of fat Food Chem. 398 2023 133913 10.1016/j.foodchem.2022.133913
12 Zhang J. Zhang Y. Wang Y. Xing L. Zhang W. Influences of ultrasonic-assisted frying on the flavor characteristics of fried meatballs Innov. Food Sci. Emerg. Technol. 62 2020 102365 10.1016/j.ifset.2020.102365
13 Dong K. Guan Y. Wang Q. Huang Y. An F. Zeng Q. Luo Z. Huang Q. Non-destructive prediction of yak meat freshness indicator by hyperspectral techniques in the oxidation process Food Chem. X 17 2023 100541 10.1016/j.fochx.2022.100541
14 Luo X. Ai M. Wu Y. Wang Q. Song H. Huang Q. Lu J. Malondialdehyde treatment reduced immunoreactivity of amandin and delayed its digestion Food Qual. Saf. 7 2023 10.1093/fqsafe/fyad013
15 Wang Q. Li X. Xue B. Wu Y. Song H. Luo Z. Shang P. Liu Z. Huang Q. Low-salt fermentation improves flavor and quality of sour meat: Microbiology and metabolomics LWT-Food Sci. Technol. 171 2022 114157 10.1016/j.lwt.2022.114157
16 Huang Q. Dong K. Wang Q. Huang X. Wang G. An F. Luo Z. Luo P. Changes in volatile flavor of yak meat during oxidation based on multi-omics Food Chem. 371 2022 131103 10.1016/j.foodchem.2021.131103
17 Yin Y. Zhou L. Pereira J. Zhang J. Zhang W. Insights into digestibility and peptide profiling of beef muscle proteins with different cooking methods J. Agric. Food Chem. 2020 10.1021/acs.jafc.0c04054
18 Al-Dalali S. Li C. Xu B. Effect of frozen storage on the lipid oxidation, protein oxidation, and flavor profile of marinated raw beef meat Food Chem. 376 2022 131881 10.1016/j.foodchem.2021.131881
19 Zhou C.-Y. Xia Q. He J. Sun Y.-Y. Dang Y.-L. Ou C.-R. Pan D.-D. Cao J.-X. Zhou G.-H. Improvement of ultrasound-assisted thermal treatment on organoleptic quality, rheological behavior and flavor of defective dry-cured ham Food Biosci. 43 2021 101310 10.1016/j.fbio.2021.101310
20 Geng L. Liu K. Zhang H. Lipid oxidation in foods and its implications on proteins Front. Nutr. 10 2023 10.3389/fnut.2023.1192199
21 Calkins C.R. Hodgen J.M. A fresh look at meat flavor Meat Sci. 77 2007 63 80 10.1016/j.meatsci.2007.04.016 22061397
22 Chen Y.P. Li W. Yu Y. Wang M. Blank I. Zhang Y. Liu Y. Elucidation of the impact of steaming on the key odorants of Jinhua dry-cured ham using the sensomics approach J. Agric. Food Chem. 71 2023 4932 4942 10.1021/acs.jafc.2c08423 36930805
23 Stetzer A.J. Cadwallader K. Singh T.K. McKeith F.K. Brewer M.S. Effect of enhancement and ageing on flavor and volatile compounds in various beef muscles Meat Sci. 79 2008 13 19 10.1016/j.meatsci.2007.07.025 22062593
24 Li X. Tu Z. Sha X. Li Z. Li J. Huang M. Effect of coating on flavor metabolism of fish under different storage temperatures Food Chem. X 13 2022 100256 10.1016/j.fochx.2022.100256
25 Roldán M. Ruiz J. del Pulgar J.S. Pérez-Palacios T. Antequera T. Volatile compound profile of sous-vide cooked lamb loins at different temperature–time combinations Meat Sci. 100 2015 52 57 10.1016/j.meatsci.2014.09.010 25306511
26 Wen R. Hu Y. Zhang L. Wang Y. Chen Q. Kong B. Effect of NaCl substitutes on lipid and protein oxidation and flavor development of Harbin dry sausage Meat Sci. 156 2019 33 43 10.1016/j.meatsci.2019.05.011 31125945
27 Zhu Z.H. Yu M.H. Zhang J. Zhang W.G. Effects of ultrasonic-assisted curing on the eating quality of lamb J. Food Eng. 361 2024 10.1016/j.jfoodeng.2023.111756
28 Wang S. Chen H. Sun J. Zhang N. Wang S. Sun B. Effects of cooking methods on aroma formation in pork: a comprehensive review Food Chem. X 20 2023 100884 10.1016/j.fochx.2023.100884
29 Berdagué J.L. Monteil P. Montel M.C. Talon R. Effects of starter cultures on the formation of flavour compounds in dry sausage Meat Sci. 35 1993 275 287 10.1016/0309-1740(93)90033-e 22061221
30 Wu L. Wang X. Hao J. Zhu N. Wang M. Geographical indication characteristics of aroma and phenolic acids of the Changping strawberry Foods 12 2023 10.3390/foods12213889
31 Zhang J. Zhang W. Zhou L. Zhang R. Study on the influences of ultrasound on the flavor profile of unsmoked bacon and its underlying metabolic mechanism by using HS-GC-IMS Ultrason. Sonochem. 80 2021 105807 10.1016/j.ultsonch.2021.105807
32 Cheng L. Li X. Tian Y. Wang Q. Li X. An F. Luo Z. Shang P. Liu Z. Huang Q. Mechanisms of cooking methods on flavor formation of Tibetan pork Food Chem. X 19 2023 100873 10.1016/j.fochx.2023.100873
33 Liu D. Du L. Huang Q. Zhou M. Xiong G. Li C. Qiao Y. Wu W. Effects of ultrasound treatment on muscle structure, volatile compounds, and small molecule metabolites of salted Culter alburnus fish Ultrason. Sonochem. 97 2023 106440 10.1016/j.ultsonch.2023.106440
34 Bianchi F. Cantoni C. Careri M. Chiesa L. Musci M. Pinna A. Characterization of the aromatic profile for the authentication and differentiation of typical Italian dry-sausages Talanta 72 2007 1552 1563 10.1016/j.talanta.2007.02.019 19071797
35 Cheng Y. Li G. Wu H. Huang L. Wang H. Identification of light-induced key off-flavors in Ponkan mandarin juice using MDGC-MS/O and GC–MS/PFPD J. Agric. Food Chem. 69 2021 14259 14269 10.1021/acs.jafc.1c05465 34784211
36 Ye Y. Wang L. Zhan P. Tian H. Liu J. Characterization of the aroma compounds of Millet Huangjiu at different fermentation stages Food Chem. 366 2022 130691 10.1016/j.foodchem.2021.130691
37 He H. Wang J. Gong P. Xiao Y. Li S. Wang J. Geng F. Structural identification and immunomodulatory effects of chicken egg white glycopeptides LWT-Food Sci. Technol. 200 2024 116195 10.1016/j.lwt.2024.116195
38 Xu J. Zhang M. Wang Y. Bhandari B. Novel technologies for flavor formation in the processing of meat products: a review Food Rev. Int. 39 2021 1 25 10.1080/87559129.2021.1926480
39 Watanabe A. Kamada G. Imanari M. Shiba N. Yonai M. Muramoto T. Effect of aging on volatile compounds in cooked beef Meat Sci. 107 2015 12 19 10.1016/j.meatsci.2015.04.004 25919931
40 Ying W. Ya-Ting J. Jin-Xuan C. Yin-Ji C. Yang-Ying S. Xiao-Qun Z. Dao-Dong P. Chang-Rong O. Ning G. Study on lipolysis-oxidation and volatile flavour compounds of dry-cured goose with different curing salt content during production Food Chem. 190 2016 33 40 10.1016/j.foodchem.2015.05.048 26212938
41 Bassam S.M. Noleto-Dias C. Farag M.A. Dissecting grilled red and white meat flavor: Its characteristics, production mechanisms, influencing factors and chemical hazards Food Chem. 371 2022 131139 10.1016/j.foodchem.2021.131139
42 Palma-Harris C. McFeeters R.F. Fleming H.P. Solid-phase microextraction (SPME) technique for measurement of generation of fresh cucumber flavor compounds J. Agric. Food Chem. 49 2001 4203 4207 10.1021/jf010182w 11559111
43 D. Karrer, V. Weigel, N. Hoberg, A. Atamasov, M. Rühl, Biotransformation of [U-13C]linoleic acid suggests two independent ketonic- and aldehydic cycles within C8-oxylipin biosynthesis in Cyclocybe aegerita (V. Brig.) Vizzini, Mycol. Prog. 20 (2021) 929–940. doi:10.1007/s11557-021-01719-3.
