Effects of the degree of oral processing on the properties of saliva-participating emulsions: using stewed pork with brown sauce as the model

Mingcheng Zhng, Xioco Zho, Dengyong Liu*, Gun Wng

a College of Food Science and Technology, Bohai University, Jinzhou 121013, China

b Cuisine Science Key Laboratory of Sichuan Province, Sichuan Tourism University, Chengdu 610100, China

c Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, Nanjing 210095, China

Keywords:Oral processing Fat Saliva Emulsif ication Stewed pork with brown sauce

A B S T R A C T This work investigated the changes in properties of saliva-participating emulsions during different oral processing stages (the whole process from intake to swallowing was divided into f ive stages, i.e., 20%, 40%,60%, 80% and 100% stage). The stewed pork with brown sauce was masticated and the emulsion was collected for the determination of emulsion stability, droplet size, ζ-potential, interfacial tension, and microstructure.The results showed that the emulsion stability increased gradually during the oral processing and reached the highest level near the swallowing point. The droplet size of emulsion showed a signif icant downward trend(P < 0.05). Microstructure observations also found different degrees of reduction in fat droplets size at different stages of oral processing. In addition, the ζ -potential of food boluses emulsion was decreased from-16.4 mV to -41.2 mV and the interfacial tension decreased by 52.6% before and after oral progressing. In conclusion, the oral processing of stewed pork with brown sauce was essentially a process in which fat was constantly emulsif ied, and saliva might act as an emulsif ier. This study provides new insights on understanding the oral processing process and sensory changes of fat.

1. Introduction

Food oral processing is a complex dynamic process of food properties and oral physiological response. Under the action of muscle activity, such as jaw movements, and tongue movements,the structure and physicochemical properties of food have been signif icantly changed from the f irst bite till f inal swallowing [1]. In the initial stage of oral processing, food is cut into some small chunks by teeth and the food particles were exposed to saliva in the mouth.At the same time, the mechanical stimulation from chewing and the chemical stimulation from food components lead to saliva secretion.The saliva secreted then coats the surfaces of food particles and lead to an increasing in bolus moisture. When the food transforms into a cohesive bolus, a swallowing will be triggered [2].

Human whole saliva is a typical colloidal system containing a variety of salivary proteins, such as salivary glycoproteins of high molecular weight (MUC5B mucins) [3]. It means that saliva could also function as an effective emulsif ier during oral processing of oil/fat [4]. Some research has pointed out that fat-containing food can be emulsified by saliva during oral processing [5-9]. Glumac et al.collected the saliva from real oral cavity and study it at a molecular level used a quartz crystal microbalance with dissipation (QCM-D),they found that some macromolecular components in saliva could be adsorbed on the oil-water interface, promoting oil-water compatibility and causing electrostatic aggregation [10]. The aggregates in the emulsion mixing with saliva help to improve the viscosity of the bolus and establish a special texture perception [11]. It is generally accepted that the viscosity enhancement and lubrication by the emulsion droplets plays an important role in oral sensory perception.In fact, at the structural level, saliva facilitates the breakdown of food structures, contributes to the formation of a cohesive bolus and changes in-mouth friction, and then alters oral sensation of oil/fat [4].At the molecular level, saliva interacts with food components, leading to the formation of new compounds, complexes and microstructures,the taste perception and texture perception of foods were likely to be influenced [12].

Stewed pork with brown sauce is one of the famous pork dishes for the Chinese family [12]. Pork rib belly is the main raw material for making stewed pork with brown sauce, therefore, the meat cubes can present obvious fat layer and lean layer. In previous studies, we took stewed pork with brown sauceas the research object and successfully reported the physical properties changes and aroma release of pork during oral processing [12]. Stewed pork with brown saucecan release a large amount of fat and protein during oral processing, which can be mixed with saliva to form emulsion in the oral cavity [13-15].As a ‘primitive state fat-dependent’ food, stewed pork with brown sauce is considered to be a suitable model to study the formation of saliva-participating emulsions. Studying the formation and changes of emulsions during oral processing can help us to understand texture perception, taste perception and aroma perception during mastication.So far, some researches were studied by preparing artificial saliva or collecting human salivain vitro. However, artificial saliva usually can’t unable to accurately characterize some real saliva features, such as lubricity and viscoelasticity [16,17]. Therefore, this work attempted to collect the emulsions from human oral cavity and analyzed the properties of saliva-participating emulsions during oral processing.

2. Materials and methods

2.1 Materials

Streaky pork with skin (pig breed was Sanyuan pig, fallow period was 5-6 months, sampling part was pork belly fleshy meat) was purchased from Darunfa supermarket (Jinzhou, Liaoning province,China), stored at -18 °C for no more than 30 days, and defrosted overnight at 4 °C before cooking. Chive, Ginger, Big batch, Cassia bark, Fragrant leaf, Fermented bean curd (Wangzhihe), Fine white sugar, Soy sauce, Soybean oil were bought from Darunfa Supermarket(Jinzhou, China). Liquor, normally with an alcohol content of 56%(by vol), was bought from Beijing Shunxin Agricultural Joint Stock Company (Beijing, China).

2.2 Methods

2.2.1 Cooking process and preparation ofstewed pork with brown sauce

The stewed pork with brown saucewas prepared according to the method of Liu et al. [12]. The specific processes were as follows:The streaky pork used to make stewed pork with brown saucewas purchased once for the duration of the study and kept at -18 °C for no more than 30 days. After washing and draining, the pork (1 kg) was cut into pieces (3.5 cm × 3.5 cm × 4 cm) and deep-fried at 150-160 °C for 2.5 min. The fried pork was transferred to a pan and 10 g of oil and accessories (20 g chive, 20 g ginger, 5 g big batch, 5 g cassia bark, 0.5 g fragrant leaf) were added and stir-fried for 1 min, then the seasonings (80 g soy sauce, 40 g fermented bean curd, 50 g fine white sugar) were added and stir-fried for 2 min. After that, one-kilogram boiling water was poured and the stew was simmered for 2 h.

Take out the prepared stewed pork with brown sauce, remove the skin and clean the juice on the meat surface with an absorbable paper.Then cut the meat chops into a bite sized ((5.0 ± 0.2) g). Samples with a fat to lean weight ratio of about 4:6 were selected for subsequent oral processing experiments, and the temperature of the samples was heated and kept between 65 °C and 75 °C prior to each experiment.

2.2.2 Panelist selection

According to our previous work [12], 20 panelists (10 females and 10 males, aged between 20 and 29) previously trained in the oral processing procedure participated in this study. They were in a good dental health and none of them were smoking or had other diseases.The panelists were informed of the observational nature of this study and signed consent. They were required to avoid food and drink at least 1 h before the test sessions. Then, all of them were further selected based on the repeatability of oral physiological parameter.This process was performed from 10:00 am to 11:00 am, twice a week for 4 weeks. Finally, eight panelists were selected for the subsequent experiment analyses, on their stable (the difference was not significant) physiological parameter such as salivary flow rate,chewing numbers and time.

2.2.3 Emulsion collection

The stewed pork with brown saucesample (one meat chop) was introduced into the panelist’s oral cavity and chewed naturally. When the panelists were read to swallow, they raised their hands, indicating that the oral processing was over. The total chewing time in one was calculated by the subjects under the supervision of the experimenter.In this study, oral processing was divided into five stages based on the chewing time, i.e., 20%, 40%, 60%, 80% and 100% of total chewing time. After completing a certain stage of oral processing, the subjects spit the pellets and saliva into a disposable plastic container and filtered them with four layers of sterile medical gauze. The collected emulsion is immediately stored at 4 °C for further analysis, and emulsion samples stored for more than 12 h are not used [4]. Subjects were allowed to take a sip of water before repeating the same process on two replicate samples (n= 3 total for each trained subject). The panelist should not chew the sample for more than 2 min, and the whole experiment must be completed within 90 min.

2.2.4 Emulsion composition detection partition

The emulsion composition detection was according to the method of Cofrades et al. [18]. The partition coefficient (PC) of the emulsion composition was calculated as follows:

Here,W0is the weight that 50 mL of covered centrifuge tube and gauze which was used to filter.W1meant the weight ofW0and food bolus samples generated in each oral processing stage.W2is the weight of emulsion filtered with two layers of sterile gauze (the mixture of fat and water).W3is the total weight of the centrifuge tube, minced meat and gauze.W4is a symbol of the evaporating dish containing the emulsion which is placed in an oven at (105.0 ± 0.5) °C to be dried at a constant weight (the weight change ranges within 0.02 g).W5referred to the weight of the evaporating dish.

2.2.5 Determination of droplet size

The particle size of emulsions was measured by a Bettersize 3000-plus laser particle analyzer (Dandong, China) at 25 °C [19].The emulsion was diluted in deionized water, and the samples and deionized water had refractive indexes of 1.52 and 1.33, respectively.The particles size was reported as the surface weighted mean diameterd32and the volume-weighted mean diameterd43.

2.2.6 Microscopic examination

2.2.6.1 Optical microscope analysis

The emulsion was dropping onto a glass slides and covered by glycerol-coated coverslips, observed by an optical microscope according to Zhao et al. [20]. Then observed with a 10 × magnification lens with the MICRO PHOTO FXA (NiKang Microscope Nikon Corporation, Japan). The distribution of the emulsion microstructure was carried out at five oral processing stages (20%, 40%, 60%, 80%and 100%).

2.2.6.2 Confocal laser-scanning microscope (CLSM)analysis

The microstructure of emulsion was evaluated using CLSM(FV1000-IX81, Olympus, Japan) at approximately 25 °C [21]. 5.0 mL of emulsion was placed in a test tube and subsequently 200 μL Nile Blue (0.1% (m/V) in ethanol) were added and mixed thoroughly. The mixture was dropped on a slide and then covered with a cover slip,which was observed under the microscope with a 60 × oil immersion objective lens. The emission was excited at 635 nm.

2.2.7 ζ-Potential measurements

According to the method of Crudden and made some modifications [22], theζ-potential was measured using a Zatasizer Nano-ZS90 (Malvern Instruments, England). The zeta-potential was recorded for 20%, 40%, 60%, 80% and 100% stages, and controlled the temperature at 25 °C.

2.2.8 Interfacial tension measurement

The determination of interfacial tension was performed according to a modified version of method described by Chivero et al. [23].The interfacial pressure of emulsion at the oil/water interface was performed at 25 °C using an optical contact angle meter (OCA25,Dataphysics Intruments GmbH, Germany). The different stages of emulsion were placed in the syringe and cuvette, respectively. Then a 10 μL drop of the emulsion was delivered into the cuvette and allowed to stand at the tip of the needle for 10 800 s to achieve emulsion adsorption. The density (the quality of the collected emulsion is divided by their volume) of the emulsions used to calculate the interfacial tension was (1.042 ± 0.003) g/mL (stage of 20%), (1.041 ±0.017) g/mL (stage of 40%), (1.031 ± 0.030) g/mL (stage of 60%),(1.027 ± 0.027) g/mL (stage of 80%) and (1.023 ± 0.019) g/mL,respectively. The data was recorded and processed with the software of instrument.

2.2.9 Emulsifying properties

The nephelometry was used to determine the emulsifying properties of the emulsion [24]. 50 μL of emulsion was put into a beaker and diluted 500 times with 1 g/L sodium dodecyl sulfate(SDS) solution. After the emulsion was placed at 4 °C for 1 h, the absorbance was recorded at 500 nm. The same concentration of SDS solution was used as the control. The emulsifying stability index (ESI)was calculated with the following equations:

WhereAtis the absorption values after standing for 1 h,A0is the absorption values after standing for 0 h.

2.3 Statistical analyses

The data were analyzed by the software IBM SPSS 19.0 (SPSS Statistical Software, Inc., Chicago, IL, USA), and the results were expressed in the mean ± standard deviation, and the data was performed using the Origin 8.6 software. Significant differences between means (P< 0.05) were determined by one–way analysis of variance (ANOVA) with Duncan’s multiple comparisons. All experiments were carried out in triplicate.

3. Results and discussions

3.1 Effect of oral processing on emulsion composition

The determination of emulsion composition was helpful for analyzing the changes of emulsion during different oral processing steps. In the process of masticating food, the continuous secretion of saliva by the salivary glands could wrap and lubricate the food particles which caused the mixture to aggregate and formed cohesive food boluses eventually [25]. Saliva is a complex heterogeneous clear fluid consisting of 2% organic and inorganic substances and roughly 98% water, which is essential for emulsion formation. As shown in Fig. 1, the moisture in emulsion increased with the oral processing and reached a maximum (12.96%) near the point of swallowing(100% stage), which was agreement with the report of Pematilleke and Aguayo-Mendoza [25,26]. It indicated that under the mechanical stimulation of mastication, the moisture from the saliva was increased continuously in the emulsion. It was worth mentioning that, at the stage of 60%, the moisture content in the emulsion increased significantly (P< 0.05) and sharply increased by 423.08% compared with the 20% stages. The need of chewing can be seen in two aspects:to fragment food particles small enough so that they were well mixed and properly lubricated by the saliva to form a coherent bolus that can be swallowed safely and comfortably [27]. In this study, we thought that the first stage corresponded to the first 40% of the oral processing process, during which, the stewed pork with brown saucewas cut off and grounded into small particles by tooth and the secretion of saliva was slow. After that, the small broken particles started to stimulate glands in the oral cavity to secrete saliva, in order to secrete more saliva that could assist in bolus formation and reduced friction between teeth and the food by acting as a lubricant. Therefore,from the 60% stage, more saliva was secreted, and the water in the emulsion increased accordingly. What’s more, the mechanical stimulation of chewing behavior was also one of the reasons to promote salivary secretion, namely, saliva could be increased with the number of mastication cycles [28]. At the same time, the structure of the adipose tissues in the stewed pork with brown sauce was damaged after chewing and the fat was constantly being released. According to literature, saliva could emulsify the fat gradually during the oral processing as an emulsifier [4]. When the released fat mixes with the constant secretion of saliva, it can clearly observe that the content of the emulsion increased sharply increased by 122.60% at the stage of 60%. Following the moisture content previously described, it was corresponding to the trend of the emulsion composition. This result was agreement with the work of Bleis et al. [29].

Fig. 1 Emulsion composition at different stages of oral processing. Values are given as the mean ± SD. Means which have different letter superscripts are significantly different (P < 0.05).

3.2 Determination of droplet size

Size distribution and droplet size were quite important for evaluating emulsion stability [30]. The particle size distribution of the emulsions stabilized by different emulsifiers in different stages of oral processing were shown in Fig. 2a. The size distribution showed a sightly bimodal pattern, the fat globule size distribution was characterized by a significantly greater width for 20% stage compared to 40% and the particle size distribution curve gradually deviated to the direction with smaller particle size with the development of oral processing. A similar result was also reported by Anwesha Sarkar et al., who believed that the emulsion showed bimodal distributions at both low and high mucin concentrations by investigating a series of mucin concentrations [5]. Mucin is an important protein in saliva,which is closely related to oral processing and digestion. As the oral processing proceeded, the secretion of saliva increased, which leads to more protein being adsorbed on the surface of fat droplets. When the oral processing had progressed to 60% stage, the width of both peaks began to narrow significantly, and the shape of these peaks was sharper than that of the first two stages. This may be due to the fact that a large amount of saliva secreted in the 60% stage can completely combine with fat and form small droplets under the shear stress of chewing. However, during the later processing, until swallowing,these changes seemed less obvious (Fig. 2).

The Fig. 2b and Fig. 2c display the surface weighted mean diameterd32and the volume-weighted mean diameterd43,respectively. In the initial stage,d32andd43were 102.77 μm and 7.37 μm, respectively, as the continuous proceeding of chewing,d32andd43gradually decreased to 32.34 μm and 2.64 μm, respectively.The results confirmed that a higher level of oral processing had a significant effect on reducing the droplet size of emulsion. As a general trend, the mean droplet size was found to decrease with the increase of mastication cycles, showing that enhanced shear condition was beneficial to produce finer saliva emulsion. What’s more, the reduction of droplet size of the emulsion was beneficial to the stability of the emulsion [31]. These results were in agreement with the pattern that the stewed pork with brown saucewere broken during the oral processing. In addition, the surface weighted mean diameterd32and the volume-weighted mean diameterd43of the emulsion changed significantly at the stage of 60% which indicated that this stage might be the key stage for the changes of physical and chemical properties during oral processing.

Fig. 2 Emulsion particle size changes at different oral processing stages.(a) Changes of particle size distribution during oral processing. (b) Changes of the surface weighted mean diameter d32 during oral processing. (c) Changes of the volume-weighted mean diameter d43 during oral processing. Values are given as the mean ± SD. Means which have different letter superscripts are significantly different (P < 0.05).

3.3 Microstructure of emulsions

Microscopic examination is an intuitionistic technique to show distributions of fat droplets in emulsion. The microstructure of emulsions observed under the microscope was shown in Fig. 3. It was shown that the droplet was polydisperse in the emulsion. In the initial stage of oral processing (Fig 3a), it was easy to find the large droplets in the emulsion were prominent in the field of vision. After 40% chewing, the large fat globules began to disappear, and the number of small fat globules increased. It may be that mastication played an important role in the preliminary stage of oral processing(20%–40% oral processing stage) and during this period, the whole food was broken into large particles and a large amount of fat was released. At this point, the nonuniform distribution of droplets was obvious (Fig. 3b). In the middle stage of oral processing (60% oral processing stage, also known as transition stage, Fig. 3c), increased mastication cycles and enhanced shear conditions (extrusion of the tongue and the teeth) broke a part of large fat globules into many small fat globules and resulted in bimodal size distribution, with a considerable proportion of the droplets in the size range 0.32–280.70 μm andd43being about 3.32 μm. Base on this, the emulsion seemed to be more uniform compared with the previous oral processing stage. At the late period of oral processing (80%–100% oral processing stage,Figs. 3d-e), emulsification continued to show bimodal distributions, but thed43values showed a gradual reduction. Some previous works had pointed out that particle size was a key factor to trigger a swallow [32].Smaller food particles were more beneficial for swallowing. This phenomenon was consistent with the data of droplet diameters and droplet size in Fig. 2.

The CSLM was used to observe the distributions of fat droplets more precisely and distinctly at high magnification. As shown in Figs. 3a2-e2, the fat droplets in the emulsion appear green and the size of fat droplets gradually decreased as the oral processing continues,which could intuitively confirm the change result ofd43andd32mentioned above. The emulsions near the point of swallowing (100%stage) showed the most uniform distribution and smallest fat droplets.This phenomenon is consistent with the changes in fat droplets observed under a light microscope (Figs. 3a1-e1).

3.4 ζ-Potential measurements

Theζ-potential, which can descript the quantity of charge carriers on the surface of particles in emulsion [33], Results ofζ-potential measurements were given in Fig. 4, which could offer an indication of the potential stability of an emulsion system [34]. From Fig. 4,zeta potential values of all the emulsions were found to be of negative charge in the range of ?16.4 mV (20% stage) to ?19.37 mV (40%stage). It was mainly attributed to the proteins adsorbed on the surface of the emulsion droplet being negatively charged. As the oral processing continues, the absoluteζ-potential value increased rapidly, and a strongly anionic emulsion was formed at the stage from 60% (–30.17 mV) to 100% (–41.2 mV). This may be the result of an increase in negatively charged mucins in saliva during this period [16]. Highζ-potential of the droplets may lead to the strong electrostatic repulsion among the droplets so that it can give electric repulsion force and protection against aggregation and increase the stability of emulsion [35]. The increased absoluteζ-potential values provided a high energy barrier among emulsion droplets, thereby providing good electrostatic repulsion for emulsion [31]. Mucin,lactoferrin, proline-rich protein (PRP) and lysozyme and other saliva proteins can adsorb on the surface of the fat droplets in the emulsion that may provide charge for the surface of fat droplets [31,36]. But mucin was the main protein in saliva which had a low isoelectric point (about 2–3) and the high absolute potential value may increase the stability of mucin in emulsion. In addition to the above effects,droplet size also affected theζ-potential, that is, smaller droplet size facilitated emulsion stability [35,36]. This result was consistent with the data of droplet diameters and particle size distributions in Fig. 2 and confirmed the analysis results of the microstructure analysis. In addition, the absoluteζ-potential value at the stage of 60% increased by 55.76% (P< 0.05) on the basis of the 40% stage. And this stage may be the key one through the process of the strew pork with brown sauce emulsification.

Fig. 3 The distribution and size images of fat droplets at different oral processing stages (a-e, 20%, 40%, 60%, 80%, 100%, respectively) by optical microscope (a1-e1)and CLSM (a2-e2). Scale bar represents 10 μm.

Fig. 4 Emulsion potential changes at different stages of oral processing.Values are given as the mean ± SD. Means which have different letter superscripts are significantly different (P < 0.05).

3.5 Interfacial tension measurement

Interface tension can be used to analyze the adsorption behavior of surface-active substances at the water-fat which referred the increment of surface potential energy when the unit surface area increased [37]. The fat-saliva interfacial tension during the oral processing was shown in Fig. 5. The interfacial tension decreased through the oral processing and compared with the stage of 20%(52.3 mN/m), the interface tension was decreased by 8.99%,46.37%, 52.24% and 52.58% at the stage of 40%, 60%, 80%,100%, respectively. It can be illustrated that the increase of salivary proteins which evidently decreased the interfacial tension of the emulsion system during oral processing, especially during 40% to 60% stages, due to proteins could be the effective interfacial tension depressors [4,38]. The decreased interfacial tension indicated that the emulsion system had low internal energy, which was beneficial for the adsorption of hydrophilic and hydrophobic amino acids and polypeptide chains thereof on the surface of the emulsion layer. They can uniformly distribute on the surface of the fat droplets to form the emulsion layer to improve the emulsion stability [39]. In other words,it was beneficial for the emulsion stability to decrease the interfacial tension [31]. Based on the above theories, the emulsion systems at the stage of 80% and 100% were more stable than other stages due to the salivary proteins being the most (from Fig. 1) and the interfacial tension being the smallest. This result was corresponding to the conclusion of theζ-potential.

3.6 Emulsifying properties

ESI was the ratio of the absorbance between fresh emulsion and standing for different time at 500 nm. The closer the ratio was to 1,the better the stability of the emulsion was [40]. As shown in Fig. 6,it was noted that the longer the chewing time of the emulsion was and the more stabilize of the emulsion was at the same standing time.It may be implied that the length of chewing had a significant effect on the stability of emulsion in the same standing time. Saliva could be the emulsifier for fat during oral processing and the long chewing time may make the saliva and fat mixed fully and also provide enough time for the emulsification reaction. During this period, the number of the chewing cycles could facilitate the emulsifiers (such as mucins) to adsorb the oil droplets and produce a layer around droplets that prevented the emulsion instability [41]. In addition, these proteins may also inhibit the formation of new interfaces by reducing the free energy, thus reducing the interfacial tension at the fat-saliva interface [42].Therefore, the fat droplets uniform distribution and the emulsion were more stable from 80% to the swallowing stage. This finding agrees with the work of Ellouze et al. [43] who investigated the effect of protein concentration on emulsion stability. They found that emulsion stability in certain depended on protein concentration.

Fig. 5 Interfacial tension of emulsion at different stages of oral processing.Values are given as the mean ± SD. Means which have different letter superscripts are significantly different (P < 0.05).

Fig. 6 Emulsification stability of emulsions at different stages of oral processing. Bars represent the means values ± standard deviations of three replicates. Means which have different letter superscripts are significantly different (P < 0.05).

4. Conclusions

This study provided a better understanding of the emulsion formation during the oral processing of the stewed pork with brown sauce. In this study, the whole oral processing was divided into five different stages. The results showed that the amount of emulsion produced in the oral cavity gradually increased with the continuous development of oral processing, saliva can be used as an important component of emulsion combined with the fat that was constantly released from the stewed pork with brown sauce. At high levels of mastication cycles, the particle size of emulsion droplets was significantly decreased, and the distribution of emulsion droplets became more uniform. Compared to the initial oral processing stage(20%–40% stage), 60% stage was a more important stage for the formation of stable emulsions in the mouth. In this stage, the meat was broken up into finer particles, and more fat could easily move from the food into the emulsion. At the same time, the amount of saliva secreted by mechanical stimulation began to increase rapidly.A further increase in the masticatory cycles (60%–100%) caused the emulsions to revert to be a more pronounced bimodal distributions and the peak value moved to the direction of smaller particle size,a further decrease ind43andd32. It can be reflected by the reduced values ford43andd32. At the same time, the increase of theζ-potential of the emulsion effectively prevented the coalescence of the droplets.

Conflict of 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.

Acknowledgement

This work was supported by the National Natural Science Foundation of China (31571861), the Liao Ning Revitalization Talents Program (XLYC1807100) and the Open Foundation Program of Cuisine Science Key Laboratory of Sichuan Province(PRKX2020Z13).