Optimization and Preparation of Tallow with a Strong Aroma by Mild Oxidation

This study was performed to extract and separate the volatiles with solid-phase microextraction (SPME), and was conducted to analyze volatile odor compounds qualitatively and quantitatively in the production of a strong aroma tallow by mild oxidation. A total of 51 odor compounds were detected in the tallow smelted under different conditions. It was found that the high proportion of aldehydes was an important feature of the aroma components in the oxidized melted tallow, such as 1-hexanal, heptanal, nonanal, octanal, benzaldehyde, etc. Through the determination of various indicators, sensory evaluation, and the gas chromatography-olfaction–mass spectrometry (GC-O–MS) analysis and, in combination with response surface methodology, the optimal process parameters for oxidative smelting of tallow were determined as follows: a reaction temperature of 149.61 °C, a reaction time of 31.68 min, and an airflow rate of 97.44 L/h. The accelerated oxidation test further verified the quality of the oxidized tallow.


Introduction
Tallow is a form of rendered fat and is classically made from beef. When the slaughtered cattle are raised to the slaughter condition, a large amount of adipose tissue will be accumulated under the epidermis, between muscles, in the abdominal cavity, intestinal region, and other parts, and the tallow adipose tissue obtained by segmentation is refined by using an appropriate processing method to obtain edible grease, which is called tallow [1].
Tallow is widely used in food processing because of its good flavor and is often used as the processing raw material of shortening, cakes, and other foods [2]. As the soul of the Sichuan and Chongqing hotpot, tallow played an important role in the formation of the hotpot flavor, both in its own flavor and the flavor changes produced during the cooking process [3]. According to the previous analysis of the aroma of 41 old hot pot tallow, it was found that the strong aroma tallow was particularly popular. Therefore, in this experiment, the strong aroma tallow was prepared through the mild oxidation of fat to meet the needs of consumers [4].
Lipid oxidation is an important pathway for the formation of characteristic meat flavor compounds. The research by Pearson et al. [5] has shown that the basic meat flavors of all kinds of meat flavors are the same, while the differences in characteristic meat flavors mainly come from the thermal oxidation degradation reaction of fat, in which heating-induced oxidation of unsaturated hydrocarbon chains in lipids is an important pathway and generates a variety of volatile compounds, including fatty aldehydes, ketones, and acids. Oxidation of fat at a normal temperature will produce a rancid taste, while oxidation under heating conditions will produce characteristic flavor substances, which are the main basis for adding fat and regulating fat oxidation in the production process tatively analyze the aroma active compounds. The changes in aroma compounds in the oxidation of beef tallow were studied. Combined with sensory evaluation, the optimal conditions were determined, which provided a scientific basis for exploring the development and utilization of Nongxiang tallow hot pot seasoning, quality control, and use safety.

Effects of Different Factors on AV, PV, and P-AV of Oxidized Melted Tallow
When the temperature is 150 • C, the time is 35 min, and the airflow rate is 108 L/h (Figures 1-3), the hydroperoxide, the primary oxidation product of tallow, reaches the maximum value. When it exceeds this value, the tallow oxidation is already in the termination stage. It can be seen in the figure that the PV and P-AV values change significantly beyond a specific node, so it is speculated that the oxidation induction period should be less than 35 min and the reaction is already in the oxidation transfer period; that is, the generation of hydroperoxide in the primary oxidation reaction and the pyrolysis of hydroperoxide in the secondary oxidation reaction are both increasing significantly, but the primary oxidation reaction is the main reaction, so both PV and p-AV values increase. At 150 • C, the primary oxidation product of tallow hydroperoxide reached the maximum value. When the temperature exceeds 150 • C, the oxidation of tallow was in the end stage and the generation of secondary oxidation products, namely the pyrolysis reaction of hydroperoxide, became the leading reaction and the generation of primary oxidation products took a back seat, so the PV value began to decline while the P-AV value increased significantly. From the aspect of the reaction mechanism, the higher the melting temperature and oxygen supply, the lower the AV value. It reached the lowest point at 150 • C and 108 L/h, which may be due to the oxidation of unsaturated fatty acid to generate hydroperoxide. Considering the influence of oxidation temperature on the PV, P-AV, and AV value, the oxidation melting of tallow at 150 • C for 35 min and an airflow rate of 108 L/h was selected.
conditions were determined, which provided a scientific basis for exploring the de ment and utilization of Nongxiang tallow hot pot seasoning, quality control, an safety.

Effects of Different Factors on AV, PV, and P-AV of Oxidized Melted Tallow
When the temperature is 150 °C , the time is 35 min, and the airflow rate is 1 (Figures 1-3), the hydroperoxide, the primary oxidation product of tallow, reach maximum value. When it exceeds this value, the tallow oxidation is already in the nation stage. It can be seen in the figure that the PV and P-AV values change signif beyond a specific node, so it is speculated that the oxidation induction period sho less than 35 min and the reaction is already in the oxidation transfer period; that generation of hydroperoxide in the primary oxidation reaction and the pyrolysis droperoxide in the secondary oxidation reaction are both increasing significantly, b primary oxidation reaction is the main reaction, so both PV and p-AV values incre 150 °C , the primary oxidation product of tallow hydroperoxide reached the max value. When the temperature exceeds 150 °C , the oxidation of tallow was in the end and the generation of secondary oxidation products, namely the pyrolysis reaction droperoxide, became the leading reaction and the generation of primary oxidation ucts took a back seat, so the PV value began to decline while the P-AV value inc significantly. From the aspect of the reaction mechanism, the higher the melting te ature and oxygen supply, the lower the AV value. It reached the lowest point at and 108 L/h, which may be due to the oxidation of unsaturated fatty acid to genera droperoxide. Considering the influence of oxidation temperature on the PV, P-A AV value, the oxidation melting of tallow at 150 °C for 35 min and an airflow rate L/h was selected.

Sensory Evaluation of the Sample
The weighted scores of each flavor type of melted tallow were different under different conditions (Table 1). Under reaction temperature conditions, the weighted total score was a maximum of 2.54 at 150 • C and a minimum of 2.14 at 120 • C. The milk flavor, sweet taste, and fruit flavor were the most intense at 150 • C, while each flavor type was relatively weak at 120 • C. In addition, the animal fat at 140 • C and 160 • C had a thick taste but, at the same time, the acid odor also increased. Under the reaction time conditions, the weighted total score for 35 min was the highest at 3.29, and the weighted score for 65 min was the lowest at 1.83. Milk flavor, animal fat flavor, and sweet taste were the most prominent at 25 min and 35 min, while each flavor type was relatively weak at 65 min. Under the airflow rate condition, the weighted total score of 108 L/h was the highest at 3.12 and the weighted total score of 138 L/h was the lowest at 1.83, of which the "shan" flavor and sweet taste at 108 L/h were higher than those under other flow rate conditions, while each flavor type at 138 L/h was relatively weak.

Sensory Evaluation of the Sample
The weighted scores of each flavor type of melted tallow were different under ent conditions (Table 1). Under reaction temperature conditions, the weighted tota was a maximum of 2.54 at 150 °C and a minimum of 2.14 at 120 °C . The milk flavor taste, and fruit flavor were the most intense at 150 °C , while each flavor type was re weak at 120 °C . In addition, the animal fat at 140 °C and 160 °C had a thick taste the same time, the acid odor also increased. Under the reaction time conditio weighted total score for 35 min was the highest at 3.29, and the weighted score for was the lowest at 1.83. Milk flavor, animal fat flavor, and sweet taste were the most inent at 25 min and 35 min, while each flavor type was relatively weak at 65 min.

Sensory Evaluation of the Sample
The weighted scores of each flavor type of melted tallow were different unde ent conditions (Table 1). Under reaction temperature conditions, the weighted tot was a maximum of 2.54 at 150 °C and a minimum of 2.14 at 120 °C . The milk flavo taste, and fruit flavor were the most intense at 150 °C , while each flavor type was re weak at 120 °C . In addition, the animal fat at 140 °C and 160 °C had a thick taste the same time, the acid odor also increased. Under the reaction time conditio weighted total score for 35 min was the highest at 3.29, and the weighted score for was the lowest at 1.83. Milk flavor, animal fat flavor, and sweet taste were the mos inent at 25 min and 35 min, while each flavor type was relatively weak at 65 min the airflow rate condition, the weighted total score of 108 L/h was the highest at 3   (Tables 2-4). Among them, twenty-nine aroma compounds, including fifteen aldehydes, four alcohols, four acids, three esters, and three heterocyclic compounds were detected in tallow melting at different reaction temperatures. A total of thirty-nine aroma compounds were detected in melted tallow at different reaction times, including seventeen aldehydes, six acids, six ketones, five esters, two alcohols, and three heterocyclic compounds. A total of twenty-nine aroma compounds, including fourteen aldehydes, eight acids, three alcohols, three esters, and one other compound were detected in tallow melting at different air flow rates.  x: RI, the retention index on capillaries DB-WAX. ND: not detected. y: the relative concentration stated as the mean ± SD and the unit is ppt (part per trillion, ng/g). a,b,c,d : Different letters represent significant data, while the same letter represents weak significance. x: RI, the retention index on capillaries DB-WAX. ND: not detected. y: the relative concentration stated as the mean ± SD and the unit is ppt (part per trillion, ng/g). a,b,c,d,e : Different letters represent significant data, while the same letter represents weak significance.
(2) Quantitative results A heat map was drawn according to the content of aroma compounds in the oxidized melted tallow under different conditions, as shown in Figure 4. In the figure, blue represents content below the average level, red represents content above the average level, and red also indicates higher content.
It could be seen that the overall content of aldehydes at 120 • C was higher than other temperatures in the reaction temperature, especially 1-hexanal (green, grassy), 1-nonanal (fatty), 1-octanal (fatty), (E)-2-nonenal (cucumber), (E)-2-decenal (fatty) and 2-undecenal (orange peel). The 1-heptanal (fatty), benzaldehyde (nutty), and 2-methylpyrazine (nutty) at 150 • C were more prominent. However, according to the sensory evaluation and determination of the oxidation index, it was found that although the overall substance content was high at 120 • C, the flavor was not very popular, largely because the temperature did not reach the melting point during the melting process, resulting in a slight fishy and greasy taste of the overall flavor and, thus, the aldehyde content was higher than that of samples at other temperatures.
The overall content of aldehydes was the highest in the reaction time of 65 min, especially 1-nonanal (fresh), (E)-2-nonenal (cucumber), (E)-2-decenal (fatty), 2-undecenal (orange peel), and (E,E)-2,4-decadienal (fried). In addition, it also contains high acid substances, such as acetic acid (vinegar), octanoic acid (putrid), nonanoic acid (waxy), etc. At the same time, it added many ketone substances, such as 2-octanone (earthy), 2-nonone (refreshing), 2-decanone (orange peel), etc. However, according to the sensory evaluation and determination of the oxidation index, it was found that although the overall substance content of beef tallow under 65 min was higher than that of samples under other conditions, the long reaction time further oxidized and degraded the fat and produced more small molecule aldehydes, ketones, and acid compounds, thus showing serious sour and rancid odor.  It could be seen that the overall content of aldehydes at 120 °C was higher tha temperatures in the reaction temperature, especially 1-hexanal (green, grassy), 1-n (fatty), 1-octanal (fatty), (E)-2-nonenal (cucumber), (E)-2-decenal (fatty) and 2-un (orange peel). The 1-heptanal (fatty), benzaldehyde (nutty), and 2-methylpyrazine at 150 °C were more prominent. However, according to the sensory evaluation an mination of the oxidation index, it was found that although the overall substance was high at 120 °C , the flavor was not very popular, largely because the temperat oxidation index, it was found that the overall substance content of the 108 L/h melted tallow was not too high and it had the characteristic flavor of tallow, such as fat aroma.

Composition Characteristics of Aroma Compounds in Oxidized Melted Tallow under Different Conditions
By calculating the proportion of various aroma compounds in the melted tallow under different conditions (Table 5), it was found that the high proportion of aldehydes was an important feature of the aroma components in the oxidized melted tallow. The reaction temperature was kept within the range of 140-160 • C, the reaction time was within the range of 25-45 min, and the airflow rate was within the range of 78-138 L/h. The proportion of aldehydes was the highest, with an average of 53.46%.

Principal Component Analysis
In order to further clarify the aroma characteristics of oxidized melted tallow under different conditions, principal component analysis was performed on 15 levels of oxidized melted tallow with different factors according to the compositions and contents of different aroma compounds, as shown in Figure 5. It could be seen from the figure that the samples of 15 levels of oxidation-smelting tallow with different factors were relatively concentrated and could be divided into three types after dimension reduction treatment with a principal component.
Type I was oxidized melted tallow samples at different temperatures, which mainly contained five kinds of samples at 120  The proportion of aromatic compounds was analyzed by principal component analysis (PCA) based on the different factors of oxidized melted tallow sample by SPME-GC-MS. It was found that the principal components had significant differences among the different factors, but there was no significant difference at each level. Finally, according The proportion of aromatic compounds was analyzed by principal component analysis (PCA) based on the different factors of oxidized melted tallow sample by SPME-GC-MS. It was found that the principal components had significant differences among the different factors, but there was no significant difference at each level. Finally, according to the comprehensive evaluation of the oxidation index and sensory evaluation, the optimal reaction temperature was determined to be within the range of 140-160 • C, the reaction time was within the range of 25-45 min, and the airflow rate was within the range of 78-138 L/h.

Response Surface Optimization
Based on the single-factor test, three factors, including reaction temperature, reaction time, and airflow rate, were selected to design the response surface test. The factor-level table is shown in Table 6 and the response surface analysis scheme and results are shown in Table 7. The results were analyzed by Design-Expert data software and the response surface diagrams for the obtained data were shown in Figures 6-8.   (1) (2)   The significance test and analysis of variance for the regression equation are shown in Table 5. The significance for regression analysis of variance indicates that the regression is extremely significant, p = 0.0001, and non-significant, p = 1.0000, for the missing term, with R 2 = 0.9726 and R 2 Adj = 0.9374. The model indicates that the equation fits the experiment well. When describing the relationship between each factor and response value, the linear relationship between the dependent variable and the independent variable is significant for the regression equation.
In the process of studying the influence of related variables on the processing results, in order to further study the interaction between variables, the quadratic regression model was analyzed by software and the response surface stereogram was drawn. The response surface isochronal diagram could intuitively reflect the effects of various factors on the response value to identify the optimal process parameters and the interaction between the parameters. The stereogram of the response surface showed that the fitting surface had the true minimum value. The center point of the smallest ellipse in the contour map was the lowest point of the response surface. The shape of the contour line could reflect the strength of the interaction effect. The ellipse indicated that the interaction between the two factors was significant, while the circle indicated the opposite. Figures 6-8 show the effect of temperature, time, and airflow rate on sensory evaluation, and the contour plot shows that the influence of factors on sensory evaluation is significant. After regression fitting of each factor, the regression equation was obtained as follows: R1 = 9.03 − 0.020A − 0.28B − 0.53C + 0.000AB + 0.000AC + 0.000BC + 3.81BD − 0.26A 2 − 0.42B 2 − 0.75C 2 . The RSM analysis system showed that the optimal conditions for oxidative After regression fitting of each factor, the regression equation was obtained as follows: R1 = 9.03 − 0.020A − 0.28B − 0.53C + 0.000AB + 0.000AC + 0.000BC + 3.81BD − 0.26A 2 − 0.42B 2 − 0.75C 2 . The RSM analysis system showed that the optimal conditions for oxidative melting of tallow were a reaction temperature of 149.61 • C, a reaction time of 31.68 min, and an airflow rate of 97.44 L/h.

Accelerated Oxidation Test
After the determination of various oxidation indexes, sensory evaluation, and SPME-GC-O-MS analysis, the optimized oxidized melted tallow was subjected to an accelerated oxidation test, so as to ensure that various indexes of the produced oxidized tallow reached the standards.
The determination of each index in the accelerated oxidation test of oxidized melted tallow is shown in Figure 9. After being placed in an oven at 62 ± 1 • C for 15 d (the equivalent of 15 months), the AV value exceeded the standard after 13 d, and all indicators were qualified but not exceeding the standard before 12 d [19]. In addition, according to the analysis results of SPME-GC-MS, as shown in the Table 8, there was no significant difference in all substances of oxidized melted tallow in 0-15 d, such as the contents of acid compounds such as propionic acid, butanoic acid, octanoic acid, and decanoic acid, to confirm the quality of oxidized melted tallow.
x FOR PEER REVIEW oxidation test, so as to ensure that various indexes of the produced oxidiz reached the standards.
The determination of each index in the accelerated oxidation test of oxidiz tallow is shown in Figure 9. After being placed in an oven at 62 ± 1 °C for 15 d alent of 15 months), the AV value exceeded the standard after 13 d, and all indic qualified but not exceeding the standard before 12 d [19]. In addition, accord analysis results of SPME-GC-MS, as shown in the Table 8, there was no signific ence in all substances of oxidized melted tallow in 0-15 d, such as the conte compounds such as propionic acid, butanoic acid, octanoic acid, and decano confirm the quality of oxidized melted tallow.   of mass spectrometry were as follows: the ion source type was the electron bombardment, the electron energy was 70 eV, the transmission line temperature was 280 • C, the ion source temperature was maintained at 230 • C, the quadrupole temperature was set at 150 • C, the mass scanning range m/z was 40~250, and the solvent delay was 4 min.

Qualitative and Quantitative Methods of Volatile Aroma Compounds
The qualitative methods of compounds specifically included matching mass spectra with the NIST 08 library to identify compounds and identifying compounds by the standardized compound retention index (RI) and smell (O).
The RI value is calculated according to the peak time of the target compound and the peak time of a series of alkane standards under the same gas quality parameters, and the formula is as follows: where t a in the formula represents the retention time of sample a and t n represents the retention time of C n in normal paraffin standard (the retention time of sample A is between two adjacent normal paraffin C n and C n + 1 ) [23]. Combined with SPME-GC-O-MS technology, a human nose was employed as a detector to smell the odor compounds separated by chromatographic column at the same time, and the odor description was recorded and compared with the RI value, and odor characteristics reported in the literature.

Qualitative Analysis
All the standards used in this study were diluted with n-hexane to a certain concentration. In this study, an internal standard was added to semi-quantitate the aroma compounds in tallow and the relative concentrations of all volatile organic compounds were calculated using 2,4,5-trimethylthiazole as an internal standard. The concentration of each compound was calculated using the following formula: where C i is the concentration of the compound, C is is the internal standard concentration of 1.013 µg/µL, A j is the chromatographic peak area of the compound, and A is is the chromatographic peak area of the internal standard.

Sensory Analysis
Sensory evaluation of oxidized melted tallow under different conditions, Samples of 7.5 g were weighted into an empty vial, put in a 60 • C water bath, and the smell after melting was evaluated. The sensory evaluation team is composed of 13 laboratory professionals. Sensory evaluation is conducted on eight aroma types, such as milky, muttony, animal fat, sweet, burnt, sour, fruit, and spoiled. The scores are from light to strong, with no smell at all being 0, a slight smell but not lasting being 1-2, and a strong smell being 3-4. The eight aroma types were weighted (Table 9), and the weighted total score of the sensory evaluation of each sample was calculated.

Accelerated Oxidation Analysis of Oil
This study used an oven heating accelerated oxidation test (the Schaal oven test). Different oil samples were placed in a colorless cover and put in a thermostat at 62 ± 1 • C for continuous heating and oxidation for 30 days and treated every 12 h [24].
Shook them and changed their positions in the thermostat at will. The acid value (AV), peroxide value (PV), and anisidine value (P-AV) were measured at 0, 3, 6, 9, 12, and 15 days after oxidation in order to understand the formation kinetics of oxidation products of the above-mentioned oils and fats.

Statistical Analysis
The obtained data were analyzed by one-way ANOVA with SPSS 13.0 (SPSS Inc, Chicago, IL, USA) software and p < 0.05 was significant. All data are expressed as mean standard deviation (SD, n = 3). Response surface optimization was analyzed by Design-Expert 8.0.6. PCA analysis was made by SIMCA-P + 11 software.

Conclusions
In this study, a qualitative and quantitative analysis of compounds with aroma characteristics was performed using SPME-GC-MS. A total of 51 aroma compounds were detected in melted tallow under different conditions. Through the determination of various indicators, sensory evaluation, and SPME-GC-O-MS analysis, the single factor range of the oxidation conditions of melted tallow was determined to be a reaction temperature within the range of 140-160 • C and a reaction time within the range of 25 min-45 min. The airflow rate was within the range of 78-138 L/h. The response surface methodology was used to determine the optimal process parameters for oxidative melting of tallow as follows: a reaction temperature of 149.61 • C, a reaction time of 31.68 min, and an airflow rate of 97.44 L/h. The accelerated oxidation test further verified the quality of oxidized tallow. In addition, because the aroma of oxidized beef tallow was complex and its flavor was unique, it was necessary to carry out the research on frying beef tallow condiments on this basis to further determine the flavor analysis of strong flavor beef tallow and the flavor performance during the actual application of beef tallow condiments. These studies would be conducive to the comprehensive analysis of the aroma characteristics and taste of a beef tallow hot pot.