1. Introduction
Porcine bone is an economical source of flavorful amino acids, peptides, nucleotides, organic acids and other substances [
1,
2]. Making soup is a good method of cooking porcine bone, which has the characteristics of balanced umami and thick flavor and is rich in protein and other nutrients [
3]. The essential amino acids and trace elements in porcine bone soup occupy an indispensable part of people’s diet, and it is favored by consumers. China is one of the major producers and consumers of livestock and poultry. According to the data reported by the Ministry of Agriculture and Rural Affairs of the People’s Republic of China, the number of pigs slaughtered in China was estimated to be 162.59 million in 2020. The porcine bones account for 10% of the carcass, and the amount of pig by-products is huge. The use of nutrients and bioactive compounds such as amino acids and peptides [
4] extracted from the porcine bone soup may achieve the maximum utilization of resources [
5].
Welsh onion (
Allium fistulosum L., Alliaceae) has been a traditional medicine and food plant in China, and it is reported to have anti-oxidative, anti-hypertensive and anti-obesity properties [
6,
7].
Allium vegetables are widely used to flavor foods and improve the flavor in foods [
8]. In addition, welsh onion is a classic combination with porcine bone in Chinese traditional cooking. Welsh onion is also used globally as a flavor enhancer that is usually cooked together with meats [
9,
10]. The utilization of welsh onion plays an important role in the industrial production of soup. Studies have shown that welsh onions are rich in bioactive compounds, including polyphenols and organic acids [
8,
11,
12]. Thus, the addition of welsh onion can not only improve the flavor of the food but also increase the nutritional value through antioxidant and antibacterial effects [
8]. With the addition of welsh onion extract, the water holding capacity of chicken rolls was increased and the oxidation of chicken fat was reduced, thereby improving the quality of chicken rolls [
13]. Additionally, mixed extracts of onion, ginger and garlic were used to preserve the freshness of stewed porcine, which prolonged the shelf life of stewed porcine [
14]. Thus, welsh onion may have an important impact on the taste of porcine bone soup during the stewing process.
Amino acids, nucleotides, organic acids, soluble sugars and metal elements contribute to the taste of food [
15,
16]. Porcine bones are rich in amino acids, including a variety of taste amino acids. For taste amino acids, most hydrophobic L-amino acids exhibit a bitter taste, such as L-Phe, L-Tyr, L-Trp, L-Leu, L-Val and L-Ile; most D-amino acids are sweet, and Gly and L-Ala also have strong sweetness [
17]. Organic acids have their own flavor, and they may also enhance other tastes. A mouse model was applied to study fish broth, and it was found that lactic acid can enhance the contribution of amino acids to the taste of soup, while citric acid and lactic acid can change the taste of divalent salt [
18]. Taste nucleotides are mainly 5′-adenosine acid, 5′-inosinic acid and 5′-guanylic acid [
19], and the synergy effect of taste nucleotides together with amino acids can increase the sensory quality of foods; for example, sodium glutamate and nucleotides together can enhance the umami taste of chicken soup [
16]. Mineral elements not only impart a particular flavor but also cooperate with other taste components to enhance the flavor [
18]. At present, there are few studies on the effect of welsh onion on the taste and components of porcine bone soup. The current analysis of porcine bones is mostly focused on cooking techniques and the content of amino acids [
3,
20], and it still lacks comprehensive studies on the contribution of organic acids, nucleotides and mineral elements to the taste of the porcine bone soup.
In this study, the effect of welsh onions on the composition and sensory characteristics of porcine bone soup was investigated. The varieties of amino acids, organic acids, nucleotides and mineral elements after adding welsh onions were explored, and the correlation between taste components and sensory characteristics was also analyzed.
2. Materials and Methods
2.1. Materials and Reagents
Porcine thigh-bones (femur) and welsh onions were purchased from Yonghui Supermarket (Beijing, China). L-(+)-Tartaric acid, formic acid, lactic acid, acetic acid, citric acid, succinic acid, L-(+)-ascorbic acid, propionic acid, potassium dihydrogen phosphate dodecahydrate phosphate (all AR grade),and malic acid (BR grade) were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). Potassium dihydrogen phosphate (KH2PO4), phosphoric acid (H3PO4), phenol (C6H5OH), concentrated sulfuric acid (H2SO4), hydrochloric acid (HCl), disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) (all AR grade), and concentrated nitric acid (HNO3) (GR grade) were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). A multi-element standard solution (50.0 μg/mL) was purchased from the National Center for Analysis and Testing of Nonferrous Metals and Electronic Materials. Inosine 5′-monophosphate (5′-IMP), adenosine 5′-monophosphate (5′-AMP), guanosine 5′-monophosphate (5′-GMP), and cytidine 5′-monophosphate (5′-CMP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Durashell AA analytical reagents, including an internal standard solution, were purchased from Tianjin Bona Agel Technology Co., Ltd. (Tianjin, China). Methanol, trifluoroacetic acid (TFA), and acetonitrile (ACN) (all HPLC grade) were purchased from Fisher Scientific (Shanghai, China). Ultrapure water was purchased from Hangzhou Wahaha Group Co., Ltd. (Hangzhou, China). Sulfosalicylic acid (AR grade) was obtained from Biochemical Technology Co., Ltd. (Shanghai, China). Salt was purchased from Zhongyan Yangtze Salinization Co., Ltd. (Beijing, China).
2.2. Sample Preparation
The fistular onion stalk was taken out of the welsh onion, cut into 5 ± 0.2 cm pieces and split in the middle. The porcine bones were shaved, split in the middle, and washed several times with water until they were clean. The porcine bones were then boiled and rinsed to remove blood foam. For the optimization of stewing time, bone-to-water ratio and the ratio of welsh onion (
Table S1), the umami intensity was ranked by using GB/T 12315-2008 [
21] and the least significant difference (LSD) between samples was calculated by the following formula. There is a significant difference if the difference of rank-sum between the two samples was not lower than LSD.
Here, p and j are the number of samples and panelists, respectively.
Based on the result in
Figure S1, the stewing conditions were optimized: the material–liquid ratio (m/V) was 1:1, the stewing time was 5.0 h, and the ratio of welsh onion was 2.5%. According to the optimized conditions, the sample of welsh onion soup (WS), porcine bone soup (PS) and porcine bone soup with welsh onion (PWS) were prepared in a nutrient soup mode using a DGD40-40DWG electric stew pot (Guangdong Tonze Electric Co., Ltd., Guangdong, China). The soups were then cooled to room temperature, then filtered and centrifuged by a TGL16M centrifuge (Hunan Xiang Yi Laboratory Instrument Development Co., Ltd., Hunan, China) at 10,000 r/min for 10 min to remove impurities. For groups WS, PS and PWS, stewing was repeated three times and the soups were mixed together as one. Samples were then collected from the three groups using the five-point sampling method [
22], and the collected sample with a certain quantity was retrieved in triplicate for each analysis. Sensory analysis was performed after sample collection, and other samples were stored in sterilized vials at −20 °C for further analysis.
2.3. Free Amino Acid Analysis
The quantitative analysis of amino acids was carried out as described in the literature [
23] with some modifications. A 1 mL sample was added to 1 mL 10% (
v/
v) 5-sulfosalicylic acid solution and diluted to 25 mL with 0.1 mol/L HCl. Then, it was mixed with an internal standard solution and filtrated through a 0.22 µm nylon filter membrane before HPLC analysis. The chromatography Durashell AA (4.6 mm × 150 mm, i.d. 3 µm) a column (Agela Technologies, Tianjin, China) and the Durashell AA analysis kit (Agela Technologies, Tianjin, China) were used to analyze the free amino acid in WS, PS and PWS. Mobile phase A (4.50 mg/mL Na
2HPO
4 and 4.75 mg/mL Na
2B
4O
7, pH = 8.2, water solution) and mobile phase B (methanol: acetonitrile: water = 9:9:2,
v/
v/
v) were used for the elution solvent. A 1260 Agilent HPLC (Agilent Technologies Inc., Santa Clara, CA, USA) coupled with a diode array detector (DAD) was used for free amino acid analysis. The gradient elution method was 6–10% B for 0–6 min, 10% B for 6–8 min and 10–16% B for 8–10 min; the flow rate of the mobile phase was 1.6 mL/min; the column temperature was 45 °C; and the detection wavelengths were 338 and 262 nm.
2.4. Organic Acid Analysis
The samples (2 mL) were filtered through a 0.22 µm nylon filter membrane before analysis. Venusil XBP C18 (4.6 mm × 250 mm, 5 μm) was used to analyze the organic acids. Identification and quantification of the organic acids were conducted according to the literature [
24]. The mobile phase comprised buffer salt I (0.01 mol/L KH
2PO
4, pH = 2.8) and methanol (5:95,
v/
v), which were used for equal gradient elution at a flow rate of 1 mL/min, with an injection volume of 20 μL. A Thermo U3000 UPLC system (Thermo Fisher Scientific, Wilmington, DE, USA) was used to analyze the organic acids. The organic acids were detected at a wavelength of 205 nm; the column temperature was 25 °C. The external standard method was used for quantitative analysis; we set the mixed standard concentrations of organic acid to 10.00, 5.00, 3.00, 1.00, 0.80, 0.50, 0.20 and 0.08 μg/mL. The test under the above conditions and the peak area of the obtained chromatogram were plotted against the concentration to draw the standard curve of organic acid.
2.5. 5′-Nucleotide Assay and Equivalent Umami Concentration
The pretreatment method of the sample was conducted based on the method described in the literature [
25]. The instrument, detector, column, column temperature, and mobile phase elution gradient were similar to those used for the analysis of the organic acids. The nucleotides were detected at 254 nm. The mobile phase comprised methanol-buffer salt II (5:95,
v/
v) at a flow rate of 1 mL/min. The mixed nucleotides (5′-AMP, 5′-GMP, 5′-IMP and 5′-CMP) were prepared into 0.1 mg/mL calibration standard solution with ultrapure water, then diluted into seven gradients. Each 5′-nucleotide was quantified according to the calibration curve of the standard 5′-nucleotide.
Since disodium nucleotide and monosodium glutamate have a synergistic effect, the equivalent umami concentration (EUC) formula was used to convert the umami intensity presented by the mixed solution into the equivalent umami concentration of monosodium glutamate (MSG).
Here, EUC is MSG equivalent (g MSG/100 g), ai and aj are the concentration of umami amino acids and nucleotides (g/100 g), respectively; bi is the relative umami concentration (RUC) for each umami amino acid versus MSG (Glu, 1 and Asp, 0.077); bj is the RUC for each umami 5′-nucleotide versus 5′-IMP (5′-IMP, 1; 5′-GMP, 2.3 and 5′-AMP, 0.18), and 1218 is a synergistic constant based on the concentration of g/100 g.
2.6. Mineral Element Analysis
Mineral elements were measured according to a previously described method [
20,
26]. For samples WS, PS and PWS, 5 mL of the sample was accurately transferred into the digestion tank, 6 mL of concentrated HNO
3 was added, and the sample was pre-digested for 30 min at 120 °C. It was then taken out and cooled to room temperature 25 °C. Microwave digestion was carried out in a JUPITER-B microwave digestion workstation (Shanghai Sineo Microwave Chemistry Technology Co., Ltd., Shanghai, China) according to the procedure detailed in
Table 1. The acid was then removed at 160 °C until the remaining sample was about 1 mL. The constant volume was then taken out to 25 mL. The blank was treated as above.
The multi-element standard solution was used to qualitatively and quantitatively analyze the metal elements in the samples by the standard curve method. The measurement was carried out using ICPE-9800 (Shimadzu, Hong Kong, China) according to the measurement conditions shown in
Table 2.
2.7. Taste Traits by Electronic Tongue Analysis
The taste traits of WS, PS and PWS were evaluated by an Electronic tongue (INSENT SA402B, Tokyo, Japan) following the reported method [
27] with some modifications. The electronic tongue was composed of six taste sensors for sourness (CA0), bitterness (C00), astringency (AE1), umami (AAE), saltiness (CT0) and sweetness (GL1). The SA-402B taste analysis system uses artificial lipid membrane sensing technology similar to the working principle of taste bud cells. The artificial lipid membrane produces changes in the membrane potential through electrostatic or hydrophobic interaction with taste-bearing substances. The electric potential is used as the output of the sensor, and the signal is transmitted to the computer for analysis to recognize the taste intensity and taste characteristics. Before analysis, the sensor was pretreated in a reference solution (30 mM KCl solution containing 0.3 mM tartaric acid) for 24 h, and then the WS, PS and PWS samples (50 mL) were filtered and degreased for measurement. The program was set with a sample taste collection time of 30 s, an aftertaste collection time of 30 s, and a cleaning time of 300 s. The test was conducted at a room temperature of 25 °C.
2.8. Sensory Evaluation
All recruited panelists were informed of the detailed steps and aims of the sensory evaluation. Then they were provided with written informed consent. According to the training methods in the literature [
28], 10 panelists (5 men and 5 women aged between 22 and 30, healthy and non-smoking with no taste/odor disorders) were trained by the ranking test for 3 weeks. The taste solutions including glucose (50.00 g/L), citric acid (5.00 g/L), sodium chloride (15.00 g/L), sodium glutamate (20.00 g/L), and quinine (6.00 mg/L), were respectively diluted in 2-fold serials of 1:2, 1:4, 1:6, 1:8, and 1:16. Each of the taste solutions with five different concentrations was presented to the panel, and they were asked to rank the orders according to taste perception intensity. Panelists were requested to participate in the 2-AFC (a two-alternative forced-choice) test according to ISO 5495. Only the panelists with correct answers (100% cut-off point) were selected to participate in further exploration.
According to “GB/T19547-2004” [
29], the soup samples (50 mL) were put into a bottle and randomly numbered with three digits. After the training, the qualified panelists were asked to take a sip of the sample and to keep it in the mouth for 10 s, then spit it out. The intensity of each attribute (sourness, sweetness, bitterness, saltiness, umami and astringency) was scored by the panelists according to the degree for sensory evaluation (
Table S2). At the interval between samples, panelists were asked to wash their mouth with 50 mL drinkable water to avoid fatigue and carryover effect. The sensory evaluation experiment was performed on different groups at a 1-hour interval.
2.9. Statistical Analysis
Statistical analysis was performed using SPSS software (version 19.0, SPSS Inc., Chicago, IL, USA) and Excel (20 10, Microsoft Co., Ltd., Redmond, WA, USA) [
30,
31]. All statistical analyses with the experimental results are expressed as means ± standard deviation. One-way analysis of variance and Duncan’s multilevel tests were applied for determining significant differences at
p < 0.05.
4. Conclusions
In summary, the addition of welsh onion can improve the sensory quality of porcine bone soup by affecting the related components. Porcine bone soups were prepared under these conditions: the material–liquid ratio (m/V) was 1:1, the stewing time was 5.0 h, and the ratio of welsh onion added was 2.5% (accounting for bone mass). Variations of amino acids, organic acids, nucleotides, and mineral elements were detected in bone soup with or without welsh onion. The results suggested that the addition of welsh onions increased the free amino acids in porcine bone soup, especially the umami amino acids (increased by 35.73%), which contribute to promote the taste quality of porcine bone soup. Moreover, the release of lactic acid, pyroglutamic acid, citric acid, and ascorbic acid was significantly promoted with the addition of welsh onion. Compared with PS, the EUC value was increased from 7.85 to 9.71 g MSG/100 g in PWS, which was due to the high content of umami amino acids and the synergistic effect with 5′-nucleotides. In porcine bone soup with welsh onion, the enhancement of umami taste was verified by sensory evaluation, and the connection between tastes and certain components was revealed according to a correlation analysis.