## 3 Results and Discussion
### Changes in the Nutritional Composition of the Fish Broth
#### 3.1.2 Nucleotides and Free Amino Acids  
To analyze the effect of thermo-ultrasonic treatment on the taste of the fish broth, 5\({}^{\prime}\)-nucleotides (5\({}^{\prime}\)-GMP, 5\({}^{\prime}\)-IMP and 5\({}^{\prime}\)-AMP) were qualitatively and quantitatively quantified using HPLC. Nucleotides are important components in the formation of fish's umami flavor, and there is a synergistic effect between nucleotides and flavor amino acids to produce a more intense umami flavor [33]. AMP and IMP are mainly produced by the degradation of adenosine triphosphate (ATP). While AMP promotes the perception of sweetness, IMP is an ideal flavor enhancer. GMP is a product of the conversion of IMP, which also possesses strong flavor-enhancing properties. As can be seen in Figure 1D-E, the thermo-ultrasonic treatment group had a higher content of all three 5\({}^{\prime}\)-nucleotides than the boiling water group at different times. The content of all three nucleotides from the fish broth that was thermo-ultrasonically treated for 60 min was the highest. Thermo-ultrasonic treatment effectively promotes the degradation of adenosine triphosphate (ATP) in fish heads, resulting in an elevated nucleotide content [34]. After thermo-ultrasonic treatment for more than 60 min, the content of 5\({}^{\prime}\)-GMP and 5\({}^{\prime}\)-IMP began to decrease, probably due to the occurrence of transformation or degradation [35]. The taste activity value (TAV) is often used to evaluate the taste contribution of compounds, and a TAV greater than 1 indicates that the TAV has a significant effect on food taste [17]. The highest content of 5\({}^{\prime}\)-GMP and all TAV values exceeded 1 in the samples, which indicated that 5\({}^{\prime}\)-GMP played an important role in the taste of the fish broth. However, the TAV values of 5\({}^{\prime}\)-AMP and 5\({}^{\prime}\)-IMP were less than 0.3, which had weak effects on the flavor of the fish broth.  
The results in Figure 2A show that free amino acids in the fish head tissue were gradually extracted into the broth as the processing time increased. Seventeen free amino acids were detected in both treatment groups, including seven essential and ten non-essential amino acids. The cavitation effect of the ultrasound and the denaturing effect of the thermal component of the treatment combined to accelerate the disruption of the fish head tissues. This accelerated the extraction of more free amino acids into the broth, resulting in an overall higher content of free amino acids in the thermo-ultrasonic treatment group. According to the study of [PERSON] et al., free amino acids provide a variety of taste sensations including umami (Asp, Glu), sweetness (Ser, Pro, Gly, Thr, Ala), bitterness (Val, Met, Ile, Phe, Lys, Leu, Arg, His, Tyr) and tastelessness (Cys) [36]. From the results in Figure 2B, it can be seen that sweet amino acids were the main types of amino acids in the fish head broth. In particular, Ala, the most abundant amino acid in the broth, is a typically sweet amino acid. The total free amino acid and variously flavored amino acid  
Figure 1: Changes in protein (**A**), fat content (**B**) and total sugar (**C**) by boiling water and thermo-ultrasonic treatment with different treatment times (0, 30, 60, 90, 120 min). The contents and TAV of 5\({}^{\prime}\)-GMP (**D**), 5\({}^{\prime}\)-AMP (**E**) and 5\({}^{\prime}\)-IMP (**F**) by boiling water and thermo-ultrasonic treatment with different treatment times (0, 30, 60, 90, 120 min).  
contents of the thermo-ultrasonic treatment group were significantly higher than those of the boiling water group. The sweet amino acid content of B5 was 117.27 mg/L, which was much higher than the sweet amino acid content of A5 of 108.75 mg/L. However, the TAV values of all the types of flavored amino acids (Table S1) were much less than 1. In particular, the increase in the content of bitter amino acids did not have a significant effect on the flavor of the fish broth, which is the same as the findings of [PERSON] et al. (2017).