Optimization of Fermentation Process of Zanthoxylum bungeanum Seeds and Evaluation of Acute Toxicity of Protein Extract in Mice

Abstract

The seeds of Zanthoxylum bungeanum seeds, a high-quality vegetable protein source, encounter application limitations due to their high molecular weight and anti-nutritional factors. This study focused on optimizing the fermentation process by investigating key parameters such as inoculation amount, inoculation ratio, material-to-liquid ratio, fermentation temperature, and fermentation time. Both single-factor experiments and response surface methodology were used to determine the optimal conditions. The effects of fermentation on particle size, surface morphology (scanning electron microscopy), water holding capacity, oil holding capacity, solubility, and emulsification properties of Zanthoxylum bungeanum seed protein were analyzed. In addition, acute toxicity was investigated at doses of 1.5 g/kg, 3 g/kg, 6 g/kg, and 12 g/kg. The results showed that the optimal fermentation conditions were an inoculum concentration of 10%, a ratio of Bacillus subtilis to Lactobacillus plantarum of 1:1, a material-to-liquid ratio of 0.8:1, a temperature of 35 °C, and a fermentation period of 4 days. Under these optimized conditions, the soluble protein content reached 153.1 mg/g. After fermentation, the functional properties of Zanthoxylum bungeanum seed protein improved significantly: the water holding capacity increased by 89%, the oil holding capacity by 68%, while the emulsifying activity and stability indices improved by 6% and 17%, respectively. The macromolecular proteins in the seeds of Zanthoxylum bungeanum were effectively broken down into smaller fragments during fermentation, resulting in a more folded and porous surface structure. In acute toxicity tests, all mice treated with fermented Zanthoxum seed protein survived for more than 7 days after injection, and there were no significant differences in body weight, organ index, and hematological tests between groups, but FZBSP of 1.5 g/kg~12 g/kg caused varying degrees of steatosis and inflammatory damage in the heart and liver. In conclusion, this study confirms that follow-up pilot studies using 1.5 g/kg FZBSP have the potential for further development and utilization.

Keywords: Bacillus subtilis; Lactobacillus plantarum; Zanthoxylum bungeanum seeds; fermented Zanthoxylum bungeanum seeds protein; security; solid state fermentation.

Figure 1

Inoculation amount (A), inoculation proportion (B), material–liquid ratio (C), fermentation temperature (D), and fermentation time (E) on the yield of soluble protein content of FZBS. All tests were performed 3 times. Each data point represents the mean ± SD of triplicates.

Figure 2

Response surface plots (A–F) and the corresponding contour plots (G–L) showing inoculation amount and material–liquid ratio (A,G); inoculation amount and fermentation temperature (B,H); inoculation amount and fermentation time (C,I); material–liquid ratio and fermentation temperature (D,J); material–liquid ratio and fermentation time (E,K); and fermentation temperature and fermentation time (F,L).

Figure 3

The particle size distribution of ZBSP (A) and FZBSP (B). All tests were performed 3 times.

Figure 4

Scanning Electron Microscopy of ZBSP (A) and FZBSP (B). All tests were performed 3 times.

Figure 5

When pH = 3, pH = 5, pH = 7, pH = 39, the solubility of ZBSP and FZBSP. Results are expressed as mean ± SD (n = 3). ZBSP means Zanthoxylum bungeanum seeds protein, and FZBSP means fermented Zanthoxylum bungeanum seeds protein.

Figure 6

Impact on body weight of mice in FZBSP toxicity. Six mice were randomly selected in each group.

Figure 7

Impact on histopathological examination of mice in FZBSP toxicity.