Response surface optimization and flavor determination of fermentation processes of orange peel tea wine

Determination of total flavonoid content

Orange peel tea liquor was diluted to prepare the sample solution, and 0.2414 mg/mL rutin standard solution, 10% aluminum nitrate, 5% sodium nitrite, and 10% sodium hydroxide solution were prepared in reagent bottles for use.

The standard curve was determined according to the methods outlined by Yang et al. (2020).

First, 1.0 mL 5% sodium nitrite solution was added to 1.0 mL of the precision suction sample solution, mixed, and then left for 6 min. Then, 1.0 mL of 10% aluminum nitrate solution was added, mixed, and left for 6 min before 10% sodium hydroxide solution was added and mixed. Absolute ethanol, as the blank, was then added until the solution reached a total volume of 25 mL. The solution was then mixed and colored for 30 min. Absorbance was measured at a wavelength of 503 nm. Then, the concentration of total flavonoids in the samples was calculated according to the standard curve equation, and parallel experiments were performed.

Determination of total phenol content

Folin-Ciocalteu (F-C) reagent: the dilution of the folin-phenol reagent was doubled.

Preparation of the standard curve: the standard curve was prepared according to the methods outlined by Li et al. (2007).

Test sample processing: 1.0 mL orange peel tea wine was added to a 50.0 mL volumetric flask with 49 mL of water and shaken well.

Sample determination: 1.0 mL sample solution was added to 5.0 mL water, 1.0 mL F-C chromogenic agent, and 3.0 mL 7.5% sodium carbonate solution, respectively, to develop color. The absorbance of the samples was measured at 760 nm wavelength after 2 h of storage. Then, the concentration of total phenols in the samples was calculated according to the standard curve equation, and parallel experiments were performed.

DPPH free radical scavenging assay

According to the methods of Wang et al. (2018) the orange peel tea wine was centrifuged at 12,000 r/min and 4 °C for 5 min, and the supernatant was taken for testing; 0.05 mg/mL 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical solution was configured. The sample was prepared and then left to react in the dark for 40 min. The absorbance was measured at 517 nm and calculated according to the following formula:

$$\mathrm{E}(\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H})(\mathrm{\%})=[1-({\mathrm{A}}_{\mathrm{I}}-{\mathrm{A}}_{\mathrm{I}\mathrm{I}})/{\mathrm{A}}_{\mathrm{I}\mathrm{I}\mathrm{I}}]\times 100$$

A_I: 2.0 mL of orange peel tea wine mixed with 2.0 mL of 0.05 mg/mL DPPH solution; A_II: 2.0 mL orange peel tea wine mixed with 2.0 mL absolute ethanol; A_III: 2.0 mL of 0.05 mg/mL DPPH mixed with 2.0 mL absolute ethanol.

·OH free radical scavenging assay

According to the methods of Xia, Shuang & Yang (2021) 6 mmol/LFeSO₄ solution, 6 mmol/L H₂O₂ solution, and 6 mmol/L salicylic acid solution were prepared. After preparing the sample, the reaction time was 10 min. Then, 2.0 mL of 6 mmol/L salicylic acid solution was added, the solution was shaken well, and its absorbance was measured at 510 nm after standing for 30 min.

$$\mathrm{E}(\cdot \mathrm{O}\mathrm{H})(\mathrm{\%})=[1-({\mathrm{A}}_{\mathrm{I}}-{\mathrm{A}}_{\mathrm{I}\mathrm{I}})/{\mathrm{A}}_{\mathrm{I}\mathrm{I}\mathrm{I}}]\times 100$$

A_I: 2.0 mL of 6 mmol/LFeSO₄ solution, 2.0 mL wine sample, 2.0 mL of 6 mmol/L H₂O₂ solution; A_II: 2.0 mL of 6 mmol/LFeSO₄ solution, 2.0 mL wine sample, 2.0 mL distilled water solution; A_III: 2.0 mL of 6 mmol/LFeSO₄ solution, 2.0 mL distilled water, 2.0 mL of 6 mmol/L H₂O₂ solution.

Sample preparation

A 5.0 mL sample of orange peel tea wine was moved to a 20 mL headspace bottle. Then, 2.0 g sodium chloride was added, and the mixture was shaken evenly. The wine sample was preheated at 60 °C for 5 min, the extraction head was inserted into the headspace port, and the adsorption was carried out at 60 °C for 30 min. The extraction head was then extracted and inserted into the GC injection port, and the thermal analysis was carried out at 220 °C for 5 min.

GC-MS conditions

The chromatographic conditions were HP-FFAP (30 m × 0.25 mm × 0.25 μm). The shunt mode was “no shunt” with a flow rate of 1.21 mL/min. The injection port temperature was 240 °C. To heat the sample, the temperature was kept at 40 °C for 3 min, then the sample was heated at 5 °C/min to 80 °C without holding, followed by 8 °C/min to 230 °C for 7 min (Feng et al., 2015).

Mass spectrometry conditions were as follows: ionization mode was set to “(EI) ionization source,” electron energy was 70 eV, interface temperature was 220 °C, and ion source temperature was 220 °C.

The effect of material volume ratio on the quality of orange peel tea wine

At 28 °C with 25% sucrose and 0.8% yeast, the volume ratio of orange peel juice to tea juice was tested at 1:5, 1:4, 1:3, 1:2, and 1:1. The sensory evaluation and the alcohol content evaluation of these ratios of orange peel juice to tea juice were performed after 7 d of fermentation.

As shown in Fig. 2, when the volume ratio of orange peel juice to tea juice was 1:3, the sensory score and alcohol content of orange peel tea wine were the highest. At this ratio, there was a good balance of tea aroma and orange aroma, the style of the tea wine was typical, and the tea wine was full-bodied. When the volume ratio of orange peel juice to tea juice was 1:5 and 1:4, the orange flavor was not obvious, the tea taste was rich and bitter, and the sensory score and alcohol level were both low. When the volume ratio of orange peel juice to tea juice was 1:2 and 1:1, the orange flavor was too rich, overpowering the tea flavor, the aroma was not balanced, and the sensory score was low. The results show that the quality of the orange peel tea wine was the highest with a volume ratio of orange peel juice to tea juice of 1:3.

Influence of sucrose content on the quality of orange peel tea wine

At 28 °C, with a volume ratio of orange peel juice and tea juice set to 1:3, and 0.8% yeast addition, the amount of sucrose added was tested at 15%, 20%, 25%, 30%, 35%, and 40%. The sensory evaluation and the alcohol content evaluation of the orange peel tea wine with these sucrose addition amounts were performed after 7 d of fermentation.

As shown in Fig. 3, as the amount of sucrose added increased, both the sensory score and alcohol content increased, which may be because an appropriate amount of sucrose can reduce the astringency of tea. Sucrose is also conducive to the production of alcohol by yeast. However, excessive sucrose made the orange peel tea wine too sweet and inhibited the activity of yeast, causing both the sensory score and alcohol content to decline. Excessive sucrose is also not conducive to the preservation of orange peel tea wine (Han et al., 2018; Jia et al., 2023). The results show that the quality of orange peel tea wine was the highest when sucrose content was 30%.

Influence of fermentation temperature on the quality of orange peel tea wine

When the volume ratio of orange peel juice and tea juice was 1:3, the added amount of yeast was 0.8%, and the added amount of sucrose was 25%, the fermentation temperatures tested were 20 °C, 24 °C, 28 °C, 32 °C, and 36 °C. The sensory evaluation and the alcohol content evaluation of the orange peel tea wine were performed after 7 d of fermentation at the tested temperatures.

As shown in Fig. 4, alcohol content and sensory scores first increased and then decreased as fermentation temperature increased. Alcohol and sensory scores were highest at a fermentation temperature of 28 °C. When the temperature was too low (20 °C), yeast growth was slow, stunting the production of alcohol. When the temperature was too high (32 °C and 36 °C), lactic acid bacteria, acetic acid bacteria, and other microorganisms began to multiply, resulting in a sour orange peel tea wine taste (Liu et al., 2022a). The results show that the quality of orange peel tea wine was the highest when the fermentation temperature was 28 °C.

Influence of fermentation time on the quality of orange peel tea wine

When the volume ratio of orange peel juice to tea juice was 1:3, the added amount of yeast was set to 0.8%, the added amount of sucrose was 25%, and the fermentation temperature was set to 28 °C, the fermentation times tested were 3 d, 5 d, 7 d, 9 d, 11 d, 13 d, 15 d, 17 d, and 19 d, after which the alcohol content and sensory evaluations were conducted.

As shown in Fig. 5, as fermentation time increased, alcohol content also increased before plateauing, and sensory scores first increased and then decreased. After 7 d of fermentation, sensory scores were the highest, with the wine having both a full taste and body. As fermentation time continued to increase, the wine took on a sour and astringent taste, and the sensory score decreased. The results show that the quality of fermented orange peel tea wine was the highest when the fermentation time was 7 d (Liu et al., 2022b).

Influence of yeast addition amount on the quality of orange peel tea wine

When the volume ratio of orange peel juice and tea juice was 1:3, the added amount of sucrose was 25%, and the fermentation temperature was 28 °C, the amount of yeast added was tested at 0.8%, 1.6%, 2.4%, 3.2%, 4.0%, 4.8%, 5.6%, and 6.4%. The sensory evaluation and the alcohol content evaluation of the orange peel tea wine with these sucrose addition amounts were conducted after 7 d of fermentation.

As shown in Fig. 6, as the amount of added yeast increased, the alcohol and sensory scores also increased. When the yeast amount was 4.8%, sensory scores and alcohol scores were the highest. When the added amount of yeast was less than 4.8%, there was not enough yeast to make full use of sugar to produce alcohol, resulting in high residual sugar content and low alcohol content. When the added amount of yeast was more than 4.8%, the added amount was too high, resulting in rapid consumption of nutrients and metabolic by-products, decreasing yeast metabolism and thus also decreasing alcohol content. Sensory scores also decreased with added yeast amounts higher than 4.8% (Novo et al., 2014). The results show the quality of orange peel tea wine was the highest when the yeast content was 4.8%.

Response surface results of orange peel tea wine process optimization

After single factor testing, fermentation temperature (A), yeast content (B), and sucrose content (C) were selected to optimize the response surface of the brewing process of orange peel tea wine. The results of the response surface experiment are shown in Table 3, and the analysis of variance of the response surface experiment is shown in Table 4.

Interaction analysis of influencing factors

As shown in Table 4, the F value of the quadratic regression model constructed by the response surface method was 60.64, and the effect was extremely significant (P < 0.0001). The lack of fit P-value was 0.2255, and the effect was not significant (P = 0.2255 < 0.05). These results indicate that all test sites could be described by this model. The determination coefficient R² = 0.9873 and the adjusted determination coefficient R_Adj² = 0.9711 prove that the model had good reliability, and the regression model could predict the response value well. The primary term, A, and its secondary terms A², B², and C² are highly significant (P < 0.01); primary items B and C and interaction item BC were all significant (P < 0.05).

On the response surface, maximum values can be observed, indicating that the selected factor levels are reasonable. The contours of fermentation temperature (A) and yeast addition (B), and fermentation temperature (A) and sucrose addition (C) were approximately circular, indicating that the effect on the alcoholic strength of orange peel tea wine was not significant (Fig. 7). Whereas, the contours between yeast addition (B) and sucrose addition (C) were elliptical, indicating that these two factors had significant effects on the alcohol content of orange peel tea wine. According to the response surface software analysis, the optimal fermentation conditions for the brewing process of orange peel tea wine were as follows: fermentation temperature 28.82 °C, yeast content 4.92%, and sucrose content 28.58%. For simplicity in production, these conditions were appropriately modified to a fermentation temperature of 29 °C, a yeast content of 4.9%, and a sucrose content of 29%. Under these fermentation conditions, three flat tests were conducted, and the average alcohol content of orange peel tea wine was 12.56% vol, which was only 0.1% vol different from the theoretical predicted value, which verified that the response surface test data were accurate and reliable.