Black Tea Extract on the Preservation of Cantonese Sausage

Corresponding Author: Dan Wu College of Biosystems and Food Science, Zhejiang University, Hangzhou, China Email: wudan2008@zju.edu.cn Abstract: Black Tea Extract (BTE) is a natural pigment with strong antioxidant activity. Its effect on the preservation of Cantonese sausage was investigated based on polyphenol components analysis in this study. The content of tea polyphenols in BTE was 26.61% by using spectrophotometry, the tea catechin in BTE was analyzed by HPLC, which included gallic acid (0.27%), caffeine (5.75%) and 8 catechins (0.87% Catechin, 1.66% epicatechin, 0.34% catechin gallate, 2.54% epicatechin gallate, 1.88% gallocatechin, 5.31% epigallocatechin, 2.69% gallocatechin gallate, 10.23% epigallocatechin gallate). The tea sausages of five treatments with the addition of 0.05, 0.10, 0.15, 0.20 and 0.25% BTE were designed, compared to the control and the sausage with the addition of 0.02% sodium Nitrite (NIT) and the addition of 0.01% NIT and 0.01% BTE. The acid value, peroxide value and sensory indices were analyzed and the shelf-life of the sausage was predicted by peroxide value. The sausage with 0.15% BTE had significant improved the product quality by reducing the acid value and lipid oxidation value. The nitrite and BTE had synergistic inhibition of acid value increase. The BTE had the positive effect on the color of the freshly prepared sausage, but this effect was not obvious during the storage. The sensory value of the control sausage was decreased by 42.12% when stored for 30 days, while sausages added with BTE was decreased by only about 13.34%. BTE showed more potential in Cantonese sausage preservation and the sausage with more than 0.15% addition of BTE had twice shelf-life (40°C) more than that of 0.02% sodium nitrite sausage and five times more than that of control group. Adding BTE in sausage could be a good potential for reducing the usage of nitrite in meat products.


Introduction
Nitrite is an important food additive in the processing of meat products due to its excellent antibacterial and antioxidant effects (Majou and Christieans, 2018;Berardo et al., 2016). It can prevent the lipid oxidation of sausage. However, excessive consumption of nitrite will cause adversary health effects such as carcinogenesis, teratogenesis and mutagenicity. It is necessary to search for a natural extract to replace nitrite partly or completely in sausage for food safety consideration.
Tea polyphenols may be the main antioxidant components of the Black Tea Extract (BTE), which was related to antioxidant, antimicrobial activity, anti-cancer etc.. The proportion of different components in tea polyphenols may affect its biological activity. There are few report about BTE effect on sausage with its tea polyphenols detailed components.
In this study, the total tea polyphenols in BTE was detected by spectrophotometry, the tea catechin in BTE was analyzed by HPLC. Cantonese sausages of five treatments with different BTE dosages were prepared. The acid value, peroxide value and sensory quality of the tea sausages were determined, compared to the control, the sausage with the addition of sodium Nitrite (NIT) and the addition of NIT and BTE. The shelf-life of the sausage was predicted and the preservation effect of BTE on Cantonese sausage was evaluated.
Preparation of Cantonese sausage: The raw materials of sausage are from the local market. 60.61% port lean and 25.97% fat were cut into a strip about 1 cm square and mixed with 1.73% salt, 2.16% rice wine, 4.33% soy sauce and 5.19% sugar. After curing for 30 min, the meat mixtures were stuffed into casings. Raw sausages were tied into knots every 15 cm and prick 3-5 pinhole every 1 cm. They were dried at 60℃ for 48 h and flipped every 6 h. The mature sausages were stored at 40℃ for 36 days in an incubator.
Eight treatments for pork sausages: Control (basic formulation); basic formulation with the addition of 0.05, 0.10, 0.15, 0.20 and 0.25% BTE; basic formulation with the addition of 0.02% sodium Nitrite (NIT) and basic formulation with the addition of 0.01% NIT and 0.01% BTE.

Physicochemical Properties Analysis
Lipid oxidation and the acid value were measured in triplicate according to the national standard GB5009 (Ministry of Health of China, 2016). Color analysis was carried out in triplicate using a portable spectrophotometer (HunterLab ColorFlex EZ, USA), by measuring a* (red intensity) values of the CIE system.

Prediction of Shelf-Life
The peroxide value was detected every 6-8 days and the prediction of sausages shelf-life at 40℃ was studied. The Arrhenius equation was used to the prediction of sausages shelf-life.

Sensory Evaluation
A panel of 10 assessors aged from 22 to 40 years was trained in descriptive evaluation of sausages. An affective acceptance test was carried out by using a 9point scale (Baldin et al., 2016).

Data Analysis
Objective measurements were replicated triplicate. The experimental design of lipid oxidation, the acid value, color value and sensory evaluation were by using Complete Randomized Design (CRD). Data Processing System (DPS) software v18.10 (Tang and Zhang, 2013) was applied to analyze the data. Means were compared using Duncan's Multiple Range Test. Differences at P<0.05 were considered significant.

The Tea Polyphenol in BTE
The polyphenol content of BTE was 26.61%. Gallic acid, caffeine and 8 catechins were identified by HPLC and the results showed that the EGCG content was the highest (Table 1). The order of polyphenol content was: EGCG> EGC> GCG> ECG>GC>EC>C>CG.
The tea polyphenol has showed antioxidant and antimicrobial activity. Wenjiao et al. (2014) reported that it had positive effect on sausage quality. The BTE used in the experiment had more EGCG percentage content than the tea samples reported (Nian et al., 2019). The polyphenol components of different kind of tea is very different (Nian et al., 2019;Wang et al., 2010).

Physicochemical Properties
The changes of acid value of sausage during the storage are showed in Fig. 1. There was no significant difference among the treatments at first. The acid value of all treatments increased with time. The higher concentration of BTE added, the slower the increase of the acid value. When the concentration of BTE reached 0.15%, statistical analysis showed that the acid value in the sausage was significantly lower compared to the control (P<0.05). The samples with addition of 0.02% NIT and the addition of 0.01% NIT and 0.1% BTE showed positive effect on the acid value too (P<0.05). The higher acidity means lower pH. This study was in good agreement those of reported by (Jayawardana et al., 2019;Lorenzo et al., 2014). The change of the acid value might due to the fat and protein breakdown by the bacterial activity. These results indicated that nitrite and black tea extract had synergistic inhibition of acid value increase.
The changes of lipid oxidation of sausage during the storage are showed in Fig. 2. The lipid oxidation of all treatments were increased with time and the peroxide value of the control group had the fastest rising rate and exceeded the national limit (show the limit value here) within 36 days, while the sausages with different concentrations of BTE and nitrite were still within the national limit. Different additives have different effects on the initial peroxide values of the sausages. This result conformed to the study by (Jayawardana et al., 2019). The addition of BTE and NIT have inhibitory effect on the lipid peroxide value. The higher concentration of BTE added, the slower the increase of the peroxide value. The ratio of the final to initial peroxide value on the 36th storage day were 29.20 (control), 3.90 (0.05% BTE), 2.38 (0.10% BTE), 1.89 (0.15% BTE), 1.82 (0.20% BTE), 1.88 (0.25% BTE), 4.42 (0.02%NIT), 3.61 (0.01%NIT+0.10%BTE) respectively. When the concentration of the BTE was increased to 0.15%, the inhibitory effect of the BTE on the peroxide value tended to be stable. Jongberg et al. (2013;Jayawardana et al., 2019) also observed the green tea and black tea extract could inhibit TBARS values increasing of sausage. Furthermore, (Jayawardana et al., 2019) showed that the sausage of 0.2% and 0.3% BTE addition had significantly lower TBARS compared to the 0.1% BHT sample. In this study, the lipid oxidation inhibitory had no significant different (P<0.05) between the samples of BTE (0.20% or 0.25%) and NIT (0.02%) and between the samples of BTE (0.20% or 0.25%) and NIT (0.01%) with BTE (0.01%) on the 36 th day. The lipid oxidation reduction might be attributed to the polyphenols in BTE (Jukić et al., 2015;Predescu et al., 2018), as detected above. Figure 3 shows the a* values of the CIE system of sausage. During the storage period, the chroma value first decreased and then increased for all samples. On the 16th day, there were no significant different between the control sample and the 0.02% NIT sample, but after that, the a* value of 0.02% NIT samples increased faster than the control samples and there were significant different (ρ<0.05) between them. This may be related to the coloration mechanism of meat products (Hu, 2016).

Prediction of Sausages Shelf-Life
According to the Arrhenius empirical formula and based on the lipid peroxidation value, the first-order dynamic equation:

Ln Lipid oxidation value a time b   
where, a is the reaction rate constant, b is natural logarithm of the initial peroxide value. By detemining the peroxide value of sausage at different times, the prediction of sausages shelf life at 40℃ under different treatment conditions can be calculated ( Table 2).
The shelf life of the control group was predicted to be 43 days (Table 2), while the sausage with 0.05% BTE had a shelf life of 117 days, which was longer than the sausages with 0.02% nitrite (107 days). The shelf life of sausage with more than 0.15% BTE had more than 240 days of predicted shelf life, which was more than twice to the shelf life of 0.02% NIT sample and five times the shelf life of the control. Therefore, the Cantonese sausage with the addition of BTE only was an acceptable meat product, in terms of shelf life.

Sensory Evaluation
Sensory evaluation data of the samples are presented in Fig. 4. The sensory value of the control sausage was decreased by 42.12% when stored for 30 days, while sausages added with BTE was decreased by about 13.34% only. Statistical analysis indicated that there is no significant different (P<0.05) among the sausage of the addition of 0.15, 0.20, 0.25% BTE, 0.02% NIT and 0.01% NIT with 0.10% BTE on the 15th day and among the sausage of the addition of 0.20% BTE, 0.02% NIT and 0.01% NIT with 0.10% BTE on the 30th day. The sausages added BTE showed less rancidity after 4 weeks' storage compared to the control group. BTE with strong antioxidant and antimicrobial properties might have affected the rate of sausage spoilage processes (Almajano et al., 2008;Chen and Ho, 1995;Friedman, 2007). The concentrations of BTE or NIT have an important impact on sensory acceptance of the Cantonese sausages.      control, the sausage with 0.15% BTE had significantly improved the product quality by reducing the acid value and lipid oxidation value. The nitrite and BTE had synergistic inhibition of acid value increase. When the BTE concentration increased to 0.15%, the inhibitory effect on the peroxide value tended to be stable. The BTE had the positive effect on the color of the freshly prepared sausage, but this effect was not obvious during the storage. The sausages added BTE showed less rancidity after 4 weeks' storage compared to the control group. The sensory value of the control sausage was decreased by 42.12% when stored for 30 days, while sausages added with BTE was decreased by about 13.34% only. Sensory evaluation had no significant different (ρ<0.05) among the sausage of the addition of 0.20, 0.25 BTE, 0.02% NIT on the 15th and 30th day. The shelf life of sausage with more than 0.15% BTE had longer predicted shelf life, which was more than twice to that of 0.02% NIT and five times to that of the control. The development of nitrite free sausage with BTE could be promising in the future processed meat product market.