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. 2020 May 22;58(1):216–222. doi: 10.1007/s13197-020-04531-1

Pasteurization of flavored shredded pork using Zno nanoparticles combined with radio frequency pasteurization technology

Jicheng Xu 1,2, Min Zhang 1,3,6,✉, Ping Cao 4, Benu Adhikari 5
PMCID: PMC7813921  PMID: 33505066

Abstract

Effects of ZnO nanoparticles combined radio frequency (ZNCRF) pasteurization on the survival of microorganism, flavor and taste of Flavored shredded pork were compared with conventional high pressure steam (HPS) sterilization. The results showed that ZNCRF pasteurization was better than HPS sterilization in terms of flavor and taste parameters and at the same time met the pasteurization requirement. GC–MS and NMR measurements were performed to explore changes in volatile compounds and status and distribution of water within the sample. The ZNCRF pasteurization when carried out for 30 min and the HPS sterilization reduced the relative contents of aldehydes by 18.8% and 19.7%, respectively, while the ZNCRF pasteurization within 20 min had less effect on aldehydes. Both ZNCRF pasteurization and HPS sterilization destroyed the vacuolar membrane of the samples caused the loss of water from the cytoplasm (T23). This work shows that ZNCRF when applied for 20 min is a mild pasteurization method that can be applied to improve the quality of Flavored shredded pork.

Keywords: ZnO nanoparticles, Radio frequency pasteurization, Flavored shredded pork, E-tongue

Introduction

Flavored shredded pork is a typical representative of traditional Chinese cuisine. It is famous for its unique flavor and taste. This dish is traditionally prepared in small scale, such as in homes and restaurants and is popular with domestic and overseas consumers. Currently, the Flavored shredded pork is marketed in simple vacuum packs and its shelf life is quite short, hence, pasteurization and/or sterilization is needed to extend its shelf life (Min et al. 2005; Wang et al. 2010). Currently, the commercialized Flavored shredded pork is autoclaved to sterilize. Because of the high temperature prevailing in high pressure steam (HPS) sterilization, the sensory quality of the sterilized product is greatly compromised (Liu et al. 2014; Suklim et al. 2014). In order to better maintain the product quality (flavor and taste) and nutritional properties, it is essential to find a pasteurization technology that is suitable for Flavored shredded pork.

Antibacterial agents obtained from organic and inorganic sources are commonly used in food industry. Organic antibacterial agents are very sensitive to heat and are easily destroyed during the manufacturing process, thus readily lose their bactericidal effect. Inorganic antibacterial agents are less sensitive to heat and maintain good antibacterial properties during food processing (Zhang et al. 2009; Suo et al. 2017; Tsagkaris et al. 2018). Nano zinc oxide is one of the most studied and used inorganic antimicrobial agents. Nano zinc oxide has good antibacterial activity against both Gram-positive and Gram-negative bacteria (Chau et al. 2007; Zhang et al. 2017). Furthermore, nano zinc oxide is known to possess good immunoregulatory function and high bioactivity (Sawai 2003; Omri et al. 2017). Studies have found that zinc oxide nanoparticle possess antimicrobial properties and are able to kill and inhibit certain pathogens in both food and pharmaceutical products (Espitia et al. 2012; Mirhosseini and Firouzabadi 2013). Referring to the standards of food additives (GB 2760-2014) and the effects of preliminary experiment, ZnO nanoparticles suspension of mass fraction 0.04 g/kg would meet standard requirements and also have the best bacteriostatic effect in the dish.

Radio frequency (RF) is an electromagnetic wave with frequency ranging from 300 kHz to 300 MHz. The RF irradiation heats materials and due to long wavelength (1000–1 m) and it has a high penetration depth (Mohamad et al. 2018; Jiao et al. 2019; Zhang et al. 2019). In contrast to conventional heating systems, the advantage of electromagnetic heating systems including RF is that heat is generated internally; hence, the temperature distribution within the material is more uniform (Duan et al. 2005; Song et al. 2009; Wang et al. 2009). When RF is used for pasteurization, it imparts both thermal and non-thermal effects. The thermal effect causes denaturation of bacterial protein. The non-thermal effect interferes with bacterial cell membrane and its transport mechanism leading to microbial inactivation and death (Luechapattanaporn et al. 2005; Yan et al. 2010). RF pasteurization is increasingly applied in food systems including vegetables and meat (Ling et al. 2018; Jiang et al. 2019). Byrne et al. (2010) designed a RF-based pasteurizing unit to pasteurize pork luncheon meat and showed that the efficacy of RF-based pasteurization depended on the product formulation.

RF pasteurization can give rise to cold spots which can result into uneven or insufficient pasteurization; thus, incorporation of small amount of ZnO nanoparticles can increase the bactericidal effect without causing thermal damage to the samples. Combination of RF heating with ZnO nanoparticles can impart synergistic pasteurizing effect and reduce the intensity of thermal treatment during RF pasteurization (Matan et al. 2015). Thus, ZNCRF pasteurization could be an innovative and mild pasteurization technology that can be readily applied in food and pharmaceutical industries.

In the above context, the aim of this research was to determine the effectiveness of ZNCRF pasteurization applied to Flavored shredded pork in terms of minimizing the loss of flavor and taste and shortening the pasteurization time.

Materials and methods

Raw materials

Pork, green pepper, mushroom, carrot and bamboo shoots were purchased from the Yonghui supermarket (Wuxi, China). ZnO nanoparticles were donated to this project by Yiqiao commerce and trade Co., Ltd. (Shanghai, China). One hundred g pork, 20 g green pepper, 20 g mushrooms, 30 g carrot and 30 g bamboo shoots were cleaned and cut into desired size. These ingredients were combined prepare Flavored shreded pork according to the traditional Chinese menu. Once cooked, the Flavored shreded pork samples were cooled to room temperature which took about 30 min. A suspension of ZnO nanoparticles (10 mL, 0.04 g/kg) was prepared and uniformly mixed with the Flavored shreded pork sample. The mixed (Flavored shreded pork + ZnO nanoparticles) sample was vacuum (0.01 kPa) packed in retort pouches (Polyamide, Blueberry Co., Ltd., China.) and stored in a refrigerator at 4 °C.

Pasteurization of flavored shredded pork using ZNCRF

Pasteurization of Flavored shredded pork samples was carried out using a RF equipment (6 kW, 27 MHz, Strayfield Co. Ltd., Berkshire, UK). The space between two RF plates was kept at 20 mm. The samples were then pasteurized for 10, 20 and 30 min. The temperature during these ZNCRF pasteurization tests was maintained at 75–78 °C. In addition, the pork samples were also sterilized using HPS (121 °C, 30 min) for comparison.

Measurement of temperature uniformity

An infrared thermal imager (IRISYS 4000, Northampton, UK) was used to measure the temperature uniformity of Flavored shredded pork samples (Xu et al. 2018). After RF pasteurization, the Flavored shredded pork samples were taken out from the RF chamber at 10 min, 20 min and 30 min. The temperature uniformity of samples was scanned immediately using the camera. The distance from the sample to the camera is 0.8 m. The whole measurement should be done within 3 s. The software associated with the camera was used to process measurement data.

Enumeration of colony forming units (CFU)

The CFU of the Flavored shreded pork samples were determined by aerobic plate counting method (Awuah et al. 2005). For this, 225 mL of sterile saline was added in a 500 mL Erlenmeyer flask and then 25 g of Flavored shreded pork was pulverized and loaded (tenfold dilution). The Erlenmeyer flask was homogenized with a homogenizer (SH-1, Nanjing Boda Co., Ltd.) for 3 min. After the sample was homogenized, a further tenfold dilution was performed. For the assay, 1 mL of the diluted sample was transferred to a Petri dish with a pipette. Nutrient agar (46 °C, 12–15 mL) was added into the Petri dish and carefully mixed with the homogenized and diluted sample. Once the nutrient agar was solidified the Petri dish was inverted and incubated at 37 °C for 48 h in an incubator. This entire process was performed in triplicate.

Measurement of volatile compounds

A gas phase chromatography—mass spectrometer was used to measure the volatile aroma compounds of Flavored shredded pork samples (Xu et al. 2018). About 50 g of Flavored shredded pork sample was crushed in a blender. The crushed sample (3 g) was placed in a 20 mL headspace extraction vial. Then 3 mL of physiological saline was added. The head of extraction cartridge was inserted into the headspace extraction flask and the extraction was carried out at 70 °C for 20 min. Once the extraction was complete, the head of the extraction cartridge was inserted into a GC–MS injector for desorption for 5 min.

Qualitative analysis of volatile components of Flavored shredded pork was performed with the aid of computerized database. Each peak in the assay results was matched with the NIST library and the Wiley library, and those with a matching degree greater than 800 were selected for qualitative analysis. Quantitative analysis of volatile components of the samples was carried out with the aid of an internal standard (4-Methyl-1-pentanol). The peak area of volatile compounds detected in the Flavored shredded pork samples was normalized to the peak area of 4-Methyl-1-pentanol, and the relative concentration of volatile organic compounds was calculated.

Measurement of taste parameters

The taste parameters in Flavored shredded pork samples were measured by a taste sensing instrument (SA402; Intelligent Sensor Co. Ltd., Kanagawa, Japan). For this test, 50 g sample and 200 mL pure water were put into a food blender and blended for 1 min to extract the taste (gustation) parameters. Then, this blended sample was centrifuged for 10 min at 3,000 rpm in a refrigerated centrifuge. After the centrifugation, the supernatant was taken out and used as a measurement sample.

Seventy mL of the supernatant was transferred into test cups (30 mm diameter and 35 mm height) to determine the taste parameters. Four samples were used and each sample was measured/analyzed at least 3 times for analysis. All sensors were automatically cleaned in the reference solution before each measurement. The sensor types of Taste Sensing instrument included include bitterness, sourness, astringency, after-taste-A, aftertaste-B, richness, umami, and saltiness.

Low field nuclear magnetic resonance (LF-NMR) measurements

The LF-NMR tests were performed using a low field pulse NMR analyzer (MicroMR20, Niumag Co. Ltd., Shanghai, China). Approximately 2 g of Flavored shredded pork sample was put into a test tube (diameter 2.5 cm, height 15 cm) and inserted into the NMR coil. Carr–Purcell–Meiboom–Gill (CPMG) sequence was selected to determine spin–spin relaxation time (T2). Each sample was repeated three times (Cheng et al. 2014).

Statistical analysis

SPSS 2015 software (IBM, Chicago, IL, USA) was used to perform statistical analysis of variance (ANOVA) of different samples. Tukey’s test procedure, at 95% confidence level (P < 0.05) was used to determine the significant difference between two mean values.

Results and discussion

Temperature uniformity of Flavored shredded pork under different ZNCRF heating time

Figure 1 was a temperature uniformity schematic diagram of infrared photographic measurement of Flavored shredded pork. From Fig. 1, we could see that in the pasteurization process of ZNCRF, the temperature distribution of Flavored shredded pork in the cooking bag was uniformity which contributed to the germicidal effect. Outside the retort pouch, the temperature suddenly dropped, indicating that the retort pouch with strong insulation properties minimized temperature non-uniformity during ZNCRF pasteurization. The above results showed that when ZNCRF was used for the pasteurization of Flavored shredded pork, the temperature distribution is uniform and a good pasteurization effect can be ensured.

Fig. 1.

Fig. 1

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Temperature uniformity in Flavored shredded pork samples as a function of ZNCRF pasteurization

Effect of ZNCRF heating time on bacterial death

Figure 2 shows the survival or death chart (CFU of samples) and indicates that bacterial survival decreased as the time of exposure during ZNCRF pasteurization increased up to 20 min beyond which (e.g., 30 min) there was no further decrease. The CFU of samples subjected to ZNCRF pasteurization for 20 min decreased significantly (P < 0.05) compared to that of control samples. No CFU was detected in HPS sterilized samples indicating that it achieved an effective sterilization. These results showed that the ZNCRF pasteurization carried out for 20 min met the requirements of commercial pasteurization (log CFU < 3) to maintain a shelf life of 6 months. Masood et al. (2017) reported a design and construction of a RF pasteurization instrument with more uniform electric field (Steinmetz treatment chamber) to inactive Escherichia coli and achieved slightly higher (3.6 log CFU reduction) inactivation level.

Fig. 2.

Fig. 2

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The colony forming units (CFUs) during ZnO nanoparticles combined radio frequency (ZNCRF) pasteurization and high pressure steam (HPS) pasteurization. a Control samples, b ZNCRF pasteurization for 10 min, c ZNCRF pasteurization for 20 min, d ZNCRF pasteurization for 30 min, e HPS sterilization samples

Volatile organic compounds change of Flavored shredded pork

Table 1 shows the types and relative content of volatile organic compounds in Flavored shredded pork. During ZNCRF pasteurization and HPS sterilization, the aldehyde contents decreased from 38.37 to 28.41%. Similarly, the heterocycle content decreased from 34.36 to 15.25%. At the same time, the hydrocarbon content increased from 5.36 to 32.18%. This level of change in the volatile organic compounds during ZNCRF pasteurization and HPS sterilization is not expected to alter the perception of aroma in Flavored shredded pork. This is because aldehydes that predominantly impart fatty aroma have a very low threshold, even at low concentration they provide sufficient meaty flavor (Mottram 1998). Heterocycles are more intimately associated with flavor of Flavored shredded pork, while hydrocarbons contribute less to its flavor.

Table 1.

Type and relative content of volatile organic compounds in ZNCRF pasteurized and HPS sterilized Flavored shredded pork

Control%
(83)		 RF 10 min%
(77)		 RF 20 min%
(79)		 RF 30 min%
(85)		 HPS%
(81)
Aldehydes

35.37 ± 2.36a

(13)

34.58 ± 1.89a

(11)

32.66 ± 1.51a

(12)

28.72 ± 1.08b

(13)

28.41 ± 1.14b

(13)
Heterocycles

34.36 ± 2.96a

(16)

31.83 ± 2.85a

(15)

31.67 ± 2.73a

(16)

22.30 ± 1.66b

(16)

15.25 ± 1.30c

(16)
Esters

3.75 ± 0.18a

(12)

2.76 ± 0.09b

(13)

3.83 ± 0.21a

(12)

3.79 ± 0.16a

(13)

3.95 ± 0.16a

(11)
Alcohols

9.53 ± 0.38a

(22)

9.67 ± 0.36a

(19)

9.68 ± 0.41a

(20)

10.19 ± 0.35a

(21)

10.56 ± 0.29a

(21)
Ketones

8.63 ± 0.45a

(8)

8.12 ± 0.58a

(7)

8.26 ± 0.67a

(8)

8.38 ± 0.49a

(9)

9.65 ± 0.62a

(8)
Hydrocarbon

8.36 ± 0.56a

(12)

13.04 ± 0.82b

(12)

13.90 ± 0.71b

(11)

26.62 ± 1.95c

(13)

32.18 ± 2.33d

(12)
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Results are mean ± standard deviation. Different lowercase letters in the same row express the significant differences (P < 0.05). Numbers in brackets are number of different (types) of volatile organic compounds of Flavored shredded pork

The ZNCRF pasteurization of 30 min HPS sterilization reduced the contents of aldehydes by 18.8% and 19.68%, respectively. The heterocycles contents were reduced by 35.1% and 55.62%, respectively. However, ZNCRF pasteurization within 20 min had less reducing effect on aldehydes and heterocycles. These observations show that ZNCRF pasteurization (within 20 min) only mildly affects the volatile aroma compounds and is a suitable method to pasteurize Flavored shredded pork.

Change in taste parameters of flavored shredded pork

Sensory evaluation based on intuition comes with shortcomings of poor repeatability, strong subjectivity and difficulty of quantifying/interpreting the data. Thus, instrument based sensory evaluation is increasingly used in food industry. The electronic tongue, which simulates the human taste sensation, is fast and reliable method to determine taste sensation (Woertz et al. 2010). Electronic tongue system detects various tastes such as umami, saltiness, sourness, and astringency by detecting the quantitative relationship between taste substances and human taste sensations (Eckert et al. 2011). The error rate in electronic tongue tests refers to the impact of different extraction and sampling protocols on the determination taste. Generally speaking, when the error rate does not exceed 50%, the sensor can effectively distinguish the taste index in the sample (Itoyama et al. 2015).

According the results of error rate, taste response of Flavored shredded pork samples measured using electronic tongue instrument is showed in Fig. 3. As can be observed, the saltiness gradually increased and sourness/bitterness/astringency showed a slow downward trend throughout the pasteurization process. The increased saltiness may be due to the increase infusion or diffusion of sodium chloride into the meat during pasteurization. The slow decline in sourness/bitterness/astringency demonstrated that the compounds that impart these taste sensations were gradually decreased compared that in control as the treatment intensity or exposure time increased. These taste sensation data also indicate that ZNCRF pasteurization is capable of preserving taste quality of Flavored shredded pork compared to HPS sterilization.

Fig. 3.

Fig. 3

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Electronic tongue taste response of Flavored shredded pork samples pasteurized using ZnO nanoparticles combined radio frequency (ZNCRF) system and sterilized using high pressure steam (HPS). a Control samples, b ZNCRF pasteurization for 10 min, c ZNCRF pasteurization for 20 min, d ZNCRF pasteurization for 30 min, e HPS sterilization samples

Water status and distribution in Flavored shredded pork

Figure 4 shows that the status or organization of water molecules (T2 curves) in the Flavored shredded pork. It shows four characteristic peaks which correspond to three different status of water: monolayer, multilayer water and free water, respectively. The first and second peaks (T21) represents the monolayer water bound to the cell wall polysaccharides (pectin, cellulose, and hemicellulose) by strong hydrogen bonds. The third peak (T22) represents the multilayered water, as in cytoplasm. The fourth peak (T23) represents free water molecules weakly bound to the sugars and small molecular substances located within the vacuole (Vicente et al. 2011; Lagnika et al. 2013). The larger the T2 curves, the greater the degree of freedom of hydrogen protons: a phenomenon that can be used to distinguish between different status of water (Doona and Baik 2007). As can be seen from Fig. 4, the T2 curves of the samples subjected to ZNCRF pasteurization for 10 and 20 min are similar to that of the control. The T2 curve of samples pasteurized by ZNCRF for 30 min and sterilized by HPS samples moved towards the left of the abscissa. These observations indicated that the change in status of water due to ZNCRF pasteurization for 30 min or HPS sterilization is caused by destruction of hydrogen bonds.

Fig. 4.

Fig. 4

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Typical distribution of T2 relaxation of Flavored shredded pork samples subjected to ZnO nanoparticles combined radio frequency (ZNCRF) pasteurization and high pressure steam (HPS) sterlization

Figure 5 shows that the values of T22 (multilayer water) of samples decreased gradually from ZNCRF 10 min to 30 min. The values of T22 between ZNCRF 10 min and HPS samples were not significantly different. The total water content in samples subjected to ZNCRF pasteurization for 10 min and 20 min was higher than those subjected to ZNCRF pasteurization for 30 min and HPS sterilization. These observations indicate that heating to varying degrees destroyed the cellular structure and which resulted in the loss of water in the cytoplasm (T22). The loss of water in the cytoplasm caused by ZNCRF 30 min and HPS was significantly greater than the loss caused by ZNCRF when carried out for 10 and 20 min (P < 0.05).

Fig. 5.

Fig. 5

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Water status of Flavored shredded pork subjected to ZnO nanoparticles combined radio frequency (ZNCRF) pasteurization and high pressure steam (HPS) sterilization

Conclusion

A well-known Chinese traditional dish, Flavored shredded pork, was pasteurized using an innovative ZnO nanoparticles combined radio frequency (ZNCRF) pasteurization technology. It pasteurization efficacy and ability to preserve the quality attributes of Flavored shredded pork were compared with those of high pressure steam (HPS) sterilization. It was found that the flavor and taste of Flavored shredded pork samples were significantly better preserved by ZNCRF pasteurization carried out for 20 min than by HPS sterilization, although the sterilization effect of HPS was better than that of ZNCRF. The ZNCRF pasteurization carried out for 20 min met the pasteurization requirement for Flavored shredded pork. The retention of characteristic taste, volatile components and preservation of status and distribution of water in Flavored shredded pork were better preserved by ZNCRF pasteurization (20 min) Flavored shredded than by HPS sterilization.

Acknowledgements

The authors acknowledged the support by National Key R&D Program of China (Contract No. 2017YFD0400501). The 111 Project(BP0719028),The research was also supported by Jiangsu Province Key Laboratory Project of Advanced Food Manufacturing Equipment and Technology (No. FMZ201803), the National First-class Discipline Program of Food Science and Technology (No. JUFSTR20180205) and the Open Project Program of State Key Laboratory of Food Science and Technology, Jiangnan University (No. SKLF-KF-201915).

Footnotes

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