‘This is the peer reviewed version of the following article:
Cheng, J.-R., Liu, X.-M., Zhang, W., Chen, Z.-Y., & Wang, X.- P. (2018). Stability of phenolic compounds and antioxidant capacity of concentrated mulberry juice-enriched dried- minced pork slices during preparation and storage. Food Control, 89, 187–195. https://doi.org/10.1016/
j.foodcont.2018.02.008
which has been published in final form at https://doi.org/10.1016/j.foodcont.2018.02.008
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Stability of phenolic compounds and antioxidant capacity of concentrated mulberry juice-enriched dried-minced pork slices during preparation and storage
Jing-Rong Cheng, Xue-Ming Liu, Wei Zhang, Zhi-Yi Chen, Xu-Ping Wang
PII: S0956-7135(18)30054-9
DOI: 10.1016/j.foodcont.2018.02.008
Reference: JFCO 5971
To appear in: Food Control
Received Date: 26 August 2017 Revised Date: 17 December 2017 Accepted Date: 08 February 2018
Please cite this article as: Jing-Rong Cheng, Xue-Ming Liu, Wei Zhang, Zhi-Yi Chen, Xu-Ping Wang, Stability of phenolic compounds and antioxidant capacity of concentrated mulberry juice- enriched dried-minced pork slices during preparation and storage, Food Control (2018), doi:
10.1016/j.foodcont.2018.02.008
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1 Stability of phenolic compounds and antioxidant capacity of concentrated mulberry juice-enriched
2 dried-minced pork slices during preparation and storage
3 Jing-Rong Chenga, Xue-Ming Liua*, Wei Zhangb, Zhi-Yi Chena, Xu-Ping Wanga
4 a Sericultural & Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences, Key
5 Laboratory of Functional Foods, Ministry of Agriculture, Guangdong Key Laboratory of
6 Agricultural Products Processing. Guangzhou 510610, PR China
7 b Department of Medical Biotechnology, Flinders University, Sturt Rd, Bedford Park, GPO Box
8 2100, Adelaide 5001, South Australia
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18 *Corresponding author.
19 Address: 133 Yihenglu, Dongguanzhuang, Tianhe District, Guangzhou 510610, PR China
20 Tel: +86 -020 37227152
21 E-mail address: [email protected] (X.-M. Liu)
22
23 Functional foods have been of increasing demand due to the growing consumer awareness of
24 the relationship between diet and health. Addition of healthy ingredients to meat products is an
25 important method for development of functional meat products. In this contribution, functional
26 dried-minced pork slices incorporated with concentrated mulberry juice (CMJ) were developed
27 and stability of phenolic compounds and antioxidant capacity during product preparation and
28 storage were evaluated. The CMJ contained high amounts of total phenolics, anthocyanin and
29 flavonoids (19.13±0.64 mg GAE/g d.m., 4.91±0.18 C3GE mg/g d.m. and 18.39±1.21 mg CE/g
30 d.m., respectively) and showed excellent antioxidant activities in all assays used. CMJ
31 incorporation drastically minimized lipid and protein oxidation of dried-minced pork slices by
32 decreasing thiobarbituric acid-reactive substances (TBARS) value and carbonyls content during
33 processing and storage. Thermal treatment significantly destroyed the main antioxidant
34 ingredients including anthocyanins, flavonoids, and phenolics in dried-minced pork slices with
35 added CMJ; however, this deterioration could be effectively counteracted by using β-cyclodextrin.
36 The redness increased but lightness decreased with CMJ added, and color of the product remained
37 stable during storage. Therefore, CMJ rich in phenolic compounds could be used as a natural
38 antioxidant and pigment in dried-minced pork slices with suitable protective strategy.
39 Keywords: dried-minced pork slice; concentrated mulberry juice; phenolic compounds; lipid and
40 protein oxidation; β-cyclodextrin
41
42 Abbreviations:
43 ANOVA analysis of variance
44 C3G cyanidin-3-glucoside
45 C3GE cyanidin-3-glucoside equivalent
46 C3R cyanidin-3-rutinoside
47 CD cyclodextrins
48 CE catechin equivalent
49 CMJ concentrated mulberry juice
50 DF dilution factor
51 d.m. dry matter
52 DPPH 1,1-diphenyl-2-picrylhydrazyl
53 fw fresh weight
54 GAE gallic acid equivalent
55 HPLC High performance liquid chromatography
56 MDA malondialdehyde
57 MQA metal chelating activity
58 ROS reactive oxygen species
59 RP reducing power
60 RSA radical scavenging activity
61 TBARS thiobarbituric acid-reactive substances
62 TAC total anthocyanin content
63 TFC total flavonoid content
64 TPC total phenolic content 65
66 1. Introduction
67 Meat and meat products occupy a prominent position in the human diet for their high quality
68 protein content, essential amino acids and excellent source of B vitamins, minerals and other
69 nutrients (Zhang, Xiao, Samaraweera, Lee, & Ahn, 2010). Nevertheless, lower antioxidant and
70 high quality nutrients leads to the problem of perishability of the meat and meat products (Das,
71 Ranjan, Nath, & Laskar, 2013). The inherent antioxidant capacity of meat products is very low
72 leading to concern about the quality and shelf life of meat and meat products (Kumar, Kumar,
73 Tripathi, Mehta, Ranjan, Bhat, et al., 2013). Many consumers also believe that meat products are
74 unhealthy due to their high animal fat, synthetic pigment, antioxidant, and antimicrobial contents
75 which may be associated with several degenerative diseases (Cross, Leitzmann, Gail, Hollenbeck,
76 Schatzkin, & Sinha, 2007). With the consumer’s growing awareness of a link between diet and
77 health, there have been increasing demands for foods with healthy characteristics, such as meat
78 products with bioactive or functional components. The effective approach of developing healthy
79 food products is decreasing the undesired substances and/or increasing desired healthier
80 components. Low molecular weight polyphenolic compounds such as flavonoids can protect the
81 cells by scavenging and inhibiting the production and initiation of free radicals, including
82 superoxide anions and lipid peroxyl radicals, and hence are useful ingredients for developing
83 functional meat products (Kumar, Kumar, Tripathi, Mehta, Ranjan, Bhat, et al., 2013).
84 Generally, meat products when subjected to processing (heat) and storage undergo changes in
85 their physical and chemical characteristic that leads to the development of oxygen free radicals
86 which initiate the oxidation of polyunsaturated fatty acids. Besides, keeping attractive color is also
87 important for food quality which affects consumer acceptance (Wang, Cao, & Prior, 1997).
88 Synthetic antioxidants and artificial colorants have been reported to have potential toxicity to
89 human health, especially when they are excessively consumed daily (Ito, Hirose, Fukushima,
90 Tsuda, Shirai, & Tatematsu, 1986). Presently, researchers are interested in the utilization of plant
91 based derivatives that have both coloring and antioxidant properties (Castañeda-Ovando, Pacheco-
92 Hernández, Páez-Hernández, Rodríguez, & Galán-Vidal, 2009; Wang & Lin, 2000). Contrary to
93 the artificial additives, natural colorants have attracted considerable interest due to their presumed
94 safety, as well as potential health effects. However, incorporation of natural colorants to food
95 systems remains to be a challenge due to their low stability when subject to processing (heat) and
96 storage.
97 The use of naturally-occurring substances instead of synthetic additives in meat and meat
98 products is an effective means of developing healthier and novel meat products. A number of
99 publications have focused on the utilization of herbs, spices, fruits and vegetable extracts, and
100 have shown that the addition of these extracts to raw and cooked meat products decrease lipid and
101 protein oxidation (Estevez, Ventanas, & Cava, 2006), and improve color stability and antioxidant
102 capacities of meat products. For examples, Estévez et al.(2008) reported that adding 0.1% sage
103 extract significantly inhibited the increase of protein carbonyls in porcine liver pâté during
104 refrigerated storage. Rosemary extract was found to be effective in inhibiting protein oxidation in
105 beef patties (Lund, Hviid, & Skibsted, 2007). Black currant extract delayed thiobarbituric acid-
106 reactive substances (TBARS) value increasing in pork patties under chilled storage conditions
107 (Jia, Kong, Liu, Diao, & Xia, 2012). The major active components responsible for the
108 pigmentation and antioxidant activity of plant derivatives are polyphenols, flavonoids, phenolic
109 diterpenes and tannins (Zhang, Xiao, Samaraweera, Lee, & Ahn, 2010). Nevertheless, information
110 concerning changes of such active ingredients in meat products during processing and storage and
111 their contribution to product quality are still limited.
112 Mulberry has been found to have large amounts of phenolic compounds and its antioxidant
113 potential has been demonstrated in vitro (Cheng, Liu, Chen, Zhang, & Zhang, 2016; Liu, Xiao,
114 Chen, Xu, & Wu, 2004). As a potential artificial additives substitute, mulberry juice has superior
115 properties in term of color, anthocyanin content, and beneficial health effects compared to the
116 commonly used vegetable or herbaceous plant extracts, such as red cabbage, eggplant, and
117 citronella, among others. (Castañeda-Ovando, Pacheco-Hernández, Páez-Hernández, Rodríguez,
118 & Galán-Vidal, 2009; Wang, Cao, & Prior, 1997; Wang & Lin, 2000). Theoretically speaking,
119 mulberry juice could be used as an alternative colorant as well as antioxidant in improving meat
120 product quality. However, literature suggests that phenolic compounds, especially anthocyanins,
121 are particularly unstable during heat processing (Cheng, Liu, Chen, Zhang, & Zhang, 2016;
122 Slavin, Lu, Kaplan, & Yu, 2013).
123 Dried-minced meat slices are snack meat products and have gained much popularity and
124 acceptance in the world. Presently, to develop healthy dried-minced meat slices is one of the hot
125 projects in this area. Natural plant extracts rich in phenolic compounds are becoming important
126 ingredients in the production of dried-minced meat slices. In this study, a new healthy dried-
127 minced pork slices was developed by incorporating concentrated mulberry juice (CMJ). The
128 stability of phenolic compounds and antioxidant capacities of CMJ-enriched dried-minced pork
129 slices were investigated.
130 Since meat products must be thermally processed prior to consumption, the thermal
131 degradation of phenolic compounds is a major problem in the application of mulberry juice as
132 natural pigment in the meat industry. Therefore, to retain more bioactive compounds in the end
133 products is one of the key techniques waiting for solving in the production of healthy dried-
134 minced pork slices. Cyclodextrins (CDs) are inexpensive cyclic oligosaccharides produced from
135 enzyme-degradation of starch, which are non-toxic ingredients, are not absorbed in the upper
136 gastrointestinal tract, and are completely metabolized by the colon microflora (Szente & Szejtli,
137 2004). There are several types of cyclodextrin, including α-, β-, γ-, and δ- cyclodextrins named
138 according to their number of glucopyranose unit. As the purification of α- and γ-CDs increases
139 considerably the cost of production, 97% of the CDs used in the market are β-CDs. β-CD
140 comprises seven glucose units, and has been on the GRAS list since 1998, as a flavor carrier and
141 protectant, at a level of 2% in many food products. CDs can be considered as empty capsules of a
142 certain molecular size that can include a variety of molecules in this cavity. Thus, CDs are capable
143 of improving the physical, chemical and biological properties of bioactive molecules (Astray,
144 Gonzalez-Barreiro, Mejuto, Rial-Otero, & Simal-Gándara, 2009). Hence, CDs are widely used in
145 the food industry as food additives, for stabilization of flavors, elimination of undesired tastes or
146 other undesired compounds and to avoid microbiological contamination and browning reactions.
147 Many reports have shown that CDs can protect active ingredients against oxidation, light-induced
148 reactions, heat-promoted decomposition, loss duo to volatility and sublimation, among others
149 (Pinho, Grootveld, Soares, & Henriques, 2014). A wide range of reports have been published
150 regarding the encapsulation of natural polyphenolic agents such as chlorogenic, ferulic and gallic
151 acids by CDs (Zhao, Wang, Yang, & Tao, 2010; Olga, Styliani, & Ioannis, 2015), and for food
152 and drug delivery proposes. Thermal stability of anthocyanin extract of Hibiscus sabdariffa L. was
153 significantly improved in the presence of β-CD (Mourtzinos, Makris, Yannakopoulou,
154 Kalogeropoulos, Michall, & Karathanos, 2008). Phenolic compounds are the most important
155 bioactive compounds in CMJ, therefore, effects of β-CD on the stability of phenolic compounds
156 and antioxidant capacity were also evaluated.
157 2. Materials and methods
158 2.1 Materials and chemicals
159 Trimmed pork was obtained from a commercial meat processing company (Guangzhou,
160 Guangdong, China). Concentrated mulberry juice, produced by vacuum concentration, was kindly
161 provided by Guangdong Bosun Health Food Company (Guangdong, China) that is involved in
162 mulberry fruit processing. All other chemicals and reagents were purchased from Qiyun Company
163 (Guangzhou, China) and Sigma Chemicals (Sigma-Aldrich, Steinheim, Germany).
164 2.2 Dried-minced pork slice preparation
165 The trimmed pork (longissimus thoracis) was minced twice (10 mm plate followed by 4 mm
166 plate) using a meat grinder. The dried-minced pork slices were prepared as follows: 850 g lean
167 meat, 150 g pork fat, 20 g sodium chloride, 3 g phosphate, and 4 g papain (2500 u/g). Ground
168 meat and other ingredients were mixed by blending for 30 min in a Kitchen Aid mixer (Suihua,
169 Guangdong, China). After blending, CMJ and β-cyclodextrin were added, followed by another 10
170 min blending. Minced pork was shaped by hand into square with approximately 4 cm side length
171 and 4 mm thickness. After that, two steps of heat treatment were conducted for the dehydration:
172 firstly, heat pump drying (temperature 55oC, humidity 60%) was employed until the sample
173 moisture content was close to 18%; secondly, all samples were baked for 3 min at 180 oC. After
174 cooling to room temperature, dried-minced pork slices were vacuum-packed in plastic bags and
175 stored at room temperature (25±3 oC) under fluorescent lights to simulate supermarket retail
176 display conditions. Three experimental groups were designed to study the antioxidant activity and
177 polyphenolic compound stability: 1% CMJ (10.0 g CMJ per kg of meat stuffing) was added; 1%
178 CMJ and 1% β-cyclodextrin (10.0 g CMJ and 10.0 g β-cyclodextrin per kg of meat stuffing) were
179 added. A control was prepared in the absence of mulberry juice and β-cyclodextrin. A strict
180 sanitation procedure was followed to avoid microbial contamination. Three independent
181 experimental trials (replications) were conducted.
182 2.3 TBARS and carbonyls analysis
183 Before experiments, dried minced pork slices were chopped up with a knife. TBARS value
184 and protein carbonyls were measured according to our previous study (Cheng, Liu, Zhang, Zhang,
185 Chen, Tang, et al., 2017).
186 2.4 Extraction of anthocyanins and phenolic compounds from samples
187 Anthocyanins and phenolic compounds in the dried-minced pork slice samples were
188 extracted according to the procedure described by Žilić et al. (2016). Total phenolic in 500 mg of
189 samples were released by alkaline hydrolysis for 4 h at room temperature using 10 mL 4 mol/L
190 NaOH. After the pH was adjusted to 2.0 by 6 mol/L HCl, 5 mL of all the hydrolyzates were
191 extracted with 5 mL of ethyl acetate and diethyl ether (1:1, v/v) four times. Five milliliters of
192 combined extracts were evaporated under N2 stream at 30 oC to dryness. Final residues were
193 redissolved in 1.5 mL of methanol. The methanolic solutions so prepared were used for the
194 analyses of total phenolic compounds, flavonoids and anthocyanins. The extracts were kept at -70
195 oC prior to analyses. All extractions were performed in triplicate for each replications of baking
196 experiment.
197 2.5 Total phenolic content and flavonoid content analysis
198 The total phenolic content (TFC) of CMJ and extract was determined by using the Folin-
199 Ciocalteu assay as described by Shi et al. (2005) and expressed as mg of gallic acid equivalent
200 (GAE) per kg of dry matter (d.m.). A hundred milliliters of CMJ or extract was transferred into
201 graduated cylinders and their volume made up to 500 mL with distilled water. After addition of
202 Folin-Ciocalteu reagent (2.50 mL) and 20% Na2CO3 solution (1.25 mL), tubes were vortexed. The
203 absorbance of the mixture was read at 750 nm after 40 min.
204 Total flavonoid content (TFC) of CMJ and dried-minced pork slice was determined as
205 described by Eberhardt, Lee, and Liu (2000) and expressed as mg catechin equivalent (CE) per kg
206 of d.m. Briefly, 250 μL of CMJ or extract was mixed with 1 mL distilled water and subsequently
207 with 150 μL of 15% (w/v) sodium nitrite solution. After 6 min incubation, 75 μL of a 100 g/L
208 aluminum chloride solution was added, and the mixture was allowed to stand for an additional 5
209 min before 1 mL of 4% (w/v) NaOH solution was added. The mixture was immediately made up
210 to 2.5 mL with distilled water and mixed well. The absorbance of the mixture was then measured
211 at 510 nm. The total flavonoid content was expressed as mg CE per kg of d.m.
212 2.6 Anthocyanin content analysis
213 The total anthocyanin content (TAC) was determined using the pH differential method
214 (Cheng et al., 2017). Briefly, 0.25 mL of CMJ or extract was diluted 20 times with a pH 1.0
215 potassium chloride buffer (0.025 M) and a pH 4.5 sodium acetate buffer (0.4 M), respectively. The
216 absorbance at 520 nm was compared with a distilled water blank after incubation in darkness for
217 15 min. Anthocyanin content, expressed as cyanidin-3-glucoside (C3G) equivalents, was
218 calculated as: anthocyanin content (mg/L) = (ApH 1.0 − ApH 4.5) × 449.29 × DF × 1000 / (26,900×1)
219 where ApH 1.0 and ApH 4.5 are the absorbance of the sample in a pH 1.0 and a pH 4.5 buffer,
220 respectively; 449.29 is the molecular weight of C3G (g/mol); DF is the dilution factor (20); 1000
221 is the factor for conversion from g to mg; 26,900 is the molar extinction coefficient of C3G
222 (M−1•cm−1); and 1 is the pathlength (cm).
223 C3G and cyanidin-3-rutinoside (C3R) are the primary anthocyanins present in mulberry juice
224 and are analyzed by high performance liquid chromatography (HPLC) according to the method
225 reported by Liu, Xiao, Chen, Xu, and Wu (2004).
226 2.7 Antioxidant activity determination
227 The antioxidant activity of the juice based on the scavenging activity of 1,1-diphenyl-2-
228 picrylhydrazyl (DPPH) radical was determined by the method described by Wu et al. (2003). The
229 Fe2+-chelating ability of the extracts was estimated according to Andres et al. (2017). The
230 reducing power was determined based on the method described by Wu et al. (2003) with ascorbic
231 acid as a positive control.
232 2.8 Color evaluation
233 Surface color measurements of dried mince pork slice were performed using a colorimeter
234 (UItraScan VIS, Chroma Meter, US) according to our previous study (Cheng et al., 2017). Before
235 each session the colorimeter was calibrated on the CIE color space system using a white tile
236 (L*=93.16, a*=−0.79, b*=1.28). Color measurements, L* value (lightness), a* value (redness) and
237 b* value (yellowness), were made on the surface of each slice in triplicate at three randomly
238 selected locations. Color measurements were made at room temperature (25 °C) with illuminant
239 D65 and a 0° angle observer.
240 2.9 Statistical analyses
241 Three dried-minced pork slices per group and per sampling time (the initial, before baking,
242 after first heat treatment, after second heat treatment, storage for 1days, storage for 7 days and
243 storage for 14 days) were sampled and three independent experimental trials (replications) were
244 conducted. The results are presented as means ± standard deviation of three replicates of
245 independent experiments, each analyzed three times (n = 3 × 3 → n = 9 per group and
246 sampling time). The analysis of variance was conducted and the differences between variables
247 were tested for significance by one-way ANOVA with a SPSS 11 (Statistical Package for the
248 Social Sciences) program. Differences at P<0.05 were considered statistically significant.
249 3. Results and discussion
250 3.1 Antioxidant characteristics of CMJ
251 Total phenolic, flavonoid and anthocyanin contents, and antioxidant capacity of CMJ are
252 summarized in Table 1. There were 19.13±0.64 mg GAE/g d.m. phenolic, 18.39±1.21 mg CE/g
253 d.m. flavonoid and 4.91±0.18 mg C3GE /g d.m anthocyanin in CMJ, respectively. Although no
254 data are found in the literature about total phenolic, flavonoid and anthocyanin contents in
255 concentrated mulberry juice, there are many reports about their contents in fresh mulberry fruits
256 and mulberry juice. We tested total anthocyanin content in fruit juice of 31 cultivars of mulberry
257 (Morus atropurpurea Roxb., Morus alba, etc.), and these ranged from 0.15 to 2.73 mg C3GE /mL
258 (Liu, Xiao, Chen, Xu, & Wu, 2004). Koca et al. (2008) reported that total phenolic and
259 anthocyanin contents in juice of wild purple mulberry (Morus rubra) grown naturally were 1.31
260 mg GAE/g and 0.19 mg C3GE/g. Özgen et al. (2009) found that the average total phenolic
261 contents of Morus nigra and Morus rubra were 2.74 and 1.60 mg GAE/g fw, and Morus nigra had
262 the highest amount of anthocyanin with an average of 0.57 mg C3GE /g fw. Kamiloglu et al.
263 (2013) showed that TPC, TFC and TAC in fresh black mulberry (Morus nigra L.) fruits were
264 14.51 mg GAE/g dw, 7.69 mg CE/g dw and 12.21 mg C3GE/g dw, respectively. Yang et al.
265 (2016) found values of 11.23 mg GAE/g, 15.1 mg RE /g fw and 11.77 mg C3GE /g fw,
266 respectively, in fresh fully matured mulberry (Morus atropurpurea Roxb.) fruits. It could be
267 concluded that TPC, TFC and TAC in mulberry fruits are highly diversified, and affected by many
268 factors such as mulberry species and cultivar, cultivation place, plant administration, growing
269 climate, harvest season and weather, and processing method, among others. (Liu, Xiao, Chen, Xu,
270 & Wu, 2004; Koca, Ustun, Koca, & Karadeniz, 2008; Özgen, Serçe, & Kaya, 2009; Kamiloglu,
271 Serali, Unal, & Capanoglu, 2013). Phenolic compounds, especially anthocyanins, are sensitive to
272 heat treatment and may be destroyed to some extent depending on heating method and conditions
273 (Fazaeli, Hojjatpanah, & Emam-Djomeh, 2013). CMJ used in this experiment was produced by
274 vacuum concentration and still contained high amounts of phenolic compounds even after the
275 concentration process.
276 Phenolic contents were highly correlated with the antioxidant capacity. The DPPH
277 scavenging activity and Fe2+-chelating ability of CMJ, expressed by IC50, were 0.67±0.08 mg/ml
278 and 0.31±0.07 mg/ml, respectively; the reducing power of 1 mg/ml CMJ expressed by A700 was
279 0.26. Nevertheless, in spite of higher phenolic content (134.79±0.8 mg GAE/g) in pomegranate
280 extract, lower antioxidant capacities (DPPH scavenging activity of 0.16±0.02 mg/ml and reducing
281 power of 0.03) were detected (Andres, Petron, Adamez, Lopez, & Timon, 2017). The conflicting
282 results may be ascribed to the existing differences in phenolic types and extract concentrations.
283 Phenolic compounds in mulberry fruits comprise anthocyanins and non-anthocyanin phenolics.
284 The former ones are mainly C3G, C3R and pelargonidin-3-O-glucoside (Liu, Xiao, Chen, Xu, &
285 Wu, 2004), while the latter ones are mainly rutin, chlorogenic acid, 4-caffeolyquinic acid,
286 quercetin, and procatechuic acid (Zhang, Han, He, & Duan, 2008). Not all phenolic compounds
287 inhibit oxidation via the same mechanism and some may be more effective than others (Brewer,
288 2011). Flavonoids with multiple hydroxyl groups exhibit stronger antioxidant property than those
289 with a lower degree of hydroxylation (Brewer, 2011). Thus, the antioxidant activities cannot be
290 attributed solely to the phenolic contents, but also to the actions of different antioxidant
291 compounds and their interactions with each other (Ahn, Grün, & Fernando, 2002).
292 3.2 Effects of CMJ addition on oxidation of dried-minced pork slices
293 Due to their rich nutritional composition, meat and meat products are susceptible to quality
294 deterioration which is mainly attributed to chemical and microbial changes. The most common
295 form of chemical deterioration is the oxidation of meat lipids and proteins. Lipid and protein
296 oxidations are complex processes, dependent on chemical composition of meat, light and oxygen
297 availability and storage temperature, and are also affected by some processing technological
298 procedures (Shah, Bosco, & Mir, 2014). They lead to the formation of several other compounds
299 which have negative effects on the quality of meat and meat products causing changes in sensory
300 (color, texture and flavor) and nutritional quality (Karakaya, Bayrak, & Ulusoy, 2011). Natural
301 plant extracts are believed to play an important role in ameliorating oxidation processes by
302 quenching free radicals, chelating catalytic metals and scavenging oxygen in food and biological
303 systems (Ahn, Grün, & Fernando, 2002; Andres, Petron, Adamez, Lopez, & Timon, 2017), and
304 they are widely used as antioxidants in the meat industry (Karakaya, Bayrak, & Ulusoy, 2011;
305 Shah, Bosco, & Mir, 2014; Kumar, Yadav, Ahmad, & Narsaiah, 2015). In this study, the oxidation
306 was assessed by comparing both TBARS and carbonyls formation in dried-minced pork slices
307 formulated with and without CMJ during heat processing and storage. Besides, the protective
308 effect of β-CD against oxidation was also studied, since it has been reported that the stability of
309 anthocyanins could be enhanced by CD (Flores, Ruiz del Castillo, Costabile, Klee, Bigetti
310 Guergoletto, & Gibson, 2015).
311 3.2.1 TBARS
312 Lipid oxidation, expressed as TBARS, in control dried-minced pork slice which prepared
313 without CMJ, increased rapidly throughout heat processing and storage, reaching 2.34±0.10
314 mg/kg at the end of the storage (Fig.1 A). The levels of malonaldehyde (MDA) equivalents for all
315 samples are lower than 5 mg MDA equivalents /kg meat which is the detectable concentration for
316 rancidity (Insausti et al., 2001). The analysis of variance with the data indicated that the TBARS
317 level was significantly affected (P<0.05) by CMJ. After heat processing, TBARS values for all
318 samples treated with CMJ were significantly lower than the control (P<0.05), indicating a
319 protective effect against lipid oxidation during processing. Interestingly, this protective effect was
320 maintained during the entire storage period. At the end of storage (Day 14), samples treated with
321 CMJ had significantly lower TBARS value than control. Addition of 1% CMJ with and without β-
322 CD inhibited TBARS in the dried-minced pork slices by 39.3% and 31.6%, respectively. These
323 results are consistent with the antioxidant characteristics of CMJ (Table 1), which contained
324 19.13±0.64 mg GAE/g d.m. phenolic and 18.39±1.21 mg CE/g d.m. flavonoids. The inhibitory
325 efficacy of CMJ on lipid oxidation of dried-minced pork slices (39.3%) was lower than that of
326 black currant extract on pork patties (90.7%) during chilled storage (Jia, Kong, Liu, Diao, & Xia,
327 2012). The main cause for this difference may be attributed to different systems in addition to the
328 different extracts. In the study of Jia et al. (2012), the pork patties incorporating black currant
329 extract were not heat-treated, and only stored at 4 °C up to 9 days. It is noteworthy that β-CD
330 facilitates the protective effect since the TBARS values of all samples were significantly lower
331 than that contained CMJ alone (P<0.05). This result was identical to that of other researchers who
332 also demonstrated that cyclodextrin facilitated the stability and bioavailability of anthocyanins
333 (Flores, Ruiz del Castillo, Costabile, Klee, Bigetti Guergoletto, & Gibson, 2015). Thus, CMJ is a
334 good source of natural antioxidants that protects meat products against lipid oxidation.
335 Natural plants and their extracts used as lipid oxidation inhibitors in meat and meat products
336 have a long history and there are several review papers on this topic (Karakaya, Bayrak, &
337 Ulusoy, 2011; Shah, Bosco, & Mir, 2014; Kumar, Yadav, Ahmad, & Narsaiah, 2015; Ahmad,
338 Gokulakrishnan, Giriprasad, & Yatoo, 2015). Methanolic extracts from the fruit of Prunus mume
339 were found to reduce TBARS values of precooked chicken breast meat patties at 4 °C for 3 days
340 (Jo et al., 2006). Pomegranate juice phenolics inhibited TBARS values in spent hen breast meat
341 samples up to 12 days of refrigerated storage at 4 °C (Vaithiyanathan, Naveena, Muthukumar,
342 Girish, & Kondaiah, 2011). A waste product from industrial tomato paste production was found to
343 yield efficient protection against lipid oxidation in pressurized chicken meat (Alves, Bragagnolo,
344 da Silva, Skibsted, & Orlien, 2012). Rose polyphenols protected lipids against oxidation in
345 naturally dry fermented sausages (Zhang, Jiang, Rui, Li, Chen, & Dong, 2017). Our results are
346 consistent with these previous findings and suggest the potential application of CMJ as an
347 antioxidant for the production of meat products with improved quality traits. The inhibitory effect
348 of CMJ on lipid oxidation may be attributed to its phenolics and other biochemical compounds
349 that contribute to antioxidant activity.
350 3.2.2 Carbonyls
351 Protein oxidation is another major cause of meat quality deterioration in addition to lipid
352 oxidation and has drawn increasing attention in recent years. The extent of protein oxidation was
353 assessed by measuring the formation of carbonyls. Carbonyl contents of all samples exhibited
354 identical increasing trend during processing and storage of dried-minced pork slices (Fig. 1B),
355 indicating the occurrence of protein oxidative reactions. This carbonyl accumulation is attributed
356 to the direct (primary) oxidation of amino acid side chains or indirect (secondary) carbonylation
357 (Rysman, Van Hecke, Van Poucke, De Smet, & Van Royen, 2016). Furthermore, carbonyl groups
358 can be formed by scission of oxidative peptides, or in the presence of oxidizing sugars or lipids
359 (Ganhão, Morcuende, & Estévez, 2010). The carbonyl content of the control sample increased
360 faster and reached 4.01±0.22 nmol/mg protein after a two-step heat treatment. Though samples
361 treated with CMJ exhibited carbonyl contents lower than 3.52 nmol/mg proteins, β-cyclodextrin
362 did not provide any additional protective effect against protein oxidation during the heat
363 processing stage. At the end of the storage, the carbonyl content of the control sample reached
364 5.56±0.14 nmol/mg protein while others were lower than 4.31 nmol/mg proteins. Jia et al. (2012)
365 found that black currant extract significantly inhibited carbonyl formation in pork patties at 6 and
366 9 days of storage. Similar results were reported by Andres, Petron, Adamez, Lopez, and Timon
367 (2017), who demonstrated good antioxidant properties for tomato and red grape extracts in lamb
368 meat patties. Parallel to the variation of TBARS, β-cyclodextrin provided decreased carbonyl
369 formation in the slices containing mulberry juice during the storage period.
370 The precise mechanisms involved in the antioxidant actions of plant phenolic on dried-
371 minced pork slices system are not well understood. Several studies have demonstrated that
372 phenols from mulberry act as efficient radical scavengers which can block the prooxidant action of
373 reactive oxygen species (ROS) on proteins (Rice-evans, Miller, Bolwell, Bramley, & Pridham,
374 1995). Additionally, some phenolic compounds such as C3G, which is abundant in mulberry,
375 display metal-chelating activities. This might be particularly relevant in heat treated meat products
376 since phenols may hinder the prooxidant action of non-heme iron by chelation. Both antioxidant
377 mechanisms can explain the results obtained since certain ROS such as hydroxyl radicals and iron
378 are directly involved in the formation of protein carbonyls and TBARS from meat products
379 (Estevez, Kylli, Puolanne, Kivikari, & Heinonen, 2008).
380 3.3 Phenolic compound stability in dried-minced pork slices during preparation and storage
381 3.3.1 Total phenol stability
382 Total phenolic content changes in dried-minced pork slices with different treatments during
383 preparation and storage are shown in Fig.2. The total phenol decreased throughout preparation and
384 storage of pork slices. Total phenolic contents in dried-minced pork slices enriched with CMJ,
385 with or without β-cyclodextrin addition dropped from 190.67±1.21 μg GAE/g to less than 160 mg
386 GAE/g dried product before heating. This notable loss was caused by the oxidation effect and
387 phenolic extractability change. On one hand, the mixing process brought in large amount of
388 oxygen which triggered the oxidation reaction; on the other hand, the minced meat stuffing was
389 rich in proteins, lipids and polysaccharides, which could interact with polyphenols and modify
390 their extractability from samples (Mazzaracchio, Pifferi, Kindt, Munyaneza, & Barbiroli, 2004).
391 Given that mulberry juices contain anthocyanins which are prone to degradation during
392 thermal processing, decreasing of total phenolic content in dried-minced pork slices may partly be
393 due to the loss of anthocyanins. With 1% CMJ added, the recovery of total phenolic content after
394 two-step heat processing was 60.7±1.21% (after heat pump drying process) and 54.26±1.32%
395 (after baking process), respectively. Though β-cyclodextrin lowered this loss, the effect still
396 existed during the heating process (Fig.2). Some authors have ascribed the phenolic reduction
397 during storage to lipids and protein oxidation reactions (Ribas-Agustí, Gratacós-Cubarsí, Sárraga,
398 Guàrdia, García-Regueiro, & Castellari, 2014). However, this was not the same in our study since
399 the total phenol contents did not undergo any significant reduction during storage (P<0.05). The
400 satisfactory stability of phenolic compounds in dried-minced pork slices was probably related to
401 vacuum packaging which increased protection against oxidation. These results are in concordance
402 with the work of Fernández-López et al. (2007), who found that hesperidin, a phenolic compound
403 from orange by-products, was very stable in dry fermented sausages over time. Furthermore,
404 though oxidation destroyed available phenolics, additional chemical substitutes that release
405 phenolics might make up for this initial loss during the storage (Moore, Luther, Cheng, & Yu,
406 2009).
407 3.3.2 Flavonoid stability
408 Total flavonoid contents in dried-minced pork slices during manufacturing and storage are
409 presented in Fig.3. The detrimental effect of mixing on flavonoid compounds might be responsible
410 for lower total flavonoid content for both groups before heat treatment than the initial values upon
411 addition (180.85±0.21 μg CE/g). Mixing may involve cellular structure damage, oxidative and
412 hydrolytic enzyme release that can degrade flavonoids and other antioxidant compounds
413 (Wojdyło, Figiel, & Oszmiański, 2009). Heat pump drying process, as a main dehydration step,
414 significantly reduced the flavonoids content in dried-minced pork slices. Based on the recovery of
415 catechin, a greater recovery of flavonoids was observed in slices made with β-cyclodextrin
416 (58.70±1.74 %) than the control (49.97±2.04%). Previous works also have indicated that heat
417 treatment of fruit juices and plant extracts results in a significant loss of flavonoids and changes in
418 isoflavone forms (Slavin, Lu, Kaplan, & Yu, 2013). Nonetheless, differences in flavonoid content
419 during the second heat process were insignificant. Two previous studies have reported a similar
420 retention of total isoflavones during the baking process of a soy and wheat bread. This stability
421 was ascribed to tautomeric effect since they had substitutes from malonyl glucoside to
422 acetylglucosides and β-glucosides during baking (Shao, Duncan, Yang, Marcone, Rajcan, & Tsao,
423 2009). After two-step heat processing, flavonoid contents in dried-minced pork slices were
424 84.01±0.54 μg/g dried product (without β-cyclodextrin) and 101.6±0.29 μg/g dried product (with
425 β-cyclodextrin). During storage, total flavonoid content of dried-minced pork slices only with the
426 addition of CMJ varied from 84.1±0.21 to 64.1±0.49 μg/g dried product, while samples containing
427 both CMJ and β-cyclodextrin were more stable. Slices with β-cyclodextrin addition had higher
428 final flavonoid contents (90.03±0.36 μg/g dried product) which was attributed to changes in
429 flavonoid form and protective effects of β-cyclodextrin (Shao, Duncan, Yang, Marcone, Rajcan, &
430 Tsao, 2009; Slavin, Lu, Kaplan, & Yu, 2013).
431 3.3.3 Anthocyanin stability
432 The effects of thermal processing on food anthocyanins stability have been reviewed (Patras,
433 Brunton, O'Donnell, & Tiwari, 2010), but the available data on the topic were limited. C3G and
434 C3R are the predominant anthocyanins in mulberry juice (Cheng, Liu, Chen, Zhang, & Zhang,
435 2016), and their stability in dried-minced pork slices during manufacturing and storage were
436 studied. Variations of total and main monomeric anthocyanins are presented in Fig.4. Before
437 baking, the difference of anthocyanin recovery between the two groups was insignificant
438 (P>0.05), and about 77.87% anthocyanins were recovered in the products. This result is consistent
439 with previous findings, which suggested that proteins, lipids and polysaccharides can interact with
440 polyphenols and finally modify their extractability (Bordenave, Hamaker, & Ferruzzi, 2014). The
441 decrease in anthocyanin content in dried-minced pork slices with supplementation of CMJ was
442 time-dependent. Both C3G and C3R degradation are noted during the whole study period, but the
443 distinction in total anthocyanin recovery was remarkable (P<0.05). Total anthocyanin recovery of
444 product with β-cyclodextrin (56.29±0.39%) was much higher than that without β-cyclodextrin
445 (45.15±0.62%). In addition, drying under lower temperatures for longer time (temperature 55oC,
446 humidity 60%, drying until the sample moisture content close to 18%) resulted in a high loss of
447 anthocyanins. Baking process under higher temperatures for limited time (180 oC, 3 min) also
448 degraded C3G and C3R in a high amount. Without β-cyclodextrin, the dried-minced pork slices
449 lost 52.88±0.12% C3G and 46.43±0.23% C3R during the first heat treatment, increased to
450 60.45±0.31% and 58.01±0.16% after the second baking period and 71.40±0.32 and 68.32±0.16%
451 losses during the storage. At the end of the storage, 4.32±0.31μg/g dried product C3G and
452 5.81±0.53μg/g dried product C3R were recovered. With β-cyclodextrin added, the anthocyanins
453 recovery was remarkably enhanced. The recoveries for C3G and C3R from dried-minced pork
454 slices prepared with β-cyclodextrin were respectively higher by 5.11% and 5.89% compared to the
455 control (without β-cyclodextrin). This was perhaps because of the thermodynamic interactions
456 between anthocyanins and β-cyclodextrin, which enhanced the stability of anthocyanins (Del
457 Valle, 2004).
458 3.4 Color stability in dried-minced pork slices during preparation and storage
459 The color stability for dried-minced pork slices with and without CMJ is shown in Fig.5.
460 Mulberry juice, which is rich in anthocyanins, enhanced the redness of the dried-minced pork
461 slices. Before baking, slices with mulberry juice displayed a distinctive purple color because of
462 anthocyanin pigments. During the manufacturing and storage, a* values showed a steady decrease
463 in all groups, and the total loss reached 4-6 units throughout the manufacturing process (Fig. 5A).
464 The decline in redness was mainly due to oxidation of myoglobin, forming metmyoglobin, which
465 is brown in color (Jia, Kong, Liu, Diao, & Xia, 2012), and anthocyanins degradation. Contrary to
466 a*-value, L*-value showed a smooth increase in all groups during manufacturing, slices with both
467 mulberry juice and β-cyclodextrin showed the highest L*-value (Fig. 5B). Jia et al. (2012) and
468 Andres et al. (2017) also reported a similar color deterioration, a steady decrease in a*-value and
469 increase L*-value in raw pork patties and lamb patties, respectively, during storage. After heat
470 processing, the color of the product became more natural than the brown color in the control and
471 retention of the initial purple color, which might be more acceptable as reported by Jia et al.
472 (2012). Although the color changes were obvious during manufacturing, they become stable
473 during the storage with mulberry juice. This can be interpreted from the protective effect of
474 phenolic compounds on dried-minced pork slices. As revealed, the discoloration in pork was due
475 to the formation of metmyoglobin and non-enzymatic browning reactions between lipid oxidation
476 products and amine groups. Therefore, antioxidant ingredients, such as anthocyanins, flavonoids,
477 and other phenolic compounds, may form products that can protect the slices against
478 discoloration. Our findings are in agreement with previous results, demonstrating that phenolic-
479 rich extracts, for example, extracts from mustard leaf kimchi (Lee, Choi, Choi, Han, Kim, Shim, et
480 al., 2010) and avocado peel and seeds (Turgut, Soyer, & Işıkçı, 2016), can effectively inhibit the
481 color change in pork during storage. Ganhão, Morcuende, and Estévez (2010) found that
482 blackberry, which is rich in anthocyanins, enhanced the color of cooked burger patties. However,
483 discoloration in slices enriched with mulberry juice was remarkably reduced by β-cyclodextrin
484 addition during heating process and storage, even though no obvious color difference was
485 observed before baking (P>0.05). This further verified the protective effect of β-cyclodextrin on
486 stability of pigments present in mulberry juice.
487 4. Conclusion
488 To develop balanced meat products is a challenge for food technologists and represents an
489 appealing research line for producing novel foods. For the first time, concentrated mulberry juice
490 rich in phenolic compounds was added to formulations of dried-minced pork slices for the
491 preparation of functional meat products. In situ testing confirmed that concentrated mulberry juice
492 is a highly effective antioxidant in dried-minced pork slice because it inhibited both lipid and
493 protein oxidation and stabilized the color during preparation and storage. Heat processing is the
494 primary factor causing loss of phenolic compounds, which could be reduced by the presence of β-
495 cyclodextrin. In spite of the protective effect of β-cyclodextrin on phenolic compounds, reducing
496 phenolic compounds loss is still a barrier that hampers the health promoting properties of the
497 product. On the basis of the present results, it may be suggested that concentrated mulberry juice
498 provides a potentially functional ingredient to be used (at level of 1.0%) for improving shelf-life
499 quality of dried-minced pork slices. Such processed meat product would be highly desirable from
500 a diet/health standpoint as they contain phenol compounds. These results establish a solid basis for
501 further studies, such as the use of plant polyphenol, to develop processed meat products with
502 higher nutritional value. We also suggest that concentrated mulberry juice be used in other meat
503 products such as sausage as natural pigments and bio-preservatives to substitute synthetic
504 pigments and preservatives. Furthermore, bioavailability of phenolic compounds and digestability
505 of protein in this functional meat product also need to be investigated for the commercialization of
506 the new formula and technology in future.
507 Acknowledgements
508 The authors gratefully acknowledge the financial support of Guangzhou Science and
509 Technology Program key projects [grant No. 201704020054], Guangdong Provincial Science and
510 Technology Program [grant No. 2014A020208040; 2014B040404059; 2016LM3167], Guangdong
511 Natural Science Foundation [grant No. 2015A030313569]. Thanks are due to Prof. Fereidoon
512 Shahidi for providing language help.
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Figure lists:
Fig.1. Influences of CMJ on TBARS values (A) and carbonyl content (B) of dried minced pork slices during heat process and storage.
Fig.2. Phenolic content variation of dried minced pork slices during preparation and storage.
Fig.3. Flavonoid variation of dried minced pork slices during preparation and storage.
Fig.4. Anthocyanin variation of dried minced pork slices during preparation and storage.
Fig.5. Lightness (L*) and redness (a*) on dried minced pork slices during preparation and storage.
Fig.1. Influences of CMJ on TBARS values (A) and carbonyl content (B) of dried minced pork slices during heat process and storage. Error bars refer to the standard deviations obtained from triplicate sample analysis. MJ: mulberry juice; β-CD：β-cyclodextrin. a-c, Values with different letters within the same sampling time are significantly different (P<0.05); A-E, Values with different letters within the same treatment are significantly different (P<0.05).
Fig.2. Phenolic content variation of dried minced pork slices during preparation and storage. Error bars refer to the standard deviations obtained from triplicate sample analysis. a-b, Values with different letters within the same sampling time are significantly different (P<0.05); A-E, Values with different letters within the same treatment are significantly different (P<0.05).
Fig.3. Flavonoid variation of dried minced pork slices during preparation and storage. Error bars refer to the standard deviations obtained from triplicate sample analysis. a-b, Values with different letters within the same sampling time are significantly different (P<0.05); A-E, Values with different letters within the same treatment are significantly different (P<0.05).
Fig.4. Anthocyanins variation of dried minced pork slices during preparation and storage. Error bars refer to the standard deviations obtained from triplicate sample analysis. A-E, Values with different letters within the same treatment are significantly different (P<0.05).
Fig.5. Lightness (L*) and redness (a*) on dried minced pork slices during preparation and storage.
Error bars refer to the standard deviations obtained from triplicate sample analysis. a-c, Values with different letters within the same sampling time are significantly different (P<0.05); A-E, Values with different letters within the same treatment are significantly different (P<0.05).
Highlights：
CMJ dramatically minimized lipid and protein oxidation of dried-minced pork slices.
CMJ is rich in phenolics and has good antioxidant activity.
Antioxidant ingredients added to products were unstable during thermal treatment.
β-cyclodextrin exhibited protective effect on phenolic compounds.
Table list:
Table 1 Antioxidant characteristics of CMJ.
Table 1 Antioxidant characteristics of CMJ.
Parameters Unit Value
TFC mg CE/g d.m. 18.39±1.21
TPC mg GAE/g d.m. 19.13±0.64
TAC mg/g d.m. 4.91±0.18
RSA mg/ml 0.67±0.08
MQA mg/ml 0.31±0.07
RP - 0.26
Values are given as mean ± SD from three determinations (n=3). TPC: total phenolic content as gallic acid equivalent; TFC: total flavonoid content as catechin equivalent; TAC: total anthocyanin content as C3G equivalent. RSA: radical scavenging activity, expressed as IC50, concentration (mg/ml) at 50% scavenging of DPPH radical; MQA: metal chelating activity, expressed as IC50, concentration (mg/ml) at 50% metal chelating activity; RP: reducing power at 1mg/ml, expressed as A700. d.m: dry matter.
