Oxidative stability
In this study, the effects of natural antioxidant ESH-enriched mushroom extract against lipid and protein oxidation occurred in emulsified sausage were detected. The TBARS value was used to evaluate the lipid oxidation in meat system. As shown in
Table 1, at day 0, 3, 7, and 14, the TBARS values of sausages from EEME group were 0.12, 0.27, 0.43, and 0.80 mg/kg sample, which were significantly lower than those in control group by 36.84%, 28.95%, 31.75%, and 34.62% at the respective storage times (p<0.05). For sausages from BHA group, the TBARS values were 0.13, 0.28, 0.48, and 0.82 mg/kg sample at day 0, 3, 7, and 14 of refrigerated storage, and no difference was found between group EEME and group BHA at each storage time. It was demonstrated that adding ESH-enriched
F. velutipes extract (0.8%) to sausage could effectively prevent lipid oxidation during refrigerated storage, and its efficacy was equivalent to 0.01% BHA. In current study, the content of protein carbonyls was used to evaluate the degree of protein oxidation occurring in sausages. As shown in
Table 2, the carbonyl values of sausages from EEME group were 0.032, 0.28, 0.76, and 0.92 nmol/mg at day 0, 3, 7, and 14 of refrigerated storage, which were significantly lower than those in control group by 33.33%, 36.36%, 25.49%, and 28.68% at the respective storage times (p<0.05). For the samples from BHA batch, at day 0, 3, 7, and 14, no difference was found between group BHA and group EEME. This data indicated that ESH-enriched
F. velutipes extract (0.8%) also could effectively prevent protein oxidation of sausage, and its efficacy was equivalent with 0.01% BHA. The results obtained in this study agreed with previous studies conducted on raw muscle food, these studies concluded that adding ESH-enriched mushroom extract was an effective method to control the oxidative reactions occurring in tuna [
9], yellowtail dark muscle and beef patties [
10], shrimp [
12], and salmon [
13].
During meat processing, many factors could affect the antioxidative activity of natural antioxidants, such as temperature, pH, processing conditions and food additives. So, for utilization in processed meat, the antioxidants should meet many requirements, among which being stable of heat, light and acid-base is important. Liu et al [
19] studied the stability of ESH from mushroom extract and found that it had excellent light, thermal and acid-base stability, this is very ubiquitous advantage for processed meat production. It is suggested that the extract of
F. velutipes contained high-level of ESH and could be a good antioxidant candidate for meat processing.
Numerous studies have revealed that extract of certain mushrooms had strong antioxidative ability, and the active substances in the extract (extracting with water or alcohol) including ESH and certain phenolic compounds [
10]. Nguyen et al [
20] demonstrated that, both ESH and total phenolic compounds (TPs) showed a significant correlation with DPPH radical scavenging capacity, and the ESH had higher correlation (R
2 = 0.84) with DPPH radical scavenging activity than TPs (R
2 = 0.52). Pahila et al [
7] also concluded that a high correlation (R
2 = 0.9984) was found between ESH content in crude mushroom extract and DPPH radical scavenging. Meanwhile, Bao et al [
10] also reported that ESH in mushrooms mainly contributed to the DPPH radical scavenging capacity. While Liu et al [
19] found that, after isolating ESH from mushrooms, the antioxidant ability of ESH accounted for about 25% of the total antioxidant ability of the extract. In current study, to clarify whether ESH in
F. velutipes extract plays a major role in antioxidation, we set up an AE group where authentic ESH (purity ≥98%) at a level of 0.12 g/kg (this amount of ESH was same as that of
F. velutipes extract) was added to sausage. As shown in
Table 1 and
2, compared to AE group, there was an increasing trend in the numerical values of TBARS and carbonyl in EEME group, but no significant difference was found between group EEME and group AE at day 0, 3, 7, and 14 refrigerated storage. These results confirmed that ESH mainly contributed to the antioxidative activity of
F. velutipes extract.
As shown in
Figure 1, chemically, ESH is an unusual thiol-histidine betaine with a sulfur atom linked to position 2 of imidazole ring [
3,
6]. It has distinctive features that are markedly different from ordinary thiols like GSH, for example, under physiological pH, ESH does not oxidize automatically as rapidly as GSH, and it does not promote the production of hydroxyl radicals by H
2O
2 and Fe
2+ ions [
21]. Another important characteristic of ESH is that the standard redox potential of thiol-disulfide couple of ESH at pH 7 is −0.06 V, while other widely occurring natural thiols typically range from −0.20 to −0.32 V [
22]. Because of these excellent characteristics, ESH revealed strong antioxidant capacity to control oxidative damage.
In the process of meat production, various physical operations (such as grinding, cooking, emulsification, and deboning) can cause muscle membrane system disruption, which is conducive to the oxidation reaction in meat. It is well accepted that polyunsaturated fatty acids in meat are beneficial to oxidation because the double bond in polyunsaturated fatty acids is an ideal initiator in the oxidation process, it reacts with oxygen and other fatty acids in the atmosphere to form hydrogen peroxide and free radicals [
2,
23]. This process continues until production of final products, such as aldehydes, ketones, and hexanes, which cannot further support the oxidation cycle [
23]. Meanwhile, lipid oxidation and free radicals could promote protein oxidation occurring in meat and result in protein carbonylation, polymerization and coagulation [
24]. In addition to the composition and content of lipids and proteins in meat, the oxidation process is also affected by other factors, such as light, heat, metal ions, heme pigments, low pH, oxidative enzymes, sodium chloride and other ingredients [
2,
23]. So, the substances which could act as free radical terminators (e.g. BHA and BHT), free radical preventors (e.g. metal complexing agents ethylenediaminetetraacetic acid) and redox compounds (e.g. cysteine and ascorbic acid) could retard the oxidative damage in meat [
23]. Many researchers did good work on antioxidative mechanism of ESH, they demonstrated that ESH is an active scavenger of free radicals, including H
2O
2, •OH,
1O
2, lipid peroxides, nitric oxide derivatives and superoxide ion [
3]. Meanwhile, earlier studies discovered that ESH could chelate divalent metal cations, including Cu
2+, Hg
2+, Zn
2+, Cd
2+, Co
2+, Fe
2+, and Ni
2+ [
25], and the Cu
2+ could form a most stable complex with ESH in the form at a molar ratio of 2:1 of ESH to metal ion [
25]. In the meat system, the ability of binding these metal ions could help to decrease the generation of reactive oxygen species (ROS). Encarnacion et al [
12], that ESH interacted directly with Cu
2+ at the putative binding sites of polyphenoloxidase, based on this reaction, ESH could prevent shrimp from melanosis. Therefore, there is a strong possibility that, during emulsified sausage processing and storage, ESH exerts the antioxidant activities through donating hydrogen to the free radicals and controlling the production of ROS through chelating metal ions, acting alone or in combination. While further studies are needed to better understand the antioxidant mechanism of ESH in meat system.
Volatile compounds
It is established that flavor is one of the most important quality attributes contributing to the eating quality of meat. In current study, using SPME-GC-MS, 36 volatile compounds were identified during the storage of emulsified sausage (
Table 3), they were classified into 6 groups including aldehydes (6), hydrocarbons (10), ketones (2), alcohols (1), esters (14), and aromatics (3). Among them, aldehydes were the main component of which hexanal dominated. As described by previous work, the level of off-flavor can be an ultimate factor determining consumer purchases [
23], and the lipid oxidation in meat matrix was associated with off-flavor [
26], furthermore, it was shown that the hexanal content was widely used as indicator of lipid oxidation in meat system [
26]. As shown in
Table 3, at day 0, 3, 7, and 14, the concentrations of hexanal in sausages from groups BHA, AE, and EEME were significantly (p<0.05) lower than those from control group, while, no significant difference was found among groups BHA, AE, and EEME. In terms with pentanal, at day 0, 3, 7, and 14, the concentrations of pentanal in sausages from groups BHA, AE, and EEME were significantly (p<0.05) lower than those from control group. These results were in line with the findings of Frankel and Tappel [
27], who concluded that the amounts of pentanal correlated well with the corresponding amounts of hexanal in meat. These data confirmed our above observations in lipid oxidation measured by TBARS, and they were also consistent with sensory evaluation of sausage.
Volatile hydrocarbons have a relatively high aroma threshold [
28], so, they may not have a major impact on meat flavor, and there is evidence that volatile hydrocarbons are produced during lipid peroxidation [
23]. In present study, at day 0, 3, 7, and 14, the total hydrocarbon amounts in sausages from EEME group was significantly (p<0.05,
Table 3) lower than other groups. These results agreed with the TBARS values shown in
Table 1. It is reported that volatile alcohols are usually associated with fresh, fruity and fatty aromas [
29], while the aroma threshold of alcohols is higher than that of aldehydes and ketones [
30]. In this study, large amounts of ethanol were found in all groups, but there was no difference among groups control, BHA, AE, and EEME. The alcohol-derived volatiles in sausages should be related to the wine added during manufacturing. It is described that most of volatile ketones have milky and fruity aroma [
16], while they poorly contribute to the flavor of sausage due to the high aroma threshold value. In current study, only two kinds of ketones were detected, they were 2,3-octanedione and acetophenone. As shown in
Table 3, at day 3, 7, and 14, the 2,3-octanedione amounts in sausages from groups BHA, AE, and EEME were significantly (p<0.05) lower than those from control group. Based on previous studies, ketones are another major product of lipid oxidation, accordingly, these results coincided with the results of lipid oxidation as shown in
Table 1. From
Table 3, it is shown that, at day 0 and 3, higher amounts of 2-pentyl-furan were detected in EEME group. It is reported that many kinds of mushroom contained 2-pentyl-furan which can provide vegetable-like, earthy and bean aroma [
31]. Therefore, the higher levels of 2-pentyl-furan identified in sausages of EEME group could be derived from mushroom extract. While, at day 7 and 14, the amounts of 2-pentyl-furan in EEME group were lower than control group. As discussed above, this phenomenon can be explained by the lower oxidative reaction occurred in sausages from EEME group, because 2-pentyl furan could be formed from deterioration of lipid and protein at low temperature storage [
31].
It is demonstrated that volatile esters are generally responsible for fruity and flowery smell of food [
16], and they are formed by the interaction of alcohol and free fatty acid produced by lipid oxidation [
32]. During storage of emulsified sausages, 14 kinds of ester compounds were identified in samples from EEME group (
Table 3), and they were dominated by hexanoic acid, ethyl ester (
Table 3), this phenomenon could be attributed to ester compounds contained in mushroom extract [
33], or these ester compounds were formed in other reactions taking place in stored sausage. Overall, above data demonstrated that adding ESH-enriched mushroom extract to sausage could be able to enrich flavor composition and increase content of characteristic flavor of emulsified sausage.