Effect of Saccharomyces uvarum on lipid oxidation and carbonyl compounds in silver carp mince during cold storage

Abstract Fish lipid is highly susceptible to oxidation, resulting in accumulation of toxic substances reactive carbonyl compounds (RCCs), the reduction of nutritional value, and the production of odorous substances. In this study, the effect of yeast (Saccharomyces uvarum) on RCCs, fat acid composition, volatiles, and sensory traits in silver carp mince stored at 4°C was evaluated. Yeast eliminated malondialdehyde, 4‐hydroxyl‐2‐hexenal, and 4‐hydroxyl‐2‐nonenal by about 80%, 68%, and 60%, which increased by about 170%, 340%, and 300% in the control, respectively. Yeast helped retain about 80% of the polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), respectively; only about 53% and 46% of EPA and DHA, respectively, were maintained in the control. Yeast removed off‐odors hexanal, nonanal, and decenal, resulting in enhanced sensory traits. These findings were economically important for improving the quality of fish products. It might present an approach to improve the flavor of fish products.

RCCs were detected in cold stored fish, pork, and beef (Sakai et al., 1997). The RCCs' content in these products increased significantly during cold storage. HHE's content in fresh carp muscle varied from 1128.4 μg/kg to 4873.2 μg/kg (Sakai et al., 1997). HHE's content in yellow herring fillets frozen at −20°C increased from 374.48 ± 238.08 μg/kg to 7541.68 ± 544.36 μg/kg in 28 weeks. Fresh saury contained 1698.8 ± 793.6 μg/kg of HHE, which increased to about 6,200 μg/kg during frozen storage at −20°C for 3 months and increased to 12,000 μg/kg during storage for 12 months (Tanaka et al., 2013). The threshold of toxicological concern (TTC) of HHE and HNE in fish meat is about 1800 μg/kg (for an adult weight of 60 kg and average fish meat intake of 50 g/d) (Papastergiadis et al., 2014); thus, the RCCs' content in a significant portion of fish products is considerably higher than TTC, and their risks of food safety need to be considered.
Numerous technological approaches have been adopted to reduce lipid oxidation in fish products (Song, Liu, Shen, Liu, Shen, You, & Luo, 2011). The use of synthetic or natural antioxidants is one of the most applied employed measures (Shi, Cui, Yin, Cui, Yin, Luo, & Zhou, 2014). However, owing to growing consumer concerns over the possible carcinogenic effects of chemical preservatives, interest in the development of bio-preservation techniques has increased, including the use of natural or controlled microflora to extend the shelf life and improve the quality of foods (Devlieghere, Vermeiren, & Debevere, 2004). Lactobacillus and yeast are the most attractive microflora.
In several reports, Lactobacillus and yeast are characterized by their outstanding antioxidant activity Kakuta et al., 1999;Vieira, Melo, & Ferreira, 2017). Baka found that L. sakei 4,413 could effectively inhibit lipid oxidation in sausages and reduce the thiobarbituric acid reactive substances (TBARS) by about 1 mg/ kg relative to that of the blank control (Baka, Papavergou, Pragalaki, Bloukas, & Kotzekidou, 2011). Zeng, Xia, Jiang, Xia, Jiang, and Yang (2013) indicated that Lactobacillus plantarum 120, Pediococcus pentosaceus 220, and Saccharomyces cerevisiae 22 could effectively inhibit lipid oxidation during fermentation of "Suan Yu," a traditional fermented fish product characterized by a sour taste. The yeast strain of Kluyveromyces marxianus KU140723-02, S. cerevisiae ATCC6037, and Kluyveromyces lactis ATCC34440 screened from kefir exhibited high antioxidant activity (Cho et al., 2018). Moreover, the Chinese traditional fish product "Zao-yu" fermented with yeast (S. cerevisiae) had a floral and fruity aroma. Its fishy and rancid off-odor were inhibited or removed. As previously mentioned, the off-odor of silver carp mince was mainly caused by aldehydes, and yeast typically contains high-activity aldehyde dehydrogenase/reductase, which transforms aldehydes into corresponding acids/alcohol with a lower odor activity (Datta, Annapore, & Timson, 2017;Wang, Xiao, et al., 2017).
Another advantage of yeast strains over Lactobacillus is that yeast is easier to culture.
We compared several strains, including Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, S. cerevisiae, and S. uvarum, about their activity of aldehyde dehydrogenase; the results showed that the S. uvarum had the highest activity (data not shown). Thus, we assumed that yeast S. uvarum could be a good bio-preservative for reducing lipid oxidation, removing RCCs, and inhibiting the production of off-odor in silver carp mince during storage. So the present study evaluates the effect of S. uvarum on preventing lipid in silver carp mince during cold storage from oxidation.
The MDA, HHE, HNE, and fatty acid components were assayed.
Gas chromatography-mass spectrometry (GC-MS) was carried out to detect volatile compounds. Sensory evaluation was further conducted, in order to help develop a new bio-preservative for fish mice.

| Preparation of silver carp mince
Live silver carp (average weight of about 2 kg/fish) was purchased from a local fishery market. The fishes were killed by knocking them on top of the head using a wooden club, and then, the fish was gutted, headed, skinned, and washed (Qin et al., 2016). Mince samples were collected manually and stored at 4°C immediately.

| Effect of yeast on lipid oxidation in silver carp mince
Wet yeast cell was carefully and evenly mixed with the fish mince. About 10 7 cfu/g mince of wet yeast cell was added. The yeasttreated sample (YTM) and the blank control samples (BCM) were packed in polyvinyl chloride bags and then stored in refrigerators at 4°C. Samples were taken randomly for analysis at selected time intervals (0, 24, 48, and 72 hr).

| Determination of HHE and HNE
HHE and HNE were measured in accordance with the literature (Steppeler et al., 2016;Surh & Kwon, 2005) and little modification. 18 ml of 0.1% ascorbic acid solution (dissolved in water) was added to the sample, homogenized at 11,000 rpm for 2 min, and centrifuged (10,000 × g for 10 min at 4°C), and the supernatant was collected. The extraction was repeated analogously, combined the supernatant. Then, the dichloromethane (10 ml) was used to extract for three times, and the organic phase was collected after centrifuged at 10,000 g for 2 min at 4°C, then filtered (Whatman, 595 1/2), and dried at 30°C with nitrogen. 200 μl of BSTFA (N,O-bis(trimethylsilyl) trifluoroacetamide) (Sigma) was added and kept at room temperature (25°C) for 3 hr to derivatize (Steppeler et al., 2016).
After derivatization, 1 μl of solution was analyzed with a gas chromatography on a capillary column (SH-Rtx-5 SIL MS, 30 m × 0.25 mm × 0.25 μm, SHIMADZU) with a MS detector (GCMS-QP2010 Ultra, SHIMADZU). The parameters were set as follows: splitless mode, injector temperature 250°C, carrier gas (Helium) constant flow of 1.0 ml/min, transfer line temperature 280°C, ion source 230°C, EI 70 eV, and oven program: 100°C held for 4 min, increased at 15°C/min to 300°C, and then kept at 300°C for 3 min. Fragment ions with m/z of 157, 186 and m/z of 157, 199 were monitored for HHE and HNE in selective ion monitoring (SIM), respectively (Surh & Kwon, 2005). Used external standard methods to quantify with the same derivatization procedure. The standard HHE and HNE was purchased from Cayman Chemical Co.
The fat acids were quantified using external standard methods (the standards were purchased from Sigma).
GC-MS was conducted in a gas chromatography system equipped with a SH-Rtx-5 SIL MS capillary column and an electron ionization ion source operated at 70 eV. Temperature programming was applied from 35°C to 220°C at a rate of 10°C/min. The volatile compounds (hexanal, octanal, nonanal, decenal, 1-hexanol, 1octen-3-ol, and 2-ethyl-1-hexanol) were quantified using external standard methods (the standards were purchased from Sigma). The results are the means of three measurements (standard deviation ＜8%).

| Sensory evaluation
The mince samples were stored in 100 ml brown bottles with caps for sensory evaluation. Ten trained panelists sniffed the samples, using a scale of 0-10 (with 10 being the strongest) to evaluate the odors (Richards & Hultin, 2000). The average was defined as the final score. In training sessions, assessors smelled the mince, discussing among themselves the most appropriate descriptions and agreeing on the meaning of their elected descriptors (Table 1).

| Statistical analysis
The data represent the mean and standard deviation from three independent experiments. The statistical significance of data was determined using ANOVA in SAS ver. 6.0 (SAS Institute Inc.).

| Effect of yeast on MDA, HHE, and HNE contents in silver carp mince
The MDA is believed to be characteristic decomposition product of unsaturated fatty acid oxidation. During cold storage of the mince, the MDA content of the control (BCM) increased rapidly in 24 hr from 1.36 ± 0.10 mg/kg to 3.51 ± 0.12 mg/kg, indicating the occurrence of serious lipid oxidation ( Figure 1). However, the MDA content of the yeast-treated mince (YTM) was significantly lower than that of BCM (p < 0.05). Notably, yeast markedly decreased the MDA content in the first 24 hr from 1.36 ± 0.10 mg/ kg to 0.31 ± 0.02 mg/kg and then almost remained constant at this low level (Figure 1). The HHE and HNE contents in BCM increased rapidly, from 220.3 ± 13.5 μg/kg and 91.2 ± 7.1 μg/kg to 974.5 ± 19.8 μg/kg and 360.4 ± 15.2 μg/kg, respectively. The HHE and HNE contents in YTM decreased throughout the period of storage and were significantly lower than those of the control (p < 0.05). In other words, the yeast eliminated MDA, HHE, and HNE by about 80%, 68%, and 60%, which increased by about 170%, 340%, and 300% in the control, respectively.

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Associated with fresh grass
Comparison between the control and the samples treated with yeast revealed large differences in the intensities of all compounds.
The intensities of all off-odor aldehydes were reduced by yeast except for octanal. The content of hexanal, nonenal, and decanal was below the minimum limit of detection in YTM after cold storage for 3 day; meanwhile, their contents in BCM increased by about 6.3, 0.8, and 2.1 times, respectively. Its fishy off-odor presents a disadvantage for silver carp and thus should be removed by yeast.
Wang also reported that fermentation with L. plantarum and P. pentosaceus significantly decreased the hexanal content of dry-cured hairtail (p ＜ 0.05) (Wang, Xiao, et al., 2017). The octanal contents in BCM and YTM were increased by about 6.5 and 0.8 times, respectively. The 1-hexanol in BCM and YTM was increased by about 28 and 9 times, respectively. The 1-octen-3-ol contents in BCM and YTM were increased by about 4.7 and 1.4 times, respectively. The 2-ethyl-1-hexanol contents in BCM and YTM were increased about 2.2 and 14.8 times, respectively.

| Effect of yeast on the sensory traits of silver carp mince
The sensory traits of fresh silver carp mince were identified as milddelicate grassy, mushroom-like, and sweet-fruity (Figure 2), which is consistent with the results obtained by Fu et al. (2015) and Zhou et al. (2016). The control mince (BCM) in cold storage for 3 days was observed as oxidized oil and fishy in odor, whereas the yeast-treated mince (YTM) was noted as slightly sweet-fruity.
During cold storage, the strength of grassy, fishy, oxidized oil-like, and mushroom-like odors of the control mince increased, whereas the sweet-fruity flavor decreased. With yeast treatment, the strength of grassy, fishy, mushroom-like, and oxidized oil odors was decreased, while the sweet-fruity aroma was increased.
Combined with the results of volatile compounds (Table 3)  Note: BCM-1, BCM-2, and BCM-3, representing blank control mince stored at 4°C for 1, 2, and 3 days, respectively. YTM-1, YTM-2, and YTM-3, representing yeast-treated mince stored at 4°C for 1, 2, and 3 days, respectively. Means in each row having different superscript letters are significantly different (p < 0.05) (n = 3). carp surimi effectively, resulting in improved flavor. The present results show that yeast could not only reduce the off-odor volatile but increase the content of aroma substances as well.

| D ISCUSS I ON
Several studies evaluated the inhibitory effect of antioxidants on the accumulation of RCCs. For instance, 0.6% VE could significantly inhibit increases in MDA and HHE contents in frozen saury (Tanaka et al., 2013). Another report found that for ice-stored yel- nonanal, and decanal with a simple inhibitor (Figure 1 and Table 3) suggested yeast strongly exhibited the capacity to react and/or convert carbonyl compounds to other compounds. Thus, an enzyme mechanism was assumed. Carbonyl compounds transforming enzymes include alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), aldehyde oxidase (AOX), and aldehyde deformylating oxygenase (ADO). ADH catalyzes the reduction of aldehydes; ALDH catalyzes the irreversible oxidation of aldehydes into their corresponding carboxylic acids; and AOX and ADO convert aldehydes into corresponding acids and alkanes, respectively (Foo, Susanto, Keasling, Susanto, & Keasling, 2017). ADH and ALDH have been purified from yeast (Datta et al., 2017;Wang, Wu, & Li, 2017). Yeast can efficiently perform chemo-, regio-and stereo-selective biotransformation of alcohols and aldehydes, affording the corresponding carboxylic acids (Svitel & Sturdik, 1995). Yeast involved in the removal of a wide range of undesirable wort carbonyl compounds during fermentation (Iersel, Brouwer-Post, Rombouts, Brouwer-Post, Rombouts, & Abee, 2000). "Green note" aldehydes were successfully reduced into their corresponding alcohols by commercial yeast ADH and yeast cells (Fauconnier et al., 1999). Furthermore, Lehto et al. (2003) reported that the ALDH-type activity present in oat efficiently decreases hexanal content and prevents the accumulation of short chain aldehydes that cause off-odors. ALDH purified from bovine liver mitochondria was used to remove the "green" odor of soybean products (Sawada, Hara, Nakayama, Hara, & Nakayama, 1982). As far as we know, no studies on the enzymatic conversion of carbonyl compounds in muscle foods have been reported.

| CON CLUS IONS
Lipid oxidation in silver carp mince during cold storage was effectively inhibited by yeast (S. uvarum). Yeast (S. uvarum) decreased MDA, HHE, and HNE and helped retain PUFA. It also removed most of the off-odor compounds. The S. uvarum might be a good bio-preserver for fish mince. These findings were significant because of the economic value of improving the quality of fish products.

ACK N OWLED G M ENT
We would like to thank the Natural Science Foundation of Hunan Province (No. 2019JJ40538) for the support.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.