干腌培根加工过程中脂质氧化调控机制研究
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摘要
干腌肉制品是传统肉制品的代表,因其独特的风味品质深受广大消费者青睐。脂质氧化是干腌肉制品加工过程中风味形成的必要途径,也是影响产品安全品质的重要因素；因此,干腌肉制品脂质氧化机理研究一直是肉品科学研究的一个重要方向,但有关干腌肉制品脂质氧化调控机制的研究才刚刚起步。本课题是在十一五国家科技支撑计划2006BAD05A15“低温肉制品与传统肉制品开发及产业化示范”传统干腌肉制品研究专题基础上的进一步延伸,旨在研究干腌培根腌制、风干成熟过程脂质分解氧化规律,明确其脂质氧化的机理并探索其调控机制,为干腌肉制品加工过程中调控脂质氧化、提高风味品质提供理论依据。具体研究内容和结果如下：
1.干腌培根腌制、风干成熟过程中脂质分解氧化规律研究
以五花肉为原料,经腌制、风干成熟制成干腌培根。跟踪测定加工过程样品肌肉脂肪组成、过氧化值(POV)和硫代巴比妥酸反应底物值(TBARS)变化规律,研究脂肪酶及脂肪氧合酶(LOX)活性变化与其相关性。结果表明：加工过程肌肉脂质中游离脂肪酸相对含量显著上升(P<0.05),与磷脂相对含量显著下降(P<0.05),二者显著负相关(r=-0.85,P=0.0004<0.01)；酸性脂酶、中性脂酶和磷脂酶活性在加工过程中总体上均呈显著下降趋势(P<0.05),酸性脂酶活性显著高于中性脂酶(P<0.05),而磷脂酶活性下降幅度最大,到成熟结束时降幅达90.9%。LOX活性在腌制过程中显著升高(P<0.01),腌制结束时达到最大(29.4 U/minxg蛋白),然后在风干成熟过程中逐渐下降,与TBARS呈正相关(r=0.94); POV和TBARS在整个加工过程中都经历一个先增后降的变化过程,分别在风干成熟第6天和腌制结束时达到最大值(0.148 meq/kg lipid和0.28 mg MDA/kg肌肉),然后在风干成熟后期(6-12天)显著下降(P<0.01)。这些结果说明：干腌培根腌制、风干成熟过程中,尤其是腌制阶段,较高盐分能够抑制酸性脂酶、磷脂酶的活性,但可促进LOX的活性；合理调控工艺时间和温度、盐分等工艺调控因子可以降低最终产品的POV和TBARS,提高产品的风味品质。
2.肌肉脂质氧化中的酶促氧化和非酶促氧化研究
采用LOX提取方法除去五花肉肌肉中的LOX,然后以去LOX后的肌肉为原料,通过设定加LOX提取液和空白对照在光照和避光条件下氧化试验,以POV、TBARS和LOX活性为考察指标,研究肌肉脂质氧化中的酶促氧化和非酶促氧化。结果表明：肌肉脂质氧化过程中非酶促氧化占主导地位,二者可共同解释氧化总变异的89%,还有11%的氧化变化未能被解释,表明二者同时存在时对肌肉脂质氧化可能具有一定协同作用；在氧化过程中酶促氧化与非酶促氧化共同存在的全氧化处理组LOX活性下降速度大于酶促氧化处理组,表明在肌肉中较高浓度氧化产物会抑制LOX活性。
3.脂肪氧合酶主要酶学特性及工艺调控因子对其活性的调控机理研究
从新鲜五花肉肌肉中提取脂肪氧合酶(LOX)粗酶,然后以亚油酸为底物研究其主要酶学特性；在单因素试验基础上,采用响应曲面试验方法(RSM)研究了温度、NaCl浓度以及缓冲液pH对LOX活性影响的交互作用,并通过分析工艺调控因子对LOX活性影响的交互作用来探究其调控机制。研究结果表明：LOX催化亚油酸氧化的最适反应底物浓度为3.47 mM、最适温度为40℃、最适pH≧9.0, Km=68μm Vmax=0.26 U/min; NaCl对LOX活性影响也存在一临界值,在20℃条件下为3.1%(w/w),高于此值会对LOX活性产生抑制作用。温度与NaCl及pH对LOX活性存在显著交互作用(P<0.05)；温度临界值随着酶液中NaCl浓度的逐渐增大呈线性下降趋势,而随着pH的升高逐渐升高,NaCl临界值随着温度的升高也呈线性降低。这些结果表明降低腌制用盐量,且在风干成熟前期提高并加快温度上升速率可促进培根中LOX活性,加快风味前体化合物—脂质氢过氧化物形成；在后期继续升高温度,盐分含量快速升高,但与此同时盐分临界值被降低,酶活被抑制,氢过氧化物形成速率降低,但温度的升高会提高氢过氧化物进一步分解的速率,从而有效降低产品氧化指标、促进风味形成。
4.干腌培根加工过程中抗氧化酶对脂质氧化的作用及工艺调控因子对其活性影响
测定干腌培根加工过程中POV、TBARS及过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)和超氧化物歧化酶(SOD)活性变化,通过分析抗氧化酶活性变化与脂质氧化的相关性来研究抗氧化酶对脂质氧化的作用,并采用响应曲面试验方法研究工艺调控因子对抗氧化酶活性的影响。结果表明：三种抗氧化酶活性与POV (P> 0.05)及TBARS (P<0.05)都呈负相关,且随温度升高相关性都逐渐减弱。三种抗氧化酶的活性在整个加工过程中都持续下降,其中GSH-Px最不稳定,其次是CAT；在影响LOX活性的温度临界值范围内(<40℃),较高温度能够促进抗氧化酶活性,使其降低幅度减小,但是盐分对抗氧化酶活性始终起抑制作用,且随浓度增大抑制作用逐渐增强；干腌培根加工过程中抗氧化酶活性是温度促进作用与盐分抑制作用交互作用的结果,增大盐分含量能够降低温度对抗氧化酶的促进作用,而升高温度能够促进盐分对抗氧化酶活性的抑制作用。这些结果表明,在干腌培根加工过程中抗氧化酶对调控肌肉脂质氧化有重要影响；较低水平腌制用盐量情况下、大幅提高工艺温度能够抑制抗氧化酶活性,加快氢过氧化物分解,降低产品氧化指标POV和TBARS,促进风味化合物形成。
5.干腌培根脂质氧化工艺调控机制研究
通过单因素实验测定不同温度和盐分处理组干腌培根的POV和TBARS值,研究肌肉中脂质一次氧化和二次氧化的动力学特性及不同盐分含量对其影响；采用响应曲面试验方法研究工艺调控因子对脂质一次氧化速率的交互作用并对脂质一次氧化速率调控方法进行优化,结果表明：在温度<40℃,NaCl添加量<5.0%(w/w)范围内,温度和NaCl对肌肉脂质氧化均有显著促进作用(P<0.05),并且温度的影响大于NaCl;肌肉脂质一次氧化所需活化能(Ea,92.35 kJ/mol)大于二次氧化(65.66 kJ/mol),添加NaCl(<5%, w/w)能够降低各级脂质氧化的Ea,其中NaCl添加量分别在3.1%和3.4%水平时,对应脂质一次和二次氧化Ea最低；温度和盐分含量对干腌培根加工过程中肌肉脂质氧化速率有极显著(P<0.001)交互作用,影响脂质氧化的NaCl临界浓度随温度升高呈线趋势降低,35℃高温及2.77%(w/w)腌制用盐量对应脂质一次氧化速率最快(Tmax=1.03天),说明加工过程中提高温度,降低腌制用盐量能加快干腌培根肌肉脂质一次氧化速率,使POV的变化拐点提前形成,为脂质二次氧化降低产品氧化指标,提高风味品质提供更多时间。
Dry-cured meat products are the representative of traditional meat products, which win warm praise from the masses of consumers. Lipid oxidation is a necessary pathway of the flavour development in dry-cured meat products during processing, furthermore, it is also an important factor influencing the safety and quality of dry-cured meat products. So, studies on lipid oxidation mechanism in dry-cured meat products have always been an important research direction in meat science reaearch area. However, studies on the regulation mechanism of lipid oxidation in dry-cured meat products during processing have just started. This topic is a further extension on the basis of special studies on traditional dry-cured meat products of national scientific supporting program 2006BAD05A15 "Product development and industrialization of low-temperature and traditional meat products". The main aim of this topic is to study the lipolysis and lipid oxidation laws during dry-cured bacon salting and drying-ripening, make clear the lipid oxidation mechanism and to explore the regulation mechanism of lipid oxidation; and through all these studies, to provide a theoretical basis for regulating lipid oxidation and improving flavour quality during dry-cured meat product processing. The detailed contents and results are as follows.
1. Studies on lipolysis and lipid oxidation in bacon during curing and drying-ripening
Dry-cured bacons were processed using bacon belly as material by dry-salting and drying-ripening. The change rules of lipid composition, peroxide value (POV) and thiobarbituric acid reactive substance value (TBARS) in bacon sample muscles were tracked and measured during processing. Furthermore, activity changes of lipolytic enzymes and lipoxygenase (LOX) as well as their correlations with lipolytic and oxidative changes of muscle lipid were investigated. The resulrs showed that free fatty acid content significantly inceased during processing, which was negatively correlated with the significant decrease (P<0.05) in phospholipids content (r=-0.85, P<0.01). All the activities of acid lipase, neutral lipase and phospholipase decreased significantly during processing (P< 0.05), and acid lipase showed higher activity than did neutral lipase (P< 0.05) throughout the whole processing. Among all the three lipolytic enzymes, the activity decrease amplitude of phospholipase was the largest, which reached 90.9% until the end of drying-ripening. LOX activity increased significantly and reached a peak (29.4 U/minxg protein) until the end of salting (P<0.01), thereafter gradually decreased during drying-ripening period, which was positively correlated with TBARS (r=0.94). Both POV and TBARS values increased initially and reached maximal values (0.148 meq/kg lipid and 0.28 mg MDA/kgmuscle) at drying ripening 6 days and end of salting, respectively, and then decreased. All these results indicated that high salt content inhibited acid lipase and phospholipase activities but promoted LOX activity during salting period. And properly controlling processing time and other regulation factors, such as temperature and salt content, could decrease the oxidative indices of final products, and thus inproving the safety and flavour quality of the products.
2. Studies on enzymatic and non-enzymatic lipid oxidation in muscle lipid oxidation
The LOX was removed from fresh bacon belly muscles by the method of LOX extraction. Then the minced muscle removed of LOX was used as material, the enzymatic and non-enzymatic lipid oxidation in the muscles were evaluated by measuring POV, TBARS and LOX activity of different experimental treatments, which set the muscles as added LOX extracts and control (not added LOX extracts) and both the samples were oxidized at two different conditions (in dark and under illumination), respectively. The results showed that, during the muscle lipid oxidation period, non-enzymatic oxidation played a dominant role in the overall lipid oxidation phenomenon of muscle lipid. Non-enzymatic and enzymatic oxidation totally explained about 89% of the total lipid oxidation variation in belly muscle and there remain have 11% of lipid oxidation variation could not be explained, suggesting that when enzymatic and non-enzymatic oxidation jointly functioned in the belly, there may be a synergistic effect on muscle lipid oxidation existed between them. In addition, during the oxidation period, the LOX activity decreased more quickly in the muscle samples of full oxidation group, in which enzymatic and non-enzymatic oxidation were functioned together, than did the LOX in the muscles of enzymatic group, indicating that the high content oxidative products could inhibit the activity of LOX.
3. Studies on main enzymatic properties of muscle LOX and the regulation mechanism of process factors on its activity
Crude lipoxygenase (LOX) was extracted from fresh bacon belly and studied of some main characteristics using linoleic acid as substrate. Based on the results of single-factor experiments, the interactions of temperature, sodium chloride (NaCl) and pH on LOX activity were investigated by response surface methodology (RSM), and the regulation method of LOX activity was explored by analyzing the interactive effects of process factors on LOX activity. The results showed that the optimum substrate concentration, temperature and pH value for LOX catalyzing linoleic acid oxidation were 3.47 mM,40℃and≧9.0, respectively, and the Michaelis-Menten constant (Km) and maximum velocity (Vmax) were 68μM and 0.26 U/min, respectively. The NaCl also existed a critical value for LOX activity, which was determined as 3.1%(w/w) at 20℃, and above which inhibited the LOX activity. Temperature had significant interactions (P<0.05) with NaCl and pH. The temperature critical value decreased linearly with NaCl content increasing in the enzyme solution, while increased with pH increasing. The NaCl critical value for LOX activity also decreased linearly with temperature increasing. All these results suggested that decreasing salt added amount at salting stage and increasing temperature during early drying-ripening period could promote LOX activity, which will accelerate lipid oxidation and promote the formation of flavour precursors (lipid hydroperoxides). In addition, during the last drying-ripening stage, the salt content quickly increases with continuous increase in temperature, but meanwhile the NaCl critical value decreases with temperature increasing, therefore LOX activity will be inhibited and the formation rate of flavour precursors will decrease, while their further decomposition will be accelerated, which could promote the flavour compounds development.
4. Effect of antioxidant enzymes on lipid oxidation during dry-cured bacon processing and the influence of processing factors on antioxidant enzyme activities.
Both the oxidative stability and antioxidant enzyme stability changes were evaluated by assessing the changes of POV, TBARS and activities of catalase, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) during dry-cured bacon processing. The effect of antioxidant enzyme activities on muscle lipid oxidation were investigated by analyzing the correlations between the changes of antioxidant enzyme activities and lipid oxidation stability during dry-cured bacon processing. And the effects of process factors on antioxidant enzyme activities were investigated by response surface methodology. The results showed that all three antioxidant enzyme activities were negatively correlated with POV (P> 0.05) and TBARS values (P<0.05), and the correlations decreased with temperature increasing. The activities of all three antioxidant enzyme continuously decreased (P< 0.05) untill the end of process, among which GSH-Px was the most unstable one followed by catalase. Within the temperature range of lower than the temperature critical value for LOX (40℃), the higher drying-ripening temperature was in favor of all the antioxidant enzyme activities. But NaCl always showed an inhibitory effect on all the antioxidant enzyme activities, and which was concentration dependent. The antioxidant enzyme activities in dry-cured bacon during processing were influenced interactively by the temperature promoting effect and salt inhibitory effect. Increasing salt content in bacon decreased the promoting effect of temperature on antioxidant enzyme activities, but raising temperature enhanced the inhibitory effect of salt on antioxidant enzyme activities. All the above results indicated that antioxidant enzymes have important influence on controlling lipid oxidation in muscle. And, under the condition of low salt (NaCl) added level, greatly increasing processing temperature could inhibit antioxidant enzyme activities, accelerate the decomposition lipid hydroperoxides, decrease oxidation indices (POV and TBARS) and promote flavour development.
5. Studies on process reulation mechanism of lipid oxidation in dry-cured bacon
Kinetic characteristics of primary and secondary lipid oxidation in dry-cured bacon and the influence of different NaCl contents on them were investigated by measuring POV and TBARS values of different bacons treated with different temperatures or NaCl levels according to temperature and NaCl single-factor experiments. The interactive effect of process factors on lipid oxidation rate was further studied using response surface methodology, and the regulation method of primary lipid oxidation was optimized. The results indicated that, within the range of temperature lower than 40℃and NaCl added level lower than 5.0%(w/w), both temperature and NaCl showed significant (P<0.05) prooxidant effects, but temperature showed relatively greater effect than did NaCl. The activation energy (Ea,92.35 kJ/mol) for POV was higher than that (65.66 kJ/mol) for TBARS. Adding NaCl (<5%, w/w) could decrease both the Ea, and when NaCl added level were 3.1% and 3.4%, respectively, the corresponding Ea for primary and secondary lipid oxidation were the lowest. Temperature and NaCl had extremely significant (p<0.001) interactive on primary lipid oxidation rate. And elevating temperature could significantly (p<0.05) and linearly (y=-0.067x+5.113) decrease the threshold value of NaCl concentration influencing lipid oxidation during dry-cured bacon processing. When temperature was 35℃and NaCl level was 2.77%, the primary oxidation rate was the fastest. These results suggested that raising temperature and decreasing salt used amount could accelerate primary lipid oxidation rate in dry-cured bacon, which will make the change inflexions of POV and TBARS advanced, and thus provide more times for decreasing oxidation indices and improving flavour quality of products by secondary or more further oxidation.
1.干腌培根腌制、风干成熟过程中脂质分解氧化规律研究
以五花肉为原料,经腌制、风干成熟制成干腌培根。跟踪测定加工过程样品肌肉脂肪组成、过氧化值(POV)和硫代巴比妥酸反应底物值(TBARS)变化规律,研究脂肪酶及脂肪氧合酶(LOX)活性变化与其相关性。结果表明：加工过程肌肉脂质中游离脂肪酸相对含量显著上升(P<0.05),与磷脂相对含量显著下降(P<0.05),二者显著负相关(r=-0.85,P=0.0004<0.01)；酸性脂酶、中性脂酶和磷脂酶活性在加工过程中总体上均呈显著下降趋势(P<0.05),酸性脂酶活性显著高于中性脂酶(P<0.05),而磷脂酶活性下降幅度最大,到成熟结束时降幅达90.9%。LOX活性在腌制过程中显著升高(P<0.01),腌制结束时达到最大(29.4 U/minxg蛋白),然后在风干成熟过程中逐渐下降,与TBARS呈正相关(r=0.94); POV和TBARS在整个加工过程中都经历一个先增后降的变化过程,分别在风干成熟第6天和腌制结束时达到最大值(0.148 meq/kg lipid和0.28 mg MDA/kg肌肉),然后在风干成熟后期(6-12天)显著下降(P<0.01)。这些结果说明：干腌培根腌制、风干成熟过程中,尤其是腌制阶段,较高盐分能够抑制酸性脂酶、磷脂酶的活性,但可促进LOX的活性；合理调控工艺时间和温度、盐分等工艺调控因子可以降低最终产品的POV和TBARS,提高产品的风味品质。
2.肌肉脂质氧化中的酶促氧化和非酶促氧化研究
采用LOX提取方法除去五花肉肌肉中的LOX,然后以去LOX后的肌肉为原料,通过设定加LOX提取液和空白对照在光照和避光条件下氧化试验,以POV、TBARS和LOX活性为考察指标,研究肌肉脂质氧化中的酶促氧化和非酶促氧化。结果表明：肌肉脂质氧化过程中非酶促氧化占主导地位,二者可共同解释氧化总变异的89%,还有11%的氧化变化未能被解释,表明二者同时存在时对肌肉脂质氧化可能具有一定协同作用；在氧化过程中酶促氧化与非酶促氧化共同存在的全氧化处理组LOX活性下降速度大于酶促氧化处理组,表明在肌肉中较高浓度氧化产物会抑制LOX活性。
3.脂肪氧合酶主要酶学特性及工艺调控因子对其活性的调控机理研究
从新鲜五花肉肌肉中提取脂肪氧合酶(LOX)粗酶,然后以亚油酸为底物研究其主要酶学特性；在单因素试验基础上,采用响应曲面试验方法(RSM)研究了温度、NaCl浓度以及缓冲液pH对LOX活性影响的交互作用,并通过分析工艺调控因子对LOX活性影响的交互作用来探究其调控机制。研究结果表明：LOX催化亚油酸氧化的最适反应底物浓度为3.47 mM、最适温度为40℃、最适pH≧9.0, Km=68μm Vmax=0.26 U/min; NaCl对LOX活性影响也存在一临界值,在20℃条件下为3.1%(w/w),高于此值会对LOX活性产生抑制作用。温度与NaCl及pH对LOX活性存在显著交互作用(P<0.05)；温度临界值随着酶液中NaCl浓度的逐渐增大呈线性下降趋势,而随着pH的升高逐渐升高,NaCl临界值随着温度的升高也呈线性降低。这些结果表明降低腌制用盐量,且在风干成熟前期提高并加快温度上升速率可促进培根中LOX活性,加快风味前体化合物—脂质氢过氧化物形成；在后期继续升高温度,盐分含量快速升高,但与此同时盐分临界值被降低,酶活被抑制,氢过氧化物形成速率降低,但温度的升高会提高氢过氧化物进一步分解的速率,从而有效降低产品氧化指标、促进风味形成。
4.干腌培根加工过程中抗氧化酶对脂质氧化的作用及工艺调控因子对其活性影响
测定干腌培根加工过程中POV、TBARS及过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)和超氧化物歧化酶(SOD)活性变化,通过分析抗氧化酶活性变化与脂质氧化的相关性来研究抗氧化酶对脂质氧化的作用,并采用响应曲面试验方法研究工艺调控因子对抗氧化酶活性的影响。结果表明：三种抗氧化酶活性与POV (P> 0.05)及TBARS (P<0.05)都呈负相关,且随温度升高相关性都逐渐减弱。三种抗氧化酶的活性在整个加工过程中都持续下降,其中GSH-Px最不稳定,其次是CAT；在影响LOX活性的温度临界值范围内(<40℃),较高温度能够促进抗氧化酶活性,使其降低幅度减小,但是盐分对抗氧化酶活性始终起抑制作用,且随浓度增大抑制作用逐渐增强；干腌培根加工过程中抗氧化酶活性是温度促进作用与盐分抑制作用交互作用的结果,增大盐分含量能够降低温度对抗氧化酶的促进作用,而升高温度能够促进盐分对抗氧化酶活性的抑制作用。这些结果表明,在干腌培根加工过程中抗氧化酶对调控肌肉脂质氧化有重要影响；较低水平腌制用盐量情况下、大幅提高工艺温度能够抑制抗氧化酶活性,加快氢过氧化物分解,降低产品氧化指标POV和TBARS,促进风味化合物形成。
5.干腌培根脂质氧化工艺调控机制研究
通过单因素实验测定不同温度和盐分处理组干腌培根的POV和TBARS值,研究肌肉中脂质一次氧化和二次氧化的动力学特性及不同盐分含量对其影响；采用响应曲面试验方法研究工艺调控因子对脂质一次氧化速率的交互作用并对脂质一次氧化速率调控方法进行优化,结果表明：在温度<40℃,NaCl添加量<5.0%(w/w)范围内,温度和NaCl对肌肉脂质氧化均有显著促进作用(P<0.05),并且温度的影响大于NaCl;肌肉脂质一次氧化所需活化能(Ea,92.35 kJ/mol)大于二次氧化(65.66 kJ/mol),添加NaCl(<5%, w/w)能够降低各级脂质氧化的Ea,其中NaCl添加量分别在3.1%和3.4%水平时,对应脂质一次和二次氧化Ea最低；温度和盐分含量对干腌培根加工过程中肌肉脂质氧化速率有极显著(P<0.001)交互作用,影响脂质氧化的NaCl临界浓度随温度升高呈线趋势降低,35℃高温及2.77%(w/w)腌制用盐量对应脂质一次氧化速率最快(Tmax=1.03天),说明加工过程中提高温度,降低腌制用盐量能加快干腌培根肌肉脂质一次氧化速率,使POV的变化拐点提前形成,为脂质二次氧化降低产品氧化指标,提高风味品质提供更多时间。
Dry-cured meat products are the representative of traditional meat products, which win warm praise from the masses of consumers. Lipid oxidation is a necessary pathway of the flavour development in dry-cured meat products during processing, furthermore, it is also an important factor influencing the safety and quality of dry-cured meat products. So, studies on lipid oxidation mechanism in dry-cured meat products have always been an important research direction in meat science reaearch area. However, studies on the regulation mechanism of lipid oxidation in dry-cured meat products during processing have just started. This topic is a further extension on the basis of special studies on traditional dry-cured meat products of national scientific supporting program 2006BAD05A15 "Product development and industrialization of low-temperature and traditional meat products". The main aim of this topic is to study the lipolysis and lipid oxidation laws during dry-cured bacon salting and drying-ripening, make clear the lipid oxidation mechanism and to explore the regulation mechanism of lipid oxidation; and through all these studies, to provide a theoretical basis for regulating lipid oxidation and improving flavour quality during dry-cured meat product processing. The detailed contents and results are as follows.
1. Studies on lipolysis and lipid oxidation in bacon during curing and drying-ripening
Dry-cured bacons were processed using bacon belly as material by dry-salting and drying-ripening. The change rules of lipid composition, peroxide value (POV) and thiobarbituric acid reactive substance value (TBARS) in bacon sample muscles were tracked and measured during processing. Furthermore, activity changes of lipolytic enzymes and lipoxygenase (LOX) as well as their correlations with lipolytic and oxidative changes of muscle lipid were investigated. The resulrs showed that free fatty acid content significantly inceased during processing, which was negatively correlated with the significant decrease (P<0.05) in phospholipids content (r=-0.85, P<0.01). All the activities of acid lipase, neutral lipase and phospholipase decreased significantly during processing (P< 0.05), and acid lipase showed higher activity than did neutral lipase (P< 0.05) throughout the whole processing. Among all the three lipolytic enzymes, the activity decrease amplitude of phospholipase was the largest, which reached 90.9% until the end of drying-ripening. LOX activity increased significantly and reached a peak (29.4 U/minxg protein) until the end of salting (P<0.01), thereafter gradually decreased during drying-ripening period, which was positively correlated with TBARS (r=0.94). Both POV and TBARS values increased initially and reached maximal values (0.148 meq/kg lipid and 0.28 mg MDA/kgmuscle) at drying ripening 6 days and end of salting, respectively, and then decreased. All these results indicated that high salt content inhibited acid lipase and phospholipase activities but promoted LOX activity during salting period. And properly controlling processing time and other regulation factors, such as temperature and salt content, could decrease the oxidative indices of final products, and thus inproving the safety and flavour quality of the products.
2. Studies on enzymatic and non-enzymatic lipid oxidation in muscle lipid oxidation
The LOX was removed from fresh bacon belly muscles by the method of LOX extraction. Then the minced muscle removed of LOX was used as material, the enzymatic and non-enzymatic lipid oxidation in the muscles were evaluated by measuring POV, TBARS and LOX activity of different experimental treatments, which set the muscles as added LOX extracts and control (not added LOX extracts) and both the samples were oxidized at two different conditions (in dark and under illumination), respectively. The results showed that, during the muscle lipid oxidation period, non-enzymatic oxidation played a dominant role in the overall lipid oxidation phenomenon of muscle lipid. Non-enzymatic and enzymatic oxidation totally explained about 89% of the total lipid oxidation variation in belly muscle and there remain have 11% of lipid oxidation variation could not be explained, suggesting that when enzymatic and non-enzymatic oxidation jointly functioned in the belly, there may be a synergistic effect on muscle lipid oxidation existed between them. In addition, during the oxidation period, the LOX activity decreased more quickly in the muscle samples of full oxidation group, in which enzymatic and non-enzymatic oxidation were functioned together, than did the LOX in the muscles of enzymatic group, indicating that the high content oxidative products could inhibit the activity of LOX.
3. Studies on main enzymatic properties of muscle LOX and the regulation mechanism of process factors on its activity
Crude lipoxygenase (LOX) was extracted from fresh bacon belly and studied of some main characteristics using linoleic acid as substrate. Based on the results of single-factor experiments, the interactions of temperature, sodium chloride (NaCl) and pH on LOX activity were investigated by response surface methodology (RSM), and the regulation method of LOX activity was explored by analyzing the interactive effects of process factors on LOX activity. The results showed that the optimum substrate concentration, temperature and pH value for LOX catalyzing linoleic acid oxidation were 3.47 mM,40℃and≧9.0, respectively, and the Michaelis-Menten constant (Km) and maximum velocity (Vmax) were 68μM and 0.26 U/min, respectively. The NaCl also existed a critical value for LOX activity, which was determined as 3.1%(w/w) at 20℃, and above which inhibited the LOX activity. Temperature had significant interactions (P<0.05) with NaCl and pH. The temperature critical value decreased linearly with NaCl content increasing in the enzyme solution, while increased with pH increasing. The NaCl critical value for LOX activity also decreased linearly with temperature increasing. All these results suggested that decreasing salt added amount at salting stage and increasing temperature during early drying-ripening period could promote LOX activity, which will accelerate lipid oxidation and promote the formation of flavour precursors (lipid hydroperoxides). In addition, during the last drying-ripening stage, the salt content quickly increases with continuous increase in temperature, but meanwhile the NaCl critical value decreases with temperature increasing, therefore LOX activity will be inhibited and the formation rate of flavour precursors will decrease, while their further decomposition will be accelerated, which could promote the flavour compounds development.
4. Effect of antioxidant enzymes on lipid oxidation during dry-cured bacon processing and the influence of processing factors on antioxidant enzyme activities.
Both the oxidative stability and antioxidant enzyme stability changes were evaluated by assessing the changes of POV, TBARS and activities of catalase, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) during dry-cured bacon processing. The effect of antioxidant enzyme activities on muscle lipid oxidation were investigated by analyzing the correlations between the changes of antioxidant enzyme activities and lipid oxidation stability during dry-cured bacon processing. And the effects of process factors on antioxidant enzyme activities were investigated by response surface methodology. The results showed that all three antioxidant enzyme activities were negatively correlated with POV (P> 0.05) and TBARS values (P<0.05), and the correlations decreased with temperature increasing. The activities of all three antioxidant enzyme continuously decreased (P< 0.05) untill the end of process, among which GSH-Px was the most unstable one followed by catalase. Within the temperature range of lower than the temperature critical value for LOX (40℃), the higher drying-ripening temperature was in favor of all the antioxidant enzyme activities. But NaCl always showed an inhibitory effect on all the antioxidant enzyme activities, and which was concentration dependent. The antioxidant enzyme activities in dry-cured bacon during processing were influenced interactively by the temperature promoting effect and salt inhibitory effect. Increasing salt content in bacon decreased the promoting effect of temperature on antioxidant enzyme activities, but raising temperature enhanced the inhibitory effect of salt on antioxidant enzyme activities. All the above results indicated that antioxidant enzymes have important influence on controlling lipid oxidation in muscle. And, under the condition of low salt (NaCl) added level, greatly increasing processing temperature could inhibit antioxidant enzyme activities, accelerate the decomposition lipid hydroperoxides, decrease oxidation indices (POV and TBARS) and promote flavour development.
5. Studies on process reulation mechanism of lipid oxidation in dry-cured bacon
Kinetic characteristics of primary and secondary lipid oxidation in dry-cured bacon and the influence of different NaCl contents on them were investigated by measuring POV and TBARS values of different bacons treated with different temperatures or NaCl levels according to temperature and NaCl single-factor experiments. The interactive effect of process factors on lipid oxidation rate was further studied using response surface methodology, and the regulation method of primary lipid oxidation was optimized. The results indicated that, within the range of temperature lower than 40℃and NaCl added level lower than 5.0%(w/w), both temperature and NaCl showed significant (P<0.05) prooxidant effects, but temperature showed relatively greater effect than did NaCl. The activation energy (Ea,92.35 kJ/mol) for POV was higher than that (65.66 kJ/mol) for TBARS. Adding NaCl (<5%, w/w) could decrease both the Ea, and when NaCl added level were 3.1% and 3.4%, respectively, the corresponding Ea for primary and secondary lipid oxidation were the lowest. Temperature and NaCl had extremely significant (p<0.001) interactive on primary lipid oxidation rate. And elevating temperature could significantly (p<0.05) and linearly (y=-0.067x+5.113) decrease the threshold value of NaCl concentration influencing lipid oxidation during dry-cured bacon processing. When temperature was 35℃and NaCl level was 2.77%, the primary oxidation rate was the fastest. These results suggested that raising temperature and decreasing salt used amount could accelerate primary lipid oxidation rate in dry-cured bacon, which will make the change inflexions of POV and TBARS advanced, and thus provide more times for decreasing oxidation indices and improving flavour quality of products by secondary or more further oxidation.
引文
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