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巴氏杀菌切片火腿冷藏期间褪色机理研究
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摘要
色泽是评价肉品质量的一个重要指标,大多数消费者都以其作为判断肉品新鲜度和决定是否购买的一个主要尺度。巴氏杀菌肉制品是肉品未来的发展方向,我国生产起步较晚,与发达国家相比,还存在许多质量问题。其中货架期褪色就是限制我国巴氏杀菌肉制品保质期和市场竞争力的关键问题之一。目前由于缺乏该类产品色泽变化的相关理论,使得部分生产者盲目加大亚硝酸盐的用量或者添加大量的人工色素。这样不仅没有真正改善产品的色泽稳定性,反而降低了产品的品质,甚至给消费者的健康带来了潜在的危害。为此,本论文以典型的巴氏杀菌牛肉切片火腿为研究对象,将产品表观色泽变化理论与产品色素变化特性相结合来揭示其冷藏期间褪色机理,为采取科学有效的护色措施提供理论依据。具体研究内容和结论如下:
     1.影响巴氏杀菌切片火腿冷藏期间褪色的主因素分析
     通过对加工和贮藏中影响色泽稳定的7个因素分别采用不同的水平同时作用,并对色泽参数进行逐步回归分析,筛选影响色泽稳定的主导因素。结果表明:包装袋的氧渗透率(OTR)、光照强度(Ill)、混合磷酸盐水平(Phos)和蒸煮温度(CT)是影响切片火腿色泽稳定性的主要因素。其中OTR的影响作用最大;3000 mg/kg的磷酸盐以及蒸煮中心温度达85℃都显著影响切片火腿色泽稳定性;1200 lx范围内不同强度的光照,显著影响亮度(L*)、黄色度(b*)和色调(H),而对红色度(a*)的变化影响不显著。10℃以下的不同冷藏温度,以及国标规定范围内添加不同水平的亚硝酸盐不是引起产品褪色的主要原因。
     2.产品贮藏期间色泽与其它品质的变化及其相互关系
     采用多元统计分析的主成分分析(PCA)和因子分析方法,研究色泽参数与其它品质指标之间的相互关系。结果表明:
     PC1的贡献率占总变异的59.82%,集中反映了切片火腿在冷藏期间的色泽变化。PC2的贡献率占总变异的22.28%,代表了引起色泽变化的环境因子。两个主成分揭示了切片火腿表观红色度的变化主要是色素NH发生了氧化还原反应所致,红色度a*与亚硝酰血色原(NH)、游离巯基、蛋白质氧化形成的羰基衍生物、脂肪氧化形成的丙二醛和双烯值之间相关极显著。其次,产品褪色与基质环境的氧化还原力和酸碱度的变化也显著相关,环境变化首先影响到亮度L*和黄度b*,进而引起表观红色度a*的变化。另外,PCA和因子分析表明产品基质环境中含氮化合物的存在形式及含量显著影响其色泽变化。色泽参数之间α*、C和NH的相关性极显著,L*,b*与H的相关性极显著,色饱和度同时综合了红色度和黄色度的主要信息,而色调以红色度为基础但主要受黄色度来调节。
     3.亚硝酰血色原(NH)的提取及结构鉴定
     根据不同溶剂对NH进行提取的紫外可见吸收光谱(UV)特性和电子顺磁共振波谱(EPR)特性建立了采用石油醚、丙酮、乙酸乙酯三步顺序提取和分离亚硝酰血色原(NH)的方法。
     利用高效液相-电喷雾离子化高分辨率质谱联用(HPLC-ESI-HR-MS)、EPR光谱、傅立叶变换红外光谱(FT-IR)和红外激光拉曼光谱(Raman)四种波谱综合分析鉴定NH的结构为一亚硝酰亚铁卟啉血色原。
     4.NH的氧化特性研究及产品褪色机理分析
     通过分析NH在光和氧的环境下的降解动力学,在具有光照的空气中自然氧化以及添加强氧化剂H202条件下氧化的HPLC-ESI-HR-MS、EPR波谱、FT-IR光谱、Raman光谱和’H核磁波谱特性,以及在p-胡萝卜素/亚油酸模型体系和加热肌肉模型体系中的氧化特性,结合前两章对产品表观色泽变化理论的研究结果,综合得出产品氧化褪色机理:切片火腿主导色素成分NH属于光敏剂,光照直接作用于NH,促使其共轭结构发生变化,向接近分子轴对称的另一种棕黄色到黄色的共轭体系转化,NO基团没有与血红素卟啉铁解离。NH具有抗氧化特性,空气中氧的存在,会加速这种变化。这种变化在无强氧化剂的条件下是可逆的,可以通过肉品体系中的还原型成分使其还原或维持一种动态平衡。随着NH被氧化,基质中的多不饱和底物也逐渐发生氧化,形成H2O2等更强的氧化剂。强氧化剂可促使NH发生不可逆的降解,使NO基团离开卟啉铁,去清除体系中的自由基。失去了NO基团的保护,卟啉Fe2+很快被氧化为Fe3+,随之从血红素分子中释放,Fe3+离子是脂质氧化的启动子,从而促使了脂质的过氧化,脂质过氧化产物反过来又会加速NH发生不可逆的氧化降解,使得产品色泽发生不可复原的变化,乃至失去商品价值。
     5.NH的稳定性研究
     为揭示其它因素对切片火腿褪色的效应,研究了不同环境因子对色素NH的稳定性以及不同含氮化合物对加热牛肉匀浆物的色泽及其稳定性的影响。结果表明:
     pH对NH的光学特性和稳定性有显著的影响,强酸和弱碱条件下有利于NH红色的保持。碱性条件下,NH由粉红色向棕黄色到黄色过渡,NH分子中NO基团仍然与卟啉铁结合,可能与空气中自然氧化NH的结构一样。弱酸性条件下NH极不稳定,是pH变化显著影响产品表观色泽的主要原因。
     8种与色泽变化相关显著的氨基酸Cys、Pro、Thr、Gly、Val、Met、Leu和Tyr无论对NH丙酮溶液的色泽,还是对加热牛肉匀浆物的色泽均产生显著的影响。其中Cys可以显著改善NH的红色度,Pro可以使NH的粉红色保持稳定,His使NH丙酮溶液产生红色沉淀,其余的不同程度地使NH丙酮溶液转化为黄绿色至黄色。这几种氨基酸在牛肉匀浆体系中与肌红蛋白或血红蛋白作用,经热处理以后形成不同色泽的血红素衍生物。其中His和Val形成近似于NH的色泽,且His形成的粉红色血红素衍生物为水溶性的。Gly、Tyr和Pro形成颜色较浅稍偏灰而透着淡粉色的血红素衍生物。Cys和Met两种含硫氨基酸形成的颜色都偏灰至褐。这些衍生物相对于NH来说,更不稳定,切片火腿中这些血红素衍生物的存在也是其褪色的一个重要原因。
     肉品相应浓度的7种无机离子(Na+、K+、Fe2+、Mg2+、Ca2+、Zn2+、cu2+)均可显著提高色素NH丙酮溶液的稳定性。其中添加Fe2+对NH的稳定效果最显著,其次是含Na+组,添加K+和Mg2+组组间差异不显著。含Zn2+和Cu2+的处理组稳定性相对最差,其次是Ca2+处理组。
     70℃以下不同温度处理对色素NH的损失率没有显著影响,冷藏和常温贮藏对NH的降解没有直接的影响。75℃和80℃处理,NH的贮藏稳定性显著低于72℃处理组。85℃处理,NH的损失率及稳定性均会受到显著影响。
     6.天然抗氧化剂对巴氏杀菌切片火腿色泽稳定效应的研究
     不同天然抗氧化剂对同一种产品以及同一种抗氧化剂对不同产品均产生不同的色泽稳定效应。苹果多酚和迷迭香在实验剂量范围内对猪肉和牛肉切片火腿的脂肪氧化均表现出了显著的抑制效应,然而对色泽稳定效应却不同。添加500 mg/kg苹果多酚可以有效地抑制猪肉切片火腿冷藏期间的褪色,而对牛肉的效果却不显著。添加500~1500 mg/kg的迷迭香在显著影响切片火腿表观红色度的同时更显著地提高了黄色度b*值,而且对b*的提高有浓度依赖性。
Color stability is one of the important characteristics of meat and meat products. It is the primary quality attribute seen by the consumer, who may use it as an indication of freshness and wholesomeness. As the way of future development of meat products, pasteurized meat products were introduced China lately and a lot of quality defects still exist currently. Discoloration is one of the important problem which will shorten pasteurized meat products' shelf-life and limit its market competitiveness. Many producers blindly increase the amount of nitrite or add many artificial pigment in absence of the knowledge on the theory of discoloration of pasteurized meat products, which did not really improve the color stability and prolong the shelf-life, instead of decreasing the quality of their products, even gave potential health hazards to consumers. Therefore, the purpose of present study was to reveal the mechanism of discoloration of representative pasteurized meat products—beef sliced ham, and to provide a theory basis for seeking a scientific method effectively to inhibit discoloration. Based on this purpose, the research were designed from two aspects including the fading theory about products and oxidation characteristics of predominant pigment. The contents and results are as follows:
     1. Study on the main influencing factor of discoloration of pasteurized sliced beef ham
     Seven factors affected color stability in processing and preservation were designed and instrumental color of ham were evaluated in this chapter. Multiple stepwise regression analysis was adopted to develop mathematical models describing the relationship between color parameters (L*, a*, b*) and seven factors. The results showed that refrigeration temperature (< 10℃) and the level of nitrite (in the range of GB) had insignificant effect on color stability; OTR had the greatest effect, both the addition of 3000 mg/kg phosphate and cooking center temperature of 85℃significantly affect the color stability. Illumination (< 1200 lx) did not significantly affect the a* value but the L*、b* and Hue value.
     2. Studies on the change of color parameters and other quality index and their relationship
     Principal component analysis (PCA) and factor analysis were adopted to research the interrelationship between color and other parameters. The PCA results showed that the first principal component explained 59.82% of the total variation, and the second explained 22.28%. PC1 was a concentrated reflection of color changes of ham during storage and PC2 was a representative index of environment factor causing the change of color. These results indicated that discoloration of products was mainly due to the oxidation of predominant pigment and greatly related to the reductive capacity of matrix. Furthermore, significant correlations were found between pigment oxidation and lipid oxidation as well as that and protein oxidation. Matrix condition significantly influenced the color stability by change of the b* and L* firstly and caused the variation of a* value then. In addition, remarkable correlations were seen between color stability and some nitrogen compounds in matrix by PCA and factor analysis. For color parameters, great significant correlations were found in the present study among a*,C and the content of nitrosylhemochromogen (NH), meanwhile L*、b* and H had great significant correlations; The information of C was a combination of a* and b*, while the H, degrees against the backdrop of a* value, was adjusted by b* value.
     3. Studies on the extraction method and structure of NH
     Extractions of NH from beef sliced ham with many different solvents were carried out. A three-step gradational extraction and isolation procedure with petroleum ether/acetone/ethyl acetate step by step was established based on the characteristics of UV and electron paramagnetic resonance spectroscopy (EPR) of NH solution.
     The structure of NH was identified as a pentacoordinate mononitrosylheme complex by four kinds of spectra, which were EPR, HPLC-electrospray ionization high resolution mass spectrometry (HPLC-ESI-HR-MS), infrared spectrum (IR) and Raman spectrum.
     4. Studies on the oxidation properties of extracted NH
     The main research content in this chapter include the kinetics of NH degradation exposed in air and/or light, the spectra characteristics of NH oxidized in air with natural light or by addition of 1.5 mg/kg H2O2 as well as the prooxidation and antioxidation properties of NH in aβ-carotene/linoleate model system and cooked muscle model systems. Based on the results of above research, discoloration mechanism by oxidation was concluded as following. The predominant pigment (NH) of sliced ham is a photosensitizer and an antioxidant and is vulnerable to be oxidized when oxygen and light are present. The changed EPR spectra of oxidized NH in acetone suggested a new proposal that the NO' group might not detach itself from iron porphyrin of NH during oxidation in air with normal lighting, but changed in conjugated structure, and the structure tended to axial symmetry of the molecule. This transformation of NH is reversible and the reductant in matrix may cause fading return to original state or maintain a dynamic balance. As the NH oxidized, the polyunsaturated substrates in matrix were also oxidized gradually and form a stronger oxidant such as H2O2. When NH was oxidized by addition of H2O2, the NO group might interact with free radicals leaving the iron porphyrin of NH to quench free radicals in the medium, and subsequently the Fe2+ was converted to Fe3+ since it lost the protection of NO, and then released from the hemoglobin molecule. Fe3+ iron is the promoter of lipid oxidation and which promoted the lipid peroxidation rapidly, and in turn the oxidative degradation of NH was accelerated by products of lipid peroxidation further. This kind of decomposition is irreversible and will make the ham fade permanently.
     5. Studies on the stability of NH under different conditions
     In order to reveal the effect of other factors on color fading, the effect of different factors on the stability of NH as well as the effects of nitrogen component on the color of cooked beef slurries were studied. The results were shown as following.
     pH had significant influence on the optical properties and stability of NH, and the conditions of strong acid and weak alkali would help to maintain the red color of NH. The pink-red color of NH turned brown yellow under strong alkali condition. For the molecular structure of yellow NH, the NO group was still bind with iron porphyrin and this structure may be the same as the one of oxidized NH exposed in air. The main reason that pH significantly influenced the color stability of ham was the remarkable instability of NH under weak acid conditions.
     By investigating Cys, Pro, Thr, Gly, Val, Met, Leu and Tyr, significant effects were observed on color stability of NH acetone solution and of cooked beef slurries for all listed amino acid. Cys could improve the red color of NH, Pro could maintain the stability of NH, His caused the NH acetone solution appear red precipitate, and the rest of amino acids made the NH acetone solution transform to yellow-green to varying degrees. In cooked beef slurries system, these amino acid could form colored complexes by reaction with myoglobin or hemoglobin. His and Val produced a pink to reddish coloration, similar to that of the nitrite containing controls, and Gly, Tyr, Pro produced slurries with a thin pink to Beige, Cys and Met produced slurries with pigments of various shades of pink and tan. The pink heme derivative produced by His were water-soluble. All of these different colored derivative were more instability than NH, which may be another important reason for discoloration of sliced cooked cured ham.
     Fe2+, Na+, K+, Mg2+, Zn2+, Cu2+ and Ca2+ enhanced obviously the stability of NH acetone solution at the concentration meet with cooked meat product respectively. The best effect was present by addition of Fe2+, the next was in the decreasing order:Na+> K+≥Mg2+>Ca2+>Cu2+>Zn2+.
     The loss percentage of NH did not have remarkable differnce treated with different temperature below 70℃. Refrigerated and ambient temperature storage had no direct impact on the degradation of NH. The storage stabilities of NH treated with 75℃and 80℃were remarkably inferior to that of NH treated with 72℃. The loss percentage and stability of NH treated with 85℃were subjected to significant influence.
     6. Effect of natural antioxidant on color stability of pasteurized sliced hams
     Effect of two kinds of natural antioxidant on color and oxidative stability of beef and pork pasteurized sliced ham were studied in this chapter. The results of the present study indicated that different antioxidant had different effects on the same stored ham, and the same antioxidant produced different effects on different ham. Both apple polyphenol (AP) and rosemary successfully inhibited lipid oxidation of both pork and beef sliced ham. However, different effect on color stability was observed. Addition of 500 mg/kg AP to the samples significantly inhibited discoloration in pork sliced ham but not in beef. Addition of 500~1500 mg/kg rosemary to the samples more significantly enhanced b* value than a* value, and there was a concentration-dependent increase on the b* value.
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