【作者】 吴潇；

【导师】 马美湖；

【作者基本信息】 华中农业大学 ， 食品科学， 2011， 硕士

【摘要】 孵化条件下禽蛋的抗菌问题研究较少,国内外一般只对受精蛋在孵化过程中的理化性质和物质交换进行研究。为了探明受精蛋在孵化条件下的抑菌机制,本文采用浸泡接种法对受精蛋与非受精蛋在孵化条件下抗菌性质的变化进行了比较分析。并且分别对分离的受精蛋与非受精蛋蛋清、蛋壳与壳膜在孵化条件下抗菌性的变化进行了比较。另外对在孵化条件下受精蛋与非受精蛋蛋清中两种主要的抗菌蛋白(溶菌酶、卵转铁蛋白)含量的变化进行了比较研究。本研究对不同孵化阶段,受精蛋与非受精蛋抗肠炎沙门氏菌的能力进行了比较研究。利用1×105 cfu/mL Salmonella Enteritidis (SE)菌悬液接种受精蛋和非受精蛋,测定在不同孵化时间蛋壳与壳膜、蛋清、蛋黄的SE的侵染比例。结果表明：孵化4d,受精蛋蛋壳与壳膜、蛋清比非受精蛋抗肠炎沙门氏菌活性强(P<0.05),两种蛋的蛋黄样本都未受到侵染；孵化8、12d,两种蛋的三个部位样品肠炎沙门氏菌的阳性比例没有显著性差异(P>0.05)；孵化17d,受精蛋蛋壳与壳膜、蛋黄抗肠炎沙门氏菌活性显著高于非受精蛋(P<0.05),此时受精蛋鸡胚的漂洗物肠炎沙门氏菌阳性率为13.78%,非受精蛋蛋清肠炎沙门氏菌阳性率为45.16%。通过测定孵化过程中受精蛋与非受精蛋蛋清干重以及含水量的变化,发现蛋清在孵化过程中被浓缩,且受精蛋蛋清的浓缩作用更大。同时测定了蛋清在孵化过程中pH的变化,发现受精蛋蛋清pH由孵化初期的8.94,在孵化2d时升至9.30,然后逐渐降低降至8.40,非受精蛋蛋清pH逐渐变大,由孵化初期的9.22升至9.69。蛋清pH的变化限制了SE在蛋清中的生长。以孵化后的受精蛋与非受精蛋蛋清为介质,对其抗菌活性进行了比较。发现蛋清对本实验所用菌株(肠炎沙门氏菌CMCC(B)50041、金黄色葡萄球菌ATCC6538、CP5型金黄色葡萄球菌、大肠杆菌8099、猪霍乱沙门氏菌C78-1)皆具有抑制作用,且不同孵化天数的受精蛋和非受精蛋蛋清的抑菌活性有差异,同一孵化天数的受精蛋与非受精蛋蛋清的抑菌活性也具有一定的差异。无论是革兰氏阳性菌还是革兰氏阴性菌,受精蛋与非受精蛋蛋清的抑菌性在孵化的第2d最强,之后两者的抗菌性均逐渐减弱,蛋清对革兰氏阳性菌具有更好的杀灭作用。对于革兰氏阴性菌来说,受精蛋与非受精蛋蛋清经过孵化0、1、2d,受精蛋蛋清与非受精蛋蛋清的抑菌活性基本等同。孵化3、4d时,受精蛋蛋清的抑菌活性比非受精蛋蛋清弱。对于革兰氏阳性菌来说,受精蛋与非受精蛋蛋清经过孵化0、1、2、3d,受精蛋蛋清与非受精蛋蛋清的抑菌活性基本等同。孵化4d时,受精蛋蛋清的抑菌活性比非受精蛋蛋清的强。孵化4d时,受精蛋的抗肠炎沙门氏菌的活性高于非受精蛋,而此时受精蛋蛋清的抗肠炎沙门氏菌的活性弱于非受精蛋蛋清。这说明：孵化前期蛋清并不是主要的抵抗SE侵入的屏障。因此,通过建立蛋壳穿透模型比较孵化前期SE穿透受精蛋与非受精蛋蛋壳与壳膜的能力。结果发现：受精蛋孵化Od与孵化1-4d的蛋壳与壳膜在抵抗SE穿透能力上具有显著性差异(P<0.05),SE穿透时间从第Od的81.6h下降到第4d的60.0h。非受精蛋孵化0-4d的蛋壳与壳膜在抵抗SE穿透上不具有显著性差异(P>0.05),蛋壳的平均抑菌时间变化不大,一直维持在58-63h之间。0-4d,受精蛋蛋壳比非受精蛋蛋壳的抑菌时间长19.2、5.4、5.4、6.5、1.2h。为了进一步揭示受精蛋与非受精蛋蛋清抗菌性差异的原因,本研究还采用分光光度法对蛋清中两种主要抗菌蛋白在孵化条件下含量的变化进行了测定和比较。结果发现：孵化过程中,受精蛋与非受精蛋蛋清中溶菌酶的含量先增大后减少,受精蛋蛋清中溶菌酶含量由孵化0d的3.78mg/mL升至最大值5.23mg/mL(孵化2d)。非受精蛋蛋清中溶菌酶的含量由孵化0d时的3.86mg/mL升至最大值4.71mg/mL(孵化3d)。孵化3、4d时的受精蛋蛋清中溶菌酶的含量高于非受精蛋蛋清,由于溶菌酶主要作用于革兰氏阳性菌,说明3-4d时,受精蛋蛋清的抗革兰氏阳性菌的能力高于非受精蛋,这与第三章的结果相吻合。孵化过程中,受精蛋与非受精蛋蛋清中卵转铁蛋白的含量先增大后减少,孵化0d时受精与非受精蛋中卵转铁蛋白含量分别为4.17mg/mL、4.31mg/mL。在孵化2d时,卵转铁蛋白含量达到最大值,分别为：6.57mg/mL和6.28mg/mL。溶菌酶和卵转铁蛋白含量的变化与蛋清抗菌性的变化存在一定的联系。溶菌酶和卵转铁蛋白含量上升时,蛋清的抗菌性增强。溶菌酶和卵转铁蛋白含量下降时,蛋清的抗菌性减弱。更多还原

【Abstract】 The mechanisms of anti-microbial properties of fertile eggs during incubation are not clear, recently. In this paper, a comparison research was carried on to determine the anti-microbial activity of fertile eggs and infertile eggs using a inoculation method of immersion during incubation. The anti-microbial activity of albumen and eggshell and shell membranes of fertile and infertile eggs were also taken into a comparison. Above all, the content of lysozyme and ovotransferrin, the two main types of anti-microbial proteins, were compared.In order to clear the anti-bacterial mechanism of fertile eggs during incubation, the fertile and infertile eggs were used to identify the anti-Salmonella enteritidis differences during different stages of incubation. They were put into inoculation in a SE suspension of approximately 1×105cfu/mL. Egg shell and membrane, albumen, yolk were sampled respectively. The results showed that it was significantly that fewer of the fertile eggs’ shell and membrane, albumen samples were contaminated on days 4 (P<0.05), the yolk samples of the two kinds of eggs were clear. There were no significant differences between fertile an infertile eggs in all the sample tested on days 8 and 12 of incubation (P >0.05). However, On days 17, it was significantly that fewer of the fertile eggs’ albumen and yolk samples were contaminated (P<0.05), moreover,13.78% of the chick rinses samples from fertile eggs were positive,45.16% of the albumen samples from infertile eggs were positive. By measuring the dry weight and water content of the albumen changes during incubation, a concentration was found and the fertile egg albumen was concentrated more. Meanwhile, the pH values of the albumen were determined to know how it changed, we found that the pH values of fertile eggs’ albumen first increase and then decrease, the pH values of infertile eggs’ albumen increase all the time. The pH effect will set obstacle for the growing of SE.The incubated albumen of fertile and infertile eggs were set as media, a comparison of the anti-microbial activity was done. The inhibition of all the strains in this experiment was found and there were some differences. The 2d’s incubation albumen had the strongest inhibition effect for both the gram-positive and gram-negative bacteria. Then, the inhibition effect would decrease. For the gram-negative bacteria, the inhibition effect was almost the same for the albumen incubated for 0-2 days. When albumen incubated for 3 or 4 days, the inhibition effect of fertile eggs’s albumen was weaker then the infertile eggs. For the gram-positive bacteria, the inhibition effect was almost the same for the albumen incubated for 0-3 days. On the 4th day of incubation, the inhibition effect of fertile eggs’s albumen was stronger then the infertile eggs.In the chapter 2, we found that on the 4th day of incubation, the anti-SE properties of fertile eggs were stronger than infertile eggs. In the chapter 3, we found that the anti-SE properties of fertile eggs’ albumen were weaker than infertile eggs’ albumen. Therefore, in the chapter 4, the eggshell penetration of fertile and infertile eggs was discussed. The results showed that:The anti-SE properties of eggshell of fertile eggs were decreased in the early incubation, the resistant time was changed from 81.6h to 60.0h, and there are significant differences(P<0.05); as for the infertile eggshell, the properties were almost the same (P>0.05), the resistant time was remain 58-63h. Most importantly, the average resistant time of fertile eggs’shell was longer than the infertile eggs on the same day of incubation 19.2h (0d),5.4h (1d),5.4h (2d)、6.5h (3d)、1.2h (4d).In addition, the study also using spectrophotometry to determine the content of two main antimicrobial egg white protein (lysozyme and ovotransferrin). The results showed that the content of lysozyme in albumen first increase then decrease, for the fertile eggs it changed from 3.78mg/mL(0d) to 5.23 mg/mL (2d) when it reached the maximum; for the infertile eggs it changed from 3.86 (0d)mg/mL to 4.71(3d) when it reached the maximum. After that, the content of lysozyme in the infertile eggs’s albumen decrease during the whole time of incubation. On the 3-4 days, the content of lysozyme in the fertile eggs’albumen was higher than infertile eggs, because the lysozyme played the major role in inhibiting the gram-positive bacteria, it means that on the 3-4 days, the albumen of fertile eggs have higher ability to inhibit growing than infertile eggs, which is consistent with the results in chapter 3. The content of ovotransferrin is just the same as lysozyme, on the Od of incubation, the content was 4.17 mg/mL and 4.31 mg/mL, respectively. then it commit a increase, on days 2, it reaches the maximum,6.57 mg/mL、6.28 mg/mL, respectively. There must be a relationship between the anti-microbial activity of albumen and the changes of lysozyme and ovotransferrin content. When the content of lysozyme and ovotransferrin increase, the anti-microbial activity gets stronger, when the content of lysozyme and ovotransferrin decrease, the anti-microbial activity gets weaker.更多还原