大蒜汁对枯草芽孢杆菌抑制作用的研究
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摘要
大蒜被称为“天然的广谱抗生素”,对食品腐败菌和致病菌有较强的抑制和杀灭作用,包括具有极强抗逆性的芽孢杆菌。它们可以引起食品变质、食物中毒和一些食源性疾病。本试验采用新鲜大蒜直接破碎取汁,对芽孢杆菌的典型代表——枯草芽孢杆菌的抑菌特性进行全面、系统地评价和研究;并通过响应面分析方法对大蒜汁热处理温度、处理时间和pH值,进行科学的优化分析;最后进行了大蒜汁对芽孢各个阶段的抑制作用特性的全面、系统地分析研究。研究主要获得了如下结论:
(1)通过对储藏过程中大蒜汁中硫含量的测定,结果表明:大蒜在4℃低温长期保藏过程中,其硫含量并未发生显著变化,稳定在0.198～0.215%之间。
(2)不同浓度大蒜汁的抑菌圈试验表明,蒜汁含量与其抑菌能力的关系呈正比例关系,蒜汁浓度越大,抑菌圈就越大,抑菌性就越强。通过固体平板菌落计数法以及液体法的验证试验,确定了大蒜汁的最低抑菌浓度(MIC)为0.4%,最低杀菌浓度(MBC)为1%。
(3)枯草芽孢杆菌在0.3%的大蒜汁培养基中的生长曲线比对照试验的延滞期延长12h以上,说明大蒜原汁对枯草芽孢杆菌的萌发有明显的阻滞作用。大蒜汁随着处理时间的延长,抑制作用逐渐下降。当大蒜汁浓度≥1%时,作用16h菌体生长依然没有增加,说明已有部分菌体被杀死。当大蒜汁与菌悬液混合后,作用时间低于16h的抑菌效果,明显好于作用时间超过24h以上。
(4)当大蒜汁的温度为35℃时,抑菌活性达到最大。当超过或低于35℃时,大蒜汁的抑菌活性逐渐下降,在75℃时,大蒜完全失去抑菌活性,说明热处理会使大蒜汁的抑菌能力降低或散失,尤其是高温。大蒜汁在35℃下静置24h抑菌活性变化幅度不大。但是大蒜破碎6h时,抑菌活性为最高,6h后大蒜汁的抑菌活性有所降低,但是比较缓慢。说明在低温条件下,其抑菌活性时效性较好。大蒜汁在35℃下,静置6h,其pH在3～8的范围内,对枯草芽孢杆菌有很好的抑菌效果;pH为6.5时,抑菌效果最好。当pH 9.5时,大蒜汁完全失之抑菌活性。以上说明大蒜抑菌的适宜pH应低于8,即在偏酸性或中性条件。
(5)利用响应面分析法,建立响应曲面模型,并采用脊岭分析,确定了大蒜汁抑制枯草芽孢杆菌的最适处理条件,即大蒜汁在pH 4.9,处理温度为35.1℃下处理7.2h,抑菌效果最佳。
(6)大蒜汁对枯草芽孢杆菌的芽孢形成有重要影响作用。当蒜汁浓度为1%,菌悬液浓度为10~4cfu/mL时,其抑制时间可持续到48h时仍有一定的抑制性。随着大蒜汁相对浓度的增加,芽孢形成的数量减少,其抑制芽孢形成的时间越长。通过芽孢形成过程中2,6-吡啶二羧酸(DPA)含量的变化表明,大蒜汁处理过得枯草芽孢杆菌中芽孢所含DPA含量的增长趋势要比对照的DPA含量要晚12h。因此,大蒜汁对枯草芽孢杆菌的芽孢形成影响主要是延缓了芽孢的形成过程。
(7)大蒜汁对芽孢的发芽影响作用不大,在芽孢发芽的2h内,反应时间长达2h,浓度高达10%的大蒜汁都对芽孢的发芽没有显著影响。
The garlic is known as "natural broad-spectrum antibiotic",has strong bacteriostatic and bactericidal effect on the food spoilage and food pathogenic microorganisms,including Bacillus.They can cause food deterioration,food poisoning and some food-borne diseases.In this experiment,by crushing the whole garlic to get the juice,the antibacterial characterization to Bacillus subtilis——typical representative of Bacillus,was evaluated systemically;and the treatment temperature,pH and the processing time of garlic juice was optimized by response surface analysis; finally,garlic juice's inhibitory action at each stages of the spore was researched systemically.The study result are as following:
(1) By the determination of the sulfur content of garlic in preservation periods,which showed that sulfur content was not obvious change with preservation in 4℃.Sulfur content was in the range of 0.198～0.215%.
(2) The experiment of zone of inhibition showed the content of garlic juice in direct proportion to its antibacterial activity.The higher concentration of garlic juice,the larger zone of inhibition and the stronger antibacterial activity.By plate count and liquid assay method,confirmed garlic juice MIC is 0.4%,MBC is 1%.
(3) 0.3%garlic juice could increase 12h of the lag phase,which showed that garlic juice obvious block germination of Bacillus subtilis.With the extension of treatment time,the garlic juice gradually decreased its antibacterial activity.When the concentration of garlic juice≥1%,the number of Bacillus subtilis did not increase reacted 16h,which showed that some Bacillus subtilis had been killed.The inhibitory effect at less than 16h was better than above 24h.
(4) The antibacterial activity of garlic juice was highest at 35℃and gradually decreased at higher or lower 35℃.The antibacterial activity of garlic juice had not effect at 35℃.The results showed that heat treatment on garlic juice can decreased or eliminated the antibacterial activity,especially high temperature.Within treated garlic juice 24h,the antibacterial activity of garlic juice to Bacillus subtilis had not obviously change at 35℃.The optimum treatment time was 6h.After 6h,the antibacterial activity of garlic juice decreased,but slowly.When the pH was in the range of 3～8,the antibacterial activity of garlic juice to Bacillus subtilis had good effect at 35℃treated garlic juice 6h.The optimum pH was 6.5.When pH was 9.5,garlic juice was not effect.
(5) The treatment conditions of garlic juice was optimized by response surface analysis.Through response surface model and ridge analysis,the result indicated that the optimum treatment conditions of garlic extract for inhibition of Bacillus subtilis was:temperature 35.1℃,time 7.2 h,pH 4.9.
(6) Garlic juice had important influence function to sporulation of Bacillus subtilis.When the concentration of garlic juice and bacterial suspension respectively was 1%and 10~4 cfu/mL,the antibacterial time still exist at 48h.The higher concentration of garlic juice,the less spores;and the longer time of inhibited sporulation.The content change of dipicolinic acid(DPA) with sporulation indicated that growth of the DPA with treatment by garlic juice is later 12h than contrast.So,the influence of garlic juice to sporulation of Bacillus subtilis mainly delayed the process of sporulation.
(7) The garlic juice had not obvious affect to germination of spore,within 2h of germination,2h of reaction time and 10%of the concentration of garlic juice.
(1)通过对储藏过程中大蒜汁中硫含量的测定,结果表明:大蒜在4℃低温长期保藏过程中,其硫含量并未发生显著变化,稳定在0.198～0.215%之间。
(2)不同浓度大蒜汁的抑菌圈试验表明,蒜汁含量与其抑菌能力的关系呈正比例关系,蒜汁浓度越大,抑菌圈就越大,抑菌性就越强。通过固体平板菌落计数法以及液体法的验证试验,确定了大蒜汁的最低抑菌浓度(MIC)为0.4%,最低杀菌浓度(MBC)为1%。
(3)枯草芽孢杆菌在0.3%的大蒜汁培养基中的生长曲线比对照试验的延滞期延长12h以上,说明大蒜原汁对枯草芽孢杆菌的萌发有明显的阻滞作用。大蒜汁随着处理时间的延长,抑制作用逐渐下降。当大蒜汁浓度≥1%时,作用16h菌体生长依然没有增加,说明已有部分菌体被杀死。当大蒜汁与菌悬液混合后,作用时间低于16h的抑菌效果,明显好于作用时间超过24h以上。
(4)当大蒜汁的温度为35℃时,抑菌活性达到最大。当超过或低于35℃时,大蒜汁的抑菌活性逐渐下降,在75℃时,大蒜完全失去抑菌活性,说明热处理会使大蒜汁的抑菌能力降低或散失,尤其是高温。大蒜汁在35℃下静置24h抑菌活性变化幅度不大。但是大蒜破碎6h时,抑菌活性为最高,6h后大蒜汁的抑菌活性有所降低,但是比较缓慢。说明在低温条件下,其抑菌活性时效性较好。大蒜汁在35℃下,静置6h,其pH在3～8的范围内,对枯草芽孢杆菌有很好的抑菌效果;pH为6.5时,抑菌效果最好。当pH 9.5时,大蒜汁完全失之抑菌活性。以上说明大蒜抑菌的适宜pH应低于8,即在偏酸性或中性条件。
(5)利用响应面分析法,建立响应曲面模型,并采用脊岭分析,确定了大蒜汁抑制枯草芽孢杆菌的最适处理条件,即大蒜汁在pH 4.9,处理温度为35.1℃下处理7.2h,抑菌效果最佳。
(6)大蒜汁对枯草芽孢杆菌的芽孢形成有重要影响作用。当蒜汁浓度为1%,菌悬液浓度为10~4cfu/mL时,其抑制时间可持续到48h时仍有一定的抑制性。随着大蒜汁相对浓度的增加,芽孢形成的数量减少,其抑制芽孢形成的时间越长。通过芽孢形成过程中2,6-吡啶二羧酸(DPA)含量的变化表明,大蒜汁处理过得枯草芽孢杆菌中芽孢所含DPA含量的增长趋势要比对照的DPA含量要晚12h。因此,大蒜汁对枯草芽孢杆菌的芽孢形成影响主要是延缓了芽孢的形成过程。
(7)大蒜汁对芽孢的发芽影响作用不大,在芽孢发芽的2h内,反应时间长达2h,浓度高达10%的大蒜汁都对芽孢的发芽没有显著影响。
The garlic is known as "natural broad-spectrum antibiotic",has strong bacteriostatic and bactericidal effect on the food spoilage and food pathogenic microorganisms,including Bacillus.They can cause food deterioration,food poisoning and some food-borne diseases.In this experiment,by crushing the whole garlic to get the juice,the antibacterial characterization to Bacillus subtilis——typical representative of Bacillus,was evaluated systemically;and the treatment temperature,pH and the processing time of garlic juice was optimized by response surface analysis; finally,garlic juice's inhibitory action at each stages of the spore was researched systemically.The study result are as following:
(1) By the determination of the sulfur content of garlic in preservation periods,which showed that sulfur content was not obvious change with preservation in 4℃.Sulfur content was in the range of 0.198～0.215%.
(2) The experiment of zone of inhibition showed the content of garlic juice in direct proportion to its antibacterial activity.The higher concentration of garlic juice,the larger zone of inhibition and the stronger antibacterial activity.By plate count and liquid assay method,confirmed garlic juice MIC is 0.4%,MBC is 1%.
(3) 0.3%garlic juice could increase 12h of the lag phase,which showed that garlic juice obvious block germination of Bacillus subtilis.With the extension of treatment time,the garlic juice gradually decreased its antibacterial activity.When the concentration of garlic juice≥1%,the number of Bacillus subtilis did not increase reacted 16h,which showed that some Bacillus subtilis had been killed.The inhibitory effect at less than 16h was better than above 24h.
(4) The antibacterial activity of garlic juice was highest at 35℃and gradually decreased at higher or lower 35℃.The antibacterial activity of garlic juice had not effect at 35℃.The results showed that heat treatment on garlic juice can decreased or eliminated the antibacterial activity,especially high temperature.Within treated garlic juice 24h,the antibacterial activity of garlic juice to Bacillus subtilis had not obviously change at 35℃.The optimum treatment time was 6h.After 6h,the antibacterial activity of garlic juice decreased,but slowly.When the pH was in the range of 3～8,the antibacterial activity of garlic juice to Bacillus subtilis had good effect at 35℃treated garlic juice 6h.The optimum pH was 6.5.When pH was 9.5,garlic juice was not effect.
(5) The treatment conditions of garlic juice was optimized by response surface analysis.Through response surface model and ridge analysis,the result indicated that the optimum treatment conditions of garlic extract for inhibition of Bacillus subtilis was:temperature 35.1℃,time 7.2 h,pH 4.9.
(6) Garlic juice had important influence function to sporulation of Bacillus subtilis.When the concentration of garlic juice and bacterial suspension respectively was 1%and 10~4 cfu/mL,the antibacterial time still exist at 48h.The higher concentration of garlic juice,the less spores;and the longer time of inhibited sporulation.The content change of dipicolinic acid(DPA) with sporulation indicated that growth of the DPA with treatment by garlic juice is later 12h than contrast.So,the influence of garlic juice to sporulation of Bacillus subtilis mainly delayed the process of sporulation.
(7) The garlic juice had not obvious affect to germination of spore,within 2h of germination,2h of reaction time and 10%of the concentration of garlic juice.
引文
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