فعاليت ضد قارچي شيرهاي تخميرشده با استفاده از كشت‌هاي منفرد يا تركيبي لاكتيك اسيد باكتري‌هاي پروتئوليتيك

Antifungal activity of fermented milks produced by single or co-cultures of proteolytic lactic acid bacteria

ﺳﺎﺑﻘﻪ و ﻫﺪف: در ﺳﺎلﻫﺎي اﺧﯿﺮ اﺳﺘﻔﺎده از ﻻﮐﺘﯿﮏ اﺳﯿﺪ ﺑﺎﮐﺘﺮيﻫﺎ ﺑﻪ ﻋﻨﻮان ﮐﺸﺖﻫﺎي ﺣﻔﺎﻇﺘﯽ در ﺗﻮﻟﯿﺪ ﻓﺮاوردهﻫـﺎي ﺗﺨﻤﯿـﺮي ﮔﺴﺘﺮش ﯾﺎﻓﺘﻪ اﺳﺖ. ﺑﺨﺶ ﻗﺎﺑﻞ ﺗﻮﺟﻬﯽ از ﻓﻌﺎﻟﯿﺖ ﺿﺪ ﻗﺎرﭼﯽ ﻻﮐﺘﯿﮏ اﺳﯿﺪ ﺑﺎﮐﺘﺮيﻫﺎ ﺑﻪ ﻣﺘﺎﺑﻮﻟﯿﺖﻫﺎي ﺗﻮﻟﯿﺪ ﺷـﺪه ﺗﻮﺳـﻂ آنﻫـﺎ ﺑﻪوﯾﮋه اﺳﯿﺪﻫﺎي آﻟﯽ ﻣﺨﺘﻠﻒ ﻣﺮﺑﻮط ﻣﯽﺷﻮد. ﺑﺎ اﯾﻦ ﺣﺎل، اﺧﯿﺮا ﭘﭙﺘﯿﺪﻫﺎ و ﻫﯿﺪروﻟﯿﺰ ﺷﺪهﻫﺎي ﭘﺮوﺗﺌﯿﻨﯽ ﻧﯿﺰ ﺑﻪ ﻋﻨﻮان ﺗﺮﮐﯿﺒـﺎﺗﯽ ﮐـﻪ ﻓﻌﺎﻟﯿﺖ ﺿﺪ ﻗﺎرﭼﯽ دارﻧﺪ ﻣﻌﺮﻓﯽ ﺷﺪهاﻧﺪ. ﻫﺪف از اﯾﻦ ﭘﮋوﻫﺶ، ﺑﺮرﺳﯽ ﻓﻌﺎﻟﯿﺖ ﺟﺪارهﻫﺎي ﻻﮐﺘﯿﮏ اﺳﯿﺪ ﺑﺎﮐﺘﺮيﻫﺎي ﭘﺮوﺗﺌﻮﻟﯿﺘﯿﮏ در ﺗﻮﻟﯿﺪ ﺷﯿﺮ ﺗﺨﻤﯿﺮﺷﺪه ﺑﺎ ﺧﺎﺻﯿﺖ ﺿﺪﻗﺎرﭼﯽ ﺑﻮد. ﻣﻮاد و روشﻫﺎ: در اﯾﻦ ﭘﮋوﻫﺶ، ﺷﯿﺮ ﺑﺪون ﭼﺮﺑﯽ اﺳﺘﺮﯾﻞ ﺷﺪه ﺑﺎ ﮐﺸﺖﻫﺎي ﻣﻨﻔﺮد و ﺗﺮﮐﯿﺒﯽ ﻻﮐﺘﻮﺑﺎﺳـﯿﻠﻮس دﻟﺒﺮوﮐـﯽ زﯾﺮﮔﻮﻧـﻪ ﺑﻮﻟﮕﺎرﯾﮑﻮس، ﻻﮐﺘﻮﺑﺎﺳﯿﻠﻮس روﺗﺮي و ﻻﮐﺘﻮﮐﻮﮐﻮس ﻻﮐﺘﯿﺲ زﯾﺮﮔﻮﻧﻪ ﻻﮐﺘﯿﺲ ﺗﻠﻘﯿﺢ )2 درﺻﺪ، وزﻧﯽ-وزﻧﯽ( و ﻓﺮآﯾﻨﺪ ﺗﺨﻤﯿﺮ ﺑـﺎ ﮔﺮمﺧﺎﻧﻪﮔﺬاري در 37 درﺟﻪ ﺳﺎﻧﺘﯽﮔﺮاد ﺗﺎ رﺳﯿﺪن ﺑﻪ pH ﻣﻄﻠﻮب 4/5-4/6 اﻧﺠﺎم ﮔﺮﻓﺖ. ﻣﺤﺘﻮاي ﭘﭙﺘﯿﺪي و ﻧﯿـﺰ ﺧﺎﺻـﯿﺖ ﺿـﺪ ﻗﺎرﭼﯽ ﻋﺼﺎره ﭘﭙﺘﯿﺪي ﺑﻪدﺳﺖ آﻣﺪه از ﺷﯿﺮﻫﺎي ﺗﺨﻤﯿﺮ ﺷﺪه در ﺑﺮاﺑﺮ آﺳﭙﺮژﯾﻠﻮس ﻧﺎﯾﺠﺮ، ﭘﻨﯽﺳﯿﻠﯿﻮم اﮐﺴﭙﺎﻧﺴﻮم و ﮐﺎﻧﺪﯾﺪا آﻟﺒﯿﮑﻨﺲ ﭘﺲ از اﻧﺠﺎم ﺗﺨﻤﯿﺮ و ﭘﺲ از 7 روز ﻧﮕﻬﺪاري در ﯾﺨﭽﺎل ﺑﺮرﺳﯽ ﺷﺪ. ﯾﺎﻓﺘﻪﻫﺎ: ﻻﮐﺘﻮﺑﺎﺳﯿﻠﻮس روﺗﺮي ﮐﻤﺘﺮﯾﻦ ﻓﻌﺎﻟﯿﺖ ﭘﺮوﺗﺌﻮﻟﯿﺘﯿﮏ را در ﺷﯿﺮ ﻧﺸﺎن داد و در ﺑﯿﻦ ﺗﻤﺎم ﻧﻤﻮﻧﻪﻫﺎ، ﺑﯿﺸﺘﺮﯾﻦ ﻣﺤﺘﻮاي ﭘﭙﺘﯿﺪي ﺑﻪ ﻣﯿﺰان 6/62 ﻣﯿﻠﯽﮔﺮم ﭘﭙﺘﯿﺪ در ﻫﺮ ﻣﯿﻠﯽﻟﯿﺘﺮ در ﻧﻤﻮﻧﻪاي ﻣﺸﺎﻫﺪه ﺷﺪ ﮐﻪ ﺑﺎ ﮐﺸﺖ ﺗﺮﮐﯿﺒﯽ ﻫﺮ ﺳﻪ ﺟﺪاﯾﻪ ﺗﺨﻤﯿﺮ ﺷﺪه ﺑﻮد. ﻋﺼـﺎره ﺣﺎوي ﭘﭙﺘﯿﺪ ﻧﻤﻮﻧﻪﻫﺎي ﺷﯿﺮ ﺗﺨﻤﯿﺮ ﺷﺪه ﺑﺎ ﮐﺸﺖﻫﺎي ﺗﺮﮐﯿﺒﯽ (S23 و S123) ﭘﺲ از ﺗﺨﻤﯿﺮ ﺑﺎ MIC ﺑﺮاﺑﺮ 20 ﻣﯿﻠﯽﮔﺮم ﺑﺮ ﻣﯿﻠﯽﻟﯿﺘﺮ از ﺑﺎﻻﺗﺮﯾﻦ ﻓﻌﺎﻟﯿﺖ ﺿﺪ ﮐﭙﮑﯽ ﺑﺮﺧﻮردار ﺑﻮدﻧﺪ. ﭘـﺲ از 7 روز ﻧﮕﻬـﺪاري ﺧﺎﺻـﯿﺖ ﺿـﺪﮐﭙﮑﯽ ﻧﻤﻮﻧـﻪ S1 در ﻣﻘﺎﺑـﻞ ﭘﻨـﯽ ﺳـﯿﻠﯿﻮم اﮐﺴﭙﺎﻧﺴﻮم و ﻧﻤﻮﻧﻪ S2 در ﻣﻘﺎﺑﻞ ﭘﻨﯽﺳﯿﻠﯿﻮم اﮐﺴﭙﺎﻧﺴﻮم و آﺳﭙﺮژﯾﻠﻮس ﻧﺎﯾﺠﺮ ﺑﻪ ﺗﺮﺗﯿﺐ ﺑﺎ MIC ﺑﺮاﺑﺮ 20 و 40 ﻣﯿﻠﯽﮔﺮم ﺑﺮ ﻣﯿﻠﯽﻟﯿﺘﺮ اﻓﺰاﯾﺶ ﯾﺎﻓﺖ. ﻫﻤﭽﻨﯿﻦ ﻫﻤﻪ ﻧﻤﻮﻧﻪﻫﺎي ﺷﯿﺮ ﺗﺨﻤﯿﺮي (زﻣﺎن ﺻﻔﺮ) ﺧﺎﺻﯿﺖ ﺿﺪ ﻣﺨﻤﺮي را ﺑﻌﺪ از 72 ﺳﺎﻋﺖ ﺑﺎ MIC ﺑﺮاﺑﺮ ﺑﺎ 40 ﻣﯿﻠﯽﮔﺮم ﺑﺮ ﻣﯿﻠﯽﻟﯿﺘﺮ ﺣﻔﻆ ﮐﺮدﻧﺪ ﮐﻪ ﭘﺲ از 7 روز ﻧﮕﻬﺪاري، اﯾﻦ وﯾﮋﮔﯽ در ﺷﯿﺮﻫﺎي ﺗﺨﻤﯿﺮ ﺷﺪه ﺑﺎ ﮐﺸﺖ ﻣﻨﻔﺮد اﻓﺰاﯾﺶ ﯾﺎﻓﺖ ﻧﺘﯿﺠﻪﮔﯿﺮي: در ﻃﯽ ﺗﺨﻤﯿﺮ، ﺑﯿﺸﺘﺮﯾﻦ ﻓﻌﺎﻟﯿﺖ ﭘﺮوﺗﺌﻮﻟﯿﺰي در ﻧﻤﻮﻧﻪاي اﺗﻔﺎق اﻓﺘﺎد ﮐﻪ ﺗﻮﺳﻂ ﺗﺮﮐﯿﺒﯽ از ﺳﻪ ﺑﺎﮐﺘﺮي ﻻﮐﺘﯿﮑـﯽ ﺗﺨﻤﯿـﺮ ﺷﺪه ﺑﻮد. ﻋﺼﺎره ﺣﺎوي ﭘﭙﺘﯿﺪ ﻫﻤﻪ ﻧﻤﻮﻧﻪﻫﺎي ﺷﯿﺮ ﺗﺨﻤﯿﺮ ﺷﺪه داراي ﺧﺎﺻﯿﺖ ﺿﺪ ﮐﭙﮑﯽ و ﺿﺪ ﻣﺨﻤﺮي ﺑﻮدﻧﺪ و اﻏﻠﺐ ﻧﻤﻮﻧﻪﻫﺎ در زﻣﺎن ﺻﻔﺮ داراي MIC ﺑﺮاﺑﺮ 40 ﻣﯿﻠﯽﮔﺮم ﺑﺮ ﻣﯿﻠﯽﻟﯿﺘﺮ ﺑﻮدﻧﺪ ﮐﻪ اﻟﺒﺘﻪ در ﺑﺮﺧﯽ از ﻧﻤﻮﻧﻪﻫﺎ، 7 روز ﭘﺲ از ﻧﮕﻬﺪاري ﺧﺎﺻـﯿﺖ ﺿـﺪ ﻗﺎرﭼﯽ اﻓﺰاﯾﺶ ﯾﺎﻓﺖ. در اﯾﻦ ﭘﮋوﻫﺶ اﺣﺘﻤﺎل ﻣﯽرود ﺑﺨﺸﯽ از ﺗﺄﺛﯿﺮ ﺿﺪﻗﺎرﭼﯽ ﺷﯿﺮﻫﺎي ﺗﺨﻤﯿﺮي ﺑﻪ دﻟﯿﻞ ﭘﺮوﺗﺌـﻮﻟﯿﺰ و ﻣﺤﺘـﻮاي ﭘﭙﺘﯿﺪي آنﻫﺎ ﺑﺎﺷﺪ.

Background and objectives: In recent years, the use of lactic acid bacteria has expanded as protective cultures in the production of fermented products. They have inhibitory activities against different pathogenic or spoilage bacteria and fungi. A significant part of the antifungal activity of lactic acid bacteria is related to the metabolites produced by these organisms, in particular the various organic acids. However, recently peptides and protein hydrolysates with antifungal activity have been produced. The aim of this study was to investigate the activity of isolates of proteolytic lactic acid bacteria in the production of fermented milk with antifungal properties. Materials and methods: In this study, sterilized skim milk was inoculated with single and co-cultures of Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus reuteri and Lactococcus lactis subsp. lactis (2%, w/w) and fermented at 37 °C to reach the optimum pH 4.5-4.6. The peptide content and antifungal properties of fermented milk against Aspergillus niger, Penicillium expansum and Candida albicans were evaluated after fermentation and 7 days of storage in the refrigerator. Results: According to the results, Lactobacillus reuteri showed the least proteolytic activity in milk and among all the samples, the highest peptide content was observed in the sample fermented by combined cultures of all three isolates, resulting in the production of 62.6 mg peptide per ml. Peptide-containing extract of fermented milk samples from combined cultures (S23 and S123) had the highest anti-mold activity with a MIC of 20 mg/ml. After 7 days of storage, the anti-mold property of sample S1 against Penicillium expansum and sample S2 against Penicillium expansum and Aspergillus niger increased with a MIC of 20 and 40 mg/ml, respectively. Also, all samples of fermented milk (zero time) retained anti-yeast properties after 72 h with a MIC of 40 mg/ml, which after 7 days of storage, this property was increased in fermented milk with single culture. Conclusion: During fermentation, the most proteolytic activity occurred in a sample fermented by a combination of three lactic bacteria. Peptide-containing extract of all fermented milk samples had anti-mold and anti-yeast properties, and most of the samples had a MIC of 40 mg per ml at day 0, although in some samples antifungal activities were increased after 7 days of storage. In this study, it is likely to be a part of the antifungal effect of fermented milks due to the proteolysis and resulted peptide content.