Reduction of Aflatoxin M1 in Milk by Microorganism Isolated from Kefir Grains
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Date
2021-03-09Author
Adriansyah, Putri Nabila Adinda
Rahayu, Winiati Pudji
Kusumaningrum, Harsi Dewantari
Osamu, Kawamura
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One of the nutritious source that widely consumed including children and elderly is milk. However milk is not always free of contamination that can endanger people who consumed it, especially to children and elderly who are more prone to contamination. One of the most toxic mycotoxin is aflatoxin B1 (AFB1) which is mainly produced by Aspergillus flavus and Aspergillus parasticus. Those molds can grow on feed that is fed to the cow and converted inside the cow to aflatoxin M1 (AFM1). AFM1 considered as potential hazard because it almost has the same structure as AFB1 although only has 10% of AFB1 toxicity. AFM1 also is highly resistant to heat so it can survive on milk heating process. Mycotoxin can be reduced by several methods, such as chemical, physical and biological methods. Biological method with microorganism antagonism is one of the emerging methods because unlike chemical methods, microbial method use microorganism that belongs to GRAS (Generally Recognize as Safe).
Decontamination method with microorganism usually done with lactic acid bacteria (LAB) or yeast. LAB and yeast can reduce AFM1 that contaminate milk. LAB and yeast can be found in kefir. Kefir is a fermented milk product which is made from cow or goat milk with kefir grains as starter. Kefir is known for its beneficial effect for health because of the metabolites that microorganism in kefir grains produce, such as lactic acid, kefiran, and bacteriocin. Kefir has complex bacterial composition that is varies according to the medium, origin of kefir grains, and storage condition. Hence microorganism from kefir grains has potential to reduce AFM1 in milk.
This research was conducted to to compare the reduction ability of microorganism isolated from kefir grains with different incubation time and identify the strain of lactic acid bacteria and yeast that can effectively reduce AFM1 In general, this research was conducted through this following stages : (1) Preparation ; (2) Enumeration and isolation of microorganism from kefir grains (3) Column Preparation (4) Evaluation of reduction of AFM1 by microorganism isolated from kefir grains (5) Identification of LAB and yeast strains from kefir grains that can reduce AFM1.
The result of the research showed that there were more aerobic lactic acid bacteria than anaerobic lactic acid bacteria in kefir grains. The means of colony count of anaerobic lactic acid bacteria, aerobic lactic acid bacteria, total yeast of kefir grains were 1.9 x 109 CFU/g, 2.1 x 109 CFU/g, 2.0 x 108 CFU/g respectively and in kefir milk were 1.0 x 108 CFU/g, 1.0 x 108 CFU/g, 7.6 x 107 CFU/g respectively.
Evaluation of AFM1 reduction was done by microorganism isolated from kefir grains, namely anaerobic lactic acid bacteria (LAN), aerobic lactic acid bacteria (LAE) and yeast (YEA). This experient successfully isolated 10 isolates of LAE, 4 isolates of LAN and 9 isolates of YEA. This study used non-viable cell, for approximately 1.0 x 108 cfu/mL of microorganism to reduce 10 ng/mL AFM1 in Phosphate Buffer Saline (PBS). Non-viable cell was used for this experiment because past studies observed that non-viable cells showed higher percentages of AFM1 removal in a relatively short contact time. The isolates and AFM1 contaminated PBS mixture were incubated in 4oC for 4 hours and 24 hours to see the reduction ability of each isolates. ANOVA test showed that interaction of isolates and incubation time factors only gave significant result on LAN, meaning that there is different pattern of AFM1 reduction on LAN influenced by both different isolates and time. The most optimum combination for LAN is LAN3 and LAN5 at 24 hours incubation at with significant result on AFM1 reduction.
AFM1 reduction ability of microorganism isolated from kefir grains ranged from 1.6% to 29.3% in both 4 and 24 hours incubation time. All LAN isolates at 4 hours incubation except for LAN5 showed significant result on AFM1 reduction, with LAN2, LAN3 and LAN4 showed non significant difference. Meanwhile at 24 hours incubation, LAN3 and LAN5 had significant result on AFM1 reduction, although AFM1 reduction percentage by LAN3 and LAN5 at 24 hours did not have significant difference. Increase of incubation time affected AFM1 reduction on LAN isolates significantly. LAE isolate with the highest reduction and significant result on 4 hours incubation was isolate LAE7 (29.3 ± 0.6 %), although the result was not significantly different than LAE1, LAE9 and LAE10. This result showed that 4 hours incubation give significant result on AFM1 reduction of LAE isolates percentage than 24 hours incubation. Meanwhile at 24 hours incubation, LAE1 showed significant result than other isolates. Incubation of both 4 and 24 hours incubation showed all YEA isolates was not significantly different. Isolate YEA2 has the highest reduction percentage on both 4 and 24 hours incubation despite has non significant result. Although in general, AFM1 reduction percentage by YEA isolates increased with longer time incubation with significant result.
In this study, LAB isolate have higher AFM1 reduction percentage than yeast. It is also known that the mean reduction in LAE has decreased after 24 hours, while the average AFM1 reduction of LAN and yeast increased after 24 hours. LAE isolates have higher mean of AFM1 reduction percentage than yeast and LAN isolates. It can be seen that LAE shows different behavior from LAN and yeast. It can be concluded that LAE is a bacterium that has most influence on AFM1 reduction among all microorganism isolated from kefir grains.
The highest AFM1 reduction percentage on each the tested microorganisms was isolate LAE7 ( 29.3 ± 0.6%) in 4 hours incubation time followed by isolate YEA2 (20.6 ± 0.8%) in 24 hours incubation time. Analysis of DNA sequencing of LAE7 Isolate using BLAST results obtained that this isolate have homology or level a similarity of 99.79% with species of Lactobacillus kefiri strain A/K Analysis of DNA sequencing of YEA2 isolate using BLAST results obtained that this isolate have homology or level a similarity of 99.49% with species of Saccharomyces cerevisiae NRRL Y-12632. This experiment proved that isolates from kefir grains can reduce AFM1 and have potential for practical use.
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