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MUTANT OF ALOCAL ISOLATE OF LACTOBACILLUS
PLANTARUMBY UV LIGHTIN COMPARISION WITH
ASTANDARD STRAIN THE EXTENT OF ITSABILITY TO LOWER
CHOLESTEROL LEVELS1
Sinan J. Abdul-Abbas*; Aswan H. Aboud Al-bayyar**;
Adnan E. Al-Badran***
* Department of Food Science ,College of Agriculture,University of ,Basrah,Iraq
**Department of Food Science ,College of Agriculture,University of Baghdad, Baghdad
,Iraq
Department of Biology ,College of Science,university of Basrah,Basrah,Iraq
(Received 9 May 2016,Accepted 26 May 2016)
Key words: Mutation, UV light, RAPD-PCR
.
ABSTRACT
Local strain bacteria of Lactobacillus plantarum IRQ12 isolated from cucumber
sativuspickle registered in (GenBank LN871447) and another standard strain, IRQ12
strain was mutated by UV light (254 nm) for (50 cm) distance for different periods 20,
25, 30, 35 and 60 second, RAPD – PCR test was done to investigate the effect of UV
light onL.plantarum genes and determine the genetic distant. The ability ofstandard and
local mutated bacteria to decrease the amount of cholesterol was determined in MRS
broth–EY mediawhich was (4.6 mg/ml), the cholesterol amount was decreased at 25s
which was0.35 mg/ml in a decreased percentage 92.3%, followed by 60s and 35s which
were 0.43and 0.45mg/ml in a decreased percentage 90.6% and 90.2%, respectively.
INTRODUCTION
In biology, a mutation is a change occurs in the genetic information - bio genetic
information encoded in DNA sequences and chromosomes that contain DNA or RNA
sequences, as in some viruses, mutation makes changes in the sequences of nucleotide
bases in different ways, it may change the arrangement sequences of nucleotides, or it
may change its number by deletion or insertion or mutations caused by viruses(2, 6,9).
Mutationcan occur as a result of exposure of mutagenic chemicals and radiation and
errors that occur during DNA doubled,or spontaneously, can be caused by organismitself
by cellular processes such as hyper mutation (26).The mutation is one of the methods
1 Part of Ph.D. thesis
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used widely to improve the bacterial strains which are used in industrial microbiology(3).
Ultra-violet (UV) light has strong effects on DNA genes, occurrence of mutations,in the
worst case it may cause the growth of cancer; sun is the main source of UV (31, 8).
Random mutations by ultraviolet rays used to improve certain properties of the strain, as
the efficiency of the formationmutations depends on the type of damage (replace the pair
of base, deletion and insertion..Etc.)Which causes a certain change in the DNA and the
mechanism to repair this damage, mutation resulted from the use of UV light is more
stable in the long time of generation (11, 25, 36). (15, 10) studied the effect of random
mutations of three genes ofL.plantarumbacteria which encode proteins related to stress,
studies were indicated that these mutations can cause the distinctive changes could be
observed on the cell surface when grow and thus affect their ability to live symbiotically
(16, 7, 10). (27)Was indicatedthatthe range of UV impact in the DNA depends on the
dose ofradiation as its intensity,time exposure and the distance between the sample and
the source.The cholesterol is the main cause of heart disease and hardening of
thearteries,(37)illustrated that there is a relationship between cholesterol and heart disease
and blood vessels.There are two types of cholesterol, one of them is because the food
contain of a high rate of cholesterol like meat, milk and eggs, and this is called the outer
cholesterol (Exogenous cholesterol) The second type is the internal cholesterol
(Endogenous cholesterol) which is synthesis within the body and is made in the liver
(24).Many studies have indicated that Lactobacillus bacteria have the ability to lower
cholesterol through many mechanisms like deconjugation (22).This is done through the
ability of therapeutic bacteria to absorb the cholesterol and decreased its level in addition
to its ability to produce ferulic acid(38, 39). (37) found that L.plantarum bacteria have the
ability to metabolic the cholesterol in a rate 28.3% in amediainclude the cholesterol in a
rate 100 Mμ gm/ml when treated for 24 hours, whilein MRS media which include the
intestinal fluid 100 Mμ gm/ml of cholesterol metabolism is 20.5%.
The aim of this studyis to investigatethe ability of the Lactobacillus plantarumbacteria to
decrease the cholesterol levels and using the mutation of bacteria by UV light to increase
its ability to decrease the cholesterol levels.
MATERIALS AND METHODS
The local strain bacteria Lactobacillus plantarum IRQ12 isolated from
Cucumbersativus pickle which registered in (GenBank LN871447) was used in this
study, while the standard strain thatused as starter was suppliedbyChr-Hansen’s Denmark
/ UK. The mutation process for these bacteriadone by using UV frequency (254nm) for
distance 50 cm with various periods (20, 25, 30, 35 and 60) seconds according to the
method of (19, 20)which wasmodified by (34) usingTryptone soy broth (TSB) media.
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Detection of the identical genes
DNA was extractedusing Automated Nucleic Acid Extraction system following
the manufacturer instructions. Theelectrophoresis gel wasprepared for the detection of
DNA molecules(1).
Random Amplified Polymorphic DNA (RAPD) byPCR
RAPD-PCR was performed as previously described (33).
Table (1) Identification RAPD – PCR primers
Primers Sequence Length Optimizing TA
1283 5'-GCGATCCCCA-3' 10 36˚C
Table (2) Materials and quantities used for the amplification of RAPD (20μl) in PCR
Reagent Volume
primer 1.5 (30 pmol)
Go Taq Green Master Mix. 2x 5μl
DNA template 2.5μl
Free nuclease water 11 μl
Total volume 20μl
Table (3) the conditions for PCR cycles for RAPD reaction
Steps Temperature Time Number of cycles
Initial denaturation 95o C 3 min. 1
Denaturation 94oC 1 min.
Annealing 36oC 1 min. 45
Extension 72oC 2 min.
Final extension 72oC 5 min. 1
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Data Analysis
- The RAPD bands of each individual strain were calculated for theirdistances based on
the ladder's bands.
- The data of the RAPD patterns of all strains according to (29) were transformed to the
Unweighted Pair Group Method with Arithmetic mean (UPGMA) algorithm program
creating and modifying by (18).
- RAPD patterns of individual strains were compared based on the index of similarity
between samples (12), providing a mathematical model by calculating a similarity
matrix and transforms similarity coefficients into a distance matrix (Distance Matrix
value "0.000" indicating identical strains) and makes a clustering to construct a
dendrogram from a set of variables, to study genetic variation especiallywith difficult
or closelyrelated RAPD patterns.
Cholesterol determination
The mediaMRS-Egg yolk (MRS-EY) was preparedas previously described(4).
Determination of cholesterol rate
The rate of cholesterolof all media used for testing the ability of Lactobacillus to decrease
the cholesterol was determinedas previously described (17).
RESULTS AND DISCUSSION
Study the rate of geneticvariation for L.plantarum IRQ12isolate before and after
induction of mutation
Figure (1) showed theresults of using RAPD – PCR between the non-mutated strain in
line 2 and the mutated strain with the exposure periods 20, 25, 30, 35 and 60s in the lines
3, 4, 5, 6 and 7 respectively, which are distinctlydifferent and there is a mismatch
between bands. The genetictree (Dendogram) (Figure.2) illustrate that the periodof
radiation exposure led tomakevarietymutations in all periods, and this indicate that the
irradiation process led to make a genetic mutation.It was also absolved the lack of any
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genetic match between the allmutation periods for IRQ12 strain, as each time showed
individual mutation genetic.
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Table (4) showed that L.plantarum IRQ12 strain exposed to irradiation for 25s was
moreaffected on bacteria DNA and this is because of ahigh mutation rate for strain which
was 2.52, while it was less affectedfor irradiation exposure (less genetic distance),at 30s
and 20s as it was 1.359.
Figure (3) indicate the local and standard strains ability to decrease the cholesterol rate of
(4.6 mg/ml) after growth on MRS broth – EY media at 37°C for 24 hours. The
cholesterol rate in broth media for IRQ12 and the standard strains before induction of
mutation was 0.52, 0.733 mg/ml.It was absolved that IRQ12 have less rate of cholesterol
(Figure4) in the media with decreasing the percentage 88.69%, while the decrease
percentage of thestandard strain was 84.06%, the results of mutation for L.plantarum
IRQ12 bacteria after grow on MRS broth – EY media at 37°C for 24 hours indicate that
the cholesterol rate in varying depending on the radiation dose which was used, the less
amount of cholesterol in the media for mutation isolate for 25s which was 0.35 mg/ml
with a high decreasing percentage 92.3%, Followed by the strain treated for 60s with
amount 0.43 mg/ml and with decreasing percentage 90.6%, then the mutation strain for
35s with amount 0.45 mg/ml and with decreasing percentage 90.2%, while the mutation
strains treated for 30s and 20s the cholesterol amount in the media were approached
IRQ12 strain before mutation which were 0.541 and 0.537 mg/ml with the decreasing
percentage 88.23% and 88.3%, respectively. It was noted by the results mentioned above,
find that L.plantarum bacteria has the ability to decrease cholesterol levels were high
when it compared with other results from previous studies.
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The current results isagreement with (32) in a study included 15 isolated bacteria
L.plantarum from different sources which observed bacteria ability to absorb and
decrease cholesterol between 56.52 - 95.65%.It is also agree with (30)when L.plantarum
strain capable to decrease cholesterol levels between 26.74 - 85.41% in growth media
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after 20 hours in incubator.While as for the isolation of mutation isolate IRQ12 which
showed a significant increase in the percentage of decrease cholesterol at 25s,35s and 60s
and this result are approach with (5), whichwas investigated the L.caesiability to
decreasethe cholesterol when grown in MRS broth - EY media, the percentage decrease
was 92.5%.The increasingof the cholesterol decreasing percentage in mutationisolates
maybe due tothe increasingability for the cell membrane of bacteria to absorb more than
the amount of cholesterol in the media(23). It must be pointed out that the bacteria's
ability to absorb cholesterol affected according to their growth rate in the media (14).
(28)Investigatedthat proposed mechanism for reducing cholesterol in the lactic acid
bacteria involves removing some of cholesterol through the cell membrane during
growth, so the useful of a high amount of live bacteria to increase ability to absorb more
amount of cholesterol,thereby reducing the rate of absorption of cholesterol in the
intestine and transfer to the blood(21). The absorb cholesterol through the cell membrane
estimated 20% (13).Whilethecholesterol as a whole did not absorbed through the cell
membrane, assome researchers believe that free cholesterol maybe compoundwith cells
so maybe disposal out throw thedead cells, this characteristic may increase due to the
occurrence of some of the changes that may lead to increase the tensile strength of the
external membrane of the bacteria, which increase the deconjugation(21).Also, the
efficiency of the binding process between the cholesterol molecules and the outside
member of bacteria are strongwhich cannot separate, even when washing several
times(35).
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بوساطة الأشعة فوق Lactobacillus plantarum تطفیر العزلة المحلیة لبکتریا
البنفسجیة ومقارنتھا مع سلالة قیاسیة لتقدیر مدى قابلیتھا على خفض الکولسترول 2
سنان جودة عبد العباس* ،أسوان حمد لله عبود** ،عدنان عیسى البدران***
*قسم علوم الأغذیة ، کلیة الزراعة ، جامعة البصرة ،البصره ،العراق.
** قسم علوم الأغذیة ، کلیة الزراعة ، جامعة بغداد ،بغداد ،العراق.
*** قسم علوم الحیاة ، کلیة العلوم ، جامعة البصرة ،البصره ،العراق.
الخلاصة
معزولة من مخلل خیار القثاء Lactobacillus plantarum IRQ استخدمت سلالة محلیة من بکتریا 12
IRQ وسلالة أخرى قیاسیة، تم تطفیر السلالة 12 (GenBank LN ومسجلة فی بنک الجینات ( 871447
50 عن النماذج وعلى مدد تطفیر cm 254 ) ولمسافة nm) بوساطة الأشعة فوق البنفسجیة بطول موجی
RAPD – مختلفة 20 و 25 و 30 و 35 و 60 ثانیة، وجرى فحص التضخیم العشوائی متعدد الأشکال
وتقدیر البعد الوراثی بین L.plantarum لبیان مدى تأثیر الأشعة الآیونیة على جینات بکتریا PCR
الجینات. جرى تقدیر قابلیة البکتریا القیاسیة والسلالة المحلیة المطفرة على خفض مستوى الکولسترول
إذ أظھرت النتائج انخفاض مستوى الکولسترول MRS – EY 4.6 ملغم کولسترول / مل) فی وسط مرق )
للبکتریا المطفرة بمدة التطفیر 25 ثانیة وبلغ 0.35 ملغم کولسترول / مل وبنسبة مئویة للانخفاض بلغت
92.3 تلتھا مدة التطفیر 60 و 35 ثانیة بمقدار 0.43 و 0.45 ملغم کولسترول / ملوبنسبة مئویة للانخفاض
بلغت 90.6 و 90.2 على التوالی.
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