Download The effects of Mentha longifolia on the growth of probiotic bacteria, B

The effects of Mentha longifolia on the growth of probiotic bacteria, Bifidobacterium bifidum and
Lactobacillus acidophilus in milk and yoghurt and investigation of biochemical factors and liver
tissue in rats
Abstract
Aim:Despite significant progress in reducing rate of cardiovascular disease death and mortality, still these
diseases are the leading cause of death in many countries and hyperlipidemia is one of the most important
factors in the pathogenesis of these diseases. The aim of the present study is to investigate the effect of
Mentha longifolia in increasing growth of probiotic bacteria Lactobacillus, Acidophilus,
Bifidobacterium bifidum in probiotics milk and Yogurt and their impact on serum lipid pattern in a highfat diet fed rats. Materials and Methods:To determine different doses of Mentha longifolia (0%, 0.2%,
0.4% and 0.6 % ) effects in the first (milk ) and the second (yogurt ) phase on growth of probiotic bacteria
Bifidobacterium bifidum and Lactobacillus Acidophilus, Separately 0/33 grams lyophilized bacteria of
Bifidobacterium bifidum and Lactobacillus acidophilus were added to a liter of ultra-heat treated skim
milk. Acidity, pH and microbial growth were examined during incubation and retention period. And on
the tenth day after production products were subjected to sensory evaluation. Followed 144 male Wistar
rats were divided into 24 groups of 6 and within a week were used to fatty food And water containing
25% milk. Then for 14 days were treated with different doses of probiotics milk and yogurt that contains
Mentha longifolia. At the end of the fourteen day in order to measure the levels of cholesterol,
triglycerides, LDL-C and highest level of HDL-C blood was taken from all the test groups. Results:In
microbial culture evaluating , Bifidobacterium Bifidum probiotic bacteria than Lactobacillus acidophilus
didn’t have good growth on MRS Agar medium. The study results showed that increasing concentration
of Mentha longifolia had positive effect on the growth of these probiotic bacteria in probiotic milk and
yoghurt. Also the group that received (0.6%) Mentha longifolia Milk and yoghurt had the lowest levels of
cholesterol, triglycerides , LDL-C and highest rate of HDL-C. (p≤ 0/05). Conclusion:In general we can
say that Mentha longifolia causes growth of Lactobacillus Acidophilus and Bifidobacterium Bifidum
probiotic bacteria in milk and yoghurt. Also the daily dose of Mentha longifolia, Lactobacillus
acidophilus and Bifidobacterium in milk and yoghurt By reducing serum cholesterol, triglycerides , LDLC and increasing HDL-C improves serum lipid pattern.
Introduction:
Heart disease, especially coronary artery disease is a leading cause of disability and death in industrialized
countries.(1,2) Several factors are effective in the development of atherosclerosis in the arteries ,
including the coronary arteries.(3,4) Nowadays ischemic heart disease is the most important indicator for
Atherosclerosis. Virtually all patients affected by myocardial infarction have coronary atherosclerosis.
And most victims of sudden death have had a history of ischemic heart disease diagnosis.(5,6)
Hyperlipidemia is elevated levels of serum lipids including total cholesterol, LDL and TG of which can
lead to increased progress in the development of atherosclerosis and ultimately lead to myocardial
infarction.(7) Probiotics are microorganisms that provide health benefits when consumed, as claimed by
some.[8] The term probiotic is currently used to name ingested microorganisms associated with beneficial
effects to humans and other animals [9]. According to definition of FAO, probiotic microbes are alive
organisms that are used by eating and condition application in repellent number and caused creation of
one or many healthy effects on host body. These include characteristic anticancer effects (10), regulating
and enhancing safety system, anti microbial effect, decrease of serum cholesterol (12, 13), improvement
of lactose intolerance, increase of nutritional value (11), and improvement of lactose intolerance. Bifidus
are the most usual species to use among microbes of probiotic Lactobacillus acidophilus and
Bifidobacterium bifidum (8,9,13). Recently design and product of probiotic productions with basic plant
are considered for both rule of health nature these food material groups (protein, fiber, vitamin, solute)
and variation creation in product and consume. It seems that probiotic product of these productions with
relevant quality property. Take considerable part of studied searches in the future (14). After the sufficient
consumption of probiotics, beneficial effects on the natural microbial population of the gastrointestinal
tract can be seen. The most common probiotic bacteria included Lactobacillus acidophilus and
Bifidobacterium bifidum.(8, 15). Based on animal and human studies , probiotics in multiple levels
regulates reduction of endogenous inflammatory mediators. The main routes are: regulating the immune
system , adjust the intestinal flora , improving intestinal tight junctions , reducing the permeability of
pathogens into the bloodstream ,anti-inflammatory and anti- fibrosis activity.(16) Now, there is no
definitive chemotherapy for Nonalcoholic fatty liver disease (NAFID). The effectiveness of probiotics has
been reported in several experimental models of NAFLD / NASH. In a study on ob / ob mice with highfat diet observed that manipulation of intestinal flora in this experimental model affects Fatty liver disease
caused by obesity. Indeed VSH # 3 probiotic improves liver histology, reduces liver total fat and serum
levels of alanine aminotransferase (ALT).(17) In another study , probiotic VSH # 3 could improve lipid
Profile and reduced Inflammation, oxidative damage and tissue levels of TNF- .(18) To date, few studies
have examined the effect of probiotics in the treatment of human NAFID. In a research , the daily
consumption of probiotic supplements for three months, decreased serum levels of liver enzymes (ALT,
AST and gamma- glutamyl trans glutamyltranspeptidase GGT) .(19) Mentha longifolia is one of Labiatae
family species, which includes 20 species which are distributed around the world. This medicinal plant is
one of the Mint species that typically is known as Pennyroyal. The natural habitat of this plant is in
Europe, North of Africa and Asia Minor and the Middle East (20). Aerial parts including Mentha
longifolia flowers normally are used as effective substance in treating colds, sinusitis, cholera, food
poisoning, inflammation of the bronchi, and tuberculosis as well (21). This medicinal plant also contains
properties such as carminative, expectorant and diuretic, antitussive and anti-menstruation (22).
Antimicrobial medicinal plant powder and oil of Mentha longifolia has been determined (23). In this
study the effects of Mentha longifolia on the growth of probiotic bacteria, Bifidobacterium bifidum and
Lactobacillus acidophilus in milk and yoghurt and Investigation of blood factors in adult male rats were
investigated.
Material and method
-
Bacteria lyophilize Lactobacillus acidophilus (CHR Hansen company, Denmark)
-
Bacteria lyophilize Bifidobacterium bifidum (CHR Hansen Company, Denmark)
-
Low-fat sterilized milk (1.5 % fat)
-
Low-fat yoghurt from supermarket.
- Mentha longifolia essence
We do an experiment to compare effect of essence and Mentha longifolia essence probiotic bacteria’s
grow which type of them is used for experiment.
The effect of Mentha longifolia on the production of probiotic Bifidobacterium Bifidum milk at first
passage
In order to produce the milk containing probiotic bacterium Bifidobacterium bifidum, four containers
each containing 1 liter of low-fat sterilized milk (%1.5 fat) were considered as our four groups. The
starter (Bifidobacterium bifidum) was added directly to all the containers, followed by adding dried
Mentha longifolia essence %0 ( to the control), 0.2%, 0.4%, and 0.6% to all the containers, respectively
and finally they were placed in hot –house at 380c. The acidity test was performed approximately every 2
hours until reaching 420 Dornic (8).
The samples were then taken out of hot –house, transferred to a refrigerator and stored at 20c. The
produced probiotic milk was evaluated once every 5 days by counting the microbes using direct counting
method.
The effect of Mentha longifolia on the production of probiotic Bifidobacterium bifidum yoghurt at the
second passage
To produce Bifidobacterium bifidum yoghurt in this stage, after providing 4 containers, 1 liter of low - fat
sterilized probiotic milk (%1.5 fat) from the control group at first passage and the (%1.5) starter of lowfat yoghurt (%1.5) were added to each container.
Different concentrations of Mentha longifolia (0%, 0.2%, 0.4%, and 0.6%) were added respectively to
the containers and mixed properly so that Mentha longifolia was uniformly dissolved. Afterwards, all the
containers were placed in the hot – house at 380 C. Approximately every 2 hours, the acidity and pH tests
were done until acidity reached 900 Dornic. Then, the samples were taken out of the hot-house,
transferred to a refrigerator and stored at 20C. The produced probiotic Mentha longifolia yoghurt was
evaluated every 5 days by counting the microbes using direct counting method and after 10 days the
yoghurt was evaluated for sensory properties (8), using questionnaires filled by 40 people. The
respondents were asked to rate the factors of scent, taste and permanence on a scale ranging from very
good, good, medium, to weak. The results were analyzed in a statistical descriptive test by SPSS version
16 software.
The effect of Mentha longifolia on the production of probiotic Lactobacillus acidophilus milk at first
passage
In order to produce the milk containing bacterium Lactobacillus acidophilus, 4 containers each containing
1 liter of low-fat sterilized milk (%1.5 fat) as our 4 groups were prepared, and the starter (Lactobacillus
acidophilus) was added directly to all of them. Then, dried Mentha longifolia essence %0 (Control), %0.2,
%0.4 , and %0.6 were added to them, and all the containers were placed in the hot-house at 380 C.
Approximately every 2 hours, the acidity test was done until it reached 420 Dornic. The samples were
then taken out of the hot-house, transferred to a refrigerator and stored at 20C. The produced probiotic
milk was evaluated every 5 days by counting the microbes using direct counting method.
The effect of Mentha longifolia on the Production of probiotic Lactobacillus acidophilus Yoghurt at
Second Passage
To produce Lactobacillus acidophilus yoghurt in this stage, after providing 4 containers, 1 liter of low-fat
sterilized probiotic milk (%1.5 fat) from the control group at first passage, and the starter of low-fat
yoghurt (%1.5) were added to each container.
The different concentrations of Mentha longifolia (%0, %0.2, %0.4, and % 0.6) were added to containers,
respectively and mixed properly so that the Mentha longifolia was uniformly dissolved. Then, all the
containers were placed in the hot-house at 380 C. The acidity of the samples was checked approximately
every 2 hours till reaching 900 Dornic, at which point the samples were taken out of the hot-house,
transferred to a refrigerator and stored at 20 C. The produced probiotic Mentha longifolia yoghurt was
evaluated every 5 days by counting the microbes using direct counting method, and after 10 days, the
yoghurts were evaluated with regard to sensory properties performed using questionnaire. The factors
including scent, taste and permanence, were ranged on a scale from very good, good, medium, and weak.
The results were subsequently analyzed in a statistical – descriptive test by SPSS software version 19.
The effect of Mentha longifolia on the production of Probiotic Lactobacillus acidophilus and
Bifobacterium bifidum milk at first passage
As previously described, four containers containing 1 liter of sterilized low-fat milk (%1.5 fats) were
considered as our four groups. The bacteria Lyophilize Bifidobacterium bifidum and Lactobacillus
acidophilus were simultaneously added to all the containers. The first container was considered as the
control group and dried Mentha longifolia essence %0.2, %0.4, %0.6 were added respectively to the other
containers. The procedures were followed as we did in the first stage.
The effect of Mentha longifolia on the production of probiotic Lactobacillus acidophilus and
Bifidobacterium bifidum yoghurt at second passage
To investigate the effect, the four containers each containing 1 liter of sterilized low-fat milk were
considered as four groups. Then, the starter of yoghurt and the probiotic milk from the control group in
the previous stage were added to all the containers. Except for the first container which was considered as
the control, the Mentha longifolia %0.2, %0.4, %0.6 were added to the other containers. Procedures of
yoghurt production were the same as those for the yoghurt with the above – mentioned bacteria.
Determining the product shelf life duration
Having produced the above-mentioned products, we stored 1000 gr of each product in a disposable
container placed in a refrigerator for 20 days. During this period, each sample was tested in days 1, 5, 10,
15, 20 for acidity, pH, and sensory properties.
Blood Sampling
160 male Wistar rats were divided into 10 groups of 16 and within a week they were adapted to the food
contains 25% milk and water. Control group 1: Milk containing Lactobacillus acidophilus, Control group
2: Milk containing Bifidobacterium bifidum, experimental groups 1, 2, 3 in order received 0.2, 0.4 and
0.6 milk containing Lactobacillus acidophilus and Mentha longifolia. Control groups 3 and 4, respectively
received yogurt contains Lactobacillus acidophilus and Bifidobacterium bifidum and the experimental
Groups 7, 8 and 9, respectively received Yogurt containing Lactobacillus acidophilus and Mentha
longifolia 0.2 , 0.4 and 0.6. The experimental groups 4, 5 , 6, 10 , 11 and 12 , respectively, received 0.2 ,
0.4 and 0.6 milk and yogurt containing Bifidobacterium bifidum and Mentha longifolia. Rats were under
tested for 40 days with different doses of Mentha longifolia, and then blood samples were taken and liver
enzymes, cholesterol, triglycerides, cholesterol-HDL, cholesterol-LDL, total bilirubin, creatinine,
Albumin, total protein in rat.
Measurements:
ALT, AST and ALP were measured using colorimetric standard methods. Serum total cholesterol was
analyzed by BIOTRON BTR 820 Auto Analyzer using enzymatic method [24]. HDL cholesterol was
measured by the above-mentioned analyzer using phosphotungstate [1]. LDL cholesterol was calculated
based on Friedwald’s equation [1]. Serum triglycerides were estimated by using Autopack Reagent Kit by
enzymatic DHBC colorimetric method. Serum creatinine and total bilirubin were determined according to
Young and Pestaner (1975), and Jendrassik (1938) methods respectively. Serum total protein was
measured according to the Biuret method as modified by Hutson et al., (1972) using bovine serum
albumin as standard [24, 25, 1]. Serum albumin were assayed by Bromocresol green method.
Histopathological examination:
Rat liver slices were collected and immersed in 10% neutral formalin as fixative. The fixative liver
samples were sent to the Cancer Institute for histopathological examination according to Bancroft et al.,
(1996)[26].
Results
Table 1 shows the acidity in Dornic degree in Lactobacillus acidophilus Mentha
longifolia yoghurt and milk at refrigerator during 20 days. Findings show that the
samples with % 0.6 concentration Mentha longifolia were ranked the best for taste,
color, insolubility. Although low fat milk was used to produce yogurt, it was tasted
full fat. Existence of probiotic bacteria were evaluated by direct count and
cultivation on MRS environment. During the 20 day period of observation, the
microbial counts were persistent and there was a significant difference between the
controls (Table 2). Also groups receiving 0.6 % of Mentha longifolia to significant
reduction of liver enzymes, bilirubin, creatinine and cholesterol-LDL (P≤0.05).
Furthermore, a significant increase was seen in triglycerides, cholesterol-HDL
levels respectively (P≤0.05 and P≤0.01) (Table 3).
Table 1-The acidity level based on Dornic degree in the chamomile Lactobacillus
acidophilus milk and yoghurt within 20-day storage in the refrigerator
Acidity level in Dornic degree
Chamomile
milk
0%
1
day
500D
5
day
10
day
15
day
20
day
Chamomile
yoghurt
1
day
5 day
10
day
15
day
20
day
530D
500D
470D
490D
0%
980D
1190D
990D
1120D
1000D
0.2%
430D
460D
420D
400D
450D
0.2%
1130D
1200D
950D
950D
970D
0.4%
500D
590D
520D
470D
510D
0.4%
970D
1110D
930D
1000D
1130D
0.6%
530D
560D
530D
430D
520D
0.6%
1050D
1170D
1020D
1140D
1160D
Table 2- The microbes grows on MRS-A cultivation environment in Lactobacillus
acidophilus Chamomile milk and yoghurt at refrigerator during 20 days
insolubility
Chamomile
milk
1 day
5 day
10 day
15 day
20 day
Chamomile
yoghurt
1 day
5 day
10 day
15 day
20 day
0%
40×107
43×107
44×107
34×107
10×107
0%
16×107
160×107
20×107
24×107
‫ــ‬
%0.2
47×107
40×107
88×107
55×107
80×107
%0.2
26×107
16×107
20×107
24×107
3×107
%0.4
60×107
92×107
30×107
30×107
54×107
%0.4
17×107
26×107
72×107
20×107
2×107
%0.6
12×107
80×107
30×107
55×107
38×107
%0.6
39×107
36×107
72×107
10×107
20×107
Table 3- The mean ±SE of ALT, AST, ALP, cholesterol, triglyceride, HDLcholesterol, LDL-cholesterol, total protein and albumin.
Albumin
Total
(g/dl)
protein
ALP (U/L)
ALT
(U/L)
AST
(U/L)
(g/dl)
4.15±0.123
8.85±0.12
4.45±0.19
8.61±0.11
3.66±0.09
8.58±0.14
4.00±0.17
8.30±0.19
520.87±49.1
1
499.25±83.9
9
461.50±56.7
5*
412.87±45.5
8*
Significant in P<0.05*
Significant in P<0.01**
Histopathological examination:
55.5±
3.09
53.25±
3.99
52.00±
4.10
45.62±
3.23*
293±
42.38
290±
11.30
277.12
±30.41
267.37
±17.53
*
LDL-
HDL-
Cholest Triglycerid Param
Choleste
Choleste
erol
rol
rol
(mg/dl)
(mg/dl)
(mg/dl)
36.25±
1.501
37.87±
0.81
39.12±
0.9
41.00±
1.12*
26.00±
1.331
25.50±
0.72
29.12±0.6
1
31.00±
0.89 **
56.97±
2.69
47.45±
2.11
55.15±
2.50
56.12±
2.61
e
ers
Group
38.99±3.12
0%
48.12±3.91
0.2%
40.9±2.12
0.4%
53.00±2.61
**
0.6%
Control group 1: Milk containing Lactobacillus acidophilus, Control group 2: Milk containing
Bifidobacterium bifidum, experimental groups 1, 2, 3 in order received 0.2, 0.4 and 0.6 milk containing
Lactobacillus acidophilus and Mentha longifolia.
Control groups 3 and 4, respectively received
yogurt
4
2
1
contains Lactobacillus acidophilus and Bifidobacterium bifidum and the experimental Groups 7, 8 and 9,
respectively received Yogurt containing Lactobacillus acidophilus and Mentha longifolia 0.2 , 0.4 and
0.6. The experimental groups 4, 5, 6, 10, 11 and 12, respectively, received 0.2, 0.4 and 0.6 milk and
yogurt containing Bifidobacterium bifidum and Mentha longifolia.
Conclusion:
This study evaluated the effect of Mentha longifolia extract on increasing the growth of
probiotic bacteria Lactobacillus acidophilus and Bifidobacterium bifidum in milk and yoghurt
and their impact on biochemical factors including liver enzymes, cholesterol, triglycerides,
cholesterol-HDL, cholesterol-LDL, total bilirubin, creatinine, Albumin and total protein.
Changes in Mentha longifolia milk and yoghurt that contains Bifidobacterium bifidum and
Lactobacillus acidophilus in terms of indices such as acidity, pH and viability of probiotic
bacteria respectively in time periods of two-hour to achieve 42 dornic degree (milk) and 90
dornic degree (yoghurt) pH in 38 ° C oven were recorded. Lactobacillus acidophilus milk
samples containing % 0.6 Mentha longifolia extract faster than the rest of the samples, reached
the 42 dornic degree pH then % 0.4 Mentha longifolia and % 0.2 Mentha longifolia samples and
ultimately control sample in lactobacillus acidophilus probiotic milk reached 42 dornic degree
and then were transferred to a 2 ° C refrigerator. Product retention was determined 21 days in the
refrigerator. During this period, % 0.6 Mentha longifolia extract milk acidity was higher than the
other samples. Probiotic bacteria to get their properties must be at least 107 CFU.ml-1 per gr and
live in food consumer body (27).
In direct microbial count, total bacteria count in the first and fifteenth day of 0.6% extract
concentration was greater than the rest of concentrations. In this study, amount of bacteria was
10 10CFU.ml-1. Thus they create the properties of the probiotic microbes in consumer. The
number of microbes on the MRS Agar was 107 CFU.ml-1 colonies of microbes. Inhibitory and
lethal effects of low pH values on Bifidobacterium bifidum was higher than Lactobacillus
acidophilus. Therefore, it seems obvious that product shelf life and increasing fermentation
which decreases the pH is one of the main factors of Bifidobacterium growth slowing down (28).
Product retention was determined in refrigerator for more than 20 days and during this period
other samples yoghurt pH was set between 115 and 152 dornic degrees. Of course, this increase
in product retention was due to the acidity of the fermentation process. In Mentha longifolia
yoghurt containing Bifidobacterium bifidum sensory evaluation and Mentha longifolia different
concentration there was a significant difference between color and flavor of products (p<0.05)
and Bifidobacterium bifidum yoghurt with 0.4 percent of Mentha longifolia had best flavor and
color in the sensory evaluation. Although basis of probiotic products is their medicinal properties
(viability) but the sensory properties of these products have an important position. In other
words, taking advantage of probiotics through food and not as a drug is enjoying their sensory
properties. Among the probiotic products, fermented products and specially probiotic yoghurt
due to the unique sensory properties is internationally accepted. Probiotic yoghurt can be
considered as the most important probiotic product (29).
Zhao studies in 2005 showed that from a total of 21 species of Lactobacillus and
Bifidobacteriums that isolated from teenagers and youth stool, 6 species were able to remove
cholesterol from the in vitro culture (30). Ashar and Prajapati in a study in 2000 on 27
hyperlipidemic person found that daily consumption of 200 ml of milk containing Lactobacillus
acidophilus for 20 days had a significant effect in reducing serum total cholesterol (31).
Study by Park et al in 2007 was carried out on 36 rats which demonstrated that food containing
Lactobacillus acidophilus ATCC 43121, reduces serum total cholesterol by 25 percent but
cholesterol in the liver tissue was not significantly different (32). Chiu et al in 2006 during a
study found that milk fermented with three strains of Lactobacillus reduced serum and liver
lipids in hamsters fed a high-fat diet respectively about 30.1 and 13.4 percent (33).
In general we can say that milk and yogurt containing Mentha longifolia and Bifidobacterium
Lactobacillus acidophilus probiotic bacteria by reducing total cholesterol and increasing
triglycerides improves serum lipid pattern and can be a good food.
Authors' Contributions
Study concept and design, acquisition of data, analysis and interpretation of data, drafting of the manuscript, critical
revision of the manuscript for important intellectual content: W K and A GR. Statistical analysis: Z SH.
Administrative, technical, and material supports: F GH
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