Hypocholesterolemic Operating Parameters of Novel Probiotics In vitro
Botany and Microbiology Dept., Faculty of Science (Boys), Al-Azhar University, Cairo, Egypt.
*Corresponding Author E-mail: amrelwaseif@azhar.edu.eg, hussein.sayed.2016@gmail.com, ewais_e@yahoo.com
The Hypocholesterolemic is an effective and friendly process for the removal of cholesterol and an important aspect of human health. Removal of cholesterol by probiotic bacteria was investigated in this study. The potential probiotic bacterium was preliminarily identified as Lactobacillus casei. In addition, the various operating parameters like cholesterol concentration, incubation time, bile salts concentration, probiotic dose and initial pH was studied. The optimum parameters for most probiotics studied are cholesterol concentration is 100 μg /ml, incubation time is 48 h, bile salts concentration is 0.5%, probiotic dose is 200μl and initial pH is 6. The maximum percentage removal of cholesterol using Lactobacillus casei is 93.6. The probiotics bacteria are favorable for Hypocholesterolemic.
KEYWORDS: Cholesterol; Probiotic; Lactobacillus; Hypocholesterolemic activity.
INTRODUCTION:
According to the definition by the World Health Organization (WHO), probiotics are ‘‘live microbial food supplements which, when administered in adequate amounts confer a health benefit on the host” or more broadly as “living microorganisms, which upon ingestion exert health benefits beyond inherent general nutrition”1.
Humans have been used lactic acid bacteria for fermentation and preservation of various food products for major health benefits. Lactic acid bacteria produce several metabolites which include antimicrobials and flavoring agents that augment the stability and aroma of the food products2. Probiotic benefits include: Increased resistance to infectious diseases3-4, particularly of the intestine, decreased duration of diarrhea5-6, reduction in blood pressure, reduction in serum cholesterol concentration, reduction in allergy, stimulation of phagocytosis by peripheral blood leucocytes7-8, modulation of cytokine gene expression, regression of tumors and reduction in carcinogen or co-carcinogen production9-10.
They are classified as generally recognized as safe (GRAS) microorganisms because of their long and safe use as starter cultures in fermented products11.
Cholesterol plays a major role in human heart health. High-density lipoprotein (HDL) is good cholesterol and low-density lipoprotein (LDL) is bad cholesterol. Excess cholesterol in the bloodstream can form plaque (a thick, hard deposit) in artery walls. The cholesterol or plaque build-up causes arteries to become thicker, harder and less flexible, slowing down and sometimes blocking blood flow to the heart. When blood flow is restricted, angina (chest pain) can result. A heart attack will result when blood flow to the heart is severely impaired and a clot stops blood flow completely. When there is too much LDL cholesterol in the blood, it deposited inside the blood vessels, where it can build up to hard deposits and cause atherosclerosis, the disease process that underlies heart attacks12.
Recently, several studies have been published on the probiotics bacteria are found to produce bile salt hydrolase (BSH) that helps to reduce serum cholesterol and hence BSH activity is also considered as an additional criterion for the selection of probiotics13. Some oral bacteria such as Lactobacillus acidophilus have been commercial available for the cholesterol lowering14.
The present study, Studies of five hypocholesterolemic parameters were used to carry out cholesterol removal using probiotics in vitro. The effect of various parameters such as cholesterol concentration, incubation time, bile salts concentration, probiotic dose and initial pH has been studied15.
Samples were collected from the normal habitats of lactic acid bacteria (LAB) such as fresh raw animal milk, fermented foods cheese, infant feces.
Milk and cheese samples were incubated at 37ºC then cultured in sterilized MRS broth16 and incubated. Samples were then streaked on over agar surface of MRS medium and were incubated aerobically at 37ºC for 48 h. infant faeces were diluted serially, then 0.1ml was spread on to MRS plates and incubated at 37ºC for 48h. White and creamy colonies were picked up randomly and purified by three successive transfers on MRS medium. The cultures were routinely checked for purity by microscopic examination. The pure cultures
Gram-positive and catalase-negative were characterized cell morphology according
to17. Various biochemical tests were performed
to evaluate the characteristic profile
of the selected isolate18.
Freshly prepared MRS broth was supplemented with 0.5
% sodium thioglycholate as a bile salt. Soluble cholesterol added to the liquid at a final concentration of 100μg/ml inoculated with samples then incubated at 30°C for 24 h. After the incubation period, cells were centrifuged and the remaining cholesterol concentration in the broth was determined using enzymatic method19 (good mix the samples and then take 10 micron from any Samples and added to 1ml from enzymatic reagent and good mix and incubate at 37ºC in water pass for 10 mints and then read residual cholesterol according to stander curve (figure 1).
Removal cholesterol = (Total cholesterol – Residual Cholesterol). Reference solutions and stander curve were prepared in identical manner as above.
Figure 1: Cholesterol stander curve
1. Determination of cholesterol removal concentration:
Determination of the maximum level of cholesterol removal for five strains can be assimilated in MRS broth media in the presence of 0.5% sodium thioglycholate at 30ºC after 24 incubation period. A series of cholesterol concentrations 80, 100, 120, 150, 180 and 200μg/ml were individually added to MRS medium, while the other ingredients of the medium were remained unchanged. Results presented in tables 1 revealed that Cholesterol assimilated increased with the increase of cholesterol concentration reaching its maximum value of cholesterol removal 93% when 100μg/ml cholesterol was added for Lactobacillus casei.
In general, our strains assimilated more cholesterol in the concentration lower than 200μg/mL compared with higher concentration. Results indicated that our strains in concentration above 120μg/mL decreased the amount of cholesterol could be assimilated. The concentration of cholesterol recorded the highest removal percentage are 120μg/ml for Lactobacillus brevis 25, 14 and 34 but 100, 80μg/ml for Lactobacillus casei, Pediococcus acidilactici respectively.
Lactobacilli were able to remove or reduce the concentration of cholesterol in vitro through different mechanisms there were five possible mechanisms for the removal of cholesterol from media, namely assimilation of cholesterol during growth, binding of cholesterol to cellular surface, disruption of cholesterol micelle, deconjugation of bile salt and (bile salt hydrolase enzyme) BSH activity. Therefore, the strains studied could be used as potential health adjunct cultures in fermented dairy products to provide additional nutraceutical benefits20.
The relation between incubation time and removal of cholesterol was determined at different incubation time intervals (12, 24, 36, 48, 60, 72, 84 and 96 h) under the previously established optimal conditions for each strain. Results given in tables 2 explain the growth curve of strains and removal of cholesterol during culture for 96 h. The cholesterol removal by all strains was increased linearly to reach its maximal removal 93.6 % at 48h for Lactobacillus casei. The rapid cholesterol removal by strains achieved during 60 to 72hrs of incubation corresponded to its exponential growth phase. In general, strains assimilated more cholesterol in the incubation time over than 48h compared with longer time. Results indicated that strains in time above 72h decreased the amount of cholesterol could be assimilated. Other result was obtained that the optimum incubation time for removal cholesterol from broth media was during 12 to 18 h21.
This experiment was designed to identify the most favorable concentration of bile salt Concentration on cholesterol removal activity of five strains. A series of bill salt concentrations of 0.1, 0.2, 0.3, 0.5, 0.7 and 1.0 % were individually added to MRS medium under the previously established optimal conditions for each strain, while the other ingredients of the medium were remained unchanged. Results presented in tables 3 revealed that cholesterol removal activity increased with the increase of bile salt concentration reaching its maximum value of 93 % when 0.5% bile salt was added for Lactobacillus casei. In general, strains assimilated more cholesterol in the bile salt concentration lower than 0.5% compared with higher concentration. Results indicated that strains in concentration above 0.5% decreased the amount of cholesterol could be assimilated. The optimum bile salt concentration was 0.5 % for Lactobacillus casei, Lactobacillus brevis 14 and Lactobacillus brevis 34 but 0.2% for Pediococcus acidilactici and Lactobacillus brevis 25 respectively.
Table 1: Effect of cholesterol concentrations on cholesterol removal
Several studies have indicated that the mechanism for in vitro removal of cholesterol is linked to the bile salt hydrolase activity of probiotic strains. Moreover, the decomposition of bile salts by BSH enzyme would disrupt the formation of the cholesterol micelle which in turn prevents cholesterol absorption22.
|
Cholesterol Conc. (μg/ml) |
Cholesterol Removal Percentage (%) |
||||
|
Lactobacillus casei |
Pediococcus acidilactici |
Lactobacillus brevis 14 |
Lactobacillus brevis 25 |
Lactobacillus brevis 34 |
|
|
80 |
91 |
83 |
66 |
58 |
66 |
|
100 |
93 |
77 |
67 |
67 |
67 |
|
120 |
78 |
72 |
67 |
85 |
72 |
|
150 |
78 |
71 |
62 |
80 |
71 |
|
180 |
78 |
76 |
68 |
80 |
68 |
|
200 |
78 |
78 |
66 |
75 |
65 |
Table 2: Effect of different Incubation Period on cholesterol removal
|
Incubation Period (h) |
Cholesterol Removal Percentage (%) |
||||
|
Lactobacillus casei |
Pediococcus acidilactici |
Lactobacillus brevis 14 |
Lactobacillus brevis 25 |
Lactobacillus brevis 34 |
|
|
12 |
0 |
0 |
0 |
0 |
0 |
|
24 |
24 |
7.5 |
53 |
8.3 |
57.7 |
|
36 |
78 |
57.9 |
65.8 |
32.7 |
64.1 |
|
48 |
93.6 |
81.2 |
80.3 |
84.1 |
72.5 |
|
60 |
91.9 |
85.0 |
81.1 |
91.6 |
80.8 |
|
72 |
91.9 |
85.4 |
80.8 |
92.5 |
81.6 |
|
84 |
91.9 |
85.4 |
80.8 |
92.2 |
81.6 |
|
96 |
91.9 |
85.0 |
80.8 |
92.2 |
81.1 |
Table 3: Effect of Bill salt Concentration on cholesterol removal
|
Bill salt Conc. (%) |
Cholesterol Removal Percentage (%) |
||||
|
Lactobacillus casei |
Pediococcus acidilactici |
Lactobacillus brevis 14 |
Lactobacillus brevis 25 |
Lactobacillus brevis 34 |
|
|
0.1 |
87 |
74 |
67 |
67 |
67 |
|
0.2 |
90 |
77 |
71 |
73 |
67 |
|
0.3 |
90 |
77 |
71 |
73 |
71 |
|
0.5 |
93 |
74 |
74 |
73 |
74 |
|
0.7 |
93 |
74 |
71 |
71 |
67 |
|
1.0 |
93 |
74 |
71 |
71 |
67 |
To select the most suitable initial pH values; which support the hypocholesterolemic at different pH values (4, 5, 6, 6.5 and 8) under the previously established optimal conditions for each strain were tested. The results in table 4 revealed that the maximum level of cholesterol removal could be obtained at initial culture pH value of 6.0. Above or below this range, a decrease in the level of the cholesterol removal was recorded. On the other hand, pH values between 5.0 and 6.5 provided in adequate cell growth but it also revealed that there is a marked increase in hypocholesterolemic at pH values of 6 and 6.5. So the optimum pH is 6.0. The decreasing pH values, glycine-conjugated bile salts will be partially precipitated without hydrolysis, and conjugated bile salts will be protonated and precipitated. Because bile salt hydrolase has an isoelectric point between 4.4 and 4.623.
Table 4: Effect of pH values on cholesterol removal
|
pH values |
Cholesterol Removal Percentage (%) |
||||
|
Lactobacillus casei |
Pediococcus acidilactici |
Lactobacillus brevis 14 |
Lactobacillus brevis 25 |
Lactobacillus brevis 34 |
|
|
4.0 |
77 |
71 |
67 |
71 |
70 |
|
5.0 |
77 |
74 |
70 |
74 |
70 |
|
6.0 |
83 |
77 |
73 |
74 |
77 |
|
6.5 |
83 |
77 |
73 |
74 |
77 |
|
8.0 |
83 |
74 |
67 |
74 |
77 |
|
9.0 |
80 |
71 |
67 |
71 |
73 |
In this experiment effect of inoculating the fermentation medium with different inoculums size ranging from 25- 200 μl/10 ml on cholesterol removal was investigated. This inoculum were prepared from a stock culture of 24 h old (10 ml/l) and used to inoculate tubes of 50 ml capacity containing 25 ml of optimized fermentation medium under the previously established optimal conditions for each strain. Results given in Table 5 indicated that cholesterol removal was positively responded to increase of inoculum size. The highest cholesterol removal 93 % was recorded using 150 μl/10ml inoculum for Lactobacillus casei. In general, strains assimilated more cholesterol in the inoculum size 200 μl compared with lower concentration. Results indicated that strains in concentration lower 200 μl decreased the amount of cholesterol could be assimilated.
Table 5: Effect of inoculum size on cholesterol removal
|
Inoculum size (μl) |
Cholesterol Removal Percentage (%) |
||||
|
Lactobacillus casei |
Pediococcus acidilactici |
Lactobacillus brevis 14 |
Lactobacillus brevis 25 |
Lactobacillus brevis 34 |
|
|
25 |
43 |
20 |
10 |
37 |
33 |
|
50 |
63 |
53 |
30 |
57 |
53 |
|
100 |
87 |
77 |
63 |
67 |
73 |
|
150 |
93 |
77 |
67 |
83 |
77 |
|
200 |
93 |
84 |
73 |
87 |
80 |
Based on our results, the analysis of operating parameters of cholesterol removal for five probiotic strains studied. Incubation period and inoculums size were the most significant factor affecting on cholesterol removal. Therefore, the optimum cholesterol concentration was 100μg/ml, incubation time 48 h, bile salts 0.5%, probiotic dose 200μl and initial pH 6. Accordingly, owing to its good probiotic properties, this strain could be potentially used in functional food and health products especially where cholesterol reduction in food is the main target. Further in vivo study is necessary to prove the hypocholesterolemic effect of the Lactobacillus casei. Moreover in vitro studies are required to determine the mechanism (s) involved in the reduction of cholesterol by such a promising isolate. These results are suggested that Lactobacillus casei is a promising probiotic functional component for reducing serum cholesterol concentration. These findings and suggested cholesterol removal parameters in this work might be opening a novel trend in dealing with hypercholesterolemia and related diseases24-25.
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Received on 13.12.2020 Modified on 26.01.2021
Accepted on 23.02.2021 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(10):5197-5201.
DOI: 10.52711/0974-360X.2021.00904