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Biol Med (Aligarh)ISSN: 0974-8369 BLM, an open access journal Volume 7 • Issue 5 • BM-149-15

Biology and Medicine Prosekov et al., Biol Med (Aligarh) 2015, 7:5http://www.biolmedonline.com

Research Article Open Access

KeywordsOncology patients; Probiotic strains; Bioactive peptides; Intestinal

microflora

IntroductionOne of the major causes of mortality all over the world is oncological

diseases regardless of the level of development of healthcare. Despite the advances in modern science and medicine, it is not possible to reduce the morbidity and mortality level. The main instrument in this fight is the prevention and early diagnosis, which can achieve positive results. Studies conducted in many countries have shown a correlation between the development of the pathological process and those or other errors in the diet [1,2]. Probiotics have a positive effect on the health of the host by improving his/her intestinal microbial balance. Probiotic microorganism should be nonpathogenic and nontoxic and resistant to low pH and salts of bile acids, to increase the chances of survival in the gastrointestinal tract. Probiotics are used for the treatment and prevention of a wide variety of diseases, including cancer [3-12]. Most probiotics are members of two genera of bacteria which produce lactic acid, Lactobacillus and Bifidobacterium, as well as Saccharomyces and Enterococcus [6,13-17].

The objective of this research is to identify the most active probiotic strains isolated from the gastrointestinal tract of humans, to create functional food products for the rehabilitation of patients with cancer.

Materials and Methods

Isolation of strains and their antagonistic activitySamples of microorganisms were isolated from the gastrointestinal

tract of 500 healthy and sick with cancer people of various age groups (Russian Federation). Cultivation was carried out under anaerobic and aerobic conditions. Incubations were performed for 24 h at 37°C in an incubator. After incubation, cultural and morphological properties of microorganisms were determined according to traditional methods, and we count the number of colony forming units (CFU) of each type of colonies and CFU recalculation per 1 g (ml) of the test material. Pure culture was accumulated for identification and further study. Isolation of the microorganisms was carried out on agar SS (Oxoid CM99) and CPSID (BioMerieux 43211), followed by segregation. If necessary, samples were reconstituted in brain, heart broth. Identification of strains

was performed using standard biochemical classification methods using API20E, API 20 Ne, API Staphy and API Step (BioMerieux), according to manufacturer’s recommendations.

Each of the 23 isolated strains of the normal flora of the gastrointestinal tract of humans was designated: No. 1 – Actinomyces meyeri, No. 2 – Actinomyces odontolyticus, No. 3 – Bacteroides ovatus, No. 4 – Bifidobacterium bifidum, No. 5 – Bifidobacterium breve, No. 6 – Bifidobacterium dentium, No. 7 – Clostridium butyricum, No. 8 – Clostridium beijerinckii, No. 9 – Enterococcus gallinarum, No. 10 – Enterococcus faecalis, No. 11 – Escherichia coli, No. 12 – Eubacterium aerofaciens, No. 13 – Eubacterium lentum, No. 14 – Lactobacillus spp., No. 15 – Leptotrichia buccalis, No. 16 – Micrococcus spp., No. 17 – Neisseria mucosa, No. 18 – Peptostreptococcus asaccharolyticus, No. 19 – Sarcina spp., No. 20 – Staphylococcus xylosis, No. 21 – Staphylococcus cohni, No. 22 – Staphylococcus capitis, No. 23 – Streptococcus agalactiae

The antagonistic activity of bacteria was determined by perpendicular grooves on the surface of the medium. We streaked the culture of the test strain in Petri dishes and incubated it at 37°C for 96 h to form a diffusion in agar and inhibitory compounds. Then perpendicularly from the edge of the dish to the stroke of growing culture, streaked exponential culture of the test strain was tucked, lightly touching the stroke bacterium. The dish was incubated at 37°C for 36 h once again. The presence and degree of the antagonist activity in the test strain were predetermined by the magnitude of the zone of inhibition test strain at the interface with the prime bacterial growth.

Identification of Probiotic Strains Isolated from Human Gastrointestinal Tract and Investigation of Their Antagonistic, Antioxidant and Antiproliferative PropertiesA. Prosekov1,*, I. Milentyeva1, S. Sukhikh1, L. Dyshlyuk1, O. Babich1, L. Asyakina1, S. Ivanova1, M. Shishin1, L. Matskova2

1Department of Bionanotechnology, Kemerovo Institute of Food Science and Technology, 47 Stroiteley Boulevard 650056, Kemerovo, Russia2Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, 1 Solnavägen 17177, Solna, Stockholm, Sweden

Abstract

We researched the potential use of microorganism strains of different taxonomic groups, derived from the gastro-intestinal tract of healthy and sick with cancer people of different age groups, as probiotics, having antitumor effects in the products rehabilitation of oncological patients. We isolated the most active ones on antimicrobial, antioxidant, hepatoprotective and anticancer activity.

*Corresponding author: Prosekov A, Department of Bionanotechnology, Kemerovo Institute of Food Science and Technology, 47 Stroiteley Boulevard 650056, Kemerovo, Russia

Received: Nov 18, 2015; Accepted: Dec 9, 2015; Published: Jan 2, 2016

Citation: Prosekov A, Milentyeva I, Sukhikh S, Dyshlyuk L, Babich O, et al. (2015) Identification of Probiotic Strains Isolated from Human Gastrointestinal Tract and Investigation of Their Antagonistic, Antioxidant and Antiproliferative Properties. Biol Med (Aligarh) 7(5): BM-149-15, 5 pages.

Copyright: © 2015 Prosekov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


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for 30 min, 2 mM l-glutamine, 100 µg/ml penicillin and 100 µg/ml streptomycin sulfate (PanEco, RF). Cells were incubated in a СО2- incubator under 37 (C in 5% CO2 for 15 days to be fully differentiated).

Adhesion of cancer cellsCancer cells were seeded with 1 ml of medium containing 5 •

104-6,5 • 104 cells/well flat-bottomed 96-well microplate (Costar), and reincubated for 24 h before the addition of the test organisms. The obtained cell cultures (20-180 µl of cell suspension) were incubated for 48 h. The control wells were added with 0.9% sodium chloride solution in an adequate amount (20 µl). The number of live cells in each well after the incubation period was determined by the MTT colorimetric method.

Antiproliferation of colon cancer cells

MTT assayAn MTT calorimetric method based on the ability of

dehydrogenases of living cells to reduce 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) to purple formazan crystals, insoluble in DMSO. Optical absorption of colored solutions of dimethyl sulfoxide was measured on a tablet photometer Multiskan MS (Lab system, Finland) at λ 5 540 NM. Cytotoxicity of testing cell cultures was evaluated according to the formula:

% cell cytotoxicity 5 (1 – the optical absorption in the test samples/ the optical absorption in control) 3 100

Trypan Blue exclusion assayThe cancer cells (106 cells/1 ml of cell suspension) were added to

a 96-well plate. The suspension was incubated at 37 (C for 24 h. cell viability was examined using Trypan Blue exclusion):

% cell viability 5 (live cell count/total cell count) 3 100

Statistical analysisAll experiments were carried out with three replications. Processing

data were carried out by standard methods of mathematical statistics. Differences between means are considered significant when the confidence interval is smaller than 5% (p (0.05)).

Results

Characteristics of probiotic strainsThe isolated strains from the gastrointestinal tract of healthy

humans and patients microorganisms were divided into normal group, conditionally pathogenic and pathogenic microflora, and only the first group showed no pathogenicity. Antimicrobial, antioxidant, hepatoprotective and antitumor properties of these microorganisms (Actinomyces meyeri, Actinomyces odontolyticus, Bacteroides ovatus, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium dentium, Clostridium butyricum, Clostridium beijerinckii, Enterococcus gallinarum, Enterococcus faecalis, Escherichia coli, Eubacterium aerofaciens, Eubacterium lentum, Lactobacillus spp., Leptotrichia buccalis, Micrococcus spp., Neisseria mucosa, Peptostreptococcus asaccharolyticus, Sarcina spp., Staphylococcus xylosis, Staphylococcus cohni, Staphylococcus capitis, Streptococcus agalactiae) have been only subsequently studied, and each of the strains was assigned a number from No.1 to No. 23, respectively.

The antioxidant properties of microorganismsOriginal β-Phycoerythrin solution was prepared by dissolving

1 mg in 5.6 ml of phosphate buffer (0.2 M, pH 7.0). The working solution was prepared by mixing 300 µl of stock solution with 13.4 ml of phosphate buffer immediately before use. A solution of 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH) was prepared before the test. For this purpose 60 mg of AAPH was weighed and dissolved in 5 ml of phosphate buffer. A solution of 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid (Trolox) was prepared by dissolving 5 mg in 200 ml of 0.2 M phosphate buffer as a stock solution (100 µ). For obtaining the working solution, 1 ml of the solution was mixed with 9 ml of a phosphate buffer. The stock solution was stored at 2°C. Phosphate buffer was prepared by mixing 0.75 M of solutions NaH2PO4 and K2HPO4 to 61.6: 38.9 v/v. The mixture was then diluted 1: 9 with distilled water, and the pH adjusted to 7.0. Working solution (0.2 M) was stored at 2°C.

To each well of the plate, 20 µl of the corresponding sample and 160 µl of working solution with β-phycoerythrin were added. Immediately before measuring, 20 µl of AAPH was added to each well to initiate the reaction. The plate was covered and placed in the analyzer. In the analysis, the ability of compounds to protect phycoerythrin β-oxidation was monitored by the decay curve. Quantitation was achieved by measuring the net protection area in accordance with the curve of β-phycoerythrin decay in the presence of AAPH. To evaluate the antioxidant activity of culture, the aliquots of samples were diluted and control buffer was added to the reaction mixture; the degree of protection from oxidation of β-Phycoerythrin was quantified by measuring the relative fluorescence at 595 NM and at 535 NM for 70-min period:

The ability to absorb the oxygen radicals5 X·K (sample – Sblank)/(Strolox – Sblank)

Wherein X is sample volume, UL; K is the dilution factor; S represents the area under the curve corresponding to the index; sample is a pattern; and blank is control.

Hemolytic activity and antibiotic resistanceHemolytic activity was determined by the presence around the

colonies of the hemolysis zone after day incubation of the studied cultures crops of microorganisms to 5% in blood nutrient agar at 37°C for 24 h. The experiment used human blood O (I) Rh1.

Resistance of testing strains to antibiotics was analyzed with a set of disks, soaked with 0.4% (v/v) of different types of antibiotics (cefuroxime, ciprofloxacin, doxycycline, ampicillin, chloramphenicol, oxacillin, tetracycline, kanamycin, penicillin) by the diffusion method [18]. Discs were applied to dense nutrient medium seeded with the culture of test strain, discs with antibiotics were superimposed and cultured overnight at 37°С. Then we measured the diameter of the zone of inhibition around the discs.

Cell cultureCancer cells (LBR2 – Burkitt lymphoma, DU145 – human prostate

cancer, MDAMB-231 MCF7 – Human breast cancer, HepG2 – hepatocellular carcinoma, U-87 – brain cancer, PANC-1 – human pancreatic cancer) provided by Cancer Center, Karolinska (Sweden) were cultured in RPMI 1640 (PanEco, Russia) with 10% (v/v) of fetal bovine serum (Hyclone Laboratories, Logan, UK), inactivated 56°C


Page 3 of 5


Maximum antimicrobial activity against considered test strains are characterized by (Table 1) the following samples No. 4, No. 5, No. 14, No. 16 and No. 23; microbial strains: Bifidobacterium bifidum, Bifidobacterium breve, Lactobacillus spp., Micrococcus spp., Streptococcus agalactiae, respectively.

Preliminary isolation, identification and selection of strains of lactobacilli that do not exhibit hemolytic, cytotoxic, catalase, lecithinase, urease, gelatinase, RNA-ase, DNA-ase activity allowed to use them as promising strains to create a functional food for the rehabilitation of cancer patients. This expands the group of selected specimens, samples No.14/1 – Lactobacillus fermentum, No.14/2 –

Lactobacillus plantarum, No.14/3 – with Lactobacillus acidophilus, No.14/4 – Lactobacillus salivarius.

The isolated strains are characterized by different antibiotic resistance (Table 2).

DiscussionImportant properties of probiotic strains in connection with their

use in the technology of food products for functional rehabilitation of cancer patients is the antioxidant activity, hepatoprotective and anticancer properties.

Strain

The diameter of the growth inhibition zones of test cultures, mm

B-6954 (–) B-5651 () B-3502 (–) ATCC 9027 (–) B-8404 () ATCC 885-653 ATCC 25923 ()

1 5.6 0.3 8.7 0.4 11.6 0.8 12.3 0.6 7.2 0.4 8.5 0.4 9.0 0.5

2 15.5 0.8 12.4 0.6 8.8 0.4 10.2 0.5 6.3 0.4 13.4 0.7 12.0 0.6

3 5.3 0.3 8.5 0.4 6.2 0.3 10.1 0.5 11.8 0.6 6.3 0.3 7.8 0.4

4 36.5 1.8 28.9 1.4 32.0 1.6 29.3 1.6 30.9 1.5 33.1 1.7 37.0 1.9

5 28.4 1.4 30.6 1.5 33.2 1.7 35.6 1.8 26.8 1.3 29.0 1.5 31.1 1.6

6 5.5 0.3 8.9 0.4 14.5 0.7 12.0 0.6 11.4 0.6 10.5 0.5 9.3 0.5

7 7.7 0.4 11.5 0.6 8.0 0.4 14.7 0.7 15.0 0.8 12.2 0.6 6.8 0.3

8 10.6 0.5 12.2 0.6 8.9 0.4 9.5 0.5 5.8 0.3 7.9 0.4 11.0 0.6

9 13.8 0.7 10.0 0.5 7.4 0.4 8.5 0.4 12.2 0.6 10.0 0.5 11.0 0.5

10 4.8 0.2 11.6 0.6 10.3 0.5 6.7 0.3 8.9 0.4 10.5 0.5 11.3 0.6

11 7.5 0.4 12.3 0.6 13.4 0.7 9.5 0.5 10.9 0.5 14.7 0.7 15.0 0.8

12 6.6 0.3 11.2 0.6 13.6 0.7 12.5 0.6 14.6 0.7 15.3 0.8 8.6 0.4

13 5.5 0.3 8.9 0.4 12.4 0.6 13.0 0.7 12.7 0.6 14.5 0.7 16.0 0.8

14 24.9 1.2 38.2 1.9 32.4 1.6 29.7 1.5 28.5 1.4 29.6 1.5 33.5 1.7

15 7.7 0.4 10.3 0.5 14.6 0.7 12.3 0.6 14.0 0.7 11.8 0.6 11.0 0.6

16 33.9 1.7 27.8 1.4 25.2 1.3 36.7 1.8 35.5 1.8 34.0 1.7 32.2 1.6

17 13.5 0.7 18.9 0.9 14.2 0.7 12.0 0.6 15.5 0.8 13.4 0.7 9.6 0.5

18 13.6 0.7 12.0 0.6 16.0 0.8 6.9 0.3 8.7 0.4 9.0 0.4 11.2 0.6

19 11.8 0.6 14.5 0.7 13.9 0.7 15.6 0.8 13.3 0.7 14.4 0.7 16.7 0.8

20 9.6 0.5 11.5 0.6 12.0 0.6 14.3 0.7 7.8 0.4 8.4 0.4 9.2 0.5

21 6.6 0.3 13.4 0.7 15.9 0.8 12.2 0.6 8.7 0.4 9.5 0.5 10.2 0.5

22 4.6 0.2 11.2 0.6 12.5 0.6 13.7 0.7 14.5 0.7 15.6 0.8 7.7 0.4

23 34.0 1.7 27.6 1.4 29.0 1.5 35.5 1.8 33.1 1.7 36.5 1.8 38.4 1.9

Keys: B-6954 (2) 5 Escherichia coli, B-5651 (1) 5 Bacillus fastidiosus, B-3502 (2) 5 Pseudomonas fluorescens, ATCC 9027 (2) 5 Pseudomonas aeruginosa, B-8404 (1) 5 Leuconostoc mesenteroides, ATCC 885-653 5 Candida albicans, ATCC 25923 (1) 5 Staphylococcus aureus

Table 1: Antimicrobial activity of microbial strains

Strain

Antibiotic

I II III IV V VI VII VIII IX

Diameter of the inhibition zone, mm

4 23.0 1.2 0 36.5 1.8 33.2 1.7 30.6 1.5 0 27.6 1.4 28.3 1.4 0

5 0 0 3.2 0.2 27.5 1.4 23.4 1.2 32.2 1.6 0 29.8 1.5 30.8 1.5

14/1 36.6 1.8 0 33.0 1.7 0 0 29.9 1.5 24.5 1.2 32.0 1.6 25.6 1.3

14/2 0 35.4 1.8 14.2 0.7 0 13.4 0.7 32.0 1.6 34.6 1.7 11.9 0.6 15.6 0.8

14/3 0 15.6 0.8 0 17.0 0.9 0 35.6 1.8 0 37.0 1.9 28.5 1.4

14/4 0 0 18.9 0.9 0 0 24.5 1.2 22.7 1.1 30.5 1.5 26.5 1.3

16 27.8 1.4 0 25.6 1.3 23.9 1.2 0 22.7 1.1 0 33.4 1.7 35.0 1.8

23 32.9 1.6 0 0 33.0 1.7 35.7 1.8 0 34.2 1.7 27.8 1.4 0

Keys: I 5 cefuroxime, II 5 kanamycin, III 5 penicillin, IV 5 ciprofloxacin, V 5 doxycycline, VI 5 ampicillin, VII 5 tetracycline, VIII 5 chloramphenicol, IX 5 oxacillin

Table 2: Antibiotic resistance of strains


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Strain

The cancer cells

LBR 2 DU 145 MDAMB-231 MCF7 HepG2 U-87 PANC-1

Выживаемость клеток (при разной концентрации микроорганизмов, 105/ 107КОЕ/мл), %

486.3 1.2/45.9

0.690.8 1.3/52.1

0.777.5 1.1/38.7

0.592.6 1.3/46.1

0.6112.2 1.6/57.0

0.895.0 1.3/53.8

0.884.3 1.2/37.2

0.5

562.3 0.9/32.4

0.473.4 1.0/38.3

0.554.3 0.8/28.9

0.465.0 0.9/34.0

0.562.9 0.9/31.3

0.470.5 1.0/36.7

0.558.9 0.8/29.6

0.4

14/1106.2 1.5/52.1

0.894.0 1.3/46.7

0.668.9 0.9/32.4

0.477.1 1.1/34.6

0.585.6 1.2/35.0

0.587.0 1.2/44.2

0.692.2 1.3/48.9

0.7

14/253.4 0.7/25.5

0.459.0 0.8/28.7

0.462.5 0.9/31.4

0.460.6 0.8/29.7

0.455.2 0.8/27.5

0.457.3 0.8/28.7

0.464.2 0.9/32.1

0.4

14/375.6 1.0/33.0

0.568.9 1.0/23.5

0.370.2 1.0/18.7

0.373.4 1.0/21.0

0.361.2 0.9/15.6

0.260.5 0.8/19.8

0.358.9 0.8/23.4

0.3

14/493.2 1.3/49.8

0.788.0 1.2/45.6

0.675.4 1.0/38.9

0.584.2 1.2/43.2

0.680.3 1.1/42.0

0.677.3 1.1/35.6

0.572.1 1.0/36.9

0.5

16105.6 1.5/55.3

0.885.8 1.2/42.6

0.680.9 1.1/38.7

0.594.5 1.3/47.8

0.779.8 1.1/39.9

0.687.3 1.2/44.5

0.689.5 1.2/46.7

0.7

2390.2 1.3/48.9

0.7113.4 1.6/65.0

0.995.7 1.3/56.7

0.8103.7 1.4/52.4

0.788.4 1.2/45.6

0.696.7 1.3/48.9

0.779.0 1.1/43.4

0.6

Table 3: Anticancer properties of microbial strains

the maximum antioxidant activity in intact cells is observed in strains of Bifidobacterium breve, Lactobacillus salivarius and Lactobacillus plantarum.

We examined the hepatoprotective properties of the strains by observing the inhibiting effect of the microorganisms on β-glucuronidase production by E. coli HGU-3 strain. The main pathological conditions wherein there is an increase of serum β-glucuronidase are liver diseases, especially malignant tumors. The data obtained (Figure 2) indicate that pronounced hepatoprotective properties of the isolated strains are characterized by maximum inhibitory ability: Bifidobacterium breve, Lactobacillus salivarius and Lactobacillus acidophilus.

To study the antiproliferative activity of the isolated strains, the MTT test was performed on various human cancer cell lines (Table 3). All the studied strains have antitumor properties in varying degrees for the different types of cancer cells, which are most pronounced at a concentration of 107 CFU/ml. The presented results allow us to distinguish two strains: Lactobacillus plantarum and Lactobacillus acidophilus.

ConclusionProbiotics have a positive influence on the immune system as the

gastrointestinal flora lead in reducing the impact of mutagenic and carcinogenic substances. The study of phenotypic, morphological properties of the isolated microorganisms and their antimicrobial, antioxidant, anticancer and hepatoprotective properties allows us to identify the eight most active strains (Bifidobacterium bifidum, Bifidobacterium breve, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus salivarius, Micrococcus spp., Streptococcus agalactiae) for their use in the creation of functional food products for the rehabilitation of cancer patients. To improve the efficiency in the rehabilitation of patients with a particular type of cancer, the most promising is a compilation of probiotic complexes.

AcknowledgementsFinancial support for this research was provided by the Ministry of

Education and Science of the Russian Federation and research partner

From Figure 1, it follows that the cell-free extracts of strains Lactobacillus fermentum, Micrococcus spp. and Lactobacillus plantarum are characterized by highest antioxidant activity. At the same time,

Figure 1: Antioxidant activity of probiotic strains (a) intact cells and (b) a cell-free extract

Figure 2: The inhibitory effect of strains for the production of β-glucuronidase E. coli HGU-3


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