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Knowledge Base

Lactobacillus plantarum

Lactobacillus plantarum is classified as a lactic acid bacterium that has an important industrial function (Parente E., Ciocia F., Ricciardi A., Zotta T., Felis GE, Torriani S. 2010. Diversity of stress tolerance in Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum: a multivariate screening study. International Journal of Food Microbiology 144, 270-279). These bacteria are commonly found in fermented foods, olives, cheese, and wine and silage (Tanganurat W., Quinquis B., Leelawatcharamas V., Bolotin A., 2009. Genotypic and phenotypic characterization of Lactobacillus plantarum strains isolated from Thai fermented fruits and vegetables, Journal Basic Microbiology 49, 377–385). Therefore, strains of this species possessing probiotic properties are used in the production of functional and therapeutic foods as well as potential oral live vaccines (Shah N.P., 2007. Functional cultures and health benefits. International Dairy Journal 17, 1262-1277).

The mechanism of action of probiotic bacteria is multifactorial and specific for individual strains (Tuohy K.M., Probert H.M., Smejkal C.W., Gibson G.R., 2003. Using probiotics and prebiotics to improve gut health. Drug Discovery 8, 692–700). One of the better understood ways of probiotics against pathogens is antagonism based on the release of bacteriostatic and / or bactericidal substances by probiotics into the environment described by Kesarcodi-Watson et al. (Kesarcodi-Watson A., Kaspar H., Lategan MJ, Gibson L. , 2008. Probiotics
in aquaculture: The need, principles and mechanisms of action and screening processes. Aquaculture, 274, 1-14). It is believed that the presence of probiotics in the intestine or on the surface, skin, host inhibits the growth of potentially pathogenic bacteria (Verschuere L., Heang H., Criel G., Dafnis S., Sorgeloos P., Verstraete W., 2000. Protection of Artemia against the pathogenic effects of Vibrio proteolyticus CW8T2 by selected bacterial strains. Applied and Environmental Microbiology, 66, 1139-1146). This antibacterial effect is caused by individual or combined substances produced by bacteria such as antibiotics, bacteriocins, lysozyme, proteases, hydrogen peroxide, ammonia, diacetyl, siderophores, single organic acids (Verschuere L., Rombaut G., Sorgeloos P., Verstraete W., 2000. Probiotic bacteria as biological control agents in aquaculture. Microbiology and Molecular Biology Review, 64, 655-671). The effectiveness of antagonism based on the release of inhibitors into the environment is largely dependent on the conditions that the experiment is conducted in.

Biedrzycka et al. (Biedrzycka E., Markiewicz L.H, Bielecka M., Siwicki A.K., 2007. Shaping the gastrointestinal microecosystem, Control of gastrointestinal development
in newborn mammals, edited by Zabielski R., Wydawnictwo Rolne i Leśne, 5, 126-140) showed that the antagonistic activity of probiotic strains is not only the production of inhibitors, but also prevents colonization by coaggregation of probiotic bacteria cells with pathogen cells and competition for a site of attachment to the host’s mucous membranes. Various mechanisms are involved in this process, e.g., electrostatic interactions, hydrophobic interactions, lipoteichoic acids (Gómez R., Geovanny D., Balcázar JL, Shen M, 2007. Probiotics as control agents in aquaculture, Journal of Ocean University of China, 6 , 76-79).
These properties enable and help bacteria maintain a significant advantage
in the digestive tract of humans and animals, but the dynamic and very complex reaction of microorganisms is extremely important for intestinal epithelial cells and the host’s immune system (Shi HN, Walker A. Bacterial colonization and the development of intestinal defenses, 2004, Can. J Gastroenterol, 18, 493-500).

To determine the antibacterial ability of L. plantarum AMT14 against selected pathogens belonging to the species Salmonella enterica subsp. enterica serovar Enteritidis, Escherichia coli and Staphylococcus aureus they were co-cultured in hydrolyzed milk providing good growth for the inhibitory strain Lactobacillus plantarum AMT14 and the inhibited Salmonella enterica subsp. enterica serovar Enteritidis KOS64, Salmonella enterica subsp. enterica serovar Enteritidis 65 / s / 10, Escherichia coli O157: H7, Escherichia coli Nissle1917, Staphylococcus aureus ATCC33862.

 

In vitro tests showed a complete reduction in the number of Salmonella enterica subsp. enterica serovar Enteritidis KOS64, Escherichia coli Nissle1917 and Staphylococcus aureus ATCC33862 during 24 hours incubation, and Salmonella enterica subsp. strains enterica serovar Enteritidis 65 / s / 10 and Escherichia coli O157: H7 after 48 hours co-culture with Lactobacillus plantarum AMT14. There was no significant negative or positive effect of the medium itself on the results of the antibacterial activity of Lactobacillus plantarum AMT14 against selected pathogens belonging to the species Salmonella enterica subsp. enterica serovar Enteritidis, Escherichia coli and Staphylococcus aureus.

The results are given in Table 1 below.

 

Table 1 In vitro study on the antagonistic activity of the strain Lactobacillus plantarum AMT14 against pathogens of the gastrointestinal tract and skin

Lactobacillus plantarum AMT14 Escherichia coli O157:H7 Escherichia coli Nissle1917 Salmonella Enteritidis KOS64 Salmonella Enteritidis 65/s/10 Staphylococcus aureus ATCC33862
Inoculum
L. plantarum AMT14 1,80x109
E. coli O157:H7 2,81×106
E. coli Nissle1917 1,41×107
S. Enteritidis KOS64 1,24x105
S. Enteritidis 65/s/10 8,43x105
Staphylococcus aureus ATCC33862 6,10×106
24 h incubation
L. plantarum AMT14 2,76x109
E. coli O157:H7 5,30x108
E. coli Nissle1917 9,22x108
S. Enteritidis KOS64 1,20x109
S. Enteritidis 65/s/10 6,0x108
Staphylococcus aureus ATCC33862 2,45×108
Lactobacillus plantarum AMT14 Escherichia coli O157:H7 Escherichia coli Nissle1917 Salmonella Enteritidis KOS64 Salmonella Enteritidis 65/s/10 Staphylococcus aureus ATCC33862
48 h incubation
L. plantarum AMT14 1,13x109
E. coli O157:H7 3,20x108
E. coli Nissle1917 9,46×108
S. Enteritidis KOS64 2,00×108
S. Enteritidis 65/s/10 3,2×108
Staphylococcus aureus ATCC33862 2,24×107
L. plantarum AMT14
+ E. coli O157:H7
L. plantarum AMT14
+ E. coli Nissle1917
L. plantarum AMT14
+ S. Enteritidis KOS64
L. plantarum AMT14
+ S. Enteritidis 65/s/10
L. plantarum AMT14
+ S. aureus ATCC33862
Inoculum
L. plantarum AMT14 1,80x109 1,80×109 1,80x109 1,80x109 1,80x109
E. coli O157:H7 2,81×106
E. coli Nissle1917 1,41×107
S. Enteritidis KOS64 1,24x105
S. Enteritidis 65/s/10 8,43x105
Staphylococcus aureus ATCC33862 6,10×106
24 h incubation
L. plantarum AMT14 1,69×109 2,51×109 1,96×109 1,42×109 3,18×109
E. coli O157:H7 1,00×101
E. coli Nissle1917 Nb
S. Enteritidis KOS64 Nb
S. Enteritidis 65/s/10 7,50×103
Staphylococcus aureus ATCC33862 Nb
L. plantarum AMT14
+ E. coli O157:H7
L. plantarum AMT14
+ E. coli Nissle1917
L. plantarum AMT14
+ S. Enteritidis KOS64
L. plantarum AMT14
+ S. Enteritidis 65/s/10
L. plantarum AMT14
+ S. aureus ATCC33862
48 h incubation
L. plantarum AMT14 5,29x108 1,30×109 1,40x109 8,67x108 1,94×109
E. coli O157:H7 Nb
E. coli Nissle1917 Nb
S. Enteritidis KOS64 Nb
S. Enteritidis 65/s/10 Nb
Staphylococcus aureus ATCC33862 Nb

*Nb – absent

The tested strain Lactobacillus plantarum AMT14 showed 100% survival
at low pH = 3 and 89% survival in the presence of bile salts.

The results showed a high resistance of the Lactobacillus plantarum AMT14 strain to low pH and bile salts, which indicates that this strain has been adapted to the conditions in the gastrointestinal tract.

Bifidobacterium animalis

The genus Bifidobacterium (Latin “bifidus” – split) – first isolated and described in 1899-1900 by Tissier – are Gram-positive rods with irregular morphology (Krieg NR, Holt JG, Bergey’s Manual of Determinative Bacteriology, Williams and Wilkins , 1984, Vol. 2). They reach sizes in the range of 0.5-1.3 × 1.5-8μm. They can be simple or split. They differ in shape depending on the species and strain and the environmental conditions in which they live. Bacterial cells freshly isolated from the gastrointestinal tract or propagation media, under optimal incubation conditions usually show characteristic forms of X, Y or V, but also club-shaped or scapular, usually with rounded ends. Occur individually or in clusters, they do not move and do not form permanent forms.

Bifidobacteria require strictly anaerobic growth conditions (Müller G., Fundamentals of Food Microbiology WNT, 1990, Warsaw). They multiply at 37-41°C, in an environment with a pH in the range of 6.0-7.0. They produce basic metabolites – lactic and acetic acid. They show activity of the intracellular enzyme phosphoketolase-fructose-6-phosphate (FKF6F).
This enzyme participates in glucose metabolism in a specific pathway of enzymatic transformations – so-called the “bifid pathway”.
It is an enzyme characteristic of the genus Bifidobacterium – on its basis they can be identified by genus.

The natural habitat of bifidobacteria are the so-called crypts: 0.7-millimeter, densely packed cavities in the epithelium of the large intestinal mucosa (Saloff-Coste CJ, Bifidobacteria. Danone World Newsletter, 1997, 16, 1-9; Schlegel HG, General microbiology, 2000, PWN, Warsaw). Bacterial cells adhere to microvilli on the surface of enterocytes without causing damage to them and to mucosal epithelial cells.

Bifidobacteria perform many beneficial functions, such as (Hoover DG, Bifidobacteria: activity and potential benefits, Food Technol., 1993, 47, 120-124; Gibson GR, Wang X, Regulatory effects of bifidobacteria on the growth of other colonic bacteria, J. Appl. Bacteriol., 1994, 77, 412-420; Gomes AMP, Malcata FX, Bifidobacterium ssp. And Lactobacillus acidophilus: biological, biochemical, technological and therapeutical properties relevant for use as probiotics, 1999, Trends in Food Science and Technology, 10 , 139-157):

– participation in digestion and metabolism

– synthesis of vitamins: B1, B2, B6, B12, nicotinic and folic acid

– gastrointestinal protection against colonization of pathogens

– inhibiting the growth of putrefactive and pathogenic bacteria.

The development and final stabilization of the feeding microecosystem is shaped by the balance between the food microflora, physiological interaction of the body, diet and external factors (Biedrzycka E., Markiewicz LH, Bielecka M., Siwicki AK, 2007, Shaping the food microecosystem, shape the digestive structure) newborn mammals, edited by Zabielski R., Wydawnictwo Rolne i Leśne, 5, 126-140). The acetic and lactic acids produced by these bacteria lower the pH of the large intestine, inhibiting the growth of pathogenic bacteria such as Escherichia coli, Shigella, and Clostridium perfringens. Due to the very important role of bifidobacteria in the required shape of microorganisms in the initial stage of the microecosystem, meals intake and the fact that later in life the size of the bifidobacteria population is seriously dependent on treatment, which results in accelerating the aging process, deteriorating health and well-being . Bifidobacterium animalis AMT30 with confirmed bacterial properties against pathogenic bacteria and fungi.

The Bifidobacterium animalis AMT30 strain has been isolated from human stool. The strain was deposited in accordance with the Budapest Treaty in the Polish Collection of Microorganisms (PCM) at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, 53-114 Wrocław, ul. Rudolf Weigel 12. The deposit was made on 31-05-2016. The deposit was given the number B / 00109

Investigations of the antagonistic properties of the Bifodobacterium animalis AMT30 strain against pathogens: Escherichia coli O157: H7 (enterohemorrhagic strain), Candida albicans 637, Candida krusei 8.

In vitro tests showed a complete reduction in the number of enterohemorrhagic Escherichia coli O157: H7 bacteria as well as Candida albicans 637 and Candida krusei 8 during 72 hours incubation of co-culture with Bifidobacterium animalis AMT30. The results are given in Table 2 below.

Table 2. Inhibition of growth of pathogenic bacteria and fungi by Bifidobacterium animalis AMT 30

strain

Joint breeding symbol

Inoculum 24 h incubation 48 h incubation 72 h incubation
cfu/ml cfu/ml cfu/ml
cfu/ml AMT30 pathogen AMT30 pathogen AMT30 pathogen
AMT30 pathogen
Bifidobacterium animalis AMT30

(monoculture)

1,8×109 2,4×109 2,4×109 2,1×109
Escherichia coli O157:H7

(monoculture)

2,9×105 1,1×109 8,8×108 5,2×108
Candida albicans 676

(monoculture)

6,1×105 1,8×107 1,5×107 1,3×107
Candida krusei 8

(monoculture)

1,2 x105 4,1×108 2,9×108 2,4×108
Bifidobacterium animalis AMT30 + Escherichia coli O157:H7 1,8×109 2,9×105 2,7×109 1,8×106 2,5×109 3,1 x102 2,1×109 Nb*
Bifidobacterium animalis AMT30 + Candida albicans 676 1,8×109 6,1×105 2,7×109 1,1×106 3,1×109 1,1 x103 2,8×109 Nb*
Bifidobacterium animalis AMT30 + Candida krusei 8 1,8×109 1,2×105 2,5×109 1,4×106 3,6×109 7,3 x102 3,8×109 Nb*

Nb*- absent in 1 ml joint culture with Bifidobacterium animalis AMT30

The survival of the Bifidobacterium animalis AMT30 strain at low pH was determined by reducing the acidity of the Bifidobacterium animalis AMT30 culture being in the stationary growth phase to a pH value of 3.
However, when determining the survival of the Bifidobacterium animalis AMT30 strain in the presence of bile salts, at first the pH of the Bifidobacterium animalis AMT30 culture was increased to a value of 6, and then bile salts in 3% of the culture were added .

Table 3 Survival of the Bifidobacterium animalis AMT30 strain at pH = 3

Number of bacteria (log10 cfu/ml) Survival after 180 minutes
before lowering the pH pH=3
0 0 40 minutes 180 minutes %
Bifidobacterium animalis AMT30 9.17 9.16 9.18 9.28 100

Table 4 Survival of the Bifidobacterium animalis AMT30 strain in the presence of 3% bile salts

Number of bacteria (log10 cfu/ml) Survival after 6 h

[%]

before adding bile salts after adding bile salts
0 h 1 h 3 h 6 h
Bifidobacterium animalis AMT30 9.07 9.10 9.00 8.88 8.50 94

Bifidobacterium animalis AMT30 strain demonstartes survival
at low pH = 3 and 94% survival in the presence of bile salts with a concentration of 3%

The results showed a high resistance of the Bifidobacterium animalis AMT30 strain to low pH and bile salts, which indicates that this strain has been adapted to the conditions of the gastrointestinal tract.

Bifidobacterium animalis AMT30 exhibits a unique broad spectrum of antibiotic resistance. In the presence of some antibiotics: colistin, toltrazuril, amprolium, levamisole hydrochloride, flubendazole, neomecin, sulfachloropyrazine sodium, excellent ability to multiply the AMT30 strain was found in relation to the number of strains achieved in the control culture. In contrast, in the presence of Trimetoprim / sulfmethoxazole, Tylmykosine and tylosin, the number of Bifidobacterium animalis AMT30 remained at the inoculum level. In the case of co-cultures with Tiamulin, Enrofloxacin, Florfenicol, Amoxicycline, Doxycycline, Amoxicycline + clavulanic acid, Phenoxymethylpenicillin, Lincomycin and Tylvalosin, a decrease in the number of bacteria of the AMT30 strain was noted from 1 to 3 orders of magnitude compared to the control.

 

RESEARCH – RESULTS

Based on microbiological analysis verifying the activity of products against selected pathogens NATURE SCIENCE’S (MYCOBIOTIC) turned out to be 5 times better than product B in activity against Staphylococcus aureus koag (+) during 24h incubation and 10 times better during 48h incubation

In the case of activity against Enterococcus faecalis L and Candida albicans 676, NATURE SCIENCE’S MYCOBIOTIC proved to be:

after 24h incubation, 1,000,000 times better and 100 times better;
after 48 h incubation, 1000 times better and 10,000 times better.

MYCOBIOTIC showed equivalent activity to Product B when it comes to other pathogens.

Tested material Incubation time
0 h (inoculum)

CFU/mL

24 h

CFU/mL

48 h

CFU/mL

PRODUCT B (multivalent) 1,9 x 109 1,4 x 109 1,3 x 109
PRODUCT A (L. rhamnosus GG) 2,2 x 108 6,2 x 108 3,8 x 108
NATURE SCIENCE PRODUCT (Lactobacillus plantarum AMT14 +
Bifidobacterium animalis AMT30
1,5 x 109 5,3 x 109 2,3 x 109
Escherichia coli O157:H7 6,0 x 107 3,1 x 108 1,1 x 108
Staphylococcus aureus koag (+) 9,8 x 107 1,1 x 109 9,8 x 108
Salmonella enteritidis KOS-64 1,7 x 107 1,6 x 108 6,2 x 107
Enterococcus faecalis L 6,8 x 107 1,5 x 108 8,4 x 107
Candida albicans 676 1,7 x 107 3,0 x 107 2,0 x 107
Probiotic product + pathogen
Escherichia coli O157:H7 + PRODUCT B (multivalent) 6,0 x 107 <1,0 x 100 <1,0 x 100
Escherichia coli O157:H7 + PRODUCT A (L. rhamnosus GG) 6,0 x 107 2,30 x 105 <1,0 x 100
Escherichia coli O157:H7 + NATURE SCIENCE PRODUCT 6,0 x 107 <1,0 x 100 <1,0 x 100
Staphylococcus aureus koag. (+) + PRODUCT B (multivalent) 9,8 x 107 5,5 x 101 <1,0 x 101
Staphylococcus aureus koag (+) + PRODUCT A (L. rhamnosus GG) 9,8 x 107 1,3 x 104 3,4 x 102
Staphylococcus aureus koag (+) + NATURE SCIENCE PRODUCT 9,8 x 107 1,0 x 101 <1,0 x 100
Salmonella enteritidis KOS – 64 + PRODUCT B multivalent) 1,7 x 107 <1,0 x 100 <1,0 x 100
Salmonella enteritidis KOS-64 + PRODUCT A (L. rhamnosus GG) 1,7 x 107 <1,0 x 100 <1,0 x 100
Salmonella enteritidis KOS-64 + NATURE SCIENCE PRODUCT 1,7 x 107 <1,0 x 100 <1,0 x 100
Enterococcus faecalis L + PRODUCT B (multivalent) 6,8 x 107 2,0 x 106 3,0 x 103
Enterococcus faecalis L + PRODUCT A (L. rhamnosus GG) 6,8 x 107 5,4 x 107 3,6 x 103
Enterococcus faecalis L + NATURE SCIENCE PRODUCT 6,8 x 107 <1,0 x 100 <1,0 x 100
Candida albicans 676 PRODUCT B (multivalent) 1,7 x 107 1,6 x 105 3,8 x 104
Candida albicans 676 + PRODUCT A (L. rhamnosus GG) 1,7 x 107 1,0 x 105 4,6 x 104
Candida albicans 676 + NATURE SCIENCE PRODUCT 1,7 x 107 8,4 x 103 <1,0 x 100
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